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Roles of CER6 and CER7 proteins in the biosynthesis and regulation of wax production in Arabidopsis thaliana Hooker, Tanya Suzanne 2003

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Roles of CER6 and CER7 proteins in the biosynthesis and regulation of wax production in Arabidopsis thaliana By  Tanya Suzanne Hooker MSc, University of Calgary, 1995 BSc, University of Calgary, 1992  A thesis submitted in partial fullfilment of the requirements for the degree of Doctor of Philosophy  in  The Faculty of Graduate Studies Department of Botany  We accept this thesis as conforming To the required standard  The University of British Columbia September 2003 © Tanya Suzanne Hooker, 2003  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e 'and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e h e a d o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  Department o f The U n i v e r s i t y o f B r i t i s h V a n c o u v e r , Canada  Date  S£PT<zM&EK  Columbia  /2 2oo3 ;  ii ABSTRACT  Plant cuticular waxes embed and overlay the cutin matrix of higher plant cuticles. By forming the primary hydrophobic barrier and interface between the plant and the environment, they play important roles in plant responses to abiotic factors, including drought, light and temperature. Cuticular waxes also play important roles in mediating biotic relations, such as plant-insect interactions, as well as fungal and bacterial host and non-host responses. Numerous wax deficient mutants have been isolated from Arabidopsis. The mutations causing wax deficiency identify gene functions required for wax deposition and allow cloning of these genes. These gene functions include biosynthetic enzymes, regulatory factors, and proteins involved in wax secretion. Thus, they are an invaluable.resource for increasing our understanding of wax production. Several wax-related genes have been cloned in recent years. One of these genes is CER6, which encodes an enzyme involved in the first step of wax biosynthesis: the elongation of fatty acids to produce very long chain fatty acid (VLCFA) wax precursors. Since the cer6 mutant has a severe waxless phenotype due to a lesion in a protein early in the wax biosynthetic pathway, we hypothesized that wax deposition could be controlled, at least in part, by the regulation of CER6 expression in plants. To determine the extent of its role in the plant, I investigated its expression pattern in detail. M y results indicate that CER6 is transcribed exclusively in epidermal cells throughout shoot development. This expression pattern, and the increase in wax load found in transformed plants over-expressing CER6 confirmed that CER6 is a key enzyme involved in wax synthesis in Arabidopsis shoots. To investigate whether wax deposition in Arabidopsis was influenced by environmental factors, and if this process was to some extent mediated by transcriptional regulation of CER6, I examined CER6 transcript levels and wax loads of plants grown in the absence of light, plants exposed to osmotic stress and different temperature regimes, as well as in wounded plants. I found that light is required for CER6 transcript accumulation, and that osmotic stress, cold temperatures, and A B A induce CER6 transcription. Changes in wax load correlated with CER6 transcript levels in cold-treated plants. However, no correlations could be established for the other conditions tested.  To further investigate the regulation of wax deposition in Arabidopsis, I isolated the CER7 gene marked by a mutation in the wax-deficient cer7 mutant, using a positional cloning approach. I first accurately determined the chromosomal location of the cerl gene using simple sequence length polymorphism (SSLP) marker analysis in an F2 mapping population, and then identified the CER7 gene by complementing the cerl mutant using genomic fragments from the chromosomal area of interest. I also characterized a Salk Institute T - D N A insertion line with an insertion site 400 bp 5' to the CER7 translational start codon, which represents a second cer7 allele. CER7 is a putative 3'-5' RNAsePH homologue of the yeast Rrp45p, a protein which is one of 10 core proteins forming an R N A processing and degradation complex, the exosome. Transcript analysis of all of the cloned wax-related genes in the cer7 mutant background revealed that none of them was affected by this mutation. Therefore, the CER7 target R N A must represent a factor which has not yet been identified. The results presented in this thesis represent a first concrete step toward elucidating the pathways of regulation of wax production in Arabidopsis, in that I have established the transcriptional regulation pattern of the only known gene coding for an enzyme involved in wax biosynthesis (CER6), and I have identified a new gene (CERT) that is likely to be involved in regulating wax production by influencing the transcript stability of wax-related genes.  T A B L E OF CONTENTS  page ii  ABSTRACT T A B L E OF CONTENTS  iv  LIST OF T A B L E S  viii  LIST OF FIGURES  ix  ABBREVIATIONS  xii  ACKNOWLEDGEMENTS  xiv  DEDICATION  xv  ;  C H A P T E R 1. I N T R O D U C T I O N A N D L I T E R A T U R E R E V I E W  1.1. THE P L A N T CUTICLE A N D C U T I C U L A R W A X E S 1.2. THE PROTECTIVE R O L E OF EPICUTICULAR W A X E S 1.3. INVESTIGATION OF W A X DEPOSITION: BIOCHEMICAL, GENETIC, A N D GENOMIC TOOLS 1.4. O V E R V I E W OF W A X BIOSYNTHESIS: V L C F A s A N D THEIR DERIVATIVES 1.5. F A T T Y ACID E L O N G A T I O N 1.5.1. Biochemical characterization of fatty acid elongation 1.5.2. Molecular characterization of F A E component enzymes Condensing enzymes In silico analysis of expression patterns of Arabidopsis K C S genes Comparison of in silico data with experimental results for some K C S genes K C S mutants in Arabidopsis Biochemical characterization of K C S activities Phylogenetic relationships of plant KCSs K C S conclusions Reductases P-hydroxyacyl-CoA dehydratase 1.5.3. Summary 1.6. THE D E C A R B O N Y L A T I O N P A T H W A Y 1.6.1. Fatty acyl-CoA reduction produces aldehydes 1.6.2. Aldehyde decarbonylation produces alkanes 1.6.3. Alkane derivatives: secondary alcohols and ketones 1.7. THE A C Y L - R E D U C T I O N P A T H W A Y 1.7.1. Fatty acyl-CoA reduction produces primary alcohols 1.7.2. Esterification of primary alcohols and fatty-acyl-CoAs  1  1 4 7 11 13 15 18 18 21 25 27 29 32 37 37 38 45 45 46 46 49 49 49 51  V  1.8. W A X SECRETION 1.8.1. Intracellular trafficking 1.8.2. Transit through the apoplast 1.9. R E G U L A T I O N OF W A X PRODUCTION 1.9.1. Mutants affecting the regulation of wax production 1.9.2. Developmental influences on wax synthesis and deposition 1.9.3. Environmental influences on wax synthesis and deposition 1.10. R E S E A R C H GOALS 1.10.1. Thesis objectives C H A P T E R 2. M A T E R I A L S A N D M E T H O D S  2.1. P L A N T GROWTH CONDITIONS 2.1.1. General 2.1.2. Plant material 2.1.3. Terminology 2.1.4. Experiments testing effects of different environmental factors on CER6 expression 2.1.5. Plant transformation 2.2. G E N E EXPRESSION A N A L Y S I S 2.2.1. R N A gel blot analysis 2.2.2. Genomic Southern blots 2.2.3. Quantitative RT-PCR 2.2.4. In situ hybridization 2.3. D N A SEQUENCE A N A L Y S I S 2.4. GUS A N A L Y S I S 2.4.1. Generation of the CER6 promoter: :GUS construct 2.4.2. GUS histochemical assay 2.4.3. GUS fluorometric assay 2.5. T R A N S F O R M A T I O N OF ARABIDOPSIS WITH A D D I T I O N A L COPIES OF CER6promoter.:CER6 2.6. W A X A N A L Y S I S 2.6.1. Gas chromatography 2.6.2. Scanning electron microscopy 2.7. Cerl M A P P I N G 2.8. Cerl C O M P L E M E N T A T I O N 2.9. S A L K T-DNA INSERTION LINE A N A L Y S I S C H A P T E R 3. C E R 6 , A W A X SPECIFIC p - K E T O A C Y L S Y N T H A S E  3.1. INTRODUCTION 3.2. RESULTS 3.2.1. Tissue specificities of CER6 and CER60 expression 3.2.2. Expression of CER6 at different stages of development 3.2.3. Epidermal specificity of CER6 expression 3.2.4. Expression of CER6 promoter-GUS fusions in transgenic Arabidopsis  53 53 54 56 56 59 60 62 63 65  65 65 66 66 66 67 68 68 69 70 72 73 74 74 75 75 75 77 77 77 78 80 85 87  87 88 88 91 93 97  vi 3.2.5. Overexpression of CER6 can increase surface wax accumulation 3.3. DISCUSSION 3.3.1. Spatial and temporal pattern of CER6 expression 3.3.2. The CER6 promoter directs high levels of gene expression in the shoot epidermis 3.3.3. The effect of CER6 overexpression of stem wax accumulation 3.4 CONCLUSIONS C H A P T E R 4. E N V I R O N M E N T A L E F F E C T S O N C E R 6 E X P R E S S I O N A N D W A X DEPOSITION  4.1. INTRODUCTION 4.2. RESULTS 4.2.1. Environmental effects on CER6 transcription Light Osmotic stress Temperature Wounding 4.2.2. Environmental effects on wax deposition 4.3. DISCUSSION 4.3.1. Light 4.3.2. Osmotic stress 4.3.3. Cold 4.3.4. Wounding 4.4. CONCLUSIONS C H A P T E R 5. C E R 7 , A P U T A T I V E E X O S O M A L RNAse I N V O L V E D IN W A X REGULATION  5.1. INTRODUCTION 5.2. RESULTS 5.2.1. Phenotypic and genetic characterization of the cer7 mutant 5.2.2. Map-based cloning of CER7 5.2.3. S A L K T-DNA insertional allele 5.2.4. Identity of CER7 5.2.5. Expression of At3g60500 gene in the cer7 mutant and SALK_003100 line 5.2.6. Tissue specificity of CER7 expression 5.3. DISCUSSION 5.3.1. CER7cloning 5.3.2. Cer7 wax profile 5.3.3. Expression of other CER genes in the cer7 mutant 5.3.4. Role of CER7 in wax production 5.4. CONCLUSIONS  100 105 105 107 108 109  110  110 111 112 112 113 115 117 117 124 124 125 126 126 127  129  129 130 130 135 139 146 152 154 156 156 157 157 158 164  vii C H A P T E R 6: C O N C L U D I N G R E M A R K S A N D D I R E C T I O N S F O R F U T U R E RESEARCH  6.1. CER6 6.2. CER7 6.3. C L O N I N G A N D C H A R A C T E R I Z A T I O N OF OTHER W A X - R E L A T E D GENES L I T E R A T U R E CITED  166  166 167 168 ...169  viii LIST O F T A B L E S  Table Table Table Table Table Table Table  1.1. 1.2. 1.3. 1.4. 1.5. 1.6. 1.7.  Page Wax deficient mutants of Arabidqpsis 9 Substrate specificities of KCS proteins 31 PSORTI predictions of cellular localization of enoyl-CoA hydrase-like genes 43 EST and MPSS hit frequency for enoyl-CoA hydratase-like genes 44 Alcohol-forming reductase-like genes in Arabidopsis 50 Wax synthase-like genes in Arabidopsis 52 Proposed and confirmed functions of wax mutants and corresponding genes 57  Table Table Table Table Table Table  2.1. A T medium composition 2.2. Arabidopsis thaliana standard nomenclature 2.3. Primers and PCR conditions used to generate probes for R N A and D N A blots 2.4. Primers and conditions used for RT-PCR 2.5. Markers used to fine-map CER7 2.6. P C R conditions used to amplify coding sequences from the target region for CER 7 cloning Table 2.7. Sequencing primers Table 2.8.Gene-specific primers used to confirm transgene presence in kanamycinresistant plants recovered from transformations Table 2.9. Primers used for confirmation of S A L K T-DNA insertions Table 5.1. Summary table showing candidate gene letter designations, identities, and results of sequencing, complementation attempts, and T-DNA insertion line pheno types Table 5.2. Positions of mutations found in coding sequences of the target region Table 5.3. Complementation of cer7 by transformation Table 5.4. Results of BLASTp of the NCBI nr database with the CER7 amino acid sequence Table 5.5. Core proteins of the yeast exosome and putative Arabidopsis homologues  65 66 69 71 79 82 83 84 86  137 138 139 148 150  ix LIST O F F I G U R E S  Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure  1.1. 1.2. 1.3. 1.4. 1.5. 1.6.  Page 1 2 4 12 14  The structure of the plant cuticle Chemical structures of aliphatic wax components derived from V L C F A s Wax microstructure of Arabidopsis stems Pathways of cuticular wax biosynthesis Schematic of fatty acid synthesis and fatty acid elongation in the cell Model of enzymes and biochemical reactions comprising the fatty acid elongase complex 16 1.7. Schematic representation of parallel F A E pathways in Arabidopsis, and sequential elongase complexes required for the different pathways. 19 1.8. Total frequency of EST and MPSS hits for Arabidopsis K C S genes 22 1.9. Distribution of EST and MPSS hits in different tissues of Arabidopsis 24 1.10. Mutant phenotypes caused by K C S disruption 28 1.11. Neighbor-joining phylogram of KCS-like derived protein sequences 33 1.12. Chromosomal locations of Arabidopsis KCS-like genes plotted on a diagram of large chromosomal duplications 34 1.13. Unrooted phylogram of P-hydroxyacyl-CoA dehydratase/enoyl-CoA hydrataselike genes of Arabidopsis 40 1.14. Structure of carnitine 41  Figure 2.1. Diagram of the T-DNA construct containing the CEi?6promoter-GUS (pCER6GUS) gene fusion used to transform Arabidopsis 74 Figure 2.2. CER6 gene structure and CER6promoter-CER6 overexpression cassettes 76 Figure 2.3. Constructs used to transform cer7 to establish complementation 81 Figure 3.1. Genomic Southern blot hybridized with CER6 and CER60 probes 89 Figure 3.2. CER6 expression is limited to shoots and is much higher than that of CER60 90 Figure 3.3. R N A blot hybridization analysis of CER6 expression in developing Arabidopsis stems and leaves 92 Figure 3.4. In situ localization of CER6 mRNA 94 Figure 3.5. In situ localization of CER6 mRNA 95 Figure 3.6. In situ localization of CER6 mRNA 96 Figure 3.7. The CER6 promoter directs epidermis-specific expression of GUS in Arabidopsis throughout the shoot from very early stages of development 98 Figure 3.8. GUS activity in plants transformed with CER6 promter-GUS was higher than that found in plants transformed with 35S promoter-GUS. 99 Figure 3.9. Wax loads on stems of the Ti generation of plants transformed with the I X cassette, 2X cassette, and 3X cassette of CER6promoter-CER6 101 Figure 3.10. GC traces showing elution profiles of wax extracted from stems of plants transformed with extra copies of CER6pvomoter-CER6. 103 Figure 3.11. Wax loads of stems and R N A blot hybridization of CER6 expression in the T2 generation of plants transformed with CER6promoter-CER6 104 Figure 3.12. S E M of Arabidopsis stem tops of WT and T2 plants from lines 2-5 and 3-10 transformed with CE7?<5promoter-C£./?6' constructs 106  X  Figure 4.1. The CER6 5' promoter region has numerous consensus sequences for cz's-acting elements involved in environmental regulation Figure 4.2. Light is required for CER6 expression in seedlings and in bolting stems Figure 4.3. Osmotic stress increases CER6 transcript accumulation in seedlings and in bolting stems of Arabidopsis Figure 4.4. CER6 transcript is increased by lower and reduced by higher ambient temperatures, but is not affected by CBF overexpression Figure 4.5. Wounding reduces CER6 transcript levels in Arabidopsis Figure 4.6. Total wax load on stem bases of plants grown at different temperatures Figure 4.7. S E M of stem tops and bases of plants grown at 10 °C and 20 ° C Figure 4.8. Total wax load on stem bases of WT and transformed lines overexpressing CBF1,2, and 3. ' " ' Figure 4.9. Total wax loads of stem bases of well-watered and unwatered Arabidopsis Figure 4.10. Total wax loads on WT and aba mutant stems bases, and S E M showing microcrystal density on WT and abal-4 stem tops  111 112 114 116 117 118 120 121 122 123  Figure 5.1. Cerl mutant stems show a glossy green, waxless phenotype which contrasts with the glaucous appearance of WT and F l plants under high light intensity 131 Figure 5.2. S E M stem tops and bases of WT and cer7 reveals a lower density of wax microcrystals in cerl compared to WT 132 Figure 5.3. Total wax load and composition extracted by chloroform dip from stem bases, siliques and leaves of WT, cerl and F l plants 133 Figure 5.4. R N A gel blot hybridization showing transcript levels of CER1 to CER6 in stems of WT and cer 1 mutants. 135 Figure 5.5. SSLP markers used for fine-mapping of the cer7 locus, represented schematically on Arabdiopsis Chromosome III, with recombination frequencies shown by percentages; BACs used to design new markers; new marker locations on a detailed map of the annotated genes with the number of recombinants indicated in parentheses 136 Figure 5 6. Transformation of cerl plants with gene S, but not gene R, restores the WT waxy phenotype 140 Figure 5 7. Wax loads and compositions of cerl plants transformed with gene S, gene N , and cerl and WT untransformed plants 141 142 Figure 5 8. T-DNA insertions in the target region of Chromosome 3 Figure 5 9. Phenotype of SALK_003100 waxless plants in comparison with cerl and WT (Ler and Col) 143 Figure 5 10 . Wax composition (by load of major components) of individual plants grown from SALK_003100 T seed obtained from A B R C 144 Figure 5 11 . S E M of wax from stem tops of 5 T3 plants from SALK_003100 seed obtained from A B R C 145 146 Figure 5 12 . Genomic sequence amplified for complementation of cerl by gene S Figure 5 13 . Alignment of CER7 (At3g60500) with yeast Rrp45p and the other Arabidopsis homologue (At3gl2990) 149 . Bootstrapped neighbor-joining phylogram of RNAse-PH-like exosomal 14 Figure 5 homologues from yeast and Arabidopsis 151 3  XI  Figure 5.15. RT-PCR analysis of CER1 from 14 day old whole seedlings of cerl mutants and SALK_003100 in comparison to WT Figure 5.16. Nucleotide sequence alignment of CER1 (At3g60500) and its sister gene (At3gJ2990) Figure 5.17. Genomic Southern blot showing hybridization with specific and non-specific probes for CER7 and At3g 12990 Figure 5.18. Northen blot showing CER1 and At3gl2990 transcript levels in different tissues of Arabidopsis Figure 5.19. A model showing possible mechanisms of CER7 regulation of wax production in Arabidopsis  153 154 155 156 162  ABBREVIATIONS  35S Cauliflower mosaic virus 35S (strong constitutive) promoter aa amino acid ABA abscisic acid aba abscisic acid synthesis mutant ABRE abscisic acid (cis-acting) response element ACBP acyl-CoA binding protein ACP acyl carrier protein AP alkaline phosphatase AT, At Arabidopsis thaliana AT-Kan A T medium containing 50(J.g/mL kanamycin BAC bacterial artificial chromosome BLAST Basic Local Alignment Search Tool BLASTn B L A S T of nucleotide sequences BLASTp B L A S T of protein sequences BSA bovine serum albumen bp D N A base pairs CaMV35S Cauliflower mosaic virus 35S (strong constitutive) promoter CAPS cleaved amplified polymorphic sequence CBF Cold binding factor cDNA copy D N A (reverse-transcribed from RNA) C carbon atom C29; CI 8, etc. hydrocarbons and derivatives with the specified number of carbon atoms cer eceriferum (waxless) mutant, and corresponding genes and proteins Col Arabidopsis, Columbia ecotype det deetiolated mutant, and corresponding genes and proteins DIG digoxygenin DNA deoxyribonucleic acid DRE drought response element DW dry weight ECR enoyl-CoA reductase EDTA ethylene diamine tetracetic acid EMS ethyl methanesulfonate ER endoplasmic reticulum EST expressed sequence tag FAE fatty acid elongase fae fatty acid elongase mutant FAS fatty acid synthetase fdh fiddlehead mutant, and the corresponding gene and protein GAPC glyceraldehyde-3-phosphate dehydrogenase C (cytosolic form) GC gas chromatography GC-MS gas chromatography-mass spectrometry GFP green fluorescent protein gl glossy mutant; and corresponding genes and proteins GUS (3-glucuronidase  xiii h HCD kb KCS KCR kDa KDEL LB Ler LTP MOPS MPSS mRNA MU MUG NCBI nr ORF PCR PEG RNA rRNA RT-PCR s SDS SEM SSC SSLP SSPE ssRNA TAE TAIR TBE TE Tris UTR VLCFA wax WT  hour(s) P-hydroxy-acyl-CoA dehydratase kilo bases (1000 base pairs) P-ketoacyl-CoA synthase P-ketoacyl-CoA reductase kilo Dalton conserved ER retention signal for proteins, lysine-aspartate-glutamate-leucine Luria-Bertani bacterial growth medium formulation Arabidopsis, Landsberg erecta ecotype lipid transfer protein 3-(N-morpholino) propane-sulfonic acid massively parallel signature sequencing messenger R N A 4-methyl-umbelliferone 4-methyl-umbelliferone glucoside National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/ non-redundant open reading frame polymerase chain reaction polyethylene glycol ribonucleic acid ribosomal R N A reverse-transcriptase PCR second(s) sodium dodecyl sulfate scanning electron microscopy salt sodium citrate buffer simple sequence length polymorphism salt sodium phosphate E D T A buffer single stranded R N A Tris acetate E D T A buffer The Arabidopsis Information Resource; http://www.arabidopsis.org/ Tris borate E D T A buffer Tris E D T A buffer 2-amino-2-hydroxymethyl-l ,3-propanediol untranslated region very long chain fatty acid wax deficient mutant, and corresponding genes and proteins wild-type  XIV  ACKNOWLEDGEMENTS  The road to PhD-dom has (thankfully) not been a lonely one, and I have many people to thank for their contributions to my progress and ultimate completion of this endeavour. First, I would like to thank my supervisor, Dr. Ljerka Kunst, for her guidance, encouragement and. support during the years I have spent in her lab. It has truly been an enriching and enjoyable experience and I have learned a lot from her mentoring. Thanks also to my committee, Dr. Carl Douglas, Dr. Beverly Green, and Dr. Linda Matsuuchi, for their valuable comments and ideas. I am also grateful to the past and present members of the Kunst and Haughn labs for their input and useful discussions, which has been the medium through which I have learned the nuts and bolts of research in molecular biology and genetics. Special thanks are due to Sabine Clemens, my first molecular biology tutor, and Sabine Zachgo, who taught me how to do in situs. Thanks also to all of my friends and family, who have enriched my life and preserved my sanity through my seemingly perpetual studenthood. Thanks especially to Ravi, my constant companion, troubleshooter and cheerleader, who has put up with my lowest and most frustrating moments of research, writing, and waiting, and is still with me making life more fun.  To my grandparents The fines are fatten unto me in pteasant places; yea, I have a goodty heritage (<Psatm 16:6)  1 C H A P T E R 1. INTRODUCTION AND LITERATURE REVIEW  1.1. T H E P L A N T C U T I C L E A N D C U T I C U L A R W A X E S  The herbaceous shoot surfaces of virtually all land plants are covered with a relatively hydrophobic and impermeable membrane: the cuticle. This protective layer helps to prevent water loss from the shoot and to defend against biotic and abiotic factors that could damage the plant. Its importance is underlined by the fact that it is exuded over the surface of the primary wall of epidermal cells from very early stages of development, at least as soon as the cells come into contact with the air (Jeffree 1996). The cuticle is made up of several layers (Fig. 1.1). The cuticular layer, adjacent to the cell wall, is formed by impregnation of the outer part of the primary cell wall with cutin, an insoluble polyester matrix of aliphatic lipids (esterified CI 6 and CI8 hydroxylated fatty acids), and some soluble lipids derived from very long chain fatty acids (waxes). The cuticle proper is composed of cutin embedded with waxes. The waxes embedding the cuticle are intracuticular waxes. The outermost layer is the epicuticular wax, which may be amorphous or have a crystalline microstructure (Jeffree 1996). Cuticular waxes (including both intra- and epi-cuticular waxes) are hydrophobic, reducing evaporation of water through the cuticle and diffusion of water-soluble substances into and out of the plant. In addition, the microcrystalline structures of the epicuticular waxes of many species scatter light and/or make the plant surface less wettable (Jeffree 1996).  CP  CL PCW SCW Figure 1.1. The structure of the plant cuticle, showing the secondary cell wall (SCW), primary cell wall (PCW), cuticularized layer (CL), cuticle proper (CP), and epicuticular wax (EW). (From Jeffree 1996).  2 Cuticular waxes are composed of very long chain fatty acids (VLCFAs - fatty acids with aliphatic chains longer than 18 carbons (C)) and their derivatives: aldehydes, primary alcohols, alkanes, ketones and secondary alcohols. Chemical structures of these common wax constituents are shown in Fig. 1.2. P-diketones and esters of fatty acids and alcohols are also important wax constituents in some species. Branched alkanes as well as hydroxy-alcohols are other commonly-found V L C F A derivatives. The most commonly found chain lengths are 2035C; however, fatty acids and alkanes with less than 20C and esters with more than 60C exist (von Wettstein-Knowles 1995). In addition to fatty acid derivatives, terpenoids, sterols, and sometimes flavonoids and anthocyanins are found as epicuticular wax constituents in some species (Baker 1982). Separate analysis of intracuticular and epicuticular waxes (accomplished by mechanical removal of epicuticular waxes followed by removal of the remaining  H  v w w w v w v w v v  .OH  VLCFA  Primary alcohol  W W W V W W W V W O  Aldehyde  VSAAAAAAAAAAAA/  Alkane  Secondary alcohol  O  w w w X w w w Figure 1.2.  Ketone  Chemical structures of aliphatic wax components derived from V L C F A s  3 intracuticular waxes by organic solvents) shows that the chemical compositions of the two layers may differ (.Tetter 2000; Jetter & Schaffer 2001). In general, intracuticular waxes tend to have a narrower range of components, including n-alkanes, fatty acids, primary alcohols and wax esters; they also have broader chain length distributions than epicuticular waxes (von Wettstein-Knowles 1995). Cuticular wax composition varies greatly between species (Gulz 1994), and also between organs and even cell types of the same species (von WettsteinKnowles 1995). However, cuticular waxes are ubiquitous in the land plants, from the bryophytes to the angiosperms (Gulz 1994). A microcrystalline wax ultrastructure tends to form over the surface of the cuticle i f there is one dominant constituent of the epicuticular wax (Post-Beittenmiller 1996). The microstructure of epicuticular waxes was observed as early as 1871 by de Bary in the light microscope (Jeffree 1996); and was first well documented in surveys done with scanning electron microscopy in the 1950's (Mueller et al. 1954; Schieferstein & Loomis 1956; Juniper & Bradley 1959). The shapes of the crystals formed are characteristic of the predominant constituent for any given wax. For example, waxes composed mostly of aldehydes or (3diketones tend to form rod or tube-like crystals, whereas those composed mostly of alkanes usually have plate-like crystals (Fig. 1.3 a, b) (Lemieux et al. 1994). The scattering of light by the microcrystalline structure of the epicuticular wax is responsible for the whitish or bluish appearance given to a plant which has this type of waxy "bloom," or glaucousness (Fig. 1.3 c) (Lemieux et al. 1994). In this review I will discuss the role of cuticular waxes in biotic and abiotic interactions of plants, biochemical, genetic and molecular genetic work done to elucidate the biosynthesis of aliphatic, VLCFA-derived components of wax, wax secretion, and the regulation of wax deposition, outlining the developmental and environmental effects on wax deposition in plants. I will also describe the focus of the research in our lab, and outline the specific objectives of the research undertaken for this dissertation.  4 1.2. T H E P R O T E C T I V E R O L E O F E P I C U T I C U L A R W A X E S  Waxes are important in several aspects of plant interactions with the environment. First, they constitute the main barrier in the cuticle to water evaporation from the plant surface. Reed and Tukey (1982) found that 79% of the permeability differences between cuticles of Brussels sprouts and carnations grown in different environments could be explained by the relationship of a measured partition coefficient (caused by the chemical properties of the constituents) divided by the cuticle thickness and the proportion of wax in the cuticle. Thus, the amount and composition of cuticular wax is important to plants' abilities to withstand drought and avoid desiccation. The importance of cuticular waxes in preventing water loss from the plant has also been shown in other species, eg. tomato (Xu et al. 1995) and tissue-culture propagated grape seedlings (Ali-Ahmad et al. 1998). The microstructure of the epicuticular wax does not play a direct role in the reduction of evaporation. However, the size of the crystals is such that they interfere with the penetration of light rays, causing scattering and reflection of incident light. Thus, plants with microcrystalline epicuticular wax blooms reflect more light, as has been demonstrated with near-isogenic wild-type and bloomless mutants of sorghum (Blum 1975; Grant et al. 1995), and between spruce needles with intact or mechanically or chemically removed epicuticular wax (Reicosky & Hanover 1978). The reflection of light gives plants growing under high light and/or high U V conditions protection from photoinhibition (Robinson et al. 1993) and U V damage (Barnes et al. 1996). U V protection may also be gained from the covalent binding of UV-absorbing pigments to the cutin matrix (Baur et al. 1998) Light scattering is not equal at all wavelengths. Reicosky and Hanover (1978) found that reflection of  Figure 1.3. (a) W a x microstructure of Arabidopsis W T stem (b) Wax microstructure of Arabidopsis cer4 stem, enriched in alkanes (c) Glaucous W T Arabidopsis stem and glossy cer2 (waxless) mutant stem (from Lemieux et al. 1994)  5 incident light in spruce needles was the greatest at wavelengths over 750 nm (infrared wavelengths), and the lowest in the photosynfhetically active wavelengths. The reduction of leaf temperature causes significant reductions of transpiration in glaucous plants. In wheat, photosynthetic tissues were up to 0.7 °C cooler in glaucous than in non-glaucous plants in drought conditions, and a 0.5 °C decrease in temperature resulted in savings of at least 60 g FfiO/W/day (Richards et al. 1986). In general, plants adapted to semi-arid environments have been found to reflect more radiation than more mesophytic plants due to the crystalline microstructure of their epicuticular wax (Billings & Morris 1951; Thomas & Barber 1974; Reicosky & Hanover 1978). However, the correlation is not absolute, and will not necessarily predict xeromorphic adaptation in different plant species (Schieferstein & Loomis 1956). The reduction of water loss by the chemical and physical properties of epicuticular wax, or simply the presence of more wax has been correlated with higher water use efficiency and yields in several different crop species including wheat (Fischer & Wood 1979; Richards et al. 1986; Uddin & Marshall 1988), oats (Bengtson et al. 1979), rice (O'Toole & Cruz 1983) and sorghum (Ross 1972; Webster & Schmalzel 1979). In addition, a poorly developed wax layer in spruce (Picea abies) and pine (Pinus cemba) was related to wintertime desiccation and death of the needles (Gunthardt-Goerg 1987). Wax microstructure is also important in determining the wettability of a plant surface. If the surface is very densely covered with microcrystals, less of it is contacted by water droplets that form on it, and the contact angle between the water droplet and the surface will be more obtuse (making the surface less wettable) than if the surface were flat. Wettability is a key factor in determining a plant's interaction with foliar applied chemicals (Whitehouse et al. 1982) and pathogenic fungi (Jenks et al. 1994a) and bacteria (Marcell & Beattie 2002). If chemicals are applied in an aqueous solution, they will be shed from the surfaces of less wettable plants, but retained on and absorbed from the surfaces of more wettable plants. This is an important consideration when thinking about the effectiveness of herbicides and pesticides, and whether surfactants (which reduce water surface tension) are required (Schreiber 1995; Schreiber et al. 1996; Kirkwood 1999; Hess & Foy 2000). Additionally, i f water is easily shed from the plant surface, as in a less wettable plant, fungal spores and other pathogens will be washed off of the plant surface, rather than being allowed to accumulate there. If, on the other hand, the plant surface is easily wet, fungal spores will germinate and pathogens will grow more easily. Furthermore, wax microstructure also directly influences fungal and bacterial attachment and attack on the plant, although the relationship is not entirely clear and is most likely influenced by species variation (Marcell & Beattie 2002). Wax structure and  6 composition may play a role in fungal recognition of host plant epidermis and stomata (Kim et al. 1999; Rubiales et al. 2001; Niks & Rubiales 2002). Waxless mutants also have different relationships with microbes. A waxless mutant of sorghum had an increased susceptibility to the fungal pathogen Exserohilum turcicum under field conditions (Jenks et al. 1994a). In maize analysis of bacterial colonization of a variety of glossy mutants showed different responses to different mutants by two different species of bacteria, suggesting that the variation was speciesspecific (Marcell & Beattie 2002). The microcrystalline nature of epicuticular wax also plays a role in plant interactions with insects (Eigenbrode 1996). Wax crystals are liable to break off when insects walk on them. This can reduce insects' abilities to attach to the plant surfaces (Stork 1980). Thus, glaucousness may increase insect herbivores' difficulty in attaching to the plant, and reduce herbivory on these plants. Indeed, it has been found in some cases that waxless variants are more susceptible to insect attack than their glaucous parents (Eigenbrode & Espelie 1995; Justus et al. 2000). However, waxless variants of some species are less subject to insect herbivory in the field than their waxy counterparts (Eigenbrode & Espelie 1995; Rutledge et al. 2003). This is probably because the wax microstructure interferes with insect predators' attachment to the plant, thus reducing their.predation efficiency on the insect herbivores (Schmaedick & Shelton 2000; Rutledge et al. 2003). The presence of wax microcrystals doesn't always interfere with insects. Some specialist insects have adaptive behaviours, such as walking along leaf margins, or anatomies which allow them to move effectively on waxy plants (Federle et al. 2000; Brennan & Weinbaum 2001). It has also been suggested that wax crystals may interfere more with smaller insects than with larger ones (Eigenbrode et al. 2000). Wax composition is another factor that may influence insect behaviour on plants. For example, in Eucalyptus species, the relative quantities of aliphatic phenylethyl and benzyl wax esters in the epicuticular wax correlated with resistance to the autumn gum moth (Jones et al. 2002). Also, the increased primary alcohol content seen in the wax of cer3 waxless mutants of Arabidopsis has been linked to the reduced fecundity of aphids living on these mutants (Rashotte & Feldmann 1997) Freezing tolerance or susceptibility is another factor that may be influenced by the microstructure of epicuticular waxes. The presence of wax microcrystals might serve to create a boundary layer of air next to the surface of the plant. This could insulate the plant in nearfreezing temperatures. Also, if water is shed from the plant surface, it cannot freeze on the plant. The presence of solid ice on the plant surface would increase the interaction of the plant cells with the cold temperature of the ice. Even i f water were to collect and freeze on the  7 surface of a plant with wax microcrystals, the space introduced by the crystals might reduce the chance of the plant cells also freezing. If there are no wax crystals, water is able to accumulate and freeze in direct contact with the plant epidermal cells, and it would be more likely to cause the cells to freeze.  1.3. I N V E S T I G A T I O N O F W A X DEPOSITION: BIOCHEMICAL, GENETIC, AND GENOMIC TOOLS  Wax biosynthetic, secretory and regulatory pathways have been investigated using both biochemical and genetic methods. A combination of the two methods has given us a reasonable picture of the biosynthetic pathways, and genetic studies are starting to unveil the secretory and regulatory mechanisms involved. However, there have been some limitations for both biochemical characterization and genetic analysis of wax deposition. Because the biosynthetic enzymes are membrane-associated, the process of solubilizing, purifying and characterizing these enzymes has been slow. The substrates of these enzymes are hydrophobic, so they are not soluble in the aqueous solutions generally used for biochemical assays. Most of the substrates are also not commercially available and have to be synthesized (Kunst & Samuels 2003). Another problem with the biochemical approach is that wax biosynthesis is limited to epidermal cells, so it is difficult to obtain enough material for enzyme purification. In many species, it is also problematic to separate the epidermis from the underlying tissue (Post-Beittenmiller 1998; Kunst & Samuels 2003). Nevertheless, quite a bit of progress has been made since investigations began with the feeding of radioactively labeled substrates to tissue slices in the 1960's. Recently, a number of genomic tools have become available that allow investigation into possible additional genes and proteins that could be involved in wax production. M y analyses of the data available in the public databases will be presented in this chapter. In many cases, genetic analysis of biochemical pathways (eg. glycerolipid synthesis; reviewed in Somerville and Browse 1991) has contributed greatly to the elucidation of the pathway. For example, accumulation of substrates and absence of products of the mutant enzyme has allowed the different steps of the pathway to be identified. Analysis of genetic interactions (including additive variance, suppression, and epistasis of different mutants) have helped to determine the sequence of biosynthetic steps. Therefore, the genetic approach has been undertaken with wax synthesis, as well. The presence of a waxy bloom in many species makes wax mutants easy to find. Wax deficient mutants lack the wax bloom and look glossier  8 or brighter green than their WT counterparts. Naturally occurring bloomless variants have led to the investigation of the inheritance and distribution of the glaucous phenotype in Ricinus (castor bean) (Peat 1928) and eucalyptus (Barber 1955). Chemical and radiation mutagenesis led to the identification of over 1560 waxless, or eceriferum {cer), mutants in barley {Hordeum vulgare) (Lundqvist & von Wettstein 1962; Lundqvist et al. 1968) that were found to comprise 85 complementation groups (Sogaard & von Wettstein-Knowles 1987). Waxless mutants have also been identified and investigated in other species including Zea mays (maize) [18 complementation groups known (von Wettstein-Knowles 1995)], Brassica oleracea (Macey & Barber 1970a), Pisum sativum (Macey & Barber 1970b), and Sorghum (Blum 1975; Jenks et al. 1994a). In. Arabidopsis thaliana, 30 wax-deficient mutants (cerJ-cer24, rst, tt5 (Dellaert et al. 1979; Koornneef et al. 1989; McNevin et al. 1993; Jenks et al. 2002a), ded (Lolle et al. 1998), kcsl (Todd et al. 1999), waxl and wax2 (Jenks et al. 1996a), and fatb (Bonaventure et al. 2003)) have been identified. A list of the wax-deficient mutants of Arabidopsis and their phenotypic features is presented in Table 1.1. Arabidopsis normally does not have any wax bloom on its leaves. This has allowed the identification of 6 mutant loci that cause increased leaf wax and a glaucous leaf phenotype (knbl-knb4, bcfl and bcf2) (Jenks et al. 1996a) Stem wax is unaffected in these mutants (Jenks et al. 1996a). Despite the abundance of mutant loci, analysis of the mutant wax compositions in this case has not led to many clear indications of blocks in the biochemical pathway. In Arabidopsis, most of the cer mutants have reductions in many of the wax components. Not many cause accumulations of metabolic intermediates that would point to a specific block in the pathway. Rather, there seems to be some flux between the two branches of the wax biosynthetic pathway, which causes complex changes in wax composition and makes it difficult to determine biochemical functions involved (Kunst & Samuels 2003). Furthermore, the visual screens used to identify waxless mutants are based on the blooms caused by the presence of epicuticular wax crystals. This usually requires large decreases in total wax load. More subtle phenotypes in which the wax composition, but not the total wax load, is affected, may not be found in visual screens since these changes may not affect the glossiness of the mutant (Kunst & Samuels 2003). Thus, the mutants identified to date probably include many loci involved in regulation of wax deposition, regulation of epidermal development, transport of wax precursors within the cell, and secretion of wax components, as well as lesions in enzymes involved in the biosynthetic pathway (von Wettstein-Knowles 1995; PostBeittenmiller 1998; Kunst & Samuels 2003). Genes encoding enzymes that catalyze steps causing more subtle phenotypic changes, such as the alkane oxidase that produces ketones or  co <D >  co  CO  co  O =3 '«  m  X  J5 -e 5  00  CD o  co CO  CJ V Q  °  1*  CD  =  =3 T3 CD  CD 03 XI  >  TO  co c co o  ^—• -i—• o c  CO  CO CD  V cO o o  E  3  TO I  15  8  CO  _.  T-  o o  u)  CD  CN  £= ^  >  CD 0 Q)  CD  XI  <  c "a co a) x: co i= x: ~ CD  <  LU O  c  a) sz o  _^ CO  co —  CO  o o  co  ..  <  o  o , o  o  , o  CD  c  g  O  co  0)  co o  g  csj  co  . _ +  co  to "5  CD  O P Q) fc.  O O  CO  CD  00  CO CO T-  Si  CO CD  C ±3  co . oo co co co  . -e t - CO o Q_ "co — CO CD ^  -Q CO  CD •> m ^ CD v> o .Q>  o  O  i.i C  CD  =  CO II  M-  - O  CD CO CO X  ro o  5s  CD  x E co co ' E co >^ > Q CD  0)  re  II a> o  o  "2 o  CiiM x I  10 the ketone reductase that produces secondary alcohols, could be identified using a gaschromatography (GC)-based screen. Despite the limitations with both the biochemical and the genetic approach, both have been used to determine the sequence of steps in the wax biosynthetic pathway. Unfortunately, species that are amenable to genetics are not necessarily amenable to biochemistry, and vice versa (Post-Beittenmiller 1998). Thus, it has been difficult to elucidate the pathway in any one species. Rather, our current understanding of the wax biosynthetic pathway is a conglomeration from different species, which have differences in wax compositions. Only more recently, molecular genetic techniques in Arabidopsis and maize have allowed the cloning of some of the enzymes involved. This allows the expression and characterization of recombinant enzymes in systems such as yeast, which are more amenable to biochemical and genetic analysis. Cloning of wax-related genes also opens the door to bioinformatic analysis, a comparison with genes from other organisms, and the use of reverse genetics to determine the biological function of an uncharacterized sequence. A growing number of bioinformatics tools are becoming available and increasingly informative. The complete genome sequence of Arabidopsis was finished in 2000 (The Arabidopsis Genome Initiative 2000). Those of 5 other eucaryotes including budding yeast (Saccharomyces cerevisiae, Goffeau et al. 1996), fission yeast (Schizosaccharomycespombe, Jenks et al. 2002b), human {Homo sapiens, Venter et al. 2001; Lander et al. 2001), nematode {Coenorhabditis elegans, The C.elegans Sequencing Consortium 1998), and fruit fly {Drosophila melanogaster, Adams et al. 2000) have also been completed. Other plant genome projects (including rice, for which a draft sequence has already been published (Yu et al. 2002)) and large-scale expressed sequence tag (EST) sequencing projects (including barley, corn, and sorghum; http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/PlantList.html), in which cDNAs are randomly sequenced, are also underway. Genome sequences and large-scale EST projects allow the identification of putative orthologs of genes of interest from other species, as well as homologues in the same species. Many genes m-Arabidopsis have been found to be members of gene families in which there may be redundancy and/or functional specialization. Thus, genomic analysis is useful in the discovery of gene sequences related to characterized genes, for which biological functions have not yet been defined. EST sequencing projects from specific tissues and treatments of Arabidopsis and other large-scale expression analysis information including the Massively Parallel Signature Sequencing (MPSS) project (Brenner et al. 2000) (details presented in section and  11 micro-array data from a number of projects are also being made publicly available. These data allow preliminary characterizations of expression of genes of interest, and comparisons of expression between members of gene families. This facilitates predictions of which members of gene families may be of interest in processes under investigation. Uncharacterized genes which seem interesting can be characterized by reverse genetic methods: in Arabidopsis, largescale T - D N A insertion projects have been undertaken by the Salk Institute (http://signal.salk.edu/cgi-bin/tdnaexpress) and GABI-Kat (http://www.mpizkoeln.mpg.de/GABI-Kat/). These projects have sequenced genomic D N A flanking the random insertion sites of T-DNA-mutagenized populations and created databases which can be searched for T-DNA insertions into genes of interest. These insertional mutants are likely to disrupt genes and cause mutant phenotypes. Mutations in genes of interest can also be identified by TILLING, a technique which identifies point mutations in a defined genomic region from an EMS mutagenized population (Till et al. 2003). Alternatively, silencing or overexpression in transformed plants can be used to investigate biological functions of unknown genes.  1.4. O V E R V I E W O F W A X BIOSYNTHESIS: V L C F A s A N D T H E I R D E R I V A T I V E S  The synthesis of cuticular waxes takes place in epidermal cells of plant shoots. It involves the synthesis of long chain saturated fatty acids (C16:0; C18:0), their elongation to V L C F A s (C20+), and subsequent modification to produce different wax components. There are two general pathways of fatty acid modification. In the acyl-reduction pathway, V L C F A s are reduced to primary alcohols, which may remain independent in the wax or undergo esterification with fatty acids to produce wax esters. In the decarbonylation pathway, V L C F A s are reduced to aldehydes, which can be decarbonylated to yield alkanes. To produce ketones and secondary alcohols, alkanes are oxidized. Species with (3-diketone derived wax components (eg. barley) probably have separate fatty acid elongation systems that produce the precursors for these components. Derivatives of both pathways can be further modified by oxidation, hydroxylation and/or esterification to yield a wide variety of other products. The different wax components are produced in varying proportions and with varying chain lengths in different plants and in different plant organs, but the basic pathway seems to be conserved over a wide variety of plants (von Wettstein-Knowles 1995). Figure 1.4 presents a schematic diagram of the pathway, with hypothesized and confirmed activities defined by wax-deficient mutants from Arabidopsis, maize and barley indicated.  12  N 03 JC  o n  in c  CD ro E  Ii= *to  ro Q. Q_  O  e 5 ° -s  T3 • CD X _L x:  ioI c ro = E "O =  W  .  o  c  ro ro  w 5 ui £ E <u ro >.  £ Q- . O  II T3in gi CD  .2 ro ro X eO o  ro x CD ._ ro _..Q c ro o ro "5 2 cu ^ .S E  U (D cp >>  •s £  O  ro  P-xi ro &  <o a>  »  ro  CL •- g O jS O  r- _ i  i  e  D)  O  CD "  13  E  13 1.5. F A T T Y A C I D E L O N G A T I O N  The elongation of fatty acids is the first step in the biosynthesis of all the major Arabidopsis wax components. Long-chain fatty acids (CI6 and CI8) are produced de novo in the plastids by the fatty acid synthetase (FAS) complex. Fatty acid synthesis is comprised of a series of four reactions with the nascent acyl chain thioesterified to the acyl-carrier protein (ACP): condensation to form the carbon-carbon bond adding 2C units from acetyl Co A and forming a (3-ketoacyl-ACP, reduction to form a P-hydroxyacyl-ACP, dehydration to form an enoyl-ACP, and a further reduction to complete the cycle (Fig. 1.5) (von Wettstein-Knowles 1993). Newly formed C16:0 and C18:0 fatty acids are cleaved from A C P by F A T B , an acylA C P thioesterase (Bonaventure et al. 2003). Recently, Bonaventure et al. (2003) showed that in afatb knockout mutant, the saturated CI6 and CI8 fatty acids were reduced by almost half, as was the wax load on the mutant stems. Leaf wax was also reduced. These results demonstrated that a supply of saturated long chain fatty acid substrates of fatty acid elongation is required for wax deposition. Long-chain fatty acids are exported from the plastids as CoA esters, then transported to the ER, possibly in association with acyl-CoA binding proteins, or by direct transfer between the membranes, where they are modified for various cellular functions (Kunst & Samuels 2003). In all cells, most unsaturated fatty acids are transferred to phospholipids for use in membrane biogenesis. In Arabidopsis seeds, CI 8:1 fatty acids are elongated to C20:l and C22:l, then transferred to a glycerol moiety to form storage oils (Lemieux et al. 1990; Ohlrogge & Browse 1995). In epidermal cells, C18:0 fatty acids undergo further elongation to form the very long chain fatty acid (C20+ V L C F A ) precursors of cuticular waxes (mostly C30 in Arabidopsis). Fatty acids may also be elongated for other uses in the plant, including the production of suberin, and sphingolipids, important membrane components. Fatty acid elongation proceeds via a series of reactions analagous to those of fatty acid synthesis, with a condensation reaction, a reduction, a dehydration and a further reduction to elongate the fatty acid by 2C per cycle. Reductions in the chain lengths of the wax components in Arabidopsis mutants cerl, cer6, and maize mutants gl8, gl2, gl3 and gl4 led to the suggestion that genes identified by these mutations could be involved in fatty acid elongation steps. The cloning of CER6 (Fiebig et al. 2000) and GL8 (Xu et al. 1997) has confirmed their involvement in the fatty acid elongation complex. Their roles will be more fully described in Sections and  14  Figure 1.5. Schematic representation of de novo fatty acid synthesis catalyzed by the enzymes of the fatty acid synthetase (FAS) and fatty acid elongation carried out by the enzymes of the fatty acid elongase (FAE) in the cell.  15 However, when the CER2 gene was cloned, it did not show sequence similarity to any known gene in the database, and seemed to encode a regulatory rather than a biosynthetic component (Negruk et al. 1996; Xia et al. 1997). More recent searches of the database suggest that it might encode a CoA-dependent acyl-transferase. GL2 has also been cloned, and shows some similarity to CER2 (Tacke et al. 1995; Post-Beittenmiller 1998). Its conservation with the acyltransferase domain, though, is not as high as that of CER2. The sequence similarity to acyltransferases suggests an accessory biosynthetic role to the elongation pathway, rather than a role in the regulation of enzyme abundance or activity. Nevertheless, the precise roles of CER2 and GL2 remain to be demonstrated biochemically.  1.5.1. Biochemical characterization of fatty acid elongation  Biochemical studies of fatty acid elongation had begun with studies of tissue slices (Kolattukudy 1966) and later microsomes (Macey & Stumpf 1968; Kolattukudy & Buckner 1972) isolated from plant epidermis and developing seeds. These studies showed that plant tissues and extracts could elongate exogenous long-chain fatty acyl-CoA "primers" i f supplied with malonyl-CoA as a C2 unit "donor." This fatty acid elongation (FAE) activity was shown to be membrane-associated (Kolattukudy & Buckner 1972; Cassagne & Lessire 1978), to use acyl-CoA, rather than A C P as a substrate (Cassagne & Lessire 1978), and to be inhibited by different chemicals than de novo fatty acid synthesis (summary, (von Wettstein-Knowles 1993)). B y all of these features, it was distinguished from fatty acid synthesis, which occurs in the stroma of plastids, and is catalyzed by a soluble enzyme complex which uses acyl-ACP and acetyl-CoA as co-substrates (von Wettstein-Knowles 1993). Solubilization of the membrane-bound activity (Lessire et al. 1985; Agrawal & Stumpf 1985a), and later purification of a fatty acid elongase from leek epidermis microsomes (Bessoule et al. 1989) showed that the F A E activity was associated with a large protein (approximately 300-350 kDa after gel filtration chromatography). Agrawal and Stumpf (1985a) suggested that the elongase was a single polypeptide chain because the entire elongation activity was elutable from a gel-filtration column in a single peak. However, further purification showed that it was more likely that elongase was a complex of proteins, because it dissociated under denaturing conditions (SDS-PAGE) into 4 different polypeptides (Bessoule et al. 1989; Lessire et al. 1990). The complex seems to be quite stable, since it was only dissociated by boiling in SDS (Bessoule et al. 1989).  16 The purification of the elongase complex made it possible to assay partial F A E activities, and it has now been shown that the plant fatty acid elongation pathway is analogous to fatty acid synthesis and similar to mammalian fatty acid elongation, involving 4 enzymatic steps: First, a condensation of malonyl-CoA with a long-chain or very long chain acyl-CoA to form a (3-ketoacyl-CoA is catalyzed by the keto-acyl-CoA-synthase (KCS; condensing enzyme). P-ketoacyl-CoA is reduced by a P-ketoacyl-CoA reductase (KCR) to form a phydroxyacyl-CoA, then dehydrated by a P-hydroxyacyl-CoA dehydratase (HCD) to form a trans-2-enoyl-CoA. The final step, catalyzed by an enoyl-CoA reductase (ECR), is a further reduction to produce the elongated acyl-CoA (Lessire et al. 1990; Fehling & Mukherjee 1991; Fehlingetal. 1992) (Fig. 1.6).  cytosol  malonyl-CoA  Figure 1.6. Model of enzymes and biochemical reactions comprising the fatty acid elongase complex. K C S , [3-ketoacyl-CoA synthase; K C R , (3-ketoacyl-reductase; H C D , p-hydroxyacylC o A dehydratase; E C R , enoyl-CoA reductase.  17 The biochemical approach to the study of fatty acid elongation has been fruitful in elucidating the sequence of elongation reactions and elongation requirements, but has not answered a number of important questions. First, there are numerous "elongases" which have been assayed, from different tissues (eg. leek epidermis (Cassagne & Lessire 1978; Agrawal & Stumpf 1985a; Bessoule et al. 1989), maize coleoptiles (Lessire et al. 1982), potato tubers (Walker & Harwood 1986), and a number of oilseeds (Pollard et al. 1979; Agrawal & Stumpf 1985b; Murphy & Mukherjee 1989) and even seemingly from the same tissue, based on substrate and product profiles, as well as reaction conditions (Cassagne & Lessire 1978; Lessire et al. 1985; Agrawal & Stumpf 1985a). However, purification of elongase complexes has not resulted in the separation and characterization of different fatty acid elongation enzymes and their tissue-specific isoforms. Second, some doubt remained as to the exact cellular localization of the F A E complex, and the pathways for the synthesis, localization and cellular use of V L C F A derivatives remained to be determined. Derivation and use of V L C F A s in cuticular waxes will be discussed in sections 1.6 and 1.7. Other uses are beyond the scope of this review. Biochemical studies of F A E localization used sub-cellular fractionation of the microsomes in which F A E was detected (Cassagne & Lessire 1978; Lessire et al. 1982; Moreau et al. 1996). These studies found that F A E is associated with the E R and Golgi membranes, and not with the plasmalemma. The E R localization of the CER6 KCS in Arabidopsis has recently been confirmed by confocal microscopic detection of a GFP (green fluorescent protein)-CER6 fusion protein that complements the cer6 mutant (Kunst & Samuels 2003). Since the F A E complexes purified so far have been tightly associated, with component proteins separating only under harsh, denaturing conditions (Bessoule et al. 1989; Lessire et al. 1990), it is likely that the whole F A E complex is embedded in the E R membrane. With the GFP-CER6 fusion, there is no indication that there is any CER6 protein in the Golgi (Hugo Zheng, pers. comm.). FAE1, the seed-specific KCS in Arabidopsis, also appears to be localized exclusively in the E R (Kunst lab, unpublished data). While it is possible that F A E complexes associated with other condensing enzymes are localized in the Golgi, it seems likely that the Golgi microsomes which showed F A E activity in the cell fractionation studies were contaminated with E R membranes. The high concentration of F A E complex proteins in the ER, estimated to be close to 1% of ER proteins found in leek epidermal cells (Bessoule et al. 1992), suggests that significant F A E activity could be found in Golgi preparations with even a small amount of ER contamination.  18  1.5.2. Molecular characterization of F A E component enzymes Condensing enzymes , The first gene isolated that encodes a fatty acid elongase (FAE) component enzyme was the Arabidopsis FAE1 (Fatty Acid Elongation 1). It was identified in independent screens for mutants lacking V L C F A s in their seed oil (Lemieux et al. 1990; James & Dooner 1991; Kunst et al. 1992). Cloning of a T-DNA-tagged allele showed that it encoded a V L C F A condensing enzyme (KCS; James et al. 1995). No mutation in any of the 3 other enzymes needed for elongation was found. This led-to the hypothesis that the 2 reductases and the dehydratase were shared with a variety of condensing enzymes for different purposes in the plant, and that mutating any of them might therefore be lethal (Kunst et al. 1992). This hypothesis was supported by the fact that expression of the FAE1 gene in yeast was sufficient to cause accumulation of V L C F A s , indicating that the plant condensing enzyme could interact with the yeast reductases and dehydratase to complete the elongation cycle (Millar & Kunst 1997). Furthermore, if F A E 1 was constitutively overexpressed behind the CaMV 35S promoter in Arabidopsis, V L C F A s accumulated throughout the plant (Millar & Kunst 1997), demonstrating that the other 3 F A E enzymes were likely present in all cells. This experiment also revealed that the condensing enzyme determined the chain-length specificity of V L C F A s produced (Millar & Kunst 1997). Biochemical analysis of a F A E complex purified from Brassica napus seeds has confirmed that the condensation step is rate-limiting (Domergue et al. 2000). Studies on a variety of F A E complexes with differing substrate specificities, product ranges, co-factor requirements, and inhibitors, together with genetic analysis of different waxdeficient mutants, led to the suggestion that there were parallel elongases, working on different substrates (eg. one elongase producing saturated V L C F A s used in the decarbonylation pathway, and one producing the P-diketones found in plants such as barley), as well as sequential elongases, each with its own specific elongation step(s), depending on substrate specificity (von Wettstein-Knowles 1993; Post-Beittenmiller 1996). Since the specificity of the elongase complex is determined by the K C S , isolation of different K C S genes and determination of the activities of different isoforms of this enzyme will help elucidate biochemical roles of different elongases. However, since V L C F A s have various uses in the plant, it is likely that different KCS genes will have different biological functions in the plant, even if they catalyze the same biochemical step (Fig. 1.7).  Parallel elongation pathways 18:0-CoA  18:0-CoA  18:0-CoA  18:1-CoA~^  20:1-CoA 22:1-CoA  24:0-CoA  26:0-CoA  28:0-CoA  30:0-CoA /  30:0-CoA  decarbonylation acyl-reduction  cuticular wax  suberin sphingolipids +suberin wax storage oils s e e d  Figure 1.7. Schematic representation of parallel fatty acid elongation pathways in Arabidopsis, and sequential F A E complexes required for the different pathways. Identical elongation steps for different pathways in the same tissue are not necessarily catalyzed by different elongase complexes - some could be shared. The different K C S proteins confer substrate/product specificity.  20 Millar et al. (1999) hypothesized that if there is a K C S specific to production of V L C F A s for seed storage oil, there should be some other KCS(s) that were involved in other functions in the plant. They used the FAE1 sequence to search the Arabidopsis expressed sequence tag (EST) database for similar sequences (using a B L A S T - Basic Local Alignment Search Tool (Altschul et al. 1990)) and identified one that resulted in a waxless phenotype when sense-suppressed. This gene was later identified as CER6 (Fiebig et al. 2000). A T-DNA insertion into the Arabidopsis condensing enzyme gene KCS1 was also found to affect cuticular wax accumulation (Todd et al. 1999). Another KCS-like gene, FDH, was cloned and found to be specifically expressed in the epidermis (Yephremov et al. 1999; Pruitt et al. 2000). Although the fdh mutant is not glossy (Yephremov et al. 1999) and has a WT wax profile, it shows post-genital fusion of different shoot organs (Lolle & Cheung 1993), indicating that epidermal cells require the F D H K C S activity for their normal function. A number of waxless and cuticle mutants (cer 10, cer 13, waxl, wax2, (Jenks et al. 1996a) ded (Lolle et al. 1998), and Icr (Wellesen et al. 2001)) also have cell fusion phenotypes. Constitutive expression of a fungal cutinase in Arabidopsis also results in such a phenotype (Sieber et al. 2000). Hie, a mutant showing a high density of stomata under high C O 2 conditions, also turned out to have a lesion in a KCS-like gene. HIC is expressed only in stomata in leaves, and was suggested to have some sort of a regulatory function (Gray et al. 2000). cerl and cer6 also have higher stomatal indices than WT (Gray et al. 2000), and wax2 has a reduced stomatal index under normal atmospheric conditions (Chen et al. 2003). Fdh has a decreased leaf trichome density (Yephremov et al. 1999). These results all suggest a link between the cuticle and/or cuticular waxes and epidermal cell differentiation and identity. The completion of the Arabidopsis genome sequence allowed a determination of the total number of KCS-like genes in this plant. There are 21. This number includes the 5 whose mutant phenotypes have been presented in the preceding paragraphs, leaving 15 genes whose biological and biochemical functions remain to be established. Fatty acid elongation in plants is required for the production of sphingolipids and suberin, as well as for cuticular wax and seed storage oils. It would be useful to be able to determine which KCSs are the best candidates for roles in cuticular wax production, and which ones are more likely to have other roles. The best way to accomplish this would be to determine expression patterns of the different K C S genes. KCSs associated with wax production should be expressed in the shoot epidermis, and possibly only in these cells. KCSs associated with sphingolipid production, on  21 the other hand, could be expected to be expressed in all plant cells. A root- and/or woundspecific expression pattern would probably characterize suberin-related KCSs, whereas seedspecificity would indicate a K C S involved in seed oil production. It is possible that some of the KCSs would have overlapping roles - especially those catalyzing early elongation steps which might be required for all of the different V L C F A products. Thus, a K C S with a general expression pattern may be used in more than one pathway. In silico analysis of expression patterns of Arabidopsis KCS genes Specific and detailed expression patterns have been examined for only a few KCSs in the Arabidopsis genome. However, additional data are available from the Arabidopsis largescale EST sequencing projects. In these projects, mRNA was isolated from a variety of plant tissues. Using a B L A S T (Basic Local Alignment Search Tool) to search the EST database at the TAIR website (www.arabidosis.org/BLAST) with the genomic KCS-like sequences as queries allows a tally to be made of the frequency with which any given gene is found (hit). Many of the sequences deposited in the database specify the tissue from which the R N A was isolated. This allows a frequency distribution to be made based on number of hits per tissue type. The overall frequency of hits, as well as the frequency of hits per tissue, can be used to get some indication of the expression level and tissue specificity of different genes in Arabidopsis. I have collected this EST frequency data for the entire K C S family of Arabidopsis. It is shown in Fig. 1.8. The MPSS (Massively Parallel Signature Sequencing) project done by the Meyers Lab at UC Davis (http://dbixs001.dbi.udel.edu/MPSS4/java.html) can also be used to get some indication of expression level and specificity for most of the expressed genes in Arabidopsis. MPSS produces short (17bp) sequence tags from cDNA libraries which can uniquely identify more than 95% of Arabidopsis genes. The relative abundance of each tag in a given library represents a quantitative estimate of expression of the corresponding gene (Brenner et al. 2000). The Arabidopsis MPSS website presently hosts a database of tag frequencies from libraries of leaf, root, flower, silique and callus cDNA. 1-2 million tags from each library have been sequenced and indexed. I have searched the database for all of the Arabidopsis K C S genes. Their frequency distributions are given in Fig. 1.8. Absence of MPSS or EST hits does not necessarily mean that a gene is not expressed. Not all tissues and conditions are represented in the databases. Thus, an absence of hits may indicate that the gene is not expressed in the tissues which have been used for the construction of the databases.  22  • E S T hits  60  1800  • M P S S hits  50  1500  40  1200 "D  1 a 3  1900 c/) UJ  20! 1600  300  10 _LL X Q U.  a. LU t< o ID v)  CO  o  co o  O <p co "3Ol to  ^  o  CM  !co £>  lO  o cn o  W> *—•  CM -•—  o  £> o TO o>  o  CO 2  < < < 1  o  •<* CO UJ  TO O  o •«t co  o co  CO  CO CM  CO CM  CM  CM  TO TO  CM  o  o  o  CM CM •tf O  CO TCM UO  T—  CO  TO TO  < <  O  o ho  o  CO T-  o  CO CM  o TO TO TO  < < <  Figure 1.8. Total frequency of E S T and M P S S hits for Arabidopsis K C S genes.  23 The most striking feature of both MPSS and EST data analysis for hits on K C S genes is that F D H and CER6 are overwhelmingly the most frequently hit (Fig 1.8). In the EST database, FAE1 is also well-represented, with about half the hit frequency of CER6. Its expression in the whole plant may be over represented, though, since there has been an EST sequencing project which focussed entirely on developing seeds (White et al. 2000), where FAE1 is specifically and highly expressed (Rossak et al. 2001). MPSS data are not helpful in this case, since FAE J was not hit at all in that project. This is not surprising, however, since siliques used in the MPSS project were harvested 1-2 days after pollination (http://dbixs001.dbi.udel.edu/MPSS4/java.html, library information), and FAE1 expression is only detectable by R N A blot hybridization at 7-15 days after flowering (Rossak et al. 2001). A third frequency class includes a number of genes which have about 25% the EST or MPSS hit frequency of that of CER6 and FDH: KCS1, KCS3, KCS4, At5g43760, CER60, At5g04530, Atlg04220, and At2g28630 (Fig. 1.8). The rest of the family members have very few hits in either database (Fig. 1.8). This could be due to low expression levels, or to expression restricted to a few cells in a tissue, as is the case with HIC (expressed only in stomata; (Gray et al. 2000)). Alternatively, they may be expressed under conditions not analyzed for the EST and MPSS projects. A very restricted expression pattern could amplify sampling error, particularly if the tissue or condition in which the gene is expressed is not represented in the database. There are also some differences in abundance suggested by EST and MPSS data. For example, judging by frequency of EST hits, KCS3 and At5g43760 have similar message abundance in the plant. MPSS data, on the other hand, suggest that the latter gene is much more abundant than the former. Again, this could be due to sampling error in the tissues used or not used for the different databases. Thus, neither the EST data nor the MPSS data can be taken as definitive. More detailed studies should be made of genes of interest. However, these data are useful to get an indication of expression levels and tissue specificities that can be used to determine which genes in the family might be most interesting for further study. Tissue specificities of expression are also important in trying to determine whether any of the family members might be specific to one function {eg., wax production) or another. Therefore I have broken down the EST and MPSS hits into categories based on tissues from which mRNA was isolated for construction of the libraries. This data is represented in Fig. 1.9.  24  (/> 25  + J  r— CO  2 0  W  15  ° FDH • CER6 o CER60 o KCS4  10 5 0 0 • • •  200  HIC At1g71160 At3g52160 At2g28630  100  R  H  Sh  nan  L  F  Si • • • B •  3 2  S  W  HIC At2g28630 KCS2 At3g52160 At2g15090  E  n  1  H  0  Si  0  R  Sh  L  F  Si  S  W  E  20 300  • At1g04220 « FAE1 • At4g34250  • At1g04220  15 200  10 5  100  0 400 300  Si  R 0 1 • • •  At5g43760 At1g 19440 KCS3 At2g26640 KCS1  0  Sh  100  2 0  L B i o 0 1 i  6 4  Si  R  8  200  0  _  R  Sh  L  J F  Si  n S  W  E  W  E  At5g43760 At1g 19440 KCS3 At2g26640 KCS1 At5g04530  F  Mj Si  S  Figure 1.9. Distribution of E S T and M P S S hits in different tissues of Arabidopsis. R, roots; Sh, 2-6 week-old shoots; L, leaves ( M P S S , 14-d-old; E S T , rosette); F, flowers; Si, siliques ( M P S S , 1-2 d post anthesis; E S T , green siliques); C, callus; S, seed; W , water or droughtstressed; E, etiolated or dark-grown.  25 Four main categories of hit distribution among different tissues arose. Some genes are well-represented in all shoot tissues, but not expressed in roots. These include CER6, FDH, CER60, and KCS4. Others were expressed mainly in flowers, including HIC, Atlg71160, At3g52160, KCS2, and At2g28630. A third pattern included expression in flowers and siliques: FAE1, Atlg04220 and At4g34250 were in this group. The fourth group included genes that were expressed in most or all tissues: KCS J, KCS3, At5g43760, Atlgl9440, At2g26440, and At5g04530. This group included all of the genes for which any ESTs were isolated from root tissue. MPSS data suggested high levels of root expression for two of them, Atlgl9440 and At5g43760. In addition to the general categories that were based on both EST and MPSS data, EST frequency suggested that a few of the genes, notably FDH, CER6, KCS1, KCS3, KCS4, Al2g28630, At5g43760 were more highly expressed in stressed (salt- and/or drought-treated) tissue than in the ESTs isolated from tissues grown under normal conditions. In general, the EST and MPSS data are consistent with the roles found for KCSs which have been characterized in more detail. FDH and CER6, both first discovered as mutants with severe phenotypes, have the highest representation in the databases, and very little expression in roots, consistent with the shoot expression and phenotypes reported (Millar et al. 1999; Yephremov et al. 1999; Pruitt et al. 2000). KCS1, which has a milder mutant phenotype (Todd et al. 1999) than CER6 or FDH, is less frequently hit in the EST and MPSS databases. It is also found in all tissues, in agreement with the northern blot presented by Todd et al. (1999). FAE1, known to be a seed-oil specific K C S (Kunst et al. 1992), has hits mostly in developing seeds and siliques. HIC, which is only expressed in stomata (Gray et al. 2000), has very little representation in the EST and MPSS databases. In the databases, HIC cDNAs were found in siliques and flowers, whereas Gray et al. (2000) only looked at expression in leaves. It would be interesting to determine whether HIC expression is restricted to stomata in these tissues as well. Comparison of in silico data with experimental results for some KCS genes Since the EST and MPSS data agree quite well with the published data on the characterized genes, it seems that the data could be useful in making predictions of which genes might be involved with wax biosynthesis, and which might have other roles in the plant. Genes expressed, especially at relatively high levels, in the root, are probably less likely to be involved in wax biosynthesis, and more likely to be involved in suberin or sphingolipid production. This would suggest that genes such as CER60, KCS3, KCS4, and At2g28360 are  26 probably more likely to have roles in wax production than some of the others, since they are all expressed at fairly high levels in various shoot tissues, but not in roots. In addition, KCS3, KCS4, and At2g28630 are represented in the EST libraries obtained from salt- and droughttreated shoots, as are CER6, FDH and KCS1. These types of stresses have been shown to increase wax production (section 1.9.3), and could accomplish this through upregulating, or at least maintaining, transcription of genes encoding enzymes (including KCSs) involved in wax biosynthesis. The prevalence of CER6 in the EST and MPSS databases, as well as the severe waxlessness found in sense suppressed and mutant plants, led me to center the first part of my thesis research on characterizing its expression pattern more fully. These results will be presented in Chapter 3. However, it was also of interest to take a preliminary look at cell specificity of expression of some of the other genes identified by the database study as potential wax-related K C S genes. Accordingly, Nicole Quenneville, an undergraduate working under my direction, made promoter-GUS constructs of KCS3 and KCS4 to further test whether these KCSs could be involved in the synthesis of wax precursors. Her results showed that the promoters of both of these genes directed GUS expression throughout the shoot, in seedlings as well as in mature plants. No GUS activity was detected in roots. Interestingly, KCS3 expression seemed to be limited to the epidermis, and KCS4 was localized specifically in stomatal guard cells, similar to that seen for HIC in leaves (Gray et al. 2000). These preliminary results suggest that at least KCS3 may be involved in wax production in Arabidopsis. Expression specific to guard cells suggests that there may be some specialization in these cells that uses specific KCSs. It has been found in other species that there are differences in wax crystals on guard cells (von Wettstein-Knowles 1995), but this has not been observed in Arabidopsis. Expression data have also been obtained for a few other K C S genes. KCS2 is expressed most strongly in flowers, consistent with the 2 of the 3 EST hits for this gene being found in flowers. GUS assays on KCS2promoter-GUS-transformed plants showed that this floral expression was limited to anthers (Scherson 2000). KCS2 was also weakly expressed in very young emerging leaves (Scherson 2000). This expression pattern suggests that, as in stomatal guard cells, there is some use for a specific K C S in these tissues. In anthers, it may be that KCS2 is involved in the production of pollen tryphine lipids, like CER6 (Fiebig et al. 2000). However, suppression experiments did not produce any obvious phenotype (eg. male sterility) in the case of KCS2.  27 A root-specific K C S has been isolated from Lesquerella fendleri (LfKCS45) (Moon et al. 2001; Chowrira et al. 2002). Its expression domain (based on promoter-GUS analysis) has a partial overlap with a region of neutral lipid accumulation in the root tip (based on Nile red staining). However, its biological function has not been elucidated. One or more of the Arabidopsis KCSs with root expression may be orthologues of this gene. Atlgl9440 is a particularly good candidate, since its EST hits are mostly from roots and stressed shoots, and MPSS data suggest that root expression is higher than shoot expression for this gene. A K C S has also been cloned from corn roots (ZmFAE) (Schreiber et al. 2000), but no expression studies were done- on this gene. KCS mutants in Arabidopsis The best way to determine biological roles of different KCSs in the plant is to find mutants and characterize their phenotypes. Some mutant phenotypes are easier to decipher than others, though. Fael, lacking seed oil V L C F A , and cer6, lacking epicuticular wax, represent two K C S genes with obvious functions. A detailed analysis of wax in kcsl has also allowed KCS1 assignment to a wax-related function. Fdh and hie, on the other hand, are less tractable. For both of these genes, some sort of developmental signaling has been suggested by the groups that cloned them. Fdh has an organ fusion phenotype, but no wax phenotype was detected either visually (Yephremov et al. 1999) or by GC (Jenks et al. 1996b). A more detailed analysis of epidermal, as well as cuticular lipids, and a biochemical analysis of the substrate and product specificity of this gene may help to pinpoint its role. In the case of hie, no wax or lipid analysis was reported. Biochemical and more detailed phenotypic analysis would also be required to elucidate its role. One of the thrusts of the Arabidopsis functional genomics projects recently has been to produce a sequence-indexed database of T-DNA insertional mutants (Salk Institute; searchable website, http://signal.salk.edu/cgi-bin/tdnaexpress), which are made available through the Arabidopsis Biological Resource Center (ABRC). Through this project, T - D N A insertions into a number of the Arabidopsis K C S genes have become available. I ordered and grew six of these insertion lines, hoping to find more K C S mutants, particularly in wax-related K C S genes. Only one of these six lines had a visible phenotype. A n insertion in At5g43760 produced a dwarf phenotype with greatly reduced fertility (Fig. 1.10). MPSS and EST hit frequency for this gene suggested that it is expressed throughout the plant (Fig. 1.9). There were also quite a few ESTs isolated from stressed shoots. The ubiquity of the expression pattern and the severity of the phenotype suggest a role in sphingolipid production. However, the phenotype requires more detailed analysis before any conclusions can be made.  vwd-type  35S-CUT1  v.,_ f  r  »•»*  >  •From Yephremov et al. 1999  c  WT  A  kcs1-1  * S*  •  •  From Todd e t a l . 1999  %  l!l|!!!!|n  Figure 1.10. Mutant phenotypes caused by K C S disruption. A, CER6 suppression. B1, fdh mutant; B2, F D H overexpression phenotype. C , kcsl mutant; D1, Dwarf phenotype of K C S At5g43760 T-DNA insertion mutant, comparison with W T (plants are the same age); D2, K C S AT5g43760 T - D N A insertion mutant, close-up of infertile siliques  29 Biochemical characterization of KCS activities Expression analysis can give some indication of which KCS proteins may be involved in producing V L C F A precursors for different uses in the plant. However, most pathways require several elongation steps to produce the chain lengths needed. Sphingolipids require C26 fatty acids, which involves 4 elongation cycles. Waxes require 6 elongation cycles to produce their C30 fatty acid precursors. These elongation cycles significantly increase the length of the hydrophobic hydrocarbon chains of the fatty acids, and it is quite likely that more than one K C S is required to catalyze the sequential elongation cycles. Most of the biochemical study to date has been done on FAE1 and on other seed-specific K C S genes isolated from other plants. The only non-seed K C S that has been characterized in detail and published is KCS1 (Todd et al. 1999). Study of other Arabidopsis KCS substrate and product specificities has been initiated in our lab, and some preliminary results are available. However, the discussion will be broadened at this point to include K C S genes and proteins isolated from other plants. FAE1 is the only seed-specific KCS in Arabidopsis. Biochemical assays in vitro (Kunst et al. 1992; Ghanevati & Jaworski 2002) and in yeast (Millar & Kunst 1997) have shown that it can use  CI6-C0A, CI8-C0A  and C20-CoA as substrates to produce the seed oil characteristic  of Arabidopsis. The FAE1 sequence has been used to isolate KCS genes from other species, which have different V L C F A chain length compositions in their seed oils. KCSs have been identified from seeds of jojoba (Simmondsia chinensis, (Lassner et al. 1996)), high- and lowerucic acid rapeseed (Brassica napus, (Barret et al. 1998; Fourmann et al. 1998), and Brassica campestris, (Das et al. 2002)); mustard (Brassica juncea, (Venkateswari et al. 1999); Sinapis alba, (Drost et al. 2001)), Brassica oleracea (Das et al. 2002), desert mustard (Lesquerella fendleri, (Moon et al. 2001)), and meadowfoam (Limnanthes douglasii, (Cahoon et al. 2000)). Characterization of substrate specificities or product profiles in transformed yeast (in vitro or in vivo) has been carried out for some of these KCS proteins. For others, data from the purification of the complex from developing seeds, and the fatty acid profile of the seed oil, is the only characterization that has been done. The assumption in these cases is that the K C S cloned from the seeds is the only one contributing to the seed oil, and that in vivo, it catalyzes the condensation reactions for all of the elongations required for the seed oil composition. This may be a valid assumption in some cases (eg. Arabidopsis), but in others (eg. B. napus) it is not necessarily so. B. napus, in particular, has an amphidiploid genome. Therefore, each plant contains two BnFAEl homologues (Barret et al. 1998; Fourmann et al. 1998). For B. napus, a number of different FAE1 alleles have been isolated and their sequences have been compared, but no definitive comparison of activities for each allele has appeared in the literature. It seems  30 to be assumed that alleles isolated from high-erucic varieties will have the characteristic activity, and those isolated from low-erucic varieties will not (ie., that they will be mutant forms) (Fourmann et al. 1998; Drost et al. 2001; Das et al. 2002). For elongases purified from epidermal or other tissues, no assumptions can be made as to substrate specificities, since more than one are probably needed in each situation. For these tissues, it would be more useful to characterize recombinant K C S proteins in heterologous systems such as yeast, as done by Blacklock and Jaworski (2002) and Ghanevati and Jaworski (2002) for F A E 1 . A summary of the available data on substrate specificities of different K C S proteins is presented in Table 1.2. Interestingly, the seed-specific KCSs seem to have quite broad specificities, elongating both saturated and mono-unsaturated fatty acids of several different chain lengths. The seed oils of the plants from which KCSs have been isolated are mainly monounsaturated. The difference between seed oil compositions and substrate specificities of the K C S enzymes isolated from oilseeds may be related to the endogenous lipid environment of fatty acid elongation. It could be that the elongase complex in the developing embryo only has unsaturated chain lengths available. Therefore these are elongated in vivo. The elongase complex purified from leek epidermis has also been found to use both saturated (with varying chain lengths) and 18:1 fatty acids as substrates (Lessire et al. 1989), unlike elongases assayed from microsomes in earlier experiments, which would only elongate saturated fatty acids (Agrawal et al. 1984). Cassagne et al. (1994) suggested that the absence of 18:1 elongation activity in microsomes was due to the organization of the membrane or because of competition from other enzymes (eg. phospholipid synthases) for the 18:1 substrate in the epidermal cells. The activity of recombinant KCS1 in yeast was similar to that found for the purified elongase (Table 1.2; (Todd et al. 1999)), which is in agreement with this hypothesis. KCS2, on the other hand, seemed to be have a specificity for shorter chain (18C) saturated fatty acids (Scherson 2000). This, together with preliminary results obtained for CER6, suggests a new hypothesis. When CER6 was expressed alone in yeast, it elongated the endogenous C26:0 to C28:0. However, when it was co-expressed with B n F A E l , which causes significant accumulation of C24:l in yeast cells, the presence of CER6 caused an accumulation of C26:l and C28:l fatty acids (Owen Rowland, pers. comm.). This suggested that although CER6 may not have a specificity for saturated vs. unsaturated fatty acids, another K C S which must be active in the production of precursors for wax synthesis (since CER6 doesn't seem to elongate fatty acids shorter than C24) could have such a specificity. Therefore, the KCSs catalyzing elongation of longer fatty acids would never encounter an unsaturated substrate (Owen Rowland, pers. comm.).  31 E  CD  £ o  CN 03 (35  T3 CO CD  E  CM  O O  CO CO  O) O •Q  CU O  CN  CU  O  o  CD  CN  .2 a:  . 03  -C C  -3  . - CD  %  T3 C  CU  "D  T3  E o  ,C o  CD  CD T3 CO  CD  CD CU ><  CD  CD °3 . y CD  co C CO CO CD C  CD O  3  CD & CD -oco  CD  CD  c  a5 fu o CD Q. 5  co 2 o ™  ^ "° 9  O £= "CD CD J2 O (/) w  CD  o  t  3  \zz ~  CD  3  CO  _0> XI  F  T3  1  c  3  CO  .3 OQ  E  •a I 8. ?  CN  03 CN "co to XI 3 (0  CO "O2 £  "co X! 13  + + +  "+" + + +  O  E  S..y -Q o"  B ~  + + + +  <  CO -p-  *S a . CL Q.  0 TO .O 3 (0  c  CD CD  + + + + + + + +  C  "» c  o  C£  (0 03  >  2 Q. 3  O  -  O > > C  Q-  CD  CO  C  .  RO  to  co <  CO  I-a cu  CD  CD CL  co ?;  XI  (O  E  — 4o  E  I_  to  Q. CU  Si  •o o c o Q.  ID  O  I "5  CU  .CD co  OJ  CU  'i  CD CU >%  CU  .co co  o  f  (0  co  E cu  o QT c  CD  (U  (0  O CD  •a  CD  CU Q.  T3~ C JO  CJ)  11  -R ">  c  co  CD  jl  CD  CU  C3  CN  CD  £ E o o  8  o o o  03 03  I'd  CD  XI  O CO 03  CD CU  c  CO CD  ^  o  + +  + + +  + + +  + + +  T" + +  + + +  a) <. o 3 O i to >. CO o co "CD  + + + +  + +  + + "T" + + _+_  U  E  >>l  LU  < LL C  ICQ  CO  o  XL o  CO  LU < LL T3  m CO  1^'  o  32 Considering only the non-seed KCSs that have been studied, it seems that there must be at least 3 involved with production of the C30:0 fatty acids used in Arabidopsis waxes. CER6 does not elongate anything shorter than C24, and only continues as far as C28. The yeast data supporting this are supplemented by the fact that CE7?(5-suppressed transgenic plants have an accumulation of 24C wax components. Some of the seed-specific KCSs start with shorter chain lengths (including C16 and CI 8), and elongate fatty acids up to C24. Most of them, however, seem to stop at shorter chain lengths. It may be that two KCSs are required for the synthesis of wax precursors up to the C24 stage. KCS2, which is expressed in anthers and very young leaves, elongates CI8. However, it is not a very abundant enzyme and not expressed in tissues where most wax synthesis is taking place. Therefore, other KCS(s) probably fulfill this role for wax production in most of the plant. Finally, CER6 does not elongate C28. Therefore, another K C S would be required to elongate this substrate to C30. The root-specific LfKCS45 does include this specificity. It elongates saturated C26 fatty acids to C30 in yeast cells. A similar K C S may be present in Arabidopsis that catalyzes a similar set of elongation condensation reactions and is expressed in shoot epidermal cells that produce wax. Phylogenetic relationships of plant KCSs The biochemical specificities, mutant phenotypes and expression patterns are available for a number of the K C S genes in Arabidopsis. It would be interesting to see whether this information together with a phylogenetic analysis involving KCS-like genes from other plant species would allow any predictions as to which K C S genes are involved in which process. This might direct research into the most fruitful direction to fully characterize K C S genes involved in wax biosynthesis, or those involved in other processes. Therefore, I aligned most of the available K C S sequences (identified from published papers and from a B L A S T p (BLAST using amino acid sequences) of the non-redundant (nr) database at N C B I , for unpublished sequences deposited to Genbank) with the Arabidopsis K C S protein sequences using Clustal X . I also constructed a bootstrapped neighbor-joining tree to compare the KCSs based on sequence similarity, and to determine whether any patterns emerge, or any functional predictions can be made (Fig. 1.11). The chromosomal locations of all of the Arabidopsis K C S genes have also been plotted on a chart of the Arabidopsis genome which shows regions of chromosomal duplication (Fig. 1.12).  33 0.05 999 1000  907 1000  1000  1000 1000  •At3g52160 —OsAAN65442 FDH • AmCAC84082 -TmAA047729 •GhAAL67993 -OsBAB91850 -CER60 •CER6 At4g34250 At2q15090 LfKCS45 LfKCS3 AtFAEI 998 rBjCAA71898 |_rBrAAM33539 BjAAM11648 1000 r-BrCAC79669 j-BnAAA96054 f1rBnAAM08350 "BnAAM08351 rBrCAC79670 BoCAC79671 1rBrAAM08352 ^BnAAK64213 -At1g19440 —KCS2 —KCS3 —ScKCS -At5g43760 -At1g04220 TmAAL.99199 -LdAAG28600 —At2g26640 -ZmCAC01441 r"At3g1028Q HIC •~"KCS1 OsAAK95678 •HXAAC34858 SDAAD27560 OsAAN06858 DSAAK11266 M  988  D  m 73  5 m  r  1000  941j  997 1000 1000  1000  705  D  I  l  998 989"t 1000  807  1000  -OsAAG16863 •At5g04530 •KCS4 -At2g28630 -GiEAA38730 —Chalcpne synthase  o  O -ft  Figure 1.11. Neighbor-joining phylogram of KCS-like derived protein sequences from Arabidopsis (Arabidopsis thaliana; At prefix), rice (Oryza sativa, Os), snapdragon (Antirrhinum majus, Ar), nasturtium (Tropaeolum majus, Tm), cotton (Gossipium hirsutum, Gh), Lesquerella fendleh (Lf), Brassica sp. (6. juncea, Bj; B. rapa, Br; B. napus, Bn; B. oleracea, Bo), jojoba (Simmondsia chinensis, Sc), meadowfoam (Limnanthes douglasii, Ld), maize (Zea mays, Zm), daylily (Hemerocallis hybrid HX), sorghum (Sorghum bicolor, Sb), Dunaliella (Dunaliella salina, Ds), and Giardia intestinalis (Gi). Arabidopsis chalcone synthase was included as an outgroup. Bootstrap values have been included for selected branches. Clades are separated by coloured shading, and labeled to the right of the phylogram.  34 o  iAt1g71160  c-t  c-i  At5g49070  At5g43760  o  |At1g04220  At5g04530  1-BKCSI  !  <At1g01120) l=  Figure 1.12. Chromosomal locations of Arabidopsis KCS-like genes plotted on a diagram of large chromosomal duplications. Coloured bars show regions of duplication; KCS-like gene loci are indicated with black marks. Scale (mega-bases) is noted on the sides. (Adapted from http://mips.gsf.de/proj/thal/; some of the coloured bars linking duplicated regions have been deleted. Dashed lines have been added to indicate K C S genes with very high similarity, which are not included in large duplicated regions.)  35 The tree defines a number of well-supported clades. I have designated these as the F D H , K C S 1 , CER6, D W A , FAE1, and KCS4 clades. The F D H clade contains two Arabidopsis sequences, F D H and At3g52160, and sequences from snapdragon (Antirrhinum majus; Am), nasturtium (Tropaeolum majus; Tm), cotton (Gossypium hirsutum; Gh) and rice (Oryza sativa; Os) that group with F D H . Nothing is known about the biochemistry of any of these proteins. However, it is interesting that F D H seems to have candidate orthologues in many of the species from which K C S sequences have been isolated. This suggests that the F D H function may be conserved in many plant taxa. CER6 groups with CER60, as well as an apparent rice orthologue. The presence of two CER6-like homologues in Arabidopsis seems to be the result of a chromosomal duplication event between the top and bottom of chromosome 1 (Fig. 1.12). The rice protein branch length is quite long; however, the grouping has good bootstrap support. It would be interesting to determine experimentally (by suppression in rice, complementation of the cer6 mutant, and/or biochemical analysis) whether the rice sequence is a functional orthologue of CER6, which could verify or nullify the significance of this grouping. The FAE1 clade is the biggest, including all of the seed-specific KCSs isolated from the Brassicaceae and 5 Arabidopsis sequences whose functions have not yet been defined. The Brassica species FAE1 homologues all have similar functions to that of F A E 1, although they allow elongation up to C24:l instead of just C22:l fatty acid, as found for A t F A E l . LfKCS3, which elongates hydroxy fatty acids of similar chain length (Moon et al. 2001), groups closely with the other F A E homologues, but outside of the main group. The hypothesis that enzymes of similar biochemical function might form distinct clades in the tree is opposed by the fact that the FAE1 clade also includes LfKCS45, which has a very different substrate specificity than the seed-specific KCSs. The FAE1 clade includes KCS2 and At4g34250. The gene encoding KCS2 is located less than lkb upstream from FAE1, and the At4g34250 gene is also very close by. A set of two KCSs, whose genes are also closely spaced on chromosome 2, KCS3 and At2gl5090, are also included in this clade. No large chromosomal duplication events seem to link their genes, but their placement is interesting and suggests small duplication events - either independently within the chromosomes, or once within and once between the two chromosomes. It will be interesting to revisit this connection when the KCSs in this family have been more fully characterized. Another chromosomal duplication event on chromosome 1 involves Atlgl9440, from the FAE1 clade, and Atlg71160. The protein encoded by the latter gene is not very closely  36 related to the former. It is quite divergent from all of the other KCSs on the tree. Similarly, At3gl0280 and At5g04530 are part of a large duplication between chromosome 3 and chromosome 5. The products of these genes are also more distantly related than At3gl0280 and HIC, which have identical amino acid sequences. It is interesting that gene products that are very closely related are not always encoded by genes present in large duplicated segments, and that genes that are generated by gene duplication often encode proteins that are not very closely related. KCS4 groups closely with At2g28630. Genes encoding these two KCSs seem to be part of a chromosomal duplication event from chromosome 1 to chromosome 2. However, At2g28630 is located quite far from the main block of duplication. Perhaps this was an early duplication event, and subsequent events distanced At2g28630 from the rest of the block. The KCS4 clade also includes another Arabidopsis K C S and a rice K C S which are more distantly related. Little is known about any member of this group. The KCS1 clade includes sequences from rice and daylily which group with good support more closely to KCS1 than to HIC and At3gl0280, which are also included in the clade. The rice and daylily sequences could represent KCS1 orthologues. Finally, the D W A clade includes three Arabidopsis KCSs which seem to be fairly similar, but not associated with any large chromosomal duplication. At5g43760, which has a dwarf phenotype in the T-DNA insertion line, is quite closely related to Atlg04220. It would be interesting to determine whether these two KCSs are functionally redundant. This could be done by crossing the two insertion lines to determine whether the At5g43760 phenotype would be more severe in the double mutant. Based on the MPSS hit and EST frequency, Atlg04220 is expressed mostly in flowers and siliques, whereas At5g43760 has a broad expression pattern. It could be that the double mutant would be sterile or even lethal, if the two KCSs are involved in V L C F A production for sphingolipids. A biochemical analysis would help determine whether the two enzymes have overlapping specificity, and an examination of tissue- and cellspecificity of expression would help in elucidating their biological roles. The other Arabidopsis sequence included in the D W A clade is At2g26640. It groups most closely with the K C S cloned from corn roots. MPSS and EST frequency of At2g26640 suggest that it is expressed in roots, but also elsewhere in the plant, including shoots, developing seeds and etiolated plants. Since no expression profile of the corn root K C S has been reported, it could be that these two enzymes are orthologues. Further characterization of both KCSs would be interesting. This clade is not well-resolved, however, and also includes  37 seed-specific KCSs from jojoba (Simmondsia chinensis; Sc), nasturtium (Tropaeolum majus; Tm), and meadowfoam (Limnanthes douglasii; Ld). The analysis of At5g43760 and its partner (Atlg04220) would likely be more productive. KCS conclusions To characterize the K C S gene family in Arabidopsis, a combination of approaches is required. V L C F A s are used in plants for the production of seed oils, waxes, suberin, and sphingolipids. Determining cell-specific expression patterns (using promoter-GUS fusions and assays, as well as in situ hybridization), and characterizing mutant phenotypes (with particular focus on changes in composition of V L C F A s and their derivatives) will be the most productive ways of determining which KCSs are involved with which pathways in the plant. Biochemical assays of recombinant enzymes will reveal which elongation step each K C S catalyzes, and allow the big picture to be stitched together. The preliminary evidence presented in the preceeding sections suggests that CER6 is the major wax-specific K C S , due to its high expression level and its severe mutant phenotype (Millar et al. 1999; Fiebig et al. 2000). Thus, this gene merits further investigation as a potential rate-limiting control point for wax biosynthesis. However, the preliminary activity assessment for CER6 suggests that other KCSs must also be involved in wax biosynthesis. A few likely candidates for this have been identified. KCS1 has been shown to be involved in wax production, mostly in leaves (Todd et al. 1999). Expression patterns of CER60, KCS3, KCS4, and At2g28630 suggest that these KCSs are also good candidates for wax-related condensing enzymes. Reductases Both reductases of the F A E complex have been cloned. Beaudoin et al. (2002) identified a p-ketoacyl-CoA reductase in yeast by screening knockout mutants for the loss of a heterologous F A E activity. The yeast YBR159W gene was found to rescue the mutant's heterologous F A E and to interact with the other yeast F A E enzymes. A n Arabidopsis orthologue (Atlg67730) was identified and shown to complement the yeast mutant, as well (Han et al. 2002). When X u et al. (1997) cloned the GL8 waxless gene from maize, similarity of its sequence to the bacterial FAS reductase led them to suggest that it encoded a F A E (3ketoacyl-CoA reductase. An Arabidopsis orthologue of this gene was cloned from a c D N A library and sequenced (Xu et al. 1997). This gene is the same as the one identified by Beaudoin  38 et al. (2002). Recently, the GL8 protein was also shown to have the predicted (3-ketoacyl-CoA reductase activity (Xu et al. 2002). A BLASTp of the Arabidopsis genome database revealed that there is an additional P-ketoacyl-CoA reductase homologue (Atlg24470) in Arabidopsis (Kunst & Samuels 2003). A yeast enoyl-CoA reductase, TSC13, was identified as a temperature-sensitive mutant that had a deficiency in V L C F A synthesis (Kohlwein et al. 2001). It was shown to interact genetically (additive phenotype) with the yeast V L C F A condensing enzymes E L 0 2 and E L 0 3 (Kohlwein et al. 2001). Its activity was confirmed by build-up of enoyl-CoA and hydroxyacyl-CoA intermediates in F A E assays of the mutant microsomes, and it was shown to coimmunoprecipitate with yeast condensing enzymes (Kohlwein et al. 2001). Kohlwein et al. (2001) identified an Arabidopsis orthologue (At3g55360) of the Tscl3p by amino acid sequence similarity, and showed that the yeast, human and Arabidopsis orthologues all have similar hydrophobicity profiles, each with 3 potential membrane-spanning domains. A B L A S T p of the Arabidopsis genome database suggests that this is the only Arabidopsis homologue for this protein. The only other Arabidopsis protein (At5gl6010) with significant similarity to Tsclop is more similar to steroid 5a reductase. Tscl3p and the human homologue, SC2, are also similar to the steroid reductase, which prompted Kohlwein et al. (2001) to investigate the possibility that Tscl3p was the enoyl-CoA reductase, since the steroid reductase also catalyzes the reduction of a double bond that is ot,P to a carbonyl group. To demonstrate that the putative Arabidopsis homologues of the acyl-CoA reductase and the enoyl-CoA reductase do have the expected activity, they should be used to complement the corresponding yeast mutants, and/or assayed biochemically. It would also be useful to examine the knockout phenotypes of all three of these genes in Arabidopsis to determine whether any of them result in wax-deficiency, as found with cer6. However, it may not be possible to find knockouts in these genes if they encode essential functions. p-hvdroxyacvl-CoA dehydratase  .  The only component of the F A E complex that still remains elusive to cloning and molecular characterization is the P-hydroxyacyl-CoA dehydratase (HCD). A n antibody to a purified rat HCD has been produced (Osei et al. 1989) and shown to cross-react with the purified leek elongase complex. This antibody identifies the 63kDa protein, of the four polypeptides observed by SDS-PAGE analysis of the purified complex, as the dehydratase  39 (Lessire et al. 1990; Lessire et al. 1993; Domergue et al. 2000). The same antibody inhibits the dehydratase reaction in fatty acid elongation assays (Lessire et al. 1993). Later studies of the rat protein showed that it was a peroxisomal protein, rather than the microsomal protein originally thought to be purified (Cook et al. 1992); however, it appears that the activity is a reversible (3-hydroxyacyl-CoA dehydration to enoyl-CoA (Knoll et al. 1999). The reversibility of the reaction and the similarity of the dehydratases isolated from leek (Lessire et al. 1993) and rat brain (Knoll et al. 1999) to the rat liver protein (Bernert, Jr. & Sprecher 1979) against which the antibody was raised (Osei et al. 1989) suggest a similarity of hydroxyacyl-CoA dehydration in F A E with enoyl-CoA hydration in P-oxidation. Two Arabidopsis proteins, AIM1 (At4g29010) and CHY1 (At5g65940) have been identified as being involved in P-oxidation, based on cloning of genes responsible for mutations at the aiml and chyl loci (Richmond & Bleecker 1999; Zolman et al. 2001). A B L A S T p of the Arabidopsis genome using these protein sequences as queries, and then re-BLASTing with the different Arabidopsis hits identified a family of 17 Arabidopsis genes that show a 35-60% sequence similarity among the different members. Fig. 1.13 shows a neighbor-joining tree constructed using these 17 sequences, and several similar protein sequences from other species identified in a B L A S T p of the NCBI database. These sequences were included because enzymatic activities have been identified for each of them, and they might help to predict which of the family members might (or might not) be involved with fatty acid elongation. The Arabidopsis sequence most similar to the E. coli FabZ, which catalyzes the dehydration step of FAS (Heath & Rock 1996), was included as an outgroup. The tree shows several clades within the family. The one with the most members, and the most closely related sequences, includes the CHY1 protein (Fig. 1.13), which has Phydroxyisobutyryl-CoA hydrolase activity (Zolman et al. 2001). This clade has also been identified by Zolman et al. (2001), in a tree based on an alignment of the region surrounding the active sites of a number of dehydratase/hydratase-like proteins. A human homologue which was included in this clade was found to complement the chyl mutant as effectively as the CHY1 WT gene (Zolman et al. 2001). In addition, the members of this hydrolase clade have a few key residues differentially conserved from the hydratase/dehydratases, which may help to distinguish these two enzyme classes (Zolman et al. 2001). Since the Arabidopsis sequences in this clade probably represent hydrolases, rather than dehydratases, they are unlikely to represent F A E - H C D .  40  p-hydroxybutyr)  C  H  Y  1  At2g30660 At3g60510  At2g30650 At1g06550 At4g 13360 / At3g24360  At4g31810  C i n n a m o y l - C o A hydratase  At4g16210  A c y l - A C P dehydratase (At5g10160) At4g 14440  EcoiiMENB At1g60550  X ^ ~ ^ A t 4 g 14430  R n D i e n o y l - C o A isomerase At5g43280  MFP:  AIM1  %^ G ,  ^•At1g65520 At4g16800 MmAUH ^  > O  Figure 1.13. Unrooted phylogram of p-hydroxyacyl-CoA dehydratase/enoyl-CoA hydratase - like genes of Arabidopsis (At prefixes; C H Y 1 ; AIM1 and MFP2). Sequences of proteins with experimentally demostrated functions have been included; species prefixes: Hs, Homo sapiens; C e , C. elegans; Rm, Rhizobium melilotum; Rn, Rattus norvegicus; Mm, Mus musculus. An Arabidopsis acyl-ACP dehydratase (from the F A S system) and cinnamoyl-CoA hydratase have been included as outgroups.  41 The AIM1 protein has been shown to be required for normal inflorescence development. Long-chain fatty acid composition of aiml mutant leaves is also somewhat different than that of the WT (Richmond & Bleecker 1999). Both this protein and its homologue MFP2, with which it groups on the tree (Fig. 1.13), have been shown to have enoylCoA hydratase activity, and both are highly conserved (57 and 76% amino acid identity, respectively) with the characterized cucumber multifunctional protein involved in fatty acid Poxidation (Richmond & Bleecker 1999). Thus it seems likely that MPF2, as well as AIM1, is involved with p-oxidation, and both are probably unlikely to represent the F A E - H C D . A third group of three proteins (At4gl4430, At4gl4440, and Atlg65520) is the carnitine-racemase-like clade (Fig. 1.13). Carnitine is a short, bulky molecule with charged groups on both ends (Fig. 1.14). It seems likely that the substrate-binding site and hydrophobic pocket that would be necessary for fatty acid elongation would be quite different than that required for carnitine binding. Thus, this group seems unlikely to include the H C D enzyme. This leaves 4 candidate genes which do not group with any clade. Three of these group with good bootstrap support with proteins that have been characterized in other organisms. Atlg60550 pairs with the E. coli M E N B protein, with a relatively short branch length. This protein is a dihydroxynaphthoic acid synthase. As such, its substrate specificity and binding site are probably somewhat differently shaped than would be required for an enzyme involved in V L C F A elongation. At5g43280 groups with the rat dienoylCoA isomerase, which is involved  H i  H \r* C  H O  H H  N  / H  H  —  H i C — C C H  C H  H  Figure 1.14. Structure of carnitine  42 with an alternative pathway of fatty acid P-oxidation degrading unsaturated fatty acids (Modis et al. 1998). This activity is somewhat similar to fatty acid elongation, leaving this protein a reasonable H C D candidate. At4gl6800 groups with mouse A U H protein that has been shown to have enoyl-CoA hydratase activity. Immunolocalization experiments indicated that the mouse protein seems to be localized in the mitochondria, suggesting that it is probably involved with P-oxidation rather than F A E (Brennan et al. 1999). However, this does not rule out the possibility that the Arabidopsis protein could be involved with F A E , and this candidate deserves further consideration. Finally, At4gl6210 does not group with any of the aligned sequences, but it is more closely related to the enoyl-CoA hydratase family than to the FAS dehydratase outgroup. This raises the possibility that it could have a function novel to the group, and is a possible candidate for the F A E HCD. To further characterize the best candidates, I ran the protein sequences through the PSORTI program (Nakai and Horton, http://psort.nibb.ac.jp/), collected predicted molecular weight and topography for the proteins (from MIPS, http://mips.gsf.de/proj/thal/), as well as EST frequency (from MIPS) and MPSS hits for estimates of expression. These data are given in Table 1.3 and 1.4. At5g43280 has a classical S K L peroxisomal target sequence. At4gl6800 and Atlg60550 also have fairly high probabilities of being peroxisomal. However, neither of these two proteins has a very clear sorting signal. At4gl6210 is predicted to have a transmembrane domain, but because it does not have a clear E R localization signal, it is predicted to be localized to the plasma membrane. It is worth mentioning that PSORTI does not provide a strong E R localization prediction for K C S genes either, yet both FAE1 and CER6 have been shown experimentally to be localized in the ER membrane (Kunst & Samuels 2003). Since the prediction of subcellular localization is based on established sorting signals, it may be that for these lipid biosynthetic enzymes, some novel sorting mechanism is involved. Thus, these predictions seem to favour At4gl6210 as an HCD, but they are only predictions, and cannot exclude the other candidates. To add one more factor to differentiate between potential H C D genes, I summarized expression data found in the databases in the form of EST frequency and MPSS tag frequency (Table 1.4). EST's have been isolated for 3 out of the 4 candidates, and MPSS data for the one candidate (At4gl6800) not represented in the EST database suggest that it is expressed throughout the plant. Atlg60550 is represented by 2 ESTs, but does not have any MPSS hits. The low frequency of ESTs suggests that it is not highly expressed. Since it contains potential  43 Table 1.3. PSORTI predictions of cellular localization of enoyl-CoA hydrase-like genes. Values indicate degrees of certainty that the protein will be localized in the given compartment. P S O R T PREDICTION  GENE  Peroxisome At4g 16800 At4g16210 At5g43280 At1g60550 At4g 13360 At4g31810 At3g06860 At4g29010 At2g30650 At2g 30660 At5g65940  0.8 0.8 0.436 0.3  Nucleus Mitochondria Chloroplast 0.6  0.32 0.1 0.36 0.44l||  0.1 0.124 0.1 0.28 0.2  Plasma membrane  ER  0.7  0.2  Golgi Cytoplasm  0.45 0.45  0.647 0.496  0.5 0.8 0.845 0.805  0.1 0.511  0.8  0.437  0.93  0.19  0.28 0.1 0.28 0.28  unique signature sequences that could be detected by MPSS, the lack of MPSS hits may also indicate a low expression or a lack of expression in the tissues represented in the MPSS database. At5g43280 is well-represented by both EST and MPSS hits, and seems to be quite highly expressed throughout the plant. At4gl6210, the only candidate with a predicted transmembrane domain, has MPSS hits only in shoots, but 2 of its ESTs were isolated from roots. However, this protein does not seem to have a very high expression level. It is enticing to think that one of the four candidates identified could represent the HCD gene. It is difficult to narrow the choice further based on predictions of sub-cellular localization and topography, or expression. While PSORTI analysis favours At4gl6210, which has a predicted transmembrane domain and is predicted to be in the plasma membrane or ER, one could also expect that the HCD gene would be quite highly expressed, similar to the other components of the F A E complex. In comparison with the EST and MPSS hit frequency found for the H C D candidates, the reductase homologues Atlg67730 (KCR) and At3g55360 (ECR) look much more highly expressed, with 31 and 33 EST hits, respectively. No MPSS hits were found for At3g55360, but Atlg67730 has about 10 times more hits than the most abundant of the H C D candidates. The most highly-expressed KCS genes, CER6 and FDH, also have similar numbers of EST and MPSS hits (Fig. 1.8). Since F A E complex components are required in stoichiometric quantities, it would seem that none of the candidates is expressed highly enough. It may be that none of them is an HCD.  44  Table 1.4.  E S T and M P S S hit frequency data for enoyl-CoA hydratase-like genes  Gene A t 4 g ' 6 8 0 0 enoyl-CoA hydratase At4g16210 enoyl-CoA hydratase-like At5g43280 enoyl-CoA hydratase-like At1g60550 enoyl-CoA hydratase-like At4g 13360 3-hydroxyisobutyryl-co A hydrolase At4g31810 enoyl-CoA hydratase-like At3g06860 multifunctional protein At4g29010 AIM1 At2g30650 3-hydroxyisobutyryl-co A hydrolase At2g30660 3-hydroxyisobutyryl-co A hydrolase At5g65940 3-hydroxyisobutyryl-co A hydrolase  ESTs 0 5 7 2 2 1 12 8 0 0 3  M P S S hits Mutant callus flower root leaf silique 54 11 48 34 57 21 0 0 0 0 97 109 22 64 88 no hits no hits 108 102 69  47 51 145  27 8 53  153 5 3  62 33 45  aim1  no hits 45 77  0 138  0 69  0 40  0 104  chyl  45 However, it would be interesting to test the candidate genes by obtaining the S A L K TD N A insertion lines which are available for all of the candidates, and evaluate the phenotypes. If the H C D is a unique (ie., non-redundant) gene used by all of the F A E complexes in plants, and therefore required for sphingolipids and suberin production as well as wax production, it is quite likely that a knock-out phenotype would be severe or even lethal. Therefore, it would be necessary to determine the genotypes of the individuals in the segregating T3 seeds supplied. If only heterozygous carriers of the T-DNA insertion are found, their siliques should be monitored to determine the progress of homozygous mutant embryo development. It could also be possible that there is some redundancy of the HCD gene. In this case, it may be possible to find a milder phenotype - for example, wax-deficiency or a reduction of V L C F A in the seed oil. Both of these possibilities could be evaluated by GC analysis, and a wax-related phenotype would likely be visible on the stems. A second way to test the hypothesis that one of these candidates is the HCD would be to make epidermis- or seed-specific promoter-RNAi constructs of the different candidates and express them in plants. If the expression is knocked out in a specific tissue, it may result in an altered wax phenotype or reduced V L C F A content in seed oil. This kind of experiment would be especially useful if a knock-out mutant is lethal.  1.5.3. Summary  Fatty acid elongation is the best characterized pathway involved in the biosynthesis of waxes and other VLCFA-derived lipids. The molecular identification of 3 of the 4 enzymes involved, opens the way for more thorough investigations of each individual enzyme and their interactions as a multi-enzyme complex. Sequence analysis has revealed the presence of many K C S genes in Arabidopsis, whereas there seem to be only one or two genes for the K C R and ECR. This is consistent with the K C S being responsible for the specificity of the F A E complex. Investigations of expression domains of the different K C S genes, together with characterization of T-DNA insertion mutant phenotypes, should soon allow the assignment of many of these genes to the cellular processes in which they are involved.  1.6. T H E D E C A R B O X Y L A T I O N P A T H W A Y  The biosynthesis of very long chain alkanes in plants was a mystery until the 1960's, when Kolattukudy added radiolabeled fatty acids to plant tissue slices and showed that they were derived from elongation and decarbonylation of fatty acids, rather than a head-to-head  46 condensation of two shorter fatty acids (reviewed in (Kolattukudy 1970b)). Inhibition of alkane synthesis by mutations or chemical inhibitors caused an accumulation of aldehydes, showing that the immediate precursors of alkanes were aldehydes, and not fatty acids (Kolattukudy et al. 1973; Mikkelsen & von Wettstein-Knowles 1978; Bognar et al. 1984).  1.6.1. Fatty acyl-CoA reduction produces aldehydes  The enzyme responsible for the first step of the decarbonylation pathway, the aldehydeforming acyl-CoA reductase, has been purified to homogeneity from young, rapidly expanding pea leaves (Vioque & Kolattukudy 1997). Vioque and Kolattukudy (1997) showed that there were actually two separate acyl-CoA reductases in pea, one forming aldehydes and the other forming alcohols from VLCFacyl-CoAs, thus demonstrating that the decarbonylation pathway is separate from the acyl-reduction pathway from the point of fatty acyl-CoA reduction. No gene has yet been isolated for an aldehyde-forming reductase. However, Arabidopsis cer8 has been suggested to be a lesion of this step, based on its accumulation of fatty acids (more than 4 times the amount found in WT; Fig. 1.4), its severe reduction in all of the components of the decarbonylation pathway, and slight increase in products of the acyl-reduction pathway (Lemieux et al. 1994). Thus, cloning of the CER8 gene could yield the aldehyde-forming reductase. Its activity could be demonstrated using the assay conditions that have been established for peas (Vioque & Kolattukudy 1997), if the recombinant protein was expressed in yeast.  1.6.2. Aldehyde decarbonylation produces alkanes  Aldehyde decarbonylases have been solubilized and partially purified from the green microalga Botryococcus braunii (Dennis & Kolattukudy 1991) and from young expanding pea leaves (Cheesbrough & Kolattukudy 1984; Schneider-Belhaddad & Kolattukudy 2000). These 66-67 kDa enzymes catalyze the decarbonylation of 18C aldehyde to 17C alkane, releasing carbon monoxide, without any cofactor requirements. The cofactors that had been thought to be required, CoA, ATP and NAD(P)H, seem to be required for the fatty acid reduction to the aldehyde, rather than for the decarbonylation (Dennis & Kolattukudy 1991; SchneiderBelhaddad & Kolattukudy 2000). The decarbonylation reaction was greatly stimulated by the addition of phospholipids, especially phosphatidyl-choline, to the purified enzyme, confirming  47 that it requires membranes for activity (Schneider-Belhaddad & Kolattukudy 2000) It was inhibited by metal ion chelators, showing that metal ions were required for the reaction. The 9-1-  decarbonylase from Botryococcus seemed to require Co (Dennis & Kolattukudy 1991), but the pea decarbonylase had a higher activity when supplied with C u  2 +  (Schneider-Belhaddad &  Kolattukudy 2000). The fractionation of the decarbonylase with a heavy membrane fraction led to the suggestion that it was localized in the cuticle (Cheesbrough & Kolattukudy 1984). Kunst and Samuels (2003) suggested that it was located on the cytoplasmic side of plasma membrane associated with cell walls, rather than in the apoplast or cuticle, based on its pH optimum of 7.0 (Schneider-Belhaddad & Kolattukudy 2000). Based on its wax composition that showed an accumulation of aldehydes and a severe reduction of decarbonylation pathway wax components, the cerl mutant of Arabidopsis was proposed to have a lesion in the decarbonylation step of the pathway (Hannoufa et al. 1993; McNevin et al. 1993; Lemieux et al. 1994). When the CER1 gene was cloned, its derived protein sequence did not show any similarity to other proteins in the databases at that time. Since no decarbonylase had yet been cloned, it was not unexpected that sequence similarities would not be helpful in assigning a biochemical function to the protein. However, the predicted protein sequence did have a number of putative transmembrane domains, suggesting that it was an integral membrane protein, and three histidine-rich motifs suggesting that it could bind a di-iron cofactor (Aarts et al. 1995). These features, as well as its predicted molecular weight of 66 kDa, were consistent with its being a decarbonylase. Since that time, two other genes whose mutations cause wax deficiencies have been cloned, both encoding gene products that show significant amino acid sequence similarity to that of CER1: GL1 from maize (Hansen et al. 1997) and W A X 2 from Arabidopsis (Chen et al. 2003). While the decarbonylation pathway-derived components (and not the acyl-reduction pathway-derived components) were greatly reduced in the wax2 mutant, there was no accumulation of aldehydes (Chen et al. 2003). Furthermore, there were changes to the cuticle structure in the wax2 mutant that could not be attributed to a decarbonylase function. The mutant phenotype of the maize gll was also not consistent with a decarbonylase function for the GL1 protein: aikanes were higher in the mutant than in the WT (Hansen et al. 1997). The GL1 sequence also lacked one of the histidine-rich motifs found in C E R l . Based on the multiple transmembrane domains found in GL1, C E R l and homologous ESTs identified from Kleinia odora epidermis (EPI23) and rice, Hansen et al. (1997) suggested that these proteins  48  were receptors or transporters, or that the GL1 protein, which is considerably shorter than CER1, had a different function. Chen et al. (2003) found that although the W A X 2 , CER1 and GL1 proteins had comparable transmembrane structures, they did not show high similarity to membrane receptors. Instead, the conserved domain found in all of these proteins showed similarity to the sterol desaturase family, which includes fatty acyl desaturases, hydroxylases, and xylene monooxygenase. Phylogenetic analysis revealed that W A X 2 , GL1 and the EST protein sequences formed a different clade from CER1 and another CERl-like gene in the Arabidopsis genome. Multiple sequence alignment shows that this is mostly due to differences in the Cterminal domains between the two clades. Based on these data and the catalytic plasticity of the enzymes to which the sequence showed similarity, Chen et al. (2003) suggested that WAX2 could encode a bi- or multi-functional protein involved in more than one aspect of wax and cuticle biosynthesis. A reduction in aldehyde content in the wax2 mutant, and limited (16%) similarity of the W A X 2 C-terminal domain to the short chain dehydrogenase/reductase family which includes many NAD(P)-dependent oxidoreductases, led Chen et al. (2003) to propose that one of the enzymatic functions of W A X 2 could be the aldehyde-forming reductase. The sterol desaturase-like N-terminal domain of CER1 and W A X 2 , on the other hand, would be responsible for the cuticle-related functions specific to W A X 2 . If this is the case, since the cer J mutation doesn't have any of these cuticle defects, its N-terminal enzymatic activity must be somehow diverged from that of W A X 2 . The scenario that emerges is that all of the homologues (CER1, W A X 2 , GL1, EPI23 and the rice W A X 2 homologue) have the N-terminal sterol desaturase domain, which may form a complete catalytic domain with similar biochemical functions in all of the proteins. The possibility remains open that the C-terminal domain of CER1 is an aldehyde decarbonylase, and that of W A X 2 is an aldehyde-forming reductase. However, at least the former proposition seems unlikely, since the His-rich motifs suggested to bind a metal ion required for decarbonylation activity are located in the N-terminal domain. The proteins begin to seem more like enzymes than regulatory proteins, though. Biochemical characterization of recombinant proteins could begin to address the questions as to their roles in wax and cuticle biosynthesis.  49 1.6.3. Alkane derivatives: secondary alcohols and ketones  The derivation of secondary alcohols and ketones from alkanes was demonstrated by feeding tritiated C29 substrates to broccoli leaf slices (Kolattukudy 1970a; Kolattukudy et al. 1973). Supplying H-C29 alkane resulted in the production of both H-C29 secondary alcohol 3  3  and H-C29 ketone, and supplying H-C29 secondary alcohol resulted in the production of the 3  3  ketone. These experiments established the order of the reactions: hydroxylation of the alkane followed by oxidation to the ketone (Kolattukudy & Liu 1970; Kolattukudy 1970a; Kolattukudy et al. 1973). Kolattukudy et al. (1973) have also shown that the introduction of the 2_|_  hydroxyl group requires oxygen and is reversed by Fe , suggesting that a mixed-function oxidase is the type of enzyme involved. These biochemical results have also been supported by genetic analysis. In Arabidopsis, all of the cer mutants (1-20) that have reduced levels of alkanes also have reduced secondary alcohols and ketones (Jenks et al. 1995; Rashotte et al. 2001). The only wax deficient mutant isolated that has a specific lesion in the pathway after the alkane forming reaction is the barley cer'  soh  mutant, which seems to control the alkane hydroxylation step (von  Wettstein-Knowles 1979). To isolate the genes and enzymes catalyzing these steps, identification of new mutants in Arabidopsis deficient in secondary alcohols and ketones could be done by a GC screen. Such a screen may be necessary, since the visual screens used to date may have overlooked mutants with subtle changes in the glaucousness of the stems. A n alternative approach would be to purify the enzymes from species such as broccoli. It would be helpful if biochemists who purified enzymes would also sequence the N-terminus of the protein so that homologues could be identified from other plants. 1.7. T H E A C Y L - R E D U C T I O N P A T H W A Y  1.7.1. Fatty acyl-CoA reduction produces primary alcohols  The acyl-reduction pathway produces the primary alcohols and wax esters found in cuticular waxes. The first step in the pathway, the reduction of the VLCFacyl-CoA to a primary alcohol, is catalyzed by a single enzyme, the alcohol-forming reductase. Two reductases were partially purified from broccoli leaf extracts. One fraction, corresponding to the alcohol-forming reductase, reduced labeled 16C aldehyde to a primary alcohol. The other  50 fraction, corresponding to the aldehyde-forming reductase, used 16C- or 18C-acyl-CoA as a substrate (Kolattukudy 1971). Since the in vivo substrate of the alcohol-forming reductase is more likely to be the 32C fatty acid (based on the broccoli wax profile), the reductase seems to have a broad substrate length specificity (von Wettstein-Knowles 1995). Vioque and Kolattukudy (1997) partially purified the alcohol-forming reductase from pea leaves, and proposed that the reaction was a two-step reaction involving the formation of an aldehyde intermediate that was not released from the enzyme, since no aldehyde could be caused to accumulate by inhibiting the reaction. Metz et al. (2000) purified and cloned the reductase from developing jojoba seeds, and showed that the recombinant enzyme caused an accumulation of fatty alcohols when it was expressed in E. coli or Brassica napus. The jojoba alcohol-forming reductase sequence was used to search the Arabidopsis genome database for similar sequences. Eight were found, including the MS2 protein required for pollen fertility, and one that maps close to the cer4 locus (Metz et al. 2000; Kunst & Samuels 2003). Table 1.5 lists all of the alcohol-forming reductase-like sequences found in the Arabidopsis genome, along with the number of EST and MPSS hits for each gene. MS2 is required for pollen fertility. Its expression is tapetum specific (Aarts et al. 1993; Aarts et al. 1997). Since pollen walls are thin in the ms2 mutant, Aarts et al. (1997) suggested that the MS2 protein is involved in production of fatty alcohols that are used for the production of sporopollenin, the major polymer in the pollen exine. Kunst and Samuels (2003) suggested that it could also be used for the production of pollen wax esters.  Table 1.5. Alcohol-forming reductase-like genes in Arabidopsis. E S T and M P S S hit frequencies indicating relative expression levels, and number of T - D N A insertion lines available from the Salk Institute and G A B I - K A T projects are indicated.  MPSS E S T s Callus At4g33790 C E R 4 ? At5g22500 At5g22420 At3g44540 At3g44550 At3g44560 At3g56700 At3g11980 MS2  2 2 0 2 1 1 0 0  0 no hits no hits 24 no hits 0 0 0  SALK Flower Leaf Root Silique lines 428  0  35  497  0  0  143  0  0 5 251  0 16 0  0 0 0  8 14 0  4 1 3 2 1 2 1 0  GABI-KAT lines 0 0 0 2 0 0 0 0  51 The cer4 mutant is very glossy, according to the classification of cer mutants by Koornneef et al. (1987). However, it has a reduction only in primary alcohols and wax esters, which is almost completely compensated for by an increase in products of the decarbonylation pathway (Jenks et al. 1995; Post-Beittenmiller 1998). Its glossiness seems to be due to a change in the shape of its wax crystals, rather than to a decrease in wax deposition (Lemieux et al. 1994). This is the only Arabidopsis cer mutant isolated to date that seems to have a specific lesion in the acyl-reduction pathway. The proximity of its map location to one of the alcoholforming reductase homologues in Arabidopsis suggests that it may have a lesion in this gene, thus implicating this reductase-like gene in wax biosynthesis. In addition, preliminary results from our lab suggest that a knockout of this reductase causes a wax phenotype similar to that of cer4, and that it may be allelic to cer4. Further characterization of the mutant and recombinant enzyme should confirm its role. The maize mutants gl5 and gl20 cause a similar phenotype to that of cer4 in the gl5gl20 double mutant (Bianchi et al. 1978; von Wettstein-Knowles 1995), suggesting that these two genes may also encode alcohol-forming reductases that are redundant in wax biosynthesis in maize. Both cer4 and gl5gl20 mutants have an increase in aldehydes which partially compensates for the reduction in primary alcohols (Bianchi et al. 1978; Jenks et al. 1995). This was thought to imply that aldehydes were the precursors of primary alcohols (Bianchi et al. 1978; Lemieux et al. 1994). The accumulation of aldehydes, however, can also be explained by an increase in flux through the decarbonylation pathway, which resolves the apparent contradiction with the biochemical results that the (presumed) aldehyde intermediate is not released from the alcohol-forming reductase (von Wettstein-Knowles 1995).  1.7.2. Esterification of primary alcohols and fatty-acyl-CoAs  Wax synthases catalyze the esterification of primary alcohols and V L C F A s . The reaction, in which a fatty-acyl-CoA is transferred to a primary alcohol, was demonstrated and characterized in broccoli leaf homogenates (Kolattukudy 1967), microsomes from Euglena (Khan & Kolattukudy 1973) and homogenates of developing jojoba seeds (Wu et al. 1981). It is membrane-bound, like all of the other wax biosynthetic reactions, and.seems to have a low chain-length specificity for the alcohol substrate (von Wettstein-Knowles & Netting 1976b; Franich et al. 1985). A wax synthase was solubilized and partially purified from developing jojoba seeds, and a cDNA clone was isolated based on the amino acid sequences of tryptic  52 peptides of the 40 kDa protein (Lardizabal et al. 2000). Expression of the gene in transgenic Arabidopsis seeds caused a high level of wax ester accumulation in the seeds, confirming that the c D N A encoded the wax synthase activity (Lardizabal et al. 2000). Lardizabal et al. (2000) found 7 sequences from the Arabidopsis genome that were similar to the jojoba wax synthase. Expression analyses of the Arabidopsis sequences revealed that 2 of them were preferentially expressed in flowers, and one was highly expressed in developing seeds (Lardizabal et al. 2000). Kunst and Samuels (2003) reported an additional 5 wax synthase-like genes in the Arabidopsis genome, making a total of 12 (Table 1.6). None of the waxless mutants isolated so far seems to have a lesion in a wax synthase. However, T-DNA insertion lines are available from the Salk Institute for most of the wax synthase genes. The abundance of MPSS signature hits on At3g51970 in root tissues suggests that this gene may have a role in synthesizing suberin-related waxes. At5g55380 is the most likely to have a role in leaf wax production, based on the frequency of MPSS hits in cDNA collected from young leaves (Table 1.6). A more detailed analysis of the expression patterns and phenotypes of insertional mutants of the different homologues may elucidate their roles in wax production in Arabidopsis.  Table 1.6. W a x synthase-like genes in Arabidopsis. E S T and M P S S hit frequencies indicating relative expression levels, and number of T - D N A insertion lines available from the Salk Institute and G A B I - K A T projects are indicated. MPSS E S T s Callus At1g34490 At1g34500 At1g34520 At3g51970 At5g51420 At5g55320 At5g55330 At5g55340 At5g55350 At5g55360 At5g55370 At5g55380  0 0 0 0 0 0 0 1 0 0 0 2  no hits no hits no hits 4 no no no no no no no  hits hits hits hits hits hits hits 5  Flower Leaf Root Silique  0  0  27  0  0  16  0  1  SALK lines  GABI-KAT lines  0 1 3 0 1 2 1 1 2 2 1 1  3 0 1 1 0 0 0 0 0 0 0 0  53 1.8. W A X S E C R E T I O N  Wax synthesis and secretion take place in shoot epidermal cells. A n E R membrane localization of fatty acid elongation has been fairly well established (Kunst & Samuels 2003). Reactions of the decarbonylation and acyl-reduction pathways are membrane-bound, and associated with microsomal fractions of cells (as mentioned in the preceding sections). However, their precise localization to ER, Golgi or plasma membrane has not yet been determined. At some point during or after their synthesis, wax components must be transported to and then across the plasmalemma. Then, these hydrophobic entities must somehow traverse the generally hydrophilic secondary and primary cell walls before they can reach the more hydrophobic milieu of the established or nascent cuticle (Kunst & Samuels 2003).  1.8.1. Intracellular trafficking  Two basic hypotheses have been advanced for intracellular trafficking of wax components. The first is the classical vesicular traffic route; ie., E R to Golgi to plasma membrane, with vesicles traveling along the cytoskeleton between them. It would be more energetically favourable for wax components in the cell to either be embedded in membranes or in hydrophobic protein pockets such as that found in acyl-CoA binding proteins (ACBP) thought to be involved in intracellular lipid transport (Kunst & Samuels 2003). Kunst and Samuels (2003) have suggested that the wax components might form "lipid rafts," discrete domains of tightly packed molecules within a more fluid membrane (Brown and London 2000), and thus go through the secretory pathway in a manner similar to that found for sphingolipids in animal cells (Moreau et al. 1998). There is no evidence that any of the Arabidopsis cerlcerlO mutants have an accumulation of Golgi vesicles or altered Golgi morphology, which could indicate a lesion in vesicular secretion (Kunst & Samuels 2003). This, however, does not rule out the possibility that such a phenotype might be found in other wax-deficient mutants. The second hypothesis suggested by Kunst and Samuels (2003) was that domains of the E R which synthesize wax components may interact directly with the plasma membrane and transfer the wax components in that manner. This hypothesis was based on regions of close apposition of the E R and plasma membrane which they and others have observed by T E M in cryo-fixed material.  54 At some point, the wax components, which seem to be synthesized on the cytoplasmic face of the E R or plasma membrane (based on data from fatty acid elongation and decarbonylation, the two steps for which there is experimental evidence), must cross the membrane. Insertion into the membrane would be energetically favourable for these hydrophobic molecules; however, extraction from it into the more hydrophilic environment of the apoplast may require an input of energy. The Arabidopsis cer5 mutant may shed some light on this process. Cer5 has reduced levels of both acyl-reduction and decarbonylation pathway derived wax components (Rashotte et al. 2001). Examination of the epidermal cell ultrastructure showed that these cells accumulate membrane-like inclusions distinct from the ER and Golgi (Pighin et al. 2003), similar to the tri-lamellar inclusions seen in cells carrying the human genetic disease adrenoleukodystrophy (ALD) (Molzer et al. 1993). A L D is caused by a defect in transport of very long chain fatty acids into peroxisomes for P-oxidation due to a mutation in an ATP binding cassette (ABC) transporter (Dubois-Dalcq et al. 1999; McGuinness et al. 2003). Members of the A B C transporter protein family transport a wide variety of lipophilic molecules across membranes, using ATP hydrolysis to supply the required energy (Kunst & Samuels 2003). The cer5 locus maps close to two A B C transporter-like genes. TD N A insertions into these two genes were examined and one of them was found to be allelic to cer5 (Pighin et al. 2003). The identification of the cer5 mutant as an A B C transporter sheds light on the mode of transport of waxes across the cell membrane.  1.8.2. Transit through the apoplast  Once the wax components cross the plasma membrane, they must find a pathway through the hydrophilic cell wall to get to the cuticle. The mechanism by which they do so has been the subject of much speculation. However, there is as yet no concrete evidence to support or exclude any of the three major hypotheses given. In 1871 de Bary proposed that wax is either secreted through pores in the primary cell wall and cuticle, or by simple diffusion through the cuticle (Jeffree 1996). Mueller et al. (1954) observed pores in their scanning electron micrographs, but subsequent S E M work has not confirmed these observations (Schieferstein & Loomis 1959; Juniper 1960). Recent work done on sorghum has given some evidence that there may be secretory pores in this species. Wax filaments were photographed over time as they were being secreted and show growth from specific spots on the cuticle,  55  which could be pores (Jenks et al. 1994b). However, when broccoli leaves were labeled with l 4  C acetate during wax synthesis, microautoradiography showed that the labeled waxes were  distributed randomly throughout the cuticle (Anton et al. 1994). This results suggests that the waxes arrive at the cuticle by diffusion, or, if there are pores, that they must form a complex network rather than being perpendicular to the cell wall (Anton et al. 1994). Kunst and Samuels (2003) suggest that there could be hydrophobic subdomains of the cell wall formed by cell wall proteins or polysaccharides through which narrow "rivers" of hydrophobic compounds could form and move to the cell surface. Such hydrophobic channels could be consistent with either type of pore, but without necessitating a physical opening in the cell wall. A more recent hypothesis is that wax constituents or their precursors are transported to the surface by lipid transfer proteins (LTPs) (Post-Beittenmiller 1996; Jenks et al. 2000). Evidence supporting this hypothesis is given by the immunolocalization of the Arabidopsis LTP to the cell wall and cuticle (Thoma et al. 1993), the identification of an LTP as the major (>90%) wax-associated protein in broccoli (Pyee et al. 1994), and by the increase in LTP expression in barley concomitant with an increase in the epicuticular wax load upon treatment with cadmium (Hollenbach et al. 1997). LTPs are small, basic proteins which form hydrophobic tunnels that could carry wax component molecules through the hydrophilic environment (Tassin-Moindrot et al. 2000). Some LTPs have high substrate specificities, but there are also a number of non-specific LTPs which can accommodate a variety of substrates from C10-C18 (Han et al. 2001). While this range of binding specificity may accommodate cutin monomers, there is some doubt as to whether it could carry the much longer wax components through the cell wall (Kunst & Samuels 2003). The study of wax secretion is still very preliminary. There are many hypotheses and little data. Progress may be made in this area in several ways. The isolation and characterization of more wax secretion mutants would allow more proteins involved with the process to be identified. The localization of more of the wax biosynthetic enzymes would be helpful in determining the exact context of each stage of wax production, and perhaps reveal pathways of wax precursor and/or component migration through the cell. Reverse genetics to examine the roles of various ACBPs and LTPs could be used to determine whether they are involved in wax component transport within the cell and within the cell wall. New techniques in microscopy including cryo-fixation, which allows the preservation of delicate membrane structures, as well as reducing the amount of lipid lost during fixation (Kunst & Samuels 2003), confocal microscopy, which allows 3-D reconstructions of fluorescently labeled cellular  56  structures, and jellyfish green fluorescent protein (GFP) fusion proteins, which allow proteins to be traced within living cells, should allow a more accurate and detailed picture of wax secretion to emerge in the near future.  1.9. R E G U L A T I O N O F W A X P R O D U C T I O N  Wax production has several potential regulation points. A l l of the enzymes involved in wax biosynthesis and secretion must be present in the wax-producing epidermal cells. Thus, the necessary proteins must be transcribed in these cells. Variations in wax synthesis rates may be fine-tuned by transcriptional regulation (ie., levels of gene expression), post-transcriptional regulation (eg. mRNA stability), or at the protein level via protein stability or activation, and by channeling of substrates by enabling or inhibiting assembly of multienzyme complexes. These regulatory mechanisms may act on certain key enzymes and/or secretory proteins, or on many or all of the proteins involved. It would be logical that the biosynthetic enzymes, rather than the proteins involved in secretion, would be the major control points for wax production, and that enzymes earlier in the pathway or at key branchpoints (ie., decarbonylation vs. acylreduction pathways) would be controlled more tightly than those later in the pathway.  1.9.1. Mutants affecting the regulation of wax production  Analysis and cloning of mutant genes is a good way to dissect the regulation mechanisms of biological processes. Of the plethora of mutants isolated from various species, only a few seem to be involved with wax biosynthesis, based on their wax phenotypes (previous sections; summarized in Table 1.7). Furthermore, only three of the genes (CER6, GL8 and CER4) have had the expected functions clearly defined when they were cloned. The rest have novel sequences whose functions which are ambiguous (eg. CER1, GL1, WAX2, CER2, GL2) due to a lack of functional characterization of the encoded proteins. These may be regulatory, rather than biosynthetic. There is also only one gene (CER5) which has a clear role in wax secretion. Cloning more genes identified by mutations in wax-deficient lines may uncover those involved in other steps of the biosynthetic or secretory pathways. However, the wax profiles of many of them suggest that it is more likely that they have lesions in regulatory genes. Regulatory mutants are more likely to cause significant decreases in the total wax load  57  to CD CD CO  t; c .p  ITS  CD to  CU  —  m  r£i  CD to JO CD 5 s  CD to  o <fl o a.  a>  T3 ><  CO 5  c  £  co or  §I  <o cn  E  _o LU "a o <  |  1 2 O ~ T3 CO  I  T3 0) T3 to CD O -~  °-  o _CD  as  o  I  >-f (I) ^ co S  OO  T3  co  cS  "o  =3 CD  CD a  CD  •e o  Q.  (fl C CO  1_  O  CO  <  E .2  to 3 co o  Ito<"  c^ 5 LU .2  CO to CD  j ? jD  c .913 to a"  IQ  co  co '  (•a "o  CD  3 J=  _co >> o  U S  CO  £=  ™ 01  O Z  CD  •ei  58 which would affect the visible phenotype (absence of a wax bloom). Biosynthetic mutants may alter wax composition without causing a visible phenotype (Kunst & Samuels 2003). Although all of the Arabidopsis wax-deficient mutants have recessive inheritance patterns, the cer-yy mutant of barley is dominant, and causes the wax on barley spikes to mimic the leaf blade wax composition, which suggests that it is likely to be a regulatory gene (von Wettstein-Knowles 1995) . Two mutants with clear regulatory roles have been cloned, one from maize and one from Arabidopsis (Table 1.7). Maize GL15 shows similarity to the transcription factor AP2 (Moose & Sisco 1996). This protein is not specifically involved in the control of wax deposition. Rather, it is a regulator of juvenile leaf epidermal cell identity (Moose & Sisco 1996) . In fact, it could be expected that many regulatory genes that affect wax deposition may affect it incidentally. That is, wax production is likely to comprise part of the epidermal development pathway, and thus be co-regulated with it. Another maize gene, CR4, has a defect in epidermal differentiation that disrupts the cuticle and possibly the epicuticular wax (Becraft et al. 1996). It encodes a protein similar to a mammalian TNFR (peptide growth factorreceptor kinase (Becraft et al. 1996). Thus, as seems reasonable, signal cascades and transcriptional regulation for epidermal differentiation are implicated in the proper formation of epicuticular wax. When Arabidopsis CER3 was cloned, it did not show similarity to any protein in the Genbank database, but putative nuclear localization and phosphorylation sites together with its broad expression pattern (including roots as well as various shoot tissues) led the authors to suggest that it was a novel regulatory protein (Hannoufa et al. 1996; Lemieux 1996). A more recent database search showed that the CER3 protein sequence is similar to E3-ubiquitin ligases involved in the N-end rule pathway (Kunst & Samuels 2003). This type of ligase confers substrate-specificity to the N-end rule ubiquitination, thus targeting particular proteins for ubiquitin-mediated degradation by proteasomes (Bonifacino & Weissman 1998). The cer3 mutant has a wax load about 20-50% that of the WT (depending on the background ecotype: Ler or Ws) for both stems and leaves (Post-Beittenmiller 1998), with reduction in decarbonylation pathway components but a lesser reduction or an increase in acyl-reduction pathway components (Lemieux et al. 1994; Post-Beittenmiller 1998). This implicates protein stability via the ubiquitin pathway in regulating wax production in Arabidopsis. In this case, as with the maize transcription factors, the regulation may not be specific to wax. The cer3 mutant seems to have perturbations of starch metabolism which occur concurrently with some  59 developmental changes (Samuels & Kunst 2001). Thus, CER3 may have pleiotropic roles that include a direct or possibly an indirect effect on wax production. The cloning of these regulatory genes thus opens the field to more inquiry. Of particular interest is to identify a functional homolog of GL15 in Arabidopsis, and to determine how it affects wax production. Does it transcriptionally affect a battery of epidermis-specific genes, including wax biosynthetic genes? This seems likely, since the GL15 protein is downstream of a number of other transcription factors that are involved in regulating developmental pathways in maize (Moose & Sisco 1996). However, it is possible that there is another regulator downstream of GL15 that activates only wax production in the differentiating epidermal cell. Similarly, it would be useful to identify targets of CER3. Enzymes of wax biosynthesis and secondary regulators are both potential targets. Isolating more components of the regulatory pathways could be done by suppressor screens and cloning of genes identified in new mutant screens.  1.9.2. Developmental influences on wax synthesis and deposition  In addition to genes that control epidermal identity, genes that regulate organ specificity and developmental maturity influence wax deposition. This is obvious despite the lack of cloned genes and defined pathways for these processes, because different organs have different wax compositions and structures. Furthermore, wax-deficient mutants often have differential effects on the wax of different plant organs. In Arabidopsis, stems and siliques appear glaucous due to the presence of epicuticular wax crystals. However, there is no crystalline wax on leaves or floral organs other than the carpel (Lemieux et al. 1994). There are also differences in wax load on different organs, and the load and composition change as the plant matures (Lemieux et al. 1994; Jenks et al. 1996b). Differences in wax on different organs of barley have also been observed (von Wettstein-Knowles 1995; Post-Beittenmiller 1996). In maize and sorghum, juvenile leaves are glaucous whereas mature leaves are glossy (Avato et al. 1987; Post-Beittenmiller 1996). Wax composition may also change as plant organs age and senesce (Prugel et al. 1994; Ju & Bramlage 2001). Thus, there is a complexity of regulation of wax production in plants that causes these developmental and tissue differences in wax loads and compositions. Many proteins are likely to be involved, and there is much work to be done to identify how many wax biosynthetic enzymes are involved, and how they are regulated in different tissues.  60  1.9.3. Environmental influences on wax synthesis and deposition  The amount and composition of epicuticular waxes produced is often influenced by environmental factors including light (Juniper 1960; Hallam 1970; Giese 1975), temperature (Giese 1975; Reed & Tukey 1982), humidity (Baker 1974), soil water content(Hunt & Baker 1982; Uddin & Marshall 1988; Jenks et al. 2001), wind (Juniper 1960), exposure to pollutants (Bystrom et al. 1968; Percy & Baker 1987) and herbicides (Wilkinson 1974; Reddy et al. 1987), as well as insect damage (Bystrom et al. 1968). Seasonal variations in wax load and composition have been observed in many species (eg. Hosta (Jenks et al. 2002b) and ivy (Hauke & Schreiber 1998)). These variations are most likely due to a number of factors, including day length, temperature, humidity, and developmental stages of leaves. Juniper (1960) observed that there was no bloom visible on the young, still-folded leaves of Pisum and hypothesized that exposure to light was necessary for the development of the wax. S E M investigation revealed that almost no wax microstructure was visible on leaves just removed from darkness, but by 24 hours of exposure to light, a microstructure becomes visible and continues to develop for approximately a week. Similarly, in barley (Giese 1975), maize (Avato et al. 1980) and leek (Maier & Post-Beittenmiller 1998) seedlings, exposure to light affects the amount and composition of wax produced. It may be argued that the light necessary for normal wax deposition in these instances is basically a developmental effect, since it occurs on young etiolated seedlings. In any case, environmental factors tend to influence only those parts of the plant that are producing wax. Hence, it is important to keep in mind that developmental and environmental factors interact (Giese 1975). Light intensity also affects epicuticular wax on non-etiolated green plants. In eucalyptus, less than 20% of full daylight intensity causes a reduction in the number of wax microstructures observed on the leaves, but 20% of full daylight intensity or more is sufficient for normal wax development (Hallam 1970). Other species have different requirements. Brassica napus needs 60% of full glasshouse light for normal development of wax microstructures (Whitecross & Armstrong 1972). The wax loads on surfaces of a number of species including cucumber (Tevini & Steinmuller 1987), pea (Juniper 1960), carnation (Correll & Weathers 2001), and Tragopogon (Upadhyaya & Furness 1994) has been shown to vary directly with the amount of light provided. As the amount of light increases, the accumulation of the wax in cucumber is faster (Tevini & Steinmuller 1987). Ultraviolet light  61 has also been found to cause changes in wax composition in some species (Tevini & Steinmuller 1987; Barnes et al. 1996; Hess et al. 2002). Hawke and Stumpf (1965) found that the presence of light during incubation of lightgrown tissue slices increased the incorporation of radiolabeled acetate into V L C F A s . Furthermore, the chain length distribution of the wax components is also influenced by exposure to light, indicating that light may activate some elongases, (Giese 1975; Tevini & Steinmuller 1987; Barnes et al. 1996). Macey (1970), however, attributed the shift in Brassica oleracea wax composition from predominantly aldehydes and esters to more alkanes, ketones and secondary alcohols to a stimulation of the utilization of the V L C F A products, rather than to the stimulation of V L C F A production. Thus, light may affect the reductases, decarbonylase, and oxidases that produce the final wax products, as well as the elongases that provide their V L C F A precursors. Light also interacts with temperature in affecting wax production. In Brassica napus grown in 100% of full daylight intensity, a temperature regime of 15°C (day)/10°C (night) causes an accumulation of less wax with a different microstructure compared to plants grown at 27°C/22°C. On the other hand, at 60% light intensity, no temperature effect was observed, and at 40% light intensity, more wax was produced at the higher temperature (Whitecross & Armstrong 1972). The light/temperature interaction varies with different species. In Brassica oleracea (Brussels sprouts), plants grown at 15 °C produced more wax than those grown at 25 °C, regardless of the light intensity provided. However, in carnation, more wax was produced at a light intensity of 440 uE/m s than at 145 u.E/m s, at both temperatures (Reed & 2  2  Tukey 1982). Temperature affected the chain lengths of alkanes and free fatty acids in barley, as well as the amount of wax produced (Giese 1975). Thus, it is important to consider the interactions of temperature and light, as well as the possible variation between different species in temperature response. The differences may be due to adaptation of different species to different environmental conditions, and related to tolerance to cold or heat stresses. The final important environmental factor to be considered in detail is water availability. Lee and Priestly (1924) observed that a decrease in available soil moisture caused the development of a thicker cuticle, and as early as 1927, Shantz recognized glaucousness as an adaptation to drought. A soil water deficit increases the epicuticular wax deposition in several crop species, including wheat (Uddin & Marshall 1988), oat (Bengtson et al. 1979), sorghum (Jordan et al. 1984) and pea (Hunt & Baker 1982). In addition, a decrease in the relative  62 humidity of the atmosphere caused an increase in epicuticular wax production in Brussels sprouts (Brassica oleracea) (Baker 1974). While the detection of the soil water deficit must be communicated to the shoot tissues by the roots in contact with the soil, a decrease in relative humidity may be sensed directly by the epidermal tissues. Thus, there may be direct and indirect signaling pathways at work in the regulation of the enzymes responsible for wax production in the epidermis.  1.10. R E S E A R C H G O A L S  Our group is interested in the control of wax deposition in Arabidopsis. Arabidopsis stem wax is comprised mostly of C29 alkanes, ketones and secondary alcohols. Aldehydes, primary alcohols and wax esters are also present in lesser quantities (Kunst & Samuels 2003). We are using Arabidopsis as a model system to try to piece together the biosynthetic, secretory and regulatory pathways of wax production in this species that is readily amenable to genetic and molecular techniques, and has the advantage of a large pool of genetic and genomic information available. In our investigation of wax production in Arabidopsis, we are using molecular genetic and biochemical tools to identify and characterize different aspects of wax deposition. We are also using both forward genetics (ie., characterizing mutant phenotypes and cloning the mutated genes responsible for these phenotypes, with the goal of identifying their molecular and cellular functions) and reverse genetics (ie., identifying a gene that is likely to be involved with wax production, based on sequence similarity to genes with known functions) to accomplish this goal. The first step in the synthesis of the major aliphatic constituents of plant waxes is the elongation of CI 6 and/or C18 fatty acid (Fig. 1.4). Thus, the regulation of fatty acid elongation is likely to be important in the control of wax production. Ectopic expression of the FAE1 condensing enzyme from Arabidopsis in yeast and in tissues of Arabidopsis and tobacco that do not normally produce significant quantities of V L C F A s resulted in an accumulation of V L C F A s in those tissues (Millar & Kunst 1997). This occurred even though the other three elongation activities involved were not ectopically expressed. In addition, V L C F A production increased when extra copies of the FAE1 gene were introduced. These results indicated that the V L C F A condensing enzyme was the rate-limiting factor for the production of V L C F A s and that the other three enzymes involved were ubiquitously present (Millar & Kunst 1997). Thus,  63 we have been looking for wax-specific V L C F A condensing enzymes in Arabidopsis, to test the hypothesis that they are important in regulating the chain-lengths of V L C F A s and amount of wax produced. To increase our chances of finding wax-specific condensing enzymes, we employed a reverse genetics approach. The derived amino-acid sequence for the seed-specific V L C F A condensing enzyme FAE1 (James et al. 1995) was used to search the Arabidopsis genome database for homologous sequences, which could also be V L C F A condensing enzymes (Millar et al. 1999). Several previously anonymous sequences were found. One of these genes was used to transform Arabidopsis and resulted in a number of plants with wax-deficient (cosuppression) phenotypes. This gene was named CUT1 (for epicuticular wax biosynthesis) (Millar et al. 1999). These results provide evidence to support the proposal that V L C F A biosynthesis and the condensing enzymes controlling it are involved in the regulation of wax production in Arabidopsis. Subsequent cloning of the CER6 gene revealed that it corresponds to CUT1 (Fiebig et al. 2000). Thus, throughout this thesis the genetic name CER6 will be used, which predates the reverse-genetic designation CUT I.  1.10.1. Thesis objectives:  Since our lab had identified a condensing enzyme involved in wax biosynthesis, my objective was to determine whether it is specific to wax production, and its relative importance to the wax biosynthetic pathway over the course of plant development and under different environmental conditions. I hypothesized that, since the condensing enzyme is the rate-limiting step of fatty acid elongation, the first step of wax biosynthesis, its expression levels could be important in regulating wax production in the plant. To test this hypothesis, I attempted to correlate wax deposition in the plant with CER6 expression levels, and I overexpressed the CER6 gene in Arabidopsis to determine whether this could increase wax production. I also characterized the cerl mutant and cloned the CERl gene by positional cloning. Since the CER7 protein seems to have a regulatory role, I investigated whether it affected CER6 expression and the expression of other cloned CER genes. The specific objectives of my thesis were to:  1) Examine the cell and tissue specificity of CER6 expression to pinpoint its role in wax production throughout the plant.  64 2) Overexpress the CER6 gene in the epidermis to establish whether or not increasing CER6 levels, in particular, and fatty acid elongation in general would be sufficient to increase wax production in Arabidopsis. 3) Examine CER6 expression and wax deposition under different environmental conditions to determine whether changes in wax production in these conditions could be correlated with, and therefore regulated by, CER6 levels. 4) Clone the CER7 gene and determine whether it is involved in regulation of CER6 and/or other wax biosynthetic genes in Arabidopsis.  The results of my study will show the requirement for CER6 in wax production in Arabidopsis under most growth conditions, and the importance of the regulation of key wax biosynthetic enzymes such as CER6 during wax deposition. Furthermore, cloning and my initial characterization of CER 7 represent a first step towards unraveling the regulatory pathways involved in wax biosynthesis.  65 C H A P T E R 2. MATERIALS AND METHODS  2.1. P L A N T G R O W T H CONDITIONS 2.1.1. General  For all experiments, seeds of Arabidopsis thaliana were stratified for 3-5 days at 4 °C on their germination medium, then germinated at 20 °C either on soil in 12 cm plastic pots (Terra-Lite Redi-Earth, WR Grace and Co, Ajax, O N or Sunshine mix 5, Sungro Horticulture, Seba Beach, A B ) or on AT-agar medium ((Somerville & Ogren 1982);composition given in Table 2.1) in Petri dishes and transplanted to soil 7 days after germination. Before planting, soil was saturated with tap water in pots, and water was added to pots from the base whenever they became dry. When Sunshine mix was used, 80 mL of liquid A T medium was added to the top of each pot before planting or transplanting. During seedling establishment (from germination if plants were germinated on soil or just after transplantation), pots were covered with plastic wrap (Resinite, AEP Canada Inc., Westhill, ON). 7 days after germination (for plants germinated on soil) or 2 days after transplantation (for plants germinated on A T medium), the plastic was slit with a razor blade, and then removed 2 days later. Plants were generally grown at a density of 9-12 per pot, and were maintained in continuous light (90-120 uEin PAR) at 20 °C from germination to senescence.  Table 2.1. A T medium composition (pH adjusted to 5.8;5g/L agar added. Kanamycin (to 50 Lig'/mL) added after autoclaving and cooling to 55°C.  Macronutrient KN0 KH P0 MgS0 Ca(N0 ) FeEDTA  52.5 2 2 0.05  3  2  4  4  3  2  Micronutrient  Concentration ( L I M ) 70  H3BO3  Mir4H 0 CuS0 ZnS0 7H 0 NaMo0 2H 0 NaCI CoCI -6H 0 2  4  4  2  4  2  Concentration (mM)  2  2  14  0.5 1 0.2 : 10 0.01  66  2.1.2. Plant material  Arabidopsis thaliana ecotypes Columbia-2 (Col), Landsberg erecta (Ler) and Wassilewskija (WS) were used as WT for expression studies and as controls for mutants and transgenics, according to their genetic backgrounds. Cer, det and aba mutants and S A L K TD N A insertion lines were obtained from the Arabidopsis Biological Resource Center ( A B R C , Columbus, OH). CBF over-expressing lines were a gift from Mike Tomashow (Michigan State University).  2.1.3. Terminology  Standard nomenclature for Arabidopsis thaliana shown in Table 2.2 is used throughout this thesis. Genes are indicated by italicized symbols; the corresponding proteins are indicated by non-italicized symbols. Mutant alleles of genes are indicated by lower-case symbols, whereas WT alleles are indicated by capitalized symbols. Genes from different loci having similar sequences or similar mutant phenotypes are designated by the same 3-letter symbol and differentiated by using a number (eg. cerl, cer2, cer3, etc.). Multiple alleles of the same gene are differentiated by hyphenating a second number (eg. cer7-l, cer7-2).  Table 2.2. Arabidopsis  thaliana standard nomenclature STYLE  EXAMPLE  W T alleles  all capitals, italicized  CER7  mutant alleles  lowercase, italicized  cerl  mutant phenotypes  Initial capital, non-italicized  Cer7  proteins  all capitals, non-italicized  CER7  2.1.4. Experiments testing effects of different environmental factors on CER6 expression  For etiolated seedlings, seeds were stratified and germinated on AT-agar plates covered with aluminum foil. For transfer of etiolated seedlings to light, aluminum foil was removed from the plates. For transfer of bolting plants to darkness, large pots covered with black polyethylene were placed over the pots containing the test plants. For bolting plants exposed to different temperatures, pots containing the test plants were transferred to growth chambers with similar light conditions and the required temperatures.  67 For drought treatment, the pots were allowed to dry out and not watered. For seedlings treated with PEG (polyethylene glycol,3350, Sigma-Aldrich Canada, Ltd., Oakville, ON), NaCl, and A B A (abscisic acid, Sigma-Aldrich Canada, Ltd.), 14 day old seedlings germinated on AT-agar plates were uprooted and floated for 10 h on liquid A T medium in Petri dishes containing the test solution. Control plants were floated on A T medium without the test substance, or, in the case of A B A controls, A T medium containing an equal volume of methanol (in which A B A had been dissolved) to that added to the medium of the treated seedlings.  2.1.5. Plant transformation  Plants were transformed using the Agrobacterium tumefaciens-mediated floral dip method (Clough & Bent 1998). Columbia-2 (WT) or cerl plants (Landsberg erecta ecotype) were grown to the bolting stage. For Columbia plants, the primary bolts were cut off and secondary bolts were allowed to grow to about 10 cm tall (with few siliques and many young buds in the inflorescences). For the. cerl plants, the plants were found to senesce quickly when the primary bolts were removed, giving a lower frequency of transformation, so these plants were dipped when the primary bolts had their first open flowers. Single colonies of Agrobacterium strains containing the binary vector with the T-DNA construct to be inserted into the plants were innoculated from L B (Luria-Bertani) -agar plates into 5 mL L B broth and grown at 28 °C overnight. The entire overnight culture was then used to innoculate 250 mL of L B broth in 1 L flasks. These cultures were incubated on shakers at room temperature for approximately 24 h. The Agrobacterium cultures were centrifuged for 20 minutes at 4 °C, 4 000 rpm in a Beckmann J2-21 centrifuge with a JA14 rotor (2 500 g), to pellet the cells. The supernatant broth was removed, and the cells were resuspended in a solution of 5% (w/v) sucrose, 0.05% (v/v) Silwet L-77 (Lehle Seeds, Round Rock TX) (prepared fresh). The Agrobacterium suspension was transferred to containers a little larger in diameter than the plant growth pots, and the inflorescences of the plants were dipped for 10-15 seconds into the suspension. The pots with dipped plants were placed on their sides in trays lined with absorbent paper and covered overnight, after which they were returned to normal growth conditions and allowed to complete their seed development.  68 Seeds harvested from transformed plants were germinated on AT-agar plates containing 50 u-L/L kanamycin (Sigma), as described by Katavic et al. (1994). Kanamycin-resistant seedlings were transferred to soil and grown under normal conditions.  2.2. G E N E E X P R E S S I O N A N A L Y S I S  2.2.1. R N A gel blot analysis  Arabidopsis 2 cm long stem segments from just below the inflorescence (stem tops) or just above the rosette (stem bases), whole leaves, unopened flower buds, opened flowers, whole siliques less than 1 cm long, whole seedling shoots or whole roots were harvested and immediately frozen in liquid nitrogen. Total R N A was extracted using T R I Z O L Reagent R  (Invitrogen Life Technologies) and isopropanol precipitation, according to the manufacturer's protocol. 10 u.g of each sample were separated by electrophoresis on a 1% MOPS-agarose gel containing 5.8% formaldehyde. Separated R N A was downward-blotted (Koetsier et al. 1993) using 20X SSC as the transfer solution onto a Hybond X L membrane (Amersham) and fixed to the membrane by baking at 80 °C for 2 hrs. Hybridization was carried out in modified Church buffer (0.5 M Na-phosphate buffer pH7; 7% SDS; 1 m M EDTA) overnight at 65 °C using a PCR-generated P-labeled D N A probe. Probe templates were amplified from genomic D N A 32  (CER6, CER60 and 18SrRNA probes) or cDNA (all other probes) using Taq polymerase (Invitrogen) with the oligonucleotide primers, annealing temperatures, and extension times listed in Table 2.3. The templates were gel-purified using QiaEX Gel Purification Kits (Qiagen). Labelled probes were amplified using these templates and the same PCR conditions, but unlabelled dATP was replaced with P-a-dATP (Amersham Pharmacia Biotech). In 32  general, amplification conditions were: 94 °C for 2 min, 30 cycles of denaturation (94 °C for 15 s), annealing (15 s) and extension (72 °C), followed by a final extension at 72 °C for 5 min, in a Perkin-Elmer themocycler (model 480). Blots were washed in 2X SSC, 0.1% SDS ( 2 x 5 min); I X SSC, 0.1% SDS (15 min); and 0.1X SSC, 0.1% SDS (2 x 10 min) at 65 °C, then autoradiographed overnight at -80 °C using Kodak X A R - 5 film, or exposed to a phosphor screen which was then scanned with a STORM 860 phosphorimager (Amersham Pharmacia Biotech). Each blot was sequentially hybridized with a probe corresponding to the gene of interest, followed by 18S rRNA or GAPC (At3g04120; glyceraldehyde-3-phosphate dehydrogenase C, cytosolic form) as a loading control. The intensity of the bands developed on  69 the autoradiogram or detected by the phophorimager were quantified by densitometry using the Imagequant software for phosphor-imaged blots and using the Alphalmager 1220 (Alpha Innotech Corporation) digital camera and densitometry software for autoradiograms.  Table 2.3. Primers and P C R conditions used to generate probes for R N A and D N A blots  F primer  Probe  R primer  Annealing  Extension  temperature  time  (°C)  (s)  ACTTCCCATTTCTCAATCCCC  58  90  CER6 ORF  TCTAGCTCGGTGAAGCTCAAG  CER6 UTR  ATATCCTTCACCTTCCC  CTCTGGCATCGGTGC  50  30  CER60 UTR  GAGAAAGAGCGTTGAGTGG  AAT CAGACATTTTG GAAGAG  50  30  CER1  GAGTGAAGGTGCTTAGTCTGG  CACTCTAGTTGCACTCATCGC  55  30  CER2  GTCCTGATCTTACCTTCTCGC  CAATCAGTGCAGCTAGTTCGG  55  30  CER3  AGTCTGAGCTGAATCATGTGC  TGAACTGCGATCAAGTCCATG  55  30  CER4  CTTCCTCTGTGATCTTGATGC  TAGAAGACATACTTAAGCAGCC  55  30  CER5  TGGAGGGTTTCCTTCTTTCA  GCTTTGCTTGTATCGCCTTC  55  30  C E R 7 non-specific  TAGTGATAGCAGATAATGGAG  CTGCTTCTTACTTCCTCC  50  60  C E R 7 3' e n d  GGAGGAAGTAAGAAGCAGTAAGG  AG AAG AAAAG T AC GAG T GAT C G G  50  30  At3g12990 3'UTR  CTATTGTAGTTGTGTAGG  GTCTTGATCATATCGACG  45  30  18S r R N A  CTGCCAGTAGTCATATGC  ATGGATCCTCGTTAAGGG  50  30  GAPC  ACTCGAGAAAGCTGCTAC  ATTCGTTGTCGTACCATG  50  30  2.2.2. Genomic Southern blots  D N A was extracted from plant material, usually leaves, but also from stems, inflorescences or siliques, using a small-scale Dellaporta method. For this method, up to 300 mg (more often 50-100 mg) of tissue was ground in 720 uL of extraction buffer (100 m M TrisHC1 pH 8.0; 50 m M EDTA; 50 m M NaCl; 10 m M p-mercaptoethanol (added fresh). 92 uL of 10% SDS was added, and the mixture was incubated for 10 minutes at 65 °C. After this, proteins were precipitated by addition of 240 uL of 3 M potassium acetate (pH 5.2) and incubation on ice for 20 minutes. The mixture was centrifuged at 14 000 rpm (16 000 g) in an Eppendorf microcentrifuge (model 5402) at 4 °C for 5 minutes, and the supernatant was removed and then mixed thoroughly but gently with 484 uL of cold isopropanol to precipitate the D N A . The precipitated D N A was centrifuged at 10 000 rpm (8 000 g) at 4 °C for 10 minutes. The pellet was rinsed with 70% ethanol and allowed to air-dry. The D N A was dissolved in 10 m M Tris-HCl pH 8.0 and stored at 4 °C where it was stable for at least 6 months.  70 For genomic Southern blots, 10 u.g of D N A extracted by this method was thoroughly digested by a restriction enzyme (each blot used several different digests), and the resulting fragments were separated by overnight electrophoresis on a 20 cm 0.8% agarose-TBE gel at 20 V . The D N A was then de-purinated by incubation of the gel in 0.25 N HC1 for 15 minutes, followed by neutralization in 0.4 M NaOH. The separated D N A fragments were transferred to Hybond X L (Amersham) membrane by downward blotting using 0.4 M NaOH as the transfer solution. The membrane was baked at 80 °C for 2 hours to fix the D N A to it, then prehybridized and hybridized to PCR generated  P-a dATP-labelled probes in Church buffer at  65°C, and stringency washed in SSC-SDS solutions, as with R N A gel blot analysis.  2.2.3. Quantitative R T - P C R  Total R N A isolated as described above for R N A gel blot analysis was used for c D N A synthesis by C. therm polymerase (Roche Diagnostics) (for CER6, CER60 and histoneHl) or by Superscript polymerase (Invitrogen Life Technologies) (for CER7, At3gl2990 and GAPC) following the manufacturer's protocol. Gene-specific, intron-spanning primers were designed to differentiate products amplified from cDNA from any product amplified from contaminating genomic D N A . Gene-specific reverse primers were used in the RT (reverse transcriptase) reaction for CER6, CER60 and histone HI, and oligo-dT was used as the reverse primer for the RT reaction from which CER7, At3gl2990, and GAPC were amplified. PCR cycle number and template amounts were optimized for all fragments amplified, to yield products in the linear range of the reaction. Primer sequences and PCR conditions for each fragment amplified are given in Table 2.4. General PCR conditions used were 94 °C for 2 minutes, followed by an optimized number of cycles (given in Table 2.4 for each fragment) of 94 °C for 15 s; annealing for 30 s; and 72 °C extension for 30 or 60 s as indicated (Table 2.4). Reactions were maintained at 72 °C for 7 minutes before separation of PCR products by electrophoresis in a 1.2% agarose-TAE gel. For comparison of CER6 and CER60, PCR products were visualized by SYBR-Green I (Molecular Probes) staining of the gel and quantified by densitometry using the Image-Quant 5.2 software (Molecular Dynamics) after fluorescence scanning by the S T O R M 860 fluorescence imager (Amersham Pharmacia Biotech) at 450 nm excitation and 520 nm emission wavelengths. For CER7 and At3gl2990, gels were stained with ethidium bromide and visualized and photographed using the Alphalmager 1220 U V transilluminator and digital camera.  0) CD  ••5  f  N CM  in ro — • N  CM CM  W CM  S CM  N CM  CM CM  LO CM  in o o o CM in in in  4in <)l  g .1  o  o  o  o  o  o  CD  CD  CO  CO  CO  CO  O  O  LU  "ro a)  CO CO 00 O  E  in in in m in in  CD  u  u  EH  15  EH  EH  EH  U  o u  15  U EH EH  u  u  E u u u  EH  < U <3 u u 15 u U  < u < u FT  0)  ro  CD  CD  1 5 15 U 15  < "=C u 15  15 u EH EH  EH  << CJ  CJ  S  CO  U  ^-=£  <  o CO  Q;  Q;  o  o  Uj Uj  q  < EH EH  U  u  EH EH CJ C5 EH CJ  CO  CD  (J EH U  15 CJ  o d d  CO  U  u  15 <  15 EH  u u cj < < CJ U < 15 U EH f£ f£  EH 15 U EH 15 EH EH 15 U EH EH  EH  <;  o  .  o  co Uj 2 ^ 5 O <C CD  72 2.2.4. In situ hybridization In situ hybridization of Arabidopsis inflorescences including 0.5 cm of the stem adjacent to the apex and 8 day old seedling shoots was carried out according to the protocol of Samach et al. (1997), with a few modifications. Briefly, plant tissues were harvested, fixed in formaldehyde-acetic acid-alcohol (4% [v/v] paraformaldehyde [Canemco], 15% [v/v] acetic acid, and 50% [v/v] ethanol) dehydrated in a graded ethanol series, infiltrated and embedded in Paraplast Plus (Sigma). Sections 8 u M thick were cut on a steel-bladed microtome and heatfixed to Superfrost Plus slides (Fisher) at 42 °C overnight, followed by 3 hours at 55 °C. Wax was removed from the sections using xylene, and sections were rehydrated through an ethanol series. Slides were incubated in 2X SSPE (70 °C) for 30 minutes, then treated with 1 p,g/mL Proteinase K in 100 m M Tris, 150 m M NaCl buffer (pH 8.0; 37 °C) for 30 minutes, rinsed with RNAse-free H 2 O , dehydrated through an ethanol series and air dried at 52 °C. Hybridization was performed overnight in 300 m M NaCl, 10 m M Tris-HCl (pH 7.5), 1 m M E D T A , 50% formamide (ultrapure), 7% Dextran sulphate, I X Denhardt's reagent, 500 ixg/u,L tRNA and 250 ug/u,L poly(A) (Sigma) at 52 °C using digoxygenin (DIG) -labeled ssRNA probes in a sense (negative control) and antisense orientation with respect to the CER6 coding region. To synthesize the probes, D N A templates were amplified by PCR from cloned CER6 c D N A using primers incorporating the T7 R N A polymerase binding site. For the antisense probe, the primer sequences were: 5'-ATG CCT C A G G C A CCG-3' and 5'-GAT A A T A C G A C T C A C T A T A G G GTT A T T T G A G T A C A C C-3'. For the sense probe, the primer sequences were: 5'-TTA TTT G A G T A C ACC-3' and 5'-GAT A A T A C G A C T C A C T A T A G G A T G CCT C A G G C A CCG-3'. R N A probes were transcribed from the PCR-generated D N A templates using T7 R N A polymerase and DIG-labeled nucleotide mix (BoehringerMannheim), according to the manufacturer's directions. The probes were then cleaved to ca. 150 base pairs by alkaline hydrolysis at 60 °C in 0.2 M sodium carbonate buffer (pH 10.4) (55 min). After hybridization, slides were washed in 2X SSC at 20 °C and in 0.2X SSC at 52 °C. The slides were then treated for 20-30 minutes with Blocking Agent (Boehringer-Mannheim) in 100 m M maleic acid, 150 m M NaCl (pH 7.5), followed by 30 minutes in freshly-made B S A Triton buffer (1% BSA, 100 m M Tris-HCl pH 7.5, 0.3% TritonX-100, 150 m M NaCl), and finally incubated with anti-DIG-alkaline phosphatase (AP) antibodies (Boehringer-Mannheim) in BSA/Triton buffer. Excess antibodies were removed by washing (20 minutes, then  73 overnight) in fresh BSA/Triton buffer, followed by color development in the A P substrate NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate) (0.34 mg/mL and 0.175 mg/mL, respectively; Boehringer Mannheim premixed stock) in 100 m M Tris-HCl (pH 9.5), 100 m M NaCl, 50 m M M g C l in darkness at room temperature for 12-24 hours. The A P was activated by incubating the slides in substrate buffer prior to introduction of the substrate, and the reaction was stopped by immersion of the slides in TE buffer. The slides were examined using an AxioskopII light microscope (Carl Zeiss, Inc), and images were captured using a SPOT digital camera (Diagnostic Instruments Inc.) attached to the microscope.  2.3. D N A S E Q U E N C E A N A L Y S I S  Gene Runner 3.05 (C 1994 Hasting Software Inc.; http://www.generunner.com/), BioEdit 5.0.9 (Hall 1999; http://www.mbio.ncsu.edu/BioEdit/bioedit.html), EditSeq 4.05 and SeqMan 4.05 (DNASTAR Inc.) and Webcutter 2.0 (Max Heiman, www.firstmarket.com/cutter/cut2.html) were used for restriction enzyme mapping and sequence manipulation and assembly of contigs from D N A sequences. The Basic Local Alignment Search Tool (BLAST, (Altschul et al. 1990)) at The Arabidopsis Information Resource (http://www.Arabidopsis.org/BLAST),  was used for similarity searches of  Arabidopsis coding sequences, genomic sequences, protein sequences, ESTs, and insertion flanking sequences. B L A S T at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST) was used for similarity searches of the non-redundant (nr) protein database. ClustalX ((Thompson et al. 1994; Thompson et al. 1997); ftp://ftpigbmc.u-strasbg.fr/pub/ClustalX/) was used for multiple sequence alignments, and bootstrapped neighbor-joining trees were constructed using NJplot (M. Gouy, ftp://pbil.univlyonl.fr/pub/mol_phylogeny/njplot). PSORTI and II (KentaNakai, Paul Horton; http://psort.ims.u-tokyo.ac.jp) were used to predict subcellular localizations of CER7 and At3g 12990 based on their amino acid sequences.  74 2.4. GUS A N A L Y S I S  2.4.1. Generation of the CER6 promoter::GUS construct  The 1208 nucleotides immediately upstream of the CER6 coding region were amplified by PCR from genomic Arabidopsis D N A using the oligonucleotide primers 5'C G T C G G A G A G T T T T A A T G - 3 ' and 5'-CTTCGATATCGGTTGTTG-3' and high fidelity Pfu polymerase (Stratagene). The 1.2 kb PCR fragment obtained was blunt-end cloned into the Hindi site of pT7T3 U18 (Pharmacia), resulting in the plasmid pCUTPRO. Sequencing confirmed that the PCR product corresponded to the upstream sequence of the CER6 gene in the Arabidopsis thaliana database. The product was oriented such that the 5' end was near the Hindlll site, while the 3' end was adjacent to the Xbal site in the polylinker of pT7T3 18U. The HindllllXbal fragment was then cleaved from pCUTPRO and subcloned into the  Hindlll/Xbal  sites of pBHOl (Clontech), resulting in the binary vector pCER6-GUS (Fig. 2.1). pCER6-GUS was introduced into the Agrobacterium tumefaciens strain GV3101 (pMP90; (Koncz & Schell 1986)) which was used to transform Arabidopsis as described by (Sargueil et al. 1999). This plasmid construction and plant transformation were done by Tony Millar.  LB  NPTII  1.2kb upstream of CER6 A T G I  ^-glucuronidase  Figure 2.1. Diagram of the T - D N A construct containing the CER6 promoter-GUS ( p C E R 6 - G U S ) gene fusion used to transform Arabidopsis to evaluate the tissue specificity of CER6 expression.  75  2.4.2. GUS histochemical assay  Tissues of pCER6-GUS transformed Arabidopsis plants and transgenic plants harboring a 35SCaMV-GUS construct (pBI121, Clontech; plants transformed by Hansik Moon) were incubated in GUS assay buffer containing 100 m M phosphate buffer pH 7.0; 10 m M E D T A ; 0.1% Triton-X-100; 1 m M potassium ferricyanide; 1 m M potassium ferrocyanide; 1 mg/mL 5bromo-4-chloro-3-indolyl-P-D-glucuronide (X-gluc; (Jefferson 1987)) at 37 °C for 0.5 hour to overnight. The reaction was stopped by removal of the assay buffer and the addition of 95% ethanol. Samples were cleared by incubation in 95% ethanol overnight.  2.4.3. GUS fluorometric assay  Wild type and pCER6-GUS-transformed seedlings were harvested and homogenized with sand in protein extraction buffer (50 m M phosphate buffer, pH 7.0; 10 m M (3mercaptoethanol; 10 m M Na EDTA; 0.1% sodium lauryl sarcosine; 0.1% Triton-X-100; 2  (Jefferson 1987). Cell debris was removed by centrifugation and protein concentration of the supernatants was quantified against a B S A standard curve using the Bradford assay (Bradford 1976) (Protein assay reagent, BioRad). The GUS assay was started by addition of 20-40 uL of this crude extract to a solution of 2 m M 4-methylumbelliferone-glucuronide (MUG) in extraction buffer at 37 °C. For each transgenic line, aliquots were removed at several timepoints and added to 0.2 M Na2CC>3 to stop the reaction. The concentration of the hydrolysis product, 4-methylumbelliferone (MU), in each aliquot was measured by fluorometer (excitation at 365+7nm; emission at 460+15 nm). To determine the rates of M U G hydrolysis for different transgenic lines, the slopes of the linear portions of the curves were determined; these values were standardized to the protein concentrations of the extracts.  2.5. T R A N S F O R M A T I O N O F ARABIDOPSIS  W I T H ADDITIONAL COPIES O F  CER6promoter::CER6  A 3.6 kb fragment, including 1.6 kb of the CER6 promoter and the CER6 coding region, was amplified from Arabidopsis ecotype Columbia-2 genomic D N A by PCR using the Expand™ High Fidelity thermostable D N A polymerase (Roche) and primers 5'- C A A A T G A C A C A A T T G TTC-3' (forward) and 5'-CCC A A A T G A A A A G C A G A G - 3' (reverse).  76 The amplified fragment was ligated into the Smal site of pGEM-7Zf (+) (Promega). The genomic fragment was then directionally subcloned into pRD400 (Datla et al. 1992) using the Xbal and BamHl sites, to produce the I X expression cassette. To introduce the second and third copies of the genomic fragment, thus producing the 2X and 3X cassettes, the original PCR-amplified fragment was ligated into the Hindi site of pBluescriptll K S +/- (Stratagene). This fragment was then excised and subcloned into the Xbal and Xhol sites of the vector pGEM-7Zf (+) to introduce a BamRl site at either end of the fragment. Finally, the second and third copies of the genomic CER6 fragment were added to the I X cassette by subcloning into the BamHl site. The orientation and number of CER6 fragments in each of the cassettes was confirmed by restriction analysis. Constructs containing either a single copy of the CER6 coding sequence with its native promoter, or two and three tandem copies of the gene (Fig. 2.2) were used to transform Agrobacterium tumefaciens strain GV3101 (pMP90; (Koncz & Schell 1986)) by electroporation. Agrobacterium lines harbouring the binary vectors were used to transform Arabidopsis (Columbia-2 ecotype) by the floral dip method (Clough & Bent 1998). Screening for transformed seed was performed on 50 fig/mL kanamycin as described previously (Katavic et al. 1994).  ^  CER6 g e n e structure 1.6 kb  B  0.4 kb intron  1.1 M> exonl  V sequence  OA kt> 0.1 kb exon2  3*UTR  1x cassette RB  LB 3.6 kb  2x cassette RB  LB  NFTTl]—j  3,SkbC£Re  |  3JBkbC£fiS  j-j*  3x cassette LB "4—I NPTII j — \ 3.6  RB 3.6 Kb CER6  |  3.6 Kb Cgftf  Figure 2.2. A. Structure of the 3.6-kb CER6 genomic clone used to construct the 1X, 2 X and 3X expression cassettes. B. T - D N A containing one, two, and three copies (1X, 2X, and 3X expression cassettes, respectively) of the CER6 genomic clone used to transform Arabidopsis plants. (Hooker et al. 2002) © 2002 American Society of Plant Biologists, used with permission.  77 2.6. W A X A N A L Y S I S  2.6.1. Gas chromatography  Wax load was determined on individual Ti transformants and on T progeny of selected 2  T| plants. Stem bases (approximately 5 cm) of mature, senesced plants were immersed in chloroform:methanol (2:1) (CER6 over-expressors) or chloroform (cer7 experiments) for 10 seconds to extract cuticular waxes. 20 jixg of 17:1 fatty acid was added to each extract as an internal standard. Samples were dried under N , redissolved in N , 0 2  bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane (Pierce), and refluxed at 80 °C for 1.5 hours. Wax components were separated in a Hewlett Packard 6890 gas chromatograph using a 30 m DB-5 ([5% phenyljmethylpolysiloxane) capillary column and helium as the carrying gas, and detected by flame ionization. Wax load was estimated by quantifying the areas of the major peaks (corresponding to C29 alkane, C29 ketone and C29 secondary alcohol) in comparison with the internal standard. Wax composition for cer 7 phenotypic analysis was determined by comparing peaks obtained from samples to retention times of standards, and to a GC-MS (gas chromatography-mass spectrometry) analysis (done by Reinhard Jetter) of representative samples. Wax load was expressed on the basis of dry stem weight and stem surface area. Surface areas were determined by scanning the extracted stems in the transparent mode using the A G F A Duoscan scanner with the Northern Eclipse image analysis software (Empix Imaging Inc.).  2.6.2. Scanning Electron Microscopy  Inflorescences were removed from the tops of bolting stems, after which the top 0.5 cm of the stem was affixed to an aluminum stub. The tissue was air-dried and then coated with gold particles using a Nanotech SEMPrep2 sputter coater (Nanotech). Specimens were visualized with a Cambridge model 250T scamiing electron microscope (Leica), at an accelerating voltage of 20 kV (CER6 over-expressing lines) or with a Hitachi S4700 scamiing electron microscope at an accelerating voltage of 2-5 kV (cer7 samples).  78 2.7. Cer7 M A P P I N G  To create an F mapping population for the fine-mapping of cer 7, a cer 7 plant of 2  Landsberg erecta ecotype was crossed to a WT Columbia plant. Fi plants were grown and allowed to self-pollinate, and F seeds were collected. Approximately 2000 F plants were 2  2  grown, from which 605 with cer7 mutant phenotypes were identified. Leaf or inflorescence tissue was collected from these plants, and genomic D N A was extracted according to the protocol of Klimyuk et al. (Klimyuk et al. 1993): Tissue (equivalent to 1 or 2 small leaves) was homogenized in 40 uL of 0.25 M NaOH and boiled for 30 s. The solution was neutralized by adding 40 uL of 0.25 M HC1, after which 20 uL of 0.25% Nonidet P-40 in 0.5 M Tris-HCl pH 8.0 was added and the solution was boiled a further 2 minutes. 2 uL of this extract was used as template D N A in 20 u,L PCR reactions for the SSLP (Simple Sequence Length Polymorphism) or CAPS (Cleaved Amplified Polymorphic Sequence) markers used for mapping. However, D N A extracted by this method was not very stable, so genomic D N A extractions for later samples were done by the Dellaporta method, as described previously. SSLP or CAPS markers were used to pinpoint the map position of the cer7 locus. To do this, markers which bracketed the rough map position of cer7 were chosen and each F plant 2  was scored for a parental (Ler) or recombinant (Col; usually heterozygote) phenotype (size of amplified fragments (SSLP markers), or presence of a restriction site (CAPS markers)). Recombination frequency for each marker was calculated by dividing the number of recombinant events (phenotypes) by the total number of possible events (the number of F  2  plants x 2, since each plant has two homologous chromosomes). Based on results of the more widely-spaced markers (with higher recombination frequencies), markers closer and closer (with lower recombination frequencies) to the cer7 locus were tested. Finally, several markers with recombination frequencies of zero were found. A l l of the F progeny showed the parental 2  phenotype for each of these markers. Thus, the chromosomal location of the cer7 gene was narrowed down to the 80 kb region between the two markers for which one (tsh4) or two (tsh7) of the F plants had recombinant phenotypes. Table 2.5 lists the names and types of markers 2  used, primer sequences and PCR conditions used for amplification, resulting fragment sizes for both ecotypes, and in the case of CAPS markers, restriction enzyme used. Positions and conditions for markers ciw4, nga707, fus6.2, and nga6 (Bell & Ecker 1994) were obtained from the TAIR website  79  0)  <D <D  <U  CO  <U  CO  0)  CO  CO  to to to to (/) to  ro _ £•  to ro n3 03 0 ) 0 ) 0 ) 0 ) 0 ) 0 ) 0  rorororororororo  -sp  s*P s p O"*  -^O  CJ"  O  v£)  vP O*"  CO  CO  c?CDroD> ro O roro (O ra£{tj sC£ s£ * -a-  00  en CM  m ™  CN  n  h-  CM  M  en ({j  N  _ , in h  J  o  ~Z ~Z r d co ^  to  CO CM  CM  d  9*2 i E  T—  T—  d  d  d  C  M  C  N  C  CM  cn in  ,o  to  M  CM  O  r-  CM  T—  cn  CM  C  N  C  M  o  CM  d  O O CNdI  C  N  C  M  C  M  C  N  CO CM  C  M  ^  T  -  5  go  ~ 3 CD CD  W  C L  II  minoooococooococoinm mmmmmmmmm'<3 mio -  CD  °>  CD  t? &  » "  5 ? S  CD  ro ro co co ro E ™ ro o 8 £ 12ro ro CD . „ -o m  g  roro Bro ^_3 8' 15 roro E _ " ro jS ro o ro£ ~Eoro R ro E ro" o ro  o  ro rooro  8 S>ro8 ^ roroTOo H  ro ro roro ro  o  Q_ CD  E  I CD  tcttt  CD  LL  ro .S' ro  CL CO CO  CL CL CO CO  CO CO  ro i ?  C L CL _J CO CO CO CO  iCM  CU  3  O CO CO  •<2  CM  CO CL < O  CD  8 ro B ro  CO D_ CO  CO  co co  ( D CM T - N CM CO ^ "  (•) CO  — I  <-  N  in n  ID  o  o  o  o  — r-  CO T—  _l  CO CO CO CO  >-  CM CM  r-  —  CO CD  E IM  m m m m o O O to  CD  o  CN  1^ O  CO 1-  ro E  C/5 Q. < O  3  CM CO  CM CN  s  CD CO  in  O  T—  CD  T3  C O  r*- co  £S t2- t . 2 . C i C CM CO CO  CL  < co O CO  o n  CM  to  CD  ro o  ro roro ro E o 2> 8 ro CD „ o ro ° c ro ro a CD ro S ro > ™ ro JJ | E ro£CD O S> g o £ -2 s CD ro o ro ro roro •t^ rororo CL CD O CD O O  teat  LU  agaa  ro  tcttt  o;  ro o ro  o  CD  "TTI  y= CO CD m LU •i cn r~-  1 'o  CM  « - CM-  - cn in <o JO  J5 j5 j5  «  w  jo  CO  CD  jo  ^  CO CD  c  or  80 (http://www.Arabidopsis.or g/servlets/Search?action=ne w_search&type=marker, Accession numbers GeneticMarker:2005404; GeneticMarker: 1945541; GeneticMarker: 1945484; and GeneticMarker:1945538, respectively), and the tshl-tsh9 markers were designed based on polymorphisms listed in the Cereon database, (http://www.Arabidopsis.org/cereon). The basic PCR protocol used was 2 minutes of denaturation at 94 °C followed by 30 cycles of 15 s denaturation at 94 °C, 15 s annealing, and 30 s extension at 72 °C in a PerkinElmer thermocycler. The same protocol, but with a 30 s annealing time, was used for some reactions that were incubated in an M J PTC-200 thermocycler. Reaction volume was 25 uL; 2 L I L of genomic D N A template and 20 pmol of each primer were used. After amplification, PCR products were separated by electrophoresis on 4% agarose-TAE or 8% acrylamide-TBE (as indicated in Table 2.5) gels. The gels were stained with ethidium bromide and destained before viewing and image capture using an Alphalmager 1220 U V transilluminator and digital camera. Each plant was scored for parental or recombinant phenotype based on the lengths of the fragments amplified from its genomic D N A .  2.8. Cer7 C O M P L E M E N T A T I O N  In order to complement the cerl mutant, genomic fragments containing 16 of the 19 annotated genes within the 80 kb target region identified by fine-mapping were amplified from WT Landsberg erecta genomic D N A . Each amplified fragment included at least 1 kb of 5' sequence and 300 kb of 3' sequence to provide a native promoter and terminator for each transgene (Fig. 2.3A). PCR primers for amplification were designed to include different restriction sites on each side of the transgene, for cloning into the pGREEN 0029 binary vector (Hellens et al. 2000). Conditions for amplification of each fragment using Expand™ LongTemplate D N A polymerase (Roche Diagnostics) are listed in Table 2.6. The P C R protocol included a 2 min denaturation at 94 °C followed by 10 cycles of 94 °C for 15 s, annealing at the specified temperature for 30 s, and extension at 68 °C for the time specified. The initial 10 cycles were followed by 20 cycles in which the extension time was increased by 20 s/cycle, and then a final extension of 7 minutes at 68 °C. In addition to amplification of the WT genomic fragments, each fragment was amplified from genomic D N A isolated from cerl mutant plants. The coding regions of WT and cerl were then sequenced from the PCR products using the di-deoxy chain termination method with  81  Native promoter (>1 kb 5')  Annotated  O R F  )>300 bp 3'  B ^(At3g60500)  I  )  R(At3g60510)  C) (A|t3g60540^  (At3g60550))  j y T S 3g60560) L (At3g60570)  C (At3g60600)  F (At3g60630)  Figure 2.3. A. General cloning strategy used to amplify all annotated O R F sequences from W T (Landsberg erecta) genomic DNA. Restriction sites for cloning into p G R E E N were incorporated into the primers. B. Genes transformed into plants ( O R F arrows point towards 3' end) y^: S A L K T - D N A insertions; Mutations detected by sequencing P C R products amplified from cerl and Ler W T genomic D N A Scale:  1kb  82 BIG dye 3.0 (Applied Biosystems) and a PRISM 377 automated sequencer (Applied Biosystems; N A P S unit, Biotech lab, UBC), and sequences compared to determine which of the genes contained a mutation. Primers used for sequencing are listed in Table 2.7. The sequencing reaction was done in a Perkin-Elmer themocycler according to the following protocol: 25 cycles of 96 °C, 30 s; 50 °C, 30 s; 60 °C, 4 min. The sequencing reaction products were purified using Centri-sep columns (Princeton Separations Inc.) following the manufacturer's protocol. The sequences obtained from WT and mutant D N A were aligned and compared using DNAstar software (DNASTAR Inc.).  Table 2.6:  P C R conditions used to amplify coding sequences from the target region for CER7 cloning 5'  3'  Gene  enzyme  enzyme  S  Kpnl  Apal  Fragment Extension Annealing length time Temperature (kb) (°C) 4' 64 3.8  Primers (F) (R) TTAATGGTACCCTACATGATGTAGTTTGTGTGTGC TTAATGGGCCCTTCTCCTCTCCGATCTACCAGC  R  Xbal  Pstl  AATAATCTAGAGGAGGAAGTAAGAAGCAGTAAGG AATTACTGCAGTGGTATTCCATGTGGCCCTCTAG  Q  Kpnl  Apal  TTATAGGTACCGACTAGAGGGCCACATGGAATAC  5.2  4'  64  3.5  2'30"  54  2.3  3'  55  2.2  3'  62  2.8  3'  60  4  3'  60  4.3  3'  60  3  2'30"  54  2.6  3'  58  3.2  3'  60  3.3  3'  57  4.3  3'  58  3.5  3'  55  2.5  2'15"  58  1.9  2'30"  54  '  TTATAGGGCCCACATTCACATACATAGCCTCTTAC  P  Kpnl  Apal  TATATGGTACCGTGAAAGACATGCTAGGTTACTG TATATGGGCCCTGGATTTGAATAACGGTGACAC  O  Xhol  Pstl  TTAACCTCGAGCTAAGCCATACCAAACCTAACTC TTAACCTGCAGCAACACTAGCCTTCTAGGGATG  N  Xhol  Pstl  TTAACCTCGAGAAAGAGACTTTGCACTACGCTG TTAACCTGCAGTGCTCGTGAACCACACTACAC  M  Pstl  Xhol  TTAACCTGCAGAGGAAACCATGATCGTGTCTAG TTAACCTCGAGACGTTGTCTGAAGAAGTCCTTG  L  Xhol  Pstl  TTAACCTCGAGACAAACTGGAGATGTTCTGCTC TTAACCTGCAGCAACAAATTTACGTAGTTAGGTAG  A  Apal  Kpnl  TATTAGGGCCCTGTCCGGTAAGAAGATCATTG TATATGGTACCAACGATTCTCAATTCTTAGAGG  B  Kpnl  Apal  TATATGGTACCAGTCAACTAAATCGGACAGCTC TATATGGGCCCAACCTTAACGTCAACCTATCAG  C  Apal  Kpnl  TAT AT GG G C C C C AG AC AT T AAC AG AG AT C C T C AC TAATTGGTACCCATACACTGACTAATGCAGACAC  D  Apal  Kpnl  TATATGGGCCCTGAACAGAGATGAACGACAACG TATATGGTACCGAATCGGATGGAGACTGTGG  E  Kpnl  Apal  TATATGGTACCTCTGAAGCTTAGAAACACTATCG TATATGGGCCCTGTAAAGAGATTGCGTAATACGAG  F  Kpnl  Apal  TATTAGGTACCATTTGTGTAAGGCAGCTTAAG TATTAGGGCCCATCTTCTCCAGAGTTCATGC  G  Kpnl  Apal  TATTAGGTACCATGTTCAGGAATGCTAGTGGG TATATGGGCCCCTCTTGTAGCTCGTGTGGTC  H  Apal  Kpnl  TATATGGGCCCCTTTTGCTCGTTTCGATGAC TATAAGGTACCTGATTAGCAGAAAACCACATG  83 Table 2.7. Gene S  R  Q P 0 N M L A B  C D E  F  G H  Sequencing primers Primer name  Primer sequence  SseqFI SseqF2 SseqR2 SR RseqFI RseqF2 RseqF3 RseqRI RseqR2 QseqR Pseq OF NseqF MseqR LseqF LR AseqF AseqR BseqFI BseqF2 BseqR CseqF CseqR DseqF DseqR EseqFI EseqF2 EseqR FseqFI FseqF2 FseqR GseqF GseqR HseqF  caagctttctctgaatcgc caaccttacaaagacagacc ctgcttcttacttcctcc ttctcctctccgatctaccagc tgtcggactgatgttagc cttctactgtgtcatggc gagatggtcatactcgcag gaaaatcgatccgccatg gctggtggtgatattgtg cctctccttcatattctccg aaacctctgaaccagtgacc ctaagccataccaaacctaactc aagtcttctcttcctcgttg tgtcatgtacaatcctccg ctttgtttgaacggtagcg caacaaatttacgtagttaggtag attggtggagaggaatgcttgtag tcagcattcattgaaacaccacag ttagtatccaaacacgtcac tagagacttcaatggtgagccaag tttacttagaagcaacagattgtgg ggtgtcacgggacgaatctaatc caaggtgggtggtttatgtcctc ^ ggtcgctgctcttcaattatcttc tcagcttcgagaaggttcctgac caccattagcatctccaccgtac atgggtattcacagtagcag atgttgctttgaacttcactcttgc caagctttcatcttcgtcttcctc taagagctcgatttcgaagg ttcgtcttaacatttccaagctgag tgtgttgggatttggtaaattatcg caccaaattaagtcattgacgatcc cgattctcgccatcttggacac tcaattatgatgaggcgggtgtg  HseqR  For cloning, amplified fragments were purified using the QiaQUICK P C R purification kit (Qiagen), digested using the appropriate restriction enzymes (as listed in Table 2.6), gelpurified using the QiaQUICK Gel Extraction Kit (Qiagen), and ligated into the digested, purified p G R E E N 0029 binary vector. The ligation products were transformed into electrocompetent E. coli cells by electrpporation (2.5 V , 25 mFD capacitance and 200 Q resistance) in 2 mm electroporation cuvettes (Biorad). The electrical pulse was delivered by  84 a GenePulser and Pulse Controller (Biorad). Plasmid D N A was prepared from overnight cultures inoculated from colonies that grew on LB-agar selection plates containing 50 Ltg/mL kanamycin, and restriction digests were done to confirm the presence and identities of the inserts. Plasmids containing the inserts of interest were electroporated, along with the pSOUP helper plasmid (Hellens et al. 2000), into electrocompetent Agrobacterium strain GV3101, and cells containing the inserts were recovered by selection for growth on LB-agar medium containing 50 u,g/mL kanamycin. The inserts were checked for integrity by restriction digests and sequencing, and cells containing the correct inserts were grown for plant transformation. Inserts for all of the constructs used for plant transformation are shown schematically in Fig. 2.3B. For complementation of the cer 7 mutants, Agrobacterium strains containing the WT genomic D N A inserts were transformed into cer7 plants using the floral dip method. Seeds were collected from transformed plants. Kanamycin-resistant seedlings were selected on A T agar plates containing 50 fig/mL kanamycin, and transplanted to soil. The phenotypes of the transformed cer 7 plants were determined visually, and for those which appeared WT, stem wax was extracted and analyzed by GC. To confirm that the kanamycin-resistant transformed plants carried the correct transgenes, genomic D N A was isolated from each kanamycin-resistant plant using the Dellaporta method, as outlined above. A reverse primer specific to the p G R E E N T-DNA right border (5'-tgttctttcctgcgttatccc-3') was used with a gene-specific primer (as listed in Table 2.8) to amplify a transgene-specific fragment from these genomic D N A templates. PCR was carried out with Taq polymerase (Invitrogen) for 30 cycles using the annealing temperatures (for 30 s) and extension times (at 72 °C) listed in Table 2.8. A 2 minute denaturation step (94 °C) was carried out at the beginning of the reaction, and there was a 15 s denaturation at 94 °C for each cycle. A final 7 minute extension at 72 °C was also performed. Table 2.8. Gene-specific primers used to confirm transgene presence in kanamycin-resistant plants recovered from transformations Gene S R O N M L F  Primer  Primer sequence  RF RseqR2 OF NseqF MseqR LseqF FseqF2  ggaggaagtaagaagcagtaagg gctggtggtgatattgtg ctaagccataccaaacctaactc aagtcttctcttcctcgttg tgtcatgtacaatcctccg ctttgtttgaacggtagcg taagagctcgatttcgaagg  Annealing temp (°C) 54 50 55 50 50 50 55  Fragment size (kb) 1.5 3.3 2.5 2.5 2.5 1.6 2.6  Extension time r 2'30" 2'30" 2'30" 2'30" 1'30" 2'30"  85 2.9. S A L K T - D N A I N S E R T I O N L I N E A N A L Y S I S  The presence of an indexed and searchable database of sequences flanking T-DNA insertions in a T-DNA mutagenized population created by the Salk Institute (La Jolla, CA) made it possible to search for inserts in CER7 candidate genes. This database, found on the website http://signal.salk.edu/cgi-bin/tdnaexpress?TDNA =S16&iNTERVAL=100, was queried :  for insertions in the genomic target region between markers tsh4 and tsh7 defined by the finemapping of cer 7. Of the available lines with insertions, 13 which had insertions within the coding sequence or close to the 5' ends of annotated genes were ordered from the Arabidopsis stock center (Columbus, OH), and germinated on AT-agar plates containing 50 jxg/mL kanamycin. Kanamycin-resistant seedlings from each line were transplanted to soil and grown to maturity for evaluation of their stem wax phenotypes. Two lines which were ordered later, SALK_100127 and SALK_003100, came with the recommendation not to germinate them on kanamycin. This was necessary because the Salk Institute had found that quite a few of the lines were losing their kanamycin resistance due to silencing, although they were still inactivated by insertions. These plants were thus germinated on AT-agar plates without kanamycin, and 12 of S A L K 100127 (all seeds that germinated) and 27 of SALK_003100 (which had better germination) were transplanted to soil and grown to maturity for phenotypic evaluation. Stem wax was extracted and analyzed by G C (as described previously) for the S A L K 003100 plants, several of which showed a waxless phenotype. To confirm the presence of the T-DNA insert in the lines which didn't show any phenotype but had insertions within the annotated coding sequence, PCR was used to amplify a fragment including the T-DNA and flanking genomic sequence. To do this, genomic D N A was isolated using the Dellaporta method (Dellaporta et al. 1983) from leaves of kanamycinresistant plants from each of the insertion lines. This D N A was used as the template for amplification. The primers used were a T-DNA left-border specific primer (5'tggttcacgtagtgggccatcg-3') and a genomic D N A primer specific to each insertion site, as listed in Table 2.9. Cycling conditions for all of the reactions were: an initial denaturation at 94 °C for 3 minutes, followed by 30 cycles of 94 °C, 15 s; 52 °C, 30 s; 72 °C, 2 min; and a final extension at 72 °C for 7 minutes. The reactions were carried out in a Perkin-Elmer fhermocycler.  Table 2.9.  Primers used for confirmation of S A L K T - D N A insertions  Line  Primer  Primer sequence  SALK_040758 SALK_016803 SALK_017859 SALK_041743 SALK_015995 SALK_014653 SALK_010684  DR ER ER IR JF KR MseqR  GAATCGGATGGAGACTGTGG T G TAAAGAGAT T GC G TAATACGAG T G TAAAGAGAT TGCGTAATACGAG AAGGAGCTCAGAAGAAGCAAG ATTCATTCTACTTACCACTTGG GTTCTTGAAGCAGCTTTGTG TGTCATGTACAATCCTCCG  Size of amplified fragment (kb) 1.5 2.4 2.4 1.7 0.4 1.1 0.8  87  C H A P T E R 3. CER6, A WAX-SPECIFIC p - K E T O A C Y L SYNTHASE  3.1. I N T R O D U C T I O N  The CER6 gene encodes a V L C F A condensing enzyme that is required for the production of cuticular waxes in Arabidopsis stems. This is shown both by the cer6 mutant phenotype and by its phenocopy caused by sense suppression of CER6 expression. In both cases, mutant plant stems have less than 10% of the WT wax loads, with reductions in both acyl-reduction pathway and decarbonylation pathway components of the cuticular wax. Cer6 mutants and sense-suppressed plants also have a conditional male sterile phenotype. Fiebig et al. (2000) showed that this was due to a lack of pollen lipid deposition in cer6 mutants, which indicated that CER6 is also required for the normal production of the pollen tryphine lipids. Thus, although there are 21 V L C F A - K C S family members in the Arabidopsis genome, it appears that CER6 is the major condensing enzyme for stem wax and pollen coat lipid biosynthesis. These severe cer6 mutant phenotypes also suggest that there is no other K C S in Arabidopsis that is functionally redundant to CER6. The residual wax found on the cer6 mutant could be due to leakiness in the cer6 mutation, or to another K C S playing a minor role in fatty acid elongation in epidermal cells. To further investigate the extent of its role in wax production in the plant, CER6 expression was examined by R N A blot hybridization through the course of development. In addition, in order to get an idea of the precise location of wax biosynthesis in the stems and anthers, the cellular location of CER6 transcripts was determined by in situ hybridization. To confirm and expand upon the R N A blot hybridization and in situ hybridization results, plants transformed with a CER6 promoter-GUS fusion were examined for GUS activity. Because KCSs catalyze a rate-limiting step of V L C F A biosynthesis, and because CER6 is a major K C S providing V L C F A precursors for stem wax and pollen lipid biosynthesis, we were interested if wax accumulation is, in part, regulated by the level of CER6 transcription. To address this question, I generated a series of transgenic Arabidopsis lines over-expressing * CER6 in the epidermis.  88 3.2. R E S U L T S  3.2.1. Tissue specificities of CER6 and CER60 expression  CER6 shares approximately 50-60% amino acid identity with the closest members of the K C S gene family that were known at the time this work was initiated. A B L A S T of the genome and EST databases using the CER6 coding sequence did not result in any hits with highly conserved blocks. Thus, it seemed that the CER6 coding sequence could be used as a probe for northern blot analysis, without fear of cross-hybridization. Confirming this, Millar et al. (1999) found that a genomic Southern blot hybridized to a CER6 coding sequence probe did not show any cross-hybridizing bands at high stringency. I repeated this Southern blot using the high-stringency conditions and full-length open reading frame (ORF) probe with similar results (data not shown). However, the completion of the genomic sequence of Arabidopsis revealed the presence of another member of the K C S family, CER60, that shares 84% nucleotide identity with CER6 (Fiebig et al. 2000). This raised the possibility that there was a gene which could crosshybridize with a CER6 ORF probe, which would confuse the results of the northern blots and in situ hybridization that had already been done. Therefore, I repeated the genomic Southern blot to re-evaluate i f a full-length CER6 ORF probe hybridizes exclusively to CER6. As shown in Fig. 3.1,5' untranslated region (UTR) probes for both CER6 and CER60 gave specific, single bands for each digest. However, the full-length ORF probe for CER6 showed a weakly crosshybridizing band that corresponded to CER60. The CER60 full-length ORF probe crosshybridized with CER6, as well as with a number of other fragments. Since the CER6 ORF probe did show some cross-hybridization with CER60, a 5'UTR probe was used in all subseqent experiments. Furthermore, in order to determine the degree to which CER60 could have been detected with CER6 in the early experiments, the expression profile of CER60, as well as that of CER6, were determined in different tissues. The tissue specificities of both CER6 and CER60 expression were examined using R N A blot hybridization and RT-PCR. Northern blots using both CER6 ORF and CER6 5'UTR probes revealed similar patterns of CER6 expression, with transcript detectable in all shoot tissues examined, including seedlings, stems, leaves, flower buds and open flowers, but no expression was detectable in roots (Fig. 3.2A and data not shown). However, CER60 proved difficult to analyze by northern blot: the full-length ORF probe hybridized to two bands with different expression patterns, and the 5'UTR probe didn't give a detectable signal (data not shown).  89  CER6 ORF ABCD '•^ pjlffi"  CER60  5'UTR ABCD  ORF ABCD *m<mm~  *  5'UTR ABCD I.. ~J  m • •  . _ mm  turn  4 _  a m .  «  M  Figure 3.1. Genomic Southern blot hybridized with CER6 and CER60 probes. Specific probes (5'UTR) did not show any cross-hybridization; however, less specific probes (ORF) did cross-hybridize to some extent. For all blots, lane A, BamHl digest; lane B, Xbal digest; lane C , E c o R V digest; lane D, Ndel digest.  & k  £  J  /  CER6 18S  <fP ^,<& ^  0  >$>  <r  ^ Ml  800 W W W  f  CER6  Histone  Figure 3.2. CER6 expression is limited to shoots and is much higher than that of CER60. A. RNA-blot hybridization analysis of CER6 expression in different Arabidopsis tissues. 10 jag R N A was loaded for each lane; the blot was hybridized with the CER6 coding sequence probe and 18S r R N A (loading control). Similar results were obtained in two additional replicates. B. S Y B R - G r e e n l-stained agarose gel showing R T - P C R products of CER6 (700bp) CER60 (600bp) and histone HI (800bp) from different tissues of Arabidopsis. Similar results were obtained in a second replicate and in a northern blot (not shown). (From Hooker et al. 2002; © 2002 American Society of Plant Biologists; used with permission) t  91 Quantitative RT-PGR using histone HI as an external control was used to confirm the blot hybridization results for CER6 and to determine the CER60 expression pattern so that it could be compared to that of CER6. Cycle and template optimization showed that a P C R protocol using 22 cycles and 25 ng of cDNA template gave a result within the linear range for all three sequences amplified (data not shown). The tissue specificity of CER6 seen in the northern blots was confirmed, with high levels of expression in all aerial tissues but none in roots (Fig. 3.2B). CER60, on the other hand, was revealed to have a somewhat different pattern of expression (Fig. 3.2B). Like CER6, it was undetectable in roots, but it had low levels of expression in mature shoot tissues. Expression of CER60 in mature shoot tissues was the highest in floral tissues and siliques, and was undetectable in stems, the tissue used for most of the other experiments. CER60 expression was the highest in seedling shoots, but even in this tissue the CER60 transcript was much less abundant than CER6 (Fig. 3.2B). This pattern of transcription of CER60, particularly its absence from roots, suggests that it may also have some role in the synthesis of wax precursors. However, its low levels in the mature shoot, together with increased transcription during early shoot morphogenesis and flowering (when pollen production occurs), are consistent with an accessory role in wax deposition at certain stages of development that require higher levels of wax production. These results also suggest that although the CER6 ORF could have cross-hybridized with CER60 transcripts, it is most likely that there was very little or no CER60 transcript present in the stem tissues used for these experiments. Therefore, the early northern blots and the in situ hybridization experients were valid. Nevertheless, northern blots done after the discovery of CER60 were done using the more specific CER6 5'UTR probe.  3.2.2. Expression of CER6 at different stages of development  Deposition of cuticular waxes is known to begin very early in plant development, likely as soon as epidermal cells are exposed to air (Jeffree 1996), and continues during subsequent organ expansion. To investigate the involvement of CER6 in cuticular wax biosynthesis during stem and leaf development, I analyzed CER6 transcript accumulation in these organs by R N A blot hybridization (Fig. 3.3). CER6 mRNA levels were already high in young 8 day old seedlings, as well as in 4 cm tall bolting stems, and increased further in 10 cm stems. It is surprising that similar CER6 transcript levels were detected in tops and bases of stems of the same height, even in old 25 to 30 cm tall stems, suggesting that CER6 transcription did not . cease once the stems had finished elongating (Fig. 3.3A).  92  4 cm 10 cm 25 cm S T B T T M B A  go  B C 1,2 3  _ _ _ .. ,  Rosette 4 5 6 7  _.  Cauline 8 9,10 1 2 3  CEf?6 18S Figure 3.3. RNA-blot hybridization analysis of CER6 expression in developing Arabidopsis stems and leaves. In each case, 10 ug/lane of total R N A was probed with CER6 coding region and 18S r R N A (loading control). A. R N A extracted from 8 day old seedlings (S) and from 2 cm segments of the tops (T) and bottoms (B) of bolting stems 4 , 1 0 and 25 cm (when flower production had ceased) tall. R N A was also extracted from the 2 cm section at the center (M) of 25 cm tall stems. Similar results were obtained in two additional replicates. B. R N A extracted from cotyledons (C), rosette (in sequence; first initiated, 1; last initiated, 10), and cauline leaves (also in sequence; numbering as with rosette leaves). Leaves were harvested when the plants had bolting stems 10 cm tall. (From Hooker et al. 2002; © 2002 American Society of Plant Biologists; used with permission)  93 In leaves, CER6 mRNA was present at every developmental stage from cotyledons to the youngest cauline leaves, but was the most abundant in rosette leaves numbers 6 through 10 (Fig. 3.3B). It is interesting that while CER6 transcript was detectable in the youngest, just initiated leaves (Fig. 3.3B: Cauline 1-3), it was more abundant in these somewhat older leavesfrom stages where they were undergoing rapid expansion to mature leaves. These results were confirmed by another northern blot in which R N A from the cotyledons and first leaves was followed from initiation through rapidly expanding phases: CER6 transcript increased from the earliest to the latest stages sampled in that blot (CER6 ORF probe of a blot made by Rosita Scherson; data not shown).  3.2.3. Epidermal specificity of CER6 expression  In situ hybridization was carried out on sections of inflorescence and seedling apices, as well as on stem cross-sections, in order to determine the cell-specificity and the point of onset of CER6 transcription. In all of the plant organs tested, stems, reproductive and vegetative meristematic regions (Fig. 3.4), floral primordia, developing carpels, ovules and stamens (Fig. 3.5, 3.6B,F), the CER6 transcript was exclusively present in the epidermal cell layer, with the exception of anthers at a specific stage of development (Fig. 3.6D). CER6 transcript was already detectable in the L I layer (the outermost layer of cells) of the inflorescence and vegetative (8 day old seedling) meristems, as well as in the epidermal cells of the earliest stages of leaf and floral primordia (Fig. 3.4). This hybridization was visible both in cross and in longitudinal sections, and was continuous throughout the epidermis of the leaf primordia and in the various floral organs as they were initiated and developed (Fig. 3.4, 3.5). Thus, CER6 appears to be important for wax deposition from the earliest stages of development of all aerial organs of the plant. The only exception to the epidermis-specific transcription was found in the anthers. Even in the anthers, CER6 was transcribed in the epidermis from the primordial stage until the time when the sporogenous tissue underwent meiosis. However, shortly after young microspores were released from the tetrads, CER6 transcripts accumulated only in tapetal cells, but not in other anther tissues (Fig. 3.5, 3.6). Transcription of CER6 in the tapetum during microsporogenesis could be expected of a condensing enzyme required for production of pollen coat lipids, as the tapetum is responsible for the production of the lipidic components of the pollen coat (Piffanelli et al. 1998). CER6 mRNA persisted in the tapetum until the breakdown of the tapetal layer, after which CER6 transcripts were undetectable in the anthers (Fig. 3.6F).  Figure 3.4. In situ localization of CER6 m R N A . Sections of Arabidopsis tissues hybridized to antisense (B,C,E,F,H) and sense CER6 R N A probes (A,D,G) (negative control). Hybridization is indicated by a purple precipitate produced as a result of an alkaline phosphatase reaction with nitroblue tetrazolium/5bromo-4-chloro-3-indolyi phosphate substrate. A - C , longitudinal sections of 8 day old seedling meristems (vm) with developing leaves (Ip), 400X; D-F, longitudinal sections of inflorescence meristems (im) with developing flower buds (fp), 70X; G - H , cross sections through young stem, 70X. In all cases, hybridization is evident in the epidermal layer (e) of sections hybridized with the antisense probes. No hybridization is visible in sections hybridized with sense probes. (C,D,E from Hooker et al. 2002; © 2002 American Society of Plant Biologists; used with permission)  95  Figure 3.5. In situ localization of CER6 m R N A . Sections of Arabidopsis tissues hybridized to antisense (B,D,F) and sense CER6 R N A probes (A,C,E) (negative control). A - B , cross sections through inflorescence meristem (m) and surrounding developing flower buds, 70X; C-D, cross sections through a flower bud with developing anthers, 400X; E-F, longitudinal section through a bud at a similar stage of development, 400X. Flower bud sections show developing floral organs including petal (p), stamen (a), and carpel (c) with ovule primordia (o) beginning to emerge. Epidermal hybridization of the CER6 R N A probe is evident throughout these stages of development. (B,D,F from Hooker et al. 2002; © 2002 American Society of Plant Biologists; used with permission)  Figure 3.6. In situ localization of CER6 m R N A . Cross-sections of maturing flower buds hybridized to antisense (B,D,F) and sense CER6 R N A probes (A,C,E) (negative control), 400X. Epidermal hybridization (e) of the CER6 R N A probe is evident in carpels (c) throughout these stages of development, but in anthers (a) the hybridization changes from epidermal (Fig 3.4, 3.5) to tapetal (t) just before the tapetum degrades (D). After this stage, CER6 transcript was not detectable in anthers, only in carpels (F). (D,F from Hooker et al. 2002; © 2002 American Society of Plant Biologists; used with permission)  97 This concentration of CER6 expression in anthers at the final stages of pollen maturation also correlates well with the conditional male sterility phenotype of the cer6 mutant, since the lipidic components of the pollen coat are required for proper pollen hydration on the receptive stigma (Fiebig et al. 2000). CER6 is required for this process, and the abundance of its transcript in the anthers at this stage is not surprising.  3.2.4. Expression of CER6 promoter-GUS fusions in transgenic Arabidopsis  To further investigate the tissue specificity and timing of expression of the CER6 gene, the 1,208-bp genomic fragment immediately upstream of the CER6 coding region was fused to a promoterless bacterial uidA gene encoding (3-glucuronidase (GUS), and was used to transform Arabidopsis. Tissue samples of five independent transgenic Arabidopsis lines were stained for GUS activity. GUS activity was found in all aerial parts of the plants, but never in roots (Fig. 3.7A, B, C), consistent with the RT-PCR and northern blot data (Fig. 3.2). Free-hand cross sections of stems, leaves, and siliques from all five lines showed a localization of GUS activity exclusively in the epidermal cells (Fig. 3.7D-F), mirroring the results of the in situ experiments (Fig. 3.4, 3.5, 3.6). Histochemical GUS staining of stem and leaf cross sections of 5 independent lines of tobacco transformed with the CEi?6pro-GUS construct showed an epidermal staining pattern similar to that observed in transgenic Arabidopsis plants (Hooker et al. 2002; data collected by Tony Millar, not shown). This demonstrated that the epidermal specificity of the CER6 promoter was retained even in plant species unrelated to Arabidopsis. Thus, it appears that the CER6 promoter will be a useful tool for targeting the expression of genes of interest to the epidermis in transgenic plants. The onset of gene expression directed by the CER6 promoter was also examined in young germinating CER6 promoter-GUS seedlings. GUS expression was detected in the cotyledons and hypocotyl as early as the beginning of radicle emergence from the seed, 1 day after transfer of the stratified seeds to 20 °C for germination (Fig. 3.7A). In agreement with the RNA-blot hybridization data (Fig. 3.3), high levels of GUS staining persisted throughout the aerial parts of the seedlings (assayed on days 3,5,8, and 14 post-germination) and in rosette leaves and bolting mature plants (assayed on day 21 post-germination; Fig. 3.7 and data not shown).  98  Figure 3.7. The CER6 promoter directs epidermis-specific expression of G U S in Arabidopsis throughout the shoot from very early stages of development. Seedlings (A: 1 day old, 20X; B: 8 day old, 8X; C: 14 day old, 8X) and free-hand cross-sections of stems (D), siliques (E) and leaves (F) (50X) of Arabidopsis plants transformed with the C£f?6promoter-GUS construct were incubated in an assay buffer containing the G U S substrate 5-bromo-4-chloro-3-indolyl-i3-D-glucuronide. G U S activity is indicated by a blue precipitate. (A,C,D,E,F from Hooker et al. 2002; © 2002 American Society of Plant Biologists; used with permission)  99 To evaluate the strength of the CER6 promoter as a tool for genetic engineering of surface traits of transgenic plants, GUS.activity in plant lines transformed with the CER6proGUS construct was compared to that found in plant lines transformed with the C a M V 35S promoter-GUS (Invitrogen vector pBI121). This was done using the quantitative fluorometric GUS assay of protein extracts of 8 day old seedlings from independent transgenic lines. The range of activities obtained for each construct is shown in Fig. 3.8. Surprisingly, the average rate of hydrolysis of the 4-methylumbelliferone-glucuronide  (MUG) substrate in the CER6pro-  GUS lines (894 nmol 4-methylumbelliferone [4MU] min" u.g protein" ) was considerably 1  1  higher than that in the 35S promoter-GUS lines (325 nmol 4-MU min" p,g protein" ). 1  1  Furthermore, the highest rate of M U G hydrolysis found in the Ci?i?6'pro-GUS lines (3, 089 nmol 4 - M U min" u.g protein" ) was also about 3-fold higher than that of the best 35S promoter1  1  GUS line (1,097 nmol 4-MU min" u.g protein" ). Thus, the CER6 promoter is highly active in 1  1  transgenic Arabidopsis.  c  2 Q. D5 •L * c E o E  <  WT  35S  CER6  Figure 3.8. G U S activity in plants transformed with CER6 promoter-GUS was higher than that found in plants transformed with cauliflower mosaic virus (CaMV)35S promoter-GUS. Activity of G U S from protein extracts of 8 day old Arabidopsis seedlings, measured by accumulation of 4 - M U . Wild-type (Columbia-2; WT) and lines transformed with C a M V 35Spromoter-GUS (35S) and with CER6promoter-GUS (CER6). Each point represents the activity from an individual transformed line. (From Hooker et al. 2002; © 2002 American Society of Plant Biologists; used with permission)  100 3.2.5. Over-expression of CER6 can increase surface wax accumulation  To more directly assess whether the extent of wax accumulation can be affected by changing the level of CER6 transcription, Tony Millar over-expressed the CER6 gene in Arabidopsis using the strong constitutive CaMV 35S promoter. R N A blot analysis revealed high levels of CER6 transcript in a number of transgenic lines. However, none of these lines had a significantly greater wax load (Millar et al. 1999; Hooker et al. 2002). These experiments suggested that higher levels of CER6 transcription in the epidermis might be required to impact wax production. I therefore transformed Arabidopsis plants with an extra copy of the CER6 gene under the control of its native CER6 promoter (lx cassette). In addition, I investigated i f introducing two copies in tandem of CER6 per T-DNA copy (2x cassette) or three copies (3x cassette) would result in a greater accumulation of wax, or generate a high-wax phenotype at a higher frequency. 74 kanamycin-resistant lines transformed with a l x cassette (35 waxy, 39 waxless), 66 plants transformed with a 2x cassette (29 waxy, 37 waxless), and 67 plants transformed with a 3x cassette (37 waxy, 30 waxless) were recovered. Wax loads of all of the waxy plants were measured. Wax load was expressed both as a function of dry weight (DW) and of surface area. Similar results were obtained with both quantification methods; plants that had more surface wax/mg D W also had more wax/mm surface area than WT (Fig. 3.9). For simplicity, in this section I will only describe results of wax load measurements expressed as a function of dry weight. The wax load in transgenic plants with visible surface wax receiving all three types of cassettes ranged between 3 and approximately 13 u.g/mg DW, with an average value similar to that of the wild type (5.6-7.2 u,g/mg DW; Fig. 3.9B). A few lines, however, had a wax load almost 100% greater than that measured for wild type plants. Interestingly, the highest wax loads achieved with l x and 2x cassettes were similar, but those generated with three copies of the CER6 transgene per insert (3x cassette) were considerably higher. However, the frequency with which transgenic lines containing 3x cassettes produced a high-wax phenotype was comparable to that obtained with l x and 2x cassettes. A large proportion (45-55%) of transgenic plants receiving l x , 2x and 3x cassettes exhibited a waxless phenotype, presumably due to sense suppression (data not shown). Gas chromatographic analysis of surface wax extracted from stems of 7 randomly selected waxless plants showed that they all had less than 10% of the wax load measured in wild type Arabidopsis (ecotype Columbia-2) (Fig. 3.9).  101  A 3-11  Transformed plant (T ) 1  Figure 3.9. W a x loads on stems of the Ti generation of plants transformed with the 1X cassette, 2X cassette, and 3X cassette of CER6 promoter-CER6. Each bar represents the wax load of a single transgenic plant. W a x loads of seven transgenic lines with no visible wax (first seven bars; indicated with an asterisk), and all of the transgenic plants recovered that had visible surface wax are shown. The type of cassette introduced is indicated. Plants with wax loads significantly increased over that of the wild type are marked with their line numbers. The range of wax loads of six wild-type control plants is shaded across the graphs. A. W a x load quantified by surface area. B. Wax load quantified by dry weight. (From Hooker et al. 2002; © 2002 American Society of Plant Biologists; used with permission)  102 I also analyzed the composition of wax on the stems of transgenic Arabidopsis lines with greater wax loads. I found that wax composition of all wax over-producing lines examined was very similar to that of the Columbia-2 wild type grown under the same conditions (Fig. 3.10 and data not shown), and in good agreement with wax composition previously reported for the Columbia-2 ecotype (Millar et al. 1999). In the waxless lines, reduction of the major C29 wax components, together with an increase in C26 primary alcohol (the only obvious change; peaks identified by comparison with a trace analyzed by GC-MS) reflect a change in weight percent of the different components (Fig. 3.10). This wax composition also agrees with the wax composition profiles reported for the cer6 mutant (Hannoufa et al. 1993; Lemieux et al. 1994) and C£i?c5-suppressed plants (Millar et al. 1999). To follow the wax phenotype in the progeny of plants overproducing wax, seed was collected from the Ti plants which had more wax than the untransformed controls, as well as from a few of the waxless plants. T seeds from these primary transformants were planted, and 2  wax load (Fig. 3.11 A), as well as CER6 mRNA accumulation (Fig. 3.1 IB) were determined for 10-20 kanamycin-resistant T progeny. Line 3-20 had the individual T progeny with the 2  2  highest wax accumulation (11.3 u.g wax/mg DW; Fig. 3.11 A). Similarly, there were several individuals originating from line 3-5 with a substantially greater wax load than the wild type. It is interesting to note that line 2-10, which was waxless in the Ti generation, also had several T progeny with higher than wild type wax loads. However, on average the wax loads of the T  2  2  progeny of the wax overproducing lines fell within the wild type range. To determine whether this variability could be reduced and whether transgenic lines which stably produced more wax than WT could be generated, T individuals with high wax 2  loads were allowed to self-pollinate and T3 seed was collected. Wax loads of T 3 , T 4 and T  5  generations of single-seed descent lines selected for high wax in each generation were measured (Wax extraction and GC analysis were done by Nicole Quenneville (T3 and T4 generations) and Nora Houlahan (T4 and T5 generations)). As found for the T generation, 2  plants with wax loads higher than WT were found in each generation; however, a stable line producing more wax than WT has not yet been obtained (data not shown). CER6 transcript accumulation was measured in the T generation of the selected lines of 2  transformants (Fig. 3.1 IB), and correlated with the wax loads found on the bolting stems (Fig. 3.11A). Lines 2-10, 3-11, 3-20 and 3-5 all had greater accumulations of CER6 transcript than the wild type. Similarly, the lines with many waxless individuals, 2-4, 3-1, and 3-10 showed extremely low levels of the CER6 transcript.  103  2-4  1040-.  3-10  30-  Figure 3.10. G C traces showing elution profiles of wax extracted from stems of untransformed (WT) Columbia-2 Arabidopsis plants and plants transformed with extra copies of CER6 promoter-CER6. Labels indicate plant number; 2-4 and 3-10 were waxless; 3-5 and 3-11 had more wax than WT.  104  A 12 10 Q  8  4-  rti  •  E  •  CO  •  •  4  1~  CD  •  t  O  i  »  v  o  co w CD E  -  <si  B 2  *  CM  V co *  CM  O  V *co  CN  *7 CO  ^ p  ^ppjp-^ip-  1 0  CM  If)  ^  |5 t* 5 5 ' ' • Transformed fine (T2) s  O  <n  O  CM ip co co  18S v>  I  1.04  £  0.5  5 K  o.oj  F i g u r e 3.11. A . Wax loads on stems of the T generation of plants transformed with the 1X cassette, 2X cassette, and 3X cassette of CER6 promoter-CEf?6. Each point represents the wax load of a single trangenic plant. Each column represents progeny from one transgenic line. Wax loads of T progeny of five of the waxless transgenic lines (first five columns; indicated with asterisks), and the T progeny of the five selected plants with increased wax loads, as well as one (2-5) which had a WT wax load in the T generation, were measured. B. R N A blot hybridization of total R N A (10 u.g/lane) extracted from the tops of 10 cm bolting stems of T progeny of lines transformed with 1X, 2X and 3X cassettes. The blot was probed with CER6 coding region and 18S rRNA (loading control). The relative intensity of CER6 transcript accumulation was calculated and standardized according to the level of untransformed WT plants. (From Hooker et al. 2002; © 2002 American Society of Plant Biologists; used with permission) 2  2  2  1  2  105 Stems of 2-3 randomly chosen transgenic plants (T generation) descended from wax 2  overproducers or waxless Ti individuals were examined by S E M . S E M analysis revealed that the transgenic plants often had waxless sectors on an otherwise waxy stem (Fig. 3.12).  3.3. DISCUSSION  3.3.1. Spatial and temporal pattern of CER6 expression  Very long chain fatty acids with chain lengths greater than 18 carbon atoms are used as substrates for the production of cuticular waxes, seed storage lipids and sphingolipids, minor but important structural components of cellular membranes. Their synthesis is controlled by the activity of the P-ketoacyl-CoA synthase enzymes (condensing enzymes) of the fatty acid elongase, that determine the amounts and the overall chain length of fatty acid products of the elongation process (Lassner et al. 1996; Millar & Kunst 1997). The CER6 condensing enzyme has been previously shown to be essential for wax production in the bolting stems of Arabidopsis, and in the anthers for the synthesis of pollen lipids (Millar et al. 1999; Fiebig et al. 2000). In this study, my aim was to more precisely define the expression domains of the CER6 gene, and to determine the timing of CER6 expression in Arabidopsis shoots during development. RT-PCR, R N A blot analyses, in situ hybridization and CERf5-promoter driven GUS activity assays demonstrated that the CER6 condensing enzyme was expressed not only in the stems, but in all the aerial parts of the plant examined, including leaves, flowers and siliques (Figs. 3.2, 3.3, 3.4, 3.5, 3.6, 3.7). Furthermore, in all tissues CER6 expression was restricted to the epidermal cell layer (Figs. 3.4, 3.5, 3.6), except in the anthers during later stages of microsporogenesis. At that time CER6 expression was localized only in the tapetal cells of the anthers (Fig. 3.6). The monolayer tapetum surrounds the maturing microspores and produces abundant lipids. At the end of microspore development, tapetal cells disintegrate, thereby releasing lipids that are deposited onto the pollen surface. Thus, CER6 expression in the tapetum is consistent with its role in the production of pollen coat lipids (Millar et al. 1999; Fiebig et al. 2000). The presence of the CER6 transcript throughout the shoot and at all stages of stem and leaf development, starting from 1 day old seedlings (Figs. 3.3, 3.7), supports the idea that CER6 is a major condensing enzyme dedicated to wax biosynthesis in Arabidopsis. Additional condensing enzymes that could have roles in wax production, such as KCS1 (Todd et al. 1999)  106  Figure 3.12. S E M of Arabidopsis stem tops of W T (A) and T plants from lines 2-5 (B) and 3-10 (C) transformed with the CER6 promoler-CER6 constructs. Plant 2-5 had a wax load similar to that of WT, and plant 3-10 was waxless. A waxless sector on the stem of plant 2-5 is indicated by an asterisk. 2  107 may be expressed only in certain tissues or during a specific developmental stage perhaps to boost the overall wax levels when necessary. Other KCSs are also likely to be involved in catalyzing steps of the elongation pathway that CER6 cannot carry out (ie. C18-C22; C28C30). Surprisingly, relatively high levels of CER6 mRNA were detected even in mature stems. The wax bloom that is mechanically removed from older parts of the stem does not appear to regenerate (unpublished data). It may be that older stems still produce V L C F A precursors for wax regeneration, but wax synthesis either does not take place or wax composition is altered. The microcrystalline structure of epicuticular wax seems to be related to its chemical composition, as well as the way and rate at which the wax is exuded through the cuticle (Hall et al. 1965; von Wettstein-Knowles 1974). Therefore, if wax produced later in development has a different composition, it may not form the rod and tube-like microcrystals, but rather take on a more amorphous form which would not be visible as a wax bloom.  3.3.2. The CER6 promoter directs high levels of gene expression in the shoot epidermis  The accumulation of the CER6 transcript exclusively in the epidermis of Arabidopsis shoots suggested that this gene might be controlled by an interesting promoter of potential value for genetic engineering applications that require epidermis-specific expression of genes. One such application is the modification of surface wax composition or accumulation in crops to increase their tolerance to environmental stresses, and/or resistance to pathogens and insects. To evaluate the CER6 promoter, a 1.2 kb fragment immediately upstream of the CER6 coding region was fused to the GUS reporter gene and this construct was transformed into Arabidopsis and tobacco. In both transgenic systems, the GUS activity was restricted to the shoot epidermis (Fig. 3.7; (Hooker et al. 2002)), demonstrating that the 1.2 kb promoter fragment used contained all the regulatory elements required to direct epidermis-specific expression, and that the same regulatory elements were recognized in tobacco. The GUS histochemical assay of plants at different stages of development also revealed an early and strong GUS activity that persisted throughout shoot development. The high level of GUS expression directed by the CER6 promoter was confirmed by comparison to the C a M V 35S promoter (Fig. 3.8). The data showing that the CER6 promoter was comparable to, if not stronger than the 35S promoter are striking in view of the fact that the 35S promoter is considered a constitutive promoter, and its expression is not restricted to the epidermal cells. Taken together, these experiments demonstrate that the CER6 promoter is very effective in directing high levels of gene expression in the plant epidermis not only in Arabidopsis, but also in unrelated plant species  108 like tobacco. Thus, it should be a useful tool for the modification of surface characteristics of crop plants.  3.3.3. The effect of CER6 over-expression on stem wax accumulation  If the level of CER6 transcription is one of the factors controlling wax deposition in Arabidopsis, over-expression of CER6 should increase wax accumulation. Over-expression of the CER6 gene using the strong constitutive CaMV 35S promoter resulted in high levels of CER6 mRNA, but failed to promote greater wax deposition (Millar et al. 1999). In contrast, high levels of CER6 expression in the epidermis using the native CER6 promoter resulted in appreciably greater wax accumulation in a number of transgenic lines (Fig. 3.9). Thus, similar to lignin modification experiments, which require accurate temporal expression of genes specifically in cells undergoing lignification (Meyer et al. 1998), effective manipulation of surface wax accumulation appears to require correctly timed epidermis-specific expression of relevant genes. Furthermore, since wax composition in the wax-overproducing lines was unchanged from that of the wild type, this increase in the wax load is likely due to increased carbon flux via both decarbonylation and acyl-reduction pathways of wax synthesis. There are number of possible explanations for the relatively wide range of wax load values observed in the T progeny of plants with increased wax accumulation in the Ti 2  generation (Fig. 3.11). For example, if the high wax phenotype in primary transformants (Ti) is caused by insertions of multiple copies of the CER6 transgene, segregation of transgenes in the T generation would result in a reduced transgene copy number in a number of individuals. In 2  addition, the remaining transgenes in each T individual would be present at a variety of 2  chromosomal locations. Genomic location affects the level of expression of the transgene, which in turn influences wax accumulation. Also, transgene silencing in the T generation is 2  frequently observed, particularly when more than one copy of the transgene is inserted. This could be due to higher expression levels in the homozygous T than in the hemizygous Ti 2  plants, which can induce silencing (De Wilde et al. 2001). Another explanation for reduced wax accumulation in T plants is offered.by the presence of waxless sectors on otherwise waxy 2  stems (Fig. 3.12). Wax synthesis and deposition has been shown to be cell-autonomous (von Wettstein-Knowles & Netting 1976a). Thus, individual cells could either over-express or silence the CER6 transgene, generating a mosaic of waxy and waxless sectors. The overall wax load of a particular T plant would depend on the number and size of waxless sectors. The fact 2  that the sectoring effect was also present in the Ti generation, could account for reduced wax  109 loads measured in many of these plants in comparison to the wild type. The fact that no line stably over-producing wax has been obtained even by the T5 generation is also not unprecedented. Plants transformed with single biosynthetic genes in attempts to manipulate metabolic pathways often show the expected over-expression phenotype in the first generation but become more like WT in subsequent generations, even when the desired trait is selected for (Mlynarova et al. 1996; De Wilde et al. 2000; De Wilde et al. 2001; Daniell & Dhingra 2002; Vain et al. 2002). Thus, although CER6 is an important enzyme which acts early in the wax biosynthetic pathway, stably increasing wax production may require the manipulation of more of the enzymes involved. This could be accomplished either by over-expressing more wax biosynthetic enzymes together with CER6, or by finding a general regulator of wax production and manipulating its expression in transgenic plants.  3.4. C O N C L U S I O N S  These results demonstrate that wax accumulation in Arabidopsis is, in part, regulated by the level of CER6 transcription. The CER6 gene is highly transcribed in the epidermis of all the shoot tissues throughout Arabidopsis development, consistent with its key role in wax biosynthesis in the plant. In addition, the CER6 promoter has been shown to be very effective in directing epidermal expression of genes in transgenic plants. Thus, it may be useful in manipulating surface properties of plants for commercial and/or academic interests.  110 C H A P T E R 4. E N V I R O N M E N T A L E F F E C T S O N CER6 E X P R E S S I O N A N D W A X D E P O S I T I O N  4.1. I N T R O D U C T I O N  The primary role of cuticular waxes is to protect the plant from desiccation (Reed & Tukey 1982), but they also play roles in protection from U V light and frost damage (Blum 1975; Reicosky & Hanover 1978; Richards et al. 1986; Grant et al. 1995; Barnes et al. 1996). Furthermore, waxes are believed to be one of the factors involved in plant defense against bacterial and fungal pathogens (Jenks et al. 1994a) and to contribute to a variety of plant-insect interactions (Eigenbrode & Espelie 1995). Wax-related differences in plant resistance/susceptibility to environmental stresses, pathogens, or insects have been linked to wax accumulation (load) and wax composition, which vary among plant species (Eigenbrode & Espelie 1995; Post-Beittenmiller 1996). In addition, environmental factors including water availability, light and temperature have been shown to influence wax load and composition in a number of species. (Thomas & Barber 1974; Bengtson et al. 1979; von Wettstein-Knowles et al. 1979; Hadley 1989). The mechanisms by which plants control wax accumulation and composition under varying environmental conditions are therefore of considerable interest. Since fatty acid elongation is the first step in the biosynthesis of the major wax components of Arabidopsis (Baker 1982; Lemieux et al. 1994; Post-Beittenmiller 1996), and since CER6 expression is required for wax accumulation, I was interested to see i f CER6 transcription levels could be a control point for wax production under sub-optimal environmental conditions. Thus, I examined the effects of light, temperature, and osmotic stress on CER6 transcript accumulation and wax load in Arabidopsis stems. To determine whether known regulatory pathways might be involved in mediating these responses, I also examined CER6 transcript accumulation and wax loads of det and aba mutants and plants overexpressing the CBF transcription factor. Additionally, I investigated the effect of wounding, which is a component of insect attack, on CER6 transcript accumulation in leaves and in whole Arabidopsis shoots.  Ill 4.2. R E S U L T S  The presence of a number of conserved regulatory elements that are known to mediate environmental responses in plants immediately 5 ' to the CER6 coding region suggests that in Arabidopsis, as in other plants, wax deposition is influenced by environmental factors. The elements detected include I-boxes and GT-1 binding sites involved in light regulation of transcription, A B A response elements (ABREs) involved in regulating ABA-mediated stress responses, and sequences similar to the DRE (drought-response element) involved in mediating water deficit, salt, and cold responses in plants (Fig. 4.1). To investigate whether CER6 is induced by any of these environmental stimuli and contributes to increased wax production, I analyzed CER6 transcript levels and stem wax loads in plants subjected to a variety of environmental conditions as described below.  -1951  TAGTGCTTTA  TATATGTTTG  ATACTTCTGT  TTGGCAATAT CAATCATAGT AGAAAAGATA TGGACTTCAT  TTGAGGTTTT  TGGTGGATTG  TGTCTATATG  -1851  TGAAATCATG GGATCTCAAG ATTTGTCTGC  ATTCAGTTTC  CAAGTCAAAC ATCGTAACTA CTGTTTGATT  TTCCCTCATG  CTTGCAGTTT  TCATGGATAT  ACTTTCCAAG TCAAACATAA AGTAACTACT GATTGATATT CCCTCGTGTA  TTACCCTCTT  TCAAATGACA CAATTGGGCC  -1751  CTCAAGATTT GTCTTCTTGC  -1651  C A A G T A G A G G A A T T T C A T A G T G A A T T C A A A A G A T T A A C T G T A T T C C A C C G TCGTATTTT|G  ATAAjCATTTA G T T A T T C C T T  -1551  CAGTTTTTTT  T T A A T A C A T T TAGTGTTGGT  TTTTTTTGGA  -1451  GGAATTGTTC  ATGCTTTTTT  GATACAATAG TATACCATTT CAAAAGATAC CATAGACCAG TTATTACATG AATCGCCAAA ACAACACTAA AATCAGAAAA  -1351  TCAGTATATT  TTGGTATAGT  C l | | C C A A C A T A ; C||AATCATAAA A C C T C T G T G A A A T T T A A A A T C T A T A T T T G A  -1251  TAATTACCTA AATTTTAAGT CAAATGTGAA TTATATTTTA  -1151  AAATAACAAA ACATGTAACT CTTGTAGATA TACATGTATC GACATTTAAA CCCGAATATA TATGTATACC TATAATTTCT  - 1 0 51  mmmmmmmj,  ABRE3  GGTGATAGGT  TTGGTTCAAT  GAAATATTAT ATGTTACTTC  C T C T T C G A T A TCGGTTGTTG  CCAAACTCAC AAGTAAAAGT TTACGTACAG TGAATTCGTC  -951  GGATTACCAA TTCTTTCATT  -851  T G C A A T A T T T AGGGATGGAC A C A A G G T A A T A T A T G C C T T T  -751  T A T A T A A A C A A A T T J [ C C A A C A AAH]fTCAAGTT T T T G C T A A A A C T A G T T T A T T  TTCTTTTTTT  TCTTCTGCAA  AATAAATTAT TCATTCTTTC  TACTATAAAA  CATTTCAAAG TTTA|[ACAACA  ACGATTAACC ATGCAAAAAA GAAACATTAA TTGCGAATGT  TTTTTGGGTA  TAAGGTATAT GTGCTATATG AATCGTTTCG  CTGATTTTCA  CGCTACCj^|  TAAACGTACA TTTAATTTAC ACGTAAGAAA  TATGGTACCA GACAGAGTTA AGGCAAACAA GAGAAACATA TAGAGTTTTG ATATGTTTTC  TGCGGGTTAT  TAGjflTCTAAA  TTG[GATAA]AT A T T A A A T T G A  CATGGGTACT A A A A T T A T T T  TTAATTACCT ATCATATTAC TTGTAATATC  GTCCTTACTT ATTCGTATGT  -651  TAACGGGTAA ACCAAACCAA ACCGGATATT GAACTATTAA AAATCTTGTA AATTTGACAC AAACTAATGA ATATCTAAAT TATGTTACTG  -551  GACCATTTTT  -551  CCGGGCAACG  -3 51  T C T C T T A C A A C G A C A A T T T T G A G A A A T A T G A A A T T T T T A T A T C G A A A G G G A A C A G T C C | T T ATCJATTTGCT  CCCATCACTT GCTTTTGTCT  AGTTACAACT  -251  GGAAATCGAA GAGAAGTATT ACAAAAACAT TTTTCTCGTC  ATTTATAAAA AAATGACAAA A A A T T A A A T A GAGAGCAAAG CAAGAGCGTT  GGGTGACGTT  -151  GGTCTCTTCA  TCGCCTTTAT  -51  GTTTTTGAGA ACCATAATAT AAATT,' A B R E motif 1 TG  9HNHI  TTAACTCCTC  A B R E motif 1 W_CST<3AC  GTACTAAGTA TTTATATCCA  CCTTTAGTCA  C A T C A A A T C A A A G T A G T T A C C A A A C G C T T T GATCTCJGATA AjAACTAAAAG C T G A C A C G T C T T G C T G T T T C  TCATCTACCC CTTCCTCTGT  ATCCTTCACC TTCCCTCTCT translational start CTAATCACAT TTTGTAACAA TAATACAATT ATACATTAAA ACTCTCCGAC G ^  CTATJGATAAJC  _________ CAGTACCAAT A T : ' :  TTAATTTATT  CATCTTCATT AACTCATCTT CAAAAATACC  Figure 4.1. The C E R 6 5' promoter region has numerous consensus sequences for cis-acting elements involved in environmental regulation. Genomic sequence 5' to the C E R 6 coding region showing M f i E s Possible D R E s (implicated in A B A , water deficit and cold regulation); l-boxes and |GT1 binding sites | implicated in light regulation). The A T G initiating translation is marked with black shading. ( K o m Hooker et al. 2002; © 2002 American Society of Plant Biologists; used with permission)  1  4.2.1. Environmental effects on CER6 transcription Light The effect of light on CER6 transcription was tested in 8 day old seedlings and in bolting stem tops (Fig. 4.2). CER6 transcript was undetectable in etiolated seedlings, but had started to accumulate appreciably by 2 days after transfer of etiolated seedlings to continuous light conditions (Fig. 4.2A). Light was also required for CER6 transcription in bolting stems, as transfer of bolting plants to dark conditions resulted in a significant reduction in CER6 transcript by 24 h, and an absence of detectable CER6 transcripts by 96 h after transfer (Fig. 4.2B). CER6 transcript accumulation was also examined in etiolated detl and det2 mutant seedlings. These mutants develop as light-grown plants even when grown in darkness, due to defects in light-regulated signal transduction pathways (Chory & Susek 1994). Detl seedlings did not accumulate CER6 transcript in the absence of light, as found with the WT seedlings (Fig. 4.2A). Thus, repression of CER6 in the dark does not depend on the DET1 pathway. However, CER6 transcription in the det2 mutant was not completely repressed by darkness (Fig. 4.2A), suggesting that the DET2 pathway may be involved in CER6 dark repression.  ^  6D detl det2 L D 2L L D L D CER6 | § m m 18S  ««**  s  B L  Dark (h) 8 24 48 96  Figure 4.2. Light is required for CER6 expression in seedlings and in bolting stems, as shown by RNA-blot hybridization. Ten micrograms of total R N A was loaded into each lane. A . R N A extracted from wild-type (Columbia-2), detl and det2 mutant 8-d-old seedlings germinated on agar plates in continuous light (L), continuous darkness (D), and dark for 6 days followed by transfer to light for the last 2 d (6D+2L; wild-type only). The blot was probed with CER6 coding region and 18S rRNA (loading control). B. R N A extracted from stem tops of plants incubated in continuous light (L) or placed in darkness for 8, 24, 48, or 96 h. The blot was hybridized to the CER6 5'-UTR and 18S rRNA probes. (From Hooker et al. 2002; © 2002 American Society of Plant Biologists; used with permission)  113 Osmotic stress CER6 transcript accumulation in Arabidopsis stems and seedlings exposed to osmotic stress was examined in comparison with rd29A, an extensively studied drought-inducible gene (Yamaguchi-Shinozaki & Shinozaki 1993). In bolting stems, CER6 transcript accumulated to a level 2-fold higher in water-stressed plants than in well-watered plants (Fig. 4.3 A). A similar trend was seen for seedlings subjected to osmotic stress: as the degree of stress increased (as indicated by the amount of PEG or salt added, and the degree of accumulation of rd29A transcript), the level of CER6 transcript also increased, again to a maximum of about 2-fold greater in the stressed seedlings than in the controls (Fig. 4.3B,C) The phytohormone A B A (abscisic acid) plays an important role in mediating the transcription of a large number of genes that respond to osmotic stress. This stress triggers the production of A B A , which in turn induces gene expression. Such genes are also induced by exogenous application of A B A and typically contain ABREs (Guiltinan et al. 1990) in their promoters. The presence of ABREs in the CER6 promoter (Fig. 4.1) suggested that CER6 transcription could be under A B A control. To investigate this possibility, CER6 transcript abundance was determined in 14 day old seedlings floated on liquid A T medium supplemented with 10" M A B A for 10 h and compared with controls floated on A T medium without A B A 4  (Fig. 4.3D). CER6 transcript accumulation was 2.5-3 fold greater in ABA-treated Arabidopsis seedlings than in control seedlings (Fig. 4.3D), suggesting that A B A can induce CER6 transcription. To further test the role of A B A in the regulation of CER6 expression, we examined CER6 transcript accumulation in stems of ABA-deficient mutants under a normal watering regime. In all of the mutant plants tested, CER6 transcription was substantially reduced (Fig. 4.3E), with different abal alleles accumulating less than 50% of the WT levels of CER6 mRNA. Thus, even under normal watering conditions, A B A appears to be involved in the regulation of CER6 transcription. These findings, together with the presence of the known A B A response elements in the CER6 promoter, indicate that the induction of CER6 seen under water deficit conditions is likely to be mediated via A B A .  II  B  C  PEG (%)  NaCI (mM) 0 50 100 150 200  ^  mmmt . | m tfiflflff  • '' 9|fP':' ••I^^W^r 'Wip^r  ^^Sff*'  :  D —  0  8  J=  c  <  O  CD  O  <  • _  o O  <D  CO -Q (0  CO 1 1— CO  n  CO  CO  CM i CM CO  CO  CO  t  n  n  CM • 00 CO  n  CO  -J  Figure 4.3. Osmotic stress increases C£f?6 transcript accumulation in seedlings and in bolting stems of Arabidopsis. RNA-blot hybridization of total R N A (10 ug/lane) probed with CER6 5'UTR, rd29A unique sequence, and 18S rRNA (loading control). A . R N A extracted from the tops of 10 cm tall bolting stems of plants grown in well-watered (W) pots and in pots allowed to dry out from the time of transplanting (D). B. R N A extracted from shoots of 14 day old seedlings germinated on agar plates, then transferred to and incubated for 10 h in liquid AT medium containing P E G (0%-20%) prior to harvest. C. R N A extracted from shoots of 14 day old seedlings germinated on agar plates, then incubated for 10 h in liquid AT medium containing NaCI (0-200 mM). D. R N A extracted from shoots of 14 day old seedlings germinated on agar plates, then incubated for 10 h in liquid AT medium containing A B A in methanol (10" M) or an equal volume of methanol (control). E. R N A extracted from the tops of 10 cm tall bolting stems of wild-type (Columbia-2 and Landsberg erecta) and A B A synthesis mutants {aba1-1 (Le), aba1-3 (Le), aba 1-4 (Le), aba 2-2 (Col), and aba 3-2 (Le)). All of the experiments were done 3 times. (From Hooker et al. 2002; © 2002 American Society of Plant Biologists; used with permission) 4  115 Temperature Like water deprivation and high salt stress, cold temperature affects the water status of plants, and has been found to affect wax deposition in a variety of species. The possibility of CER6 induction in response to cold was tested by measuring CER6 transcript accumulation after transfer of bolting Arabidopsis plants from 20 °C to colder or warmer growth temperatures, 10 °C or 30 °C (Fig. 4.4A). 4 days after transfer to 10 °C, CER6 transcript was almost 2-fold higher than that found in plants maintained at 20 °C, and it was reduced by about half that of the control level 4 days after transfer to 30 °C conditions (Fig. 4.4A). Interestingly, cold treatment also seems to mitigate the effect of darkness on CER6 transcription. Whole plants transferred to darkness had reduced CER6 transcript abundance, whether they were kept at 20 °C or transferred to 4 °C (Fig. 4.4B). However, the reduction in CER6 transcript was considerably less in the plants transferred to 4 °C than in those kept at 20 °C (Fig. 4.4B). Similarly, although 96 h of darkness reduced the CER6 transcript to undetectable levels in stems of plants maintained at 20 °C (Fig. 4.2B), CER6 transcript was not obviously reduced after 96 h in darkness at 4 °C (Fig. 4.4B). These results suggest that some other regulatory pathway active in cold conditions may override the pathway responsible for dark repression of CER6 transcription. One of the key regulatory pathways for cold responses in plants is the C-repeat/DRE pathway. CBF/DREB transcription factors bind to the DRE (drought response element) to mediate drought, salt, and cold responses in an ABA-independent manner. To determine whether this pathway might be involved in the regulation of CER6 transcription in response to cold, CER6 mRNA accumulation was measured in bolting stems of plants constitutively overexpressing CBF1, CBF2, and CBF3. CER6 transcript accumulation was slightly higher in the G26 line over-expressing the CBF1 transcription factor (Fig. 4.4C) than in the WT control, but the lines over-expressing CBF2 and CBF3 did not show any increase in CER6 transcript levels relative to the WT (Fig. 4.4C). These results suggest that the C B F / D R E pathway is not involved in regulation of CER6 transcription, or that the cooperation of other factors is required.  Temperature (°C) 10 20 30 18S Rl  1.8  0.5  Whole plants Stems (24h) (96h) 20°C 20°C 4°C 20°C 4°C light dark dark light dark  B  CER6 m  m  m  18S  C  CBF1 CBF2 Ws G26 E2 E8  CBF3 A28 A30  Figure 4.4. C E R 6 transcript is increased by lower and reduced by higher ambient temperatures, but is not affected by C B F overexpression. R N A blot-hybridization probed with CER6 coding sequence and 18S r R N A (loading control). Ten u,g of total R N A was loaded into each lane. A . R N A extracted from tops of bolting stems of plants grown at 20 ° C until their primary stem bolts reached 10 cm in height, then transferred to 10 ° C , 20 ° C or 30 ° C for 96 h prior to harvest. Similar results were obtained in two additional replicates. B. R N A extracted from whole shoots of flowering plants transferred to darkness at 20 ° C or 4 ° C for 24 h and the top 2 cm of bolting stems transferred to darkness at 4 ° C for 96 h prior to harvest. C. R N A extracted from the tops of bolting stems of W T (Ws ecotype) and trangenic plants overexpressing the C B F 1 , 2 and 3 transcription factors involved in regulation of cold and dehydration responses in Arabidopsis. Similar results were obtained in two additional replicates.  117 Wounding Insect herbivory has been shown to affect wax deposition in several species. In order to test whether wounding, the major plant physiological effect of insect herbivory, could affect this process in Arabidopsis, CER6 transcript levels were measured in wounded whole plants and leaves (Fig. 4.5). Plants or leaves were cut into pieces, then incubated on filter paper moistened with dFbO before R N A extraction for northern blot analysis. CER6 transcript was noticeably reduced as early as 30 minutes after wounding of leaves (Fig. 4.5). By 24 hours after wounding of whole plants, CER6 transcript was reduced to undetectable levels (Fig. 4.5).  Whole plant (h) o  CER6  m  24  Leaf (min) o  10 30  *  Figure 4.5. Wounding reduces C E R 6 transcript levels in Arabidopsis. R N A blot hybridization of R N a extracted from whole plants 0 and 24 hours after wounding and from leaves 0 , 1 0 and 30 minutes after cutting up with a razor blade and incubating on wet filter paper in Petri dishes. 10 mg of total R N A was loaded for each treatment, and the blot was hybridized to the C E R 6 coding sequence probe and to 18S r R N A (loading control).  4.2.2. Environmental effects on wax deposition  Temperature variation, especially cold treatment, had the most marked effect on wax load of all growth conditions tested, although this effect was noticeable only if the wax was quantified on the basis of surface area, rather than dry weight of the stems (both measures are commonly used in the literature). Stems of plants grown at 10 °C for their entire lifespan had 6 times greater wax load/surface area than stems of plants grown at 20 °C (Fig. 4.6A, continuous). The wax load/surface area of stem tops of plants grown to bolting stage at 20 °C, then transferred to 10 °C, was not significantly different from that of plants maintained at 20 °C (Fig. 4.6A, 4 days; transfer). However, transfer from 20 °C to 30 °C caused a significant reduction in wax load/surface area (Fig. 4.6A, 4 days; transfer).  118  10  20 30 Growth temperature ( C)  Figure 4.6. Total wax load on stem bases of plants grown at different temperatures for their entire lifespan (continuous), or maintained at 20 °C until primary bolting stems were 10 cm tall, then transferred to different temperatures for 4 days prior to harvest (4 days, transfer). A . W a x standardized on the basis of surface area. B. W a x standardized on the basis of dry weight. Bars represent means + standard error of wax extracts pooled from 3 pots of each line (6 plants/pot).  119  Conversely, when wax load was quantified on the basis of dry weight, the opposite relationship applied: wax load/dry weight was higher at high temperatures, and somewhat lower at low temperatures (Fig. 4.6B). Observation of the epicuticular wax crystals by S E M favoured the surface area quantification: Wax crystals appeared bigger and were covering the stem more densely on both tops and bases of stems of plants grown continuously at 10 °C than on those of plants grown at 20 °C (Fig. 4.7). Although CER6 transcript level was not affected in the CBF over-expressing plants, the plants were deeper green and more robust looking than the WT, so I extracted their wax for quantification. Interestingly, the CBF1G26 line had a wax load almost twice that of the WT control, whereas the others had wax loads similar to those of the WT (Fig. 4.8). I also performed wax analysis on stem bases of plants deprived of water, and on the aba mutants under a normal watering regime, to determine whether wax accumulation would correlate with CER6 expression patterns under these conditions. Although plants deprived of water had increased CER6 expression (Fig. 4.3), their wax loads were unchanged or slightly decreased from control levels in two experimental trials (Fig. 4.9). Wax loads of aba mutants also failed to correlate well with the CER6 transcript levels. Although the aba mutants had reduced levels of CER6 transcripts in their bolting stems (Fig. 4.3), their wax loads were not significantly different from, or were actually increased in relation to those of the WT controls (Fig. 4.10A). S E M analysis of the abal mutants showed that wax crystals of abal-1 and abal3 were similar to those of WT (data not shown), in agreement with the GC data. Abal-4, however, had a more sparse cover of epicuticular wax crystals than WT (Fig. 4.1 OB), indicating that its wax load may be reduced from that of the WT. To determine whether light was essential for wax deposition in bolting stems, wax was extracted from the top 5 cm of stems of plants transferred to darkness and from control plants maintained in light, 4 days after the transfer. The plants transferred to dark conditions had continued to elongate from the 10 cm height which they had attained by the time of transfer; thus, both the dark-treated and light-grown stems were more than 15 cm long at the time of harvest. Nevertheless, GC analysis of the wax extracts showed that the wax loads on stem tops after 4 days of darkness were not significantly different from those of the control plants maintained in light (data not shown).  4um Figure 4.7. S E M of stem tops (A,B) and bases (C,D) of Arabidopsis plants grown continuously at 10 ° C (A,C) and 20 ° C (B,D) shows that wax microcrystals of plants grown at the lower temperature are bigger and more robust.  121  35 30 Q o>  25  E "><  03  jj  20 i  !  O)  H.  TJ CO O X CO  •Sllll  15 10 5 0 CO  CD CM O CD O  CM LU CM LL m o  00 LU CN LL CD O  CO CN < CO LL CD O  o co < co UL m o  Figure 4.8. Total wax load on stem bases of W T (Ws) and transformed lines over expressing C B F 1 , 2 and 3. Bars represent means + standard error of wax extracts pooled from 3 pots of each line (6 plants/pot).  122  Figure 4.9. Total wax loads of stem bases of well-watered and unwatered Arabidopsis plants. Bars represent means + standard error from two separate trials each pooling data from three replicate pots.  Figure 4.10. A. Total wax load on W T (Col and Le) and aba mutant stem bases. Bars represent means + standard error of 3 plants from each line. B. S E M showing wax microcrystal density on W T and abal-4 stem tops  124 4.3. DISCUSSION  Wax accumulation is known to be affected by a variety of environmental factors, especially light (von Wettstein-Knowles et al. 1979) and water deficit due to lack of soil water or freezing temperatures (Thomas & Barber 1974; Bengtson et al. 1979; Hadley 1989). The presence in the CER6 promoter of I-box-like and GTl-like sequences previously found in lightresponsive genes (Terzaghi & Cashmore 1995), and A B R E elements (Guiltinan et al. 1990) identified in drought- and cold-inducible genes responsive to A B A , suggested that CER6 expression may be induced by these stimuli.  4.3.1. Light  M y results presented here clearly demonstrate that light plays a pivotal role in the expression ofCER6 (Fig. 4.2), and that in the absence of light CER6 transcript levels quickly decline. A complete repression of CER6 transcription in the dark did not, however, occur in the det2 mutant, suggesting that this response may be mediated by a DET2-dependent signal transduction pathway. The maintenance of high CER6 transcript levels in darkness in coldtreated plants suggests some interplay between regulatory pathways, or at least a slowing down of regulatory processes that repress CER6 transcription in the dark (or turnover of the CER6 transcript). Surprisingly, darkness did not affect the wax loads of bolting stems, even though under normal light conditions, CER6 is essential to wax deposition ((Millar et al. 1999); Chapter 3). It could be that CER6 protein was present long enough for wax to be synthesized in the new epidermal cells, or that sufficient V L C F A wax precursors were already present in the stems for wax deposition to continue for the period of darkness tested. However, 24h of darkness is long enough to reduce CER6 transcript to almost undetectable levels (Fig. 4.2), and the stems had continued to elongate, which would dilute the CER6 protein even i f it is more stable than the transcript. Alternatively, other condensing enzymes may be present or are induced in darkness, which can produce sufficient wax precursors to maintain wax biosynthesis in the dark. A n examination of EST abundance for different Arabidopsis condensing enzymes showed that a few of them are expressed in dark conditions (Chapter 1), and could fill this role. It has also been shown that in barley and maize, the rate of V L C F A synthesis is reduced in dark-grown leaves, and the chain lengths of the V L C F A s produced are reduced (Hawke & Stumpf 1965; Giese 1975; Avato et al. 1980). It would be interesting to determine which condensing  125 enzymes in Arabidopsis are expressed in dark-grown plants, and whether these enzymes contribute to the synthesis of V L C F A wax precursors.  4.3.2. Osmotic stress  CER6 transcript accumulated in response to water deficit and other osmotic stresses (Fig. 4.3), suggesting that dehydration may enhance wax deposition by increasing V L C F A production. Furthermore, significantly greater CER6 transcript accumulation in ABA-treated seedlings, and reduced CER6 transcript levels in A B A deficient mutants implicate A B A in the induction of CER6 transcription in response to water deficit. It is also interesting that even under normal watering conditions, the presence of A B A seems to maintain CER6 transcript at its normal level. Even though CER6 transcript levels are clearly affected by water deficit and A B A , it has not been possible under the experimental conditions used for this study to demonstrate an increase in wax load in unwatered plants (Fig. 4.9). This lack of correlation between CER6 transcript accumulation and wax loads in osmotically-stressed plants may be attributable to an inappropriate experimental setup. For example, several early attempts at growing plants under drought conditions failed as water deficit was never accomplished, measured by rd29A transcript accumulation. In subsequent experiments CER6 transcript increases correlated well with rd29A transcript accumulation under water deficit conditions. However, there may not have been enough time for the changes in transcript accumulation to affect wax deposition under my experimental conditions. Indeed, Jenks et al. (2002), using different soil and growth conditions, were able to measure a 25% increase in wax load in Arabidopsis stems grown in water deficit conditions, as compared to the well-watered controls. Thus, CER6 may be involved in increasing wax deposition under water stress conditions. Most of the aba deficient mutants had wax loads similar to those of WT controls (Fig. 4.10), even though their CER6 transcript levels were reduced (Fig. 4.3). It is possible that the decrease in CER6 accumulation was not sufficient to significantly affect the wax loads in these mutants, or that other condensing enzymes were induced under low A B A conditions. However, one of the mutants, abal-4, did have a reduced epicuticular wax crystal density (Fig. 4.10) and possibly a reduced wax load. The abal-4 mutant also has the most severe phenotype (Koornneef et al. 1982; Leon-Kloosterziel et al. 1996) and the lowest CER6 transcript level of all of the aba mutants. This raises the possibility that the CER6 level in the other aba mutants was sufficient to allow normal wax production, whereas in the abal-4 allele, the reduction was severe enough to reduce wax accumulation.  126 4.3.3. Cold  The higher levels of CER6 transcripts at lower temperatures (Fig. 4.4) correlated well with the higher wax accumulation in plants grown at lower temperatures (Fig. 4.6; 4.7). This indicates that the regulation of wax production at colder temperatures involves the induction of wax biosynthetic enzymes, including CER6. The lack of induction of CER6 transcripts in the CBF over-expressing plants (Fig. 4.4C) suggests that the regulatory pathway involved, at least for CER6, is independent of CBF. Although the CER6 promoter has sequences similar to the full D R E originally reported (Yamaguchi-Shinozaki & Shinozaki 1993), a more detailed analysis of the binding kinetics and in vitro mutation analysis showed that the minimal sequence required for binding of CBF1 is C C G A C (Yu et al. 2002). The DRE-like sequences found in the CER6 promoter, T A T C G A C A T and T A C C A A T A T , lack this core. Thus it is not surprising that the CBF over-expressors do not exhibit any increase in CER6 transcript levels. It is interesting, however, that the CBF1G26 line has an increased wax load (Fig. 4.8). This suggests that some components of wax biosynthesis other than CER6 may be influenced by the C B F / D R E pathway. The difference in wax loads of plants grown at 10 °C and 20 °C depending on whether they are quantified on the basis of dry weight or surface area is striking. A quantification by surface area shows a dramatically higher wax load on stems of plants grown at the colder temperature, whereas a quantification by dry weight indicates that cold treatment has no effect on wax load. Of the two quantification methods, the surface area quantification seems to be more reasonable, since wax is distributed over the surface of the plant. Its accuracy is also supported by the S E M analysis showing that the epicuticular wax crystals formed on the stems of cold-grown plants were bigger and more robust than those of the controls grown at 20 °C (Fig. 4.7). A factor that could have contributed to the observed difference in the cold-grown plants when the two quantification methods were applied, is that the stem diameters of coldgrown plants were much bigger than those of controls grown at 20 °C (data not shown). This would reduce the surface:volume ratio, and thus the surface:dry weight ratio of the cold-grown plants, which would cause a skewed result in the dry weight quantification.  4.3.4. Wounding  It was interesting that CER6 transcript levels were quickly reduced by wounding (Fig. 4.5). The physiological effect of wounding approximates to some extent the damage caused by insect herbivory. While fungal pathogens wound the plant in penetrating the cells, their  127 elicitors often cause different responses than those of wounding alone. Wax blooms tend to decrease the efficiency of insect attachment to plants, as well as visual attractiveness of the plant to the herbivore (Eigenbrode 1996). This would seem to favour wax induction by wounding. However, under field conditions, glossy variants of several Brassica species are more resistant to insect herbivory than their waxy counterparts, possibly due to increased difficulty of adhesion of insect predators, or even due to chemical changes in the waxless plants which experience more water stress (Eigenbrode 1996). The rapid reduction of CER6 transcript levels by wounding may therefore be an adaptive response to wounding. This response would involve reducing wax load to counter insect attack by downregulating major wax biosynthetic enzymes, including CER6. The destructive wounding method used for this study rendered the investigation of wax load changes in wounded plants impossible. However, this hypothesis could be tested further by using a milder wounding technique which would allow subsequent wax extraction from the plants, or by measuring wax loads on plants challenged by insect predators.  4.4. C O N C L U S I O N S  This study established a correlation between CER6 transcript levels and wax production in plants subjected to temperature changes. These results agree with temperature-dependent changes in wax deposition found in other species (Whitecross & Armstrong 1972; Baker 1974; Giese 1975). Although this study could not establish a correlation between wax deposition and CER6 transcript levels for light or osmotic effects, changes in CER6 transcript abundance correlate with changes in wax production found in other species. Furthermore, water deficit has been found to increase wax deposition in Arabidopsis under another experimental setup (Jenks et al. 2002a). It is quite possible that the conditions in this study did not impose a water deficit severe enough to affect the wax load. Also, other enzymes are required for wax deposition; their transcription might not have been induced even if CER6 transcription was. Further study should reveal whether CER6 induction under water deficit conditions does contribute to an increase in wax production. Light is required for wax deposition in germinating pea seedlings (Juniper 1960) and increases wax deposition in mature leaves of other plants (Macey 1970; Whitecross & Armstrong 1972). However, light affects different wax components differently (Macey 1970; Giese 1975; Avato et al. 1980; Tevini & Steinmuller 1987), and it is likely that CER6 transcript repression in darkness is only part of the regulatory mechanism for wax deposition in darkness.  128 Nevertheless, the responsiveness of CER6 transcription to light, water deficit, temperature and wounding, as well as to the phytohormone A B A , suggests that these factors cause changes in wax production by altering the abundance of key wax biosynthetic enzymes, including CER6, in the epidermal cells. This study has also shown that light regulation of wax production is likely to involve the DET2 regulatory pathway. Wax deposition in response to water deficit seems to be regulated via A B A , but is independent of the C B F / D R E pathway. Similarly, cold-induced wax accumulation is also not mediated by the C B F / D R E pathway.  129 C H A P T E R 5. C L O N I N G A N D C H A R A C T E R I Z A T I O N O F CER7  5.1. I N T R O D U C T I O N  Over-expression of CER6 in transgenic plants has not yet led to a heritable, stable increase in wax load in any line. The fatty acid elongation pathway is the initial step required for wax biosynthesis, but many other enzymes and regulators are involved in this process. Evidence for this is found in both the biochemical complexity of cuticular waxes, and the abundance of wax-deficient mutants identified, even in the non-saturating screens done so far in Arabidopsis. Thus, a more successful approach to increasing or modifying wax load may be to identify key regulators in the pathway and manipulate their expression, rather than to simply over-express one of the biosynthetic enzymes. I hypothesized that mutants defective in proteins involved in regulating transcription of wax biosynthetic enzymes would have reduced transcript levels of these enzymes. At the time when I started this work CER6 was the only enzyme with a clear biochemical function in wax production, and it catalyzes an early step in wax biosynthesis. Thus, I examined CER6 transcript level in stems of 20 wax-deficient mutants (cerl-cer20) to determine i f any of the mutated CER gene products is involved in transcriptional regulation of CER6, and possibly other wax biosynthetic enzymes. Preliminary results suggested that CER6 transcript abundance was reduced in a number of the cer mutants (data not shown), suggesting that the genes identified by these mutations may encode regulators of wax biosynthesis. Cerl was one of the mutants which seemed to have a reduced CER6 transcript level, and it was chosen as the first candidate to be cloned because it had a very glossy stem, and thus an easily recognizable phenotype (unlike cer 14, 16, 19 and 20), and it was fully fertile (unlike cerlO). Koornneef et al. (1989) isolated 5 alleles of cerl (4 EMS and 1 fast neutron), mapped it to chromosome 3 at 87.1 c M , and found that cerl has a recessive inheritance pattern. S E M analysis showed that the tube- and plate-like structures of the epicuticular wax were much reduced from the WT (Koornneef et al. 1989), and GC results have established a reduction in wax load to about 50% of that of the WT (Lemieux et al. 1994; Jenks et al. 1995). However, the studies cited, as well as other studies of wax composition in cer mutants have not focused extensively on cerl. Therefore cerl wax composition has not yet been rigorously examined.  130 The objectives of this study were: (1) to more fully characterize cerl pheno typically and genetically, (2) to determine whether transcript levels of any other known CER gene were changed in the cerl mutant stems, and (3) to clone and characterize the gene mutated in the cerl line. I characterized the cerl mutant phenotype by S E M and GC analysis, and confirmed the recessive inheritance pattern by examining the wax profile of the F l progeny of a backcross. I studied the expression of CERl, CER2, CER3, CER4, CER5 and CER6 genes in the cerl mutant background by real-time PCR and R N A blot hybridization. I positionally cloned the CERlgene, and found and characterized a second, T-DNA insertional allele of the same gene. Finally, I analyzed the CER7 protein sequence by bioinformatics, and determined the transcript abundance of the CERl gene in the mutant and in different WT tissues by RTPCR and by R N A blot hybridization.  5.2. R E S U L T S  5.2.1. Phenotypic and genetic characterization of the cer7 mutant  Since the characteristics of the cerl phenotype have only been presented in a very general way to date in the literature, a more detailed phenotypic and genetic characterization of this mutant was required. The mutant stem has a bright green, glossy appearance under high light intensity (Fig. 5.1), in contrast to the glaucous appearance of the WT stems. The siliques also appear to be slightly more glossy than those of the WT (Fig. 5.1). S E M analysis of airdried stems shows that the wax microcrystals on the tops and bases of cerl mutant stems are more sparse and have a slightly different shape than those of the WT (Fig. 5.2). Wax extraction and GC analysis revealed that the cerl mutants have a reduction in stem wax load to about 50% of that of the WT (Fig. 5.3 A). In contrast, silique and leaf wax loads were only slightly reduced in the cerl mutants (Fig. 5.3A). The wax composition of the cerl mutant was also somewhat different in comparison to the WT, in that components derived from the decarbonylation pathway (notably, C29 alkane, ketone and secondary alcohol) were reduced more than those derived from the acyl-reduction pathway (the primary alcohols). Thus, the proportion of primary alcohols, and particularly of the C30 alcohol (which was roughly double in the mutant stems and silques than in the WT), was higher in the mutant than in the WT cuticular wax (Fig. 5.3B,C). Even in leaves, primary alcohols were maintained close to WT levels, whereas alkanes, C30 fatty acid and other unidentified compounds were reduced (Fig. 5.3C). In stems and siliques, although the levels of the major wax components (derived via the  Figure 5.1. Cer7 mutant stems show a glossy green, waxless phenotype (A, B) which contrasts with the glaucous appearance of W T (Ler) and F1 plants under high light intensity. (B) and (C) show close-up images of stem bases and siliques, respectively. A , 0.8X; B, 3X; C, 4 X  Figure 5.2. S E M of stem tops (A-D) and bases (E-H) of W T (Ler) and cerl reveals a lower density of wax microcrystals in Cer7 (B,D,F,H) compared to W T (A,C,E,G). The shape of the crystals is also changed somewhat: cer 7 crystals look thinner and more wormlike than the W T crystals.  133  12  A  •  stem  CD s i l i q u e  10  E3 leaf  WT  cer7  F1  B • W T stem F1 stem B Cer7 stem S W T silique • F1 silique • Cer7 silique  ?/ / / / / J"  of  cf  '  ^ -<t  p  o* / c&>  / n»  /  /  /  C>  C?  O  ^  v  6* G  V  /// &  C?  ^  o,^ cv?  cy  .r*  /// ^  >°  cr  #  Figure 5.3. A. Total wax load extracted by chloroform dip from stem bases, siliques, and leaves of W T (Ler), cerl, and F1 plants. Bars represent means + standard deviation of 3 samples each, containing extracts from 3 plants. B, C Composition of stem, silique (B) and leaf (C) wax of WT, cer7, and F1 plants.  134 decarbonylation pathway) were reduced, the level of C31 alkane was increased to approximately double the WT (Fig. 5.3B). It is also noteworthy that the amount of C30 fatty acid in the wax was reduced in all three tissues examined. Thus, the cer7 mutation appears to negatively affect the synthesis of wax components by the decarbonylation pathway, but does not have the same effect on the products of the acyl-reduction pathway. Fatty acid elongation does not seem to be affected by cer7, either, since there is an increase in C31 alkane. This also points to a specific effect on the decarbonylation pathway, slowing the use of V L C F A s for the production of alkanes such that the fatty acids can be further elongated. Cer7, like all of the other known wax-deficient mutants of Arabidopsis, has been reported to be a recessive mutation (Koornneef et al. 1989). To verify that the mutation is truly recessive, I examined the macroscopic phenotype and wax loads of the F) progeny of a backcross to the WT. I found that the macroscopic phenotype of the F| progeny was indistinguishable from the WT (Fig. 5.1). Furthermore, the wax loads of the stems, siliques, and leaves were approximately the same as those of the WT (Fig. 5.3A). Thus the cer7 mutation is indeed recessive. To determine whether the cer 7 mutation caused any cellular defect which could provide a clue as to the function of the affected gene product, cer 7 mutant stems were high-pressure frozen and examined by transmission electron microscopy in collaboration with Lacey Samuels and with the technical assistance of Minako Kaneda. Preliminary surveys of toluidine blue Ostained thick sections by light microscopy and thin sections of the same material by T E M did not show any obvious changes in cell morphology (data not shown). Therefore the ultrastructural studies were not pursued any further. The recessive nature of the cer7 mutant phenotype is consistent with the hypothesis that the CER7 protein may be involved in regulating the production of wax. To explore this possibility, I performed real-time PCR (data not shown) and northern blot analysis (Fig. 5.4) of CERl, CER2, CER3, CER4, CER5 and CER6 in the cer7 mutant. Results from both techniques indicated that there was no difference in transcript levels of any of these genes between WT and cer7 plants. This was surprising in the case of CER6, because in the preliminary experiment I found reduced CER6 transcript levels in the cer7 mutant. However, this result was not reproducible.  Figure 5.4. R N A blot hybridization showing transcript levels of CER1 to CER6 in stems of W T (Ler) and cerl mutants. All genes tested showed similar transcript levels in W T as in mutant stems. 10 ug of R N A extracted from the top 2 cm of stems of W T and cerl plants was loaded for each hybridization. 18S rRNA was used as a loading control. The blot was repeated once with the same R N A and twice more with a fresh R N A extraction. R T - P C R and real-time P C R experiments also showed similar results.  5.2.2. Map-based cloning of CER7 Although 5 alleles of cer 7 were isolated (Koornneef et al. 1989), only one, isolated from an EMS (ethyl methanesulfonate) mutagenized population, has been maintained. We obtained this line from the Arabidopsis stock center for positional cloning. I generated a mapping population of approximately 2000 F progeny from a cross between the cer 7 mutant in 2  a Landsberg erecta ecotype background and a WT Columbia ecotype. I isolated D N A from 605 F plants showing the mutant phenotype and fine-mapped the cer7 mutation by scoring the 2  population for recombination between the mutant phenotype and the parental (Ler) and recombinant (Col) forms of SSLP or CAPS markers. Marker positions on the chromosome and recombination frequencies for each of the markers are shown in Fig. 5.5. These mapping steps narrowed down the possible location of the mutation to a region of chromosome 3 spanning 80 kb, which included 19 open reading frames annotated by the Arabidopsis genome database (Fig. 5.5C). The open reading frames were assigned letters for convenience in discussion. Candidate genes, their identities and letter designations are given in Table 5.1.  cer7  A  tsh2tsh4 (3) (1)  tsh9 (0)  tsh5 (0)  tsh6 tsh7tsh3 (0) (2) (1)  Figure 5.5. (A) S S L P markers used for fine-mapping of the cer7 locus, represented schematically on Arabidopsis Chromosome III, with recombination frequencies shown by percentages; (B) B A C s used to design new markers; (C) new marker locations on a detailed map of the annotated genes (with nicknames A-S) with the number of recombinants indicated in parentheses .  Q.  E  m ro c o c  CD  E  Cl  E o o CD C  =5  CU  cr <D CO  o  CO CO CD  c ro CO  CU  CO  C  o ro c  CD Q5  CU  c CU  cm a) ro  "O  73  c ro o  CD  o  CD Q. >. O CD  _CU  ^3  (U  ro  E E  c o € <u  w a.  Q H  re  — I  c CD  138 I undertook a three-pronged approach to determine which gene was affected by the mutation. The first approach involved PCR-amplification of each candidate gene from the 80 kb region from the WT and mutant, followed by sequencing to find any putative mutations. I found differences in the coding sequences of 4 genes, 3 of which would result in amino acid changes in the corresponding proteins (Table 5.2). Simultaneous to the sequencing, I cloned the fragments amplified from WT D N A into the pGREEN binary vector for transformation into mutant plants and complementation of the mutant phenotype. I transformed 8 of the genes from the region of interest into cer 7 plants before finding the complementing fragment. The third approach was to examine the surface wax phenotypes of plants with insertional mutations in the target 80 kb region of the genome. Of the 13 insertional lines grown, one had a waxless phenotype.  Table 5.2. Positions of mutations found in coding sequences of the target region Gene  Mutation  E N  C->T G->C C->A A->C A->G C->T 15 bp insertion C->T  0 S  Position Cds length 1002 37 54 97 114 478 98 532  1200 700  276 1318  Consequence to protein silent silent T->N silent K->E silent insert G G G A G G->STOP (aa. 178/439)  I obtained kanamycin-resistant seedlings when 8 different WT genomic fragments were transformed into cer 7 plants, including all of the genes for which mutations affecting the amino acid sequence were found (Table 5.3). A l l of the kanamycin-resistant plants obtained from plants transformed with genes C, F, L, M , N , O, and R showed a waxless phenotype, indicating that none of these genes complemented the mutant. In order to confirm that these plants did indeed receive the WT transgenes, the transgenes were amplified from a number of the Ti plants using a T-DNA-specific right-border primer and a gene-specific primer (Table 5.3). In contrast, all 10 of the kanamycin-resistant seedlings recovered from plants transformed with gene S from which the transgene was amplifiable showed a complemented, waxy phenotype similar to that of the WT (Table 5.3; Figure 5.6). GC analysis of wax extracted from the stem bases of these complemented mutants confirmed that WT wax loads (Fig. 5.7 A) and wax  139 composition (Fig. 5.7B) were restored in the complemented mutants. These data support the conclusion that gene S, At3g60500, corresponds to CER7.  Table 5.3. Complementation of  cer? by transformation  Introduced gene # kanamycin-resistant plants C F L M N O R S  1 1 9 2 11 9 1 10  # from which transgene was amplified 0 1 2 0 10 4 0 10  Phenotype waxless waxless waxless waxless waxless waxless waxless WAXY  5.2.3. S A L K T - D N A insertional allele  In parallel to the attempt to identify the CER7 gene by complementation using wild type genomic fragments, I examined a number of lines with T-DNA insertions into genes in the target 80 kb region for altered wax phenotypes. These T-TNA insertion lines were generated by the S A L K laboratory, which has been sequencing D N A flanking insertion sites in a T - D N A mutagenized population and cataloguing them in a public database. I searched this database for insertions in the genes that fell within the 80 kb region identified by mapping and obtained seed for 13 different lines shown in Fig. 5.8. The seed from each of these lines is from a T3 segregating population (http://signal.salk.edu/tdna_FAQs.html). Of the 13 lines screened, only SALK_003100 showed a waxless phenotype in 7/27 of the unselected plants. This line has an insertion point 408 bp 5' to the translational start codon of gene S. A comparison of the phenotype with that of cer7, and with WT Ler and Col is shown in Fig. 5.9. The green, glossy phenotype of the SALK_3100 stems is quite similar to that observed on cer 7 stems. I characterized the phenotype of this line further by S E M and GC analysis of the stem wax. GC analysis showed that the wax load and composition of 5 wax-deficient plants was similar to that of the cer 7 mutants (Fig. 5.10). One of the plants exhibited an intermediate phenotype, with an increase in the 30C primary alcohol but with a lesser reduction in the other components, and thus in the overall wax load. The rest of the plants were waxy and had GC wax profiles similar to WT plants (Fig. 5.10).  WT  cer7 cer7-S cer7-R  Figure 5.6. Transformation of cerl plants with gene S, but not gene R, restores the W T waxy phenotype. (A) W T , cerl, cerl transformed with gene S, and cerl transformed with gene R, whole plants, 0.35X; (B) Stem base close-up, 7X (dissection micrograph)  141  10  Q E "S ra  o n  CD CO  Z  - A C N C N C O C O C O C O C O V Y W c y j W C O W t O t O C / J c o r o  5  B  • 29-alkane H 29-20H • 30-1OH  4 5  4 3.5  3  WT  Cer7  Cer7-N  Cer7-S  Figure 5.7. A. W a x loads of cer7 plant transformed with gene S (S1-1 to S3-11), gene N (N2-2; N3-6; N38), and cerl and W T (Ler) untransformed plants, showing that W T wax loads are restored in the cer7 plants transformed with gene S but not gene N. W a x was extracted from the stem bases of senesced plants. B. Average wax composition (mean + standard deviation) of the same plants, showing that the complemented mutants {cer7-S) have similar wax composition to the WT, but the uncomplemented mutants (cer7-N) have similar wax composition to cer7.  cu hcu  ALK_014653  Q_  SALK_015995  H '  o c  ALK 041743  0) tn  co  SALK_043675 SALK 017859 SALKI016803 SALK_040758  | § o > E  £ 1  SALK_006242 SALK 069209  Is i »P  re  SALK_010684 SALK 004600  o  0  V -J  C  L  ® CO t 0J c .= £ >. S E o o 5 tr *= ro a) CO 5 ro c < o J= 2: CO ro  I  1 >  Q. "-  SALK 100127  cd cu 10 t -  SALK 003100  u. CM  0 1  S o _g 3 ro 0 Q. CU  Figure 5.9. Phenotype of SALK_003100 (SALK) waxless plants in comparison with cer7 and W T (Ler and Col). Whole plants (A, 0.35X) and dissection micrographs of stem bases (B. 7X) are shown.  144  T-  CM CO  5 3 5 Figure 5.10. W a x composition (by load of major components) of individual plants grown from SALK_003100 T seed obtained from A B R C . The wax composition of the waxless plants is similar to that found in cer7. 3  S E M observations of surface wax correlated well with the GC results. The wax crystals from the tops of stems of wax-deficient plants 1,5, 10 and 21 were less dense and smaller than those of WT, and were similar in appearance to those seen on the cer 7 mutants (Figs. 5.2, 5.11). The wax crystals on the stem top of plant 3, which had a waxy appearance and a WT wax profile, also had wax crystals similar to those of WT (Fig. 5.11). The similarity of the SALK_003100 wax to that of the cer 7 wax, and the proximity of the T-DNA insertion site to gene S which complements the cer7 mutation, suggested that the gene tagged in the S A L K 003100 line represents a second mutant allele of CER7. Therefore I performed a complementation cross, and confirmed that in the Fi generation, the S A L K 003100 line indeed does not complement the original cer7 mutant. Since the genes mutated in these two lines represent two cer 7 alleles, I will refer to the original EMS mutant allele of cer7 as cer7-7, and to the SALK_003100 allele as cer7-2 from this point on.  5um Figure 5.11. S E M of wax from stem tops of 5 T plants from SALK_003100 seed obtained from A B R C . Numbers denote the plant number, and are the same as those in Figure 5.10. Plant 3, which has similar wax crystals to those of W T (Col), has a W T wax load (Fig. 5.10); the others have sparser wax crystals than that seen on W T , corresponding to their cerl -like wax composition (Fig. 5.10). Some of the lines (10, 21) have sparser wax crystals than seen on cer7 stems (cf. Fig. 5.2). 3  146 5.2.4. Identity o f C E R 7  The sequence of the genomic clone used to complement the cerl mutant is given in Fig. 5.12. It is a 3.8 kb fragment which includes a 2.2 kb coding sequence (At3g60500) comprised of 9 exons and 8 introns, as well as 1.4 kb of 5' non-coding sequence and 0.3 kb of 3' noncoding sequence. Comparisons with ESTs and a cDNA clone sequence confirmed the splice sites in the annotated genomic sequence, and have revealed the presence of another intron in the 5'UTR. Thus, the spliced sequence would produce an mRNA approximately 1.6 kb long, encoding a protein of 439 amino acids. This coding sequence is annotated in the Arabidopsis genome database at MIPS (http://mips.gsf.de/proj/thal/db/index.html) as a nucleolar autoantigen - like protein, showing similarity to the known human protein nucleolar 75K autoantigen PM-Scl (PIR:G01425). Three ESTs are assigned to the gene, AA713250, AU236953, AU227961 (MIPS website). However, the latter two represent the 5' and 3' ends  ccaacatgtt gttttttttt aagacatggt _tagaaata itaatcgt aatac aatctaaaat tactaetcat taatgtctgt tgtttgttga  tcttgttcat tcttttctct gacgaaaaga tagttctcgt ttaaagtctt aaa tctagta acaatttatc tgactttctt aagcccafcgg tccagaacga  ctacatga tagtaaacaa ttcgatgtgg gcaaggcatc aaatagctga aatgttgaag catgatcttc caaaeatgtg ccaaaaaaat gcttatatcc attataattt cagcgttttt agttattcac aattaagtaa ttttaaagaa tccacaagag tgaagacttt aattagtcca agtgtacfttg  »' i' g agattggcta atatgtggag gttgactgtt tt ategcaaget tactattaag tttggcaal  tgtagtttgt tccaaattaa ttccaagttt tegeggaate tgattcttga aaagtacaga aattctitgag cgtcaaagtt aaaataaaaa tttttgtcaa cctctcgaat f  j  gtgtgcgtga gatgatcata aggaaagagg tgtaaacttt aatagagatc aaacgatata gattttgtgg aatctttcca actcattgac ataacaaata  agcaaacata aag^acaac aci^mactta ttt"Kjgctgc gaaggattga tttgagtggt  aacatggttc catatcttct agaagagaca tcataacaga aagcttgetc gctgctggaa gaaaactg..a  tatcagtttc tagattcatc tgacccttac caaataagca ctaattaagt gacttaaacc ettttacget tgatagtttc aaaattctct cattgttaat C a a g t a g g a t a a a a t a c gt a ctggicttgtt gaatattcta al. U :caaaa agcatcaaga rctaaa acgacgccgg  ttggtttaat ttacacraatt gtttactagt gatagcagH ta agta aattcgtgga aactgcgttg caatctgagc tcaga  agaatatg ggtctctctc tcgtggtcta a .ggacaat ggag tgcgttctag ctggaaagat ggtttggtct gttcgcattg accttca' at gaact tagttgatgc gacatttagg agacccgact o : a c t g t a a -i a • v; < i •gg'c - • •lagaag • > tgatcataca tccacta atgtaatggg tttcgtgact gctcagttgg t teegtctttt gagcctggtc gtcctggcga ate  atgaag Eattatcga  acatcatctc ccaatagcct tcacgtttgg attttttaat aaaggcaaca gtgat cagttaatgc caatggegat atatgegcaa tccaaaaacc gggggaagaa "getgetgea acaacaaaga tcattagaga agaa(| gttgaa gcatataact gtgagagaag .,- -. --, .- - : j :aacata ggaagtcctcagatcaggaa gaaga agetgecaat ttcaaaggcg  gtagctcgga agttcaactt ggtcagactc gatcttcacg gaattttctc caatggctga a gaaagccgtg cagtagatac agagtcactc tgcaaatatt getgetttag cagctctca  ttgti ggacccaact tatgttgaag aagcagttat gtgtgggaga atgactgtga ggcgtgaacc agagtgtaat ccttcattgc ctgcgtcttg cttcttcaag cttacagaag gtgaagegae atcccacttt ggctaaatct gaagtttct agagctgeag agatatctcg agaacatgtt gaaagactaa agctctctac gtccctcaaa tt. ~~  *  i ' ii  aagcagggee ttattaaggc agtaactaaa tgggagagag gaggcttggc ggegg igctg gtagatcgga gaggagaa  Figure 5.12. Genomic sequence amplified for complementation of cerl by gene S (At3g60500) UTR  ggg|  P C R primers  ~~ (cerl-1 h a s C A T which changes a G i n ( C A A ) to a stop ( T A A ) ) \ceflA mutation site." < 3 A L K insertion line with w a x l e s s phenotype!  147  (respectively) of a single R I K E N full-length cDNA, so there are effectively only two EST clones from this gene. These were cloned from R N A isolated from 3-day-old seedling hypocotyls (AA713250) and from a mixture of flowers and siliques (the R I K E N clone; AU236953; AU227961). There are three other ESTs that show significant similarity to At3g60500 in a B L A S T n search of the T'AIR Arabidopsis EST database. However, these are better matched to a similar Arabidopsis gene, At3g 12990. To further investigate potential functions for this protein, the amino acid sequence was submitted to a BLASTp of the non-redundant database at N C B I (http://www.ncbi.nlm.nih.gov/BLAST/). This search revealed that the CER7 protein has two conserved RNAse_PH domains: the first, 131 residues in length, from aa36 to aal68 (100% aligned) and the second, 67 residues from aal97 to aa262 (95.5% aligned). The RNAse_PH domain is a 3'-5' exoribonuclease, with family members involved in rRNA, tRNA, or mRNA processing or degradation. The closest matches to the CER7 protein are a family of proteins from a number of species (Table 5.4) which are similar to a functionally characterized S. cerevisiae protein: Rrp45p. This protein forms part of the exosome, a protein complex involved in R N A processing in yeast (Butler 2002). Another Arabidopsis protein encoded by At3gl2990 ORF is very similar to CER7, with 88% identity and 91% similarity over amino acids 1-307. Figure 5.13 shows an alignment of the protein sequences of CER7, At3gl2990, and the yeast Rrp45p, with the two conserved RNAse P H domains indicated by lines under the alignment. The yeast protein similarity to the two Arabidopsis proteins is fairly evenly distributed through the conserved part of the protein, not concentrated only in the two RNAse domains. Interestingly, the C-terminal 130 amino acids of the CER7 protein do not show similarity to the yeast protein, to the other Arabidopsis homologue, or to anything else in the nr database at NCBI. There are 10 core proteins which make up the yeast exosome, 6 of which have similarity to the E. coli RNAse PH. I used each of the yeast protein sequences in a B L A S T p search of the Arabidopsis genome database at the TAIR website. The results of this B L A S T are given in Table 5.5, and show that there are clear homologues for several of the yeast exosome proteins. Other yeast exosome proteins do not have clear homologues in Arabidopsis (Table 5.5). In order to investigate this further, I aligned all of the Arabidopsis and yeast RNAse PH-like phosphorolytic exonucleases, along with the E. coli RNAse PH, using ClustalX. I manually removed gaps from the alignment, and constructed a bootstrapped  148  00  CO  CO  i — r— i —  CO  CO  T—  co  CN CM CM CN CN CN  CN  m  oo i-  T-  1-  CM  T~  co CM  to Q.  TO  CD  o _  E CO _ C CD T3  T-  CO  O0  oj co co  CD  co m co  co T - 1 -  CM  oo r-- oo  00  CN  Tj-  CO  O c  a:  o  CD CO TO _CD  CD CO  •g "o TO O  o  T—  CO  c  c  CD  CD CD • ~  Ql DC  CD TO O O  "E  Q .  0  3  CD  o  U  (0  a) a  CD  o  3 CD  3 CD  CD  CD  CD CO TO _CD  O 3  E  ^  O X  CD  CD T3  •a  CD  CD L.  CD  O o  CD  X a)  f  O O CO CO  o  CO CD CO 03 • _Q TO TO T3  to O  o  a. H  CO  a:  CD 3 -  UJ  CO  CO  uo  CD  01  OO  ro  ><  ro  CD  o > cu •£ o -2 3  =3  c  0.  E  E  •o  LL  in  CO  o  >^ o o Q .  X  CD a > •2 -2 S £ "3 O) CO CD  CD  ai  CD  ^ •E  E ,52 E  TO  o  •co o o CO  S CD TO" O CO TO Q . E o a 3 C to E J Z  <  _1  CO Q)  „, £  CD  -2 .22  Q .  Q3  co  o  C ^  S o  . CO  co o CO  CO CD  ^.2 re cn  CO  a> CO  cr: ai  CM  id 4) .Q  <0  oo  0  CD CO  5  i  < O  co ^  cn  CD CD CD  J CD  »  Oo  LU LU  a  149  Y e a s t Rrp45p At3gl2990 At3g60500  1 1 1  MAKDIE. . . . I S A S E SKFILEALRQNYRLDGRS FDQFRDVEITFGKEFGDVSVKMGNTKV MEGRIMSMWRLTVOTSKFVESALQSELRVDGRGLYDYRKLTIKFGKEYGSSQVQL MEGF%LANMWRLTVNESKFVETALQSELRVDGRGL^  Y e a s t Rrp45p At3gl2990 At3g60500  57 61 61  HCRISCQIAQPYEDRPFEGLFVISTEISPMAGSQFENGNITGEDEVLCSRIIEKSVRRSG MAFVTAQLVQPYKDRPSEGSFSIFTEFSPMADPSFEPGH.PGESAVELGRIIDRALRESR MGFVTAQLVQPYKDRPNEGSLSIFTEFSPMADPSFEPGR.PGESAVELGRIIDRGLRESR  Y e a s t Rrp45p At3gl2990 At3g60500  117 120 120  Y e a s t Rrp45p At3gl2990 At3g60500  174 180 180  Y e a s t Rrp45p At3gl2990 At3g60500  234 VLTVTLNKNREWQVSKAGGLPMDALTLMKCCH 228 228  Y e a s t Rrp45p At3gl2990 At3g60500  274 288 288  Y e a s t Rrp45p At3gl2990 At3g60500  282 307 348  Y e a s t Rrp45p At3gl2990 At3g60500  305 307 408  EAYS . . . H E  . DQILQL.  Figure 5.13. Alignment of C E R 7 (At3g60500) with yeast Rrp45p and the other Arabidosis homologue (At3g 12990). Regions of similarity and identity are shaded. The two conserved R N A s e _ P H domains are indicated by grey bars under the sequence. The nuclear localization signals (4 overlapping 4aa consensuses) found by PSORTI at the C-terminal end of At3g60500 are indicated by highlighting.  150  03  CN o o CN  O O O CM  — 5 E  ^= C  ro  o o  ro cL> ro > o aro  CD  ro ^—•  ro  c o>  CD  E  — !  c ro  jx.  CD CL  CD JZ  CD JZ  O  O  ro c QJ  i-  O Q.  -t—•  •<r  CJ)  i-  CO CO  co a> CN CN CJ)  <  CN O  CO CO  co co  N O  CM  CJ) T CO •^3CN CN  CN CN CO  T^ CO  CJ) CO CM  CJ) CO CM  CD CO CM  1-  CJ) O  CJ) O  1-  cj>  CN  -q-  CM  LO CO CJ)  00  CO CO CJ)  CO  CO  oo r*-  M— O  co,  CD  to CD  =3  O) o o E o  ro ro M—  JZ CO CO  I ro CL CD JZ  o  LO  CO  h-  C0 < _i CD  0 0 0 0 0 0 T- CM  CO  a.  o _ JD  CO  CO CD CM TCO CD ^J" CJ) CJ) CO CO  ro — s CD  g  _  O i_ Q. CD  1  O  O 10 10  o  o  LO  o  o  LO  CD ( S CM  N-  1-  N-  CO CO  CN  CO TI  CO T" I  CL  CL LO CL  CC  o 00 O  N O  CD CO  1-  o IO N O CO^ CO  < < <  CO D-| CN  CO  I  U)| LO  T  LO I  T-  UJ UJ LU UJ LU LU O O O O o o O O O O o o CM CO CD OO CN  LO  < < <  cj)  CN  1—  cc  cv 0- o o o CN TCM LO  CM CD T-  •sr  CD  CO CO  CO  C0|  < < CL CD • * CL  1—  cc  CD  CD CJ) CO  CL CO  CO  CM  O  LO  CD LO CO CO CO LO O CM CD D)  CL  CL O  CL  CL  cc cc  1—  CD  o •  o  JZ CL CO  £Z  so  o  o a E CD o = aCO >  X CD  o  CO  CO CO  LO  CM LO  00 r~  _CD  o  CD  O  <  T3  c= !  £  a:  x CD  LO  LO  O  r^— CM CO CJ)  00  CD  LU LU o o o o  O TLO  CJ) CO TCN •4—> -t—•  o CJ) CO CO CO CO LO  < <  o o CJ) -<3LO T CO L O CO LO  CL CL  CC  co  o  CD  E  >. N C  0  a  o — JZ CD  ro ro o 3  o >  n o  CO T-  < <  CL  CO  CL CO  LO  LU o Q  < < <  ro  o  o T-  UJ LU oUJ o  CO CD CO  _>-. CO  LO I  CO  o  CO  CD >• CD O  o  1-  CO O X CD  ro  N T-  J Z  o HI UJ UJ 0  CD  4—1 CO  00  0O  CD  T3 C CO  E o  CO N  "5) c  CD  "3  00 00 T - CO 00 00 CJ) hCN CN CM T -  CN CJ) S B T-  1-  O  I  fa >  <tf CM  CL CD CL  i_  CC  151 neighbor-joining phylogenetic tree (Fig. 5.14). This tree shows that many of the RNAse PHlike proteins in yeast do not have any obvious Arabidopsis homologues. However, the Arabidopsis homologue of Rrp41p, which has been demonstrated to complement the yeast mutant and interact with other exosome subunits in vitro and in vivo (Chekanova et al. 2000), does form a well-supported branch. The branch grouping CER7 and At3g 12990 with the yeast Rrp45p is the only other well-supported feature of the tree (Fig. 5.14). Interestingly, two other Arabidopsis RNAse PH-like putative exosomal subunits (Atlg60080 and At3g07750) also group with this branch with good bootstrap support (Fig. 5.14).  no?,  -AJ2ge45C -Rrp42p -Mti3p 3il AllgSKKO  5'.rj>45i! - Al3gl2»Q -CER? -Rlp43p  -AtSgbloM Pjp41p -At3g*2l0 -ALV14580  Figure 5.14. Bootstrapped neighbor-joining phylogram of RNAse-PH-like exosomal homologues from yeast (Rrp designations) and Arabidopsis (At designations). Two wellsupported branches stand out: one links yeast Rrp41p with At3g61620, which is also supported by experimental evidence (Chekanova et al. 2000) (lightest grey box), and one linking C E R 7 , Ateg12990 with yeast Rrp45p (darkest grey box). Two other Arabidopsis proteins which showed high similarity to these homologues, At3g07750 and At1g60080, also group with this Rrp45p branch in a fairly well-supported lineage (medium grey box)  152  To get some idea of the subcellular localization of the CER7 protein, I ran the amino acid sequence through the PSORT signal peptide prediction program. The prediction for CER7 was compared to that of At3gl2990. The check for the 4 residue pattern for nuclear targeting in PSORT I revealed the presence of 4 possible signals at the C-terminus of the protein (position indicated on Fig. 5.13). No nuclear targeting signal was found for At3gl2990. Analysis by PSORT II predicted a 30.4% probability of nuclear localization and a 26.1% probability of cytoplasmic localization for CER7, as well as a 34.8% probability of mitochondrial localization. At3gl2990, on the other hand, was predicted by PSORT II to be a cytoplasmic protein (73.9% probability; 17.4% probability of nuclear localization).  5.2.5. Expression of At3g60500 gene in the cer7 mutant and S A L K 003100 line  The cer7-l allele sequence has a C->T transition at nucleotide 532 of the At3g60500 coding region, which results in a premature stop codon being introduced at amino acid 178. This truncation of the protein eliminates one of the RNAse domains, and could result in a nonfunctional protein, a less efficient protein, or in a protein with a changed function. Four other mutations are present in the genomic sequence of the cer7-l mutant, 5' to the translational start of At3g60500: an A->C transition at -307; an A->C transition at -674; an A->G transition at 897; and a G->C transition at -1135 as indicated on Fig. 5.12. The T-DNA insertion site in the cer7-2 (SALK) allele is at -408 from the translational start, or approximately 60 bp upstream of the 5' U T R sequenced from ESTs. There are sequences resembling T A T A and C A A T boxes 30 to 70 bp upstream of the S A L K allele T-DNA insertion site (Fig. 5.12), suggesting that the insertion could interfere with transcription of the coding sequence for the At3g60500 gene and thus reduce or eliminate the protein it encodes, leading to the mutant phenotype. To obtain more evidence that CER7 is indeed the gene annotated as At3g60500,1 tested the expression of the At3g60500 gene by northern blot and RT-PCR in the mutant plants. I found that the transcript level of At3g60500 was reduced in stems, siliques and seedlings of cer7-l mutant plants, as well as in seedlings of the wax-deficient SALK_003100 line (cer7-2) (Fig. 5.15 and data not shown). This result is consistent with At3g60500 being the CER7 gene.  153  CER7  GAPC  Figure 5.15. R T - P C R analysis of CERl from 14 day old whole seedlings of cer7 mutants and SALK_003100 in comparison to W T (Le and Col), showing that CER7 expression is lower in cerl than in W T , and extremely low in SALK_003100. GAPC (cytosolic glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control.  154 5.2.6. Tissue-specificity of CER 7 expression To learn more about the role of the CERl gene product in wax production in Arabidopsis, I determined the tissue specificity of CERl expression by northern blot and RTPCR. The presence of a homologue in the genome which has 89% nucleotide identity with the CERl coding sequence (Fig. 5.16) prompted me to closely evaluate the sequences used for RTPCR primers as well as for probes for the northern blot. I tested 3 probes, one specific to the 3' end of CERl (which is not conserved), one specific to the 3' UTR of the homologue (At3gl2990), and one non-specific probe covering the conserved region of the two genes. I generated this last probe to determine whether it would hybridize to both transcripts and allow me to detect both on the same R N A blot. This was a possibility since, based on the length of their respective coding regions, the CERl mRNA should be about 400 bp shorter than the At3gl2990 mRNA. Hybridization of the probes to a genomic Southern blot (Fig. 5.17) showed that the two specific probes hybridized to different fragments. The non-specific probe, which was generated from the 5' end of the CERl cDNA, did not seem to cross-hybridize with the At3gl2990 D N A fragments or mRNA. Hybridization to a total R N A blot showed that CERl was expressed in all of the tissues tested (Fig. 5.18), both aerial tissues and roots. At3gl2990 showed a similar pattern of expression, and, as expected, had a shorter transcript than that of CERl (Fig. 5.18). A t 3 g l 2 990 At3g60500  A T G G A G G G GA G G T T G A A T A A T A T G T G G A G G T T G A C T G T G A A T G A G A O C A A A T T C G T T G A A T C T G C G T T G C A A T C T G A G C T C A G A G T C G A T G G T C G T G G C C  A t 3 g l 2 990 At3g60500  T T T A T G A T T A C C G C A A G C T T A C T A T T A A G T T T Q G C A A G G A A T A T G G C A G C T C A C A A G T T C A A C T GOG T C A G A C T C A T G T A A T G G C T*E"t T G T Q A C T G C T C A  At3gl2990 At 3g60500  GCTAGTACAACCTTACAAAGACAGAGCTAG  A t 3 g l 2 990 At3g60500  A T G G A G G WGAGAT T G G C T A A T A T G T C G A ^ T T O A C T G T T A A C G A A A G T A A A T T C G TG G A A A CT G C GT T G C A AT C T G A G C T C A G A G T A G A T G G T C G T G G C C  TTTATGATTATCGCAAGCTrACmrrAAGTT^^  GTTGGTACAACCTTACAAAGACACACCTAAT^AAGGGTC^  301 301  GG T G A A T C T G C T G T T G A G C T T G G C C G T A T T A T A G A C C G T G C T C T A C G A G A A A G C C G T G C A G T A GA TA C A G A G  GG C G A A T C T G C A G T G G A G T T GGGA C G C A T T A T C G f t T C G T G G T C T A A G A G A A A G C C G T G C A G T A G A T A C A  A t 3 g l 2 990 At3g60500  TTTGGTCTGTTCGCATTGATCTTCACATTTTOGACAAT  A t 3 g l 2 990 At3g60500  ACCTGATTGCACTGTAGGAGGGGACAACAGT^  A t 3 g l 2 990 At3g60500  GC C T T C A C G T T T G G A T T T T T T A A T A A A G GCA G T A T C T T G G T O A T G O A C C C A A C T T A C G T T G A A G A A G C TG T T A T G T G T G G <5 A G A A T G A C T G T G A C A G T C A  A t 3 g l 2 990 At3g60500  A T G C C A A T G G C O A T A T A T G C GC A A T C C A A A A A C C A O GAGAACIAAGG C<3 T G A A C C A G A G TQ T A A T C C T T C A T T GC C T G C G T C T T S S C T T C T T C A A G AG C T ' r C A T G C C A A T G G C G A T A T A T G C GC A A T C C A A A A A C C C G O G G A A G A A G G C G T G A A C C A G A G T G T A A T C C T T C A T T G C C T G C G T C T T G C T T C T T C A A G A G C T G C  A t 3 g l 2 990 At3g60500  TGCAACAACAAAGATAATTAaAGATGCAGTT^  A C C C GA C T G C A C T G T A G O A G G A G A GAA T G G T G A A GAAG T G A T C A T A C A T C C A C T AG A G G A A A G G G A A C C A C T T C C A*  GC C T T C A C G T T T G G A T T T T T T A A T A A A G G C A A C A T T G T G G T G A T G G A C C C A A C T T A TG T T G A A G A A G C A G T T A T G T G TGG G A G A A T i S A C T G T G A C A G T T A  T G C A A C A A C A A A G A T C A T T A G A G A A G A A G T T C A A G C A T A T A A C TG T G A G A G A A G C T T A C A G A A G G T G A A G C G A C A T C C C A C T T T G G C T A A A 7 C T G A A G T T  At3gl2990 At3g60500  901 901  A t 3 g l 2 990 At3g60500  924 1001  TCTGGACCTACTGTAOCT^aAAOGAGGAACATAGGAAGTCCTCAGATCAGGAAAGAGCTGCAGAGATATCTCGAGAAC^  At3gl2990 At3g6050 0  924 1101  C T A C GG A G G A A G T A A G AA GC A G T A A G GAA GAA GAAG C T G C C A A T T T C A A A G G C G G T C C C T C AAA T T G G GA C C C T T A C T C A G A A G C T A T G G A T G T T G A T T C  At3gl2990 At3g60500  924 1201  A C T G A A A G T T TC T C T T G C T T C A A G A G G G G A T C CC G T T A C T A A G T C A T C GAGC A C GAAG A A A A T G A A C G G C T C T G G A A A T G C G C A G A A G G T T G G T G T T G A G  A t 3 g l 2 990 At3g60500  924 1301  ATTTCAGTTGAGGAAGTAACCGGAGAATTAGGGAAGAAGGATACAAAACACAAGGATGGAGAAATGACACTTAAAGATGCTGTCAAACCTAAAAAGAAGC  TTGGGACCTATTGTAGTTGTG TAG  G G A A A A A C A A A A G C TGA  Figure 5.16. Nucleotide sequence alignment of CER7 (At3g60500) and its sister gene (At3g12990) (89% identity over 924 bp). Identities are shaded.  155  CER7 3'end B E X  CER7 non-specific B  E  X  At3g 12990 3'UTR B  E  X  GAPC  B E X  Figure 5.17. Genomic Southern blot showing hybridization with a C£R7-specific probe (CER7 3' end), an At3g12990-specific probe (At3g12990 3' UTR), a CER7 non-specific probe (using conserved sequence), and GAPC (the probe used for a loading control in the northern blot). For each lane, 10u.g of genomic D N A was digested with BamHI (B), EcoRI (E) or Xbal (X). Probes and hybridization conditions were the same as those used for the northern blot  CER7  CER7  3' end  non-specific  At 3g12990 3' UTR  0) O 4*2 ci)  tr \n  _  J  3 S  m </)  CER7mKHm  Figure 5.18. Northern blot showing CER7 and At3g12990 (SIS) transcript levels in different tissues of Arabidopsis. The blots were hybridized to specific (3') probes for both genes and to a less specific probe for CER7, as in the Southern blot (Fig. 5.17). GAPC was used as a loading control.  5.3. DISCUSSION  5.3.1. CER7 cloning  Complementation of the cer7 mutation by transformation with a WT genomic fragment containing the At3g60500 ORF (Figs.5.6, 5.7), sequencing that revealed the presence of a premature stop codon in the mutant coding sequence (Fig. 5.12), and the discovery of a plant line with a T - D N A insertion 60 bp upstream of the putative transcriptional start site (Fig. 5.12) that is allelic to cer7 establish solidly that At3g60500 corresponds to the CER7 gene. This is further supported by the fact that the transcript abundance of this gene was reduced in both allelic mutants (Fig. 5.15). A premature stop can reduce stability of mRNA transcripts, so it is not surprising that the steady-state levels of the CER7 transcript were reduced in cer7-l. The CER7 transcript level was also quite low in S A L K 003100 line (cer72), but some transcript was detectable by RT-PCR. Nevertheless, a severe reduction in transcript is required for a 5' T-DNA insertion to have a phenotypic effect, and such a reduction was observed in this mutant.  157 In contrast, 7 other putative genes found within the 80 kb region identified by finemapping as the location of cerl failed to complement the mutation when they were transformed into the mutant (Table 5.3; Fig. 5.7).  5.3.2. Cer7 wax profile  The cerl wax profile determined in this study generally agrees with the analyses by Lemieux et al. (1994) and Jenks et al. (1995). The stem wax load was reduced to about half of the WT load, whereas the leaf wax load was essentially unaffected (Fig. 5.3A), as described by Jenks et al. (1995). Jenks et al. (1995) limited their report on wax composition to a statement that cerl wax was similar to that of cer3, and Lemieux et al. (1994) did not analyze wax on different plant organs. In this study, I have presented wax load and composition separately for stems, siliques, and leaves, thus adding to the information so far available. The most striking feature of the cerl stem wax composition was the increase in C30 primary alcohol to approximately double that of the WT, in both stems and siliques. This is similar to the wax profile for cer3 stems as indicated by Jenks et al. (1995). Even in leaves, which exhibited only slight reductions in total wax load, products of the decarbonylation pathway were affected, but primary alcohols were maintained at WT levels. These results indicate that cerl mutants show specific reductions in the decarbonylation pathway, which could be partially compensated for by an increase in flux through the acyl-reduction pathway.  5.3.3. Expression of other CER genes in the cer7 mutant  Although an initial northern blot examining the transcript levels of CER6 in the cer mutants showed that CER6 expression was low in cerl (data not shown), this result was not reproducible in subsequent attempts (Fig. 5.4). Thus, reduced expression of CER6 was not required for the cerl phenotype. Furthermore, the wax load of cerl was only reduced to about half of the WT load (Fig. 5.3 A ; (Lemieux et al. 1994)), whereas cer6 mutants have less than 10% of the wax load of WT plants (Lemieux et al. 1994). Thus, similar to cer6 mutants, one would anticipate that a dramatic reduction in CER6 transcript levels, as detected in the initial experiment, would result in a much more severe waxless phenotype than that observed in the cer 1 mutant. Additionally, there was no chain length reduction in cerl mutant wax (Lemieux et al. 1994), and the primary alcohols (products of the acyl-reduction pathway), were either unchanged (C26 and C28 primary alcohols) or even increased (C30 primary alcohol) (Fig.  158 5.3B). Thus, CER7 may be required only for the synthesis of wax components via the decarbonylation pathway. In addition to the lack of a cerl effect on CER6 expression, transcript levels of other CER genes were not reduced in the cerl mutant (Fig. 5.4). CER4, encoding a primary alcoholforming reductase (Kunst & Samuels 2003; unpublished data) would be specific to the acylreduction pathway. Thus, it is not surprising that CER4 mRNA levels were unaffected. The functions of CER1, CER2 and CER3 have not been established with certainty. Therefore, it is difficult to predict their relationship to CER7. Finally, CER5 seems to be involved in wax export from the cell (Kunst & Samuels 2003; Pighin et al. 2003), a function which would most likely be downstream of CER7 (if CER7 regulates wax secretion). Thus, CER5 transcript levels might be regulated by CER7. Since CER5 expression is not reduced in cerl mutants, other regulatory pathways must be involved in regulating this gene. Even though it does not control mRNA levels of characterized CER genes, CER7 could still affect transcript levels of wax biosynthetic enzymes, secretory proteins, and/or transcriptional regulators that have not yet been identified. It could also affect enzyme activity indirectly, for example by modifying transcript levels of a post-transcriptional regulator or protein activity modulator (eg. kinase or phosphorylase).  5.3.4. Role of C E R 7 in wax production  CER7 is annotated to be a "PM-Scl75 nucleolar auto-antigen-like" protein (MIPS Arabidopsis thaliana database). This is a human protein linked to an auto-immune disease, polymyosistis-scleroderma overlap syndrome (Allmang et al. 1999b). The CER7 protein sequence is more similar to the human protein than to the yeast homologue Rrp45p (Table 5.4). The human homologue has been demonstrated to complement the yeast mutant and to be associated with the human functional exosome homologue (Allmang et al. 1999b). This functional characterization gives more weight to the probability that CER7 is also a homologue of the yeast protein, as suggested by similarity searches of the nr database at N C B I (Figs. 5.13, 5.14). Rrp45p is a 3'-5' exoribonuclease which is one of the 10 core proteins comprising the yeast exosome, an R N A processing complex (Mitchell et al. 1997). Six of the ten core exosome subunits show similarity to RNAse P H from E. coli (Mitchell et al. 1997). A B L A S T p query of the Arabidopsis genome with all of the known subunits from the yeast exosome results in identification of reasonably clear homologues for many of them (Table 5.5).  159 CER7 is one of the two Arabidopsis proteins which show much greater similarity to Rrp45p than any of the other putative Arabidopsis exosome subunit homologues (Table 5.5, Fig. 5.14). The two Arabidopsis proteins identified by BLASTp as homologues of the yeast Rrp4 and Rrp41 exosome subunits have been shown to complement the yeast strains with mutations in those proteins (Chekanova et al. 2000; Chekanova et al. 2002). In order to firmly establish CER7 as an Arabidopsis homologue of Rrp45, it should also be used to complement the yeast mutant lacking this protein. However, even in the absence of yeast complementation, the evidence is sufficient to warrant a hypothetical investigation of the potential function of CER7 as an exosome subunit.  v  In yeast, exosomes exist in two forms, a nuclear form and a cytoplasmic form (Butler 2002), and function in R N A processing and degradation. There are specific roles for each form. The main functions of the nuclear form are the 3' end processing of the 5.8S rRNA precursor (which stabilizes the rRNA subunit) and degradation of the internal ribosomal spacer (Allmang et al. 1999a), snRNA and snoRNA processing (Allmang et al. 1999a), and recognizing and degrading improperly processed mRNAs (Mitchell et al. 1997; BousquetAntonelli et al. 2000; Hilleren et al. 2001; Butler 2002). It may also have a role in processing of mRNAs which affect the release of mRNAs from the site of transcription and export from the nucleus (Custodio et al. 1999). Thus, the nuclear exosome plays an essential role in the maturation of stable mRNAs (Butler 2002). The cytoplasmic exosome, on the other hand, is one of the routes of degradation for turnover of mRNAs (van Hoof et al. 2000b). This process is probably involved in post-transcriptional regulation of mRNA levels in the cell (Jacobs et al. 1998). The importance of post-transcriptional regulation of mRNA levels has gained prominence in recent years, since the realization that the regulation of many plant genes cannot be explained simply by the control of transcription (Green 1993). As a first step towards determining the cellular function of CER7, it is necessary to establish which exosome it is associated with, or whether it is present in both. This could be done by subcellular localization of the CER7 protein. A PSORT analysis of the protein sequence gave quite similar probabilities for nuclear and cytoplasmic localization of CER7. Therefore its localization will have to be determined experimentally. The simplest way to accomplish this would be using a GFP reporter gene fusion, which would allow both tissue localization in living plants, as well as the subcellular localization. It may be that the protein is present in both the cytoplasmic and the nuclear exosomes. This is the case for all of the yeast exosomal subunits (Jacobs et al. 1998; Allmang et al. 1999a; van Hoof et al. 2000a) except  160 Rrp6, which is exclusively associated with the nuclear exosome (Allmang et al. 1999a; Butler 2002). However, this may not be the case in plants. The presence of a second Rrp45p homologue in Arabidopsis, At3gl2990, (Figs. 5.13, 5.14) could allow for functional specialization. It is possible that one of the two is associated with the nuclear and the other with the cytoplasmic form of the exosome. Alternatively, CER7 and At3gl2990 could be interchangeable, with the exception of specialization in CER7 which is exclusive to wax production. This seems to be a more likely scenario as discussed below. The core of the exosome seems to be a fairly general ribonuclease with its functional specificity conferred by associated proteins (Jacobs et al. 1998). Accordingly, the loss of any exosome subunit in yeast is a lethal mutation (Mitchell et al. 1997; Allmang et al. 1999a). In contrast, neither cer7-l nor cer7-2 shows any obvious phenotype besides waxlessness (Figs. 5.6, 5.9). These results suggest that there is redundancy in essential functions between CER7 and At3gl2990 proteins. This is also supported by the facts that the expression domains of the two proteins are very similar (Fig. 5.18), and that there is no obvious phenotype in the S A L K insertion line 300 bp 5' to At3gl2990 (data not shown). However, the wax-deficient phenotype, which is a unique feature of cer7 mutants not observed in the S A L K line with a TD N A insertion into the At3gl2990 gene, shows that there is specialization in CER7 with respect to wax-related functions. There is a small degree of sequence divergence over the length of the proteins, but the biggest difference between CER7 and At3gl2990 is the 130aa C-terminal extension of CER7 (Fig. 5.13). Although this part of the protein does not show any significant similarity to any domain or protein in the nr database of Genbank, it may be the source of the divergent function. Another possibility is that the cellular expression domains of the two proteins differ somewhat. This is the case with HEN2, the exosome-associated helicase in Arabidopsis (an apparent homologue of the yeast protein Mtr3p), which is expressed only in specific domains of the flower, although like CER7, it shows expression in all of the tissues when examined by northern blot (Western et al. 2002). CER7 could, for example, only be expressed in epidermal cells, and At3gl2990 in other tissues. This question could be addressed by in situ analysis, as well as by a reporter gene study. It would also be interesting to determine whether At3gl2990 or a truncated CER7 gene driven by the CER7 promoter or by the 35S promoter could complement a cer7 mutant, to assess the significance of the C-terminal extension of CER7 for its wax-related functions. Due to a difference in expression domain, a functional specialization of a redundant essential gene, or some other reason, CER7 seems to have developed a specific regulatory role  161 in wax production. Given the putative RNAse function of the protein, this function must somehow relate to degradation or stabilization during maturation of certain R N A species. It is more likely that mRNA, rather than rRNA, is the target, and that specific mRNAs involved with wax production are processed. These considerations lead to two possible models for the mechanism of action of the CER7 protein, depending on whether it is nuclear or cytoplasmic (Fig. 5.19). Both models also assume that it is either acting as part of the exosome or as a free RNAse in the cytoplasm. In the first model, CER7 would be associated with the cytoplasmic exosome or act as a free RNAse in the cytoplasm. The mRNA targets would have to be involved with repressing (transcriptionally or post-transcriptionally) the production of wax components via the decarbonylation pathway. When these targets are degraded, production of the major wax components (alkanes, secondary alcohols and ketones) would continue. In cer 7 mutants, a defective or missing RNAse would allow the target mRNAs to accumulate, thus repressing wax production. In the second model, CER7 would be associated with the nuclear exosome and act in mRNA processing to stabilize specific transcripts (targets). These stabilized mRNAs would then either act in wax biosynthesis (ie., enzymes of the decarbonylation pathway) or activate the decarbonylation pathway (by transcriptional or posttranscriptional means). The cer7 mutant would lose this exosome function and therefore the decarbonylation pathway would not be activated. This raises the question: which mRNA species could be the normal degradation target of the exosome in the epidermis? As mentioned before, the CER7/exosome target is most likely the mRNA encoding an enzyme or regulator of the decarbonylation pathway. It would be useful to identify the target or targets of CER7. This would lead to a better understanding of the regulation or biosynthesis of waxes, or both. The most direct way to find the target(s) of CER7 is probably a microarray analysis, by looking for transcripts that are more abundant in cer 7 than in WT. Alternatively, a suppressor/enhancer screen could be used to find downstream or upstream regulatory components of wax production. Microarray analysis would compare transcript levels of all genes expressed in WT and cer 7 plants. Transcripts which are absent, greatly reduced, or greatly increased in the cer 7 mutant could represent direct and indirect targets of the CER7 protein. Since the only apparent cer 7 phenotype is wax deficiency, it is likely that many or most of the CER7 targets found in this way would be either downstream regulators (transcriptional or post-transcriptional) or possibly biosynthetic enzymes of the decarbonylation pathway.  Figure 5.19. Model showing possible mechanisms of C E R 7 regulation of wax production in Arabidopsis.  163 A suppressor screen would involve mutagenizing cerl plants and screening the M 2 generation for waxy variants in the wax-deficient population. As with microarray analysis, such a screen could be expected to detect downstream regulatory targets of CER7, particularly if the repressor degradation model is correct. Mutation of a repressor would be the simplest way to ensure activation of the decarbonylation pathway, and thus a suppression of the cerl mutation in the double mutant. A suppressor screen would also detect other types of mutations, for example mutations in cerl that restore its function (this is less likely, since cerl-1 has an early stop codon, and cerl-2 is an insertional mutant), mutations in parallel pathways which would compensate for the loss of CER7, and dominant mutations in downstream regulators or biosynthetic enzymes which cause constitutive expression, stability, or activity. A mutagenized cerl population could also be screened for enhancers. CER7 enhancers would have greater wax deficiencies than cerl mutants. However, a GC screen might be required to detect them. They could also have male sterility or organ fusion phenotypes, like a number of other wax-deficient mutants, but screening for additional phenotypes such as these could also lead to unrelated mutations. A n enhancer screen may not be the most productive approach, since it would be more likely to detect regulators working in parallel pathways than gene products downstream of CER7. These could be expected to give synergistic effects. For example, new cer4 alleles could be detected in such a screen: cer4 affects only the acylreduction pathway, and cerl affects mainly the decarbonylation pathway; thus, a double mutant could be expected to have a more severe phenotype than either of the single mutants. It would be more labour-efficient to cross cerl mutants with other known wax-deficient mutants to look for enhancer effects. It would be interesting to determine, for example, whether a cerl/cer 3 double mutant would enhance the phenotypes of both single mutants. Cer3 has a wax phenotype similar to that of cerl. If the two are working in different pathways of wax regulation, a double mutant could be expected to have a more severe phenotype than either of the parents. However, if they are both in the same pathway, the double mutant would probably have a cer3-like phenotype (with conditional male sterility in addition to the cerl/cer3 wax profile). Since both cerl-1 and cerl-2 probably reduce the amount of CER7 protein available for incorporation into the exosome, rather than producing a non-functional protein, it seems likely that the entire exosome is disrupted in the mutants. Exosome assembly needs a stoichiometric ratio of all of the subunits, and few free subunits have been found in yeast (van Hoof & Parker 2002). This suggests that CER7 may be specific to the cytoplasmic exosome in epidermal  164 cells, as it is likely that a more severe phenotype would be observed i f CER7 was required for the nuclear exosome which processes rRNA. These functions would probably be indispensable for young, differentiating epidermal cells (even if CER7 is not the only Rrp45p homologue that is expressed in epidermal cells). It needs to be pointed out that there is also another mechanism of mRNA degradation in yeast: a 5'-3' exoribonuclease activity that actually plays a more important role in mRNA turnover (Muhlrad et al. 1994; Muhlrad et al. 1995; van Hoof & Parker 2002). It could be that this is also the case in Arabidopsis, and that cytoplasmic exosomes are only required for wax-related targets in developing epidermal cells. A n alternative hypothesis is that it is only At3gl2990 that forms part of the exosome, and that CER7 has diverged from that role. It is possible that in plants, some of the redundant exosomal subunits have developed accessory functions apart from the exosome, and exist in free forms in the cytoplasm, rather than in the exosomal complex. The proteins would still function as a 3'-5' exoribonucleases, but would perhaps have unique targets for a specific regulatory role. This hypothesis could be tested by immunoprecipitation of the CER7 protein and determining whether other proteins (particularly, exosomal subunit homologues) coprecipitate with it.  5.4. C O N C L U S I O N S  In this study I have performed a detailed characterization of the cerl phenotype by the analysis of the cer7 wax load, composition and surface features by S E M . I have also shown that CER7 is not involved in regulation of transcription of the known wax-related CER genes. I have isolated the gene identified by the mutation in the cer 7 line using positional cloning, and found an allelic T - D N A insertional mutant which shows a similar wax-deficient phenotype. As initial steps in determining the cellular function of the CER7 gene product, I have examined its expression pattern in plants and investigated its putative function based on database similarity searches. The ubiquitous expression of CER7 and similarity to the yeast exosome ribonuclease Rrp45p suggest a role in mRNA degradation or processing which could be involved in posttranscriptional regulation of proteins involved in wax deposition. In order to establish the activity and role of CER7 in wax production, and differentiate between roles of the two different putative Arabidopsis homologues of Rrp45p, these gene products should be used to complement the yeast knockout and/or assayed in vitro. A cellular and sub-cellular localization of the transcripts and proteins is also necessary. This could be  165 accomplished by transforming Arabidopsis with a CER7-GFP fusion protein and localizing the fluorescence by confocal microscopy, or by raising antibodies to a recombinant CER7 protein and using them to immunolocalize the native protein in thin sections by T E M . Finally, to extend the study further, other components of the wax biosynthetic and/or regulatory pathway controlled by CER7 should be identified. This could be done by looking for genes with increased or decreased steady-state transcript levels in the cerl mutant using microarray analysis, as well as by suppressor screens, which could reveal the target(s) of CER7, including downstream regulators and/or biosynthetic enzymes of the decarbonylation pathway. Crosses with other waxless mutants with apparent regulatory functions, such as cer3, would also be useful in determining whether these mutants act in the same or different pathways of wax regulation as CER7.  166 C H A P T E R 6. C O N C L U D I N G R E M A R K S AND DIRECTIONS F O R F U T U R E R E S E A R C H  Understanding wax deposition in plants requires three avenues of research: 1) Elucidating the biosynthetic pathways involved, 2) Establishing the mechanism(s) of wax secretion, and 3) Determining the regulatory pathways for both of these processes. The biosynthetic pathways have been extensively studied using both biochemical and moleculargenetic approaches. Although the decarbonylation pathway remains elusive, genes encoding most of the enzymes involved in fatty acid elongation and the acyl-reduction pathways have been cloned from Arabidopsis and other species. Secretion remains the biggest mystery, with much speculation and little data. The major contributions of my thesis research are to the area of regulation of wax production in Arabidopsis, and constitute the first concrete steps toward unraveling its complexity. First, my work has established that transcript accumulation of CER6 is involved in the regulation of wax production under normal growth conditions, as well as with variations in temperature and water status. Second, I have cloned and begun to characterize CER 7, which encodes a protein most likely involved in affecting transcript stability of some as yet unidentified wax-related gene product(s).  6.1. C E R 6  A l l evidence collected to date, including cer6 mutant and sense-suppression phenotypes, CER6 epidermal expression throughout the shoot, and correlations between wax load and CER6 expression levels, supports the conclusion that the CER6 condensing enzyme is a major player in fatty acid elongation for the synthesis of V L C F A wax precursors, and thus for wax biosynthesis. However, there is some indication that other condensing enzymes are also involved in this process. The maintenance of wax production in darkness in the absence of CER6 transcription (Chapter 4) suggests that another K C S may be able to functionally replace CER6 when light is absent. The reduction in leaf wax in the kcsl mutant (Todd et al. 1999) suggests that it plays a role in wax production in at least some organs of Arabidopsis. In addition, preliminary evidence from our lab suggests that CER6 can accept C24,C26, and possibly C22 fatty acyl-CoAs as substrates, but not CI 8, C20 or C28. If this is the case, at least two more K C S enzymes would be required for wax production in Arabidopsis: one elongating CI8 fatty acyl-CoAs to C24, and one specific to the last elongation step, from C28 to C30. It  167 would be useful, both for understanding wax biosynthesis and its regulation, to identify these KCSs and to define their biochemical roles. A survey of the entire K C S family to identify members with epidermal expression patterns may reveal which ones are most likely to be involved in wax biosynthesis. This can be accomplished by promoter-reporter gene fusions and/or northern blot or RT-PCR analysis coupled with in situ hybridization. Additionally, T-DNA insertions into many of the K C S genes are available. Visual and GC analyses of wax loads and compositions on these insertional mutants may reveal whether any more kcs mutants have wax phenotypes. This would be the best indicator of a role in wax biosynthesis. Finally, the different biochemical roles of the various KCSs involved in wax biosynthesis could be determined by expression of recombinant KCS enzymes in yeast and determining fatty acid profiles of the transformed yeast (in vivo assays), as well as isolating microsomes from the transformed yeast and doing in vitro assays.  6.2. C E R 7  I have used map-based cloning to identify the CERl gene. It encodes an RNAse-PHlike protein that is a putative orthologue of the yeast Rrp45p protein, a core subunit of the exosome. Exosomes in yeast are involved in R N A processing and degradation. This information, together with the wax profile of the cerl mutant, suggests that CER7 is involved in degrading a repressor or stabilizing an activator of the decarbonylation pathway. A second Rrp45p-like protein is also present in the Arabidopsis genome. Both CER7 and its homologue are expressed throughout the plant and show similar variations between tissues.  CERl,  however, is 400 bp longer than its homologue, suggesting a functional specialization. The activities and expression domains of both genes should be examined and compared, to determine whether they have redundant functions in addition to the wax-specific function of CERl. Before CERl was cloned, the only CER gene with a predictable function in regulation of wax production was CER3. It encodes a putative E3-ubiquitin ligase, and is probably involved in degradation of a wax-related protein. The cloning of CERl has provided preliminary evidence that transcript stability is also involved in the regulation of wax deposition in Arabidopsis-. Thus this project has opened a door that will allow a number of experiments to be carried out that should greatly expand our knowledge of the regulation of wax production in Arabidopsis. First, the apparent activity of the protein must be demonstrated experimentally by using it to complement a yeast deletion mutant defective in the CERl  168 homologue, or by in vitro assays. Second, the cellular and sub-cellular localizations of the CER7 protein should be established by GFP fusions and/or immunolocalization. Raising antibodies to a recombinant CER7 protein would also allow immunoprecipitation of the native protein to determine whether it is associated with other putative exosomal proteins, or whether it acts independently. Finally, the wax regulatory pathway involving CER7 can be dissected further using microarray analyses and suppressor screens to identify interacting proteins and downstream targets of CER7.  6.3. C L O N I N G A N D C H A R A C T E R I Z A T I O N O F O T H E R W A X - R E L A T E D G E N E S  Most of the genes identified by mutations that result in wax-deficient phenotypes have not yet been cloned. This represents a large genetic resource for the continuing investigation of wax production in Arabidopsis. The reverse genetics approach may yield a more predictable and tractable result in the short term, with its focus on functional characterization of homologues of already well-researched genes. Forward genetics leads to the discovery of previously uncharacterized genes and proteins whose functions may be more difficult to determine. It is clear that many genes encoding enzymes, secretory and regulatory proteins are involved in wax deposition. 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