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Genetic analysis of At4CL gene regulation and AtMyb subfamily 14 functional characterization in Arabidopsis… Soltani, Bahram M. 2006

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Genetic Analysis of At4CL Gene Regulation and AtMyb subfamily 14 Functional Characterization in Arabidopsis thaliana by Bahram M . Soltani B . S c , Ferdowsi University, Mashhad-Iran, 1990 M . S c , Tarbiat Modarres University, Tehran-Iran, 1994 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E E N T F O R T H E D E G R E E OF D O C T O R O F PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Genetics) T H E UNIVERSITY O F BRITISH C O L U M B I A A pr i l 2006 © Bahram M . Soltani, 2006 ABSTRACT Lignin is an important biopolymer that is developmentally deposited in secondary cell walls of specialized plant cells (e.g. tracheary elements and fiber cells) and also in response to stresses such as wounding. Lignin biosynthesis occurs via the phenylpropanoid pathway, in which the enzyme 4-coumarate:CoA ligase (4CL) plays a key role by catalyzing the formation of hydroxycinnamoyl-CoA esters. These esters are subsequently reduced to the corresponding monolignols. Four At4CL genes have been identified in Arabidopsis thaliana (At4CLl-At4CL4). At4CLl and At4CL2 genes are developmentally up regulated and co-expressed with other genes involved in lignin biosynthesis. Also, they are co-expressed in response to stresses such as wounding. This co-expression is probably through the engagement of common regulatory elements and cognate transcription factors such as Mybs and their recognition sites. In this thesis, I undertook three projects with the goals to identify components of the signaling pathway(s) regulating developmental expression and wound responsiveness of the At4CL genes, to localize cis regulatory elements controlling developmental and wound responsiveness of At4CLl and At4CL2 genes, and to investigate the functions of a subfamily of Arabidopsis Myb transcription factors. First, Arabidopsis transgenic lines containing At4CLl::GUS or At4CL2'.:GUS transgenes were mutagenized in order to find and map A t 4 C L signaling pathway mutants. Several lines with reproducible patterns of reduced GUS-expression. were identified. However, the GUS-expression phenotype segregated in a non-Mendelian manner in all of the identified lines. Also, GUS expression was restored by 5-azacytidine treatment suggesting DNA methylation of the transgene. Southern analysis confirmed DNA methylation of the proximal promoter sequences of the transgene only in the mutant lines. In addition, retransformation of At4CL::GUS lines with further At4CL promoter constructs resulted in a comparable GUS-silencing phenotype with higher frequency. Taken together, these results suggest that the isolated mutants are epimutants. Apparently, two specific modes of silencing were engaged in At4CLl::GUS and At4CL2::GUS (trans)genes silencing. While silencing in the seedlings of the At4CLl::GUS line was root-specific, it was global in the At4CL2::GUS line. Also, At4CLl::GUS transgene silencing was confined to the transgene but At4CL2::GUS . silencing was extended to the endogenous At4CL2 gene. In the second project, we generated a series of transgenic Arabidopsis plants containing promoter fragments and part's of the transcribed region of the At4CL2 gene fused to the GUS reporter gene, in order to localize cis regulatory elements which are involved in developmental and'wound responsiveness of this gene. We found that positive and negative regulatory elements effective in modulating developmental expression or wound responsiveness of the gene are located both in the promoter and transcribed regions of the At4CL2 gene. Also, histochemical GUS assays and molecular studies indicated a biphasic wounding response of the At4CL2 gene, attributing early or late response to distinct cw-regulatory elements involved in the response, suggesting that different signaling pathways may be involved in these different responses. In the third project, I initiated strategies to knock down/out multiple members of AtMyb subfamily #14 genes in Arabidopsis in an attempt to find phenotypes related to loss of function of these genes, since functional redundancy within the subfamily appears to. have hampered previous studies. Single AtMyb gene knock down, or knock out lines did not reveal any mutant phenotypes but RNAi generated AtMyb84 knockdown lines in the AtMyb68 knock out background showed small rosettes and a delay in shoot development. T A B L E OF C O N T E N T S A B S T R A C T ii T A B L E O F C O N T E N T S iv LIST O F T A B L E S vii LIST O F FIGURES viii LIST O F ABBREVIATIONS xiii A C K N O W L E D G E M E N T S xvi C H A P T E R 1 1 G E N E R A L I N T R O D U C T I O N 1 1.1 PLANT SIGNAL TRANSDUCTION 1 1.2 PLANT TRANSCRIPTION FACTORS__ _ _2 1.2.1 Plant Myb transcription factors_ 4 1.2.2 Methods of studying plant transcription factors '_ : ' 5 1.3 EPIGENETIC CONTROL OF GENE EXPRESSION ' 6 1.3.1 DNA methylation in plants .7 1.3.2 (Trans) gene silencing in plants . ; 9 1.3.3 Factors leading to TGS •_ . 1 11 1.3.4 Modifiers of TGS . ; • 13 1.4 THE PHENYLPROPANOID P A T H W A Y _ ' 14 1.4.1 At4CL genes • - 17 1.4.2 Control of gene expression in phenylpropanoid pathways ' 18 1.5 WOUNDING RESPONSE IN PLANTS ; 19 1.5.1 Plant stress responses are interrelated ; \ 19 1.5.2 Role of plant hormones in the wounding response • .•• 19 1.5.3 Duration of activated plant gene expression in the wounding response • 20 1.5.4 Biphasic phenylpropanoid gene expression in the wounding response 20 1.6 RESEARCH GOALS - . 22 1.6.1 Research Objectives for three research projects ___ 22 C H A P T E R 2 23 M A T E R I A L S AND M E T H O D S 23 2.1 PLANT GROWTH CONDITIONS • 23 2.1.1 General • - ' ' - 23 2.1.2 Plant material ._ 23 2.1.3 Agrobacterium and plant transformation 24 2.2 GENE EXPRESSION ANALYSIS 25 2.2.1 RNA 'gel blot analysis . : ; 25 2.2.2 Genomic Southern blots ' ' 26 2.2.3 Quantitative RT-PCR \ ; : 27 2.2.4 Histochemical GUS assays . 28 2.3 DNA SEQUENCE ANALYSIS . 28 2.4 PLASMID CONSTRUCTS 30 - iv -2.4.1 Strategies to generate At4CL::GUS plasmid constructs 30 2.4.2 Strategies for RNAi construct generation ; 32 2.4.2.1 AtMyb68, AtMyb84, and GUS RNAi constructs 32 2.4.2.2 Cloning of double RNAi constructs 35 2.5 MUTANT ANALYSIS •  36 2.5.1 Screening for GUS expression mutants . 36 2.5.2 Screening for dhlA expression mutants ' 36 2.5.3 Phenotypic and genetic analysis of putative mutants • 36 C H A P T E R 3 38 G E N E T I C ANALYSIS O F At4CL EXPRESSION A N D E P I G E N E T I C SILENCING O F 4CL EXPRESSION 38 3.1 INTRODUCTION : 38 3.2 RESULTS . _40 3.2.1 NEGATIVE SELECTION OF AT4CL SIGNALING PATHWAY MUTANTS 40 3.2.1.1 Generation of transgenic Arabidopsis lines containing 4CL::Reporter transgenes __40 3.2.1.2 Negative selection system optimizing for At4CL::dhlA lines 42 3.2.1.3 At4CL::GUS transgenes are silenced in At4CL::dhlA lines 44 3.2.2 ALTERNATIVE SCREEN FOR At4CL SIGNALING PATHWAY MUTANTS 47 3.2.2.1 Screen for mutants with reduced At4CLl-driven GUS expression 47 3.2.2.2 Phenotype analysis of putative mutants lines . 47 3.2.2.3 Genetic analysis of putative mutants lines ; : 50 3.2.2.4 Treatment of mutant lines with 5-aza ___ 52 3.2.2.5 Southern Blot Analysis of Mutant Lines_ - 54 3.2.2.6 Southern Blot Analysis of Transgene Copy Number : ' 58 3.2.2.7 Northern blot analysis of mutant lines 59 3.2.2.8 Effect of two silencing modifier mutants on 4CL::GUS transgene silencing . 61 3.3.1 At4CLl and At4CL2 promoters direct similar GUS expression patterns in the vascular tissues. ; ' 61 3.3.2 Evidence that mutants affected in A't4CL::GUS expression are epigenetically silenced epimutants. • 62 3.3.3 At4CL::GUS transgene silencing is specific and occurs naturally. 63 3.3.4 Silencing of At4CLl 1 and At4CL2 (trans) genes is best explained through a 5'-UTR threshold mechanism. . ' 64 3.3.5 Unanswered aspects of At4CLl 1 and At4CL2 (trans) gene silencing . 67 3.3.6 Conclusion , . ' 68 C H A P T E R 4 69 D E V E L O P M E N T A L AND WOUNDING RESPONSE CIS E L E M E N T S IN AT4CL2 G E N E 69 4.1 INTRODUCTION__ 69 4.2 RESULTS _ _ 71 4.2.1 Developmental and wound-induced expression patterns directed by At4CL Promoter::GUS fusions _ : r \ 71 4.2.2 Evidence for positive developmental and negative wound inducible cis-regulatory elements in .the At4CL2 promoter . , 74 4.2.3 Cis-elements that specifying developmental and wound-induced expression in the At4CL2 transcribed regions ._ 76 4.2.4 Biphasic wound induction of At4CL2 Expression •_. 78 4.3 DISCUSSION • 80 4.3.1 Differential and biphasic wound responsiveness of 4CL gene family members in Arabidopsis 80 - V -4.3.2 Multiple cis-regulatory elements are involved in developmental regulation of A.4CL2 gene expression 82 4.3.3 Multiple cis-regulatory elements are involved in modulating At4CL2 early wound responsiveness 85 4.3.4 Intronic cis-regulatory elements are involved in the late wounding response of At4CL2 gene expression. 86 4.3.5 Conclusion • 87 C H A P T E R 5 89 R E V E R S E G E N E T I C ANALYSIS O F A t M Y B S U B F A M I L Y 14 89 5.1 Introduction : 89 5.2 RESULTS 90 5.2.1 In-silico analysis of AtMyb subfamily #14 90 5.2.2 The AtMyb84 knock down line preparation 92 5.2.3 AtMyb84 knock down line preparation using a multiple arm-RNAi construct 93 5.2.4 Transformation of AtMyb68 knock out line using the AtMyb84 RNAi construct 96 5.2.5 The AtMyb84 knock out line preparation . 99 5.3 DISCUSSION 102 5.3.1 Bioinformatics data indicated high homology and overlapping expression of AtMyb68 and AtMyb84 genes. 102 5.3.2 No phenotype has been observed for AtMyb84 knock down and knock out lines. 102 5.3.3 Preliminary phenotype for double AtMyb68 - AtMyb84 knock down/out lines. 103 C H A P T E R 6 106 G E N E R A L DISCUSSION AND F U T U R E DIRECTIONS 106 6.1 At4CL::GUS TRANSGENE SILENCING 106 6.2 At4CL2 WOUND RESPONSE ELEMENTS 107 6.2 FUNCTIONS OF AtMYB SUBFAMILY 14 MEMBERS 108 R E F E R E N C E S 109 - vi -LIST O F T A B L E S Table 1-1 Sizes of major Arabidopsis transcription factor gene families in comparison to other species Page 3 Table 2.1 Sequences of PCR primers used to generate DNA hybridization probes and investigate the T-DNA insertion in the Myb84 Salk knock out line— Page 26 Table 2.2 Sequences of primers used in semi-quantitative PCR reactions. Page 27 Table 2-3 Sequences of primers used to prepare plasmid constructs and also for sequencing reactions. r — ~ Page 29 Table 2-4 Sequences of PCR primers used to prepare RNAi constructs.— Page 33 Table 3-1 Genetic analysis of 4CL1::GUS mutants Page 51 - vu -LIST O F FIGURES Figure 1-1 General Phenylpropanoid Metabolism — Page 16 Figure 1-2 Lignin biosynthetic pathway - Page 16 Figure 2.1 At4CL promoter fragments fused to G U S Page 30 Figure 2-2 At4CL promoter and transcribed region fused to G U S — —Page 31 Figure 2-3 R N A i constructs for AtMyb68, AtMyb84 and G U S in pHannibal Page 34 Figure 2-4 Double R N A i constructs for AtMyb68, AtMyb84 and G U S in pHannibal — - -- Page 35 Figure 3-1 Developmentally regulated G U S expression in At4Cll::GUS, At4CL2::GUS and At4CL3::GUS lines -- — -—Page 41 Figure 3-2 Treatment of At4CL::dhlA lines with D C E and comparison of their sensitivity to the 35S : :dhlA line. - — -- — - ------—Page 42 Figure 3-3 Semi quantitative R T - P C R in order to compare of dhlA expression levels in At4CL::dhlA lines and the 35S::dhlA line. - - —- Page 43 Figure 3-4 G U S expression in the several lines of At4CL::dhlA/At4CL::GUS transgenic plants — -Page 44 Figure 3-5 GUS expression from At4CL::GUS transgenes in At4CL::dhlA lines with or without treatment by 5-azacytidin (Aza). - — - — Page 45 - vi i i -Figure 3-6 D C E sensitivity of At4CL::dhlA lines with or without treatment by 5-aza compared with positive (35S::dhlA line) and negative (Wt.Ler) controls —Page 46 Figure 3-7 Histochemical analysis of G U S activity in representative putative At4CLl::GUS expression mutants — Page 47 Figure 3-8 Histochemical analysis of G U S activity in the 1-A putative mutant is compared with G U S activity in the progenitor background At4CLl::GUS line'.'—Page 49 Figure 3-9 Histochemical analysis of G U S activity in putative At4CL2::GUS expression mutant 2-8 is compared with G U S activity in the progenitor background At4CL2::GUS line. — - — —- — — — :— - — Page 49 Figure 3-10 Growth and development of putative mutants.— Page 50 Figure 3-11 GUS expression in At4CL::GUS mutants in the presence or absence of 5-azacytidin. — —-—Page 53 Figure 3-12 GUS expression in T5 generation seedlings of the At4CLl::GUS line in the presence and absence of 5-azacytidine. - — — - — — — r —Page 54 Figure 3-13 Southern blot analysis of methylation status of the M4CL1 promoter in mutant 1-A seedlings. — - — Page 56 Figure 3-14 Southern blot analysis of methylation status of the KX4C21 promoter in mutant 2-8 seedlings. - - — — — -Page 57 Figure 3-15 Investigation of At4CL::GUS transgene copy number in the transgenic lines. — Page 58 Figure 3-16 Northern analysis of At4CLl gene expression in At4CLl::GUS lines - ix -- - - - Page 59 Figure 3-17 Northern analysis of At4CL2 gene expression in At4CL2::GUS lines - - - - - - : - -- — -Page 60 Figure 4-1 Wounding response and developmental expression of At4Cl promoters. — — Page 72 Figure 4-2 Schematic presentation of positive cis element(s) in the 4CL2 promoter — — - —- -- Page 73 Figure 4-3 Developmental and wounding response expression of 950bp At4CL2 promoter fused to GUS Page 75 Figure 4-4 Schematic presentation of the At4CL2 gene showing the deduced locations of wound responsive elements Page 76 Figure 4-5 Developmental and wound-induced expression in transgenic lines containing 950-pb At4CL2 promoter and different transcribed regions fused to GUS. Page 77 Figure 4-6 Developmental GUS expression in mature leaves of nine independent At4CL2 cDNA:: GUS lines Page 78 Figure 4-7 Wounding response of At4CL genes analyzed by semi-quantitative RT-PCR--Page 79 Fig 4-8 Summary of deduced locations of positive and negative regulatory elements in the At4CL2 gene affecting developmentally regulated expression. Page 84 Figure 4-9 Summary of deduced locations of positive and negative regulatory elements in the At4CL2 gene affecting early wound inducible expression. Page 86 - x -Figure 4-10 Schematic presentation of the locations of putative regulatory elements in the At4CL2 gene Page 88 Figure 5-1 Alignment of AtMyb subfamily #14 members and subfamily #14 phylogenetictree. Page 92 Figure 5-2 RT-PCR on AtMyb84 RNAi knock down lines Page 93 Figure 5-3 Seedling growth and morphology of AtMyb84-KNAi knock down lines, compared with wild type (Col) plants. Page 93 Figure 5-4 Morphology of rosette stage AtMyb68 and AiMyb84 knock out lines compared to wild type plant. Page 94 Figure 5-5 Seedling growth and morphology of AtMyb84 RNAi knock down lines in the AtMyb68 knock out background. Page 95 Figure 5-6 Rosette stage growth and development of four Myb84 RNAi lines in the Myb68 knock out background. — Page 95 Figure 5-7 Rosettes with emerging primary shoot in seven Myb84 RNAi lines in the Myb68 knock out background. Page 96 Figure 5-8 GUS expression in transgenic plants generated by transformation of a 35S::GUS line with a Myb84+GUS double RNAi construct. Page 96 Figure 5-9 RT-PCR analysis of AtMYB84 expression in Myb84+GX)S double RNAi lines. : — Page 97 - xi -Figure 5-10 Seedling growth and morphology of transgenic lines containing double RNAi AtMyb68 +AtMyb84 constructs. Page 98 Figure 5-11 Seedling growth and morphology of F2 generation plants (10 days old) derived from a cross between Myb84 Myb68 knock out lines Page 99 Figure 5-12 Rosette stage growth and morphology of selected F2 generation plants derived from a cross between Myb84 Myb68 knock out lines. Page 100 Figure 5-13 Schematic of T-DNA insertion within AtMyb84 knock out lines from Salk collection. - — Page 101 Figure 5-14 Comparison of AtMyb68 and AtMyb84 knock out lines and wild type plant.— . . . —Page 102 - xii -LIST OF ABBREVIATIONS ACC synthase Aminocyclopropane-1 -carboxylate synthase Ago4 Argonaute4 AP2 APETALA2 ARF Auxin response factors At Arabidopsis thaliana AtPTR3 peptide transporter bHLH helix-loop-helix BCAT5 branched-chain amino acid transaminase 5 CAD Cinnamyl alcohol dehydrogenase CaMV3 Cauliflower mosaic virus CCOMT Caffeoyl-CoA 3-O-methyltransferase C4H Cinnamate 4-hydroxylase CHS Chalcone synthase CMT3-DMTase Chromo-domain containing methyltransferase) Col Arabidopsis Columbia ecotype DCE 1,2-dicholoroethane Dcl3 Dicer-Uke3 DDM1 Deficient in DNA Methylation 1 dhlk Dehalogenase A DMT DNA methyltransferase DRM-DMTases Domains Rearranged Methyltransferases dsRNA Double stranded RNA EMS Ethyl-Methyl- Sulfonate EREBP Ethylene responsive binding element protein ERF1 Ethylene Response Factor! 5-aza 5-azzacytidine 4CL 4-coumarate::CoA ligase 5mC Cytosine methylation GFP Green Florescence Protein - xiii -GUS B-glucuronidase HDGS homology dependent gene silencing HB Homeobox JAs Jasmonic acids Kan Kanamycin Kb, Kilo base LB Luria Bertani Broth Ler Landsberg erecta Arabidopsis ecotype LWRE Late Wound Response Element MAPK Mitogen Activated Protein Kinase METl-DMTs DNA Methylase Metl M O M 1 Morpheus' Molecule 1 MOPS 3-(N-Morpholino)propanesulfonic acid MPSS Massive Parallel Signature Sequencing MS Murashige and Skoog NAPS Nucleic acid and protein Service Unit at UBC NPT Neomycin Phosphotransferase ORF Open Reading Frame PAL Phenylalanine ammonia-lyase PCR polymerase chain reaction PLACE Plant cis-acting regulatory DNA elements PTGS Post transcriptional gene silencing RdDM RNA directed DNA Methylation RdRp RNA Dependent RNA Polymerase rdr2 RNA-dependent RNA polymerase2 RNAi RNA interference RT Reverse transcription SAIL collection Syngenta Arabidopsis Insertion Library SDS Sodium Dodecyl Sulfate siRNAs Short inhibitory RNAs STP4 Sugar transporter - xiv -SSC Standard Saline Citrate Buffer SUP SUPERMAN gene TBE Tris, Boric Acid, EDTA T-DNA transferred -DNA TGS Transcriptional gene silencing 3 L E 4CL3 like element UTR Un-translated Region W P K 1 Phytochrome-Regulated kinase! X-gluc 5-bromo-4-chloro-3-indolyl-B-D-glucuronide Zm ZEA mays - xv -ACKNOWLEDGEMENTS The work described here was achieved with the abundant endeavor, encouragement and support from my friends, mentors and families. M y supervisor, Dr. Carl Douglas, supported me throughout these years. His enormous patience and teaching skills made him unique in my mind. He never disagreed with all kinds of support that I asked for. - M y committee members, Drs. George Haughn, James Kronstad and Joerg Bohlmann continually supported me to complete my degree. I would like to specially thank Dr. Haughn for all o f his precious intellectual supports. - M y sincere gratitude goes to all present and past fellow lab members who have advised, encouraged and helped me throughout these years. In particular, many scientific conversations with Juergen and Bjorn made my research attitude different. I thank Dae-Kyun, Lee, A l i Samaeian, Zahra Kazemi and Nahid Delroba who helped me do all these experiments. I was so lucky to have lab and department members who were friendly and will ing to help and devote whatever they could. Ravi , Owen, Shelly, Dave, June, Eryang and Michael were awesome. - I also thank Drs. A n n Rose, Carolyn Brown, Tony Griffith, Tom Grigliatti, Craig Berezowsky and Elaine Humphrey for their diverse support. - I thank Tarbiat Modarres University and ministry of Science & Technology officials who gave me 4 years scholarship to do my Ph.D. - To start my Master and Ph.D program many reliable friends helped me and I thank all of them specially, Azizol lah Dabbaghi, Majid Zehtab, Ramazan Hajheidari, Hassan Hajhashemi, Mehdi Hajjarzadeh, Gholamali. Hajheidari, Mohammad Taheri and Dr. Mohammad Malakooti. - M y father, mother, sisters and brothers stayed close and spiritually supported me all these years specially, Mohammad who has been supportive all the time. - M y wife Aram and kids, who are my number one and brought me joy through their enormous trust, patience, support and love for the past 10 years. - xvi -To all who taught me To my family - x v i i -C H A P T E R 1 G E N E R A L I N T R O D U C T I O N 1.1 P L A N T SIGNAL T R A N S D U C T I O N Plants respond to a large number of developmental and environmental signals through changes in gene expression. This requires that signals reach the nucleus in order to regulate the expression of target genes. First, perception of the signal by an appropriate receptor molecule is necessary and then the signal will be transduced to the nucleus via intermediates, often targeting transcription factors. The cell membrane separates the protoplasm of the cell from its surrounding environment and presents a barrier to many signals that may alter gene expression. Only small lipophilic molecules such as steroid hormones are able to diffuse into the cytoplasm but the cell membrane is impermeable to large water-soluble molecules. Therefore, responses of the cell to extra-cellular hydrophilic signaling molecules (ligands) is often through the specific interaction of these ligands and the extra-cellular domain of plasma membrane receptor proteins. Receptors may physically transport the ligand inside the cell or binding of the ligands may convert the receptor from an inactive to an active form or vice versa and activate or inactivate its cytosolic domain. The process, from signal perception to target gene activation, is called signal transduction (Lewin 2004). After signal perception by an extra-cellular or intracellular receptor, the signal may be propagated through a number of different signal transduction pathways. A common pathway relies on the activation of serial protein kinases of the Mitogen Activated Protein Kinase (MAPK) class (Buchanan et al, 2000). The signal leads to the activation of effectors, some of which may act in the cytosol (for example to affect-the cytoskeleton), and some may carry the signal into the nucleus and affect activity of the transcription factors. Ultimately, a given transcription factor interacts with a specific DNA sequence in the promoter of target gene and increases or decreases the expression level of the target gene (Lewin 2004). To elucidate the molecular mechanisms underlying several signaling pathways in plants, researchers have utilized the genetically facile plant Arabidopsis thaliana to isolate mutants that confer altered responses to various stimuli. An example of a well -characterized signal transduction pathway in Arabidopsis analyzed through, mutant analysis is the ethylene-signaling pathway. Ethylene is a gaseous plant hormone that affects many developmental and stress processes such as germination, senescence, fruit ripening, and pathogen response (Bleecker 2000). Ethylene (the ligand) interacts with a specific membrane-associated receptor resulting in activation o f ethylene responses by inhibiting a negative regulator of the response. In the absence o f ethylene this receptor is functionally active and constitutively activates a serine/threonine (Ser/Thr) kinase, which in turn is a negative regulator of ethylene responses (Wolanin et al, 2002: Potuschak et al, 2003). Downstream of this negative regulatory kinase are several positive regulators of ethylene responses, which are not all fully characterized. One of these positive regulators is a transcription factor that controls the expression of its immediate target genes such as Ethylene Response Factorl (ERF1) (Solano et al, 1998). E R F 1 is itself a transcription factor that binds to a GCC-box present in the promoters of many ethylene-inducible, defense-related genes (Guo et al, 2004). Thus, in the presence of.ethylene, a signal transduction pathway is activated that relies on the repression of the negative regulator of the pathway, allowing ethylene induced gene expression to occur. 1.2 PLANT TRANSCRIPTION FACTORS The R N A polymerase II complex alone is only able to catalyze transcription at a very low (basal) level. Transcription at higher rates requires that other proteins such as transcription factors bind to the c/s-regulatory elements in the D N A around the gene. Plant transcription factors are modular proteins typically composed o f D N A binding domain and effector domains that regulate the frequency of transcription o f target gene(s) (Pabo et al, 1992: L i u et al, 1999). In eukaryotes, multi-protein complexes that mediate gene expression are commonly formed through the combinatorial action of transcription factors and co-activators. Complexes are bound to the conserved promoter elements in precise spatial orientations and on the basis of both specific protein-DNA and protein-protein interactions (Griffiths et al, 2004). A fraction of all genes in the sequenced eukaryotic genomes, encode transcription factors. According to the sequence of their D N A binding domain, the majority of transcription factors can be assigned to specific families (Pabo et al, 1992, Ulker et al, 2004) (Table 1-1). Table 1.1 Sizes of'major Arabidopsis transcription factor gene families in comparison to other species (according to Riechmann et al, 2000) Predicted Gene Number Gene Family Arabidopsis Drosophila C, elegans S.cerevisiae M y b -130 35 16 19 A P 2 / E R E B P 150 0 0 0 N A C 105 0 0 0 b H L H / M Y C 100 61 38 8 bZIP 100 24 18 15 H D 90 113 88 10 Z - C 2 H 2 85 352 138 47 M A D S 80 2 2 4 W R K Y 75 0 0 0 A R F 42 0 0 0 Dof 41 0 0 0 More than 1,600 putative transcription factor genes have been identified in the Arabidopsis thaliana genome. This represents about 6% of the total -26,000 Arabidopsis genes (Riechmann et al, 2000). Some of the transcription factor families (e.g Mybs, bZIPs and Homeodomain) are found in plants, animals and fungi. Transcription factor families such as A R F (auxin response factors) are plant specific (Rubin et al, 2000). 1.2.1 Plant Myb transcription factors The first M y b transcription factor gene was identified as a chicken oncogene derived from the avian myeloblastoma virus and was called v-Myb (Klempnauer et al, 1982). V-. Myb is a truncated version of c-Myb, which is well-conserved gene in all vertebrates examined to date (Weston et al, 1998). The plant M y b transcription factors were first discovered by similarity of their D N A binding domain to v -Myb . The presence of a M y b binding domain is the common feature of all M y b proteins that is conserved amongst animals, plants and yeasts (Lipsick et al, 1996). The M y b domain typically consist of one to three imperfect repeats called R l , R2 and R3. However, R2 and R3 repeats alone are necessary and sufficient for sequence-specific D N A binding (Howe et al, 1990; Saikumar et al, 1990; Gabrielsen et al, 1991). Each repeat is about 53 amino acids long and contains three helices which form a helix helix-turn-helix structure with the second and third helix forming the helix-turn-helix (HTH) DNA-binding structure (Frampton et al, 1991). Although M y b proteins are common to all the characterized eukaryotes, in higher plants this protein family is extraordinarily amplified. In contrast to animals, most plant Myb genes belong to the R2R3-Myb category and about 126 putative R2R3-type Myb genes in the Arabidopsis genome have been identified, which are classified in 22 phylogenetic subfamilies (Stracke et al, 2001). Plant Mybs are involved in a variety of cellular processes such as phenylpropanoid (Sablowski et al, 1994) or tryptophan biosynthesis (Stracke et al, 2001) and regulation of the phosphate starvation response (Rubio et al, 2001). Also they are involved in control of cell fate determination, regulation of the cell cycle and circadian clock-regulated gene expression (Meissner et al, 1999; Martin et al, 1997: Jin et al, 1999: Zimmermann et al, 2004). There are also reports of Mybs that function with other transcription factors such as B H L H and BZIP by protein-protein interaction in controlling flavonoid biosynthesis (Hartmann et al, 2005). Thus, Mybs form a relatively large family of plant transcription factors with potential roles for controlling gene expression, some of which have been shown to be important in regulating the phenylpropanoid bio synthetic pathway (Jin et al, 2000). 1.2.2 Methods of studying plant transcription factors After sequencing of the Arabidopsis thaliana genome, a variety of reverse genetic approaches have been used to analyze the function of genes with unknown specific functions. Creation of large populations of mutants by either T - D N A insertion or transposon tagging is a well-known method (Maes et al, 1999; Walbot et al, 2000; Krysan et al, 1999). Originally, these populations were used to screen for insertion mutations in a particular gene using a PCR-based strategy with a gene specific primer in combination with T - D N A (or transposon) border primers. Today, transposon and T - D N A collections such as the collections generated at the Salk Institution (http://signal.salk.edu/tabout.html) are compiled in databases with bordering plant D N A sequences, which provide individual mutant lines with T - D N A insertions in genes of interest to researchers. R N A interference ( R N A i ) naturally occurs in almost all the eukaryotes examined to date, including plants in which its biochemistry has been characterized (Tang et al, 2003; Matthew 2004). R N A i in plants has multiple consequences such as transcriptional and post-transcriptional gene silencing through R N A degradation and/or D N A methylation respectively (Bender 2004). R N A i has been frequently used as an alternative method to generate loss-of-function mutants for specific plant genes (Abbott et al, 2002; Zamore et al, 2002; Tomari et al, 2005). For example, R N A i has been used to down regulate the tomato Blind gene that encodes a M y b transcription factor. In the blind mutant of tomato, initiation of lateral meristems during the shoot and inflorescence development is blocked leading to reduction of the number of lateral axes, which is manifested in reduction of shoot and inflorescence branching. Using positional cloning, the Blind gene was isolated and it was shown that encodes an R2R3 class Myb gene. R N A i induced blind phenocopies confirmed the identity of the isolated gene. In this experiment 17 out of 19 independent R N A i transgenic plants showed reduction in the number of lateral shoots and the number of flower per inflorescence (Schmitz et al, 2002). In order to generate R N A i silenced lines, a D N A construct containing complementary 150-500 bp sense and antisense inserts which are separated by an artificial intron is I i typically made, and introduced into the plant genome (Helliwell et al., 2003; Mattew et a/., 2004). Transcription of this construct results in self-complementary double stranded RNA (dsRNA). Presence of the dsRNA activates the RNAi silencing system in which dsRNA is recognized and specifically cleaved by the Dicer enzyme to give small siRNAs of 21-26 nucleotides. These small siRNAs then generate short single stranded RNAs that hybridize to the target RNAs or DNA by sequence complementarity and direct a protein complex to cleave target mRNAs or methylate DNA (Bender 2004; Gendrel et al., 2005). For some genes without inverted sequences it is accepted that increased transcript copy number may trigger dsRNA formation through the function of known RNA dependent RNA polymerases and activate RNAi machinery (Shubert et al., 2004). [ RNAi transformants usually exhibit mutant phenotypes at a much higher frequency than the plants transformed with either the antisense or the sense gene alone (Chuang et al., 2000). However, as with the use of other loss-of-function approaches, a gene that shares function with related genes is difficult to functionally characterize unless potential redundant genes are knocked -out or -down at the same time (Zhang 2003). Overall, interruption of gene expression by RNAi has been often utilized in plant genetics. This method provides the flexibility necessary for the characterization of genes of diverse function and complements the T-DNA insertion reverse genetics approach. i 1.3 EPIGENETIC CONTROL OF GENE EXPRESSION The process of transcription is affected by a number of different factors including pre-initiation and RNA polymerase II complex formation, transcription factor binding and by factors required for chromatin remodeling (Griffiths et al., 2004). As a result of histone modification and/or DNA methylation chromatin structure may change and result in gene silencing (Fagard et al., 2000). Also, in many organisms including plants, different kinds of RNAs are involved in chromatin alteration and gene silencing (Gendrel et al, 2005; Bender 2004). The study of chromatin change and its effect on inheritance is generally equated with epigenetics (Henikoff et al, 2004; Gendrel et al, 2005), which is defined as the study of mitotically and/or meiotically heritable changes of gene expression without any change of DNA sequence (Wu et al, 2001). Also, epigenetics is defined as the memory of transcriptional activity, which is regulated through the binding of specific -6-chromosomal proteins and the covalent modification of chromatin (Lippman et al, 2005). Heterochromatin is the condensed area of chromatin and is generally considered as transcriptionally inactive or silenced, whereas euchromatin represents the transcriptionally active form of chromatin. As well as DNA methylation, a variety of post-translational modifications of histones such as acetylation, phosphorylation and ubiquitylation have been reported. during chromatin modification. However, only methylation of DNA has been reported so far to directly result in silencing (Jenuwein et al, 2001). The level of DNA methylation, diverse covalent modifications of histones, histone variants and specific associations with noh-histone proteins are different between hetero- and euchromatin (Richards et al, 2002). The functional relationship between plant DNA-methylation and histone modifications that lead to chromatin compaction and gene silencing is still under investigation (Tariq et al, 2004). There are also reports indicating that RNAi silencing machinery is involved in chromatin remodeling. Three pathways of RNAi silencing have been considered in recent reports; two pathways target mRNAs for either degradation or translational repression and the third pathway is chromatin-based in several organisms including Arabidopsis, ending in DNA methylation and histone- modifications (Lippman 2004,. Gendrel 2005). In summary, in many j organisms heterochromatin is silenced by conserved mechanisms of epigenetic modification of histones and DNA. This epigenetic silencing, as well as higher order of packing of chromatin into heterochromatin is believed to prevent illegitimate and harmful recombination and transposition and deleterious over-expression of genes (Lippman 2004). 1.3.1 DNA methylation in plants DNA methylation is widespread among plants and vertebrates and is widely considered as the mechanism for defending genomes against selfish DNA sequences such as transposable elements and retroviruses (Bird 2002; Martienssen et al, 2001; Chan et al, 2005). It has been shown that transposons are mobilized as a result of reduction in the level of DNA methylation in Arabidopsis genome (Miura et dl, 2001). Plants also use DNA methylation to modulate the expression of repeated gene families (Lawrence et al, 2004). DNA methylation regulates gene expression through inhibition of transcription - 7 -initiation or arresting transcription elongation, acts as an imprinting signal and suppresses recombination between homologous D N A molecules (Colot et al, 1999; Chan et al, 2005). Cytosine methylation (5mC) is always present in the transcriptionally silent chromatin in plants (Bird 2002). Cytosine methylation in symmetrical C G dinucleotide sites, is an evolutionarily conserved D N A modification in vertebrates, plants and some fungi (Bird 2002; Finnegan et al, 2000). As well as preferred symmetrical C G sites (Bender 2004), plants show significant levels of cytosine methylation in non-CG sequences, which include symmetrical C N G and asymmetric C N N sequences (where N = A, T or G) (Finnegan et al, 1994; Gendrel et al, 2005; Chan et al, 2005). D N A methyltransferase (DMT) enzymes are responsible for D N A methylation and are conserved among protists, plants, fungi and animals (Colot et al, 1999). In the Arabidopsis genome at least 10 of these genes have been found and some of them are conserved with animal genes (Gendrel 2005). These genes are categorized into three types of DMTases, two of which are plant specific (Bender 2004). The plant specific DRM-DMTases (Domains Rearranged Methyltransferases) family are involved in methylation of asymmetric cytosines. The plant specific CMT3-DMTase (Chromo-domain containing methyltransferase) maintains methylation of C N G cytosines, and METl-DMTase is related to its mammalian counterpart and maintains methylation of C G sites (Wada et al, 2003; Cao et al, 2002; Cao et al, 2003). Many of these DMTases are engaged in R N A directed D N A Methylation (RdDM) (Bender 2004; Gendrel et al, 2005). In this RNAi system, dsRNA, from different sources such as viroids, cytoplasmic viruses, transcribed inverted repeats, and overabundant or aberrant RNAs from genes or transgenes will be cleaved into 21-26 nucleotide small interfering RNAs (siRNA). These siRNA molecules may guide D N A methylation to homologous sequences. In other words, D N A methylation is one of several downstream mechanisms that siRNA can use to down-regulate gene expression during transcriptional gene silencing (Chan et al, 2005; Bender 2004; Gendrel et al, 2005). In some cases of transposon and transgene silencing in plants, a connection between R N A i and D N A plus histone methylation has been established based on several kinds of evidence. First, it was reported that RNA-induced silencing in plants often -8-! results in DNA methylation (Wasseneger et al, 1994). Then,.in Arabidopsis and tobacco, it was shown that dsRNAs targeting the promoter of transgenes induce DNA methylation and transcriptional silencing (Mette et al, 2000). Finally, it has been shown that, members of conserved gene families such as Argonaute4 (ago4), dicer-like3 (dc!3), and RNA-dependent RNA polymerase2 (rdr2), are responsible for the initiation of silencing on the transcriptional level in plants (Zilberman et al, 2003; Chan et al, 2004; Xie et al, 2004). In conclusion, different cytosine residues in the DNA are methylated by different DMTases. Also, the RNAi system apparently specifies methylation of certain parts of the genome by sequence complementarity and engaging/guiding DMTases. 1.3.2 (Trans) gene silencing in plants It seems that there is a common theme for the silencing of developmental genes, viroids and viral DNAs, transgenes and transposons. Silencing of developmental genes plays an important role during development. For example the SUPERMAN (SUP) gene of Arabidopsis encodes a transcription factor and is expressed during flower development (Sakai et al, 2000). Epigenetic mutant alleles of SUP have been described1, in which the SUP transcribed region becomes heavily methylated (Ito et al, 2003). The flower of wild type Arabidopsis contains six stamens (male reproductive organs) and two fused central carpels (female reproductive structure). Mutations in the SUPERMAN gene (sup-5 mutants for example) increase the number of stamens to 12 and carpels to 3 on average (Jacobsen et al, 1997: Schultz et al, 1991; Bowman et al, 1992). Several heritable but unstable sup epi-alleles have also been recognized in which the level of SUP RNA is reduced. In these epimutants, cytosines in a specific area within the SUP gene are hyper methylated. Epimutants show a similar phenotype to, but weaker than, that of sup mutants with an average of 8 stamens and 3 carpels. Demethylation of the SUP gene is associated with the reversion of epialleles and restores the levels of SUP RNA (Jacobsen etal, 1997). Silencing of transposons is important for the protection of genome integrity. For example, the sequence of maize (Zea mays) is over 30 times of the size of Arabidopsis, and 83% of it consists of transposable elements. The much smaller Arabidopsis genome - 9 -has a limited number of transposable elements (Bevan et al, 1998). To control the activity of transposable elements, Arabidopsis and maize have silenced transcription of the majority of these elements via methylation (Martienssen 1998). Plants also use gene silencing as a mechanism to defend their genomes against viruses (Ratcliff et al, '1997, Covey et al, 1997). It is known that plant recovery from viral disease involves viral R N A silencing through viral d s R N A degradation (Baulcombe 2004). Often, the expression level of the same transgene construct varies in between independent lines, and this variation can sometimes be attributed to gene silencing, especially, when the transgene has been active for a few generations and then is epigenetically inactivated (Fagard 2000). This suggests that transgenes can be perceived as plant genome invaders and that plant defensive methylation systems are activated in response to the introduction of transgenes (Matzke et al, 1998). Two possible molecular mechanisms have been considered for gene and transgene silencing: Transcriptional gene silencing (TGS) which blocks transcription of the (trans) gene and post transcriptional gene silencing (PTGS) which involves degradation of the R N A product of the (trans) gene (De Carvalho et al, 1992; Meyer et al, 1993; Fagard i 2000). PTGS, which is initiated during the development, may result in systemic silencing of the transgene but after meiosis, it is reset. T G S on the other hand, is both meiotically and mitotically heritable although, reversion of transgene expression is possible (Schubert et al, 2004). In Arabidopsis, T G S is mainly correlated with the methylation of the promoter (Kumpatla et al, 1997; Chandler et al, 2001) whereas in P T G S , mainly coding sequences are methylated (Vaucheret 1998; Stam et al, 1998; Chandler et al, 2001; Fagard 2000; Schubert et al, 2004). It is not clear whether methylation is a cause or a consequence of silencing (Fagard et al, 2000). Results gathered from the impairment of two nuclear proteins, which are involved in T G S suggests that T G S could operate in methylation-dependent or -independent pathways (Chandler et al, 2001). Impairment of D D M 1 , a chromatin remodeling protein, results in the release of both T G S and methylation of transgenes and silent retrotransposons (Jeddeloh et al, 1999), while the impairment of another novel protein called M O M 1 only releases T G S but not the methylation of transgenes (Amedeo et al, 2000). - 10-Researchers have suggested several models to explain T G S and P T G S . In one model, T G S occurs when there are identical sequences to the newly introduced promoter of the transgene in the genome, while P T G S occurs when the transcribed region of the transgene shows sequence identity with another sequence in the genome (Dudley 2003). In a favored RNA-mediated silencing model, the common theme is that silencing is triggered by the formation of d sRNA molecules. If d s R N A is homologous to the promoter sequence of the transgene, T G S may occur and i f it is homologous to the transcribed region, P T G S may occur (Chandler et al, 2001). In support of this model, s i R N A molecules matched with transcribed region of the transgene have been found which are considered as hallmark of P T G S (Hamilton et al, 1999). Also , discovery of s i R N A specific for the promoter sequence of the transgene correlates T G S with R N A i systems (Mette et al, 2000; Sijen et al, 2001). Double stranded R N A s , which are required in R N A i based silencing system, could be formed through the transcription from the inverted repeat sequences (Waterhouse et al, 1998) or through the function of R N A Dependent R N A Polymerase (RdRp) enzymes on aberrant or over abundant R N A s (Gendrel et al, 2005). D s R N A molecules in turn, trigger the R N A i system and finally methylation of the transgene (Chandler et al, 2001; Gendrel et al, 2005). If T G S only affects the transgene itself and not the unlinked homologous endogenous sequences, it is called m-si lencing and i f silencing affects both transgene1 and homologous D N A sequences simultaneously, it is called cis and trans silencing. Also , i f T G S affects the homologous D N A but not the transgene it is called trans silencing (Fagard et al, 2000). 1.3.3 Factors leading to T G S Several factors are known to be involved in T G S in eukaryotes. The expression of an active gene in the euchromatin may be affected by the presence of heterochromatin in the vicinity. This is manifested as mosaic or variegated expression of an active gene as seen in Drosophila melanogaster (Wakimoto 1998). In plants it has been proposed that the variability of the level of transgene expression among the transformants could be attributed to the integration position of transgenes in different lines (Jones et al, 1985; Peach and Velten 1991; Day et al, 2000). That means that proximity of the transgene to heterochromatin (Pf'ols et al, 1992) or methylated repetitive elements (Lohuis M , et al, 1995) may lead to transgene silencing simply by the position of integration/Also, integration of multiple copies of a transgene in a particular spatial arrangement may lead to methylation and T G S (Davies et al, 1997; Mittelsten et al, 1998). Based on these reports, a chromatin-level silencing model has been suggested in which repressive chromatin may spread from adjacent sequences into the transgene. Then, the silenced transgene may pass the silencing state to another homologous transgene or to endogenous genes, which are linked to or independent from original silenced transgene (Chandler et al, 2001). Consistent with this model it has been documented i n Arabidopsis and tobacco transgenic plants harboring C a M V 3 5 S promoter::GUS constructs that different transgenic lines show several hundred fold G U S expression level differences (Hobbs et al, 1990; Holtorf et al, 1995). In spite of these notions, recent research in Arabidopsis has assessed the influence of the integration position of 35S: :GUS transgene and no significant transgene expression differences between these lines in different positions of integration was reported. Also in the same report, stable expression was observed for all the lines in which the weaker nopaline synthase promoter was fused to N P T (Neomycin Phospho- Transferase) that confers kanamycin resistance (Schubert et al, 2004)'. Silencing has frequently been associated with repetitive T - D N A insertions in the transgenic plants (Muskens et al, 2000). On the other hand, it is documented that tandem arrangement of T - D N A inserts and / or inverted T - D N A repeat configurations, were not sufficient to trigger transgene silencing (Lechtenberg et al, 2003, Schubert et al, 2004). There has been a favored idea that silencing is triggered by a threshold concentration of a specific transgene transcript (Lindbo et al, 1993). In agreement with this idea', the frequency of silencing has been positively correlated with the promoter strength of transgenes (Que et al, 1997). Also, homozygous transgenic plant lines show stronger silencing than hemizygous plants (Vaucheret et al, 1998). In the most recent report, for less than a certain number of transgene copies in the genome, a positive correlation between copy number and gene expression was observed while more than a certain number of identical transgene copies triggered gene silencing (Schubert et al, 2004). Overall, position effects, repetitive elements and repeat arrangements of T - D N A s do not - 12-account for all the variability of transgene expression observed in Arabidopsis and other plants, and a key trigger for silencing appears to be the surpassing of a threshold level of gene expression. The nature of the transgene coding sequence apparently determines the gene specific threshold for silencing (Schubert et al, 2004). 1.3.4 Modifiers of TGS Transgenic Arabidopsis plants containing extra copies of the Chalcone synthase (CHS) gene have shown homology dependent CHS silencing (Davies et al, 1997). Mutagenesis of these transgenic plants and screening for modifiers of CHS (trans) gene silencing resulted in the isolation of mutants such as hogl and sill. These silencing modifiers were able to restore the expression of silenced CHS (trans) gene plus kanamycin (npt) and hygromycin (hpt) resistant genes on the same T - D N A (Furner et al, 1998). H o g l is a partial loss of function mutation in the S-adenosyl homocysteine hydrolase enzyme that generally inhibits trans-methylation metabolism (Rocha et al, 2005) In the hogl-1 mutant, CHS, npt and hpt transgenes are reactivated) and hypomethylated. The s i l l mutation is an allele of H D A 6 (histone deacetylase), which has been found to confer partial demethylation of C G sequences at specific regions of the genome (Probst et al, 2004)._In the sill-1 mutant, the functions o f transgenes are also restored but transgenes are not hypomethylated, very similar to a nonallelic mutant called moml, which is involved in silencing, probably through chromatin remodeling (Amedeo et al, 2000). Introduction of such mutant alleles into transgenic plants with silenced transgenes may reveal the nature of transgene silencing in those plants. - 13 -1.4 THE PHENYLPROPANOID PATHWAY The phenylpropanoid pathway is required for the biosynthesis of a series of natural products based on a phenylpropane skeleton derived from L-phenylalanine. The flow of carbon from primary metabolism into an array of secondary phenylpropanoid products is through the general phenylpropanoid pathway (Figure 1-1). Specific branch pathways derive their basic phenylpropanoid unit from this core series of reactions (Hahlbrock and Scheel 1989; Douglas et al, 1996). The functional significance of phenylpropanoid compounds in plants has been further corroborated through genetic and molecular analysis of mutants that are defective in phenylpropanoid regulatory or structural genes (e.g., Jin et al, 2000; Landry et al, 1995), analysis of phenotypes of transgenic plants with altered expression of key phenylpropanoid structural or. regulatory genes (e.g., Elkind et al, 1990; Tamagnone et al, 1998), and through generation of transgenic plants engineered for novel phenolic pathways (Hain et al, 1993; Mayer et al, 2001). The general part of this pathway is composed of three enzymes, phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate::CoA ligase (4CL). PAL is a tetrameric enzyme that catalyzes the first step, the de-amination of L-phenylalanine to produce cinnamic acid (Bolweil et al, 1986). Cinnamic acid is then hydroxylated at the para-position by C4H, a cytochrome P450 mono-oxygenase (Teutsch et al, 1990). The product of C4H is p-coumarate, which is further modified by other hydroxylases and O-methyltransferase to generate other derivatives of cinnamic acid such as caffeic acid, ferulic acid and sinapic acid. The enzyme 4CL catalyzes the formation of CoA esters of hydroxy-cinnamic acids, and these activated intermediates serve as the substrates for specific branch pathways, such as those leading to the synthesis of flavonoids and lignin (Noel et al, 2005). In the lignin biosynthetic pathway, the p-coumaryl-CoA substrate is converted to three types of lignin monomers; 4-coumaryl, coniferyl, and sinapyl alcohol which constitute jc-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) moieties of lignin, respectively (Fig 1-2). In this pathway, enzymes such as coumaryl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) and caffeoyl-CoA 3-O-methyltransferase (CCOMT) are involved (Peter et al, 2004). The composition of three lignin monomers in - 14-i lignin varies between species, at different developmental stages or in response to stresses (Lewis and Yamamoto, 1990). Lignin provides mechanical strength and hydrophobicity to the cell wall. Biosynthesis and deposition of lignin is developmentally regulated, and occurs in tracheary elements, vessels and fibers during the xylem differentiation (Peter et al, 2004). Lignin synthesis is also induced in response to environmental stresses such as wounding and pathogenic infection. Deposition of flavonoid pigments in plant organs also occurs in tissue and cell type specific manner, such as in the epidermal cells of petals and in the aleurone layer of maize kernels. Free phenolics acids, or their conjugates and esters, are 'other phenylpropanoid products that in combination with flavonoid pigments act as efficient sunscreens and antioxidant chemicals in epidermal cell layers of leaves. These compounds protect plants against damaging UV light (Landry et al, 1995; Li et al, 1993). The biosynthesis of phenylpropanoid compounds is also induced upon environmental stimuli such as wounding, pathogen infection, and UV irradiation (Hahlbrock and Scheel, 1989; Douglas, et al, 1992; Dixon and Paiva, 1995), After .wounding, the accumulation of lignin may seal off the wound sites to protect the plants from the loss of water and from pathogenic infection. Evolution of lignin biosynthesis is thought to have been a major adaptation that allowed vascular plants to successfully colonize the terrestrial environment (Whetten and Sederoff 1995). Genes encoding the enzymes in the general phenylpropanoid pathway and several branch pathways have been isolated from many plant species (Dixon and Harrison, -1990 Raes et al, 2003). In Arabidopsis thaliana, four genes have been annotated as members of the AXPAL gene family and AXPAL1, 2 and 4 have demonstrated de-amination activity (Cochrane et al, 2004). Only one AtC4H gene is present in the Arabidopsis genome (Mizutani et al, 1997; Urban et al, 1997; Raes et al, 2003), although multiple family members have been detected in other plants (Betz et al, 2001). - 15-FLAVONOID COUMARIN SOLUBLE ESTER General Phenylpropanoid Metabolism C4H ^ 4CL Clnnamicacid p-coumarlcacid 4-Coumaroyl CoA SUBERIN LIGNIN OTHER WALL-BOUND PHENOLICS — • Figure 1-1 General Phenylpropanoid Metabolism (according to Hahlbrock et al, 1989). Thicker arrows indicate branch pathways emanating from the general pathway. P A L , phenylalanine ammonia-lyase; C 4 H , cinnamate 4-hydroxylase; 4 C L , 4-coumarate:CoA ligase. Phenyl- Cinnamate alanine C4H p-Coumarate 4CL I H C T / C S T P-Coumaroyi C3H HCT/CST C a f f e o y | p-Coumaroyl >- shlkimate *- shiklmate »- CoA CoA quinate quinate i CCR p-Coumar-aldehyde CAD p-Courmaryl alcohol H lignin CCaOMT Feruloyl CoA CCR F5H/ ' Cald5H 5-Hvdroxv- COMT C o m f e r y l a , n l ! S y __^Sinapy l ' aldehyde -aldehyde CAD " aldehyde Coniferyl alcohol j 6 lignin F5H/ ,• Cald5H s . H y ^ : ' COMT — - c o n i f e r y l ' '• - — alcohol ,SAD/1 CAD Slnapyt alcohol J S lignin Figure 1-2 L ignin biosynthetic pathway according to Peter et al, (2004). - 16-1.4.1 At4CL genes In all of the plant species studied so far, small gene families encode for 4 C L enzymes and corresponding 4CL genes have been cloned from several plants (for example, Douglas et al, 1987; Ehlting et al, 1999; Raes et al, 2003; Hamberger et al, 2004). In. vitro, 4 C L enzyme can catalyze CoA-esterification of multiple substrates, including p-coumaric acid, caffeic acid, ferulic acid 5-hydroxyferulic acid and sinapic acid (Lee et al, 1997; H u et al, 1998, Ehlting et al, 1999). In Arabidopsis, At4CLl, At4CL2, At4CL3 and At4CL4 genes have been cloned. It is known from the published Arabidopsis genome sequence that these are the only four 4CL genes in the Arabidopsis 4CL gene family. A t 4 C L l and A t 4 C L 2 share 83% amino acid identity while A t 4 C L 3 and A t 4 C L 4 share 61% and 66% identity with A t 4 C L l , respectively (Hamberger et al, 2004). At4CLl, At4CL2 and At4CL4 are considered as more closely related class I genes, while At4CL3 is in the less closely related class II clad in the 4CL phylogenetic tree (Ehlting et al, 1999). Class I isoenzymes have been associated with the biosynthesis of lignin and structurally related soluble or cell wall-bound phenylpropanoid derivatives, whereas class II isoenzymes have been associated with flavonoid biosynthesis (Ehlting et al, 1999) and are in a single copy in plants studied so far. Different 4CL genes often show different expression patterns throughout plant development. In the mature plant, At4CLl and At4CL2 are strongly expressed in bolting stems and root respectively. Also, these two genes are expressed throughout the inflorescence stem development and their expression increases during the later stages of development (Lee et al, 1995; Mizutani et al, 1997; Ehlting et al, 1999). At4CL3 is most highly expressed in non-vascular tissue in leaves and flowers (Ehlting et al, 1999). After wounding or infection by Peronospora parasitica, the expression of At4CL3 is not affected but the other hX4CL genes (class I) are expressed in response to these stresses (Ehlting et al, 1999). - 17-1.4.2 Control of gene expression in phenylpropanoid pathways It has been shown that transcripts of genes encoding enzymes in the phenylpropanoid pathway coordinately accumulate in a tissue/cell type specific manner during plant development. Coordinate expression is also activated in response to different environmental stimuli (Hahlbrock and Scheel, 1989; Douglas et al, 1996; Dixon et al, 1995). In Arabidopsis, expression of PAL, C4H and 4CL, is coordinately activated during development and also in response to the wounding (Lee et al, 1997, Bel l -Lelong et all., 1997; Mizutani et al, 1997, Koopmann et al, 1999). It is believed that the phenylpropanoid pathway is regulated primarily through control of transcription of the corresponding genes, possibly by a common signaling pathway and transcription factors. According to this model, the presence or activity of common transcription factors is modulated by developmental and environmental signals, and these factors in turn regulate the expression of sets of genes encoding enzymes in the phenylpropanoid pathway', and thus the activity of the pathway (Hahlbrock and Scheel 1989; Douglas et al, 1996). In support of this model, coordinated expression of genes encoding phenylpropanoid pathway enzymes requires common transcription factors and also common c.^-regulatory elements. The P and L promoter boxes are characteristic of all genes involved in general phenylpropanoid metabolism investigated so far and have been described for a wide range of plant species. These boxes contain a putative M y b transcription factor binding-site. At least one copy of P and L promoter boxes has been found in the promoter proximal region of all four At4CL genes and also parsley 4 C L genes (Lois et al, 1989; Logemann et al, 1995, Ehlting et al, 1999, Hamberger et al, 2004). The presence of three perfect W-box sequences (Eulgem et al, 2000) only in the At4CL4 gene promoter has been reported, two of which fulfill the operational W-box criterion of W R K Y transcription factor binding (Hahlbrock et al, 2003). These common cw-elements provide a basis for the coordinate regulation of the transcription of structural genes in the phenylpropanoid pathway in response to developmental and environmental stresses such as wounding. - 18-1.5 WOUNDING RESPONSE IN PLANTS 1.5.1 Plant stress responses are interrelated Compared to mobile animals, non-mobile plants cannot escape biotic and abiotic stresses and are in constant danger of wounding by environmental stresses like wind, sand, and herbivores. Open mechanical wounds are potentially a passage for infectious pathogens to enter the plant tissues. Plants have developed defensive responses against wounding to combat this danger. For example, lignin and phenolic compounds, which originate from the phenylpropanoid pathway, rapidly accumulate at the sites of attack by pathogen or after wounding (Bowles 1990). It is suggested that stresses such as mechanical wounding, drought, freeze and osmotic stress are interrelated by using partially overlapping signaling pathways (Cheong et al, 2002). For example, wounded tissues may induce local osmotic stress responses (Reymond et al, 2000; Denekamp et al, 2003). Also, it has been shown that wound and pathogen induced signaling pathways share common intermediates (Romeis et al, 1999). 1.5.2 Role of plant hormones in the wounding response \ Plant hormones are involved in the activation of wounding responses. For example, jasmonic acid and related compounds (JAs) have a central role in the early wound response (Farmer and Ryan 1990; M c Conn et al, 1997; Schaller 2001, Turner et al, 2002). Transcription of J A biosynthetic enzymes is up regulated, active JAs level is increased and defense-related genes are activated shortly after wounding (He et al, 2005). Like the systemin peptide (Pearce et al, 1991) and its receptor (Scheer and Ryan 2002) that only found in Solanaceae plants, JA may also serve as a systemic signal in the wound-induced systemic responses of the other plants ( L i et al, 2002). 1 There are also reports that ethylene (O'Donnell et al, 1996) and abscisic acid (Pena-Cortes et al, 1989) contribute to changes in gene expression during wounding responses. J A and ethylene-responsive elements and S boxes, all responsive to elicitor, wounding, and pathogen stimuli, have been found in the genes involved in lignin biosynthesis (Rushton et al, 2002). J A induces 4CL gene expression in tobacco but the mechanisms and the signal transduction pathways leading to wound-induced - 19-phenylpropanoid gene activation are still not clear (Ellard and Douglas 1996). 1.5.3 Duration of activated plant gene expression in the wounding response It has been reported that steady-state m R N A levels of - 8 % of the 8,200 genes present on an Arabidopsis microarray are altered in response to wounding, and many of these genes are also osmotic stress- and heat shock-regulated (Cheong et al, 2002). The expression levels of many defensive genes transiently increase to the maximum levels within 90-120 min post wounding, followed by decreases towards the baseline (Reymond et al, 2000), while other genes are activated much later. For example, the Wound-Responsive and phytochrome-Regulated kinasel (WPK1) gene in maize (Zea mays L. ) is transiently activated immediately after wounding. This gene is also up-regulated rapidly and transiently after jasmonic acid treatment. TheAtPTR3 peptide transporter gene on the other hand is activated 4-h post wounding and gradually increases the response up to 24 h (Karim et al, 2005). Sugar transporter (STP4 and AtSUC) genes are also activated within 3 h post wounding (Truernit et al, 1996; Meyer et al, 2004). Transgenic poplar plants containing the Eucalyptus gunnii CAD2 gene promoter fused to G U S showed wound responsiveness of the transgene only after 24h post wounding and no response'was detected 0 or 48 h post wounding (Lauvergeat et al, 2002). Wounding also induces the expression of a large number of transcription factor genes including members of the A P 2 , W R K Y , and M y b transcription factor families (Cheong et al, 2002). For example, AtMyb32 (Preston et al, 2004) and AtMyb4 (Jin et al, 2000) are known to be involved in the wounding response. 1.5.4 Biphasic phenylpropanoid gene expression in the wounding response Wounding or infection may induce deposition of lignin to protect plant tissues against invading pathogens. To do so, wounding coordinately induces many phenylpropanoid pathway genes such as PAL, C4H, 4CL, CCR, COMT (Dixon & paiva 1995; Lee et al, 1995; Bell-Lelong et al, 1997Meyer et al, 1998; Mizutani et al, 1997: Ehlting etal, 1999; Reymond et al, 2000). Several reports show that At4CLl m R N A accumulates rapidly but transiently in the - 2 0 -wounded Arabidopsis leaves 1-2 h post wounding (Lee et al, 1995; Ehlting et al, 1999). The wound-induced expression pattern of At4CLl and At4CL2 are similar although the maximum amount of At4CL2 mRNA is considerably lower than that of At4CLl mRNA. At4CL3 mRNA levels do not change after the wounding (Ehlting et al, 1999) and the wound responsiveness of At4CL4 has not been tested. A report has shown two phases of coordinated wounding response by PAL, 4CL, and C4H in parsley within 24 hours. In this report, PAL, 4CL, and C4H transcript levels reach a maximum by about 10 h post wounding of leaves, followed by a decrease in RNA levels before rising to a second peak around 24 hours, after which transcript levels declined (Logemann et al, 1995). Such a biphasic wounding response has also been reported by others and suggests that multiple signaling routes may exist for phenylpropanoid gene transcriptional activation in response to wounding (Batard et al, 2000). -21 -1.6 R E S E A R C H G O A L S 1.6.1 Research Objectives for three research projects Objective # 1- Screen mutagenized populations of Arabidopsis lines expressing 4CL-reporter gene fusions for mutants defective in developmental activation of 4CL expression In an attempt to identify genes required for activation of 4CL expression, transgenic Arabidopsis thaliana lines containing 4CL.GUS transgenes were mutagenized and putative mutants affected in 4CL expression were sought. Mutant phenotypes were not inherited in a Mendelian fashion, suggesting that gene silencing had been activated. The objective of this work shifted to understanding the mechanisms of 4CL (trans)gene silencing in these lines. Objective # 2- To search for m-regulatory elements involved in developmental and stress activation of 4CL2 gene expression in Arabidopsis 1 At4CL2 gene expression is developmental^  regulated and also is regulated in response to wounding. In an attempt to identify cw-regulatory elements responsible for developmental expression and wounding response of the gene, transgenic plants containing fusions of different parts of the At4CL2 gene to GUS were prepared and At4CL2:GUS expression monitored in transgenic plants relative to the expression of the endogenous gene. . -Objective # 3- To determine the potentially redundant functions of AtMyb sub-family #14 transcription factors by reverse genetics Attempts to find a phenotype in an AtMyb68 knock out line had previously failed, possibly due to the presence of related, partially redundant AtMyb genes in AtMyb subfamily # 14. I identified AtMyb84 as the most likely gene that could have redundant f function to AtMyb68, and used reverse genetics (RNAi and T-DNA knockouts) to investigate the functions of AtMyb68 and AtMyb84. - 2 2 -C H A P T E R 2 M A T E R I A L S AND M E T H O D S 2.1 P L A N T G R O W T H CONDITIONS 2.1.1 General In all of the experiments, Arabidopsis thaliana seeds were cold treated for 1-2 day at 4°C on germination medium. Then, seeds were germinated at 20 0 C either on soil (SunsHine mix 5 plug, Sungrow Horticulture, Saba Beach, A B ) or in Petri .dishes containing '/ 2x M S (Murashige and Skoog) salts (Sigma-Aldrich), supplemented with 1% sucrose and 0.6% agar medium. At the first time of watering, soil in pots was saturated with distilled water containing Miracle Gro powder (4 g/1) (Scotts Canada Ltd. Mississauga, ON) , subsequently tap water was added to the base of pots. To help seedling establishment, pots were covered with plastic wrap (Resinite, A F P Canada Inc., West hi l l , ON) after sowing seeds on saturated soil or soon after transplantation of seedlings from plats to the pots. One week after germination of seeds on the soil or 3 days after transplantation of seedlings from the plate to the pots, the plastic wrap was cut with a razor blade, and 2-3 days later was completely removed. Plants were maintained in long day conditions (18h light) at 20° C from germination to senescence. • j . f 2.1.2 Plant material At4CLJ::GUS, At4CL2::GUS, At4CL3::GUS and all of '}. the At4CL::dhlA+At4CL::GUS transgenic lines were provided by Dr. Ehlting (Max Planck Institute, Germany) in Arabidopsis thaliana ecotype Landsberg erecta (Ler). For At4CLlr.GUS and At4CL2::GUS transgenic plants, lines with stable GUS expression over several generations were chosen for further work. At4CL4::GUS lines were provided by Dr. Hamberger in the Columbia background. Two homozygous silencing modifier mutant lines (hogl-1 (0/0), and Si l 1-1 (0/0), (Furner et al, 1998) were obtained from the Nottingham stock center (http://arabidopsis.info/). Wild-type Arabidopsis Columbia-1 (Col) was used in the At4CL2 wounding response and AtMyb knock down/out projects. - 2 3 -A Basta resistant AtMyb68 T - D N A knock out. line from the S A I L collection (http://www.tmri.org/en/partnership/sail collection.aspx) in the Columbia background (Wang 2003) was transformed with AtMyb84 R N A i construct containing kanamycin resistance gene. A n AtMyb84 T - D N A insertion line (Salk #, http://signal.salk.edu) was obtained from the S A L K population (Alonso et al, 2003). T - D N A insertion locations and genotypes were determined by P C R using genomic D N A from each line. Table 2.1 shows the primers used to confirm the T - D N A insertion in the AtMyb84 knock out line. Genomic D N A was obtained from 1-2 leaves of plants using the miniprep method of Edwards et al. (1991). Five ul of D N A was used in P C R reactions. 2.1.3 Agrobacterium and plant transformation We used several different binary vectors including p B A R (Becker et al, 1992), p A R T (Gleave 1992), and p C A M B I A (Hajdukiewicz et al, 1994) for the transformation of Agrobacterium tumefaciens strain GV3303 in different projects. After heat shock transformation of competent Agrobacterium using binary vectors carrying the gene of interest, single antibiotic-resistant colonies were selected on L B plates containing kanamycin (lOOmg/L), gentamycin (25mg/L), and rifampicilin (25mg/L). Then a few colonies were inoculated into 5 ml of L B broth and grown at 28°C overnight. These bacterial cultures were tested for the presence of the appropriate transgene using P C R followed by sequencing of P C R products. This 5-ml culture was also used to inoculate 500 ml of L B broth supplemented with gentamycin, rifampicilin, and kanamycin in 1 L flasks. These cultures were incubated with shaking at 28°C, 24 h or longer to OD6oo~0.8. Agrobacterium cultures were precipitated by centrifugation for 15 min at 4°C and 5000 rpm. The pellets were resuspended in a solution of 5% (W/v) sucrose, 0.05% ( V / V ) Silwet L-77 (Lehle Seeds, Round Rock T X ) . Plants containing about 10-cm tall flowering stems with many young buds in the inflorescences were used for transformation. The inflorescences of the plants were dipped for 1 second into the Agrobacterium suspension (Clough and Bent, 1998). The pots with dipped plants were kept dark overnight, after which they were returned to normal growth conditions and to complete their seed development. Seeds harvested from transformed plants were germinated on MS-agar -24-plates containing kanamycin (50 ug/L) or hygromycin (50 ug/L) plus Vancomycin (40 (j.g/L) (all antibiotics from Sigma) (Katavic et al, 1994). Kanamycin or hygromycin resistant seedlings ( T l generation). were transferred to soil for selfing to produce T2 populations, which would contain individuals homozygous for the transgenes. The ratio of antibiotic sensitive seedlings/ resistant seedlings showed the number o f transgene loci and T3 generation individuals with 100% resistance were used as homozygous lines for. further studies. 2.2 GENE EXPRESSION ANALYSIS 2.2.1 RNA gel blot analysis Arabidopsis 10-cm tall stems, 10 day-old seedlings, roots from plants grown in liquid media (supplemented with 3% sucrose), and wounded leaves from 35 day-old plants were harvested and immediately frozen in liquid nitrogen to provide plant material for R N A or D N A analysis experiments. Using T R I Z O L R Reagent, total R N A was extracted and purified as described by manufacturer (Invitrogen Life Technology). The R N A quality samples was assessed by running 1 \ig of R N A on a 1% agarose gel and visualizing r R N A bands. About 12-15 \ig of high quality total R N A per sample was separated by electrophoresis on 1.2% agarose gels containing 10 % M O P S and 5% formaldehyde. Blots were prepared as described by Koetsier et al. (1993) onto Hybond-N +membranes (Amersham Bioscience) using 2 0 X SSC as a transfer solution followed by baking at 80°C for 2h to fix the R N A on the membrane. Pre-hybridization was carried out in ( I M N a C l , 5%SDS, and 10% g/v Dextran sulfate) at 65°C overnight. PCR-generated or midi prep plasmid D N A preparations were used to prepare labeled probes. To purify D N A templates and labeled probes, P C R Purification or Gel Extraction kits were used (Qiagen). D N A probes were prepared with P - A T P (Amersham) and the Random Primers D N A Labeling System (Invitrogen) using > 50ng template D N A per 50 ul i reaction. Blots were washed in 2 X SSC, 0.5% SDS, 65°C for 30 min and then in 0.2 SSC, 0.1%o SDS. 65°C, 3 X for 15 min. Membranes were exposed overnight at room temperature using phospho-screens then scanned with a S T O R M 860 Phospho-imager (Amersham Pharmacia Biotech). Most of the blots were sequentially hybridized, starting with probes corresponding to the gene of interest followed by actin probe as a loading - 2 5 -control. The intensity of the bands detected by the phospho-imager was quantified, using the Imagequant software. 2.2.2 Genomic Southern blots Total genomic D N A was extracted from 10 day-old 4CL1::GUS, 4CL2::GUS, wild type Arabidopsis (Ler), and epimutant line (1-A and 2-8) seedlings grown on M S media using the DNAeasy Plant M i n i kit (Qiagen) and Nucleon P H Y T O pure Plant D N A extraction (Amersham Biosciences) kit according to the manufacture's protocol.,Total genomic D N A samples (15 p.g) were thoroughly digested by appropriate restriction enzymes, and D N A fragments were separated on 20-cm long 1.0-1.5% agarose-TBE gels at 25 volts. The D N A fragments in the gel were denatured using 1.5 M N a C l , 0.5 N N a O H , for 45 min with gentle agitation, followed by gel neutralization using three times (15-min each) washes of ( I M T r i s - H C l , p H 7.4, and 1.5M NaCl ) , and gels were blotted onto Hybound- N + membrane (Amersham) using 10 X SSC as the transfer buffer based on a standard protocol (Sambrook et al, 1989). The membrane was baked at 80°C for 2h to fix the D N A fragments, and pre-hybridized and hybridized to the probes as described with R N A gel blot analysis. Table 2.1 shows the primers used to generate probes for northern and Southern analysis. Primer name Primer sequence - l kb4CLl -Fo rward 5' - G G T C T C C A A A G T T G A A T T A A A T G G T T G T A G GUS-Reverse 5' - T C G C G A T C C A G A C T G A A T G C C C A C 4CLl /ORF-Forward 5' - C T C T T G T A A A A C A C A A C C T G T T T C G A 4CLl /ORF-Reverse 5' - G T T T C C T A A C G C C A A G C T T G G T C A G G G C -2504CL2-Forward 5' - A T G T C G A C A G C A T G G A T A A T G A T A G T A G A G T A G C 84Eco-F 5' - A A G A A T T C G G A T C C A A G A T C G A A G A T C A A G A A C T G G 84 Rev primers 5' - A T G G T A C C A A T C G A T T T G A A T C A G A A T A A A C A A G A G A G C L B b l 5 ' - G C G T G G A C C G C T T G C T G C A A C T 4CL1 /RT-F 5' - C T A A T G C C A A A C T C G G T C A G G G A T A C Table 2.1 Sequences of P C R primers used to generate D N A hybridization probes and investigate the T - D N A insertion in the Myb84 Salk knock out line. - 2 6 -2.2.3 Quantitative RT-PCR Total R N A samples were isolated, qualified, and quantified as described for R N A gel blot analysis. R N A samples (2 ug R N A / 20 u.1 reaction), were used to generate first strand c D N A using Superscript II Reverse Transcriptase (Invitrogen Life Technology) following the manufacturer's protocol. Gene-specific and intron-spanning primers (Table 2.2) were used in P C R reactions to amplify corresponding c D N A sequences. Template ( cDNA) amounts and P C R cycle numbers were optimized using actin cDNA-specif ic primers in serial P C R reactions. General P C R conditions were 95°C for 2 min, followed by 25-31 cycles of (94°C for 3o"sec, 58°C for 30 sec, and 72°C for 1 min), followed by 72°C for 5 min, using Taq-polymerase/ 25ul total reaction. P C R products were separated on 1% agarose gels, stained with ethidium bromide, visualized, and photographed under U V transluminator and a digital camera using Alphalmager 1220. Primer name Primer sequence 4CL1 /RT-F 5 ' - C T A A T G C C A A A C T C G G T C A G G G A T A C 4 C L 1 / R T - R 5 ' - C T C T T G T A A A A C A C A A C C T G T T T C G A C 4 C L 2 / R T - F 5' - C C T A A C G C C A A G C T T G G T C A G G G C T A T G G G 4 C L 2 / R T - R 5 ' -C A C C A G C A T C T T C T T C C T T C A T G G C G A C G 4CL3 /RT-F 5' - C T C A A T T C A C T C C G A T C C G G C G C 4 C L 3 / R T - R 5' - C T G G A T C G T T C A A G T A C T C T T T C A T G A T 4CL4 /RT-F 5' - C G A G G T C A T C A G C T C A T G A A A G G T T A T 4 C L 4 / R T - R 5' - C G C T T G T A G T G A A C C A C C T G T T T G T T T A C Actin-F 5' - G C G A C A A T G G A A C T G G A A T Actin-R 5' - G G A T A G C A T G T G G A A G T G C A T A C C Table 2.2 Sequences of primers used in semi-quantitative P C R reactions. (Primer sequences were adapted from Hamberger et al, 2004) In order to study changes in At4CL gene expression in response to wounding,!four week-old leaves of two Arabidopsis (Col-01) rosettes grown in separate pots under long day conditions (16 h day at 20°C, 90% humidity), and were each punched 40 times using - 2 7 -pipette tips. A t 0-72 hours after in planta woundingj leaves were harvested and frozen in liquid nitrogen and 200 mg leaf material was used for total R N A extraction. Leaves from parallel control plants grown under the same conditions were harvested without wounding. 2.2.4 Histochemical GUS assays Seedlings or organs of transgenic plants expressing G U S , in parallel with positive (35S::GUS) and negative (wild-type) controls were incubated in G U S assay buffer containing lOOmM phosphate buffer p H 7.0, 0.1% Triton-X-100, I m M potassium ferricyanide, I m M potassium ferrocyanide, and lmg/ml 5-bromo-4-chloro-3-indolyl-B-D-glucuronide (X-gluc; Jefferson, 1987) at 37C for 4 h (or longer i f noted). The assay buffer was removed and 95% ethanpl was added to stop the reaction and remove the chlorophyll. Plant materials were kept in water 15 min before photography. 2.3 DNA SEQUENCE ANALYSIS For restriction enzyme mapping and sequence manipulation, Seqpup software (http://iubio.bio.indiana.edu/soft/molbio/seqpup/java/seqpup-doc.html) was used. At4CL gene sequences were found at (http://www.arabidopsis.org/info/genefamily/Raes.html), and AtMyb gene sequences : were found at (http://w-ww.arabidopsis.org/info/genefamilv/Myb.html). For homology searches and alignments, the B L A S T algorithm (http://www.ncbi.nlm.nih.gov/BLAST/) was used. The P L A C E software program (http://www.dna.affrc.go.jp/PLACE) was used to search for putative cis regulatory elements. D N A sequencing was performed using the di-deoxy chain termination method with B I G dye 3.0 (Applied Biosystems) and a P R I S M 377 automated sequencer (Applied Biosystems; N A P S unit, N A P S Unit, U B C ) . Primers used for sequencing are listed in table 2.3. - 2 8 -Primer name Primer sequence 2Pr-R 5' - T T G T G T C G C C ATGG A T C A G A A G T T A A T A T C - 110 F 5' - A T G T C G A C A T A T G G A A A C A C T G A T C A T C A T G C - 250F 5' - A T G T C G A C A G C A T G G A T A A T G A T A G T A G A G T A G C - 4 2 0 F 5' - A T G T C G A C C T A C T C A T A A T G A C C A A T G A A T G - 750 F 5' - A T G T C G A C G T C T T G G A A G A G T A C T G T T A A A G A G . -1.6kF 5' - A T C C C G G G G A A T T C C A T C A T T T C A G T A G A G A G G A T C 2Cod-R 5' - ATCC ATGGGGTTC A T T A A T C C A T T T G C T A G T C T T G C T C T T A G 2 0 R F - F 5 '-GATCCATGGCGA C A C A A G A T G T G A T A G T C A A T G + 500 cod- F 5' - C A C C C A T G G A C T C A A T A C C G G A G A A G A T T T C G + 1000 cod-F 5' -T A C C A T G G T T A A G T C T G G A G C A G C T C C T C + 1500 cod-F 5' - T G A C C A T G G A C T G T T T T A A C T T T T A G A G C G T C T A C + 2000 cod-F 5' - G G G C C A T G G G A A T G A C A G A A G C A G G T C C G G - l k b 4 C L l - F o r 5'- G G T C T C C A A A G T T G A A T T A A A T G G T T G T A G GUS-Reverse 5' - T C G C G A T C C A G A C T G A A T G C C C A C 4 C L l / O R F - R e 5' - G T T T C C T A A C G C C A A G C T T G G T C A G G G C pHannibal-F 5 - C C C A C T A T C C T T C G C A A G A C C C pHanibal-R 5 - C A A C G T G C A C A A C A G A A T T G A A A G C Hanni-intron reverse 5 ' - C A T A C T A A T T A A C A T C A C T T A A C Hanni-intron forward | 5 - C A T G T C A T T G T G T T A T C A T T G T C Table 2-3 Sequences of primers used to prepare plasmid constructs and also' for sequencing reactions. - 2 9 -2.4 PLASMID CONSTRUCTS 2.4.1 Strategies to generate At4CL::GUS plasm id constructs Various At4CL2 promoter fragments were P C R amplified using (-1.6 kF), (-750F), (-420F), (-110F), (2Pr-R) (Table 2.3) and fused to G U S in the pBTIO vector (Figure 2.1). Inclusion of the Ncol restriction site in the (2Pr-R) reverse primer enabled fusion of amplified promoter fragments to the GUS gene. The 1.6-kb construct was digested with Xbal to eliminate 600 bp of the upstream promoter sequences to prepare the 950bp construct. 2Pr-R At4CL2 promoter ^ At4CL2 transcribed region ! I 'I . . = = — — • • — M — ^ Xbal ^ ^ ^ El E2 E3 E4 -1.6kF -750F -420F -110F Figure 2-1 Relative position of the primers used to amplify At4CL2 promoter fragments. At4CL2 promoter fragments of different lengths were amplified using the indicated primers and were fused with G U S in PBT10. Empty bars represent promoter and solid black bars represent exon (E l -E4) . The Expand Long Template P C R system (Roche) was used to amplify the full length At4CL2 gene from Arabidopsis thaliana (Col) genomic D N A using (1.6k-F) and (2Cod-R) primers and 4.3kb full-length P C R products were generated (Figure 2.2-A). These P C R products were cut by Xbal and Ncol restriction enzymes and the resulting 3.7-kb fragment (containing the 950-bp promoter plus transcribed region) was fused in-frame with the G U S - O R F that was already installed in pBluescript. This clone was designated the full-length 4CL2::GUS construct (Figure 2.2-A & -B) . Using Xbal and Sac\ restriction enzymes, the full-length 4CL2:;GUS construct was transferred to' the p B A R binary vector. - 3 0 - i B) At4CL2 promoter At4CL2 transcribed region F X b a l •1.6kF Full length Construct 2Cod-R 950bp promoter At4CL2 transcribed region G U S Xba l cDNA Construct Intron # 1 Construct SacII Nhe'I A v R I I Ncol At4CL2 c D N A Pstl Xbal SacII Nhel A v R I I N c o l E l E2+E3+E4 Pstl Intron # 2&3 Construct E 1 + E 2 A v R I I 12 13 Nhel Nco l Figure 2-2 Schematic representation of At4CL2 promoter and transcribed regions fused to G U S . A) Locations of primers used to amplify At4CL2 promoter plus transcribed regions. B) Structure o f At4CL2::GUS constructs in pBluescript. Empty bars represent promoter fragments and solid bars represent exon (E1-E4). The name of each clone referred to in the text is indicated at the left. In order to delete the introns from the full-length 4CL2::GUS construct and generate a At4CL2 promoter: :At4C_2-cDNA::GUS construct, the At4CL2-cDNA was amplified using 2 0 R F - F and 2Cod-R primers (Figure 2.2-A), both containing Ncol sites (Table 2.3). Using Ncol and SacII restriction enzymes, a 2-4 kb D N A fragment containing all three introns was removed from the full-length 4CL2::GUS construct in pBluescript and substituted by 1.3 kb of the 4CL2-cDNA (Figure 2.2B). Using Sacl and Xbal restriction enzymes, the 950 bp promoter::4CL2cDNA::GUS construct in pBluescript was partially digested and the 4.6 kb fragment was sub-cloned into pCambia 1300 (http://www.cambia.au) binary vector. To add introns # 2 and 3 to the 4CL2-cDNA::GUS construct in pBluescript, an 850-bp D N A fragment containing introns 2 and 3 was cut out from full length 4CL2::GUS construct in pBluescript clone using Ncol and Nhel. This fragment was used to substitute the corresponding area (625 bp) in the 4CL2-cDNA::GUS construct in pBluescript that had been cut with the same enzymes (Figure 2.2-B). A n Ncol-Xbal restriction fragment containing this construct was transferred to the pCambial300 binary vector. To prepare the intron 1 construct, a 2950-bp Avrll-Pstl restriction fragment containing intron 1 from the full-length construct was substituted for corresponding fragment in the c D N A construct (Figure 2.2-B). A s described above, based on the presence of convenient restriction enzyme sites, D N A constructs were transferred to different binary vectors for transformation of Arabidopsis plants. The pCambia 1300 was used for intron 1, intron # 2 & 3, and c D N A constructs. The p B A R binary vector was used for the full length 4CL2::GUS construct and the 125 bp, 500 bp, and 750 bp constructs and pCambia 1305 was used for the 950 bp construct. The sequence integrity of all constructs was confirmed by sequencing ( U B C , N A P S unit). Agrobacterium strain GV3101, Arabidopsis thaliana plants were transformed with the constructs as described above. A t least 10 transgenic lines for each construct were selected for subsequent analysis. 2.4.2 Strategies for RNAi construct generation 2.4.2.1 AtMyb68, AtMyb84, and GUS RNAi constructs A s a sense arm for the R N A i construct, 400 bp of AtMyb84 coding sequences were P C R amplified using (M84-Ec-Ba-F) and (M84-Kp-Cl -R) primers (Table 2.3). These products were cloned into pHannibal (Wesley et al., 2001) obtained from (CSIRO Plant Industry, Canberra, Australia) using Bamlil and Kpnl restriction enzymes whose sites were engineered in the primers. A 315 bp fragment as antisense arm was P C R amplified using (M84-Xba-F) and (M84-kp-Cl-R) primers, was cut by Xbal and Clal< and cloned into pHannibal which already had the 400-bp sense arm (Figure 2.3-B). 32 Primer name Primer sequence M 6 8 - E c X b H - F 5 - T T G A A T T C T A G A A G C T T C A G C A C A A A C T C A C C A T T A C C A A A C M68-__m-F 5 - A A G G A T C C T T T G C T A T C A T G A G C A G C A G C A C M 6 8 - K p - C L R 5 - A T G G T A C C A C A T C G A T T T G G C G C A T T G A A G T A A C T T G C M84-Ec-Ba-F 5 - A A G A A T T C G G A T C C A A G A T C G A A G A T C A A G A A C T G G M84-Xba-F 5 - G A T C T A G A A C T G G A G A A A A C A A A C C T C A T C M 8 4 - K p - C l - R 5 - A T G G T A C C A A T C G A T T T G A A T C A G A A T A A A C A A G A G A G C Gus-Xb-Ec-F 5 - A C T C T A G A A T T C A A A A A A C T C G A C G G C C T G T G Gus-Xho-F 5 - A A C T C G A G G G C C T G T G G G C A T T C A G T C T G Gus-Kp-Cl -R 5 - T C G G T A C C T A T G G A T T A T T G A C C C A C A C T T T G C C G M84-R-Kpn 5 ' - T A C G G T A C C C C C A A T T A A T A A T A A T G A T G T A C G Table 2-4 Sequences of P C R primers used to prepare R N A i constructs. In similar steps, 355-bp sense and 350-bp antisense arms for AtMyb68 were P C R amplified using (M68-EcXbaH-F) or (M68-Bam-F) and ( M 6 8 - K p - C L R ) primers and cloned in pHannibal using Hindlll and Kpnl restriction sites for cloning sense arm and BamWl and Clal sites for cloning the antisense arm (Figure 2-4, A ) . I chose ~ 350 bp of the down stream region of these two genes in order to provide D N A for the R N A i constructs. Two genes in these areas did not show any identity except for a domain of 50bp (with 83% identity) but even in this domain no more than 12 bp were identical in each sub domain. The amplified area is the least similar between AtMyb68 and AtMyb84. In order to prepare the G U S R N A i construct, 270 bp of the G U S - O R F was amplified using (Gus-Xho-F) and (Gus-Kp-Cl-R) primers (Table 2-4) and cut by Xhol and Kpnl restriction enzymes and cloned into pHannibal as the sense arm. The 312-bp antisense arm was also amplified using (Gus-Xbal -Eco-F) and (Gus-Kp-Cl-R) primer. P C R products were cut by Xbal and Clal and joined them to the pHannibal vector already containing the sense arm (Figure 2-3 C). - 3 3 -Xhol, EcoRI, Xbal, Hindlll Kpnl 68-S Notl Clal BamHl,XbaI | 68-A INotl AtMyb68 RNAi construct Xhol, EcoRI, BamHl Kpnl Clal Xbal 84-S 84-A INotl AtMyb84 RNAi construct Xhol Kpnl GUS-S Clal EcoRI, Xbal . GUS-A J4 >C Notl Notl GUS RNAi construct Figure 2-3 RNAi constructs for AtMyb68, AtMyb84 and GUS in pHannibal 34 2.4.2.2 Cloning of double RNAi constructs Using Xhol and £coRI restriction enzymes, GUS R N A i and AtMyb84 R N A i constructs in pHannibal were cut and GUS R N A i was ligated upstream o f the AtMyb84 R N A i construct in pHannibal (Figure 2-4, B) . Using a similar strategy, AtMyb68 and GUS R N A i constructs were fused (Figure 2-4, A ) . Also , using EcoRI and BamHI restriction sites, AtMyb68 and AtMyb84 R N A i constructs were fused to each other to make a single construct containing both R N A i constructs (Figure 2-4, C) . Sequences o f the all of these R N A i constructs were verified, transferred to p A R T binary vector using Notl and transformed into Agrobacterium for Arabidopsis (Col) transformation as described above. Xbal Notl. Notl X b l A. MMyb68 + GUS RNAi construct G U S - A ^ 84-S Xbal EcoRI B . AtMyb84 + GUS R N A i construct 68-A 84-S 84-A Xbal C. AtMyb68 + AtMyb84 RNAi construct Figure 2-4 Double R N A i constructs for AtMyb68, AtMyb84 and G U S in pHannibal 2.5 M U T A N T ANALYSIS ; 2.5.1 Screening for GUS expression mutants Five thousand seeds from the selected homozygous At4CLlr.GUS line were mutagenized by E M S (0.25%, 8 h) and were grown in 100 pots to set their seeds and collect these seeds as 100 pools containing M 2 generation. These M 2 seeds were grown in liquid media containing M S salts. Ten day-old seedlings were screened for changes of developmental G U S expression in the root and shoot. 2.5.2 Screening for dhlA expression mutants Basta resistant T l generation plants containing double transgenes (At4CLlrGUS plus At4CLl::dhlA), (At4CL2::GUS plus At4CL2::dhlA), or (At4CL3::GUS plus At4CL3::dhlA) were selected by Basta and -15 independent homozygous lines were identified in the T3 generation for all three cases. The Xanthobacter autotrophics GJ10 Dehalogenase A (dhlA) gene (Janssen et al, 1989) works as a negative selection marker in Arabidopsis (Naested et al., 1999) when d/z/A-expressing plants are treated with a haloalkane pro-toxin substrate, such as 1,2-dicholoroethane (DCE) (Janssen et al., 1994). In order to optimize the selection system for D C E treatment, 0-6 day-old seedlings of wi ld type Arabidopsis (Ler) plants grown in M S media were treated with 5- 40 (al of D C E (1.256 g/ml; Sigma-Aldrich). Plants were treated once/day for three days. I found that treatment of 3 day-old Arabidopsis seedlings on solid M S medium with 10 ul of D C E for 3 days showed the least amount of toxicity in wi ld type plants, but was toxic to a 35S::dhlA control line. 2.5.3 Phenotypic and genetic analysis of putative mutants In order to analyze the phenotype of putative At4GLlr.GUS expression mutants, overnight G U S assays were performed on 10 day-old seedlings of putative mutants grown on M S media. The morphology of putative mutants grown in pots was compared with wild type plants. ' • In order to genetically analyze the inheritance of the mutant phenotype, reciprocal crosses were made between three selected putative At4CLl r.GUS expression mutants and the T5 4CL1 r.GUS non-mutated progenitor line. In order to avoid pollen contamination, the top flowers of each mutant and non-mutant plant were emasculated and cross-- 3 6 -pollinated after 2 days. The G U S assays were performed on the F l and F2 generation of these crosses. 2.5.4 Azacytidine treatment of mutant plants The demethylation agent 5-azacytidine (5-aza), a cytosine analog, was used to prevent D N A methylation (Prakash and Kumar, 1997). Seedlings from putative mutant lines were grown on solid media supplemented with 40 mg/L of 5-aza (Sigma-Aldrich) and 50 Hg /ml kanamycin. Ten day-old seedlings treated in this way were stained for G U S expression in parallel with control lines. - 3 7 -C H A P T E R 3 G E N E T I C ANALYSIS OF At4CL EXPRESSION AND E P I G E N E T I C SILENCING OF 4CL EXPRESSION 3.1 INTRODUCTION 4-coumarate::CoA ligase (4CL) (EC 6.2.1.12) enzymes catalyze the formation of C o A esters from cinnamic acids in the plant general phenylpropanoid pathway. These esters are intermediate substrates for specific branch pathways, such as those leading to the synthesis of flavonoids and lignin (Hahlbrock and Scheel, 1989; Dixon and Paiva 1995; Douglas 1996). 4CL genes are recognized as small gene families in all the plant species studied so far and the genes encoding for 4 C L s have been cloned from several plants (reviewed by Raes et al, 2003). At4CLl, At4CL2, At4CL3 (Lee et al, 1995; Ehlting et al, 1999 and At4CL4 (Hamberger et al, 2004) encode the complete 4CL gene family in Arabidopsis. A t 4 C L l and A t 4 C L 2 share 83% amino acid sequence identity and 74% c D N A identity in c D N A coding regions (Ehlting et al, 1999; Hamberger et al, 2004). A . 4 C L 1 and A t 4 C L 2 isoenzymes both are associated with biosynthesis of lignin and structurally related soluble or cell wall-bound phenylpropanoid. derivatives in the xylem tissues of the plant vasculature (Lee et al, 1995, Mizutani et al, 1997, Ehlting et al, 1999), similar to the parsley {Petroselinum crispum) 4CL1 gene, the first 4CL gene cloned (Hauffe et al, 1991). From the analysis of organ-specific expression of At4CLl and At4CL2 genes in Arabidopsis it is known that both At4CLl and At4CL2 have .high expression levels in seedling roots. In adult plants, At4CLl is the only gene family member strongly expressed in bolting stems (Ehlting et al, 1999). At4CLl and At4CL2 promoters contain multiple common cis regulatory elements that • are also found in other genes encoding enzymes in the general phenylpropanoid and lignin-specific biosynthetic pathways (Ehlting et al, 1999; Hamberger et al, 2004). Considering the high identity of A . 4 C L 1 and A t 4 C L 2 plus common cis regulatory elements, a common mode of transcriptional regulation and partial functionalredundancy is a strong possibility. - 3 8 -Epigenetic mechanisms can alter the transcription patterns of endogenous genes as well as transgenes, and control tissue specific gene expression (Fagard and Vaucheret 2000). D N A methylation causes gene inactivation in eukaryotes (Bird 1992; Martienssen and Richards, 1995). Also , it is widely considered as a mechanism for defending genomes against the effects of transposable element and virus proliferation (Ratcliff, et al, 1997; Martiensen, 2001; Miura et al, 2001). It is also a controlling factor for developmental gene expression (Sakai et al, 2000; Ito et al, 2003), a modulator of the expression of duplicated gene family members (Lawrence 2004), and a silencer of transgenes (Ki lby et al, 1992; Stam et al, 1997; Fagard, 2000; Chandler et al, 2001). Transgene expression in plants is controlled by incompletely understood r gene silencing mechanisms. There are examples of single copy transgene silencing but often, homology between the interacting multiple (trans) genes is critical in silencing (Meyer and Saedler 1996). Transgenes can be silenced after a few generations of activity and may also silence the homologous endogenous gene (Vaucheret and Fagard, 2001). Two molecular mechanisms have been proposed to mediate H D G S that are not necessarily exclusive. In transcriptional gene silencing (TGS) promoters of two (trans) genes are homologous. This form of silencing blocks transcription of the (trans) gene and is both meiotically and mitotically heritable (Fagard and Vaucheret, 2000). T G S is mainly associated with heavy methylation of the promoter sequences (Meyer et al, 1993; Neuhuber et al, 1994; Park et al, 1996). Post-transcriptional gene silencing (PTGS) involves degradation of the cytosolic m R N A product of the (trans) gene (Meyer et al, 1993; de Carvalho et al, 1992), based on homology that is confined to the coding regions (Muskens et al, 2000). Double stranded R N A (Waterhouse et al, 2001), high R N A expression levels (Napoli et al, 1990), and otherwise aberrant R N A s may trigger P T G S (Balcombe, 2004; Gendrel et al 2005). In PTGS, mainly coding sequences are methylated (Vaucheret et al, 1998, Stam et.al,199%, Chandler et al, 2001) and this methylation is triggered by d s R N A with an identical sequence (Bender, 2004). PTGS can also be initiated by a single transgene (Muskens et al, 2000). Here, we show that At4CLl and At4CL2 promoters drive G U S reporter gene in overlapping expression patterns in the Arabidopsis vascular system. I used mutagenized - 3 9 -populations of At4CL::GUS transgenic lines to isolate multiple mutant lines with reduced GUS expression. All mutant lines showed non-Mendelian inheritance of the GUS expression phenotypes and the majority were sensitive to treatment by the demethylating agent, 5-aza that restored the original At4CL::GUS transgene expression in the roots, implicating alterations in DNA methylation in the loss of GUS expression. Southern analysis confirmed DNA methylation was confined to the promoters of the transgenes in the mutant lines, supporting methylation-mediated TGS. Increasing the number of transgenes in the non-mutated lines resulted in similar phenotypes at a high frequency. Taken together our data suggest that epigenetically silenced At4CL epimutants had been isolated. At4CLl :;GUS epimutants differed from At4CL2::GUS epimutants with respect to organ-specific gene silencing, suggesting two different silencing modes for two closely related family members. 3.2 RESULTS 3.2.1 N E G A T I V E S E L E C T I O N O F AT4CL SIGNALING P A T H W A Y M U T A N T S 3.2.1.1 Generation of transgenic Arabidopsis lines containing 4CL::Reporter transgenes I, intended to screen for mutants in genes required for activation of developmentally regulated At4CL expression in mutagenized transgenic lines containing At4CL::GUS or At4CL::dhlA transgenes. Transgenic Arabidopsis thaliana (Ler) lines containing 1.0-kb At4CLl, 1.6-kb At4CL2 and 0.6-kb At4CL3 promoter fused to GUS transgenes, were generated by Dr. J. Ehlting. The At4CLl::GUS and At4CL2;:GUS transgenic lines showed GUS expression throughout the plant vasculature while At4CL3::GUS line showed the expression of GUS in areas other than the vasculature (Figure 3-1). Both representative At4CLl::GUS and At4CL2::GUS transgenic lines'(T5 generation) were crossed to wild type (Ler) plants and segregation of kanamycin resistance (encoded on the T-DNA) in the F2 generations indicated a single transgene locus in both cases. These At4CLl::GUS and At4CL2::GUS transgenic lines were used in -40-two parallel genetic screens aimed at identifying mutants affected in developmental regulation of At4CLl and At4CL2, using GUS expression as a visual phenotype. At4CLI:.-GUS M4CL2::GUS At4CL3::GUS • ^ If A At ______ w \ _______ ' w / 4 . I J • 1 ) * Figure 3-1 Developmentally regulated GUS expression in At4Cll::GUS, At4CL2::GUS mdAt4CL3::GUS lines (data from Dr. J. Ehlting, unpublished). The dehalogenase-A (dhlA) gene, when driven by the CaMV35S promoter, has been shown to be an effective negative selectable marker in Arabidopsis plants fed with the pro-toxin substrate 1,2-dichloroethane (DCE) (Janssen et al, 1994; Naested et al, 1999). In collaboration with Dr. Ehlting, I explored the use of dhlA to select for mutants affected in developmental regulation of At4CL promoter activity. To establish a negative selection system for At4CL promoter activity, Dr. Ehlting fused the dhlA gene to At4CLl, At4CL2 and At4CL3 promoters, and introduced the At4CLl::dhlA, At4CL2::dhlA and At4CL3::dhlA fusions into At4CLlr.GUS, At4CL2::GUS and At4CL3rGUS transgenic plants, respectively, to generate lines harboring both At4CL::GUS and At4CL::dhlA transgenes. This would allow dual screening for mutants impaired in At4CL promoter activity by negative selection upon feeding with DCE, and histochemical assay of GUS expression in mutagenized populations derived from such lines. From the primary transformants, I identified -15 independent homozygous lines for each of At4CLlrdhlA, At4CL2rdhlA, and At4CL3::dhlA transgenic lines. The -41 -majority o f these lines showed monohybrid ratios for the At4CL::dhlA transgenes inheritance. Using At4CL promoter specific and dh lA/ORF specific reverse primers, the presence o f At4CL::dhlA transgenes in the plants was confirmed. 3.2.1.2 Negative selection system optimizing for At4CL::dhlA lines I first optimized the system to establish the highest amount of D C E that was not toxic for wi ld type Arabidopsis plants after 3 days o f treatment. Treatment o f wi ld type plants (10 ul of D C E / day) three times over three days, was not toxic to wi ld type plants. To test this system, multiple (at least 15) homozygous At4CL::dhlA lines in the appropriate At4CL::GUS background were treated with D C E under conditions optimized for negative selection of a control 35S::dhlA line and wi ld type plants. In multiple experiments, D C E treatment caused delay o f wi ld type seed germination and growth, but was lethal for the 35S::dhlA seeds or seedlings. Germination and growth of At4CL::dhlA lines in the At4CL::GUS background, were also delayed but D C E treatment was not lethal as their growth resumed after treatment. In all o f the At4CL::dhlA lines with At4CL::GUS background, plants responded similarly to wi ld type plants (data not shown), and negative selection did not work regardless o f whether plants were grown in M S media or soil (Figure 3-2). Figure 3-2 Treatment of At4CL::dhlA lines with D C E and comparison o f their sensitivity to the 35S::dhlA line. - 4 2 -Northern blot analysis performed on all At4CL::dhlA lines failed to detect dhlA transcripts (data not shown) although semi-quantitative R T - P C R did detect low levels of dhlA m R N A in a few tested lines (Figure 3-3). This suggested that, unexpectedly, the At4CL promoters were ineffective in multiple lines to drive sufficient dhlA expression for negative selection to be effective. Wt 4CL2 35S 4CL1 ::dh\A "dh lA ::dhlA line4 line2 Figure 3-3 Semi quantitative R T - P C R in order to compare of <MA expression levels in At4CL::dhlA lines and the 35S::dhlA line. Act in was amplified as a control for R N A amount. -43 -3.2.1.3 At4CL::GUS transgenes are silenced in At4CL::dhlA lines I examined At4CL::GUS transgene expression in the At4CL::dhlAIAt4CL::GUS lines and found that more than 50% of the lines showed down regulation o f At4CL::GUS transgenes (Figure 3-4). Figure 3-4 G U S expression in the several lines o f At4CL::dhlA/At4CL::GUS transgenic plants. A ) At4CLl::dhlA lines in the At4CLl::GUS background. B) At4CL3::dhlA lines in the At4CL3::GUS background. To test i f D N A methylation could play a role in the unexpected loss o f GUS expression, selected At4CL::dhlA lines showing down regulated GUS expression were treated with 5-aza to inhibit D N A methylation. This treatment restored developmentally regulated GUS expression to the visible levels (Figure 3-5). B - A z a +Aza At4CLl::dhlA Line 2 At4CL2::dhlA Line 2 At4CLl::GUS At4CL2::GUS Figure 3-5 GUS-activity in At4CL:.reporter transgenic lines and wi ld type controls. A ) G U S activity in At4CL::dhlA/At4CL::GUS lines with or without treatment with 5-aza. B) G U S activity in At4CL::GUS progenitor lines and wi ld type plants. Selected At4CL::dhlA lines were also co-treated with DCE and 5-aza to investigate potential methylation of At4CL::dhlA transgenes. Although both of these chemicals delayed the growth of wild type plants, treatment of At4CL::dhlA lines with 5-aza made them more sensitive to DCE treatment, as shown by chlorotic leaves of the seedlings treated with both DCE and 5-aza (Figure 3-6). Overall, these data suggest that in At4CL::dhlA/At4CL::GUS lines, both At4CL::GUS and At4CL::dhlA transgenes had been epigenetically silenced by DNA methylation and that as a result of transgene silencing, or in combination with other unknown reasons, the negative selection system using At4CL::dhlA was not functional in the Arabidopsis. No treatment +Aza +DCE +Aza &+DCE At4CLl -dhlA Line 2 A.4CL2 -dhlA Line 2 35S-dhlA Wt.Ler Figure 3-6 DCE sensitivity of At4CL::dhlA lines with or without treatment by 5-aza compared with positive (35S::dhlA line) and negative (Wt.Ler) controls. 3.2.2 ALTERNATIVE SCREEN FOR At4CL SIGNALING PATHWAY MUTANTS 3.2.2.1 Screen for mutants with reduced At4CLl-driven GUS expression A s an alternative to negative selection for mutants affected in At4CL promoter activation, I used GUS expression as a screen for loss o f At4CL promoter activation. 5000 seeds of transgenic Arabidopsis (Ler) plants containing a At4CLl::GUS transgene were mutagenized and grown in soil (50 seeds in each pot) and selfed to make the M 2 generation. M 2 seeds were harvested as 100 M 2 pools by Dr. Ehlting and I screened >100 seeds from each pool (total of 8000 M 2 seeds from 80 pools), looking for ectopic or reduced developmental GUS expression in the leaves o f two-week old plants. Overall, I found 14 putative mutants. In parallel, 8000 M 2 seeds of mutagenized At4CL2::GUS line were screened by Dr. Ehlting and >10 putative mutants were identified. 3.2.2.2 Phenotype analysis of putative mutants lines Representative phenotypes of mutants affected in At4CLl::GUS expression showing weak, patchy, or complete lack of GUS expression in the vasculature are shown in Figure 3-7. Wild-type Weak Gt/S Patchy GUS No GUS Figure 3-7 Histochemical analysis of G U S activity in the roots of representative putative At4CL::GUS expression mutants. A l l of the putative mutants in the 4CL1::GUS background (e.g. mutant 1-A) were severely affected in GUS expression in the root, with slight or no reduction of GUS expression in the veins of the cotyledons. At maturity, these mutant plants showed global loss of GUS expression in all organs (Figure 3-8). The strongest mutants in the 4CL2::GUS background (e.g. mutant 2-8) showed global loss of developmental GUS expression in both seedlings and mature plants (Figure 3-9). -48 -Seedling Bolting stem Flowering bud Figure 3-8 Histochemical analysis of GUS activity in the 1-A putative mutant is compared with GUS activity in the progenitor background At4CLl::GUS line. Seedling Bolting stem mature leaf Figure 3-9 Histochemical analysis of GUS activity in the putative mutant 2-8 is compared with GUS activity in the progenitor background At4CL2::GUS line. Mutant (2-8), was originally identified by Dr. Juergen Ehlting. No morphological differences were detected in putative mutants (1-A, 1-B, & 1-C) relative to the progenitor line (At4CLl::GUS) and wild-type plants (Figure 3-10 left). Only one of the mutants (1-C) showed a longer hypocotyl compared to non-mutant plants (Figure 3-10 right) but further genetic analysis showed that the long hypocotyl phenotype was not linked to the GUS down regulation phenotype. Figure 3-10 Growth and development of putative mutants. Left, Five-week old mutants and control plants. Right, long hypocotyl phenotype in mutant 1-C 3.2.2.3 Genetic analysis of putative mutants lines Individuals of all 14 putative 4CL1::GUS mutant lines were crossed to the non-mutant 4CL1::GUS transgenic parental line both as pollen donors or recipients. Inheritance of the mutant phenotype (loss of histochemically detectable GUS activity) was analyzed in the Fl and F2 generations. In all the cases, the phenotype was inherited in a non-Mendelian manner as shown in Table 3-1 for three of these mutants (1-A, 1-B and 1-C). -50-Table 3-1 Genetic analysis of 4CL1::GUS mutants Male" Female F l phenotype F2 phenotype parent parent #GUS+ #GUS- #GUS+ #GUS-W T 1 1 - A - l " 30 70 0 80 1-A-l W T 25 10 0 50 W T l - A - 2 10 30 0 45 " l - A - 2 W T 25 35 0 50 W T 1-B-l 0 70 0 42 1-B-l W T 25 35 0 47 W T l - B - 2 10 20 0 • 45 l -B-2 W T 0 40 0 40 W T 1-C-l 0 10 0 55 1-C-l W T . 1 115 0 35 W T l - C - 2 0 20 0 50 l -C-2 W T 0 3 0 45 1 The wild-type line used was the 4CL 1::GUS progenitor line 2 Two separate individuals from each of the mutant lines 1-A, 1-B, and 1-C were crossed in a reciprocal manner to W T (4CL1 r.GUS) plants Similar results were observed by Dr. Ehlting in crosses of more than 10 putative 4CL2::GUS expression mutants (such as mutant 2-8). To exclude the possibility of mutations in the At4CL::GUS transgenes, we sequenced the 4CL1 r.GUS and 4CL2::GUS transgenes in the 1-A and 2-8 mutant lines and confirmed their integrity. Crosses of non-mutagenized At4CL2::GUS lines from more advanced generations to wi ld type plants showed Mendelian segregation of both kanamycin resistance and GUS expression phenotypes, in leaf veins, as expected. However, in spite of the Mendelian inheritance of the kanamycin resistance phenotype, 83% of analyzed F2 plants derived -51 -from the At4CLl::GUS line (T14) crossed to wi ld type showed root specific lack of GUS expression. These results suggest that the putative mutants affected in At4CL::GUS expression may be epigenetic mutants affected in transgene expression through transgene silencing, and that such silencing can occur without E M S mutagenesis in these lines: 3.2.2.4 Treatment of mutant lines with 5-aza To examine the possibility of At4CL::GUS transgene D N A methylation in the mutant lines, I grew three mutant lines in the At4CLl::GUS back ground (1-A, 1-B, 1-C) and one mutant in the At4CL2::GUS background (mutant 2-8) in M S media supplemented with 5-aza and kanamycin. Histochemical analysis o f G U S expression in 10 day old seedlings in each case showed restoration o f GUS expression in > 90% of seedling roots treated with 5-aza while non-treated control seedlings showed the mutant phenotype. Representative results for 1-A and 2-8 mutants are shown in Figure 3-11. - 5 2 -i Putative mutant 1-A Putative mutant 2-8 Figure 3-11 GUS expression in At4CL::GUS mutants in the presence or absence o f 5-aza. Representative phenotypes from treatment o f 10 ten-day old seedlings from each line are shown. W i l d type and non-mutagenized controls are shown in Figure 3-5 B. These results strongly suggest that transgene D N A methylation plays a role in the loss o f GUS expression observed in mutants, and suggests that the mutants are in fact epimutant in which the transgene has been epigenetically silenced. During the course of mutant screening, the non-mutated At4CLl::GUS line was also selfed and used as positive controls for the G U S assays. I observed an increasing frequency of G U S down regulation in this non-mutated line after the T7 generation. This spontaneously occurring G U S down-regulation phenotype o f the At4CLl::GUS line was - 5 3 -similar to that of mutant 1-A shown. Such variation was not observed in the At4CL2::GUS line. When I grew seeds from the original T5 generation of the At4CLl::GUS line, 10 day-old seedlings also showed variation of GUS expression which was removed by 5-aza treatment (Figure 3-12). This is also supportive of my suggestion that the At4CLl::GUS mutants we identified are epimutants that arose in the population by epigenetic silencing of the transgene. -Aza + Aza MB 4 Figure 3-12 GUS expression in T5 generation seedlings of the At4CLl::GUS line in the presence and absence of 5-aza. 3.2.2.5 Southern Blot Analysis of Mutant Lines To investigate the methylation status of the transgene promoters in the mutant lines, I used methylation sensitive isoschizomer restriction enzymes such as MspVHpall and Mbo\/Sau3A\ to restrict the genomic DNA from mutants 1-A and 2-8 respectively. Mspl is only inhibited when the outermost cytosine in its recognition site (CCGG) is methylated, allowing detection of methylation in a non-CG context, and is otherwise is -54-able to cut at the recognition site. However, the Hpall is inhibited by methylation of either cytosine o f this recognition site allowing detection of C p G and G p N p G methylation (Cao and Jacobsen 2002). A s shown in Figure 3-13, there is a single MspllHpall ( T C C G G T ) restriction site in the 1 kb At4CLl::GUS promoter, whose cleavage in combination with cleavage in the T - D N A and GUS MspllHpall sites would result in generation of 266-bp and 870-bp promoter fragments. Cleavage of the promoter of endogenous At4CLl gene is predicted to yield 1200-bp and 228-bp promoter fragments. Several other recognition sites in the transcribed regions of GUS and At4CLl result in ~ 88-245 bp fragments. I hybridized Southern blots of Mspl or Hpall restricted genomic D N A from mutant and control lines with two different probes spanning different areas of the 4CL1 r.GUS transgene. A s shown in Figure 3-13, probe #1 hybridized only to 1200-bp fragments in wild-type D N A cut with either enzyme, while the probe hybridized to 1200-bp and 870-bp fragments in the At4CLl::GUS control line after both Hpall and Mspl digestion, showing that the MspllHpall restriction site was not methylated in this line. In the line 1-A also, probe #1 detected a 1200-bp band but this fragment would co-migrate with a smaller 1136-bp band, resulting from the fusion of 266 and 870 bp bands due to lack of cleavage at the methylated Mspl/Hpall site. The ~1136-bp band in Hpall restricted D N A of line 1 - A is clearly thicker and more intense than the band of similar size of the same D N A restricted by Mspl, suggesting the presence o f the predicted doublet band. This indicates C G methylation of the recognition site and partial C N G methylation o f the same site. Similarly, probe #2 hybridized strongly to the expected 870-bp and 260-bp fragments in the D N A from At4CLl::GUS line and weakly to the 1200 bp fragments of the endogenous gene generated by both Hpall and Mspl digestion of At4CLl::GUS control D N A . In contrast, hybridization of #2 to digested genomic D N A isolated from mutant 1-A roots and seedlings revealed a 1136-bp band that was stronger in the Hpall restricted D N A and also a band bigger than 1200 bp that was unique to the Hpall restricted D N A (bands marked with asterisks, Figure 3-13). The sizes o f these fragments suggest that they arose as a result of the inability of Hpall to cut its recognition site within the At4CLl::GUS and/or endogenous At4CLl promoters. Methylation of other - 5 5 -Mspl sites upstream of the At4CLl::GUS transgene in the T - D N A may created a >1200-bp band. Wt A14CL1 .:• GUS 1-A Seeding 1-A 1-A At4CLl Root Seeding ;.• GUS M H M H M H ,—I 136 bp J 870 bp Probe #1 266bp 1200 bp Probe #2 M / H 1200 bp M / H 2 2 8 M / H . 8 5 J L At4CLl promoter At4CLl transcribed region (Endogenous gene) 128 88 M / H 8 7 0 bp M / H 266 M / H 2 4 5 T - D N A sequences G U S - O R F Probe #1 Probe #2 Figure 3-13 Southern blot analysis of methylation status of the At4CLl promoter in mutant 1-A seedlings. The positions of radiolabeled probes #1 and #2 are shown below restriction maps of the endogenous At4CLl gene and the At4CLl::GUS transgene. M / H - 5 6 indicates Mspl and Hpall respectively. Sizes of predicted restriction fragments are given in bp. Probe #2 was more biased towards the transgene sequences including the GUS gene. I conducted a similar experiment to investigate the methylation states of the At4CL2 promoter in the mutant 2-8. In this case, I used the MboI/Sau3Al isoschizomer pair, with Sau3A being methylation sensitive and its specificity determined by bases flanking the G A T C core recognition site. A s shown in Figure 3-14, there is a single G A T C MbollSaubAl recognition site in the At4CL2 promoter. This analysis showed that digestion of mutant 2-8 genomic D N A with Sau3A generated an At4CL2 promoter fragment unique to the mutant. The size of this fragment (1165bp, Figure 3-14) is consistent with failure of Sau3A to cut at the MbolSaulAl recognition site in At4CL2 promoter, resulting of the fusion of 979 bp and 186 bp fragments to yield a 1165 bp Sau3A fragment in the mutant. This fragment was not observed in Sau3A digested D N A from the A4CL2::GUS control line. These data are consistent with increased methylation of the At4CLl and At4CL2 promoters in epigenetic mutant lines in which GUS expression is silenced. mutant 2-8 4CL2::GUS s s s i I ™ 1116 1191 M M AJ4CL2 endogenous gene probe]  1165 b p -S S S 1 S ' ''L___ 3 0 1 I 1 5 7 I 979 | 186 I 20 I ^ 979 J-p -Mfff At4r.T.7 nrnrnnter O U S S=Sau3Al , M=MboI Figure 3-14 Southern blot analysis of methylation status of the A\4CL2 promoter in the mutant 2-8 seedlings. The position of the radiolabeled probe is shown between restriction maps of the endogenous At4CL2 gene and the At4CL2::GUS transgene. S, MboIISauiAl restriction sites. Sized of predicted restriction fragments are given in bp. - 5 7 -3.2.2.6 Southern Blot Analysis of Transgene Copy Number In order to determine the copy number of transgenes in the At4CL::GUS transgenic lines, Southern blot analysis was conducted. Restriction enzymes indicated in Figure 3-15 do not cut in the At4CL promoter::GUS transgene contained in the T - D N A , ensuring only one band to be detected per T - D N A molecule using G U S sequences as probe. Multiple bands present in each digest suggest the presence of ~ six 4CL1::GUS and two 4CL2:: GUS transgenes 4CL1::GUS Wt 4CL2..GUS Hindlll Spel Hindlll BamHl Sad 5kb Figure 3-15 Investigation of At4CL::GUS transgene copy number in the transgenic lines. Southern analysis was performed using the enzymes indicated. Bands corresponding to the At4CLl::GUS transgene should be larger than 6 kb and those corresponding to the At4CL 1::GUS transgene should be larger than 5.5 kb. - 5 8 -3.2.2.7 Northern blot analysis of mutant lines To investigate the expression status of the endogenous At4CLl gene in the mutant 1-A and a representative 4CLl::dhlA/At4CLl::GUS line, both of which showed transgene silencing, northern blot analyses were conducted using R N A extracted from 10 day old seedlings and a At4CL 1 -specific probe. The results, consistent among several northern blot experiments, showed that the endogenous At4CLl gene was not down regulated (lanes 1 and 4, Figure 3-16) relative to expression in control plants. 1 2 3 4 5 6 7 8 ft mm 4CL/Act in I I4CL/Act in 1 2 3 4 5 6 7 8 Figure 3-16 Northern analysis of At4CLl gene expression in At4CLl::GUS lines. Total R N A was extracted from 1) mutant 1-A, 2) Wild-type, 3) At4CLl::GUS, 4) At4CLl::dhlA, 5) mutant s i l l - 1 , 6) mutant hogl-1, 7) Mutant 2-8, 8) Wild-type. The blot was hybridized to an /l /¥CZJ-specific probe, then stripped and hybridized to an actin probe to control for R N A loading. Intensities of At4CLl signals relative to actin signals are shown at the bottom. - 2 0 % difference differences between the samples were not - 5 9 -significant in repeated tests as indicated by variability in two wi ld type samples #2 and # 8. I also investigated the expression status of the endogenous At4CL2 gene in the mutant 2-8 and a representative 4CL2: :dhlA/At4CL2: :GUS line, using an At4CL2-specfic probe. Again, the results were consistent among multiple experiments, but in contrast to the At4CLl lines, showed that the endogenous At4CL2 gene was significantly down regulated in the silenced lines (lanes 1 and 4, Figure 3-17). Silencing of the endogenous gene was specific to mutant 2-8, since At4CL2 expression was not affected in mutant 1-A , affected in At4CLl::GUS expression (Figure 3-17, lane 5). This suggests that cis-silencing (affecting the transgenes alone) occurred in the mutant 1-A and the 4CL1::dhlA/At4CLl::GUS line, while both cis- and trans- silencing occurred in the mutant 2-8 and the At4CL2::dhlA/At4CL2::GUS line. 1 2 3 4 5 r>- l _ _ ! • I , t __ 1 2 3 4 5 Figure 3-17 Northern analysis of At4CL2 gene expression in At4CL2::GUS lines, Total R N A was extracted from 1) mutant 2-8, 2) wild-type, 3) At4CL2::GUS, 4) At4CL2::dhlA, 5) mutant 1-A lines. The blot was hybridized to an _4.4CZ,2-specific probe, then stripped and hybridized to an actin probe to control for R N A loading. Intensities of At4CL2 signals relative to actin signals are shown at the bottom. Band intensities were measured in this experiment, and two replicates. The average intensities ± standard error are shown at the bottom 3.2.2.8 Effect of two silencing modifier mutants on 4CL::GUS transgene silencing To better understand the silencing mechanisms of the At4CL::GUS transgenic lines, I crossed the mutants 1-A and 2-8 to the Arabidopsis hogl and sill mutants that are defective in homology-dependent gene silencing of the Arabidopsis CHS gene (Furner et al, 1998). The hogl (homology dependent gene silencing 1) is a partial loss of function mutation in the S-adenosyl-L- homocytosine hydrolase enzyme that generally inhibits trans-methylation metabolism. In the hogl-1 mutant the activity of this hydrolase enzyme is reduced (Rocha et al. 2005). The s i l l mutant (modifiers of silencing 1, Furner et al., 1998) is a new allele of histone deacetylase (AtHDA6) and mutations in the this gene influence histone acetylation levels and reactivate silent and methylated transgenes and endogenous repeats and confer partial demethylation of C G sequences at specific regions of the genome (Probst et al, 2004). Crossing of mutants l -A and 2-8 as male parents into the hogl-1 and sill-1, did not restore the expression of At4CLlr.GUS or At4CL2::GUS transgenes in the kanamycin resistant F2 generation. These results suggest the existence of distinct gene silencing pathways for homology dependent CHS and At4CL gene silencing pathways. [ 3.3 D I S C U S S I O N ' 3.3.1 At4CLl and At4CL2 promoters direct similar GUS expression patterns in the vascular tissues. Fusion of At4CLl and At4CL2 promoters to the GUS reporter gene and generation of transgenic Arabidopsis plants showed that the promoters direct overlapping patterns of vascular-specific GUS expression in different organs (Figure 3-1), similar to the pattern described for Arabidopsis plants expressing a parsley ^CZipromoter-GL / iS fusion (Lee et al, 1995). The overlapping patterns of vascular-specific expression directed by At4CLl and At4CL2 promoters were predictable considering the presence of multiple common conserved cis regulatory elements, and similar expression patterns characterized by northern analysis (Ehlting et al, 1999). Consistent with previous northern analysis, I * -- 6 1 -found the strongest GUS expression of At4CLl::GUS and At4CL2::GUS transgenes in the roots of the seedlings, and strong GUS expression in the vasculature of bolting stems. Taken together with the high identities of the At4CLl and At4CL2 coding sequences and the similar substrate specificities of the recombinant enzymes (Ehlting et al, 1999), the similar expression patterns dictated by their promoters suggest functional redundancy of these two genes. Since 4CL plays an essential role in lignin biosynthesis, loss of endogenous 4CL expression would be predicted to lead to a severe phenotype. However, functional redundancy is a likely explanation for the lack of an obvious phenotype in mutant ,2-8, in which the endogenous At4CL2 gene was down regulated (Figure 3;-17) together with the At4CL2::GUS transgene (Figure 3-9). . 3.3.2 Evidence that mutants affected in At4CL::GUS expression are epigenetically silenced epimutants. We screened EMS mutagenized M2 populations and found multiple putative mutants impaired in 4CL1::GUS or 4CL2::GUS transgene expression. These mutants showed different intensities and patterns of reduced developmental GUS expression (Figure 3-7), initially suggesting potential lesions in genes required to activate developmentally regulated GUS expression. Selected mutants were crossed to' the progenitor wild-type transgenic lines, and the inheritance of the GUS expression phenotype was analyzed in Fl and F2 generations. The results of the analysis of three mutants in the At4CLl::GUS background (summarized in Table 3-1) showed non-Mendelian inheritance of the phenotypes. I focused on the mutant 1-A as a representative mutant of the At4CLl::GUS line showing root-specific lack of GUS expression in seedlings, but global loss of GUS expression in the mature plants (Figure 3-8) and mutant 2-8 as a representative mutant in the 4CL2..GUS transgenic background showing apparent complete loss of developmental GUS expression (Figure 3-9). Genetic analysis of this mutant also showed non-Mendelian inheritance (Dr. J. Ehlting, personal communication). Based on the non-Mendelian inheritance of the mutant phenotypes we hypothesized the involvement of transgene silencing in generating the GUS expression phenotypes, rather than mutations in second genes required for 4CL promoter activation. - 6 2 -Sequencing of transgenes in the mutant plants confirmed sequence integrity, and after several generations in which the lack of GUS expression was stably inherited, treatment of mutant lines with 5-aza efficiently restored GUS expression, suggesting reversible' silencing of the transgenes, associated with changes in methylation status of DNA (Figure 3-11), since this reagent is a well-characterized inhibitor of cytosine methylation (Fieldes 1994). Consistent with this interpretation, Southern blot analysis of mutants confirmed increased methylation of the respective 4CL promoters in the 1 - A and 2-8 mutants (Figure 3-13 and 3-14). Taken together, these data strongly suggest that the mutants we isolated are epigenetic mutants (epimutants) of the At4CL::GUS transgenes, and that epigenetic silencing is mediated by methylation of promoter DNA. 3.3.3 At4CL::GUS transgene silencing is specific and occurs naturally. i GUS expression was stable in the At4CL2::GUS line from the T1-T16 generation, but introduction of an At4CL2::dhlA construct into At4CL2::GUS transgenic plants in the T5 generation resulted in GUS down regulation in >50% of the lines similar to what we saw for At4CLl::dhlA lines (Figure 3-4). Also, none of the lines were sensitive to DCE treatment (Figure 3-2), which would be predicted if the At4CL2 promoters were inactive in driving dhlA expression. I hypothesized that both of the At4CL2:\dhlA and At4CL2::GUS transgenes were silenced in these lines, triggered by introduction of the At4CL2:\dhlA transgene. Treatment of these lines with 5-aza made them more sensitive to DCE treatment (Figure 3-6) and also restored GUS expression (Figure 3-5). Similar results were observed for At4CLl::dhlA lines. Also, the occurrence of GUS down regulation similar to the mutant 1 -A phenotype in the non-mutated original T5 generation of At4CLl::GUS transgenic plants became apparent after three years reservation of seeds, and this phenotype was reversed by 5-aza treatment (Figure 3-12). These data suggest that silencing of 4CL::GUS transgenes occurred in At4CLl::GUS and At4CL::dhlA lines in the absence of EMS mutagenesis, and that'mutants such as 1-A and 2-8 represent pre-existing epigenetically silenced variants. Epimutants were isolated from seedlings grown oh kanamycin containing media. This probably selected against epimutants in which the entire T-DNA was methylated. On the other hand, the occurrence of kanamycin resistant epimutants indicates that the -63 -region of t ie T - D N A containing transgene had been specifically silenced. Apparently; the silencing machinery specifically targeted the At4CL::GUS transgene. Southern analysis showed that methylation of the transgene was restricted to their promoters (Figures 3-13 and 3-14). \ H D G S related mechanisms have been reported to be involved in the regulation of gene expression of gene family members in non-transgenic plants (Martienssen, 1998; Luf f et al, 1999; Chandler, 2000). The down regulation of endogenous At4CL2 gene expression in the mutant 2-8 was specific to At4CL2 and At4CLl was not affected (Figure 3-16 line 7). Conversely, At4CL2 gene expression was not affected in the mutant 1-A, which affected At4CLlr.GUS expression (Figure 3-17 line 5). To confirm the specificity of At4CL::GUS silencing, I crossed mutant 1-A to the At4CL2r GUS'line and mutant 2-8 to the At4CLl::GUS'\\VLQ to see i f silencing of the At4CLl::GUS transgene could trigger silencing of At4CL2::GUS and vice versa. FT seedlings showed GUS expression in both crosses suggesting that silencing of At4CLl::GUS may not affect the expression of the At4CL2r.GUS transgene and vice versa (data not shown). However, an alternative explanation for these results cannot be ruled out. In summary, these data suggest that transgene-triggered gene silencing occurred in the lines investigated, and that this silencing is gene family member specific. j 3.3.4 Silencing of At4CLll and At4CL2 (trans) genes is best explained through a 5 ' -UTR threshold mechanism. Although T G S and P T G S are mechanistically related methods of silencing (Fagard and Vaucheret 2000; Sijen et al, 2002), specific methylation of multiple identical promoters of (trans) genes and 5-aza-restorable expression point to T G S versus PTGS of At4CL::GUS transgenes. To evaluate the critical characteristics of the transgene loci that could have directed T G S of At4CL::GUS and At4CL::dhlA transgenes we considered variables such as transgene locus structure, chromosomal environment of transgene and transgene expression level that have been shown to affect T G S (Fagard and Vaucheret 2000). ; The presence of inverted repeat sequences in transgenes (Wang and Waterhouse, 2000, Chuang and Meyerowitz, 2000; Mette et al, 2000), and direct or inverted repeat ' . i -64- . . ' i arrangements of multiple T-DNAs (Hobbs et al, 1990, Assaad et al, 1993, Jorgensen et al, 1996) have been associated with silencing. However, tandem T-DNA configurations and arrangements may not be sufficient to trigger TGS (Lechtenberg et al, 2003, Shubert et al, 2004). Since TGS of At4CLl::GUS line became apparent spontaneously in the T7 generation, the arrangement of multiple T-DNAs carrying the At4CLl::GUS transgene could play a role. However, regardless of transgene copy number and arrangement, TGS of the At4CL2::GUS transgene was detectable only after introduction of an independent At4CL2::dhlA transgene .into this line. Therefore, it is hard to consider locus arrangement of the transgenes to be the primary cause of At4CL2::GUS transgene silencing. TGS has also been attributed to the chromosomal environment of transgenes (Jones et al, 1985; Peach and Velten 1991; Lohuis et al, 1995; Jakowitsch et al, 1999: Day et al, 2000). Also, abrupt change in GC content of flanking sequences is considered to be a factor affecting silencing (Meyer et al, 1993; Matzke and Matzke 1998). However, similar to other reports (Jorgensen et al, 1996; Hobbs et al., 1993; Schubert et al, 2004) it seems that transgene chromosomal position may not have played a primary role in triggering At4CL2::GUS or At4CL2::dhlA silencing. If chromosomal sequences flanking the At4CL2::GUS transgene locus, for example, had a role in its silencing, this should have been effective before the insertion of the second transgene (At4CL2::dhlA) or during the 14 generations of stable transgene expression in this line. However, we may attribute the low rate (0.125%) of silencing in the At4CL2::GUS line (represented by mutant 2-8) to the spontaneous TGS (Prols and Meyer, 1992). Then, increased frequency of transgene silencing At4CL2::dhlA line could be attributed to the trans-silencing through D N A pairing interaction (Matzke et al, 1994; Bender et al, 1995; Park et al, 1996). For the At4CLl::GUS line, we cannot rule out the involvement of transgene locus environment in TGS, since transgene expression was less stable in this line. Some reports (Lindbo et al, 1993; Jorgenson et al, 1996; Wassenegger et al, 1998) suggest that surpassing a threshold level of transgene expression may result in silencing. This hypothesis is supported by a correlation between frequency of silencing and transgene promoter strength (Que et al,. 1997) and also the report of stronger silencing in homozygous transgenic plants compared with hemizygous siblings (Vaucheret et al, 1998). In a recent report it is suggested that i f transgene expression - 6 5 -levels exceed a specific threshold, P T G S wi l l be triggered, but at the sub-threshold expression levels, the expression from mult icopy transgenes is additive (Schubert et al, 2004). Only after retransformation of the At4CL2::GUS line with the additional At4CL2::dhlA construct, the At4CL2::GUS transgene was silenced, suggesting trans-inactivation phenomena (Matzke 1993) has happened. Based on the threshold hypothesis, this could be explained on the basis that the transgene transcript copy number may not have surpassed the hypothetical threshold for silencing in At4CL2::GUS plants until the introduction of the second transgene (At4CL2::dhlA) with a common At4CL2 promoter and 5 ' - U T R sequence. We suggest that 5 ' - U T R common to both transcripts was detected by a At4CL2 gene-specific threshold sensing system, causing trans-s\\zncmg of At4CL2:.reporter transgenes and the endogenous At4CL2 gene in the At4C2::dhlA and mutant 2-8 lines (Figure 3-17). Sporadic up regulated At4CL2::GUS transgene expression in the vicinity of enhancers may have elevated transgene expression above the threshold and resulted in epimutants like mutant 2-8. Based on this hypothesis we predict that the i endogenous At4CL2 promoter as well as the At4CL2 r.GUS transgene promoter should be hyper-methylated, but the analysis shown in Figure 3-14 could not distinguish between the endogenous and transgene promoters. This hypothesis could also be further tested by introduction of At4CL2 5 ' - U T R sequences in At4CL2::GUS plants under the controfof a strong promoter other than At4CL2 promoter. We would predict this to result in a co-suppression like phenomenon (Napoli et al, 1990). Contributions of aberrant promoter transcripts to trans-TGS of un-linked transgene has also been reported (Mette et al, 1999, and 2000; Sijen et al, 2001) and it is widely accepted that aberrant R N A species direct, some, i f not all o f the heterochromatin formation in the plants (Bender 2004). The 5 ' -UTRs of At4CLl and At4CL2 genes are not predicted to form d s R N A structures, but we-cannot rule out the involvement of chimeric transgene transcripts in the silencing. Taken together, our data are not consistent with the involvement of transgene structure or chromosomal environment in the T G S we observed. Rather it seems At4CL2::GUS and At4CL2::dhlA T G S is mostly consistent with a transcript threshold mechanism, which is able to detect common 5 ' -UTRs in transcripts derived from different genes. - 6 6 -3.3.5 Unanswered aspects 6iAt4CLll and At4CL2 (trans) gene silencing A n alternative explanation for the observed silencing is that At4CL2 5 ' - U T R R N A sequences may have surpassed the threshold and/or were involved in the formation of an aberrant R N A , resulting in d s R N A triggered activation of the P T G S system and simultaneous promoter sequence methylation. This would explain the silencing of the endogenous At4CL2 gene. This scenario may not apply for the silencing of the At4CLl::GUS line, as the endogenous At4CLl gene was shown to be unaffected (Figure 3-16). • • . ' There is no clear explanation for the lack of endogenous At4CLl gene down regulation in lines silenced for At4CLl::GUS expression. The 1-kb At4CLl promoter sequence used to generate At4CLl::GUS transgene has 27% G . C pairs content including 15 C G dimers and 18 C N G trimers that could be substrates for methylation. In the 1.55-kb At4CL2 promoter, there are 37% G.C pairs including 19 C G dimers and 54 C N G trimers. If promoter methylation has been triggered by d s R N A produced from the transgenes transcripts, methylation of the target At4CLl promoter would not be as dense as methylation of the target At4CL2 promoter simply because of lower number of C G and C N G sequences. Therefore, less At4CLl promoter methylation may have resulted in weaker T G S manifested by unaffected endogenous At4CLl gene expression. The root-specific silencing of At4CLl::GUS in seedlings is also puzzling. However, the At4CLl gene has been shown to have the highest expression level in the root (Ehlting et al, 1999). This may have made it easier for At4CLl::GUS transgene expression to surpass the threshold level of 5 ' - U T R containing R N A in the root, resulting in preferential At4CLl::GUS silencing of the transgene in that organ. Elevated 4CL1 gene expression in the bolting stem and elsewhere later in development (Elthing et al, 1999) may have resulted the threshold of expression being surpassed, resulting in T G S (Figure 3-8). Therefore lack of At4CLl r.GUS line silencing in the veins of cotyledons may be attributed to the lower expression level of At4CLl gene in this area. However, it is not clear why silencing At4CLlr.GUS transgenic in the mature plants is not meiotically heritable to the cotyledons of the next generations. - 6 7 -3.3.6 Conclusion At4CLl and At4CL2 genes are closely related and show overlapping expression patterns. Although there are reports of silencing between alleles (paramutation; Brink 1973, Matzke and Matzke, 1993) and duplicated members of gene families (Matzke 1996), cross silencing between these two genes did not occur, suggesting that At4CL transgene silencing mechanisms are able to distinguish between related family members possibly through non homologous promoter sequences. Mechanisms underlying transgene silencing still are not fully known. The hogl and sill mutants, that affect the pathway used for CHS silencing, did not affect At4CL::GUS transgene silencing and At4CL gene expression (Figure 3-16), suggesting that that distinct mechanisms underlie silencing of these genes, both involved the phenylpropanoid pathway. It is now clear that dsRNA acts as a sequence specific signal for TGS by promoting promoter methylation (Mathieu and Bender, 2004). If such a hypothetical dsRNA were GUS specific, we should have seen signs of GUS down regulation in F l plants derived from crossing mutant 1-A to At4CL2::GUS plant's or mutant 2-8 to At4CLl::GUS plants. The lack of such down regulation, and other clues lead us to favor a role of gene-specific 5-UTRs in triggering an R N A i system and subsequent TGS of At4CL genes. However, regardless of the mechanism of silencing, At4CLl::GUS and At4CL2::GUS lines illustrate the diversity of silencing phenomena that may be encountered upon transgenes insertion and expression. - 6 8 -C H A P T E R 4 D E V E L O P M E N T A L A N D WOUNDING RESPONSE CIS E L E M E N T S IN AT4CL2GENE 4.1 INTRODUCTION The general phenylpropanoid pathway in plants channels carbon flow to different branch pathways via sequential actions of the phenylalanine ammonia-lyase ( P A L ) , cinnamate-4-hydroxylase (C4H) and.4-coumarate:CoA ligase (4CL) enzymes. Genesthat encode these enzymes are coordinately activated in response to developmental cues^and to non-developmental signals such as wounding or irradiation with U V light (Dixon) and Paiva 1995). Members of 4-coumarate:CoA ligase (4CL) (EC 6.2.1.12) gene families encode isoenzymes that catalyze the formation of C o A esters of cinnamic acids. These esters may be used as substrates in specific branch pathways, such as those leading to the synthesis of flavonoids and lignin (Hahlbrock and Scheel, 1989; Dixon.and Paiva, 1995; Douglas,. 1996). 1 Four members of the At4CL gene family, At4CLl, At4CL2, At4CL3 and At4CL4 have been identified and cloned in Arabidopsis (Lee et al, 1995; Ehlting et al, 1999; Hamberger et al, 2004). A t 4 C L l and A t 4 C L 2 are the closest family members with 83% identity at the amino acid level (Hamberger et al, 2004). At4CL genes appear to have specialized developmental and biochemical functions. At4CLl and At4CL2 and the isoenzymes encoded by these genes are associated with the biosynthesis of lignin, and structurally related soluble or cell wall-bound phenylpropanoid derivatives in' the xylem tissues of the plant vascular system (Lee et al, 1995, Mizutani etal, 1997, Ehlting et al, 1999). The A t 4 C L 3 enzyme may participate in the biosyhthetic pathway leading to flavonoids and the At4CL3 gene is mostly expressed in aerial organs like flowers1 and mature leaves (Ehlting et al, 1999) where flavonoids play a major role in U V protection. At4CL4 is expressed in low level in aerial parts of the plants (B. Hamberger, unpublished). In addition to developmental expression, expression of 4CL genes in different plants is also activated by external stimuli such as wounding, pathogen infection, and U V irradiation (Douglas et al, 1987;. Schmelzer et al, 1989). In Arabidopsis, At4CLl and At4CL2 transcripts accumulate rapidly but transiently in response to wounding while At4CL3 m R N A levels are not affected (Ehlting et al, 1999). The response of At4CL4 to wounding is yet unknown. . ' A typical approach used to identify cis-regulatory elements controlling plant gene expression is to fuse target gene promoter sequences to a reporter gene, and assay reporter gene expression in transgenic plant lines. Commonly, enhancers and core promoter elements are located in the promoter proximal region. When promoters containing these elements are fused to the reporter gene, they typically drive reporter gene expression in specific tissues, organs and cells and/or drive expression in response to external stimuli. There are also examples of the presence of regulatory elements within exons, introns or other areas (Douglas- et al, 1991; Zhang et al, 1994; Gidekel ei al, 1996; de Boer et al, 1999; Ito e. al, 2003). • ! Using this approach, the locations of cis-regulatory elements in the parsley (Petroselinum crispum) 4CL1 promoter that direct developmental expression have been defined (Hauffe et al, .1991; Hauffe et al, 1993; Neustaedter et al, 1999). Some of these 4 C L elements, termed A C elements or P and L boxes are highly conserved in phenylpropanoid gene promoters (Logemann et al, 1995; Douglas, 1996; Raes ei, al, 2003), are binding sites for M Y B transcription factors (Feldbrugge et al, 1997), and are also found in the promoters of At4CL genes (Ehlting et al, 1999; Hamberger 1 and Hahlbrock, 2004). Here, I show that Arabidopsis thaliana (Ler) At4CLl and At4CL2 promoter::GUS fusions direct developmental G U S expression to the vasculature of leaves, stems,: and roots, whereas the At4CL4 promoter does not direct detectable developmentally regulated G U S expression. I report the locations of cis-regulatory elements controlling, developmental and wound-induced expression of the At4CL2 gene. Using promoter deletions, I identified the locations of c/s-elements directing developmental expression in upstream promoter regions where they were expected. R N A analysis has shown rapid wound responsiveness of the. At4CL2 gene (Ehlting et al, 1999), and data presented here show that At4CL2 expression is activated in a biphasic manner, with early and late responses. However, At4CL2 promoter::GUS expression in wounded transgenic plants did not mimic this response. In contrast, fusions including both the promoter and transcribed region of the At4CL2 gene to G U S directed wound-induced expression, but - 7 0 -only the late wound response was detected. Through elimination of different introns I found multiple domains within the transcribed region containing positive and negative regulatory sequences involved in wound responsiveness of the At4CL2 gene. We also report lack of developmental G U S expression driven by the At4CL4 promoter but show its strong early and persistent wounding response. 4.2 RESULTS 4.2.1 Developmental and wound-induced expression patterns directed by At4CL Promoter::GUS fusions ! Transgenic plants containing At4CLl, At4CL2, At4CL3, and At4CL4 promoter::GUS constructs (Figure 4-1, C) were generated and introduced into Arabidopsis plants by Drs. J. Ehlting and B . Hamberger (unpublished), and I performed histochemical G U S assays on multiple transgenic homozygous lines for each construct. Ten-day old seedlings containing At4CLl::GUS and At4CL2::GUS constructs showed similar patterns of developmentally regulated G U S expression throughout the plant,J and largely restricted to the vasculature (Figure 4-2, f & g; Figure 3-1). Expression of At4CL3::GUS was not restricted to the vascular system, but instead appeared to be associated with leaf and stem surfaces, and was especially high in young leaves i and upper part of hypocotyls (Figure 3-1) consistent with a function hypothesized for 4CL3 in flavonoid biosynthesis (Ehlting et al., 1999). Developmental G U S expression iri the mature leaves of At4CL4::GUS line was not detectable in multiple line's. • Seedlings and leaves from three to four-week old mature plants of representative transgenic lines for the constructs in Figure 4-1, as well as a 35S: :GUS control line, were examined in parallel for developmentally regulated and wound-induced G U S expression. Leaves were wounded in planta for 2 to 72 hours before assays were carried out for G U S expression. The 35S control line showed predicted constitutive developmental G U S expression and lack of wound-induced expression (Figure 4-2, a). The At4CL4::GUS line responded to wounding by less than 2 h post-wounding, but did not show! any developmentally regulated expression (Figure 4-2, h). The At4CL3::GUS line showed no wound response while the At4CLlr.GUS line showed developmental expression' and responded to wounding only after 72 h, with no wound-induced expression detectable after 2 h (Figure 4-2, g). A) At4CL2 promoter X b a l At4CL2 transcribed region E l E2 E3 E4 B) C) 110bp At4CL2 promoter r_ 420bp At4CL2 promoter 750bp At4CL2 promoter 950bp At4CL2 promoter 1.6 kb At4CL2 promoter X b a l At4CL3 promoter At4CLl promoter At4CL4 promoter GUS HObp construct 420bp construct 750bp construct 950bp construct =£> 1.6kb construct At4CL3::GUS At4CLl: :GUS At4CL4::GUS Figure 4-1 Schematic representation of At4CL promoter-Gt/S' fusion constructs, A) At4CL2 gene B) At4CL2 promoter-GUS fusion constructs and C) Structure of At4CLl, At4CL3 and At4CL4 promoter-GfJS' fusions in transgenic plants provided by Drs. J. Ehlting and B. Hamberger. Empty bars represent promoter fragments and solid bars represent exons (E1-E4). The name of each clone that is referred to in the text, is indicated at the right. - 7 2 -2 h post- 72 h post developmental Wounding Wounding expression • • • • — J . ; • • -. ™ r ' • ml 2h 24h 48h 72h f Figure 4-2 Wounding response and developmental expression o f At4Cl promoters. Histochemical assays for G U S expression were performed on representative lines with the following transgenes: a) 35S::GUS line b) 1 lObp of_4^CZ2promoter.v GUS line , c) - 7 3 -420bp of At4CL2 promoter::GUS line, d) 750bp of At4CL2 promoter::GUS line, e) 950bp of At4CL2 promoter::GUS line, f) 1.6kb of At4CL2 promoter::GUS line, g) 1 kb o f , 4 ^ C Z 7 p r o m o t e r : l i n e , h) l kb o f ^ C Z 4 p r o m o t e r . \ G L # l i n e . ; Finally, while the 1.6kb At4CL2 promoter directed developmental G U S expression as expected in this line, I did not see any G U S expression in response to wounding by 2 i or 72 h directed by this promoter (Figure 4-2, f). These results indicate that, while At4CLl and At4CL2 promoters direct developmental^ regulated expression as predicted from previous experiments, wound induced expression directed by the promoters is distinct from patterns of endogenous wound-induced expression (rapid up-regulation of transcript levels by l h post-wounding) previously observed (Ehlting et al, 1999). . • .• • • i 4.2.2 Evidence for positive developmental and negative wound inducible cis- | regulatory elements in the At4CL2 promoter I generated a series of truncated At4CL2 promoter fragments, fused them to G U S (Figure 4-1, B) and 10 day-old seedlings of 8-12 transgenic lines made from each construct were assayed for G U S expression. Developmental expression and wound-induced G U S expression in seedlings and leaves from three to four week-old mature plants of representative lines for each construct are shown in Figure 4-2,,b-e. ' M y data showed that the 110-bp At4CL2 promoter (Figure 4-1, B) is not sufficient i to drive G U S expression on a visible level (Figure 4-2, b). The 420-bp promoter fragment (Figure 4-1, B) directed weak G U S expression only in some o f the lines (Figure 4-2, c). However, lines containing the 750bp construct (Figure 4-1, B) showed clear visible developmental G U S expression in the vascular tissues of the seedlings and mature leaves (Figure 4-2, d) indicating the presence of strong positive regulatory element(s) in the -750 to -420 bp interval of the At4CL2 promoter region. A l l the lines containing the 950bp construct (Figure 4-1, B) showed strong developmental G U S expression in the root but apparent ectopic expression in the cotyledons and mature leaves outside of the vascular tissues (Figure 4-2, e)- in a pattern reminiscent of At4CL3::GUS expression (Figure 3-1). This expression !was distinguishable from 35S::GUS expression, especially in seedlings (compare Figures 4-2, a and e). Thus, -950/-750 bp regions of the At4CL2 promoter appear to harbor one or i -74-more positive regulatory element(s) that drive expression very similarly to the At4CL3 gene and was called At4CL3-\\Ve positive cis -element (3LE). A s shown above, transgenic lines carrying a 1.6 kb At4CL2 promoter: :GUS fusion (Figure 4-2, f) showed strong vascular-specific expression in both seedlings and mature leaves. The G U S expression intensity o f this construct seemed stronger than that of the -750 bp construct and was clearly specific to the vasculature system (Figure 4-2, d and f). It seems, elements that enhance developmental expression or repress the ectopic expression are located between -950 bp and -1.6 kb sequences. Wounding treatment o f these lines showed that only the 950-bp At4CL2 promoter: :GUS construct directed wound-induced G U S expression (Figure 4-2, e). Wound-induced G U S expression was evident at 2 h post wounding, but appeared transient and had disappeared by 72 h after wounding (Figure 4-2, e). Other promoter::GUS lines only showed stronger expression in some cases, but this was restricted to the veins in the wounded area (Figure 4-2, c, d, f). To examine the dynamics o f wound-induced expression driven by the 950-bp At4CL2 promoter::GUS construct in more detail, single leaves were wounded for different times over a period of 72 h, followed by staining for G U S expression. A s shown in Figure 4-3 for a representative leaf, wound-induced G U S expression peaked by 2-4 h, and was no longer detectable by 48 h. Taken together, these data suggest the presence o f a cis element(s) in the -950/-750 bp region of the At4CL2 promoter responsible for an early wound response while the region between -950 bp and -1.6 kb apparently contains element(s) that negatively affect the wound response. Figure 4-3 Developmental and wounding response expression o f 950bp At4CL2 promoter fused to G U S . The same leaf was wounded for the times shown before histochemical assay of G U S expression. 4.2.3 Cw-elements that specifying developmental and wound-induced expression in the At4CL2 transcribed regions To test whether elements directing developmental and/of wound responsiveness of At4CL2 are located within the transcribed regions of the gene, the At4CL2 gene including 950-bp of promoter and the entire transcribed region (except the 3 ' -UTR) were fused in-frame to G U S (Full length construct; Figure 4-4). 1.6 kb At4CL2 promoter At4CL2 transcribed region 3 ' - U T R E l E2 E3 E4 Full length Construct cDNA Construct 950bp promoter At4CL2 transcribed region . G U S At4CL2 c D N A Intron # 1 Construct Intron # 2&3 Construct E l •E1-+ E2 II E2+E3+E4 12 13 Figure 4-4 At4CL2 promoter (950 pb) plus At4CL2 transcribed region fragments fused to G U S . Empty bars represent promoter and solid bars represent exon (E1-E4). The name of each clone is indicated in front of it and is used in the text.. - 7 6 -Transgenic Arabidopsis lines containing this construct showed developmental GUS expression that was very similar to the 1.6-kb At4CL2 promoter::GUS line (Figures 4-5, a). a) ho 1,2 h r L y I -\ 1 -b) J * z11'" ^__^^-,-iJ^*,M_r ____ - A. T C) I'/ Am 2h A \ 48 h 1 24 h ( Figure 4-5 Developmental and wound-induced expression in transgenic lines containing 950-pb At4CL2 promoter and different transcribed regions fused to GUS. a) Full length At4CL2::GUS, b) At4CL2 cDNA::Gt/S, and c) At4CL2 intron #2&3::GUS lines. Developmental expression in seedlings is shown at the left. Four-week old leaves were wounded and GUS assays performed 2-72 h post-wounding, right-hand panels In contrast to the 1.6-kb promoter, however, the full-length construct directed wound-induced GUS expression that was detectable after 48-72 h, but no early wound response such as that observed for the -950-bp At4CL2 promoter GUS fusion was observed in these lines (Figures 4-5, a). Based on these results, we postulate the presence of a Late-Wound Response Element in the transcribed portion of the At4CL2 gene. To locate the area where the LWRE resides, I removed all of the introns from the construct by substituting the transcribed region of the At4CL2 full-length construct with the At4CL2 cDNA sequence (Figure 4-4). All of the transgenic lines containing the At4CL2-cDNA::GUS construct showed strong developmental GUS expression, mostly restricted to vascular tissue, but showed ectopic GUS expression in surrounding tissues as well (Figures 4-5, b). This developmental expression pattern was highly reproducible, being observed in most of the At4CL2-cDNA::GUS lines (Figure 4-6). However, wounding did not induce GUS expression in any of these lines (Figures 4-5, b). * Line 1, 4 3 4 '7 'f 5 6 7 a , ,0 Figure 4-6 Developmental GUS expression in mature leaves of nine independent At4CL2 cDNA:: GUS lines (2-10), All lines showed ectopic GUS expression outside of veins except line 1 with no GUS expression. These data suggest that a LWRE might be located in one of the At4CL2 introns. To test this, intron # 1 of the At4CL2 gene was inserted into At4CL2-cDNA: :GUS construct (Figure 4-4) but none of the Tl transgenic lines showed developmental or wound induced GUS expression. RT-PCR analysis showed expression of the transgene and sequence analysis did not reveal any mutation in the transgene transcript or promoter sequence. Introns # 2 and # 3 were also inserted into the At4CL2-cDNA::GUS construct (Figure 4-4) and transgenic plants containing this construct were assayed for developmental and wound-induced GUS expression. These lines showed strong vascular specific developmental GUS expression and activation of GUS expression in response to wounding at 72 h, although this response was weaker than the response seen in lines harboring the full-length line construct (Figures 4-5, c). We conclude that a LWRE resides in intron # 2 or # 3. 4.2.4 Biphasic wound induction of At4CL2 Expression Our results suggested that the kinetics of wound induced At4CL expression might be more complex than it was previously realized (Ehlting et al, 1999). I used semi-quantitative RT-PCR to test the expression of the endogenous At4CL gene family members in response to wounding in leaves. At4CLl and At4CL2 m R N A levels transiently increased 2.5 h post-wounding and then returned to a basal level, before again increasing to a maximum 48-72 h after wounding (Figure 4-7). At4CL4 transcript levels also increased by 2.5 h post-wounding but remained elevated for at least 12 h. At4CL3 expression was rapidly down regulated in response to wounding, but recovered to basal levels by 4 h post wounding. Thus, At4CL gene family members display a diverse set of responses to wounding. control Omin 15 min 2.5 h 4h 12 h 24 h 48 h 72 h Actin 25- cycle M4CU 26- cycle M4CI2 27- cycle A14CL3 29-cycle A14CIA 29-cycle ^ m m tggg . ^ j p mmm mmm mmm mmm '" m m m " "HP ^ f ^ ^ ^ w WMRffll ______ M l W H W i'.»WWilk*^ pi <•*»*• - — — — -. Accumulation of At4CL mRNA after wounding •Actin average - 4CL1 average 4CL2 average - 4CL3-Average •4CL4 average ,c> .sp sj> , jo Kx> x> -c-XT ^ tf <o *• VV A V*. co^ o \ < 5 V y *v »r "1 Time after wounding -79-Figure 4-7 Wounding response of At4CL genes analyzed by semi-quantitative RT-PCR. Wounding was performed on 3-4 week old Arabidopsis leaves, and RNA isolated at the given times after wounding. Cycle numbers were adjusted empirically for each gene so that amplification was in the linear range, and amplification of actin was used to control for variations in RNA amounts between samples. The experiment was repeated three times using leaves of independent plants and the averages of band intensities (pixel number as measured by Alpha Imager software) over the course of the experiment are shown at the bottom as mRNA level.' i 4.3 DISCUSSION • ' , 4.3.1 Differential and biphasic wound responsiveness of 4CL gene family members in Arabidopsis i Induced expression of phenylpropanoid genes plays a key role in plant responses to many environmental stresses by activating the biosynthesis of defensive compounds (Dixon and Paiva 1995). Wounding coordinately induces many phenylpropanoid pathway genes such as PAL, C4H, and 4CL (Dixon and Paiva 1995; Ohi et al, 1990; Lee et al, 1995; Bell-Lelong et al, 1997; Meyer et al, 1998; Mizutani et al, 1997). Arabidopsis offers a system to study mechanisms underlying stress activated phenylpropanoid gene expression,, and the At4CL gene family provides an opportunity to test whether different gene family members with potentially different biochemical functions (Ehlting et al, 1'999) respond differentially to environmental stimuli. At4CLl andv4^CZ2.mRNAs have been reported to rapidly but transiently accumulate in an hour post-wounding ini the detached Arabidopsis leaves while At4CL3 gene expression is reported to be not affected (Ehlting et al, 1999). In this study, I focused my attention on wound-induced At4CL gene expression, with an emphasis on At4CL2. Wound-induced gene expression may be activated by signaling pathways that partially overlap with those activated by other stresses such as drought, freeze'and osmotic stress (Reymond et al, 2000; Denekamp et al, 2003) and this overlap has been shown for pathogen-induced signaling pathways as well (Romeis et al, 1999). Plant hormones such as jasmonic acid (JA) and related compounds play a central role in rapid localized and systemic wound responses in plants (Farmer and Ryan 1992; Schaller 2001, -80-Turner et al. 2002; L i et al. 2002; He et al, 2005). J A - and ethylene-responsive elements and the S box, all of which confer responsiveness to elicitation, wounding, and pathogen infection have been found in the genes that are involved in l ignin biosynthesis (Rushton et al, 2002). It has been shown that parsley 4CL1 gene expression is activated by J A treatment (Ellard and Douglas, 1996) and stresses such as wound, U V , and pathogen infection activate At4CL gene expression in Arabidopsis (Ehlting et al, 1999). The activation of At4CL gene expression by wounding is part of a complex global response. Wounding induced up-regulated expression of 8% of the 8000 genes on an Arabidopsis microarray (Cheong et al, 2002), and wound-induced genes and proteins exhibit different induction kinetics in many plants. For example, a GFP-Nitri lase 1 fusion protein aggregates in the cells directly abutting the wounded area in 30-60 min post mechanical wounding (Cutler and Somerville, 2005). In wounded tomato plants, Phospholipase A activity has increased systemically in a biphasic manner, peaking at 15 min and again at 60 min post wounding (Narvaez-Vasquez et al, 1999). The peptide transporter (AtPTR3) gene on the other hand is activated in 4 h and gradually increases in response up to 24 h (Karim et al, 2005). The sugar transporter (AtSTP4) gene is also activated in 3 h post-wounding (Truernit et al, 1996; Meyer et al, 2004). Wounding also induces the expression of transcription factors that have recognition sites on the phenylpropanoid pathway genes (Hara et al, 2000, Cheong et al, 2002). It has been shown that the expression of AtMyb32 (Preston et al, 2004) and AtMyb4 (Jin et al, 2000), regulators of phenylpropanoid gene expression, was altered by wounding and may play roles in regulating wound-induced expression. I used R T - P C R to assay changes in m R N A abundance of At4CL gene family members up to 72 h after in planta wounding of the leaves. This analysis indicated the presence of a biphasic wounding response for At4CLl and At4CL2 genes (Figure 4-7). Similar to a previous report (Ehlting et al, 1999) At4CLl and At4CL2 expression was rapidly, coordinately, and transiently activated (by 2.5 h) by wounding. Our analysis shows that At4CL4 expression is also rapidly and coordinately activated, but is not as transient as At4CLl and At4CL2, perhaps reflecting the distinct biochemical function of At4CL4 , which specifically activates sinapic acid (Hamberger and Hahlbrock, 2004). In contrast to these genes, our results show that At4CL3 expression was rapidly down - 81 -regulated, possibly again reflecting a specialized biochemical function for A t 4 C L 3 , which appears to be primarily involved in flavonoid biosynthesis (Ehlting.e. al, 1999). The second phase o f wound activated At4CLl and, At4CL2 expression started between 24 and 48 h post-wounding and lasted up to 72 h after the onset of wounding (Figure 4-7). The biphasic response of the At4CL2 gene is consistent with late GUS expression directed by the full-length and intron 2&3 lines (Figures 4-5, a and c), and the early response directed by the 950-bp promoter::GUS construct (Figure 4-3). A biphasic wounding response for phenylpropanoid pathway genes has been reported in parsley and also in Jerusalem.artichoke (Helianthus tuberosus) tubers (Logemann et al, 1995; Batard et al, 2000). However, both responses occurred within 24 h after, wounding. Based on their results, those authors suggested. multiple signaling routes might exist for phenylpropanoid gene transcriptional activation (Batard et al, 2000). Similarly, in Arabidopsis, it appears that multiple wound-generated signals may affect different At4CL gene family members differently^ and that a single gene such as At4CL2 is regulated by multiple wound-generated signals, leading to a complex pattern of wound-induced transcriptional regulation that could be reflected i n a complex array of wound responsive cis-regulatory elements associated with the gene. • . ! " 4.3.2 Multiple cis-regulatory elements are involved in developmental regulation of At4CL2 gene expression The At4CLl and At4CL2 promoters (Figure 4-1) directed developmentally regulated G U S expression in the vasculature (Figure 3-1 or 4-2), as reported for other 4CL genes (Hauffe et al, 1991; Lee et al, 1995). In contrast, the At4CL4 promoter fused to G U S (Figure 4-1) did not direct detectable developmental expression in the mature leaves (Figure 4-2, h). Coupled with the rapid wound-induced expression of At4^CL4 (Figure 4-2, h, and 4-7) this suggests that A t 4 C L 4 may play a primarily defensive role. Consistent with a hypothesized role for A t 4 C L 3 specific to flavonoid biosynthesis (Ehlting et al, 1999), At4CL3::GUS was expressed most highly in young organs I and tissues, possibly in epidermal cells (Figure 3-1). , , In order to map the locations of elements specifying At4CL expression patterns, several different At4CL2 promoter fragments (Figure 4-1, B) were fused to GUS,and developmental and wounding response expression of transgenes was assayed (Figure 4-2, - 8 2 -b-f). Expression results showed that a minimum of 750 bp of the At4CL2 promoter is required for strong vascular- specific developmental G U S expression (Figure 4-2, d), although a few lines with the 420-bp promoter: :GUS construct were able to drive weak developmental G U S expression (Figure 4-2, c). Accordingly, critical cis-regulatory element(s) are likely to reside in the -750/-420 bp fragment of the At4CL2 promoter, in addition to the P and L boxes located more proximally (Ehlting et al, 1999) (Figure 4-8). Using a Database of Plant Cis-acting Regulatory D N A Elements ( P L A C E , Higo ei al, 1999) (http://www.dna.affrc.go.jp/PLACE) several putative cis-regulatory elements were found in this fragment including potential M y b , W R K Y , M y c , and G A T A elements. , Developmental expression directed by the 950-bp construct (Figure 4-2, e) was distinct from that directed by both shorter and longer promoter fragments (750-bp! and 1.6-kb constructs; Figure 4-1, B) , and the observed pattern was more similar to that of At4CL3::GUS expression (Figure 3-1) in which expression extends to cell types outside of the vascular system in leaves. I used the same 950-bp promoter and fused it with At4CL2 transcribed regions, to generate At4CL2-cDNA::GUS and At4CL2-mtxor\ #2&3::GUS constructs (Figure 4-4). The above described G U S expression pattern for the 950 construct, was not observed in At4CL2-mXxow # 2&3::GUS lines (Figures 4-5, c); but was evident in At4CL2-cDNA::GUS lines (Figures 4-5, b). Thus, this pattern is likely intrinsic to the truncated 950-bp promoter by itself or in combination with the cDNA.jI t is possible that the junction between the flanking vector sequences and. the -950 bp promoter created novel regulatory sequences responsible for the observed phenotype. To avoid this potential problem, future experiments could substitute deleted promoter sequences with other sequences. Regardless of this possibility, we suggest that the -950/-750 bp promoter fragment may have positive regulatory element(s) (3LE; Figure 4-8) and this element(s) direct gene expression in a very similar manner to the gene expression directed by the 635-bp At4CL3 promoter (Figure 3-1). I f so, there appear to be other negative elements in the -1.6-kb/-950 fragment and in introns 2 or 3 that negatively affect the 3 L E function and restrict expression to the vascular-specific patterns directed by the 1.6kb promoter At4CL2::GUS (Figure 4-2, f), _4^CZ2-intron #2Sc3::GUS (Figure 4-5, c), and At4CL2::GUS full length constructs (Figure 4-5, a). Figure 4^8 represents all the deduced developmental cis elements and their relationship in the At4CL2 gene. Searching - 8 3 -the -950/-750 sequence for potential cis-elements using PLACE revealed multiple tandem ARR1 c/s-elements (involved in positive transcriptional regulation; Sakai et al, 2001) and also multiple GT-1 motif and GT1 consensus sequences, which play roles in pathogen and salt- induced gene expression (Park et al, 2004). Compared with shorter promoter fragments lines, the 1.6 kb At4CL2 promoter (Figure 4-1, B) directed the strongest xylem specific GUS expression (Figure 4-2, f). Therefore, we suggest another positive regulatory element is located between the -1.6-kb /-950 bp sequences (Figure 4-8). Several potential c/'s-elements including an I-box that was shown to be a recognition site for the LeMybl transcriptional activator in tomato (Rose et al, 1999) were found in this region using the PLACE program. + Developmental expression t 3LE •1600 I -95ot -750 Negatively affect 3LE function Figure 4-8 Summary of deduced locations of positive and negative regulatory elements in the At4CL2 gene affecting developmentally regulated expression. Positive elements are indicated by blue ovals, negative elements by open ovals, and the 3LE element specifying ectopic expression by a turquoise oval, Solid boxes labeled El to E4 represent exons, open boxes represent 5' promoter sequences upstream of the ATG start codon. Base pair coordinates upstream of the ATG are given. Addition of introns #2 and #3 to the cDNA construct (Figure 4-4), restricted 950-bp promoter-driven GUS expression to the vascular system (Figures 4-5, c) and a negative regulatory element in either of these two small introns was deduced to be responsible for the inactivation of 3LE element function in the -950/-750 bp fragment (Figure 4-8). Introns #2 and #3 both are about 110 bp in length, and at +30 and +40 positions of the - 84-intron #2 and the +80 position of intron #3 a tetra-nucleotide ( C A C T ) cis element was found that was shown to be a cis-regulatory element for mesophyll-specific gene expression in Flaveria trinervia (Gowik et al., 2004). Mutagenesis of these putative elements could be used to test their roles in tissue specific expression of the At4CL2 gene. When added to the At4CL2-cD\SA::GUS construct (Figure 4-4), intron #1 had the apparent and unexpected effect of blocking G U S expression in all o f the 20 tested lines. R T - P C R confirmed the expression of the transgene, at least at low levels, and sequencing of R T - P C R product confirmed the integrity of the sequence. Even though a yet un-detected mutation may have been generated during the cloning process, it is possible, that intron #1 contains a negative regulatory element with a repressive effect on developmental At4CL2 m R N A accumulation or on translation., ' • • 4.3.3 Multiple cis-regulatory elements are involved in modulating At4CL2 early wound responsiveness The 950-bp At4CL2 promoter fused to GUS was the only one of several At4CL2 r.GUS constructs that directed the early and transient wound response (Figures 4-2, e and 4-3) characteristic of the endogenous At4CL2 gene (Ehlting et al, 1999). G U S t expression in response to wounding directed by smaller promoter fragments such as 750-bp At4CL2 promoter, was restricted to some of the veins in the wounded area (Figure4-2, d). This G U S expression could be attributed to the greater accessibility, o f the X - G l u c substrate from the wound site to these veins. This suggests the presence of a positive regulator of the early wound response in the .-950/-750 region of the At4CL2 promoter (Figure 4-9). \ Other transgenic lines with the same promoter, such as those harboring At4CL2-cDNA::GUS, At4CL2-intxon # 2&3::GUS, and.full-length constructs (Figure 4-4), did not show such an early wound response (Figure 4-5). Furthermore, such an early response was not observed in lines harboring the 1.6-kb At4CL2 promoter fused to G U S ; Figure 4-9 represents the deduced cis elements involved in the early wound response and their relationship. Based on the results, elements that negatively regulate the early wound response appear to exist in the promoter and transcribed regions of the gene. One negative regulatory element may reside in the -1.6kb/-950 fragment, while another - 8 5 -appears to be in an exon, since early wound responsiveness was absent in both At4CL2-cDHAv.GUS and At4CL2-vati(m 2&3::GUS lines (Figures 4-2 and 4-5). Negative effect -1600 -950T-750 E l E2 E3 E4 t + Early wound response + Late wound response Figure 4-9 Summary of deduced positions of positive and negative regulatory elements in the At4CL2 gene affecting wound inducible expression. Approximate positions for the positive regulatory elements, are indicated by red and purple stars and position of negative elements are indicate by yellow stars. Solid boxes labeled El to E4 represent exons, open boxes represent 5' promoter sequences 5' to the ATG start codon. Base pair coordinates upstream of the ATG are given. In spite of non-detectable developmental GUS expression, At4CL4::GUS transgenic lines (Figure 4-1, C) showed a strong early wounding response, which was sustained for 72 h (Figure 4-2, h). This is consistent with the kinetics of wound induced At4CL4 mRNA accumulation in wounded leaves (Figure 4-7). In both experiments levels of wounding induced At4CL4 mRNA accumulation and At4CL4-drivm GUS expression were distinguishable from the control even after 10 min post wounding (data not shown). This characteristic distinguishes At4CL4 from the other At4CL genes, and suggests the presence of strong wound responsive c/s-regulatory elements in the 1-kb AtCL4 promoter used in these experiments. 4.3.4 Intronic cis-regulatory elements are involved in the late wounding response of At4CL2 gene expression. The line expressing the 1-kb promoter At4CLl::GUS construct (Figure 4-1, C) showed wound-activated GUS expression by 72 h post-wounding (Figure 4-2, g), a response characteristic of the endogenous gene clearly evident by RT-PCR (Figure 4-7 - 86-). On the other hand, none of the At4CL2 promoter fragments directed the At4CL2 late wounding response (Figure 4 - 2 , b-f). These data suggest the absence of positively and negatively acting cw-elements for the late wound response in the At4CL2 promoter fragments tested, and that the At4CL2 transcribed region might contain such an element ( L W R E ) directing the late wound response. While the y 4 ^ C Z 2 - c D N A : : G 6 ' 5 ' line did not show any wound responsiveness (Figures 4 - 5 , b) G U S expression in lines harboring the full-length construct or the At4CL2-'mtwn # 2&3::GUS construct showed clear wound-induced G U S expression by 7 2 h or earlier (Figures 4 - 5 , a & c). This late response was weaker in At4CL2-'mtron # 2&3::GUSlines (Figures 4 - 5 , c) than in the full-length lines (Figures 4 - 5 , a). Thus, this suggests that a L W R E resides in intron # 2 and/or # 3 of the At4CL2 gene (Figure 4 - 9 ) and that there may be another L W R E in intron 1 , directing a stronger late wound response as strong as seen in full-length lines (Figures 4 - 5 , a). Interestingly, there is an A G - m o t i f at position + 6 1 o f intron 3 , which was found in the promoter of the NtMyb2 gene, a wounding and elicitor stress induced regulator of tobacco PAL gene expression (Sugimoto et al, 2 0 0 3 ) . 4.3.5 Conclusion The wound-induced accumulation of At4CL2 m R N A is in agreement with work of others, showing an early and transient wounding response of this gene (Ehlting et al, 1 9 9 9 ) , and a second, later wound response characteristic of other phenylpropanoid genes (Logemann et al, 1 9 9 5 ; Batard et al, 2 0 0 0 ) . A s well , At4CLl and At4CL2 promoter-G U S fusions directed developmental expression to predicted sites in the vascular system, where the 4 C L enzymes play roles in the biosynthesis of l ignin precursors. A simple model would predict the localization of positive regulatory elements that direct developmental and stress-induced expression of these genes in the respective upstream promoter regions. Instead, our dissection of the regulatory elements associated with the At4CL2 gene suggests a highly complex regulatory structure, with positively and negatively acting elements scattered at different locations both upstream of and within the transcribed region (Figure 4 - 1 0 ) . This unexpected complexity may reflect the interplay of multiple signals that direct both developmental expression to specific cell types and a complex pattern of stress-induced expression of this gene. Further work wi l l be required - 8 7 -to fully characterize the specific c/s-regulatory elements that direct these complex expression patterns. -1600 -950 -750 E l E2 E3 l_-<|_> ) I • I Positive developmental Ectopic expression Negative developmental <^> Positive early wound response Negative early wound response <^> Positive late wound response Figure 4-10 Schematic presentation o f the locations o f putative regulatory elements in the At4CL2 gene -88-CHAPTER 5 REVERSE GENETIC ANALYSIS OF AtMYB SUBFAMILY 14 5.1 Introduction M Y B transcription factors belong to some of the largest plant transcription factor families (Romero et al, 1998; Riechmann et al, 2000). The M Y B gene family is comprised of more than 125 members divided in 24 subfamilies in Arabidopsis (Stracke et al, 2001). There is evidence that M Y B transcription factors play important roles in the regulation of phenylpropanoid metabolism in Arabidopsis and other plants (Sablowsky et al, 1994; Borevitz et al, 2000, Jin et al, 2000; Preston et al, 2004), as well as regulating i other biochemical and developmental pathways. Some plant M Y B transcription factors recognize a core-binding site (Grotewold et al, 1994) that is found in the promoters of PAL, 4CL and other genes encoding enzymes in phenylpropanoid metabolism (Douglas, 1996) leading to lignin biosynthesis. Dr. Wang in our lab, studying AtMYB68, showed that this gene has a strong root-specific expression pattern. Fusion of the AtMyb68 promoter to G U S revealed the predominant expression of this gene in the xylem pole pericycle cells of the root in the transgenic Arabidopsis .-seedlings. She also identified and characterized an AtMyb68 T - D N A knock out line. However, a phenotype has not been detected so far in this line (Wang, 2003). Functional redundancy with a related AtMyb gene is a possible reason for the lack of an obvious phenotype. From a phylogenetic tree the A t M y b transcription factors, it is known that AtMyb36, AtMyb68, AtMyb84, and AtMyb87 are closely related in subfamily #14 (Figure 5-1) (Stracke et al:, 2001). In'this chapter, I focused on AtMyb84, since it was found to be the closest subfamily member to AtMyb68.1 generated AtMyb84 R N A i knock down and T - D N A knockout lines to look for potential AtMyb84-velaXed phenotypes. In addition, I developed a method for creating double R N A i knock down lines, and created double AtMyb68/AtMyb84 knock out / knock down lines to study their potential functional redundancy. .! - 8 9 -5.2 RESULTS 5.2.1 In-silico analysis of AtMyb subfamily #14 According to the alignments shown in Figure 5-1, and phylogenetic analysis done by Stracke et al. (2001) for all the Arabidopsis M y b genes, AtMyb68 and AtMyb84 are the most closely related AtMyb genes in the subfamily #14 and their amino acid sequences show more than 82% identity. Using the Arabidopsis genome sequence, I also identified the genes surrounding Myb sub family # 14 genes on their linkage groups. I found that the genomic regions around AtMYB68 and AtMyb84 show the highest levels of apparent synteny. The gene immediately upstream of AtMyb68 encodes branched-chain amino acid transaminase 5 ( B C A T 5 ) while immediately downstream in reverse orientation is a putative aminocyclopropane-l-carboxylate synthase ( A C C synthase) encoding gene.'The gene upstream of AtMyb84 also encodes a branched-chain amino acid transaminase ( B C A T 3 ) and, likewise in reverse direction downstream of AtMyb84, there is again a putative A C C synthase gene. Such a strong synteny was not observed between these two AtMyb genes and the others in the subfamily. I found signatures of AtMYB84 gene expression in germinating seedlings andjalso in the inflorescence as well as root and leaves using the M P S S (http://mpss.udel.edu/at/GeneOuery.php) database. Signatures of AtMYB68 gene expression were found in the root and inflorescence. I also searched Arabidopsis Functional Genomics Tools (http ://bbc.botany.utoronto.ca) web site at the University of Toronto for expression data related to Myb subfamily # 14. The results showed that i f the expression level of AtMyb genes in the rosette is considered zero, the expression levels of AtMyb36, AtMyb68 and AtMyb84 are first down regulated (-4 to -6) in the germinating seedlings and then highly up regulated (+3 to +8) in the seedling root. Based on these data AtMyb68 and AtMyb84 are also up regulated (+2 to +4) in the hypocotyl and up regulated (+2) in the flowers. Meanwhile both are down regulated in the leaf (- 0.75). Exceptionally and in several array reports, AtMyb84 is up regulated (+1) in the shoot apex. Overall, compared with AtMyb36 and AtMyb87, data collected from more than 80 microarray experiments show that expression of AtMyb68 and AtMyb84 genes is more coordinately regulated than that of other AtMyb subfamily #14 genes, supporting the possibility that they have similar functions. -90-AtMyb36 1 M G R A P C C D K A N V - : G F S P E E D V I. D I D :•• G T G G N \ I A L P Q K I G AtMyb68 1 M G R A P C C D - .A N V K K G P W S P E E D A P C L K D I E N S G T G G N i ' I A I i P Q K I G l C AtMyb84 1 M G f A P C C D A N V G P S P E E D A L S I E N S G T G G N W I A I i P Q K I G L C AtMyb87 1 m a - V K K G P W S T E E D A V L K S I E V : : G T ' G N N I S I _ P Q : - I G I . C AtMyb36 5 1 G K S C . L - L N L R P N I G G S E E E D I I L S L . I S I G S V S I I A A Q L P G P AtMyb68 5 1 G K S C R L R L N G G S E E E D N I I C N L V T I G S 7 - S I I A A Q L P G P . AtMyb84 51 G K S C R L J L N L . P H I G G S E E E E N I I C S L . L T I G S S I I A A Q L P G P . AtMyb87 42 G K S C • L E L N ; L R P N L K H G G T D E E D I I C S L I T I G S . S I I A S Q L P G P AtMyb36 1 0 1 T D N D I K N - ' W N T K L K K K L L G P . Q k q m n r q d s i t d s t e n n l s n n n n n k s p q n l AtMyb68 1 0 1 T D N D I N N L N K Q R K E ' Q E A - M • Q E M V M M K R Q AtMyb84 1 0 1 T D N D - N N I N K Q P . K E L Q E A C M E Q Q e m M V M M K P Q AtMyb87 9 2 T D N D I K N A t M y b 3 6 1 5 1 s n s a l e r l q l HMQLQNLQSP i ' S S ! : VNN P I L WPKL- gjPLLfl AtMyb68 1 4 1 Q Q G Q g q g q s n g s t d l y l n n m f g s s p V P L l p q l p p p H H Q I P AtMyb84 1 4 3 H Q Q Q Q I Q T S r MMRQDQTMf'T W P L H -AtMyb87 1 1 7 H Q Q L n A t M y b 3 6 A t M y b 6 8 A t M y b 8 4 A t M y b 8 7 1 9 0 1 8 1 1 7 2 1 2 2 S T T T N Q N p k l a s q e s f h p l g L G M M E P T S C l j V Y q t t p s C N L E Q | V P A L F M N Q T S S F B D Q MD' - N V D B Q H n n t L K N M V K I E E J Q B P . N M V • : . E : . • • . A t M y b 3 6 A t M y b 6 8 A t M y b 8 4 A t M y b 8 7 A t M y b 3 6 A t M y b 6 8 A t M y b 8 4 A t M y b 8 7 2 7 0 2 2 8 2 1 4 1 2 2 3 2 0 2 2 8 2 1 4 1 4 8 v s k e l f q v g n e f e l t n g s s w w s e e V E L E F S S w g s a s v l d q t t e g r a G T T S S S S s q n q i q i f h d e n t k v m l q — ————— - D : A Q D S V T N F - D - S F S Q L L L D P N Q D S M T N A F D - l l S f ' S Q L L L D P N s n q t l y n q v v d p s m r a f a m e e q s m i K N Q I L E P F S . . - E P N K V L "DVD D A A A t M y b 3 6 3 2 7 •jKS I ' H S V A t M y b 6 8 2 4 9 - w____ A t M y b 8 4 2 3 6 A t M y b 8 7 1 9 7 A S S - H H H A S P A t M y b 3 6 3 3 4 A t M y b 6 8 2 9 2 SSN QTEAIN A t M y b 8 4 2 7 0 --N f'QAETVN A t M y b 8 7 2 4 7 EN FQAELFD A t M y b 3 6 3 3 4 G G E G D : A I M S S S T N S P -E G S M N S I L S A N T N S P -- L P N T S S D Q H P S Q Q Q E I - L L N T S N D n - • - Q W F G --PMDG A t M y b 6 8 A t M y b 8 4 A t M y b 8 7 3 4 2 T S T S A D Q S T I S V ' E D I T S L V N S E D A S . F n a p n h v 2 8 4 T S T S A D Q S T I S . E D I S S L V . S D S K Q F ( 2 8 3 D I S S ' I D y p l y d n e I AtMyb36 - AtMyb87 L I AtMyb84 I AtMyb68 AtMyb37 AtMyb38 Figure 5-1 Alignment of A t M y b subfamily #14 members and related phylogenetic tree, taken from Stracke et al. (2001) -91 -5.2.2 The AtMyb84 knock down line preparation Based on a revised phylogenetic tree (Figure 5-1) (Stracke et al, 2001), and as discussed above, AtMyb84 was considered the best candidate for a gene with hypothesized functional redundancy to AtMyb68. Since a T - D N A insertion line was not available at the time, I generated an AtMyb84-specific R N A i construct (Figure 5-2) to repress the expression of this gene (gene knock down). 35S promoter 84-S 84-A Figure 5-2 R N A i construct for AtMyb84 More than 10 independent homozygous lines were generated and semi-quantitative R T - P C R was performed on 10 days old seedlings of these lines to find those with down regulated AtMyb84 gene expression (Figure. 5-3). Line-2 3 6 7 8 9 10 11 12 13 Wt Actin 26 cycles AtMyb84 30 cycles Figure 5-3 R T - P C R on AtMyb84 R N A i knock down lines. Cycle numbers for the target gene and actin control genes that generated products in non-saturated levels are given. Multiple independent lines were tested and those with reduced AtMyb84 m R N A levels relative to the actin control are indicated by checks. Three of these lines (lines # 6, #8 and #13) reproducibly showed lower gene expression in several repetitions of the experiment. Seedlings of lines 3, 6, 12, 13 and wild type controls were grown vertically in order to identify possible phenotypes at the seedling level. At this stage (Figure 5-4) and later developmental stages, no morphological difference between the lines with lower AtMyb84 mRNA levels (#6 and #13) and wild type plants or RNAi lines with normal AtMyb84 expression levels (#3 and #12) were detected. AtMyb84 RNAi line 3 line 6 Wt line 12 line 13 * * 1 Figure 5-4 Seedling growth and morphology of AtMyb84-RNAi knock down lines. 5.2.3 AtMyb84 knock down line preparation using a multiple arm-RNAi construct I prepared dual RNAi constructs containing two arms specific to separate genes in order to simultaneously down regulate both genes. Constructs are shown in Figure 2-4. To test the functionality of this strategy, test dual constructs were generated containing RNAi specific to GUS and to AtMyb84 (Figure 5-5). 35S promoter G U S - S intron G U S - A 84-S intron 84-A 315 bp • 270 bp 312 bp 400 bp Figure 5-5 Dual R N A i construct specific for GUS and AtMyb84 genes A transgenic 35S::GUS line was transformed with this dual R N A i construct. More than 20 independent T l lines were tested for G U S expression using the histochemical assay and many of them showed apparent G U S down regulation (Figure 5-6). F igure 5-6 G U S expression in transgenic plants generated by transformation o f a 35S::GUS line with a Myb84+GUS double R N A i construct. Top, histochemical assay of G U S expression in leaves o f a control 35S::GUS line. Bottom leaves of similar age from 13 independent lines transgenic for the Myb84+GUS double R N A i construct in the 35S::GUS background. I performed R T - P C R on these lines to estimate AtMyb84 expression levels and found both GUS and AtMyb84 down regulation in some of the lines (Figure 5-7) with lines #2 and #10 showing strong down regulation o f the AtMyb84 gene along with strong reduction in G U S activity. N o ^My684-associated phenotype has been detected in these knock down lines so far. + G U S lines 35S: :GUS AtMyb84 - 9 4 -AtMyb84+G\JS line #2 #3 #4 #6 #10 #11 #12 # 13 35: :GUS Act in AtMyb84 G U S assay - + + - - + + Figure 5-7 R T - P C R analysis o f AtMYB84 expression in Myb84+GUS double R N A i lines. Results o f histochemical G U S assays performed on 21 days old plants are shown below; " - " indicates substantially reduced G U S expression. P C R reactions were performed for 28 cycles, within the linear range of AtMyb84 amplification. Ten transgenic lines were prepared containing the dual AtMyb68 and AtMyb84 R N A i construct shown in Figure 2-4. N o morphological phenotype was detected in the vertically grown seedlings o f these lines (Figure 5-8) or later stages of plant development (data not shown). Line-1 - 2 -3 - 4 Figure 5-8 Seedling growth and morphology of transgenic lines containing double R N A i AtMyb68 +AtMyb84 constructs. Plants were 10 days old. 5.2.4 Transformation of AtMyb68 knock out line using the AtMyb84 RNAi construct The AtMyb68 knock out line previously characterized by Wang (2003) was transformed with the AtMyb84 specific RNAi construct. Vertical growth of homozygous transformants in parallel to other controls did not reveal any morphological differences at the seedling stage (Figure 5-9). Athiyb84 RNAi in AtMyb6S knock out line Wt line-1 line-2 line-4 Figure 5-9 Seedling growth and morphology of AtMyb84 RNAi knock down lines in the AtMyb68 knock out background. Seven representative RNAi lines are shown. However, rosettes of some of these lines such as line # 1 and #3 were smaller than others in the first round of phenotypic analysis and lines 1, 2, and 3 were delayed in development of the primary inflorescence shoot (Figure 5-10). In this figure, while line 4 has developed a bolting stem and even siliques, line 2 just started to develop and lines 1 and 3 are severely delayed in bolting. -96-Figure 5-10 Rosette stage growth and development of four Myb84 R N A i lines in the Myb68 knock out background. Plants were homozygous for both transgenes and o f the same age but their rosettes showed different sizes and stages of development. Preliminary analysis of more individuals of these transgenic lines after another week of growth suggested that in lines #1, #3 and #7 development of the primary inflorescence shoot was delayed relative to control plants and other R N A i lines (Figure 5-11). While lines 4, 5 and 8 had developed primary inflorescence shoot with developing siliques, lines 1 and 3 were still at the rosette stage and line 7 had just started to develop an inflorescence. Mature plants of these lines did not show any phenotypic difference, compared to wi ld type plants (Figure 5-12). Overall, a consistent pattern o f delay in emerging inflorescence was observed in this phenotypic analysis. -97-Myb84 R N A i lines in AtMyb68 knock out background Line 3 Line 4 Line 5 Line 7 Line 8 Figure 5-11 Rosettes with emerging primary shoot in seven Myb84 R N A i lines in the Myb68 knock out background. These lines are compared to Wt, single Myb68 knock out, and single Myb84 knockout control lines one week younger in age. - 9 8 -Line 1 Line 2 Line 3 Figure 5-12 Mature plants of AtMyb84 RNAi lines in the AtMyb68 knock out background compared to Wt control. 5.2.5 The AtMyb84 knock out line preparation Subsequent to the above work, a potential AtMyb84 T-DNA knock out line became available in the Salk collection and was obtained. This line was no longer kanamycin resistant, but the presence of a T-DNA insertion in the AtMyb84 gene (Figure 5-13) was confirmed by PCR, using T-DNA left border specific (LBbl) and AtMyb84 specific (84Eco-F) primers (Table 2.1). T-DNA L B 84Eco-F Primer -// 5' 3' Intron 1 intron 2 84-Rev Primer Figure 5-13 Schematic of T-DNA insertion within AtMyb84 knock out lines from Salk collection. Blue boxes indicate exons and arrows indicate the primers used in the PCR reactions. LB indicates left border of the T-DNA. This PCR reaction generated the expected 100-bp PCR product from the third exon of the gene into which the T-DNA had been integrated. However, using two AtMyb84 specific primers, 84Eco-F and 84-Rev (Table 2.1), an additional 300-bp wild-type PCR product indicated that the original line was heterozygous. A homozygous AtMyb84 knock out line was identified in the next generation and was confirmed by multiple PCR tests. Preliminary RT-PCR confirmed AtMyb84 knock out phenotype (data not shown). Seedlings of this AtMyb84 knock out line were vertically grown in parallel with AtMyb68 knock out line and wild type plants but no significant morphological difference was detected at this stage (Figure 5-14, A). Also no morphological difference was detected at the rosette stage (Figure 5-14, B) and mature plants (Figure 5-14, C) of this line compared to wild type and AtMyb68 knock out lines. The obvious next step to cross AtMyb68 and AtMyb84 knock out lines to generate a double knock out has been initiated, but analysis of progeny from this cross is beyond the scope of this thesis. - 100-AtMyb68 AtMyb84 A Wt (Col) knock out knock out Wt AtMyb68 knock out AtMyb84 knock out Wt AtMyb68 knock AtMyb84 knock Figure 5-14 Comparison of AtMyb68 and AtMyb84 knock out lines and wild type plant, A) 10 days old seedlings, B) Rosettes, C) Mature plants - 101 -5.3 DISCUSSION 5.3.1 Bioinformatics data indicated high homology and overlapping expression of AtMyb6S and AtMybS4 genes. Based on a the Phylogenetic tree of the AtMyb sub-family # 14 shown in Figure 5-1 (Stracke et al, 2001), AtMyb84 was considered the best candidate for a gene with hypothesized functional redundancy to AtMyb68. Conserved synteny and also highly similar cDNA sequences and high homology of protein sequences of these two genes (Figure 5-1) indicate that the DNA blocks containing AtMyb84 or AtMyb68 belong to duplicated regions of the Arabidopsis genome and that AtMyb68 and AtMyb84 may be functionally redundant. We were thus expecting overlapping expression patterns for both of these genes, and the MPSS database as well as Arabidopsis Functional Genomics Tools on the University of Toronto BBC web site (http://bbc.botany.utoronto.ca/) indicated such an overlapping expression pattern for these two genes in several organs and developmental stages. These two genes also showed overlapping expression with other members of AtMyb subfamily #14, especially with AtMyb36 that is co-expressed with AtMyb68 and AtMyb84 in the root. This suggests that AtMyb68 and AtMyb84 may have similar functions in the shoot. On the other hand, these two genes as well as AtMyb36 could encode partially redundant root functions. 5.3.2 No phenotype has been observed for AtMybS4 knock down and knock out lines. In the AtMyb68 knock out line no phenotype has been detected so far (Wang, 2003; Figure 5-14). From RNA blots and GUS assays performed on AtMyb68::GUS transgenic plants (Wang, 2003) it is known thaX AtMyb68 has a root preferred expression pattern: From the microarray data it is also known that AtMyb36, AtMyb68 and AtMyb84 show overlapping expression in the root. Thus, we hypothesized that if only AtMyb68 and AtMyb84 genes are functionally redundant, we may be able to see a phenotype in the root when both are knocked down/out in the same line. If AtMyb84 is not functionally redundant to any other Myb gene in the subfamily, a single gene knock down/out of AtMyb84 should show a phenotype. - 102-I generated more than 10 independent homozygous AtMyb84 R N A i knock down lines and also AtMyb84+GUS double R N A i knock down lines, performed semi-quantitative R T - P C R , and identified lines with down regulated AtMyb84 gene expression (Figure 5-3 and 5-7). Consistent with our hypothesis, no morphological differences between vertically grown seedlings of the candidate AtMyb84 knock down lines (line #6 and #13) and the control plants were detected (Figure 5-4). Also , AtMyb84 + GUS double R N A i lines (#2 and # 10) in the Figure 5-7 were grown vertically on M S media and soil and compared with the recipient 35S::GUS line as control but no phenotypic differences between these two lines and the control were found (data not shown). I later found a putative AtMyb84 T - D N A knock out line in the Salk collection and a homozygous AtMyb84 knock out line was identified in the next generation. Although we still need to confirm this knock out phenotype through analysis o f AtMyb84 R N A levels, vertically grown seedlings of this line also did not show any differences with control plants (Figure 5-14, A ) . I also detected no differences between the rosettes and mature plants of these knock out line and those of control plants (Figure. 5-14, B and C). The tomato Blind gene is an R2R3 class MYB gene that is closely related to members of A t M y b subfamily #14 (Schmitz et al, 2002). In the blind mutants, the initiation of lateral meristems during the shoot and inflorescence development is blocked leading to reduction of the number of lateral axes, which is manifested in reduction of shoot and inflorescence branching. I also examined the Arabidopsis shoot branching in the AtMyb84 knock down and knock out lines and did not see differences between the candidate lines and the controls (Figure 5-14, C). 5.3.3 Preliminary phenotype for double AtMyb6S -AtMyb84 knock down/out lines. A s the accumulated data indicated that AtMyb68 and AtMyb84 have at least partially redundant functions, I used different strategies to knock both AtMyb68 and AtMyb84 genes down and/or out in the same plant. First, The AtMyb68 knock out line was transformed using the AtMyb84 specific R N A i construct (Figure 5-2). Second, I considered that double mutants may be lethal. To avoid full knock out of both genes I made AtMyb68 plus AtMyb84 double gene knock down lines using a double R N A i construct. In advance, functionality of dual R N A i constructs was first examined using - 103 -dual test construct of G U S plus AtMyb84 R N A i (Figure 5-5). In terms of the technical advance, I found that using two R N A i molecules in a tandem arrangement to down regulate two genes at the same time works at a reasonably high level, such that one R N A i arm (AtMyb84 specific) w i l l not prevent second R N A i arm ( G U S specific) from functioning to initiate R N A i mediated R N A degradation (Figure 5-6 & 5-7 line 2 and 10). We plan to use this method to down regulate all the genes in the subfamily #14 to further investigate the redundancy relationships of its members. Vertical growth of homozygous lines generated using the first and second strategies did not reveal any mutant phenotypes at the seedling stage (Figure 5-8 and 5-9). The lines of AtMyb84 R N A i knock down line in the AtMyb68 knock out background were transferred to soil in order to search for the possible phenotypes in the aerial parts of the mature plants. In some of these lines, such as lines #1 and #3,1 found smaller rosettes (Figure 5-10). They also were delayed in shoot development (Figure 5-11). Comparison of these lines (Figure 5-10), showed that when lines 1 and 3 were still at an early stage of rosette development, line 2 was more advanced and line 4 had already developed siliques. A l l these lines were transplanted from agar plates at the same age and grown in identical conditions. Also plants from the same homozygous line grown in each pot showed the same phenotypes. Overall, the severity of the rosette-stage phenotype varies between the individual lines generated by this approach, as judged by the sizes of mutant plants shown in Figure 5-10, and timing of the development of inflorescence meristems (Figure 5-11) relative to control plants. This variability could be due to the variability in the degree of AtMyb84 knock down between lines, which wi l l be tested by measurement of relative R N A levels by R T - P C R . Based on the phenotype of tomato blind mutants, I searched for the reduction of shoot and inflorescence stem branching in the Arabidopsis AtMyb68, AtMyb84, and AtMyb68- AtMyb84double knock out/down lines. I did not see a difference between the candidate lines and the controls (Figure 5-12 & 5-14). Once it is confirmed AtMyb84 R N A is down-regulated or absent in double knock out/down lines, this data would indicate either that AtMyb68 and AtMyb84 do not share analogous functions with the BLIND gene, despite their close phylogenetic relationships (Schmitz et al, 2002), or that - 104-additional genes within A t M y b subfamily 14 have redundant functions with AtMyb68 and/or AtMyb84 with respect to control of lateral bud formation. No phenotype was detected in the roots of double knock down/out lines. Assuming that AtMyb84 expression knock down can be confirmed, we may consider AtMyb36 as a third gene that shares redundant functions with AtMyb68 and AtMyb84 in the root, where all three genes show high expression. If so, triple knock outs of AtMyb36, AtMyb68, and AtMyb84 w i l l be required to test this hypothesis. A s mentioned above, in order to reach final conclusions it is necessary that AtMyb68 and AtMyb84 expression in the knock out/down lines to be confirmed through R N A analysis. A lack of root and lateral branch phenotypes in the AtMyb68 knockout/and AtMyb84 R N A i line could result from insufficient repression of AtMyb84 expression to generate root or lateral bud mutant phenotypes, and knock outs of both genes may be required to reveal clear phenotypes. On the other hand, in many cases and also in the case of AtMyb68 and/or AtMyb84, mutations may cause no obvious morphological defect under normal growth conditions nonetheless confer phenotypes that can be detected with biochemical/molecular analysis, or by testing the mutants under a battery of stress conditions. In that sense, further genetic and phenotypic analysis of AtMyb68 and/or AtMyb84 mutant plants may be required to test this hypothesis. In summary, as predicted by close phylogerietic relationships and partially overlapping expression patterns, and supported by the lack of obvious phenotypes associated with single AtMyb68 and AtMyb84 knockout/down lines, AtMyb68, AtMyb84 AtMyb36 may play similar and partially redundant functions in plant development. Initial information from double AtMyb68 knockout/^tMyb84 knockdown lines suggests redundant roles of these two genes in rosette development and timing of inflorescence stem development. Further genetic, molecular, and phenotypic analyses of double, and possibly triple mutants w i l l be required to draw definitive conclusions regarding such functions. Also it is necessary to back cross the mutant lines to the wi ld type plant and try to segregate away potential unlinked mutations. - 105-C H A P T E R 6 G E N E R A L DISCUSSION AND F U T U R E DIRECTIONS 6.1 At4CL::GUS T R A N S G E N E SILENCING At4CLl::GUS and At4CL2::GUS transgenic plants showed predictable, overlapping patterns of xylem abundant G U S expression similar to the pattern described in transgenic Arabidopsis plants containing the parsley 4CL1 promoter fused to GUS (Lee et al, 1995). Considering the presence of multiple common cis regulatory elements in their promoters, high identity of their c D N A sequences and overlapping expression patterns seen in northern analysis (Ehlting et al., 1999), At4CLl and At4CL2 genes are likely to show at least partial functional redundancy. Our finding that epimutant 2-8, in which the endogenous At4CL2 gene was silenced, showed no phenotypic differences to non-mutant plants supports this conclusion, and this was recently confirmed by generation and analysis of a At4CLl and At4CL2 double knock out (Hamberger personal communication). Our results show specific and different silencing modes for At4CLl::GUS and At4CL2::GUS transgene silencing. This silencing phenomenon in the At4CL::dhlA lines made our negative selection system unsuitable for mutant selection, and silencing of At4CL::GUS transgenes also impaired the ability to identify Mendelian mutants in components required for At4CL activation. To overcome these problems we suggest preparation o f At4CL::GUS or At4CL::dhlA transgenic plants containing a single transgene in each line confirmed by Southern analysis, and exclusion of 5 ' - U T R sequences. In terms of the modes of silencing, At4CLl::GUS transgene silencing was cis and root specific in seedlings, while At4CL2::GUS transgene silencing was trans and global. In keeping with a widely accepted model of double stranded R N A mediated transgene silencing (Bender 2004), overabundant messages containing At4CL 5 ' -UTRs was likely the trigger for silencing in both At4CL:;GUS and 4tCL::dhlA lines. This could be tested by cloning of At4CL 5 ' -UTRs down stream of the 35S promoter to investigate the effect of this overabundance on transgene silencing. Investigating the presence of s i R N A in the - 106-silenced lines also may confirm the involvement of R N A i machinery in the silencing of At4CLl::GUS or At4CL2::GUS or both. To avoid transgene silencing, lines with single copy of At4CL::GUS transgene is more suitable for the mutant screen. Also screens for mutants with increased or ectopic transgene expression may avoid silencing artifacts. Alternatively, different regions of the endogenous At4CL promoter could be specifically targeted for promoter methylation with transcribed inverted repeat transgenes to map the regions necessary for expression. Regardless of the mechanism o f the observed silencing, the root specific silencing of the At4CLl::GUS transgene is difficult to explain, using published silencing models. 6.2 At4CL2 WOUND RESPONSE ELEMENTS Based on northern analysis, rapid but transient accumulation o f At4CL2 m R N A is induced in response to wounding (Ehlting et al, 1999). For other phenylpropanoid genes a second, later wound response has also been reported (Logemann et al, 1995; Batard et al, 2000). Our expression results showed early and late phases of both At4CLl and At4CL2 wound responses while an immediate but longer lasting At4CL4 response was observed. At4CL3 expression was rapidly down regulated in response to wounding at the time that At4CLl and At4CL2 were up regulated. We positioned the wound response regulatory elements associated with the At4CL2 gene in both promoter, and in the transcribed regions. Our results suggest a highly complex regulatory structure, with positively and negatively acting elements scattered at different locations both upstream and within the transcribed region. To fully localize and characterize the specific cis-regulatory elements that direct these complex expression patterns, it w i l l be necessary to create serial deletion constructs, or to mutagenize candidate c/'s-elements, and determine the effects of such specific changes, alone or in combination, on wound inducible expression. We used At4CLl::GUS and At4CL4::GUS transgenic plants containing only 1 kb of these At4CL genes promoter sequences. While At4CLl r.GUS lines only showed a late wound response, At4CL4::GUS lines showed a more immediate wound response. Shuffling of the At4CLl or At4CL2 promoter fragments with At4CL4 promoter sequences may help to localize the cis elements that are responsible for the biphasic or immediate - 107-responses of At4CL genes. 6.2 FUNCTIONS OF AtMYB SUBFAMILY 14 MEMBERS N o phenotype related to AtMyb68 knock out has been found so far. Functional redundancy of this gene with the closest family member in the subfamily is a possible reason for the lack of detectable phenotype. AtMyb84 was found to be the closest family member to the AtMyb68. N o phenotype was detected in AtMyb84 knock out line, supporting the functional redundancy hypothesis. Double AtMyb68 knock out / AtMyb84 knock down lines were generated using an A tMyb8'4 R N A i construct. Preliminary results showed that rosette and primary shoot development was delayed in these knock down / out lines. Considering the lack of detectable phenotype in AtMyb68 and AtMyb84 single knock out lines, these putative phenotypes, not seen in either single mutant, are most likely related to the functional redundancy of these two genes. In the future, it w i l l be essential to analyze AtMyb84 expression in these lines (AtMyb68 expression is already known to be almost abolished in the knock out line). It w i l l also be necessary to document the morphological phenotypes in subsequent generations. Since AtMyb36 and AtMyb87 genes in the subfamily 14, could also be functionally redundant with AtMyb68 and AtMyb84 genes, it may be necessary to create triple knockout lines in order to observe strong phenotypes. AtMyb68 expression is primarily associated with roots and AtMyb36 also shows root abundant expression (Wang, 2003), but these genes are also related to the tomato Blind gene, which encodes a M Y B transcription factor that plays a role in lateral shoot formation (Schmitz et al, 2002). 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