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The 4-Coumarate:coenzyme A ligases from Nicotiana tabacum and Arabidopsis thaliana : characterization.. Lee, Diana 1996-12-31

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The 4-Coumarate:Coenzyme A Ligases from Nicotiana tabacum and Arabidopsis thaliana: Characterization of c D N A Clones, Gene Families, Recombinant Proteins, and Antisense Transgenic-Plants by DIANA L E E B.Sc, McGill University, 1989 M . S c , The University of Waterloo, 1991 A T H E S I S SUBMITTED IN PARTIAL F U L F I L L M E N T O F T H E R E Q U I R E M E N T S FOR T H E D E G R E E O F DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept this thesis as conforming /tfy the required standard>  T H E UNIVERSITY O F BRITISH C O L U M B I A October 1996 © Diana Lee, 1996  In  presenting this  degree at the  thesis  in  partial  fulfilment  University of British Columbia,  of  the  requirements  I agree that the  freely available for reference and study. I further agree that  for  permission for extensive  this thesis for scholarly purposes may be granted by  the  department  or  that  his  or  her  representatives.  It  is  understood  head of my  publication of this thesis for financial gain shall not be allowed without permission.  Department of  BoTftrti  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  NOM ember  13, i ^ ^ ^  advanced  Library shall make it  copying of  by  an  copying  or  my written  Abstract  The c D N A s encoding 4-coumarate:coenzyme A ligase (4CL), an enzyme in the general phenylpropanoid pathway, were cloned from Arabidopsis  thaliana.  Nicotiana  tabacum  and  In tobacco, 4 C L was encoded by a gene family and northern  blot analysis demonstrated that the steady-state R N A levels were highest in stems, ovaries, and non-pigmented portions of the corolla. Two 4CL c D N A s , which were 8 0 % identical to each other at the nucleotide level, were expressed in E.  coli.  The relative  abilities of the recombinant-4CL proteins to utilize 4-coumarate, ferulate, and caffeate as substrates were comparable to that of the 4 C L activity found in tobacco-stem extracts. Both recombinant-4CL proteins utilized cinnamate as a substrate, an activity not observed in tobacco extracts. This activity towards cinnamate was inhibited by a modifying-component found in tobacco extracts and the evidence suggests that the substrate specificity of 4 C L is, in part, determined by post-translational modification such as phosphorylation. In Arabidopsis,  4 C L was shown to be encoded by a single gene. Northern blot  analysis indicated that, like tobacco, 4CL steady-state R N A levels were highest in the bolting stem. The Arabidopsis-4CL behind the C a M V 35S or parsley  cDNA was inserted in antisense orientation 4CL1  promoters and introduced into  Arabidopsis.  Transgenic plants were analyzed by western blot analysis and plants with severely suppressed-4CL protein levels were further analyzed. One transgenic->4ra6/'cfops/'s line had greater than 9 0 % decrease in 4 C L enzyme activity and accumulated significantly less (50%) lignin in the bolting stem as compared to wild-type untransformed plants. Despite the decrease in 4CL mRNA, 4 C L protein, and 4 C L enzyme activity, anthocyanin accumulation was unaffected in the antisense-4CL Arabidopsis-Wnes.  Mature, fully-expanded  Arabidopsis-\eaves  were wounded and this  resulted in a coordinated increase in R N A transcripts from genes encoding enzymes in the oxidative pentose phosphate pathway, the shikimic acid pathway, and the general ii  phenylpropanoid pathway. The coordinated activation of gene expression was observed in the wild-type and antisense-4CL transgenic lines suggesting that woundinduced gene expression is not dependent on the carbon flow through the 4CLcatalyzed step.  i ii  Table of Contents  Abstract Table of Contents List of Tables List of Figures Abbreviations Acknowledgments 1. Introduction  1.1 Phenylpropanoid Metabolism 1.1.1 Introduction 1.1.2 General Phenylpropanoid Metabolism 1.2 Biosynthesis of Phenylpropanoid Products 1.2.1 Lignin 1.2.2 Flavonoids 1.2.3 Other Phenylpropanoid Molecules 1.3 Carbon Flow into Phenylpropanoid Metabolism 1.4 Research Objectives and Approaches 2. Materials and Methods 2.1 Plant Growth-Conditions, Tissues, and Experimental Treatments 2.2 Hybridization Probes 2.3 Cloning of 4CL c D N A s 2.4 Restriction-Map Analysis, Subcloning, Sequencing and Sequence Analysis of Tobacco and Arabidopsis cDNA Clones 2.5 Southern Blot and Northern Blot Analysis 2.6 Cloning of Tobacco-4CL c D N A s into E. coli Expression-Vectors 2.7 Protein Extraction 2.8 Western Blot Analysis 2.9 4 C L Enzyme Assay 2.10 Generation of Constructions to Antisense and Sense 4CL iv  2.11 Plant Transformation 2.12 Lignin and Anthocyanin Extraction 3. Cloning and Characterization of the 4 C L cDNAs from tabacum  and Arabidopsis  thaliana  Nicotiana  52  3.1 Cloning and Characterization of the Arabidopsis-4CL cDNA 3.2 Expression of the Arabidopsis 4CL 3.3 Cloning and Characterization of the Tobacco-4CL cDNAs 3.4 Inheritance of the Tobacco-4CL Genes 3.5 Expression of the Tobacco-4CL Genes 3.6 Discussion 4. Recombinant 4 C L  76  4.1 Expression of Tobacco-4CL cDNAs in E. coli. 4.2 Enzymatic Characterization of 4 C L 4.3 Modification of Recombinant-4CL Activity 4.4 Discussion 5. 4CL-Suppressed Transgenic Plants  98  5.1 Introduction 5.2 4CL-Suppressed Tobacco Plants 5.2.1 Generation of 4CL-Suppressed Tobacco Plants 5.2.2 Discussion 5.3 4CL-Suppressed Arabidopsis Plants 5.3.1 Generation of 4CL-Suppressed Arabidopsis Plants 5.3.2 Characteristics of 4CL-Suppressed Lines 5.3.3 Wound-Activated Gene Expression in 4CL-Suppressed Lines 5.3.4 Discussion 6. Conclusions and Future Directions  123  Bibliography  127  List of Tables  2.1  c D N A and gene fragments used for generating hybridization probes . . . . 41  2.2  E S T s used for generating hybridization probes  3.1  Comparison of At4CL, Nt4CL-1 and Nt4CL-19 nucleotide and predicted amino acid sequences to each other and to other 4CL sequences 54  3.2  Summary of Tobacco-4CL cDNAs cloned  4.1  Estimated K  m  and Vmax values for recombinant 4CL1 and recombinant  4CL2 from crude bacterial-extracts  41  61 80  5.1  Characteristics of primary-transgenic tobacco plants and their seeds . . . . 102  5.2  The proportion of kanamycin-resistant (KanR) to kanamycin-sensitive (KanS) T1 tobacco-seedlings  103  5.3  Summary of 4CL-suppressed transgenic-tobacco lines  104  5.4  Summary of transgenic  108  Arabidopsis  plants generated  vi  List of Figures 1.1 The general phenylpropanoid pathway  5  1.2 Schematic diagram of the biosynthesis of flavonoid- and ligninprecursors  17  1.3 Flavonoids  21  1.4 Reaction catalyzed by chalcone synthase  26  1.5 Biochemical relationship between classes of flavonoids  28  1.6 Phenylpropanoid compounds  31  1.7 Schematic diagram of the shikimic acid pathway  33  1.8 Carbon flow through the general phenylpropanoid pathway  35  2.1 Plasmids used in generating 4CL sense- and 4CL antisense- D N A constructions  48  2.2 Media used in plant transformation and regeneration  50  3.1 Comparison of the deduced amino acid sequences with that of 4CL from other plants 3.2  Arabidopsis  55  genomic Southern blot analysis  3.3 Northern blot analysis of 4CL RNA levels in  57 Arabidopsis  3.4 Northern blot analysis of 4CL R N A levels in mature  seedlings  58  Arabidopsis  59  3.5 Restriction maps of representative tobacco-4CL cDNA clones  61  3.6 3'-Sequences of representative tobacco-4CL cDNA clones  62  3.7  64  Plasmid Southern blot analysis  3.8 Tobacco genomic Southern blot analysis  64  3.9 Northern blot analysis of 4CL RNA levels in tobacco 3.10 Steady-state levels of 4CL RNA, PAL R N A and 4CL-proteins in the developing petals and sepals of tobacco  67  3.11  75  67  Diagram showing the relatedness of the orders in the angiosperms  4.1 Western blot analysis of recombinant tobacco-4CL expressed in E. 4.2 Chemical analogs used as substrates and inhibitors of 4 C L activity v ii  coli  77 80  4.3 Recombinant-4CL1 and recombinant-4CL2 activities as a function of substrate concentration  81  4.4 4 C L enzyme activity from crude tobacco-stem extracts plotted as a function of substrate concentration  82  4.5 Substrate specificities of tobacco-4CL and recombinant-4CL proteins . . . 8 2 4.6 Inhibition of recombinant-4CL2 activity by phenylpropanoid metabolites . 8 2 4.7 Effect of tobacco-stem extracts on recombinant -4CL2 activity towards cinnamate as a substrate  86  4.8 Characterization of the 4CL-modifying activity in tobacco-stem extracts . . 8 7 4.9 Distribution of the 4CL-modifying activity in tobacco organs 4.10 Distribution of endogenous tobacco-4CL activity  87 87  4.11 Affect of alkaline phosphatase and NaF on the 4CL-modifying activity from tobacco-stem extracts  88  4.12 Predicted phosphorylation-sites of polypeptides encoded by Nt4CL-1 and Nt4CL-19  4.13 Three models describing the involvement of phosphorylationdephosphorylation in the regulation of 4CL 5.1 Summary of potato-4CL cDNA constructs  88 97 102  5.2 An example of a western blot used to screen transgenic tobacco plants . . 104 5.3 An extreme case of altered leaf morphology in 4CL-suppressed transgenic tobacco 5.4 Summary of Arabidopsis-4CL cDNA constructs  105 108  5.5 An example of a western blot used to screen transgenic Arabidopsis plants  108  5.6 Phenotype of antisense-4CL suppressed transgenic Arabidopsis  110  5.7 Genomic Southern blot analysis of transgenic Arabidopsis  111  5.8 4 C L enzyme activity in transgenic and wild-type Arabidopsis  111  5.9 Lignin content in transgenic and wild-type Arabidopsis  111  5.10 Lignin content plotted as a function of 4CL enzyme activity viii  112  5.11 Anthocyanin levels in transgenic and wild-type hour high-intensity white-light treatment  Arabidopsis  after 24  114  5.12 Steady-state levels of CHS RNA, 4CL R N A and 4CL-protein in highintensity white-light treated transgenic and wild-type Arabidopsis leaves  114  5.13 Wound-induced R N A accumulation in wild-type and antisense-4CL suppressed Arabidopsis leaves  116  ix  Abbreviations 35S  Cauliflower m o s a i c virus 3 5 S promoter  4CL  4 - C o u m a r a t e : c o e n z y m e A ligase  6PGDH  6-phosphogluconate dehydrogenase  At4CL  c D N A e n c o d i n g 4 - C o u m a r a t e : c o e n z y m e A ligase from Arabidopsis  BSA  B o v i n e s e r u m albumin  C4H  Cinnamate 4-hydroxylase  CAD  C i n n a m y l alcohol d e h y d r o g e n a s e  CAF  Caffeic a c i d  CCR  C i n n a m o y l - C o A reductase  CHS  Chalcone synthase  CIN  C i n n a m i c acid  CIP  Calf intestinal alkaline p h o s p h a t a s e  COL  C o l u m b i a e c o t y p e of  COU  4 - C o u m a r i c acid  DAHPS  3-Deoxy-D-arab/noheptulosonate 7-phosphate synthase  DFR  Dihydroflavonol 4 - r e d u c t a s e  EPSPS  5-Eno/pyruvylshikimate-3-phosphate  F3H  Flavanone 3-hydroxylase  F3'H  Flavonoid 3'-hydroxylase  FER  Ferulic acid  G6PDH  Glucose-6-phosphate dehydrogenase  IPTG  Isopropyl-B-D-thiogalactopyranoside  kb  K i l o b a s e pair  kDa  Kilodalton  Km  M i c h e a l i s - M e n t e n constant  MJ  Methyl j a s m o n a t e  MS  Murashige and Skoog  Nt4CL  c D N A e n c o d i n g 4 - C o u m a r a t e : c o e n z y m e A ligase from Nicotiana  PAL  Phenylalanine ammonia-lyase.  PC4CL-P  P a r s l e y 4CL1 promoter  pQE-1  Q I A e x p r e s s i o n i s t p l a s m i d containing the N t 4 C L - 1  pQE-19  Q I A e x p r e s s i o n i s t p l a s m i d containing the N t 4 C L - 1 9  RLD  R L D e c o t y p e of  SDS  S o d i u m d o d e c y l sulphate  v  max  thaliana  Arabidopsis  Arabidopsis  M a x i m u m velocity X  synthase  tabacum  Acknowledgments  I would like to thank my supervisor Dr. Carl Douglas for his patience, his supervision, and his inspiration. I would like to thank my committee members Dr. Brian Ellis and Dr. John Carlson for their continuous advice and guidance.  Encouragement  and support from members of the Douglas, Haughn, and Kunst labs is acknowledged and was very much appreciated. Special thanks to Elizabeth Molitor and David Neustaedter who supplied advice on science and advice on life. This work was performed in the spirit of my father, a hard-working man of integrity, Yuk Bing Lee (1930 - 1995).  xi  Chapter 1 Introduction 1.1 Phenylpropanoid Metabolism 1.1.1 Introduction 1.1.2 General Phenylpropanoid Metabolism 1.2 Biosynthesis of Phenylpropanoid Products 1.2.1 Lignin 1.2.2  Flavonoids  1.2.3 Other Phenylpropanoid Molecules 1.3 Carbon Flow into Phenylpropanoid Metabolism 1.4 Research Objectives and Approaches  1.1 1.1.1  Phenylpropanoid Metabolism Introduction The flow of carbon from primary metabolism into the biosynthesis of an array of  phenylpropanoid secondary products involves a minimum of three enzymatic steps, catalyzed by the actions of phenylalanine ammonia-lyase (PAL), cinnamate 4hydroxylase (C4H) and 4-coumarate:CoA ligase (4CL) which collectively form the general phenylpropanoid pathway (Figure 1.1). The phenylpropanoid products formed by the action of specific downstream branch pathways include coumarins, flavonoids, lignin, suberin, tannins, and other phenolic compounds which serve diverse functions as phytoalexins, UV-protectants, floral and fruit pigments, structural components of cell walls, and signaling molecules (Hahlbrock and Scheel, 1989; Dixon and Paiva, 1995). The biosynthetic pathways leading to the formation of flavonoids and lignin have been relatively well characterized and they involve key 1  enzymes like chalcone synthase (CHS) and chalcone isomerase (CHI) for flavonoid biosynthesis; and cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) for lignin biosynthesis. Phenylpropanoid metabolic pathways are coordinately regulated. In bean hypocotyls, mechanical wounding and infection with pathogenic fungi causes an increase in PAL, CHS, and CHI m R N A transcripts (Lawton and Lamb, 1987). Similarly, treatment of bean cell cultures with elicitor causes a coordinate activation of PAL,  CHS,  and CHI expression (Cramer  etal.,  1985a,b) and in some studies, gene  activation was initiated as rapidly as 5 minutes post-elicitation (Lawton and Lamb, 1987). The increase in phenylpropanoid-specific enzyme activity is correlated with the increase in isoflavonoid phytoalexins (Robbins  1985). In parsley cell cultures  etal.,  (Chappell and Hahlbrock, 1984) and parsley protoplasts (Dangl era/., 1987), treatment with UV-light resulted in an increase in PAL, 4CL and CHS R N A levels followed by an increase in flavonoids. Treatment with elicitor caused an increase in  PAL,  4CL and BMT (bergaptol O-methyltransferase, an enzyme involved in  furanocoumarin biosynthesis) R N A levels concomitant with increases in coumarins (Chappell and Hahlbrock, 1984; Dangl era/., 1987).  In situ  hybridization  demonstrated the presence of PAL, 4CL, and CHS R N A s in the epidermal layer of young parsley-leaves where flavonoids accumulate. In the epithelial cells of oil-ducts where furanocoumarins accumulate, parsley PAL, 4CL, and BMT" R N A transcripts were detected (Wu and Hahlbrock, 1992). These results demonstrate that phenylpropanoid gene expression is coordinately regulated and is temporally- and spatially- specific to the accumulated end-products. Coordinate up-regulation of phenylpropanoid genes or enzymes has also been demonstrated in other plant systems (Hahlbrock and Scheel, 1989) such as soybean (Borner and Grisebach, 1982), potato (Fritzemeier (Kubasek  etal.,  etal.,  1987), alfalfa (Dalkin  etal.,  1990),  1992), poplar (Moniz de Sa, 1992), cactus (Pare  etal.,  Arabidopsis  1992), and  tobacco (Ellard-lvey and Douglas, 1996). The coordinate up-regulation of many of the 2  genes in the general phenylpropanoid and specific branch pathways may be an efficient way of controlling carbon flow through these pathways into product formation. Phenylpropanoid metabolism is developmentally regulated. Genes encoding phenylpropanoid enzymes have been shown to be activated in a cell-specific manner in cells of the developing xylem, epithelial cells of oil-ducts, and the epidermal cells of flower petals (Schmelzer  etal.,  1989; Wu and Hahlbrock, 1992; Drews  1992;  etal.,  Reinold era/., 1993). The activation of phenylpropanoid genes in these cell-types and tissues may be important for the biosynthesis of lignin, furanocoumarins, and anthocyanins respectively. Promoter-Gl/S fusion analysis in transgenic tobacco and transgenic al.,  Arabidopsis  showed that the promoters of PAL (Bevan  1989; Ohl etal., 1990),  (Schmid  et al.,  4CL  1990; Drews  (Hauffe  etal.,  etal.,  1991; Lee  etal.,  et al.,  1989; Liang  1995a), and  et  CHS  1992) direct complex patterns of G U S staining in the  flower, the leaf vein, and the root vasculature. These results show that phenylpropanoid expression occurs in a tissue-specific and temporally-regulated manner that correlates with the time and place where phenylpropanoid compounds accumulate. Wounding and pathogen attack activates phenylpropanoid metabolism. In bean hypocotyls, infection with an incompatible fungus causes an increase in PAL and CHS  R N A (Cramer  etal.,  1985a,b; Lawton and Lamb, 1987) and enzyme activity  (Robbins era/., 1985). In potato leaves, infection with a fungal pathogen results in the accumulation of  PAL  and  4CL  transcripts (Fritzemeier  etal.,  1987). In parsley,  infection with a non-pathogenic fungus and wounding both cause the accumulation of phenylpropanoid gene transcripts at the infection/wound site (Schmelzer Similarly,  Arabidopsis  PAL  (Ohl etal.,  1990; Wanner  etal.,  1995) and 4CL (Lee  1995a) R N A levels increase during wounding and during infection by syringae.  etal.,  1989). etal.,  Pseudomonas  The induction of phenylpropanoid gene expression at the wound or  infection site is believed to be for the biosynthesis of phytoalexins, phenolic compounds, and lignin-like subunits which are deposited at wound/infection sites (reviewed in Nicholson and Hammerschmidt, 1992). These phenylpropanoid products 3  prevent pathogen invasion by inhibiting pathogen growth (phytoalexins) or by inhibiting pathogen spread (deposition of cell wall components). It should be noted that pathogen attack also activates a systemic response which includes the expression of the PR (Pathogenesis-Related) genes. Some of the P R proteins are of unknown function while others encode chitinases, hydroxyproline-rich proteins, and protease inhibitors. These enzymes/proteins increase the plants systemic resistance against pathogens (SAR; systemic acquired resistance). The systemic response may be mediated in part by salicylic acid, a compound derived from the product of the PALcatalyzed reaction. Thus, in addition to the activation of phenylpropanoid gene expression, pathogen attack may also activate another class of genes, the PR genes, via salicylic acid which is derived from phenylalanine. Phenylpropanoid metabolism has also been shown to be induced by other conditions such as ozone (Sharma and Davis, 1994; Eckey-Kaltenbach era/., 1994), cytokinins (Deikman and Hammer, 1995), nutrient starvation (Dixon and Paiva, 1995), and cold treatment. (Leyva  etal.,  1995). The roles of phenylpropanoid compounds  under these stresses have not been clearly defined (reviewed in Dixon and Paiva, 1995). In vivo  footprinting and deletion analysis have defined putative c/'s-elements  that are conserved in the promoters of phenylpropanoid genes. In particular, conserved sequences like the P-, A-, and L- boxes (Lois et al, 1989; Logemann era/., 1995), and putative Myb-binding sequences (Sablowski  etal.,  1994; Douglas, 1996)  have been detected in the promoters of PAL, 4CL, CHS, and CAD.  BPF-1, a DNA-  binding protein from parsley (da Costa e Silva et al., 1993), and P B P , a flower-specific Myb protein from tobacco (Sablowski era/., 1994) have been shown bind to the P-box. Gel-retardation assays have identified nuclear proteins from parsley and tobacco which bind to the parsley 4CL1 promoter (Hauffe et al., 1993). Thus, the presence of these conserved c/s-elements and the activities of the frans-acting factors provide a mechanism in which the phenylpropanoid genes may be coordinately regulated. 4  COOH  COOH  *C-S-CoA  COOH 4CL-.  CoA-SH + ATP Phenylalanine  Cinnamic acid  4-Coumaric acid  4-Coumaroyl:CoA  Figure 1.1: The general phenylpropanoid pathway. The three core reactions in the general phenylpropanoid pathway are catalyzed by phenylalanine ammonia-lyase (PAL), cinnamate 4hydroxylase (C4H) and 4-coumarate:coenzyme A ligase (4CL).  1.1.2  General Phenylpropanoid Metabolism The three enzymes PAL, C 4 H and 4 C L comprise the general phenylpropanoid  pathway (Figure 1.1). Products derived from the general phenylpropanoid pathway enter branch pathways which synthesize specific end-products such as furanocoumarins, flavonoids and lignin. Thus, PAL, C 4 H and 4 C L are important in that they are the core enzymes from which all other phenylpropanoid compounds are made. These enzymes and the genes encoding them are described below. P A L is the first enzyme in the general phenylpropanoid pathway and it is one of the most well characterized enzymes in phenylpropanoid metabolism (Jones, 1984). P A L catalyzes the deamination of phenylalanine to form cinnamic acid (Figure 1.1). Cinnamic acid can be used for the biosynthesis of other phenolic acids (coumaric acid, caffeic acid), phenolic esters, benzoic acid derivatives (salicylic acid, gallic acid), coumarins, B-ring-deoxy flavonoids and other cinnamyl derivatives (Lewis, 1993; Lee et al., 1995b; Liu et al., 1995). A s the first enzyme in general phenylpropanoid metabolism, P A L has been suggested to catalyze the rate-limiting step which controls  the flux of carbon compounds into phenylpropanoid biosynthesis. Chemical analogs of phenylalanine like a-aminooxy-p-phenylpropionic acid (AOPP) and 1-amino-2(phenylethyl)phosphonic acid (APEP) inhibit PAL activity. In buckwheat seedlings, red-cabbage seedlings, morning-glory flowers, and other tissues, the use of A O P P causes a decrease in anthocyanin accumulation (Amrhein and Hollander, 1979). Mungbean seedlings treated with A O P P have collapsed xylem vessels and lack ligninlike components as demonstrated by phloroglucinol/HCL staining and nitrobenzene oxidation (Amrhein et al., 1983; Smart and Amrhein, 1985). In tobacco leaves, sense suppression of PAL causes a decrease in chlorogenic acid and rutin accumulation (Bates et al., 1994). In contrast, lignin content in tobacco stems decreases only when P A L activity was suppressed by 7 5 % to 8 0 % compared to wild-type levels (Bates et al., 1994). These results confirm that the biosynthesis of phenylpropanoid compounds require P A L activity and that it is the major control point in the biosynthesis of chlorogenic acid and rutin. However, with respect to lignin biosynthesis, the endogenous levels of P A L in tobacco stems are in excess relative to the amount of lignin made in normal development. The genes and c D N A s encoding PAL have been isolated from over 25 plants (Genbank) including bean (Cramer  et al.,  1989), parsley (Lois  (Subramaniam era/., 1993), tobacco (Pellegrini  et al.,  etal.,  1994) and  1989), poplar  Arabidopsis  (Wanner  et al., 1995). In most plants, P A L is encoded by a small gene family of 3-5 genes; however, notable exceptions include the P A L from loblolly pine which may be encoded by a single gene (Whetton and Sederoff, 1992) and the P A L from potato which is represented by a family of 40-50 genes (Joos and Hahlbrock, 1992). Members of the  PAL  gene family are differentially expressed. In parsley,  expressed constitutively in the roots while  PAL3  PAL2  interaction). The bean  is  is expressed in response to wounding  (Lois and Hahlbrock, 1992). In potato (Joos and Hahlbrock, 1992), more rapidly than  PAL2  PALI  is expressed  in response to infection by a virulent fungus (compatible PAL2  gene is expressed after elicitor treatment whereas the 6  PAL3  gene is not (Cramer  etal.,  1989). In agreement, transgenic studies with the  bean PAL promoters fused to GUS demonstrate that the bean PAL promoters direct different patterns of tissue-specific G U S staining and direct differential GUS-staining in response to wounding and elicitor treatment (Shufflebottom et al., 1993). Like other phenylpropanoid genes, PAL expression is developmental^ regulated, stress inducible and coordinately regulated. In parsley (Wu and Hahlbrock, 1992),  in situ  localization shows that  PAL  R N A levels accumulate in a tissue-specific  fashion that is coordinately regulated with the presence of R N A from 4CL, CHS (in the epidermis) or BMT(m  the oil ducts). In parsley cell cultures, treatment with UV-light or  elicitor cause a coordinate increase in PAL and 4CL R N A levels (Ragg et al., 1981; Chappell and Hahlbrock, 1984; Schmelzer era/., 1985). Inoculation of parsley leaves with a non-pathogenic fungus causes a localized increase in PAL R N A at the site of attempted infection and this expression is coordinated with the expression of 4CL and other pathogen-related (PR) and elicitor-related (ELI) genes (Schmelzer era/., 1989). Developmental and stress-regulated PAL expression also occurs in bean cell cultures (Edwards  etal.,  1985), potato (Fritzemeier  1992) and poplar (Subramaniam  etal.,  etal.,  1987), Arabidopsis  (Kubasek  etal.,  1993).  The second enzyme in the general phenylpropanoid pathway is C 4 H . C 4 H is a cytochrome P-450 monoxygenase and it catalyzes the hydroxylation of cinnamate at the para-position to produce 4-coumarate using O2 and N A D P H . Because of its instability and its microsomal localization, purification and characterization of C 4 H has been limited to a few plants. The C 4 H enzymes from potato (Tanaka et al., 1974), wheat (Maule and Ride, 1983), Jerusalem artichoke (Gabriac  etal.,  1991), and  soybean (Kochs et al., 1992) have been examined. In Jerusalem artichoke tubers, C 4 H enzyme activity increases after wounding (Benveniste et al., 1977) and pea seedlings which have been adapted to darkness exhibit increases in C 4 H activity when exposed to light (Benveniste  etal.,  1978). In sweet-potato roots, wound-induced  C 4 H activity is paralleled by the increase in P A L activity (Tanaka 7  etal.,  1974) and in  wounded wheat-leaves, the increase in C 4 H activity is coordinated with the increase in 4 C L activity (Maule and Ride, 1983). Full-length c D N A s encoding C 4 H have been cloned from mung bean, Jerusalem artichoke, alfalfa, and Catharanthus etal.,  roseus  1993; Farhendorf and Dixon, 1993; Hotze  etal.,  (Mizutani era/., 1993; Teutsch 1995) and the isolation of  partial-length C4H clones from other plants has been reported (Genbank). The expression pattern of C4H has not been well characterized but in the few plants in which it has been examined, C4H expression appears to be inducible and occurs in a manner analogous to that of PAL and 4CL. In parsley cell cultures, parsley leaves, and parsley roots, UV-light treatment, elicitor treatment, and wounding cause an increase in C4H R N A levels with very similar kinetics as the induction of parsley PAL2,  PAL3,  4CL1  and  4CL2  (Logemann era/., 1995). In Catharanthus  roseus  PALI,  cell  cultures, C4H R N A levels increase after elicitation (Hotze et al., 1995) and in alfalfa cell cultures, elicitation causes an increase in C4H R N A that is coordinated with the increase in CHS transcripts (Fahrendorf and Dixon, 1993). Although limited, these results suggest that C4H is induced by the same treatments as those which activate  PAL  and other phenylpropanoid genes. 4 C L is the last enzyme in the general phenylpropanoid pathway and it has  been detected in many higher plants including monocotyledons, dicotyledons and gymnosperms (Gross  etal.,  1975; Wallis and Rhodes, 1977; Kutsuki  etal.,  1982b).  Using A T P and coenzyme A (CoA), 4 C L converts 4-coumaric acid and other hydroxyor methoxy- derivatives of cinnamic acid, such as caffeic acid, ferulic acid, and sinapic acid, to form the corresponding C o A esters (Figure 1.2). These esters serve as substrates for entry into various branch-pathways (Hahlbrock and Scheel, 1989). Because of its terminal position in the general phenylpropanoid pathway, and its ability to utilize a number of differently substituted hydroxy-cinnamic acids, it has been hypothesized that 4 C L could control the partitioning of carbon into different branchpathways through the activity of distinct 4CL-isoforms (Knobloch and Hahlbrock, 1975; 8  Grand et al., 1983). For example, 4 C L may direct 4-coumaroyl:CoA into flavonoid biosynthesis via C H S and CHI. 4 C L may regulate the amounts of 4-coumaroyl, feruloyl and sinapoyl:CoA esters that enter lignin biosynthesis via the C C R and C A D reactions (Figure 1.2). In support of this, physically-distinct 4 C L isoforms have been reported from soybean, petunia, poplar, maize, and parsley (Knobloch and Hahlbrock, 1975; Ranjeva  etal.,  1976; Grand  1983; Vincent and Nicholson, 1987; Lozoya  etal.,  et  al., 1988). Furthermore, in soybean, petunia, and poplar, partially-purified 4 C L isoforms exhibit different substrate specificities towards substituted cinnamic acids. The soybean 4CL1 isoenzyme is able to use sinapate as a substrate whereas 4 C L 2 can not (Knobloch and Hahlbrock, 1975). Based on their substrate specificity, the three petunia 4CL-isoforms are named caffeate:CoA ligase, sinapate:CoA ligase, and ferulate:CoA ligase for their high activities, respectively, towards caffeate, sinapate and ferulate as substrates (Ranjeva et al., 1976). In poplar, 4CL1 is able to react with 4coumarate, ferulate, and sinapate as substrates; 4 C L 2 can use 4-coumarate and ferulate as substrates; and 4 C L 3 can use 4-coumarate and caffeate as substrates (Grand et al., 1983). The distribution of the poplar 4CL-isoenzymes correlates with the types of phenylpropanoid products formed, suggesting that the activities of the 4 C L isoenzymes control the types of products made (Grand et al., 1983). In contrast, a single 4 C L form was purified from loblolly pine (Voo et al., 1995), and two slightly different 4 C L isoforms with similar substrate preferences are encoded by two parsley  4CL genes (Lozoya era/., 1988), suggesting that 4 C L does not participate in the selective distribution of metabolites in these species. The genes and cDNAs encoding 4 C L have been cloned from 9 plants (Douglas etal.,  1987; Lozoya  etal.,  and Ebel, 1993; Voo etal.,  1988; Zhao  etal.,  1995; Yazaki  1990; Becker-Andre  etal.,  etal.,  1991; Uhlmann  1995; Allina and Douglas, 1994; Lee  et  al., 1995a; Lee and Douglas, 1996b). 4 C L is encoded by multiple, divergent genes in some plants like rice, soybean, poplar, (Zhao  etal.,  Lithospermum  erythrorhizon,  and tobacco  1990; Uhlmann and Ebel, 1993; Allina and Douglas, 1994; Yazaki 9  etal.,  1995; Lee and Douglas, 1996b); by very similar duplicated genes as in the case of parsley and potato (Lozoya era/., 1988; Becker-Andre era/., 1991); and by apparently single genes as is the case in Arabidopsis  and pine (Lee  et al.,  1995; Voo et  al.,  1995).  In conjunction with earlier biochemical studies, it is possible that the divergent 4CL genes in plants such as soybean and poplar may encode 4 C L enzymes with distinct enzymatic properties. This hypothesis has not yet been tested. Other than the study of two nearly-identical parsley-4CL genes and their encoded proteins (Lozoya era/., 1988), there has been no published information on the properties of recombinant-4CL proteins from divergent members of 4CL gene families.  4CL gene expression, like that of many of the phenylpropanoid genes, is regulated developmentally and is activated by external stimuli such as pathogen infection, elicitortreatment, wounding, and UV-light irradiation (Douglas Schmelzer  et al.,  1989; Wu and Hahlbrock, 1992). In tobacco flowers,  in  etal.,  1987;  situ  hybridization shows that endogenous tobacco 4CL transcripts and those of an introduced parsley 4CL1 gene accumulate in a cell-type specific manner, and that the patterns of accumulation are generally consistent with the sites of phenylpropanoid natural-product accumulation (Reinold era/., 1993). A s well, 4CL expression in tobacco is activated by wounding, light, and methyl jasmonate treatment (Douglas et  al., 1991; Ellard-lvey and Douglas, 1996). The 4CL genes from parsley and soybean are differentially regulated.  4CL-2  from parsley is preferentially expressed in the  flowering stem and is light inducible, whereas 4CL-1 is wound inducible in roots (Lois and Hahlbrock, 1992). In soybean, 4CL-14  4CL-16  is inducible by fungal infection whereas  is not (Uhlmann and Ebel, 1993). The 4 C L reaction converts the carboxylic acid group into a thioester group,  thereby "activating" the molecule for further biochemical reactions. In the biosynthesis of lignin, the C o A esters are reduced to form alcohols whereas in flavonoid biosynthesis, the C o A esters are condensed with malonyl CoA. Some of the products made from hydroxy- and methoxy- cinnamoyl:CoA esters are described below. 10  1.2 1.2.1  Biosynthesis of Phenylpropanoid Products Lignin Lignin is a three-dimensional polymer composed of cinnamyl alcohol  derivatives linked in covalent bonds. Lignin is deposited in the walls of specialized cells of the xylem and phloem including the tracheids, vessel members, sclereids, and fibers. Lignin provides rigidity and hydrophobicity to the cell walls, properties which are particularly important for cells which conduct water. Thus, the evolution of lignin has been considered a major step for the emergence of terrestrial vascular plants (Lewis and Yamamoto, 1990; Whetten and Sederoff, 1995). The analysis of lignin is relatively difficult because the polymer is large and resists extraction. The methods used to extract lignin usually degrade the polymer such that analysis of the polymer in its native form is near impossible. Many methods of lignin detection and extraction are available and each have advantages and disadvantages (reviewed in Lewis and Yamamoto, 1990). The deposition of lignin is developmentally regulated. Lignin is deposited in the developing vasculature of leaf veins and roots. In perennials, lignin deposition in secondary growth is activated by favorable environmental condition (Raven era/., 1987). Lignin deposition is also inducible. During infection by pathogens, plants may deposit lignin and lignin-like polymers at the infection site. It is believed that lignin may serve as a physical barrier to attenuate pathogen invasion. The deposition of lignin and lignin-like components during wounding and pathogen attack is relatively rapid and is likely derived from pre-existing phenolic compounds (Nicholson and Hammerschmidt, 1992). Zinnia elegans (Fukuda and Komamine, 1982) and pine cell cultures (Campbell and Ellis, 1992) have been useful in studying the biochemistry of lignin formation since these cultures can be induced to undergo lignification. Deposition of lignin is a multi-component procedure consisting of: 1) biosynthesis of lignin monomers, 2) storage or transport of lignin monomers, and 3) polymerization of monomers. Sequential hydroxylation and methylation of the 11  aromatic ring in the C 3 and C 5 positions of 4-coumaric acid produces caffeic acid (3, 4-dihydroxycinnamic acid), ferulic acid (3-methoxy 4-hydroxycinnamic acid), 5hydroxyferulic acid (3-methoxy 4, 5-dihydroxycinnamic acid), and sinapic acid (3,5dimethoxy 4-hydroxycinnamic acid). These ring modifications are catalyzed by coumarate 3-hydroxylase (C3H), bispecific caffeic acid/5-hydroxyferulic acid O methyltransferase (COMT), and ferulate 5-hydroxylase (F5H). 4 C L converts some, or all of these modified cinnamic acids to form their corresponding C o A esters (Figure 1.2). An alternative pathway in which the hydroxylation and methylation steps occur after C o A activation has been suggested (Figure 1.2). In support of this, an enzyme activity (CCo3H) from parsley cell cultures which hydroxylates 4-coumaroyl:CoA to form caffeoyhCoA has been described (Kneusel  etal.,  1989). In carrot cell cultures,  caffeoyhCoA 3-O-methyltransferase (CCoAOMT) which methylates caffeoyhCoA to feruloyhCoA has been described (Kuhnl era/., 1989) and this enzyme activity has been purified and characterized from parsley cell cultures (Pakusch et al., 1989). The c D N A encoding C C o A O M T has been cloned from parsley and 1991; Y e etal.,  1994). In parsley,  CCoAOMT  Zinnia  (Schmitt  et al.,  RNA levels increase in a manner  analogous to the increase in PAL R N A levels during elicitation. Crude extracts from Zinnia  have been shown to methylate caffeoyhCoA and 5-hydroxyferuloyl:CoA to form  feruloyl:CoA and sinapoyhCoA respectively (Ye era/., 1994). The temporal and spatial expression of CCoAOMT  from  Zinnia  coincides with tissues undergoing lignification  (Ye era/., 1994). These results suggests that cinnamoyhCoA derivatives may be generated by at least 2 pathways and in Zinnia,  both the COMT- and CCoAOMT-  mediated pathways appear to be functional (Ye and Varner, 1995). 4-coumaroyl:CoA, feruloyhCoA, and sinapoyl:CoA are then reduced to their corresponding alcohols via the actions of C C R and C A D . C C R and C A D use N A D P H as an electron donor to reduce the thioesters to form alcohols. The ability of C C R and C A D to reduce 5-hydroxyferuloyl:CoA to its corresponding alcohol  in vivo  has been  implied by the presence of 5-hydroxyferulyl lignin subunits in transgenic plants 1 2  (Antanassova et al., 1995) and the C C R from soybean uses 5-hydroxylferuloyl:CoA as a substrate the  trans  in vitro  (Wengenmayer era/., 1976). In most plants, lignin monomers are of  conformation; however, lignin from the bark of the American beech tree is  composed of the c/'s-isomers (Lewis era/., 1988). Radiolabeled feeding experiments show that  trans/cis  isomerizatibn occurs at the level of the alcohol (Lewis  Although UV-light induced isomerization occurs monolignols probably involves an isomerase  in vitro, trans/cis  in vivo  (Lewis  et al.,  etal.,  1987).  isomerization of 1987). Once made,  the monolignols are glycosylated at the 4-hydroxy position and then transported to the site of lignin deposition or storaged as monolignol glucosides. Monolignols are glycosylated by UDP-glucose:monolignol glucosyltransferases and de-glycosylated by 3-glucosidases. The mechanism of monolignol transport is not well understood; however, it can be partially explained with the endomembrane theory. The theory suggests that the lignin precursors are mobilized from the cytoplasm in phospholipid vesicles that are derived from the Golgi body or the endoplasmic reticulum and then deposited outside the cell membrane in a manner similar to exocytosis (Pickett-Heaps, 1968). In conifers, it has been hypothesized that monolignol glucosides are stored in the vacuole, transported out of the cytoplasm, and then de-glycosylated in the cell walls (Whetten and Sederoff, 1995). In pine, a coniferin B-glucosidase has been localized to the differentiating xylem where peroxidase activity is also found (Dharmawardhana et al., 1995). Peroxidases and/or laccases convert the monolignols into free radicals and the free radicals undergo a free-radical polymerization reaction. Evidence suggests that free-radical polymerization may be ordered and controlled since polymerization appears to begin at the middle lamella and progress inwards towards the lumen. Polymerization also appears to start at the "corners" of cell walls first and then progress away from the corners, suggesting that a mechanism exists to direct the polymerization event (reviewed in Takabe et al., 1989). There is a general trend in the types of lignin found in plants. Lignin from gymnosperms contains predominantly guaiacyl subunits (from feruloyl:CoA). In 13  angiosperms, the lignin is composed of guaiacyl and syringyl (from sinapoyhCoA) subunits and lignin from grasses contains syringyl, guaiacyl and 4-hydroxyphenyl subunits. How lignin composition is regulated is unclear; however, it is likely controlled by the enzymes involved in the biosynthesis or transport of monolignols. The substrate specificity of lignin biosynthetic enzymes may control the types of monolignols formed. For example, C O M T enzymes from gymnosperms are more active towards caffeic acid whereas C O M T enzymes from angiosperms are more active towards 5-hydroxyferulic acid (reviewed in Whetten and Sederoff, 1995). In soybean (Knobloch and Hahlbrock, 1975), petunia (Ranjeva  etal.,  1976), and poplar  (Grand et al., 1983), 4 C L isoforms with different substrate specificities have been identified and the activities of these isoforms may define the kinds of monolignols formed (see section 1.1.2). In a similar manner, the preferential reduction of different cinnamoyl:CoA derivatives by C C R and C A D may control which kinds of monolignols are made for polymerization. C C R can reduce sinapoyhCoA, feruloyhCoA, and 4coumaroyl:CoA (Sarni  etal.,  1984; Goffner  etal.,  1994); however, C C R has a  preference for feruloyhCoA as a substrate (Wengenmayer et al., 1976; Luderitz and Grisebach, 1981). Luderitz and Grisebach (1981) demonstrated that C C R from spruce is predominanty active towards feruloyl:CoA whereas C C R from soybean uses both feruloyhCoA and sinapoyl:CoA, suggesting that the activities of C C R may control the kinds of lignin found in gymnosperms and angiosperms. C A D activities with different substrate specificities have been found in wheat (Pillonel gunnii(Goffner  etal.,  1992). Kutsuki  etal.  et al.,  1992), and  Eucalyptus  (1982a) examined C A D activity from a  number of gymnosperms and angiosperms and showed that C A D enzymes from gymnosperms prefer the ferulyl substrate whereas C A D enzymes from angiosperms use both ferulyl and sinapyl substrates. These results suggest that the types of lignin found in plants may be regulated by the substrate specificities of C O M T , 4CL, C C R , and C A D ; however, more targeted studies are required before the role of each of these enzymes in regulating lignin quality can be assigned. The transport of monolignol 1 4  glucosides either for storage or for polymerization is not well characterized. However, conceptually, it is possible that lignin composition may be regulated by the selective activity of UDP-glucose:monolignol glucosyltransferases and B-glucosidases. The purified R-glucosidase from lodgepole pine uses both coniferin and syringin  in vitro,  suggesting that the activity of this particular enzyme does not control lignin quality in pine (Dharmawardhana  etal.,  1995).  The isolation of mutants and reverse genetics using cloned genes has demonstrated the importance of certain steps in the lignin biosynthetic pathway. Sense suppression of PAL in tobacco results in lower levels of lignin, demonstrating that the amount of lignin made can be controlled in the early steps of phenylpropanoid metabolism (Elkind  etal.,  1990). However, quantitative differences in lignin  accumulation are observed only when PAL activities were suppressed by >75%, suggesting that under normal conditions, PAL activity is not a limiting factor (Bates et  al., 1994). Antisense-RNA technology has been used to down-regulate COMT activity in tobacco (Dwivedi et al., 1994; Atanassova era/., 1995), and poplar (Van Doorsselaere et al., 1995) resulting in lignin with higher guaiacyl or 5-hydroxyferulyl subunits relative to the wild-type counterparts, suggesting that lignin composition is regulated, in part by, COMT expression. It is interesting to note that the syringylsubunit levels were lowered when COMT activity was suppressed, suggesting that carbon flow into sinapate synthesis is preferentially blocked. Significant differences in the S:G (syringyl to guaiacyl) ratio were observed when COMT activity was suppressed by >80% (Atanassova era/., 1995; Van Doorsselaere et al., 1995). The bm3 mutants of maize are disrupted at the COMT gene and possess altered lignin quality (Vignols et al, 1995) and quantity (Grand era/., 1985). These results suggest that COMT activity has a role in controlling lignin quality, and perhaps a role in controlling lignin quantity as well. In poplar, F5H activity was preferentially localized in sclerenchyma, a tissue which also has high levels of syringyl lignin (Grand, 1984). The fa/? 7  (sin1)  mutant of Arabidopsis,  which lacks F5H activity, has been identified and 15  these plants did not accumulate sinapate derivatives including syringyl lignin (Chappie era/., 1992). Thus in Arabidopsis,  the deposition of syringyl lignin requires  F5H function and it has been suggested that the lack of F5H activity in gymnosperms may be the reason for the high guaiacyl and low syringyl lignin in these plants (reviewed in Campbell and Sederoff, 1996; Grand, 1984). The reduction of C A D activity by antisense R N A resulted in tobacco plants with higher ratio of aldehyde/alcohol lignin monomers as compared to control plants (Halpin et al, 1994). The increase in aldehyde/alcohol monomers was evident when C A D activity was decreased by >84%. The increase in sinapyl aldehydes was more pronounced than the increase in coniferyl aldehydes. The  bmrQ  mutant of  Sorghum  has lower levels of  C A D activity and also deposits the aldehyde forms of the lignin subunits. However, the phenotype of the bmr6 mutant should not be compared directly to the antisense- CAD plants since bmr6 mutant also has decreased C O M T activity. In the bmr6 mutant, there was a pronounced increase in the coniferyl (and not sinapyl) aldehyde monomers (Pillonel era/., 1991). Overproduction of a lignin-specific anionic peroxidase resulted in an increase in lignin accumulation in transgenic tobacco (Lagrimini, 1991) and transgenic tomato (Lagrimini et al., 1993). The authors suggest that in pith tissues, the low levels of lignification is due to the limiting levels of endogenous anionic peroxidase (Lagrimini, 1991).  16  COOH  COOH  COOH  COOH  C3H 1 —-fea- «^>|  COMT OCH OH Ferulic acid  OH  OH 4-Coumaric acid  J  Caffeic acid  4CL  4CL *C-CoA  3  0CH3 H3C0 f "0CH3 OH OH Sinapic acid 5-Hydroxyferulic acid HO  |4CL  *C-CoA  J4CL  |4CL O,  'C-CoA  k  C-CoA  C-CoA  CCoOMT OCH3 HO 4-Coumaroyl:CoA CHS/  Caffeoyl:CoA  Feruloyl:CoA  y  I  C H H3CO' OCH3 OH 5-Hydroxyferuloyl:CoA SinapoyhCoA  CCR  CCR  CCR  J  CAD  CH OH  CAD  CAD  CH OH  CH OH  2  2  3  2  CH OH 2  OCH3 HO X OCH3 H3CO' "OCH3 OH OH OH Coniferyl Sinapyl 5-Hydroxyferulyl alcohol alcohol alcohol  4-Coumaryl alcohol  Flavonoids  Lignin  Figure 1.2: Schematic diagram of the biosynthesis of flavonoid- and lignin- precursors. The identification of novel enzymes suggests that the biosynthesis of CoA esters from 4-coumarate may proceed through a number of enzymatic steps potentially resulting in a metabolic grid rather than a linear pathway. Genetic engineering and the isolation of mutants demonstrate the flow of phenolic compounds through the metabolic grid (see section 1.2.1). Enzymes are: PAL, phenylalanine ammonia-lyase; C 4 H , cinnamate 4-hydroxylase, C 3 H , 4-coumarate 3-hydroxylase, COMT, caffeic acid/5-hydroxyferulic acid Omethyltransferase; F5H, ferulate 5-hydroxylase; 4CL, 4-coumarate:CoA ligase; C C o 3 H , 4-coumaroyl:CoA 3-hydroxylase; CCoOMT, caffeoyhCoA O-methyltransferase; ?, uncharacterized metabolic step; CHS, chalcone synthase; CHI, chalcone isomerase; CCR, cinnamoyl-CoA reductase; and CAD, cinnamyl alcohol dehydrogenase. Dashed arrows represent enzymatic steps which have not been clearly demonstrated.  17  1.3.2  Flavonoids Flavonoids are compounds structurally-based on the flavanone-skeleton  (Figure 1.3A). One benzene ring (A ring) is derived from the condensation of three malonyhCoA units and the second benzene ring (B ring) is derived from 4coumaroyl:CoA. The oxidation state of the central heterocyclic ring (C ring) is used to group the flavonoids into classes (Figure 1.3A). Within each class of flavonoids, members differ by the hydroxylation or methoxylation on the A- and B- rings (Figure 1.3B). For example, flavanones and flavones are two classes of flavonoids which differ by the absence and presence, respectively, of a double bond between carbons 2 and 3 of the C-ring (Figure 1.3A). Pelargonidin, cyanidin and delphinidin are all members of the anthocyanidins and they differ by the hydroxylation on the 3', 4' and 5' carbons of the B-ring (Figure 1.3B). Naturally-occurring flavonoids are usually glycosylated or modified at one or more of the hydroxyl groups. Sugars can be hexoses or pentoses and can be monosaccharides, disaccharides or trisaccharides in a or R linkages (Swain, 1965). The unglycosylated flavonoids are referred to as aglycones. In particular, anthocyanins are the glycosylated forms whereas the aglycones are called anthocyanidins. The term "leucoanthocyanidins" is sometimes used to refer to flavan-3,4-diols since they are the colorless precursors to anthocyanidins (Haslam, 1982). Condensed proanthocyanidins (also called condensed tannins) are composed of oligomeric polymers of flavan-3-ols and these are referred to as "proanthocyanidins" because treatment of the polymer with acids results in the releases of anthocyanidins (Haslam, 1982). In addition to the flavonoids listed in Figure 1.3, other minor flavonoids such as neoflavonoids, biflavonoids and triflavonoids exist (Geiger and Quinn, 1982) and will not be discussed here. Flavonoids absorb light, the quality of light absorbed depending on the number and location of double bonds, hydroxyl groups, and ketone groups. Flavonoids with few double bonds and few hydroxyl groups (Figure 1.3A, flavan-3-ols) absorb high energy UV-light and are colorless, but they may confer a cream or ivory hue. 18  Flavonoids with many double bonds and hydroxyl groups (Figure 1.3, anthocyanidins and aurones) absorb UV-light and visible light and can be red, blue or violet in color. The color that flavonoids impart largely depends on the presence of other copigments, the pH of the environment, their compartmentalization, and the presence of chelating metals (Swain, 1965). Flavonoids are usually found in the epidermal or subepidermal layers and accumulate in the vacuole (Salisbury and Ross, 1978). Flavonoids are most obvious and predominanty found in the flowers and fruits of plants where they attract pollinators and creatures which aid in seed dispersal. Flavonoids are also present in leaves, roots and tubers; however, the high levels of chlorophyll in the leaves tend to mask the flavonoids. The colors contributed by flavonoids are sometimes evident in very young leaves and senescing leaves where chlorophyll content is lower than that found in mature leaves (Swain, 1965). The flavonoids in leaves likely have a role in absorbing UV-light thereby protecting proteins and nucleic acids from UV-induced damage (Caldwell  etal.,  1983; Kootstra, 1994; Stapleton and Walbot, 1994). Some  flavonoids such as the condensed proanthocyanidins (condensed tannins) may also serve as feeding deterrents since they have an astringent taste and have the ability to precipitate proteins and polysaccharides (Haslam, 1982). Flavonoids have been implicated as signal molecules between plants and symbiotic microorganisms (Long, 1989). For example, flavonoids released from alfalfa have the ability to induce the expression of nodulation genes (Peters era/., 1986) and enhance the growth of Rhizobium  meliloti  (Hartwig  et al.,  1991). Some flavonoids, especially the  isoflavonoids, have antimicrobrial activities and may serve as phytoalexins (Adesanya and Roberts, 1995). Recently, it has been determined that flavonols are required for proper pollen and pollen tube development in maize, petunia, and tobacco (Mo et al., 1992; Ylstra  etal.,  1992; Ylstra  etal.,  1994). However, this physiological role is not  ubiquitous to all plants since mutants of Arabidopsis fertile (Burbulis  etal.,  which lack flavonoids are self  1996). The first enzyme in the flavonoid biosynthetic pathway, 1 9  chalcone synthase (CHS) will be discussed in detail and subsequent enzymes will be mentioned briefly.  20  OH Pelargonidin OH ,0H  +  HO,  o  Dihydrochalcones  Chalcones OH  ^  "OH  OH  .OH  Cyanidin OH  O Flavones  o  .OH  +  HO,  Flavanones  o_  'OH "OH  OH  o Flavonols  O OH Isoflavones  Dihydroflavonols  o ^ - O H Isoflavanones  OH Flavan-3,4-diols  OH OH Anthocyanidins  O Aurones  Malvidin  21  Figure 1.3: Flavonoids. A. Classes of flavonoids are listed with a representative member of each class. Note that the oxidation state of the C-ring defines the flavonoid class. Modifications on the A- and B- rings are not shown. B. Anthocyanidins. Note that members are differentiated by the hydroxylation and methoxylation on the B-ring.  C H S catalyzes the first committed step in flavonoid biosynthesis and it condenses 4-coumaroyl:CoA sequentially with three acetate units derived from malonyl:CoA to produce a chalcone (Figure 1.4). The enzyme has been purified and characterized from a few sources. In parsley cell cultures, C H S has been reported to only use 4-coumaroyl:CoA as a substrate (Hrazdina era/., 1976); however, in soybean, C H S uses 4-coumaroyl:CoA and caffeoyhCoA as substrates (Welle and Grisebach, 1987) and the C H S from petunia anthers uses 4-coumaroyl:CoA, caffeoyhCoA, and feruloyhCoA as substrates (Sutfeld C H S from  Haplopappus  etal.,  1978). Characterization of  and parsley cell cultures at different pHs  gracilis  demonstrated that both 4-coumaroyl:CoA and caffeoyhCoA can be used as substrates when the reaction was performed at the appropriate pH-optima (Saleh er al., 1978). More recently, C H S from and  Pinus  Cephalocereus  senilis  (old-man-cactus; Pare  (Scots pine; Sandermann Jr.  sylvestris  etal.,  et al.,  1992)  1989; Fliegmann eta/., 1992)  have been shown to use cinnamoyhCoA as a substrate. Expression of Scots pine and Sinapis  alba  CHS  in  E. coli  resulted in C H S proteins which use both 4-coumaroyl:CoA  and cinnamoyhCoA as substrates (Fliegmann et al., 1992). The use of cinnamoyhCoA by C H S is likely for the biosynthesis of B-ring-deoxy flavonoids (Pare et al., 1992; Liu era/., 1995) such as pinocembrin (a flavanone), cephalocerone (an aurone), and baicalein (a flavone). Because of the presence of B-ring specific hydroxylases and Omethyltransferases, it has been suggested that C H S probably only uses 4coumaroyhCoA as a substrate  in vivo  (Ebel and Hahlbrock, 1982); however, there is  increasing evidence suggesting that C H S can metabolize other cinnamoyhCoA derivatives in addition to 4-coumaroyl:CoA (Welle and Grisebach, 1987; Sutfeld et al., 1978; Sandermann Jr. era/., 1989; Pare era/., 1992; Fliegmann  etal.,  1992).  Genes and c D N A s encoding C H S have been cloned from more than 30 plants (GenBank). C H S appears to be encoded by a single gene in parsley, and  Arabidopsis  thaliana  Antirrhinum  (Sommer and Saedler, 1986; Herrmann  majus, et al.,  1988;  Feinbaum and Ausubel, 1988), by a small gene family of 2 - 3 gene members in maize 22  and tomato (Franken  etal.,  1991; O'Neill  etal.,  1990), and by a large gene family of 5 -  10 members in bean, petunia, soybean, pea, and clover (Ryder al.,  1989; Akada  1990; An  etal.,  etal.,  1993; Howies  etal.,  etal.,  1987; Koes  et  1995). In petunia,  members of the CHS gene family are developmental^ regulated. CHS A and CHS J expression are observed in the corollas, the tubes, and the anthers but not in the leaves, the roots and the pistils (Koes et al., 1989). Petunia CHS genes are differentially expressed such that only CHS A and J a r e expressed in the flowers whereas  CHS  A, J, G  and  Bare  inducible by UV-light (Koes  etal.,  1989). The  CHS  members from bean are also differentially expressed during light treatment, elicitor treatment, wounding and infection with compatible and incompatible fungi (Ryder et  al., 1987). The developmental and cell-specific expression of CHS has been demonstrated by in  situ  hybridization and promoter- GUS fusion analysis.  In situ  hybridization showed that CHS transcripts are localized in the epidermal layer of parsley leaves where flavonoid end-products also accumulate (Schmelzer era/., 1988; Wu and Hahlbrock, 1992). In tobacco, CHS is highly expressed throughout the anthers, and CHS transcripts, localized to the epidermal cells, were 10 times higher in the limb of tobacco flowers (where anthocyanins accumulate) as compared to the flower tube (Drews  et al,  1992). In transgenic tobacco, the bean  CHS8  promoter  directed high GUS expression in the inner-epidermal layer of pigmented petal regions and in the apical tip of roots (Schmid et al., 1990). The CHS promoter from Antirrhinum  directed highest  GUS  expression in the developing seeds (3-6 weeks  post-fertilization) and in callus from transgenic tobacco. G U S activity was also high in the pigmented portion of flower petals and in 4-week-old roots; however, comparative studies in tobacco and  Antirrhinum  showed no endogenous  CHS  transcripts  associated with roots (Fritze et al., 1991). The activity of the petunia CHS- A, J, B, and  G promoters was examined by promoter-Gl/S fusions in transgenic petunia (Koes et al.,  1990).  CHS-A  and  CHS-J  directed  GUS  23  expression in corollas, flower tubes,  flower stems, ovaries, and seedpods. Only anthers.  CHS-B  CHS-A  directed  GUS  expression in  directed low expression in the same floral tissues whereas no G U S  staining was observed in CHS-G::GUS the inner epidermis as directed by  transgenics. G U S staining was observed at and  CHS-A  J  promoters and was correlated with  the location of anthocyanin accumulation. G U S staining was also observed in cell types that did not accumulate anthocyanins and it was suggested that in these tissues C H S functions to synthesize colorless flavonols such as kaemferol and quercetin (Koes  etal.,  1990).  Like the genes of general phenylpropanoid metabolism, expression of CHS is developmental^ regulated and inducible. Treatment of parsley cell cultures with UVlight causes a temporal increase in CHS R N A levels that was coordinate with the expression of Arabidopsis,  CHS  PAL  (Kreuzaler  etal.,  1983; Chappell and Hahlbrock, 1984). In  irradiation with blue light, white light, or UV-light induces endogenous  expression and GUS expression in CHS promoter-Gl/S transgenic plants  (Feinbaum and Ausubel, 1988; Feinbaum bean (Ryder  etal.,  1984; Cramer  etal.,  etal.,  1991).  CHS  expression is induced in  1985ab) and alfalfa (Dalkin  etal.,  1990) cell  cultures after elicitation and in bean hypocotyls after wounding and infection with fungi (Lawton and Lamb, 1987). In these leguminous species, C H S activity is probably used for the biosynthesis of isoflavonoids which have antimicrobial activities (Robbins etal.,  1985, Dalkin  etal.,  1990).  Many of the CHS promoters from clover have the putative UV-inducible and elicitor-inducible c/s-elements (P-box; Howies in other phenylpropanoid genes (Lois detected in the parsley (Lois  CHS  etal.,  etal.,  promoters from bean, 1989). The maize  CHS  etal.,  1995) which have been described  1989). P- and L- boxes have also been Antirrhinum,  maize,  Arabidopsis  and  promoters also contain putative Myb- and  Myc- binding c/s-elements as well as two UV-boxes which may confer UV-light responsiveness (Fraken et al., 1991). Some of the genes which regulate maize CHS expression have been cloned (reviewed in Holton and Cornish, 1995) and they 24  include the genes at the C1 (Cone et al., 1986) and R (Ludwig et al., 1989) loci. The product of the C1 locus has homology to Myb transcription factors (Paz-Ares et al., 1987) while products of R gene members have homology to Myc transcription factors (Ludwig et al., 1989; Consonni et al., 1993). Members of the Myc and Myb gene families are both required for anthocyanin biosynthesis and evidence suggests that these regulatory gene products interact with one another (Goff era/., 1992). Suppression of CHS by antisense R N A (van der Krol et al., 1988) and sense suppression (Napoli et al., 1990; van der Krol era/., 1990) resulted in transgenic plants with lower pigmentation in the flower petals. Some flowers completely lacked pigments while other flowers acquired a pattern of alternating colored and uncolored sectors (van der Krol  etal.,  1988; Napoli  etal.,  1990; van der Krol  etal.,  1990). White  flower sectors regained pigmentation when naringenin-chalcone was supplied to the tissue suggesting that the suppression was specific to the C H S step of anthocyanin biosynthesis (van der Krol  etal.,  1988). The c 2 a n d  whp  mutants of maize (reviewed  in Holton and Cornish, 1995), the nivea mutant of Antirrhinum Forkmann, 1982), and the  tt4  mutant of Arabidopsis  defective CHS genes.  25  (Shirley  (Spribille and et al.,  1995) all have  o  o  OCCH C-S-CoA 2  OH O  Chalcone Figure 1.4: Reaction catalyzed by chalcone synthase. Structures in square brackets represent reaction intermediates.  The product of the C H S reaction is a chalcone. Once made, the chalcone is modified at the A-, B-, and C-rings to produce a variety of different flavonoids. The biochemical relationship between the different classes of flavonoids is shown in Figure 1.5; however, in some cases, the enzyme catalyzing the given reactions has not been well characterized. Chalcones can be converted into flavanones by the action of 26  chalcone isomerase (CHI; reviewed in Holton and Cornish, 1995). Flavanones can be modified to form three different classes of flavonoids. Oxidation of flavonones by a proposed flavonoid oxidase (Ebel and Hahlbrock, 1982) produces flavones. Mobilization of the aryl group, a reaction catalyzed by isoflavone synthase  (IFS;  reviewed in Barz and Welle, 1992), produces isoflavones. Isoflavones in turn can be reduced by isoflavone reductase (IFR; reviewed in Barz and Welle, 1992) to produce isoflavanones. Hydroxylation of flavanones at the number 3 position by flavanone 3hydroxylase (F3H; reviewed in Holton and Cornish, 1995) produces dihydroflavonols. The action of flavonol synthase (FLS; reviewed in Holton and Cornish, 1995) produces a double bond between carbons 2 and 3 of dihydroflavonols to give flavonols. Dihydroflavonols can also be reduced by dihydroflavonol-4-reductase (DFR;  reviewed  in Holton and Cornish, 1995) to produce flavan-3,4-diols. Flavan-3,4-diols can then oxidized and dehydrated by the actions of anthocyanidin synthase (ANS; reviewed in Holton and Cornish, 1995) to produce anthocyanidins. Members of each class of flavonoids can also be hydroxylated, methoxylated and glycosylated to produce a large number of compounds. For example, the actions of flavonoid 3'-hydroxylase (F3'H) and flavonoid 3'5'-hydroxylase (F3'5'H) results in hydroxylation of the B-ring of flavonoids. By far the most well characterized class of flavonoids is the anthocyanin class, probably because they are colored and therefore the most obvious. The genetics and biochemistry of anthocyanin biosynthesis have recently been reviewed by Holton and Cornish (1995) and will not be discussed here.  27  I  Figure 1.5:  CHS  V  OH ^  Aurones  Chalcones  o  O  T  Flavones  Isoflavones  ^Y^OH Isoflavanones  o  Dihydrochalcones  FLS  o  ^f^OH u  T  Flavonols  Dihydroflavonols DFR  OH  Biochemical relationship between classes of flavonoids. Only the C-ring is shown. Enzymes are: CHS, chalcone synthase; CHI, chalcone isomerase; IFS, isoflavone synthase; IFR, isoflavone reductase; Oxi, flavonoid oxidase; F3H, flavanone 3hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol reductase; and ANS, anthocyanidin synthase.  'OH  Flavan-3,4-diols ANS  + o  xx  0H Anthocyanidins  1.2.3  Other Phenylpropanoid Molecules In addition to lignin and flavonoids, many other phenylpropanoid compounds  are found in plants. These will be described briefly (Figure 1.6). Lignans are "dimeric" compounds composed of two monolignol subunits linked in a stereospecific manner (Figure 1.6). Lignans have antimicrobial, antifungal, and antifeedant properties and 28  may have defense related roles  in planta  (Davin and Lewis, 1992). The biosynthetic  pathway leading to lignan synthesis has not been well characterized; however, since lignans are optical active, it is likely that the mechanism of synthesis is different from that of lignin biosynthesis which is non-stereospecific (Davin and Lewis, 1992). Suberin is a waxy, protective layer composed of phenolic compounds linked in ester bonds to long-chain fatty acids (Salisbury and Ross, 1985). The phenolic component of suberin can be phenolic acids (like ferulic acid) or phenolic alcohols (like 4-coumaryl alcohol) and the lipid-like component can be 16 to 28 carbons long (Davin and Lewis, 1992). Suberin is deposited at wound-sites, in the Casparian strips of roots, in the walls of bundle-sheath cells of grasses, and other cell types (Salisbury and Ross, 1985). Stilbenes are similar to flavonoids in that they are synthesized from 4coumaroykCoA and three molecules of malonyl:CoA; however, the cyclization mechanism is different resulting in resveratrol instead of naringenin (Figure 1.6). Stibene synthase and chalcone synthase have relatively high sequence homology and can be differentiated by the products of the catalyzed reaction (Fliegmann  etal.,  1992). It has been proposed that the activities of stilbene synthase and chalcone synthase depend on a conserved cysteine residue. Site-directed mutagenesis of amino acids prior to the conserved cysteine did not convert chalcone synthases into stilbene synthases or vice versa suggesting that the inherent enzymatic activities are defined by more than the few amino acids examined (Schroder and Schroder, 1992). Stilbenes have antifungal properties and are capable of preventing fungal spore germination and mycelial growth (Dercks  etal.,  1994).  Coumarins are volatile lactones derived from hydroxylated phenylpropanic acids (Figure 1.6). The early steps in coumarin biosynthesis have not been well characterized, but involve hydroxylation at the C2- (ortho) position of the phenolic ring, trans/cis  isomerization of the propane side-chain, followed by spontaneous cyclization  (Lewis, 1993). Coumarins can be further modified by hydroxylation, methylation, and 29  glycosylation. The glycosylated forms are nonvolatile (Salisbury and Ross, 1985). Furanocoumarins are of particular interest since they have been shown to have antifungal properties (Beier and Oertli, 1983). Furanocoumarins can be linear or angular (Figure 1.6) and, in both cases, the furan ring is derived from dimethylallyl pyrophosphate. Furanocoumarins are also modified by hydroxylation and methylation and in parsley, the activity of bergaptol O-methyltransferase (BMT) has been used as a marker for furanocoumarin biosynthesis (Wu and Hahlbrock, 1992). In parsley cell cultures, treatment with fungal elicitors resulted in an accumulation of furanocoumarins (Tietjen  etal.,  1983) and in parsley leaves, infection with fungi caused an  accumulation of furanocoumarins at the site of infection (Jahnen and Hahlbrock, 1988). These results suggest that, in parsley, furanocoumarins may have a role in pathogen defense. In uninfected parsley plants, furanocoumarins accumulate in the lumen of oil ducts and these compounds may represent storage forms of the defense molecules (reviewed in Hahlbrock and Scheel, 1989). Benzoates (C-6-C-i) are not strictly phenylpropanoids since they lack the threecarbon propane side-chain (Figure 1.6); however, they may be derived from phenylpropanoids and are therefore sometimes considered products of phenylpropanoid metabolism. The biosynthesis of benzoates is still under debate; however, three mechanism have been proposed. Benzoates may be converted directly from dehydroshikimate (an intermediate of the shikimic acid pathway) or benzoates may be converted from phenylpropane units. The latter mechanism may be comparable to "8-oxidation", using C o A esters of hydroxycinnamate derivatives, or "non-oxidative", using the hydroxycinnamic acids themselves (reviewed in Lewis, 1993). Radiolabeled feeding-experiments with C-phenylalanine (Yazaki et al., 14  1991) and C-cinnamate (Yalpani era/., 1993) suggest that benzoates can be 14  derived from phenylpropanoids. Furthermore, benzoates can be synthesized in the absence of A T P and coenzyme A, suggesting that the non-oxidative mechanism is occurring and that CoA-activation of the cinnamic acid derivatives is not required 30  (French etal., 1976; Yazaki etal., 1991; Schnitzler etal., 1992). More recently however, using an improved assay system, Loscher and Heide (1994) provide evidence that the biosynthesis of 4-hydroxybenzoate occurs via 4-coumaroyl:CoA. Hydroxylated benzoates like 4-hydroxybenzoate, salicylic acid (2-hydroxybenzoate), and gallic acid (3,4,5-trihydroxybenzoate) are of particular interest. 4hydroxybenzoates are deposited in plant cell walls and are biosynthetic intermediates (Schnitzler et al., 1992). Salicylic acid has been implicated as the intercellular signal in systemic acquired resistance (Malamy etal., 1990; Metraux etal., 1990). Gallic acids are often conjugated to sugars or other phenolic molecules and are components of "hydrolysable tannins" (Lewis, 1993).  Figure 1.6:  OH  Phenylpropanoid Compounds.  OH  Resveratrol (Stilbene)  Enterodiol (Lignan)  Psoralen (Linear Furanocoumarin)  COOH  2-Hydroxybenzoate (Salicylic Acid)  Angelicin (Angular Furanocoumarin)  COOH  COOH  OH  OH  4-Hydroxybenzoate 3,4,5-Trihydroxybenzoate (Gallic Acid) 31  1.3  Carbon Flow into Phenylpropanoid Metabolism Phenylalanine, the primary metabolite from which all phenylpropanoid  compounds are derived, is made via the shikimic acid pathway (Ireland, 1990). The first enzyme in the shikimic acid pathway, 3-deoxy-D-arab/'no-heptulosonate-7phosphate synthase (DAHP synthase), condenses phosphoeno/pyruvate (from glycolysis) with erythrose 4-phosphate (from the oxidative pentose phosphate pathway) to produce DAHP (Figure 1.7). D A H P is then converted to chorismate by the actions of 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, shikimate dehydrogenase, shikimate kinase, 5-eno/pyruvylshikimate 3-phosphate synthase ( E P S P synthase) and chorismate synthase (Herrmann, 1995). Chorismate can then enter two branch-pathways for the biosynthesis of tryptophan (via anthranilate synthase) or tyrosine and phenylalanine (via chorismate mutase). A number of studies have shown that the actions of D A H P synthase and chorismate mutase increase after elicitor treatment and wounding (McCue  etal.,  1989; Muday and Herrmann, 1992;  Kuroki and Conn, 1988); however, it is only recently that the genes encoding D A H P synthase, E P S P synthase, and chorismate synthase have been shown to be transcriptionally activated during similar stress conditions including light-treatment and pathogen attack (Dyer  etal.,  1989; Keith  etal.,  1991; Henstrand  etal.,  1992; Gorlach  al., 1995). These results suggest that environmental stresses which induce the production of phenylpropanoid compounds also cause the up-regulation (gene expression and extractable enzyme activity) of components of the shikimic acid pathway.  32  et  Phosphoeno/pyruvate  Erythrose 4-phosphate  Chorismate ,  8  9  /  N  \  Tryptophan  Phenylalanine & Tyrosine  Figure 1.7: Schematic diagram of the shikimic acid pathway. The aromatic amino acids are synthesized from the shikimic acid pathway. Abbreviations and enzymes are: DAHP, 3-Deoxy-Dararj/no-heptulosonate 7-phosphate; EPSP, 5-enolpyruvyl shikimate 3-phosphate; 1, DAHP synthase; 2, 3-dehydroquinate synthase; 3, 3-dehydroquinate dehydratase; 4, shikimate dehydrogenase; 5, shikimate kinase; 6, EPSP synthase; 7, chorismate synthase; 8, anthranilate synthase; and 9, chorismate mutase. Dashed arrows represent multiple enzyme steps. Adapted from Herrmann (1995).  The oxidative pentose phosphate pathway produces two intermediates which are needed in phenylpropanoid metabolism: erythrose 4-phosphate, a substrate for D A H P synthase, and N A D P H , reducing equivalents which are used in a number of reactions. N A D P H is a key player in phenylpropanoid metabolism and N A D P H is associated with enzymes in the general phenylpropanoid pathway (C4H), enzymes in the flavonoid biosynthetic pathway (IFS, IFR, F3'H, F3'5'H, DFR), and enzymes in the lignin biosynthetic pathway (CCR, CAD). The majority of the N A D P H reducing-power 33  used in lignin biosynthesis has been shown to come from the oxidative pentose phosphate pathway (Pryke and Rees, 1977). N A D P H is produced in the first-two steps of the oxidative pentose phosphate pathway: the steps catalyzed by glucose 6phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Studies have shown that G 6 P D H enzyme activity increases during pathogen attack (Borner and Grisebach, 1982) and elicitation (Robbins era/., 1985; Daniel etal., 1990). Very recently, the genes encoding G 6 P D H and 6 P G D H have been shown to be transcriptionally induced during elicitation of alfalfa cell cultures (Fahrendorf etal., 1995). Wounding and parasitic infection causes an increase in respiration that is referred to as "wound respiration" and "infection-induced respiration" (Uritani and Asahi, 1980). Increase in oxygen consumption and the breakdown of carbohydrates is probably to supply A T P and precursors for secondary metabolism (Uritani and Asahi, 1980). There is evidence that phosphofructo kinase and other enzymes from glycolysis are enzymatically induced during wounding (Kahl, 1974; Uritani and Asahi, 1980); however, the wound-induced expression of genes encoding glycolytic enzymes has not been well characterized. Taken together, these results suggest that genes encoding key enzymes in the oxidative pentose phosphate pathway, the shikimic acid pathway, the general phenylpropanoid pathway, and the branch pathways leading to specific secondaryproduct formation may all be coordinately regulated (Figure 1.8). The flow of carbon through these metabolic pathways may depend on 1) the activities of the enzymes leading to product formation and 2) the activities of branch-point enzymes which divert carbon away from product formation (Stephanopoulos and Vallino, 1991). Stephanopoulos and Vallino (1991) proposed the concept of "network rigidity" and suggested that successful redistribution of carbon via genetic manipulation ("metabolic engineering") depends on understanding the regulation of carbon flow, particularly at biochemical branch-points ("nodes"). Kholodenko and Westerhoff (1995) suggested 34  that regulation of cellular metabolism cannot be easily predicted since subcellular ("microworld") interactions render the systems more complex ("non-ideal") than that proposed by traditional biochemical studies. Two examples of molecular interactions which affect flux through a pathway, but are not apparent in purified enzyme ("ideal") systems, are 1) metabolic channelling between enzymes, and 2) compartmentalization of enzymes or metabolites (Kholodenko and Westerhoff, 1995). The metabolic control theory (Reder, 1988; Kell and Westerhoff, 1986) has been used to describe the role of P A L in carbon flux through phenylpropanoid metabolism (Bate et al., 1994). There is a direct relationship between P A L activity and chlorogenic acid accumulation in sensesuppressed tobacco plants, pointing to P A L as a major control point in regulating carbon flux into this compound. However, the accumulation of rutin (a flavonoid) and lignin is relatively unaffected by the same perturbations in P A L activity in these plants. Other control points downstream of P A L (e.g. C H S , or the release of glycosylated lignin monolignols from vacuolar pools) are likely important for the regulation of carbon flux into these products. Primary Metabolism r  Phenylpropanoid Metabolism >k  r  >  Benzoate Glycolysis^  Oxidative Pentose Phosphate Pathway  ^  Coumarins ^  Shikimic Acid | — G e n e r a l Phenylpropanoid Pathway —\S Pathway  Lignin Lignans Flavonoids Stilbenes  Phenolic Acids  Phenolic Esters  Figure 1.8: Carbon flow through the general phenylpropanoid pathway. Phosphoeno/pyruvate, from glycolysis, and erythrose 4-phosphate, from the oxidative pentose phosphate pathway, enter the shikimic acid pathway which, through a serious of metabolic steps is converted to phenylalanine. Phenylalanine enters the general phenylpropanoid pathway via the action of PAL and is converted into diverse phenylpropanoid products. Adapted from Dixon and Paiva (1995).  35  1.4  Research Objectives and Approaches 4 C L may have a pivotal in controlling the flow of carbon from primary  metabolism into secondary metabolism. I will examine 1) the nature of the 4CL c D N A s and gene-families in tobacco and Arabidopsis,  2) the potential role of 4 C L in  regulating carbon-flow into phenylpropanoid branch-pathways, and 3) the consequences of blocking 4 C L function. Chapter 3 describes the cloning and characterization of c D N A s encoding 4 C L from tobacco and  Arabidopsis.  Using the  c D N A s as hybridization probes, the size of the 4CL gene-families and the expression patterns of 4CL are characterized. To determine if divergent 4CL c D N A s from tobacco encoded proteins with distinct enzyme activities, the cDNAs were expressed in E.  coli.  Chapter 4 describes the enzymatic characterization of the endogenous and recombinant tobacco 4 C L proteins. In chapter 5, antisense-RNA technology was used to suppress 4CL-expression in Arabidopsis. molecular consequences of suppressing  The morphological, biochemical, and 4CL  in  Arabidopsis  is described.  Antisense R N A technology can be used to down-regulated the expression of specific genes in transgenic organisms. The mechanism by which antisense-RNA suppression occurs is still not clearly understood. Accumulated evidence suggests that, in plants, the mode of action is post-transcriptional and may involve the formation of R N A : R N A duplexes (Simons, 1988; Bourque, 1995). In the nucleus, the R N A duplex may be inefficiently processed, it may be unstable and rapidly degraded, or it may be poorly transported into the cytoplasm for translation. In the cytoplasm, the R N A duplex may again be rapidly degraded, or it may not be efficiently translated due to interference with ribosome binding. In animals, there is evidence that antisense R N A may affect transcription by forming localized RNA:DNA triplexes (Helene and Toulme, 1990; Bourque, 1995). In general, inhibition of gene expression by antisense R N A does not appear to target one specific step in the flow of information from DNA to R N A to protein, but rather to affect any one of a number of steps necessary for gene expression. 36  Tobacco and Arabidopsis  are chosen as the experimental organisms for this  work. Transgenic studies with the parsley 4CL-1 promoter fused to GUS (Hauffe et al., 1991; 1993) and in  situ  hybridization (Reinold  et al.,  1993) have demonstrated the  expression-pattern of 4CL in tobacco. However, the nature of the tobacco 4CL genefamily has not been analyzed and the work described here contributes to the existing knowledge of tobacco 4CL genes and the tobacco-4CL proteins. Tobacco is also chosen as the experimental organism because it is easy to transform and regenerate. Arabidopsis  is a powerful model-system for genetic and molecular analysis because it  has a small genome, it has relatively little repetitive DNA, it is self fertile (each plant producing thousands of seeds), it is has a short generation-time, and it is also relatively easy to transform and regenerate (Estelle and Somerville, 1986; Meyerowitz, 1987). An additional advantage with using  Arabidopsis  is that there is a large  scientific community working on this crucifer and resources (electronic newsletter, mutant lines, seed stocks, linkage maps, expressed sequence tags) are readily available through the Arabidopsis  thaliana  Database (AtDB) in the internet  (http://genome-www.stanford.edu/arabidopsis/). Virtually nothing is known about the Arabidopsis 4CL  in  4CL  and the work described here represents the first detailed analysis of  Arabidopsis.  37  Chapter 2 Materials and Methods 2.1 Plant Growth-Conditions, Tissues, and Experimental Treatments 2.2 Hybridization Probes 2.3 Cloning of 4CL c D N A s 2.4 Restriction-Map Analysis, Subcloning, Sequencing and Sequence Analysis of Tobacco and Arabidopsis cDNA Clones 2.5 Southern Blot and Northern Blot Analysis 2.6 Cloning of Tobacco-4CL cDNAs into  E. coli  Expression-Vectors  2.7 Protein Extraction 2.8 Western Blot Analysis 2.9 4 C L Enzyme Assay 2.10 Generation of Constructions to Antisense and Sense 4CL 2.11 Plant Transformation 2.12 Lignin and Anthocyanin Extraction  2.1  Plant Growth-Conditions, Tissues, and Experimental and  Nicotiana  Arabidopsis  Treatments  plants were grown in soil in growth chambers at  2 3 ° C with -120 uE s- nr of light, and an 8 h dark /16 h light regime. Under tissue1  1  culture conditions, seeds were germinated and grown at 2 3 ° C under -150 JLIE s nrv _1  1  of constant light. Light source was from General Electric catalogue number F20T12/CW.  Tobacco whereas  {Nicotiana  Nicotiana  sylvestris  tabacum  and  L. cv. Xanthi SR1) seeds were from Dr. C . Douglas Nicotiana  tomentosiformis  seeds were kindly  donated by S.E.l.T.A. (Institut Experimental du Tabac, Bergerac, France). Protein extracts for enzyme assays were prepared from tobacco-stem sections approximately 38  1 cm in diameter, between the third- and sixth- node above the base of mature tobacco plants. Protein extracts used for western blot screening of transgenic tobacco were prepared from 17-day-old seedlings that had been grown in M S plates supplemented with 100 mg/L kanamycin. Tobacco R N A was extracted from tissues described below. Tobacco young shoot tips were between 4 cm and 8 cm in length. Young stems, approximately 2 cm in length, were harvested just below the apical meristems. Old stems, greater than 1 cm in diameter and approximately 6 cm in length, were harvested from the base of mature plants. Floral tissues were harvested from developmental-stages 1 to 6 as described by Reinold era/. (1993). The floral tissues were divided into sectors as described by Drews et al. (1992): the limb (corresponding to the pigmented part of the corolla), the tube (corresponding to the white part of the corolla), the base (corresponding to the corolla that is surrounded by the sepals) and the sepal. Seeds of the R L D ecotype of Arabidopsis  were kindly donated by Dr. L. Kunst  (Department of Botany, University of British Columbia, British Columbia, Canada) whereas the Columbia ecotype was supplied by Dr. J . Dangl (Max-Delbriick Labor, Max-Planck-lnsititut, Koln, Germany). Protein extracts (for western blot analysis and 4 C L enzyme assays) and lignin were extracted from cm in height.  Arabidopsis  Arabidopsis  stems that were 15  R N A was isolated from tissues described below.  seedlings, Columbia ecotype, were grown on M S plates and harvested  Arabidopsis  after 2, 3, 4, 5, 7, and 10 days post-germination. Seedlings were harvested either as whole-seedlings, or dissected into shoot- and root- sectors before freezing in liquid nitrogen.  Arabidopsis  leaves were mature and fully expanded, and bolting-stems  were 3 cm, 6 cm, 12 cm, and 20 cm in height.  Arabidopsis  flowers, prior to anthesis,  were harvested as a cluster containing one open flower with the remaining flower buds still enclosed by the sepals. Wound treatments were performed as follows. Mature, fully-expanded tobacco or  Arabidopsis  leaves were wounded by slicing the leaves into 1-2 mm-wide strips and 39  placing them on a piece of Whatman filter paper moistened with MS liquid media. Tobacco leaves were wounded for 24 h before they were used for R N A isolation, whereas  Arabidopsis  leaves were wounded for 0.5 h, 1 h, 2 h, 4 h and 6 h before they  were used for R N A isolation. For 0 h of wounding, leaves were detached from the plants and immediately frozen in liquid nitrogen. Methyl jasmonate treatments were carried out by spraying tobacco plants with 1 mM methyl jasmonate (in 1% Triton-X-100) until the solution ran off the leaves. The treated plants were covered with a bell-jar and placed under constant light for 24 h. Mature, fully-expanded leaves were then excised and used for R N A isolation. For untreated R N A samples, mature, fully-expanded tobacco leaves were detached from the plants and immediately frozen in liquid nitrogen for control, untreated R N A samples. High-intensity light treatments were performed by exposing 25-day old Arabidopsis  plants to high-intensity white-light (-900 u\E s m- ) for 24 h. The leaves _1  1  were harvested and used for anthocyanin and RNA isolation.  2.2  Hybridization Probes Hybridization probes used in Southern and northern blot analysis were  generated from cDNAs, genes, or expressed sequence tags (ESTs, Newman  etal.,  1994) as described in Table 2.1 and 2.2. The E S T s were selected from the Arabidopsis  E S T database (dbEST) based on the sequence homology between the  E S T and previously cloned genes. The 5' and 3' portions of the E S T s were sequenced to confirm their identity.  32  P-radiolabeled probes were generated using  the Random Primers DNA Labeling System (Gibco BRL, Gaithersburg, MD) according to the manufacturer's specifications.  40  Table 2.1: cDNA and gene fragments used for generating hybridization probes. Enzyme for Fragment Fragment Size Probe Excision (kb) Reference St4CL StPAL  EcoRI EcoRI of pCP61.13 EcoRI of pCP63.15 EcoRI of pBK 5.70-R  AtDSHI AtPAL AtC4H rRNA  2 0.6 0.9 1.8 0.5 2 8.7  HinrM of PAL10-3  Sal\, A/ofl HinrM of pHA2  Becker-Andre etal., 1991 Joos and Hahlbrock, 1992 Keith etal., 1991 Wanner etal., 1995 gift from C. Chappie Jorgensen etal., 1982  Table 2.2: ESTs used for generating hybridization probes. Probe  AtHEXO AtPFK AtG6PDH At6PGDH AtEPSPS AtCAD AtCHS  Putative Encoded Enzyme  Homology (%, organism)  Hexokinase Phosphofructo kinase Glucose 6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase 5-Eno/pyruvyl-shikimate 3phosphate synthase Cinnamyl alcohol dehydrogenase Chalcone synthase  98%, A. thaliana 77%, B. vulgaris  82%,  S. tuberosum 79%, M. sativa  Size (kb)  EST Accession Number  1.3 1.3 1.3  84G1T7 146I12T7 35D3T7  2  11B4T7P  98%, A. thaliana  1.3  131D24T7  95%, A. thaliana  1.3  90C21T7  99%, A. thaliana  1.2  YAP097T3  2.3 Cloning of 4CL cDNAs A tobacco c D N A library was constructed in AZAPII (Stratagene, La Jolla, CA) using poly(A)+ R N A isolated from tobacco young shoot-tips (S. Lee and C. Douglas, unpublished). The primary library, consisting of approximately 1 0 independent 7  recombinants, was amplified and approximately 10^ plaque forming units (p.f.u.) were screened using XL1-Blue cells. Putative 4CL cDNA clones were generated as pBluescript I S K plasmids via  in vivo  excision using the helper bacteriophage R408 as  specified by the manufacturer. The  Arabidopsis  A Y E S c D N A library, kindly donated by Dr. R. W. Davis  (Stanford University, Stanford, California), was made with poly(A)+ R N A from aerial portions of plants ranging in size from those which had just opened their primary leaves to plants which had bolted and were flowering. The primary library, containing 41  1 0 independent recombinants was amplified and approximately 2 x 1 0 plaque 7  5  forming units (p.f.u.) were screened using LE392 pMC9 cells. Putative clones were generated as plasmids via  in vivo  excision using BNN132 cells and the  Arabidopsis  4CL c D N A was then subcloned into the Xho\ site of pBluescript I KS. Tobacco- and Arabidopsis-  cDNA libraries were screened using a 2-kb potato  4CL c D N A (Becker-Andre et al., 1991) as a hybridization probe. The hybridization filters were washed at low stringency (2 x S S C , 0 . 1 % S D S , 65°C) as described by Sambrook  2.4  etal.  (1989).  Restriction Map-Analysis, Subcloning, Sequencing and Analysis of Tobacco and Arabidopsis cDNA Clones  Sequence  All putative tobacco-4CL cDNA clones were partially sequenced at the 5' and 3' ends using the T7 Sequencing Kit™ (Pharmacia, Piscataway, NJ). Clones with high sequence-homology to the parsley and potato 4CL cDNAs (Lozoya era/., 1988, Becker-Andre et al., 1991) were classified into groups according to their restriction maps. Overlapping subclones of the two longest cDNA clones, Nt4CL-1 and Nt4CL19, were constructed in pBluescript II K S for sequencing. A restriction map was determined for the putative  Arabidopsis  4CL cDNA, At4CL, and overlapping subclones  were constructed in pBluescript I KS. Nt4CL-1 and Nt4CL-19 subclones were sequenced by the University of British Columbia Nucleic Acid-Protein Service Unit using the P R I S M Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Foster City, CA). At4CL subclones were sequenced manually using the T7 Sequencing Kit™. Overlapping nucleotide sequences were aligned and contiguous cDNA-sequences were reconstructed. Accuracy of the sequencing results was confirmed by the complementarity between the top- and bottom- strand DNA-sequences and by translation of the sequences into amino acid sequences comparable to those from previously cloned 4 C L s . 42  DNA and predicted amino acid sequences were analyzed using B E S T F I T and P I L E U P computer programs from the University of Wisconsin Genetics Computer Group software. Predicted phosphorylation- and glycosylation- sites, isoelectric points, and molecular weights were determined using computer programs PRO-SITE and pl/MW from the University of Geneva (Geneva, Switzerland).  2.5 Southern Blot and Northern Blot Analysis Genomic DNA was isolated using C T A B (hexadecyltrimethylammonium bromide) as described by Doyle and Doyle (1990). Ten jxg of genomic DNA were digested with restriction enzymes, electrophoresed in a 0 . 8 % agarose gel, and blotted onto Hybond™ nylon-membranes (Amersham, Oakville, Ont, Canada).  Hybond™  nylon-blots were hybridized in 6 x S S C , 0 . 5 % SDS, 5 x Denhardts, and 0.1 mg/mL denatured salmon-sperm DNA (Sambrook et al., 1989). Southern blots were washed at high (0.2 x S S C , 0 . 1 % SDS,  65°C) or low (2 x S S C , 0 . 1 % S D S ,  65°C) stringency.  RNA was extracted using guanidinium hydrochloride as described by Logemann et al. (1987). Ten u.g of RNA were electrophoresed in 1.2% agarose gels containing 2.2 M formaldehyde, the gel was rinsed in water for one hour, and then blotted onto Zeta-Probe® G T Genomic Tested Blotting Membranes (Bio-Rad Laboratories, Hercules, CA) or Hybond™ nylon-membranes. Wound-induced and high-intensity white-light induced RNA samples were blotted onto Zeta-Probe® membranes. All other RNA samples were blotted onto Hybond™ membranes. ZetaProbe blots were hybridized in 0.5 M Na2HP04 pH 7.2, and 7 % S D S as specified by the manufacturer. Hybond nylon-blots were hybridized as described above.  Northern  blots hybridized to the tobacco ACL probes were washed at moderate stringency (0.5 x S S C , 0 . 1 % S D S , 65°C). Northern blots hybridized to the potato PAL probe were washed at low stringency (2 x S S C , 0 . 1 % SDS, 65°C). All  Arabidopsis  northern blots  were washed at high stringency (0.2 x S S C , 0 . 1 % SDS, 65°C). Blots were stripped by 43  pouring boiling 0 . 1 % S D S onto the blots and allowing the solution to cool to room temperature. This process was done twice.  2.6 Cloning of Tobacco-4CL cDNAs into E. coli  Expression-Vectors  P C R was used to engineer Sph\ restriction-sites into the 5' ends of the Nt4CL-1 and Nt4CL-19 c D N A s using a 17 primer and cDNA-specific oligonucleotides ( 5 ' - G G G G C A T G C C A A T G G A G A C T A C T A C - 3 ' for Nt4CL-1 and 5 ' - G G G G C A T G C A T G G A G A A A G A T A C A A A A C A G - 3 ' for Nt4CL-19). Nt4CL-1 and Nt4CL-19 P C R products were digested with Sph\ and Xho\ and cloned into the Sph\ and Sa/l restriction-sites of the expression-plasmids pQE-32 and pQE-30 respectively (QIAexpressionist™ Kit, QiaGen Inc., Chatsworth, CA). The 5" and 3' portions of the DNA constructs were sequenced to ensured fidelity of the P C R and to confirm that the c D N A s were in frame with the A T G from the p Q E expression-vectors. The center portion of each P C R product was excised and replaced with the corresponding portion from the original cDNA  {Sac\IKpn\  fragment of Nt4CL-1 and the  BsB\  fragment of Nt4CL-19) to  eliminate the possibility of single base-pair changes from the P C R . All D N A manipulations were performed in XL1-Blue cells. Expression of the c D N A s were performed in Escherichia  coli  strain M15 as recommended by the manufacture. The  expression-plasmid (pQE-30), without a cDNA insert, was generated in M15 as a negative control.  2.7 Protein  Extraction  Recombinant-4CL proteins were generated by inducing 10 mL of a bacterial culture ( O D  6 0 0  ~ 0.7) containing the expression-plasmids with 2 mM IPTG for 4 h. After  centrifugation the bacterial-pellet was resuspended in 2 mL of 200 mM Tris, pH 7.8 and the cells were disrupted in a French Press. Cellular debris was removed by centrifugation and then glycerol was added to the supernatant to a final concentration  44  of 3 0 % . Samples were frozen in liquid nitrogen and stored at -80°C until further analysis. Plant proteins, for western blot analysis, were extracted by first grinding the tissue in liquid nitrogen and then resuspending the powder in 50 mM Tris, pH 8, and 5 mM MgCl2- The homogenate was centrifuged twice at 4°C to remove cellular debris and the final supernatant was used for western blot analysis. For enzyme assays, plant extracts were prepared as described by Knobloch and Hahlbrock (1977). Briefly, stem- and leaf- tissues were ground into a fine powder in liquid nitrogen; the powder was resuspended in 200 mM Tris, pH 7.8 and 15 mM 13mercaptoethanol; the suspension was rotated, in the presence of 1 0 % (w/w) Dowex, at 4°C for 15 minutes; the mixture was centrifuged to remove debris; the supernatant was concentrated using Microsep™ 10K cut-off micro-concentrators (Filtron, Northborough, MA); the protein extract was made to 3 0 % glycerol, frozen in liquid nitrogen, and then stored at -80°C until further analysis. Protein content was quantified by the Bradford (1976) method using the BioRad Protein Assay Kit™ (Bio-Rad, Hercules, CA) with B S A as a standard.  2.8  Western Blot Analysis Western blot analysis was performed as described by Sambrook era/. (1989).  Plant (25 |ig) or bacterial (5 |ig) proteins were electrophoresed in 1 0 % SDSpolyacrylamide separating gels (Laemmli, 1970) and then blotted onto Hybond™ nylon-membranes at 4°C for 3 h using 25 mM Tris and 250 mM glycine. The blots were blocked with 5 % (w/v) non-fat powdered milk, reacted with a 1:5000 dilution of a rabbit antiserum raised against the parsley 4 C L (Ragg era/., 1981), reacted with a 1:2500 dilution of goat anti-rabbit IgG conjugated to alkaline phosphatase, (Gibco BRL, Gaithersburg, MD), and then visualized using 4.5 mM Fast-Red R and 2.5 m M Naphthol AS-MX phosphate dissolved in 50 mM Tris, pH 8 as substrates (Sigma, St. Louis, MO). 45  2.9  4CL Enzyme Assay 4 C L enzyme activity was measured spectrophotometrically at room temperature  as described by Knobloch and Hahlbrock (1977). The 4 C L reaction mixtures contained protein extracts, 5 mM ATP, 5 mM MgCl2, 0.33 mM coenzyme A, and 0.2 mM cinnamic acid derivatives. The blank (reference) mixtures contained the same components but without the coenzyme A. The change in O D of the reaction mixtures relative to the blank mixtures was monitored at wavelengths of 311 nm, 333 nm, 346 nm, 345 nm, and 352 nm, which are the absorption maxima for cinnamoyhCoA, 4coumaroyhCoA, caffeoyhCoA, feruloyhCoA, and sinapoyhCoA, respectively (Stockigt and Zenk, 1975). Calculations were performed using 22000 L m o l " c m " , 21000 L 1  1  m o l " c m " , 18000 L m o l " c m " , 19000 L m o l " c m " , and 20 000 L m o l " c m " as 1  1  1  1  1  1  1  1  extinction coefficients for cinnamoyhCoA, 4-coumaroyl:CoA, caffeoyhCoA, feruloyhCoA, and sinapoyhCoA, respectively (Stockigt and Zenk, 1975).  2.10  Generation of Constructions to Antisense and Sense All plasmids were propagated in E.  coli  4CL  strain DH5a, and manipulations were  performed by conventional molecular biology methods. The plasmids used to generate the 4CL sense and antisense constructs are as follows. The 2-kb potato 4CL c D N A used in some constructions was propagated in pBluescribe, whereas At4CL was cloned and propagated in pBluescript I KS. pRT101 is a plasmid containing the cauliflower mosaic virus 35S promoter, a convenient multicloning-site followed by the 35S polyadenylation/termination sequences (Topfer et al., 1987). Plasmid 35-31 contains the 1.5 kb parsley  4CL1  promoter in pBluescribe (Hauffe  etal.,  1991) and  pBin19 (Bevan, 1984) is a binary vector containing the right- and left- borders used in Agrobacterium-mediaXed  plant-transformations. Relevant portions of selected  plasmids are shown in Figure 2.1. pRT101-Pc4CL-P, in which the 35S promoter of pRT101 was replaced with the parsley-4CL7 promoter, was constructed in three steps. First, plasmid 35-31 was 46  digested with  Psti  and EcoRI to release the 1.5-kb parsley-4CL 1 promoter-fragment.  Second, the 0.4 kb 35S promoter was excised from pRT101 by complete EcoRI and partial Psfl digestion (Topfer era/., 1987). Lastly, the parsley-4CL7 promoter-fragment was ligated into the modified pRT101 to produce a DNA construct consisting of the parsley-4CL7 promoter, a convenient multicloning-site followed by the 35S polyadenylation/termination sequences. Both pRT101 and pRT101-Pc4CL-P were used to generate antisense- and sense- 4CL DNA constructs. The full-length potato 4CL cDNA (St4CL) was inserted in sense- and antisenseorientations behind the 35S promoter by ligating the 2-kb EcoRI cDNA-fragment into the EcoRI site of pRT101. The two orientations were distinguished by digestion with SamHI. A partial-length potato-4CL cDNA, the EcoR\/Kpn\ sense orientation into the  EcoR\/Kpn\  fragment, was cloned in  sites of pRT101. For a partial-length potato-4CL  c D N A in antisense orientation, St4CL was completely digested with Xba\ and partially digested with Kpn\ and then the released 1.6-kb fragment was ligated into the Xba\/Kpn\  sites of pRT101. The partial-length St4CL constructs lacked the polyA tail  and the equivalent of 23 amino acids at the carboxy-terminal of the predicted potato 4 C L polypeptide. The  Arabidopsis  4CL  cDNA was inserted in sense- and antisense- orientation  behind the 35S or the parsley-4CL promoters in plasmids pRT101 and pRT101Pc4CP-P, respectively. Sense orientation constructs were made by digesting At4CL with  XhoUBglH,  Kpn\/BamH\  or  Kpn\/Bgl\\,  and ligating the fragments into the Xho\/BamH\,  or  sites, of pRT101 and pRT101-Pc4CL-P respectively. Antisense constructs  were made by first generating an At4CL cDNA clone in the opposite orientation as the one diagrammed in Figure 2.1 with respect to the polylinker. This construct, At4CL*, had the Xba\ restriction-site on the 5' end of the cDNA. At4CL* was digested with Xba\IBgh\  and subcloned into the XbaUBglW  sites of pRT101 and pRT101-Pc4CL-P.  The antisense At4CL DNA constructs were digested with Xba\ and Cla\, and then Klenow was used to blunt-end the termini. Re-ligation of the plasmid produced a 47  construct which had the At4CL cDNA in antisense orientation and this construct lacked the multicloning site derived from Bluescript I KS. Digestion of the Arabidopsis 4CL cDNA with BgW\ removes the polyA tail and the equivalent of 10 amino acids at the carboxy-terminal of the predicted Arabidopsis 4 C L polypeptide. The above DNA constructs were subcloned as a cassette into the Hind\\\ site of pBin19. The potato-4CL constructs were partially digested with Hind\\\ since there are internal Hind\\\ sites within the cDNA. All DNA constructs were verified by diagnostic restriction-digest analysis after each subcloning step to confirm the orientation of the c D N A and to insure that all portions of the construct were not lost or rearranged.  EcoRI i  B a  ,  m H I  BamHI  K p  i _  ,  n l  EcoRI  Xhol  i  St4CL in pBluescribe  S a  I  ,  c l  Pvull , B  I  g l M  I  Xhol I  At4CL in pBluescript I K S  pRT101 (35S promoter)  pRT101-Pc4CL-P (Parsley 4CL1 promoter)  Figure 2.1: Plasmids used in generating 4CL sense- and 4CL antisense- DNA constructions. Abbreviations are: St4CL, potato 4CL cDNA; At4CL, Arabidopsis 4CL cDNA; and 35S promoter, cauliflower mosaic virus 35S promoter. Open bars represents cDNAs, stippled bar represents the 35S promoter, slashed bar represents the parsley 4CL1 promoter, and gray bars represents the 35S polyadenylation/termination sequences.  48  2.11  Plant Transformation Agrobacterium-med\a\ed  transformations of tobacco and Arabidopsis  were  done as described by Lee and Douglas (1996a). The pBin19 constructs were mobilized from E.  coli  into  Agrobacterium  tumefaciens  strain LB4404 by tri-parental  mating using the helper-plasmid pRK2013. For transformation into tobacco, overnight Agrobacterium  cultures were diluted 1:10 with Murashige and Skoog (MS) liquid  media while, for  transformations, overnight  Arabidopsis  Agrobacterium  cultures were  diluted 1:20 with callus induction media (CIM). Details of media components are listed in Figure 2.2. Nicotiana  tabacum  SR1 seeds were surface-sterilized and grown under axenic-  conditions in Magenta G A 7 boxes containing M S media until the tobacco leaves were 4-5 cm in length. Tobacco leaves were cut into pieces, approximately 1 cm x 1 cm in size, incubated in the diluted  cultures for 1-5 minutes, dabbed dry on a  Agrobacterium  piece of sterilized Whatman filter paper, and then placed upside-down on M S media supplemented with 1 mg/L 6-benzylaminopurine and 0.2 mg/L a-napthaleneacetic acid. After 2 - 3 days, the leaf-pieces were transferred onto M S media supplemented with 1 mg/L 6-benzylaminopurine, 0.2 mg/L a-napthaleneacetic acid, 100 mg/L kanamycin, and 300 mg/L carbenicillin. Approximately 2 - 3 weeks later, shoots protruding from the calli were excised and transferred into Magenta G A 7 boxes containing M S media supplemented with 100 mg/L kanamycin and 300 mg/L carbenicillin. When roots were visible, the plants were transferred into soil and were maintained until seed set. Arabidopsis  RLD and Columbia seeds were surface-sterilized and grown under  axenic-conditions in a 250 mL flask containing 50 mL liquid M S media. The flasks, containing 5 - 1 0 seeds, were shaken at 22°C, on a rotary shaker at 50 rpm under constant-light for 3 - 4 weeks until a mass of roots accumulated. The roots were excised from the shoots, spread onto CIM plates and pre-incubated for 3-days after which they were incubated in the diluted  Agrobacterium  49  culture for approximately 5  minutes. The roots were then dabbed dry on a piece of sterilized Whatman filter paper and placed on CIM plates. After 2 days, the bacteria were removed by washing the roots in liquid CIM supplemented with 500 mg/L carbenicillin. The roots were then dabbed dry, cut into 0.5 cm pieces, spread onto shoot induction media (SIM), and maintained by transferring them onto fresh SIM plates weekly. After 3 - 4 weeks, green calli were visible and, after a number of weeks, shoots emerged from the calli. These shoots were excised from the calli, placed in a sterile, 15 cm, glass test-tube containing 2 mL root induction media (RIM). The transformants were allowed to mature and set seed in the test-tubes.  1000  x MS Vitamins  10 g myo-inositol 0.1 g nicotinic acid 0.1 g pyridoxine HCI 1.0 g thiamine HCI adjust to 100 mL filter sterilize  MS Media  B5  1 M S Salt Mixture  Media  1 Gamborg's B-5 Medium  1L package Gibco, Cat.# 11117 30 g sucrose 1 mL 1000 x M S vitamins K O H to pH 5.7 adjust to 1L 8 g phytoagar autoclave  1L package Gibco, Cat. #21153 0.5 g M E S K O H to pH 5.7 adjust to 1L 8 g phytoagar autoclave  CIM  SIM  RIM  B5 media 0.5 mg/L 2,4-D 0.05 mg/L kinetin  B5 media 5.0 mg/L 2ip 0.15 mg/L IAA 50 mg/L kanamycin 500 mg/L carbenicillin  M S media 1 mg/L N A A 50 mg/L kanamycin  Figure 2.2: Media used in plant transformation and regeneration. Liquid media were made by omitting the phytoagar. Phytohormones are: 2,4-D, 2,4-dichlorophenoxyacetic acid; 2ip, N -(2isopentenyl)adenine; IAA, indole-3-acetic acid; and NAA, a-napthaleneacetic acid. Abbreviations are: MS, Murashige and Skoog; CIM, callus induction media; SIM, shoot induction media; and RIM, root induction media. 6  50  2.12  Lignin and Anthocyanin Extraction Lignin was extracted as described by Bruce and West (1989). Approximately  50 mg of tissue was accurately weighed and then homogenized in methanol. The homogenate was filtered through GF/A glass-microfiber discs (Whatman International Ltd., Maidstone, England) and allowed to air-dry overnight. The dried alcoholinsoluble residue (AIR, approximately 10 mg) was weighed and then transferred to a 13 mL Sarstedt tube (Newton, NC). The sample was incubated in 1 mL 2N HCI and 0.5 mL thioglycolic acid (Fisher Scientific, Fair Lawn, NJ), with intermittant shaking, at 100°C for 4 h. The mixture was centrifuged at maximum speed; the pellet was rinsed with 1 mL water and then transferred to a 2-mL screw-cap eppendorf tube; and then the solid material was extracted by shaking in 1 mL 0.5 M NaOH for 18 h at room temperature. The mixture was centrifuged and the thioglycolic acid-derived lignin was precipitated from the supernatant at 4°C for 4 h with 0.2 mL concentrated HCI. The sample was centrifuged 10 minutes at maximum speed and then the pellet was dissolved in 1 mL 0.5 M NaOH and quantified spectrophotometrically at 280 nm. Absorbance readings were calculated per mg AIR or per mg fresh weight starting material. Anthocyanins were extracted as described by Feinbaum and Ausubel (1988). Briefly, 0.5 mg of frozen leaf-tissue was homogenized in 2 mL 1 % HCI (w/v in methanol) and shaken overnight at room temperature. After the addition of 1 mL of water, the mixture was extracted with chloroform and the pink, aqueous-phase was quantified spectrophotometrically at 530 nm and 657 nm. Anthocyanin content was calculated by subtracting the absorbance due to chlorophyll using the equation - OD 76 5  51  O D 5 3 0  Chapter 3 Cloning and Characterization of the 4 C L cDNAs from Nicotiana  tabacum  and  3.1 Cloning and Characterization of the 3.2 Expression of the  Arabidopsis  Arabidopsis  thaliana  cDNA  Arabidopsis-4CL  4CL  3.3 Cloning and Characterization of the Tobacco-4CL cDNAs 3.4 Inheritance of the Tobacco-4CL Genes 3.5 Expression of the Tobacco-4CL Genes 3.6 Discussion  3.1  Cloning and Characterization of the c D N A s encoding  Arabidopsis  thaliana  4CL  were cloned from  Arabidopsis-4CL Arabidopsis  cDNA library using the potato  1991) as a hybridization probe. Five putative  4CL  Arabidopsis  cDNA  by screening an cDNA (Becker-Andre 4CL  et  al.,  clones were isolated  but plasmid Southern blot analysis of the cDNA inserts showed that only one insert strongly and reproducibly hybridized to the potato 4CL probe. This clone, At4CL, contained an insert approximately 2-kb in size and sequencing of the cDNA demonstrated that it had high ( 6 3 % to 71 %) homology to other known 4CL sequences (Table 3.1). A putative initiation ATG-codon from the At4CL insert was located only five nucleotides from the 5' end of the cDNA insert. It is possibile that additional inframe methionine codons are located further upstream; however, because the putative initiation ATG-codon is in a favorable sequence-context for the initiation of translation (Joshi, 1987; Kozak,1986) and.because amino acid homology to other 4 C L proteins begins shortly after this methionine (Figure 3.1), we consider it likely that this is the  52  initiator codon and that the c D N A is missing most of the 5' untranslated portion of the Arabidopsis  4CL  gene.  The predicted amino acid sequence from At4CL was between - 6 0 % to - 7 0 % identical to the predicted amino acid sequences from tobacco, potato, erythrorhizon,  parsley, soybean, pine and rice  amino acid identity (78%) with the 4CL14  4CL  Lithospermum  cDNAs. At4CL shared highest  clone from soybean. The At4CL c D N A  sequence had an open reading-frame predicted to encode a protein of 562 amino acids with a molecular mass of 61.0 kDa and an isoelectric point of 5.25 (not shown). The predicted polypeptide (Figure 3.1,  Arab-~\)  contained the conserved "GEICIRG"  amino acid motif believed essential for 4 C L activity (Becker-Andre et al. 1991) and an AMP-binding signature ([LIVMFY]-X -[STG]-G2-[ST]-[STE]-[SG]-X -[PALIVM]-K; 2  Schroder, 1989). The predicted  2  4 C L peptide also contained four of the  Arabidopsis  five cysteine-residues reported in other cloned  4CLs  (Figure 3.1, Uhlmann and Ebel,  1993). The high sequence homology, the conserved amino acid motifs, and the good sequence-aligment suggest that the cDNA cloned, At4CL, encodes 4 C L from Arabidopsis.  In order to estimate the number of 4CL genes in the Arabidopsis genomic Southern blots were hybridized to the Arabidopsis hybridization pattern was observed when  Arabidopsis  4CL  genome,  cDNA. A simple  genomic D N A was digested  with several different enzymes (Figure 3.2) which, with the exception of EcoRI, did not cut within the cDNA. In each case, a single hybridizing genomic restriction-fragment was observed when the blot was washed under stringent conditions. The c D N A contains two EcoRI sites which are approximately 150 bp and 300 bp from the 5' end of the c D N A clone. Based on the location of conserved exon-intron boundaries in the parsley (Lozoya  et al.,  1988), potato (Becker-Andre  etal.,  1991), and rice (Zhao  etal.,  1990) 4CL genes, these EcoRI sites would be within the first exon. It is likely that a genomic fragment containing the flanking DNA 5' to the first EcoRI site was not detected on the blot due to a weak signal from this small portion of the probe, and that 53  the predicted 150-bp EcoRI fragment was too small to be detected on the blot. Identical patterns of hybridization were observed when blots were hybridized under nonstringent conditions (not shown). Thus, consistent with previously reported results (Trezzini et al., 1993), 4 C L appears to be encoded by a single gene in Arabidopsis.  Table 3.1: Comparison of At4CL, Nt4CL-1, and Nt4CL-19 nucleotide (coding region) and predicted amino acid sequences to each other and to other 4CL sequences.  At4CL % DNA Iden. Tobacco-1 67 Potato-1 67 Tobacco-19 70 Lithospermum-1 67 Parsley-1 69 Soybean-14 71* Arabidopsis Soybean-16 70* Pine-12 64 Lithospermum-2 66 Rice-2 63  % % Amino Amino Acid Acid Iden. Sim. 66 80 71 85 69 82 70 82 69 82 78* 87* 70* 84* 65 81 61 77 60 78  Nt4CL-1  Nt4CL-19  % % % Amino Amino DNA Acid Acid Iden. Iden. Sim. 88 82 . 75 75 73* 67 66* 65 64 63  * Comparisons based on partial sequences.  54  _  _  86 81 80 73 73* 66 62* 63 62 60  92 89 89 83 83* 80 80* 79 80 80  % DNA Iden. 82 80 _  75 76 73* 70 69* 66 64 59  % % Amino Amino Acid Acid Iden. Sim. 81 89 80 88 _  _  81 72 73* 69 65* 65 62 61  90 81 83* 82 79* 80 79 80  Tob-1 Pot-1 Tob-19 Lith-1 Par-1 Arab-1 Pine-12 Lith-2 Rice-2  MPMETTTETKQSGDLIFRSKLPDIYIPKHLPLHS M D-MEKD -VI N MD Q K D K - I MGDCVAPKE T MAPQEQAVSQVMEKQSNNNNS V N S D MANGIKKVEHLY E SD MLSVASPETQKPELSSIAAPPSSTPQNQSSISGDNNSNETI P SNN T MITVAAPEAQPQVAAAVDEAP PEAVTV D S E  Tob-1 Pot-1 Tob-19 Lith-1 Par-1 Arab-1 Pine-12 Lith-2 Rice-2  (1) (2) YCFENISEFSSRPCLINGANDQIYTYAEVELTCRKVAVGLNKLGIQQKDTIMILLPNSPE N D R S C K D NS A H Q P G QF SRV I S A H H K T E L C KVGDKS TGETF SQ LS S G L I Q FATK PTGHV SD HVIS Q l ANFH VN N W L C RVA FAD D T RT CFS IS A A L GQW L CI Q A YPN T I DSKTGKQ FS TDSI A SN KG V V Q CA ARAA LPDA AA TGRT F TR L RA AA HR VGHG RV V Q CV  34  94  Tob-1 FVFAFMGASYLGAISTMANPLFTPAEWKQAKASSAKIIITQSCFVGKVKDYAS 148 Pot-1 I V A A I Tob-19 I V A H N F Lith-1 L I V T F SS I I KT L V TT P L FSQ Par-1 Y F L R F S I L Q L AYD A Arab-1 LS LA FR TA A F IA NT L EARY D I PLQN Pine-12 . A V M VR V T FYK G IA AG R V LAAY E LA LQ Lith-2 T M I I VI TG FY T IF VNV NT L NY D LRNTTINESDNK Rice-2 AV FA F VT A FCT Q IH FKG GV L L VY D LRQHEAFPRIDA +++++  Tob-1 Pot-1 Tob-19 Lith-1 Par-1 Arab-1 Pine-12 Lith-2 Rice-2  ENDVKVICIDS APEGCLHF-SELTQSDEHE IPEVKIQPDDWALPYSS L V V V I D I - V AN D E HV IM D KID S D EN TT L D E R KNIQI D QD - K MEA S M W NS DDGWIV DNESVP I R -T TTEASEV DSVE SP H VL T D APK Q I - V EA TQC A H YPKLGE F T TP N P SLLIENTQ NQ VTS S DSN PI F CTVGDDTLT T DDE--AT KA P WDLIADA GS V A S P F  Tob-1 Pot-1 Tob-19 Lith-1 Par-1 Arab-1 Pine-12 Lith-2 Rice-2  +++++++ (3) (4) GTTGLPKGVMLTHKGLVTSVAQQVDGENANLYMHSEDVLMCVLPLFHIYSLNSILLCGLR D V A P I ML V D H V T M D P MI AV C P F D IL M A M s P F D IL V A I S I D P LKHD VL V S A V RSV SG HE P H GAG AL F V SRV  55  195  255  Tob-1 Pot-1 Tob-19 Lith-1 Par-1 Arab-1 Pine-12 Lith-2 Rice-2  VGAAILIMQKFDIAPFLELIQKYKVSIGPFVPPIVLAIAKSPIVDSYDLSSVRTVMSGAA 315 P H T L N Q R VS T M D R T L H E VT NV Q A VT T V V K T P E NLL RC TVA M SETEK I V K A T NLTTC TVA I D T SQ V II A VL E GAL SHR VAAV L L N M K I V L PAP VAL PR EMGAM GA ERWR TV AV L V L N F ERH I I L  Tob-1 Pot-1 Tob-19 Lith-1 Par-1 Arab-1 Pine-12 Lith-2 Rice-  (5) PLGKELEDAVRTKFPNAKLGQGYGMTEAGPVLAMCLAFAKEPFDIKSGACGTWRNAEMK A T A E S A E S A YE NA S G PV L ER K IF N N PV S Ql R L LLNRV H IF S SPS H YPA S DL L ARL Q IF S P TPA S L  375  (6) +++++++  Tob-1 Pot-1 Tob-19 L i th-1 Par-1 Arab-1 Pine-12 L i th-2 Rice-2  IVDPDTGCSLPRNQPGEICIRGDQIMKGYLNDPEATTRTIDKEGWLHTGDIGFIDEDDEL 435 A E D K N S A Y Y D I TE A S E Y D E NA R S RT E D D S H N A A E D L D L TE E H A PE S AA E VEY D E I V I E S G E A V I Y V D V V F G L P A A V N Y V D V  Tob-1 Pot-1 Tob-19 Lith-1 Par-1 Arab-1 Pine-12 Lith-2 Rice-2  FIVDRLKELIKYKGFQVAPAEIEALLLNHPNISDAAWPMKDEQAGEVPVAFWRSNGS494 L I D I L P L VP V S G L T T I K T FL IG D T V A E A K KD V I L VA S A Q H E K -- V F P L IS A Q AA P DGF V F P L S IA S R Q DV AAD -  Tob-1 Pot-1 Tob-19 L i th-1 Par-1 Arab-1 Pine-12 Lith-2 Rice-2  AITEDEVKDFISKQVIFYKRVKRVFFVETVPK SPSGKILRKDLRARLAAG 544 T T I DAI K TT I Q V IN G DSI V K TT E I Q V V IF DAI I S ELS D Q V V INK T SI A K N E S Q I E VA KIH Y DAI . S K EL EA E V LHK Y PLYS VAVRQDFEER Q QTGR RL LNSYCSI D ESI E V LHK H IHAI A RE K C  56  Tob-1 Pot-1 Tob-19 Lith-1 Par-1 Arab-1 Pine-12 Lith-2 Rice-2  L i th-A  VPN* IS * L * FL GPTTNWPNGGNVAKDNVPNGVSNGVSKANGGVAKEGVANGVPTDGD DLPK* L* * A* *  547  YGVATKGVANGISNGVYKQVSNGWSNGVANGIVSNGIANGVHN*  Figure 3.1: Comparison of the deduced amino acid sequences with that of 4CL from other plants. Amino acid sequences from Nt4CL-1 (Tob-1), Nt4CL-19 (Tob-19), and At4CL (Arab-1) are aligned to those from potato 4CL1 (Pot-1), Lithospermum 4CL1 (Lith-1), parsley 4CL1 (Par-1), pine 4CL12 (Pine12), Lithospermum 4CL2 (Lith-2), and rice 4CL2 (Rice-2). Amino acids are only shown when they differ from the predicted amino acid sequence of Nt4CL-1. Dashes indicate gaps introduced to maximize alignment. Putative initiation methionines are in bold; conserved cysteine-residues in 4CL proteins are underlined and are numbered above the sequence; conserved motifs postulated to be involved in 4CL enzyme activity are marked (+++) above the sequence; translational stops are indicated with an asterisk; and numbers on the far right indicate the predicted amino acid position of Nt4CL-1. S 8  12.2  % •£  p «  > tr 8  2  -  10.2 - jLm  " W  6.1 —  3.2  4.0  -  2.0  -  E x p r e s s i o n of t h e Arabidopsis  J=  Figure 3.2: Arabidopsis genomic Southern blot analysis. Arabidopsis Columbia genomic DNA (10 jig) was digested, separated on an agarose gel, transfered to a nylon membrane, hybridized to the At4CL probe, and washed at high stringency (0.2 x SSC, 0.1% SDS, 65°C). The migration of size standards is shown to the left of the blot.  4CL  The developmentally-regulated expression of the Arabidopsis 4CL gene was examined by northern blot analysis. Arabidopsis seeds were germinated and grown on M S plates under constant light at 23°C. Under these conditions, the seedlings  57  underwent normal growth and development and exhibited very little anthocyaninpigmentation (indicative of light-induced stress) in the hypocotyls and cotyledons. Seedlings were harvested after 2, 3, 4, 5, 7, and 10 days of germination and, by 3days post-germination, it was possible to separate the seedlings into root- and shootsectors. These tissues were used for R N A isolation. Northern blot analysis (Figure 3.3) showed that  Arabidopsis  4CL mRNA accumulation was not detectable in 2-day  old seedlings but was detectable subsequent to 3-days post-germination. Low, but detectable, levels of 4CL mRNAs were found in the shoots whereas the roots contained higher levels of 4CL transcripts compared to the shoots. 4CL mRNA levels appeared to remain fairly constant in these tissues from day-3 to day-10 seedlings. In mature  plants (Figure 3.4), northern blot analysis indicated that  Arabidopsis  4CL was expressed at low levels in mature leaves and in flower buds (prior to anthesis). In contrast, 4CL transcripts accumulated to high levels in bolting stems that were between 3 cm to 20 cm in height. 2  3  4  5  7  10  days  seedling  shoot  root  58  Figure 3 . 3 : Northern blot analysis of 4CL levels in Arabidopsis seedlings. Seedlings were harvested 2-10 days postgermination and RNA was extracted from whole-seedlings, seedling-roots and seedling-shoots (hypocotyls and cotyledons). Total RNA (10 ug) was separated on a formaldehyde gel, transferred to a nylon membrane, and hybridized to an At4CL probe. The gel was stained with ethidium bromide before transfer to the nylon membrane to ensure equal loading of RNA, and the blot was washed at high stringency (0.2 x SSC, 0.1% SDS, 65°C).  R  N  A  4?  3.3  r  Figure 3.4: Northern blot analysis of 4CL RNA levels in mature Arabidopsis. RNA was isolated from mature, fully-expanded leaves, bolting stems (3 to 20 cm in height), and flower buds. Total RNA (10 u.g) was separated on a formaldehyde gel, transferred to a nylon membrane, and hybridized to the At4CL probe. The gel was stained with ethidium bromide before transfer to the nylon membrane to ensure equal loading of RNA, and the blot was washed at high stringency (0.2 x SSC, 0.1% SDS, 65°C).  ?  Cloning and Characterization of the Tobacco-4CL cDNAs Tobacco 4CL cDNAs were cloned by screening a tobacco shoot-tip c D N A  library using the potato 4CL cDNA (Becker-Andre et al., 1991) as a hybridization probe. Eleven clones encoding 4 C L were isolated and preliminary sequence-analysis and restriction digests showed that the cDNAs could be placed into four groups, represented by Nt4CL-1, Nt4CL-5, Nt4CL-17, and Nt4CL-19 (Table 3.2). The c D N A s within each group had identical 3'-sequences suggesting that although they were of different size, they were likely products of the same gene. Detailed restriction-maps of clones 1,5, 17, and 19 (Figure 3.5), showed that the four cDNAs fell into 2 classes. One class (Nt4CL-1 and Nt4CL-17) had distinctive Psfl and Kpn\ sites while the other class (Nt4CL-19 and Nt4CL-5) had /Wall, SamHI, and S a d sites. Sequencing of the 5' region of these clones showed that restriction-site polymorphisms between Nt4CL-17 and Nt4CL-1 and between Nt4CL-19 and Nt4CL-5 were due to single base-pair changes. The 3' sequences of the four cDNAs were compared (Figure 3.6) and the results demonstrate that the four cDNAs again fell into two classes with Nt4CL-1 and Nt4CL-17 in one class ( 9 4 % sequence identity) and Nt4CL-19 and Nt4CL-5 in a second class ( 9 7 % sequence identity). In contrast, the 3' sequences of Nt4CL-1 and Nt4CL-19 were only 6 7 % identical; comparison of any other pair of clones between the two classes gave similar results (<70% sequence identity, not shown). It should be noted that although Nt4CL-19 and Nt4CL-5 were 9 7 % identical at the 3' end, there is a 36 base- pair region within Nt4CL-5 which is absent in Nt4CL-19. One clone from 59  each of the two 4CL-cDNA classes was selected for further analysis. The two longest clones, Nt4CL-1 and Nt4CI_-19, were subcloned and completely sequenced. The nucleotide and predicted amino acid sequences of Nt4CL-1 and Nt4CL-19 were compared to each other and to other 4CL sequences available in the E M B L database (Table 3.1). In general, the predicted amino acid sequences from Nt4CL-1 and Nt4CL-19 had the highest identity (> 80%) to the predicted 4 C L amino acid sequences from potato and  Lithospermum  erythrorhizon.  The tobacco 4 C L amino  acid sequences were also homologous, but with decreasing percent identity (<73%), to the predicted amino acid sequences from the parsley, soybean, and rice  4CL  sequences. An exception is the  4CL-2  clone from  Arabidopsis,  Lithospermum  pine which  shared significantly lower amino acid identity (62%) to the tobacco 4 C L polypeptides as compared to the  Lithospermum  4CL-1  clone (-80%). Interestingly, Nt4CL-1 shared  a slightly higher amino acid homology to the potato 4 C L sequence (85%) than to the prediced amino acid sequence from Nt4CL-19 (81%). A putative initiation ATG-codon was identified in the 5' end of Nt4CL-1 and Nt4CL-19. Upstream of this ATG-codon was approximately 70 nucleotides which included translational-stop codons that were in-frame with the predicted polypeptides (not shown); therefore, these c D N A s likely encode the entire tobacco-4CL proteins. The Nt4CL-1 cDNA had an open reading frame predicted to encode a protein of 547 amino acids with a molecular mass of 59.8 kDa and an isoelectric point of 5.40. The predicted polypeptide from Nt4CL-19 was 542 amino acids in length with a molecular mass of 59.4 kDa and an isoelectric point of 5.69. Both predicted peptides (Figure 3.1) contained the conserved "GEICIRG" amino acid motif believed essential f o r 4 C L activity (Becker-Andre  etal.  1991), an AMP-binding signature ([LIVMFY]-X 2  [STG]-G -[ST]-[STE]-[SG]-X -[PALIVM]-K; Schroder, 1989), and five other 2  2  characteristic cysteine-residues that have been reported in other predicted 4 C L amino acid sequences (Figure 3.1, Uhlmann and Ebel, 1993).  60  The high sequence homology, the conserved amino acid motifs, and the good sequence-aligment suggest that Nt4CL-1 and Nt4CL-19 encode tobacco 4 C L proteins. Clones Nt4CL-1 and Nt4CL-19 were both used for further analysis since they represent members of the two divergent classes of tobacco 4CL c D N A s cloned. It is interesting to note that tobacco is an allotetraploid which arose from the hybridization of two  species related to present-day  Nicotiana  N. sylvestris  and  N.  Whether the two divergent classes of 4CL c D N A s originated from the  tomentosiformis.  two progenitor-species and how the 4CL genes are inherited is addressed in the following section.  Clone Grouping  Homologous Clones  Size (kb)  Nt4CL-1  1 6 11 14 16  2 2 1.7 1.7 0.9  Nt4CL-5  5 13 18  1.5 0.7 1.4  Nt4CL-17  17 4  1.6 1.3  Nt4CL-19  19  2  Restriction Map  c D N A Clone Rl  Nt4CL-17 Nt4CL-1  Nt4CL-19 Nt4CL-5  Table 3.2: Summary of tobacco- 4CL cDNA clones. The eleven isolated 4CL cDNA clones can be placed into four groups.  Rl  >  I  Rl I  J  BRI P II  I  HII L_ I  u_  HII HII  HII I  Accl Sail HII HII I I  HII  x  J_L Al  J_  I I  HII _l_ Rl  KK  Rl HII HII  All _l All  _J_  B L_  K  X J  J J  x  x  Figure 3.5: Restriction maps of representative tobacco-4C/. cDNA clones. Results demonstrate that the 4 cDNAs fall into two classes. Restriction enzymes are: Al, Aval, All, AvaW; B, BarrfrH; HII, HincH; K, Kpn\; P, Psrl; Rl, EcoRI; S, Sad; and X, Xho\.  61  J  c D N A Clone 4CL-17  % Identity  T^ATTCCTTTTGGCTTGGGATTTGACGGTGGGTGTAATAAGCTAAACAAGTCCACCATTATTGAAAATATTATATGTTT  I. i 1 . ! 1 : 1 1 1 ; . 111 -! I! I: I - 1 1 1 1  1 1 ; I -11 - 1 1 1 1 • 11 ! . , I I I . .  IIIIIMIIIIIIIIIII*  I III * l l l l * * l * l l * * * * l l l * l l * l l l  1*11*11****111*111*1*11  11111111*1*1*111  I III IIIII^IiII.* III' Ii  lllllllllllllllllllllll  1  4CL-1  3' Sequence 1  TAATTCCTTTTGGCTTGGTATTTGATGGTG. . . GTAACAAACTAAACAAGTCCACC . . . ATTGAAGATATTATATGTTC  4CL-19 XM.T • ACTTTCAGTTTCAGCTTTAATAGTG. . . GCAATAACTATAACCAGTTCGCC . . . ATTGAAGACAATTTAT . . . . :  4CL-5  III  I I II I I I I I I I I I I I I  T&AT. ACTTTCAGTTTTAGCTTTAATAGTG. . .GCAATAACTATAACCAGTTCGCC. . . ATTGAAGACAATTTATTTTT  4 CL -17 TTTGAGT  TTTACCTTCACCCTA.  .ATGTTCGGTATAATTTC  4CL-1  TTTACCTTCACCTAA.  CGGTATAATTTC  111*1*1  111111111111**1  TTTAAAT  4CL-19 4CL- 5  Illlllllllll  1111 :1  Illlllllllll  - 1 1 1 •. 1 1 i I i :  . GAATTAATTTAC  TATTAAAATGTTACATAATATGTTCTTTTGATTGTACCTTCAACTACGTGCCTCTTCGGTCA.  4CL-17 CAGTTTGGC. . AACGGGCGAAATGTCTGTAAATTAATTTATAA. GACTTCTTTATTCATCTTT 4CL-1  ***** . GAATTAATTTAC  TGTACCTTCAACTACATGCCTCTTCGGTCA.  I II II I II I  lllllimillllllllllllllllllllll  IIMIIIIIIIIIIIIIII  1***11111  ll*l*l**llllll*IMIIIII****l*lll  111111**1*11**  CAGTTTGGC.. AACGGGCGAAATGTCTGTAAATTAATTTATAA. GACTTCTTTATTCATCTTT  ]  94% J  ]  67%  4CL-19 CGAATTGGCAAAAGGAGGAAAATGTATGTAAATTTGACTGTAACGACTTCAATTTTTT  ]  4CL-5  J  IIIIIIIIIIIIMIIIIIIIIIIMIIIMIIIIIIIIMII**IIIIIIIIIIIM  '•  CGAATTGGCAAAAGGAGGAAAATGTATGTAAATTTGACTGTAAGAACTTCAATTTTTT  J  Figure 3.6: 3' sequences of representative tobacco-4CZ. cDNA clones. Translation stop codons are underlined. Vertical lines indicate identical nucleotides, asterisks indicate different nucleotides and dots indicate gaps introduced to maximize alignment. All sequences terminate with a poly(A) tail (not shown) with the exception of Nt4CL-17 which extends for an additional 136-bp and Nt4CL-19 which extends for an additional 6 nucleotides prior to polyadenylation. The percent sequence identity between pairs of clones is shown at the bottom right.  3.4  Inheritance of the Tobacco-4CL Genes Southern blot analysis was used to assess the size of the 4CL gene-family in  tobacco. Cytogenetic (Smith, 1968), biochemical (Gray etal., 1990; Takahashi etal., 1992; Pellegrini etal.,  species of Nicotiana  etal.,  1991), and more recently, molecular studies (van Buuren 1993; Hua etal.,  suggest that N. tabacum  1974; Obokata  1993; Kroneberger etal.,  etal.,  1993) strongly  (tobacco) arose from the hybridization of gametes from two  follwed by chromosome duplication. The gametes are thought to  be from ancestors of N. sylvestris  and N. tomentosiformis.  Thus, we also used  Southern blot analysis to determine if the 4CL genes corresponding to the cloned c D N A s , Nt4CL-1 and Nt4CL-19, were present in the progenitor species and how they 62  were inherited into tobacco. In a preliminary experiment, restriction-digested Nt4CL-1 and Nt4CL-19 c D N A s were blotted and hybridized separately to probes generated from the insert of each plasmid. Figure 3.7 shows that at moderate stringency (0.5 X S S C , 0 . 1 % S D S , 65°C), little cross-hybridization was observed, demonstrating that the c D N A s could be used as gene-specific probes for distinguishing the presence of Nt4CL-1-like and Nt4CL-19-like sequences in tobacco. Genomic Southern blots prepared with DNA from tomentosiformis,  and N.  N. sylvestris,  N.  were hybridized sequentially to Nt4CL-1 and Nt4CL-  tabacum  19 probes. Figure 3.8 shows that both probes hybridized to restriction fragments from the progenitors and from tobacco, and that each probe hybridized to a unique set of restriction fragments in the three species. This demonstrates that at least two divergent 4CL genes, represented by the Nt4CL-1 and Nt4CL-19 c D N A clones and which we designate  4CL1  and  4CL2,  are both present in the progenitors as well as in  tobacco. The small number of fragments which hybridized to each probe in N. and  suggests that there are 1 -2 tobacco  N. tomentosiformis  4CL  7-like and  sylvestris 4CL2-\\ke  genes in these progenitor species (Figure 3.8). The D N A hybridization-patterns in N. tabacum  were more complex, as would be predicted from the acquistion of a set of  4CL 7-like and 4CL2-like genes from each progenitor. Indeed, Nt4CL-1-hybridizing (4CL1)  restriction fragments in AV. tabacum  can be directly traced from the restriction  fragments of the parental species. For example, a single 9-kb was observed in the  kb in size) were observed in N. observed in the N.  tabacum  hybridizing fragments combination of the about half of the  genome, three  N. sylvestris  4CL1  fragment  fragments (1.3 kb, 3 kb, and 4  and all four E c o R V fragments were  genome (Figure 3.8, probe Nt4CL-1). Nt4CL-19-  [4CL2)  N. sylvestris  N. tabacum  tomentosiformis,  EcoRV  EcoRV  in N.  tabacum  and 4CL2  also appeared to be derived from a  N. tomentosiformis  4CL2  gene-complements, but  restriction fragments were polymorphic with respect  to fragments in the progenitor species (Figure 3.8, arrows). 63  Restriction-fragments hybridizing to both 4CL1 and 4CL2 were detected when the genomic Southern blot was hybridized to Nt4CL-19 and washed at low stringency (Figure 3.8, Nt4CL-19 low stringency).  In addition, however, additional hybridizing  restriction fragments were observed (Figure 3.8, asterisks) which were not apparent after high-stringency washes with either probe. These bands may represent additional 4CL-genes divergent from 4CL1 and 4CL2.  - 2 ^  probe:  1  ^2 ^^ |S  19  sylv. torn tab. BEHXBEHXBEHX  probe:  Figure 3.7: Plasmid Southern blot analysis. Southern blot analysis of tobacco- 4CL cDNAs hybridized to either Nt4CL-1 or Nt4CL-19 probes (1 or 19). Blots were washed at moderate stringency (0.5 x SSC, 0.1% SDS, 65°C).  Nt4CL-1  sylv. torn tab BEHX BEHXBEHX  Nt4CL-19  sylv. torn. tab BEHX BEHXBEHX  Nt4CL-19 low stringency  Figure 3.8: Tobacco genomic Southern blot analysis. Southern blots of genomic DNA (10 ug) from N. sylvestris (sylv.), N. tomentosiformis  (torn.), and N. tabacum (tab.) digested with different restriction-  enzymes. A single blot was hybridized sequentially with Nt4CL-1 and Nt4CL-19 probes and washed at high stringency (0.2 x SSC, 0.1% SDS, 65°C). Arrows indicate hybridization restriction-fragments that are unique to N. tabacum and not found in the progenitors. Asterisks indicate unique hybridization restriction-fragments that are observed when the blot was probed with Nt4CL-19 cDNA and washed at low stringency (2 x SSC, 0.1% SDS, 65°C) but not observed at high stringency washes. Restriction enzymes are: B, BgH\; E, EcoRV; H, HindlU; and X, Xba\.  64  3.5 Expression of the Tobacco-4CL G e n e s Characterization of the tobacco 4CL c D N A s demonstrated that there were at least two classes of divergent 4CL genes. To determine if these divergent forms of  4CL were differentially expressed, northern analysis was performed using R N A isolated from wounded tobacco-leaves, methyl jasmonate treated tobacco-leaves, and various organs from tobacco plants. Blots were hybridized to Nt4CL-1 or Nt4CL-19 probes under conditions which allowed little cross-hybridization between probes (Figure 3.7). Subsequent stripping and re-hybridization of the blots to a pea rRNA gene probe confirmed equal RNA-loading between samples and between gels. Figure 3.9 demonstrates that the expression of both classes of 4CL genes was inducible by wounding and methyl jasmonate treatment. In this and similar experiments, however,  4CL1,  4CL2  was less responsive to methyl jasmonate treatment than  but was approximately equivalent in its response to wounding. The steady-  state levels of both  4CL1  and  4CL2  mRNA transcripts were highest in old stems  followed by young stems and ovaries. RNA levels were lowest in shoot tips, the pigmented limb of the petals, and untreated, mature leaves (Figure 3.9). No major differences in the expression of  4CL1  and  4CL2  were observed, although  4CL2  RNA  was reproducibly less abundant in young stems and ovaries relative to 4CL1. The low expression of 4CL in the pigmented limb of tobacco petals was unexpected since transgenic studies using the parsley 4CL-1 promoter, bean PAL and  CHS  promoters, and a tobacco CHS promoter fused to the GUS reporter-gene  showed high GUS-activity in the floral limbs (Bevan Hauffe  etal.,  1991, Drews  etal.,  etal.,  1989; Schmid  1992). This led us to examine  4CL  etal.,  1990;  expression in  petals more closely. Tobacco flowers from developmental-stages 1 through 6 (at stage 1, the corolla is just emerging from the sepals; pigmentation of limb-tissue is  65  detectable at stage 4, and at stage 6 anthers are dehiscing; Reinold et al., 1993) were harvested, and R N A was isolated from the sepals, and the base, tube, and limbsectors of the corolla (see section 2.1). Figure 3.10 shows a northern blot of this R N A hybridized to an Nt4CL-19 probe.  4CL2  mRNA accumulation was highest in the  unpigmented portions of the petals (tube and base), where its accumulation was temporally regulated. High transcript-levels were first evident in stage 4 flowers, and 4CL2  m R N A levels had decreased significantly in stage 6 flowers. An identical pattern  of expression was observed when blots were hybridized to a Nt4CL-1 probe (results not shown). To determine whether PAL gene expression was coordinately regulated with that of 4CL in petals, a northern blot was hybridized to a potato PAL probe (Figure 3.10, StPAL). Although the heterologous probe did not hybridize strongly, an identical pattern of PAL expression was observed, with high expression in tube- and basesegments of stage 4 and stage 5 flowers. To complement the results from the northern blots, western blot analysis was performed using an antiserum raised against the parsley 4CL. Similar to the R N A levels, 4 C L protein was most abundant in the tube- and base- segments of tobacco petals (Figure 3.10, anti-4CL). 4 C L protein levels were highest in stage 5 and stage 6 petals, consistent with the accumulation of 4 C L protein subsequent to 4CL R N A accumulation. Western blot analysis also revealed that 4 C L proteins were also present in the limbs and in the sepals, although R N A was only weakly detectable in these organs. These results show that, during flower development, phenylpropanoid gene expression and enzyme accumulation are very active in the unpigmented portions of tobacco-petals and appears to be much higher than in pigmented florallimbs.  66  E  you  CO E  c  MJ  =5  E  old ste  ec  CD  O)  WOL  to  pui  a)  «  Q_  |  O  sho  rd CO  > O  probe Nt4CL-1 rRNA  Figure 3.9: Northern blot analysis of 4CL RNA levels in tobacco. RNA was isolated from fully-expanded leaves (mature leaves), or fully-expanded leaves which had been wounded (wounded leaf) or treated with methyl jasmonate (MJ) and other tissues as indicated. Duplicate blots of total RNA (10 ug) from various organs were separated on formaldehyde gels, blotted to nylon membranes, hybridized to Nt4CL-1 or Nt4CL19 probes and washed at moderate stringency (0.5 x SSC, 0.1% SDS, 65°C). The blots were subsequently stripped, and hybridized to rRNA probes to demonstrate evenness of loading.  Nt4CL-19  rRNA  Tissue  Limb  Stage  2 3 4 5 6  Tube  2 3 4 5 6  Base  Sepal  2 3 4 5 6  1 2 3 4 5 6  tf  mm  probe Nt4CL-19  rRNA  • St PAL  25 "H  rRNA  a n t i - 4 C L  Figure 3.10: Steady-state levels of 4CL RNA, PAL RNA and 4CL proteins in tissues from developing petals and sepals of tobacco. Tobacco flowers in developmental stages 1 through 6 were harvested and dissected into four sectors: the limb, the tube, the base and the sepals (see section 2.1). Duplicate northern blots prepared using 10 u.g total RNA from these samples were hybridized to Nt4CL-19 and potato PAL (StPAL) probes and washed at moderate (0.5 x SSC, 0.1% SDS, 65°C) and low (2 x SSC, 0.1% SDS, 65°C) strigency respectively. The blots were subsequently stripped and re-hybridized to rRNA probes to demonstrate evenness of loading. The bottom panel shows western blot analysis of protein extracts from the petal and sepal samples described above. The blot was reacted with a polyclonal antibody raised against parsley 4CL.  67  3.6  Discussion High nucleotide and predicted amino acid homology, and good alignment of the  putative initiation-methionine to known 4 C L sequences, suggests that At4CL, Nt4CL-1, and Nt4CL-19 are near full-length cDNAs encoding 4 C L from tobacco. In Arabidopsis,  Arabidopsis  and  4 C L appears to be encoded by a single gene, a result also  suggested by Trezzini era/. (1993), who identified a putative, although poorly characterized,  Arabidopsis  4CL  genomic clone. 4 C L is encoded by a single  functional gene in pine (Voo era/., 1995) and by very similar, duplicated genes in parsley (Lozoya Arabidopsis  etal.,  1988) and potato (Becker-Andre  etal.,  1991). Since  4 C L is encoded by a single gene, it is likely that the promoter of the  4CL  gene is responsive to all developmental signals and environmental signals which dictate the need for 4 C L activity. In contrast, the tobacco 4 C L proteins are encoded by a divergent gene-family analogous to that found in soybean (Uhlmann and Ebel, 1993),  Lithospermum  (Yazaki  etal.,  1995), rice (Zhao  etal.,  1990; E M B L accession  number L43362), and poplar (Allina and Douglas, 1994). Nt4CL-1 and Nt4CL-19 were only - 8 0 % identical at the amino acid level and the nucleotide level; however, despite their divergence, the corresponding genes were apparently not differentially expressed. To date, differential expression of 4CL genes has only been demonstrated in soybean and parsley. In soybean, fungal infection and elicitation of cell cultures caused the increase in 4CL-16  while the R N A levels of 4CL-14  remained the same.  The soybean 4CL c D N A s are partial-length and the truncated polypeptides are predicted to be 6 5 % identical to one another (Uhlmann and Ebel, 1993). In parsley, 4CL-1  is wound-inducible in the roots whereas  4CL-2  is also preferentially expressed in the flowering stem (Lois and Hahlbrock,  4CL-2  is light-inducible in cell cultures.  1992). The predicted amino acid sequences from the parsley 4CL cDNAs are 9 9 . 5 % identical (Lozoya  etal.,  1988).  Genomic Southern blot analysis (Figure 3.8) demonstrates the presence of tobacco  4CL  gene-family members in  Nicotiana  68  sylvestris  and  N. tomentosiformis,  the  species most closely related to the presumed progenitors of tobacco,  Nicotiana  strongly suggesting that 4CL gene-duplication and gene-divergence had occurred in prior to the emergence of these species. The sequences of near full-length  Nicotiana  c D N A clones specific to two of these genes, which we call  4CL1  and  4CL2,  show that  they are relatively divergent, sharing about 8 0 % nucleotide and amino acid sequence identity (Table 3.1). Consistent with the allotetraploid nature of the  N.  tabacum  genome, genomic Southern blots (Figure 3.8) show that the tobacco 4CL gene-family contains copies of 4CL1 and  4CL2  from both parental species, for a total of at least 4  genes. This is further supported by the existence of two classes of polymorphic cDNA clones, each class containing two cDNA types (Figures 3.5 and 3.6). Formally, it is possible that Nt4CL-1 / Nt4CL-17, and Nt4CL-19 / Nt4CL-5, are allelic to one another and represent the 4CL genes inherited from one or the other progenitor species. However, since we isolated eleven 4CL cDNA clones and they all fell into 4 groups (Table 3.2), it is more likely that Nt4CL-1 & 17 and Nt4CL-19 & 5 are not allelic but are the  4CL1-  and  4CL2-  genes originating from the two progenitor species. In this case,  the polymorphism observed between clones 1 &17, and clones 19 & 5, are due to the different orgin of these genes from the two progenitor species. Similar evolutionary schemes have been proposed for the origins of tobacco gene-families encoding nitrite reductase (Kroneberger Buuren  etal.,  etal.,  1993), psbP (Hua etal.,  1993) and endochitinases (van  1992).  When the tobacco genomic Southern blot was washed at low stringency, the Nt4CL-19 probe hybridized to numerous restriction-fragments in tobacco and its progenitors which were distinct from the hybridization fragments observed at highstringency washes (Figure 3.8, low stringency, asterisk). These DNA sequences may represent a third class of 4CL genes, distinct from  4CL1  and  4CL2.  Three tobacco  4CL cDNA sequences have recently been deposited in the E M B L data bank. The sequence of one partial-length clone, T O B T C L 2 (EMBL accession number D50033), is 9 9 % identical to Nt4CL-1 at the nucleotide level. A second clone, full-length in size, 69  T 0 B 4 C C A L (EMBL accession number D43773), has high homology to the 5' ( 9 7 % identical) and 3' ( 1 0 0 % identical) end of Nt4CL-5 and includes the 36-bp stretch specific to Nt4CL-5 (Figure 3.6). T O B T C L 2 and T O B 4 C C A L are likely the same clones as Nt4CL-1 and Nt4CL-5 with single base-pair differences due to sequencing descrepencies or natural polymorphism between individual plants. In contrast, a third partial-length tobacco 4CL clone, T O B T C L 6 (accession number D50034) is only 7 5 % and 7 4 % identical to Nt4CL-1 and Nt4CL-19 respectively. Thus, T O B T C L 6 may represent a member of the third, highly-divergent class of tobacco,  4CL3,  which I had  detected under low-stringency hybridization. The tobacco 4CL nucleotide and amino acid sequences (Table 3.1) deduced from Nt4CL-1 and Nt4CL-19 were most similar to the potato 4CL, followed by the 4CL1  sequence from  Lithospermum.  Polemoniales) and Lithospermum  Tobacco (order Polemoniales), potato (order (order Lamiales) are all in the Asteridae class of  plants and thus, it is not surprizing that the 4CL sequences reflect this relatedness (Figure 3.11). In addition, tobacco and potato are in the same family of plants (Solanaceae) and, as expected, have 4CL sequences of even greater homology to each other. The tobacco 4CL sequences are increasingly divergent (Table 3.2) from the 4CL sequences in plants of the orders Umbellales (parsley), Fabales (soybean), Papaverales  {Arabidopsis),  Pinales (pine), and Poales (rice). Although there are but a  few 4CL sequences available, this pattern of sequence-divergence is consistent with proposed taxonomic relationships between these groups (Heywood, 1978; Figure 3.11). An exception is the 4CL-2  clone from  Lithospermum  the expected taxonomic ranking (Table 3.2). Yazaki from  Lithospermum  etal.  which does not comply to (1995) suggests that  4CL-2  may be a highly-divergent form of 4 C L which has an organellar  localization. Whether a similar situation is found with members of the putative gene-family from tobacco (detected by genomic Southern analysis under lowstringency washes) is unknown and further analysis is required before the evolutionary relationship between these divergent 4CL genes is understood. 70  4CL3  Interestingly, the Nt4CL-1 cDNA similar to potato  4CL  than to Nt4CL-19  {4CL1) (4CL2).  and amino acid sequences are more This suggests that  4CL1  may be  homologous to an ancestral 4CL-gene from which all Solanaceous 4CL-genes (including potato  4CL)  evolved, while  duplicated in the  Nicotiana  4CL2  may represent a  gene which had  4CL  evolutionary line and has undergone greater divergence  from the ancestral 4CL-gene. 4CL1 restriction-fragments are conserved in N. with respect to its proposed progenitors  tabacum  N. sylvestris  and  N.  tomentosiformis  (Figure 3.8, Nt4CL-1 probe) whereas many of the 4CL2-specific restriction-fragments are polymorphic (Figure 3.8, Nt4CL-19 probe, arrows). This is consistent with a more rapid divergence of the 4CL2-\ocus  than the 4CL 7-locus subsequent to the  hybridization event giving rise to tobacco. The interspecies hybridization event is thought to have occurred within the last 6 million years (Okamuro and Goldberg, 1985). Northern blot analysis was used to examine the expression pattern of 4CL in Arabidopsis  and tobacco. In young  Arabidopsis  seedlings,  4CL  was developmental^  regulated and accumulation of the mRNA was not visible until the third day postgermination (Figure 3.3). The timing of this expression-pattern, as demonstrated by basic fuchsin staining (Lee era/., 1995a) and confocal laser scanning microscopy (Dharmawardhana et al., 1992), correlates well with the onset of lignin deposition in the differentiated tracheids of the cotyledons and roots. In other studies, high expression of  Arabidopsis  PAL,  CHS  and other anthocyanin biosynthetic genes was  reported in 3- to 4-day-old seedlings, and was correlated with the accumulation of anthocyanin pigments (Kubasek et al., 1992; Ohl era/., 1990]. Here however, under the light conditions in which the seedlings were grown, no anthocyanin pigmentation was observed, suggesting that 4CL gene expression is not associated with anthocyanin biosynthesis, rather, it is associated with the biosynthesis of lignin during vascular differentiation. The higher levels of 4CL transcripts in seedling-roots relative to shoots may be important for the biosynthesis of additional phenylpropanoid-derived 71  to shoots may be important for the biosynthesis of additional phenylpropanoid-derived compounds in roots such as suberin. 4CL and PAL promoter- GUS fusion studies also report high GUS-activity in non-vascular root tissues as compared to shoot tissue (Lee etal.,  1995a; Ohl  et al.,  1990). In mature  Arabidopsis  plants,  4CL  expression was  highest in bolting stems (Figure 3.4). A comprehensive characterization of lignin deposition in  demonstrates that the stem tissues are highly lignified  Arabidopsis  (Dharmawardhana  etal.,  1992). Therefore,  Arabidopsis  4CL  expression appears to  be associated with lignin deposition and may be useful as a marker for xylemdifferentiation during vascular system development. Analogous to tobacco  4CL1  and  4CL2  Arabidopsis,  expression is highest in old stems where it likely functions for  the biosynthesis of monolignol-precursors (Figure 3.9). Tobacco  4CL1  and  4CL2  are both wound inducible and methyl jasmonate  inducible (Figure 3.9). Methyl jasmonates have been implicated as the intracellular signal in response to wounding (Farmer and Ryan, 1992; Ellard-lvey and Douglas, 1996) and, in agreement with the results presented here, studies utilizing heterologous probes in tobacco have shown that 4CL genes are activated by wounding and methyl jasmonate treatment (Douglas et al., 1991; Ellard-lvey and Douglas, 1996). The high levels of induced 4CL transcripts likely have a role in the biosynthsis of defense-related compounds such as phenylpropanoid-derived phytoalexins and cell wall components which may serve as physical barriers against pathogen invasion (reviewed in Hahlbrock and Scheel, 1989; Nicholson and Hammerschmidt, 1992). While both levels of  4CL2  4CL1  and  4CL2  appeared to be inducible, the  (Nt4CL-19) m R N A appeared to be lower than that of  4CL1  (Nt4CL-1) in  methyl jasmonate treated leaves as well as in the ovaries and young stems (Figure 3.9). Although these differences were reproducible, the significance of the 2 - 3 fold  72  lower levels of 4CL2 4CL1  and 4CL2  transcripts is unclear. Other than these quantitative differences,  does not appear to be differentially regulated despite the high  divergence between members of the 4CL1 and 4CL2 suggest that  4CL1  and 4CL2  gene-family. These results  gene-families may be functionally redundant.  However,  since the ovary is a complex organ and 4CL is expressed in the ovules, the carpel walls, and in the nectaries (Hauffe  et al.,  1991; Reinold  1993), we cannot  etal.,  exclude the possibility that the two genes are differentially regulated at the tissue-level in this organ. Further experiments using gene-specific probes and in  situ  hybridization  will be necessary to resolve this issue. The high levels of  4CL1  and 4CL2  expression in the unpigmented corolla tube  and base-portions of tobacco flowers, and the very weak expression in the pigmented limb (Figure 3.10) are in striking contrast to the heterologous and  4CL-GUS  PAL-GUS,  CHS-GUS,  fusions which direct cell-specific expression in the limb-portions of  transgenic-tobacco flowers (Bevan  etal.,  1989; Liang  et al.,  1989; Schmid  etal.,  1990,  Hauffe et al., 1991). The spacial accumulation of tobacco 4CL transcripts in the flowers does not correlate with the accumulation of tobacco CHS transcripts and does not correlate with the G U S activity (directed by tobacco  CHS-GUS  fusions) found in  the epidermal cells of the limb where anthocyanin pigments accumulate (Drews et al., 1992). However, the temporally-regulated expression of 4CL in the petal tube and base is consistent with previous results which showed, by  in situ  hybridization, that  tobacco 4CL m R N A accumulates to high levels in epidermal and mesophyll cell-layers of stage 4 and stage 5 tobacco flowers (Reinold  etal.,  1993). Furthermore, the results  in Figure 3.10 show that tobacco PAL mRNA, detected using a heterologous potato  PAL  probe, accumulates in a similar manner, and that 4 C L protein accumulates to  higher levels in the tube and base of the flower than in the limb of the flower. The presence of 4 C L proteins in the limbs and sepals may represent the basal-level of preexisting 4 C L that is present before the developmentally-induced  4CL1  and  4CL2  transcripts observed during stage 4 and stage 5 of tobacco flower development. It is not clear whether theses pre-existing 4 C L proteins are products of the 4CL1 and  4CL2  genes or products of other divergent 4CL genes. The biosynthesis of anthocyanins in the pigmented portion of tobacco flowers may be due to these pre-existing proteins. Taken together, these results clearly indicate that phenylpropanoid metabolism is strongly activated in the unpigmented petal during flower development. 73  A possible function of 4 C L in the unpigmented portions of tobacco petals is to synthesize colorless flavonols like kaempferol and quercetin which accumulate in tobacco-flower petals (Holton et al., 1993). Recent observations indicate that colorless flavonoids are abundant in the base- and tube- regions of tobacco flowers (Reinold, 1995) and, in petunia, CHS expression in unpigmented petal cells may be correlated with the accumulation of uncolored flavonoids (Koes et al., 1990). The white petals of Arabidopsis  also accumulate kaempferol and quercetin (Shirley era/., 1995) yet, 4CL  m R N A levels were low in the flower buds (Figure 3.4). The reason for this discrepancy may be because the flower buds used for northern analysis corresponded to an early developmental stage (see section 2.1) in which flavonoid biosynthesis may not have yet begun. In agreement with this, PAL promoter-G|/S fusion studies reported G U S staining in mature  Arabidopsis  flowers but no G U S activity in early flower development  (Ohl et al., 1990). Another possiblity is that 4CL transcripts may be diluted out since the  Arabidopsis  flowers used for northern analysis were still enclosed by sepals. A  more detailed study of Arabidopsis  flower development and 4CL expression is  required before this can be clarified. Another role for P A L and 4 C L in the unpigmented corolla tube of tobacco flowers may be to synthesize yet uncharacterized phenylpropanoid-products such as colorless flavonoids (see section 1.2.2). Currently, there is no evidence of this, nonetheless, it is a formal possiblity that should be considered and investigated. In summary, the expression pattern of At4CL and Nt4CL, as demonstrated by northern analysis, is consistent with the expression of other phenylpropanoid genes and with the expression as described by promoter- GUS studies. A striking difference between the endogenous tobacco-4CL expression-pattern as compared with GUS reporter-gene analysis is the high levels of 4CL transcripts in the unpigmented portion of the petal. We have shown 4CL to be encoded by a single gene in Arabidopsis by divergent gene-families in tobacco. We predict that the Arabidopsis  and  4CL gene  would be responsive to all developmental and environmental signals which direct 4CL expression. Potentially, the divergent 4CL genes from tobacco may respond uniquely to developmental and environmental signals; however, we have not shown the tobacco 4CL genes to be differentially regulated at the level of R N A accumulation. The divergence of the tobacco 4CL genes may confer different enzymatic properties to the encoded 4CL-isoforms. The possiblity of divergent 4CL-isoforms regulating 74  c a r b o n f l o w into p h e n y l p r o p a n o i d  m e t a b o l i s m at t h e l e v e l of s u b s t r a t e s p e c i f i c i t y  e x a m i n e d in t h e f o l l o w i n g c h a p t e r u s i n g r e c o m b i n a n t t o b a c c o - 4 C L  is  proteins.  Figure 3.11: Diagram showing the relatedness of the orders in the angiosperms. Of particular interest are the orders Polemoniales (tobacco and potato), Lamiales (Lithospermum), Umbellales (parsley), Fabales (soybean), Papaverales (Arabidopsis), and Poales (rice). Taxa are: 1, Nymphaeales; 2, Sarraceniales; 3, Aristolochiales; 4, Trochodendrales; 5, Cercidiphyllales; 6, Didymeleales; 7, Eupteleales; 8, Eucommiales; 9, Casuarinales; 10, Leitneriales; 11, Juglandales; 12, Batales; 13, Plumbaginales; 14, Lecythidales; 15, Salicales; 16, Diapensiales; 17, Podostemales; 18, Haloragales; 19, Cornales; 20, Rafflesiales; and 21, Rhanmales. From Heywood, 1978.  75  Chapter 4 Recombinant 4CL 4.1 Expression of Tobacco-4C/_ cDNAs in E.  coli.  4.2 Enzymatic Characterization of 4 C L 4.3 Modification of Recombinant-4CL Activity 4.4 Discussion  4.1 Expression of Tobacco-4CL cDNAs in E. coli. Nt4CL-1 and Nt4CL-19, two cDNAs encoding tobacco-4CL proteins, were - 8 0 % identical at the nucleotide and predicted amino acid sequence levels. To determine if the encoded enzymes had different biochemical characteristics, recombinant 4CL1 and 4 C L 2 were produced in Escherichia  coll.  The cDNA-inserts  were cloned into p Q E expression-plasmids, designated pQE-1 and pQE-19 for Nt4CL1 and Nt4CL-19 respectively, and transformed into E.  coli.  pQE-1 and pQE-19 each  contain an engineered translational start codon, a histidine-tag and a multicloning site such that the recombinant proteins have 13 ( M R G S H H H H H H G I R ) and 14 ( M R G S H H H H H H G S A C ) additional amino acids prior to the initiation methionine encoded by the cDNA-inserts. pQE-19-transformants expressed 4 C L protein efficiently and a protein-band corresponding to the recombinant 4 C L 2 was detected by Coomassie Blue staining on a SDS-polyacrylamide gel after 2 hours of IPTGinduction. pQE-1-transformants expressed 4 C L protein less efficiently and a Coomassie Blue stained protein-band corresponding to the recombinant 4CL1 was visible only after 4 hours of IPTG-induction (results not shown). In addition, the bacterial strain harboring pQE-19 was grown in L B media whereas the bacterial strain 76  containing pQE-1 was grown in Super Media, a more nutritious medium recommended for generating higher levels of recombinant proteins (QIAexpressionist™ Kit, QiaGen Inc., Chatsworth, CA). After 4 hours of IPTG-induction, pQE-19-generated protein (4CL2) was present in both cytosolic fractions and membrane bound fractions whereas pQE-1 -generated protein (4CL1) was localized in the cytosol (results not shown). The recombinant proteins were approximately 60 kDa in size, reacted with the antibody raised against parsley-4CL, and migrated to the same location in an SDS-polyacrylamide gel as the 4 C L protein(s) found in tobaccostem extracts (Figure 4.1). No 4 C L protein (Figure 4.1, lane 1) or 4 C L activity (not shown) was detected in the bacterial strain containing the pQE-30 plasmid lacking a  4CL cDNA insert.  1  2  3  4  k D a  [-111  - 74 - 45.5  - 29.5  Figure 4.1: Western blot analysis of recombinant tobacco-4CL expressed in E. coli. Crude, bacterial protein-extracts (5 ug) were separated by SDS-PAGE, blotted onto a nylon membrane, and the blot reacted with an antibody raised against parsley-4CL. Lane 1, extract from bacteria harboring the expression-plasmid pQE-30 without an insert; lane 2, extract from bacteria harboring pQE-1, expressing Nt4CL-1-encoded protein (4CL1); lane 3 extract from bacteria harboring pQE-19, expressing Nt4CL-19-encoded protein (4CL2); lane 4, extract of total protein (25 ug) from tobacco old-stem. Molecular mass standards (in kDa) are shown on the right.  4.2 Enzymatic Characterization of 4CL The recombinant proteins present in crude bacterial-extracts expressing pQE-1 and pQE-19 were tested for their relative abilities to utilize differently-substituted hydroxycinnamic acids as substrates. Initially, enzyme activities of recombinant 4CL1 and recombinant 4 C L 2 were determined using 0.025 mM to 0.5 m M cinnamic acid, 4coumaric acid, caffeic acid, ferulic acid, and sinapic acid as substrates (Figures 4.2 and 4.3). Double-reciprocal transformation of the data (Lineweaver-Burke plot; Table 4.1) was used to estimate the Michealis-Menten constant ( K ) and the maximum 77 m  velocity of the reaction ( V  m a x  ) . The recombinant proteins did not react with sinapate  and exhibited enzyme kinetics comparable to one another when cinnamate, 4coumarate, caffeate, and ferulate were used as substrates (Figure 4.3). Both recombinant proteins experienced substrate-inhibition at concentrations above 0.2 mM of 4-coumarate, caffeate, or ferulate and these data points were not used in K and V  m a x  determination. The K  m  and V  m a x  m  values of recombinant 4 C L 2 towards  caffeate could not be estimated with reasonable accuracy from the data obtained. In parallel, crude extracts prepared from tobacco-stems were assayed for 4 C L activity and found to have an approximate K for 4-coumarate (Figure 4.4). The K coumarate is comparable to the K  m  m  m  and V  m a  x of 0.0095 mM and 0.45 mmol s  _ 1  kg  - 1  of endogenous tobacco 4 C L towards 4-  of recombinant 4CL1 (0.017 mM) and 4 C L 2  (0.0087 mM) towards 4-coumarate (Table 4.1). 4 C L enzyme activity from crude tobacco-stem extracts was also inhibited by concentrations of 4-coumarate above 0.2 mM (Figure 4.4, arrow). K  m  and V  m a x  values for other substrates were not determined  because the 4 C L enzyme activity in crude tobacco-stem extracts was low, especially when low concentrations of substrate were used, and approached the limit of detection. Since preliminary data (above) suggests that the enzymatic properties of the recombinant proteins appear relatively similar to one another, the substrate specificities of these enzymes were characterized more thoroughly at one concentration of substrate. A substrate concentration of 0.2 mM was chosen since at this concentration, there was little or no apparent substrate-inhibition of the recombinant 4 C L proteins and of the endogenous tobacco-stem 4 C L proteins (Figures 4.3 and 4.4) and, with the exception of cinnamate, this concentration represents nearsaturating levels of substrate (Figure 4.3). Crude extracts prepared from tobacco stems had relative enzyme-activities of 1 0 0 % , 1 7 % , and 6 0 % when 4-coumarate, caffeate, and ferulate (respectively) were used as substrates; no detectable activity towards cinnamate as a substrate; and a small amount of activity (4%) towards 78  sinapate as a substrate. Recombinant 4CL1 had relative 4CL-activities of 1 0 0 % , 2 1 % , 1 7 % , and 7 3 % when 4-coumarate, cinnamate, caffeate, and ferulate were used as substrates (respectively), while extracts of recombinant 4 C L 2 had relative activities of 1 0 0 % , 2 9 % , 2 5 % , and 6 2 % (Figures 4.2 and 4.5). Taken together, these results show that the two recombinant-4CL proteins have nearly identical substrate specificities, and that these are comparable to those of the 4 C L from tobacco-stem extracts. However, the lack of activity when sinapate was used as a substrate and relatively high activity when cinnamate was used as a substrate (cinnamate is not generally regarded as a cellular target for the 4 C L enzyme) distinguished the recombinant enzymes from the native 4CL-protein(s) in stem extracts. Subsequent experiments focused on recombinant 4 C L 2 since the two recombinant proteins appeared to be relatively similar to one another and 4 C L expression from pQE-19 was more efficient than expression from pQE-1 (section 4.1). The ability of chemical analogs (Figure 4.2) and down-stream phenylpropanoidproducts to inhibit recombinant-4CL2 activity was analyzed. When 4-coumarate was used as a substrate, recombinant-4CL2 activity was strongly inhibited by 0.2 mM sinapic acid and 3,4-methylenedioxycinnamic acid and weakly inhibited by the same concentration of 5-hydroxyferulic acid. Coniferin (coniferyl alcohol glucoside), derived from feruloyhCoA, did not inhibit recombinant-4CL2 activity whereas 1 mM naringenin, a flavanone derived from 4-coumaroyl:CoA, did (Figure 4.6). Similar results were observed with crude 4CL-preparations from tobacco stems (not shown).  79  COOH  A  COOH  B  COOH  COOH  C  COOH  D  COOH  E  COOH  F  G  Figure 4.2: Chemical analogs used as substrates and inhibitors of 4CL activity. The chemical structures of cinnamic acid derivatives used to characterize 4CL is listed: cinnamic acid (A); 4-coumaric acid (B); caffeic acid (C); ferulic acid (D); 5-hydroxyferulic acid (E); sinapic acid (F); and 3,4-methylenedioxycinnamic acid. Table 4.1: Estimated K bacterial-extracts.  m  and V  m a  x values for recombinant 4CL1 and recombinant 4CL2 from crude  Recombinant 4 C L 2  Recombinant 4CL1  Substrate  Vmax Km(mM) (mmol s" kg" )  Cinnamic acid 4-Coumaric acid Caffeic acid Ferulic acid  0.70 0.017 0.38 0.044  1  1  21 25 13 22  Vmax m * /K  30(1) 1500 (50) 34(1.1) 500 (17)  * relative ratios are presented in parenthesis.  80  Vmax Km(mM) (mmol s" kg' ) 1  Vmax/Km *  1  0.27 0.0087  17 26  63(1) 3000 (48)  0.017  17  1000 (16)  -  -  -  Recombinant 4CL2  Recombinant 4CL1  0.1  0.2  0.3  0.4  0.1  0.5  0.1  B  0.2  0.3  0.4  0.5  [4-Coumaric Acid] (mM) „  0.2  0.3  0.4  0.5  0.6  [Cinnamic Acid] (mM)  [Cinnamic Acid] (mM)  0.1  0.6  0.2  0.3  0.4  0.5  I  I  [4-Coumaric Acid] (mM)  5  U> 7  I  0.(  -  0.1  0.2  0.3  I  '  0.4  0.5  I  I  [Caffeic Acid] (mM)  16  _ i  2/ o .o >  0.6  0.1  I 0.2  I 0.3  i 0.4  i 0.5  [Caffeic Acid] (mM)  0.6  -  W 12 O  E  o >  0  I 0.1  I 0.2  I 0.3  I 0.4  I 0.5  [Ferulic Acid] (mM)  0.6  H  [Ferulic Acid] (mM)  Figure 4.3: Recombinant-4CL1 and recombinant-4CL2 activities as a function of substrate concentration. Recombinant 4CL1 (A-D) and recombinant 4CL2 (E-H) were assayed for enzyme activity using varying concentrations (0.025 mM to 0.5 mM) of cinnamic acid (A, E), 4-coumaric acid (B, F), caffeic acid (C, G), and ferulic acid (D, H). Enzyme activity was measured at room temperature spectrophotometrically. Arrows indicate data points which were not used for K and V x determination. m  81  m a  Figure 4.4: 4CL enzyme activity from crude tobaccostem extracts plotted as a function of substrate concentration. Crude tobacco-stem extracts were assayed for enzyme activity using varying concentrations (0.025 mM to 0.5 mM) of 4-coumaric acid. Enzyme activity was measured at room temperature spectrophotometrically. Arrow indicate the data point which was not used for K and V x determination. m  0.1 0.2 0.3 0.4 0.5 [4-Coumaric Acid] (mM)  0.6  m a  Figure 4.5: Substrate specificities of tobacco-4CL and recombinant-4CL proteins. 4CL enzyme activity was measured from crude tobacco-stem extracts (tobacco), crude bacterial-extracts expressing Nt4CL-1 (pQE-1) and crude bacterial-extracts expressing Nt4CL-19 (pQE-19). 4CL activity is expressed as a percentage of the activity of the preparation using 4-coumarate as a substrate. One hundred percent activity  120  represents 0.33 mmol s" kg" (tobacco), 1  23 mmol s" kg" (pQE-1) and 25 mmol s" kg" (pQE-19). Results are averaged from three determinations using 0.2 mM of the hydroxycinnamate substrates. Error bars represent standard deviation. 1  1  o o  120 100 80  o  <  ill  60  •  40 20 0 C  o  •g o < o  "D O <  o  CL CO  c CO  ~5  S i | CO  o  1  u.  k—  c <u  c  H— 'c o O  CO  o  -a  82  1  1  Figure 4.6: Inhibition of recombinant4CL2 activity by phenylpropanoid metabolites. Recombinant-bacterial extracts expressing Nt4CL-19 (pQE-19) were assayed for 4CL enzyme activity using 0.2 mM 4-coumarate as substrate and the presence of 0.2 mM sinapic acid, 0.2 mM 3,4-methylenedioxycinnamic acid, 0.2 mM hydroxyferulic acid, 1 mM coniferin, or 1 mM naringenin. Activity is expressed as a percentage of the 4CL activity in the absence of added inhibitors. Results are averaged from three determinations; error bars represent standard deviations.  4.3  Modification of Recombinant-4CL Activity Comparisons between recombinant 4 C L 1 , recombinant 4 C L 2 and 4 C L from  crude tobacco-stem extracts suggested that the recombinant proteins have enzymatic characteristics like those of native tobacco-4CL. However, a striking difference between the recombinant-4CL proteins and endogenous tobacco-4CL is the ability of the recombinant proteins to use cinnamate as a substrate (Figure 4.5). We considered this phenomena particularly enigmatic and considered possible explanations for this discrepancy. Conceivably, the bacterial extracts which were used to assay recombinant-4CL activity could contain an activity capable of modifying cinnamate (e.g. by hydroxylation), leading to an apparent activity towards this substrate. This was tested by assaying 4 C L activity, using cinnamate as a substrate, in tobacco extracts to which crude extracts of the bacterial strain harboring the empty expression-plasmid (pQE-30) had been added. No 4 C L activity towards cinnamate was detectable, indicating that the bacterial extract itself was not the cause of the apparent 4 C L activity towards cinnamate (results not shown). We next considered the possibility that recombinant-4CL protein has bona fide activity toward cinnamate as a substrate, but that the native tobacco-protein is posttranslationally modified, or interacts with other proteins, repressing its ability to use cinnamate as a substrate in planta. To test this, crude tobacco-stem extracts were incubated together with crude bacterial-extracts containing recombinant 4 C L 2 and the mixture assayed for activity towards cinnamate as a substrate. Figure 4.7 shows that the ability of recombinant 4 C L 2 to use cinnamate as a substrate decreased exponentially in the presence of increasing amounts of tobacco-stem extract, consistent with the hypothesis that such extracts indeed contain a component capable of modifying the substrate specificity of recombinant 4CL. Furthermore, this apparent modification was specific to the activity towards cinnamate as a substrate. The ability of recombinant-4CL2 to use 4-coumarate, caffeate or ferulate as substrates was unchanged (Figure 4.8). Boiled tobacco-stem extract was no longer capable of 83  modifying the activity of recombinant 4CL2 (Figure 4.8). In contrast, desalting the tobacco-stem extract by chromatography through a G-50 Sephadex column did not abolish its ability to modify recombinant-4CL2 activity towards cinnamate as a substrate (Figure 4.8). Neither plant-extraction buffer alone (Figure 4.8) nor B S A (not shown) decreased the ability of recombinant-4CL2 to use cinnamate as a substrate. While these experiments were performed using pQE-19 extracts containing recombinant-4CL2 protein, a similar effect of tobacco-stem extracts on recombinant 4CL1 in extracts of the pQE-1-expressing E.  coli  strain was also observed (results not  shown). Taken together, these results suggest that tobacco-stem extracts contain a large heat-labile component, possibly a protein which inhibits the ability of recombinant-4CL to use cinnamate, thereby modifying its substrate specificity. The distribution of the 4CL-modifying component was determined. Preliminary results (Figure 4.9) show that crude tobacco-extracts from old leaves, wounded leaves, methyl jasmonate treated leaves, and shoot tips were also able to decrease the ability of recombinant-4CL2 to use cinnamate as a substrate. This decrease in activity towards cinnamate was not observed when equivalent levels of B S A was added to the reaction mixture (not shown). In general, it appeared that less than 50 u.g of tobaccostem protein was required to decrease the recombinant-4CL2 activity (towards cinnamate as a substrate) by 5 0 % whereas in excess of 200 u,g of protein from the other tobacco-extracts was required for 5 0 % inhibition (Figure 4.9). 4 C L in these tobacco extracts did not use cinnamate but did use 4-coumarate as a substrate. 4 C L activity was highest in tobacco stems and significantly lower in shoot tips, wounded leaves, methyl jasmonate treated leaves, and old leaves (Figure 4.10). Although these experiments were performed once and the results should be considered as preliminary data, there appears to be a good (but not absolute) correlation between the concentration of the modifying-component and the levels of endogenous tobacco4 C L activity.  84  The identity of the modifying-component and the modification it performs is unknown; however, since the 4 C L enzyme-assay mix includes A T P , it was postulated that the modification event may involve phosphorylation. To test this, recombinant 4 C L 2 , tobacco-stem extracts, and alkaline phosphatase were incubated together prior to assaying for 4 C L activity towards cinnamate as a substrate (Figure 4.11). In the presence of alkaline phosphatase, the inhibition of recombinant-4CL2 activity towards cinnamate by tobacco-stem extract was partially alleviated (Figure 4.11, x x x x). This suggests that the modifying-component in tobacco functions through phosphorylation since the presence of a phosphatase reduces its affect. The complementary experiment using NaF, a phosphatase inhibitor, showed that the inhibition of recombinant-4CL2 activity towards cinnamate by tobacco-stem extracts was intensified with added NaF (Figure 4.11,  ). The affect of NaF was specific since  an equivalent amount of NaCl did not elicit the same response (not shown) and control assays, performed in the presence of NaF and alkaline phosphatase, demonstrate that the concentration of NaF used was effective in blocking exogenously-applied alkaline phosphatase (Figure 4.11, • • • •). Although the affect of NaF was small, it was reproducible and suggests that, under the physiological state from which the stems were harvested, endogenous tobacco-phosphatases are a minor component of the proposed 4 C L phosphorylation-dephosphorylation regulatory-system. Under normal conditions, 4 C L from tobacco-stem extracts do not use cinnamate as a substrate (Figures 4.5, 4.10 and 4.11); however, preliminary results show that in the presence of 20 units of alkaline phosphatase, 4 C L from crude tobacco-stem preparations displayed a slight activity towards cinnamate as a substrate (not shown). These results suggests that tobacco extracts contain an activity, possibly a kinase, which regulates 4 C L substrate-specificity through phosphorylation and that this regulation occurs  in vivo  with the endogenous tobacco-4CL as well as  recombinant 4 C L proteins.  85  in vitro  with the  Computer analysis of the encoded Nt4CL-1 and Nt4CL-19 peptide-sequences predicted numerous potential sites of phosphorylation ([S/T]-X-[R/K], [S/T]-X -[D/E], 2  [R/K]-X -3)-[D/E]-X( . -Y; Patschinsky etal., 1982; Hunter, 1982; Woodgett etal., 2 3)  (2  1986). Eleven putative serine-threonine phosphorylation-sites and one tyrosine kinase phosphorylation-site were found in both Nt4CL-1 and Nt4CL-19 predicted amino acidsequences (Figure 4.12, underlined). Furthermore, two putative serine-threonine phosphorylation sites were conserved in all predicted 4CL-sequences analyzed in Table 3.1 (Figure 4.12, +++). To determine if the recombinant-4CL proteins were directly phosphorylated by a putative kinase found in tobacco extracts, radiolabelling experiments using  3 2  P y-ATP were performed. Crude bacterial-extracts expressing  pQE-19 were incubated with tobacco-stem extracts in the presence of  3 2  P y-ATP and  then chromatographed through a nickel column to enrich for the recombinant 4 C L by virtue of the engineered histidine-residues at the amino terminus (see section 4.1). The eluant was electrophoresed on an SDS-polyacrylamide gel and then exposed to autoradiographic film. Results showed three radioactive signals, one of which was approximately 60 kDa in size; however, the intensity of the 60 kDa signal was conspicuously weak (results not shown) and since the nickel column did not purify the recombinant 4 C L to homogeneity, we cannot yet conclude that 4 C L is directly phosphorylated.  20 0  0  10  20  30  40  50  Tobacco Stem Protein (ng)  86  Figure 4.7: Effect of tobacco-stem extracts on recombinant-4CL2 activity towards cinnamate as a substrate. Recombinant bacterial-extracts expressing Nt4CL-19 (pQE-19) were assayed for 4CL enzyme activity using 0.2 mM cinnamate as substrate in the presence of different amounts of a tobacco-stem extract. Activity is expressed as a percentage of the 4CL activity towards cinnamate as a substrate in the absence of added tobacco extract. Results are averaged from three determinations; error bars represent standard deviations.  120  -  100  -  CAF  cou  CIN  JL  •  80 -  Figure 4.8: Characterization of the 4CLmodifying activity in tobacco-stem extracts. Recombinant 4CL2 (pQE-19) was assayed for 4CL activity towards cinnamate (CIN), 4coumarate (COU), caffeate (CAF), or ferulate (FER) as substrates in the absence (- Extract) or presence (+ Extract) of 25 ug tobacco-stem extract. The tobacco-stem extracts used in the assays with cinnamate as a substrate were either untreated (CIN + Extract), heated at 100°C for 15 minutes (CIN + Boiled Extract), or passed through a Sephadex G-50 column (CIN + Desalted Extract) before being added to the enzyme assays. Activity is expressed as a percentage of the 4CL activity towards each hydroxycinnamic acid in the absence of added tobacco extract. Results are averaged from three determinations; error bars represent standard deviations.  FER  60 40 20  "  O  £  <D  nJ i t  S  O  O  ffl  «I  S mS  CO  5  LU LU  LU LU ~[ LU LU  Z'  2  +ZZ ffl" D  o jj o  ~° <D CO  §3  o + D  Z  CO  u  +  O  •G T3 CO CO  LU LU  ' + Irf u-  T5 B CO CO  •K *  LU LU  • +  CC fx  O  o z o 125  100  200  300  400  500  600  Plant Protein (jj.g)  Figure 4.9: Distribution of the 4CLmodifying activity in tobacco organs. Recombinant-bacterial extracts expressing Nt4CL-19 were assayed for 4CL enzyme activity using 0.2 mM cinnamate as substrate in the presence of different amounts of tobacco-stem extract (-O-O-), tobacco shoot-tip extract (-•-•-), wounded tobacco-leaf extract (-A-A-), old tobacco-leaf extract (-•-•-), or methyl jasmonate treated tobacco-leaf extract (-XX-). Activity is expressed as a percentage of the 4CL activity towards cinnamate as a substrate in the absence of added tobacco extract. Figure 4.10: Distribution of endogenous tobacco-4CL activity. Crude tobacco-extracts from tobacco stems, tobacco shoot-tips, wounded tobaccoleaves, old tobacco-leaves, or methyl jasmonate treated tobacco-leaves (MJ) were assayed for 4CL enzyme activity using 4-coumarate (COU) or cinnamate (CIN) as substrates.  87  *  o E c < o o o E  .1 O CO  Figure 4.11: Affect of alkaline phosphatase and NaF on the 4CL-modifying activity from tobaccostem extracts. Recombinant-bacterial extracts expressing Nt4CL-19 (pQE-19) were assayed for 4CL enzyme activity using 0.2 mM cinnamate as substrate (A A A). Recombinant-4CL activity was assayed in the presence of 25 ug of tobacco-stem extract (• • •); in the presence of 25 ug of tobaccostem extract and 20 units of calf intestinal alkaline phosphatase (X X X); in the presence of 25 ug of  4  2 ,A  1  X  X  >  <  tobacco-stem extract and 50 mM NaF ( ); or in the presence of 25 ug of tobacco-stem extract, 20 units of calf intestinal alkaline phosphatase and 50 mM ooo6ooooo6ooooo&ooooo&oooQOg> NaF (••••). Open circles (ooo) represent 4CL 2 3 enzyme activity of 25 ug tobacco-stem protein Time (minutes) toward cinnamate as a substrate. The data represent the results of a typical experiment. v  X  Nt4CL- 1 Nt4CL- 19  MPMETTTETKQSGDLIFRSKLPDIYIPKHLPLHSYCFENISEFSSRPCLI MEKDTKQ-VDIIFRSKLPDIYIPNHLPLHSYCFENISEFSSRPCLI  50 45  Nt4CL- 1 Nt4CL- 19  NGANDQIYTYAEVELTCRKVAVGLNKLGIQQKDTIMILLPNSPEFVFAFM NGANKQIYTYADVELNSRKVAAGLHKQGIQPKDTIMILLPNSPEFVFAFI  100 95  Nt4CL- 1 Nt4CL- 19  GASYLGAISTMANPLFTPAEVVKQAKASSAKIIITQSCFVGKVKDYASEN GASYLGAISTMANPLFTPAEWKOAKASSAKIIVTQACHVNKVKDYAF EN  150 145  Nt4CL- 1 Nt4CL- 19  DVKVICIDSAPEGCLHFSELTQSDEHEIPEVKIQPDDWALPYSSGTTGL DVKIICIDSAPEGCLHF S VLTQANEHDIPEVEIQ PDDWAL PYSSGTTGL  2 00 205  Nt4CL- 1 Nt4CL- 19  PKGVMLTOKGLVTSVAQQVDGENANLYMHSEDVLMCVLPLFHIYSLNSIL 250 PKGVMLTHKGLVT S VAQQVDGENPNL YIH S EDVMLC VL PLFHIYS LNS VL 245  Nt4CL- 1 Nt4CL- 19  LCGLRVGAAILIMQKFDIAPFLELIQKYKVSIGPFVPPIVLAIAKSPIVD LCGLRVGAAILIMQKFDIVSFLELIQRYKVTIGPFVPPIVLAIAKSPMVD  3 00 295  Nt4CL- 1 Nt4CL- 19  SYDLSSVRTVMSGAAPLGKELEDAVRTKFPNAKLGQGYGMTEAGPVLAMC DYDLSSVRTVMSGAAPLGKELEDTVRAKFPNAKLGQGYGMTEAGPVLAMC  350 345  Nt4CL- 1 Nt4CL- 19  LAFAKEPFDIKSGACGTWRNAEMKIVDPDTGCSLPRNQPGEICIRGDQI LAFAKEPFEIKSGACGTWRNAEMKIVDPKTGNSLPRNQSGEICIRGDQI  400 395  Nt4CL- 1 Nt4CL- 19  MKGYLNDPEATTRTIDKEGWLHTGDIGFIDEDDELFIVDRLKELIKYKGF MKGYLNDPEATARTIDKEGWLYTGDIGYIDDDDELFIVDRLKELIKYKGF  450 445  Nt4CL- 1 Nt4CL- 19  QVAPAEIEALLLNHPNISDAAWPMKDEOAGEVPVAFWRSNGSAITEDE QVAPAELEALLLNHPNISDAAWPMKDEQAGEVPVAFWRSNGSTITEDE  500 495  Nt4CL- 1 Nt4CL- 19  VKDFISKQVIFYKRVKRVFFVETVPKS PSGKILRKDLRARLAAGVPN * VKDFISKQVIFYKRIKRVFFVDAIPKSPSGKILRKDLRAKLAAGLPN*  547 542  +++.  +++  .  .  .  .  .  .  .  .  .  Figure 4.12: Predicted phosphorylation-sites of polypeptides encoded by Nt4CL-1 and Nt4CL-19. Computer analysis of the predicted amino acid sequence from Nt4CL-1 and Nt4CL-19 revealed numerous putative phosphorylation-sites, some of which are found in both Nt4CL-1 and Nt4CL-19 (underlined) and two of which (+++) are found in all the 4CL sequences listed in Table 3.1. Dashes indicate gaps introduced to maximize alignment, translational stops are indicated with an asterisk, and numbers on the far right indicate the predicted amino acid position.  88  Discussion  4.4  The expression of two divergent tobacco-4CL cDNAs, Nt4CL-1 and Nt4CL-19, in  E. coli  and V  m a  allowed us to examine the properties of recombinant-4CL proteins. The K  m  x values of recombinant 4CL1 and recombinant 4 C L 2 were estimated;  however, these kinetic characteristics should be considered as preliminary data since the experiments were performed only once and were performed with unpurified enzymes. Given these limitations, the K  m  of recombinant-4CL activity towards 4-  coumarate (-10 u.M to -20 pJvl) is within the range of that reported for the 4 C L enzymes from  Brassica  (14 uivl; Rhodes era/., 1973), soybean (17 |xM; Knobloch and  Hahlbrock, 1975), parsley (14 u.M; Knobloch and Hahlbrock, 1977), spruce (11 jiM; Luderitz  etal.,  1982), and poplar (10 uivl for4CLII and 13 uivl for 4CLIII; Grand  1983). However, the K  m  etal.,  values towards other cinnamic acid derivatives were not  directly comparable between the recombinant-4CL proteins and the 4CL activities from other plants. The endogenous concentration of cinnamate in bean cell cultures is between 4 u.M to 25 uM (Mavandad et al., 1990) and is much lower than the estimated K  m  of the recombinant 4CL proteins towards cinnamate. However, uptake 14  experiments using  C-cinnamic acid showed that cinnamate concentrations after  elicitation can be as high as 550 |iM (Mavandad This suggest that although the K  m  etal.,  1990; Edwards  etal.,  1990).  values of the recombinant 4CL towards cinnamate  is high, it is not impossible that endogenous cinnamate concentratrions reach these levels. Authors also suggest that the presence of specific metabolic pools and interactions between cinnamate and proteins may cause high, localized, concentrations of cinnamate (Mavandad et al., 1990). The relative substrate-specificities as determined by the V  m a  x/K  m  ratios (Table  4.1, values in parenthesis) demostrate the almost identical substrate-specificities of recombinant 4CL1 and recombinant 4CL2. The relative substrate-specificity as measured by the percent enzyme activity using 0.2 mM cinnamic acid derivatives (Figure 4.5) also demonstrates the similarity in enzyme activites of 4CL1 and 4 C L 2 . In 89  general, the activities of the recombinant-4CL proteins were most similar to the activities of the parsley-4CL proteins which had maximal activity towards 4-coumarate, followed by ferulate, caffeate, and cinnamate as substrates (Lozoya et al., 1988). By using a series of chemical analogs as substrates and inhibitors (Figure 4.2), the nature of the substrate binding site of recombinant 4 C L 2 was investigated. 4 C L activity was higher towards 4-coumarate as compared to cinnamate as substrates, suggesting that the hydroxyl group on position 4 of the aromatic ring may be important for electrostatic interactions between the substrate and enzyme. In contrast, a hydroxyl group on position 3 of the aromatic ring seemed to decrease the activity of recombinant 4 C L towards caffeate as a substrate; however, methylation of the 3hydroxyl group caused ferulate to be more efficiently metabolized. This suggests that the substrate binding site of 4 C L may contain a hydrophobic pocket such that nonpolar groups at the number-3 position are better tolerated. Since the chemical group modified by 4 C L is the carboxylic acid group on the propane chain distal to the aromatic ring, it is likely that the hydroxylation and omethylation modifications at the aromatic ring affects substrate binding and the observed differences in K  m  values is in  support of this (Table 4.1). However, it should be noted that the carboxylic acid group is apart of the conjugated double-bond system so that its chemical reactivity may be affected by ring modifications. One might predict that sinapic acid, 3,4methylenedioxycinnamic acid, and 5-hydroxyferulic acid may act as competitive inhibitors that bind to 4 C L and prevent catalysis of 4-coumarate (Figure 4.6). However, preliminary kinetic-studies using sinapate as an inhibitor and 4-coumarate as a substrate showed that the mechanism of sinapate inhibition was quite complex. Increasing the 4-coumarate concentration did not alleviate the inhibition caused by sinapate and the K  m  and V m a x of recombinant 4 C L 2 towards 4-coumarate as a  substrate were both altered by the presence of sinapate (results not shown). It has been hypothesized that, in some plants, different forms of 4 C L may play a role in controlling the biosynthesis of different phenylpropanoids by their substrate 90  specificities (Knobloch and Hahlbrock, 1975; Grand  etal.,  1983). The results reported  here suggest that the recombinant proteins do not have different enzymatic properties and, in fact have kinetic properties very similar to one another (Table 4.1, Figures 4.3 and 4.5). Thus, it is unlikely that the proteins encoded by the Nt4CL-1 and Nt4CL-19 c D N A s have a role in modulating the types of phenylpropanoid-products made in tobacco. This conclusion is consistent with the expression patterns of Nt4CL-1 and Nt4CL-19 which show no differential expression of the two genes at the level of m R N A accumulation (Figure 3.9). 4 C L is encoded by a divergent gene-family in soybean (Uhlmann and Ebel, 1993) and two isoforms of 4 C L with different substrate specificities have been identified in soybean (Knobolch and Hahlbrock, 1975). The soybean-4CL c D N A s were partial-length so that the enzymatic activity of the encoded proteins were not tested. In poplar, three 4CL-isoenzymes with distinct enzyme activities have been found and two significantly different 4CL c D N A s have been cloned (Allina and Douglas, 1994). Preliminary evidence suggests that the poplar-  4CL c D N A s encode enzymes with indistinguishable activities (S. Allina and C. Douglas, unpublished). Two parsley  4CL  c D N A s were expressed in E.  coli  and the  recombinant proteins had enzymatic properties very similar to one another. This however, was not unexpected since the predicted amino acids sequences were 9 9 . 5 % identical. Thus, despite a number of reports in which 4 C L forms exhibiting distinct substrate specificities were partially purified (Grand 1977; Ranjeva  etal.,  etal.,  1983; Wallis and Rhodes,  1976; Knobloch and Hahlbrock, 1975), there is yet to be a  documented example of recombinant-4CL isoforms having different substrate specificities. Preliminary results show that the recombinant-4CL activities were inhibited by substrate concentrations above 0.2 mM (Figure 4.3). Substrate inhibition of 4 C L by 4coumarate was also reported in soybean and parsley cell cultures (Knobloch and Hahlbrock, 1975; 1977) whereas the 4 C L purified from loblolly pine xylem was not inhibited by cinnamate, coumarate, ferulate, caffeate or sinapate (Voo et al., 1995). 91  Inhibition of 4 C L by naringenin has been reported here with recombinant 4 C L 2 (Figure 4.6) as well as in the 4 C L proteins from loblolly pine and petunia (Voo et al., 1995; Ranjeva era/., 1976). Naringenin, made from 4-coumaroyl:CoA, is one of the first compounds produced in the flavonoid biosynthetic pathway and, as suggested by Voo et al. (1995) and Ranjeva et al. (1976), its ability to inhibit 4 C L activity towards 4coumarate as a substrate may represent regulation by feedback-inhibition. Similiar to the 4 C L in  Vanilla  planifolia  (Funk and Brodeolius, 1990), recombinant 4 C L 2 and  endogenous tobacco-stem 4 C L were inhibited by 3,4-methylenedioxycinnamic acid. Recombinant-4CL activity towards 4-coumarate as a substrate was also inhibited by sinapate. Thus not only does recombinant 4 C L 2 not use sinapate as a substrate, sinapate also serves as an inhibitor. Recently, it has been shown that tobacco stems contain syringyl lignin (Halpin era/., 1994). How is this type of lignin made when the two recombinant-4CL proteins examined here do not use sinapate as a substrate? One possibility is that a third form of 4 C L exists in tobacco and this form is capable of converting sinapate into sinapoyhCoA for syringyl lignin biosynthesis. If this is true, then the ability of sinapate to inhibit recombinant 4 C L 2 suggests that sinapate may be a key metabolite in the fine-tuning of 4 C L activity in tobacco. For example, during developmental stages where sinapyl alcohol is in high demand, the actions of hydroxylases and omethyltransferases may generate relatively high levels of sinapate which in turn down-regulate the 4 C L isoforms which do not use sinapate as a substrate. A s the sinapyl alcohol demand is supplied, the metabolic-pool of sinapate would decrease and susceptible forms of 4 C L would be released from sinapate inhibition. Currently there is no evidence to support this hypothesis; however, in addition to the 4CL-cDNAs cloned here, there appears to be another class of 4CL genes in tobacco (Figure 3.8 and Genebank sequences) and crude tobacco-stem extracts do exhibit low 4 C L activity towards sinapate. This data suggest that alternate forms of 4CL genes and 4 C L activities exist in tobacco but whether these forms are the ones which metabolize sinapate will be the topic of future research. 92  The inability of crude tobacco-stem extracts to utilize cinnamate was unexpected since recombinant-4CL proteins used cinnamate as a substrate (Figure 4.5) and northern analysis demonstrated high levels of Nt4CL-1 and Nt4CL-19 transcripts in this organ (Figure 3.9). One formal possibility is that this unique property of the recombinant proteins is due to the presence of the additional amino acids at the amino terminus of the recombinant proteins, a consequence of the bacterial system we used to express Nt4CL-1 and Nt4CL-19 c D N A s (see section 4.1). However, we considered this possibilty unlikely since several kinetic properties of the recombinant proteins were similar to those of endogenous 4 C L from tobacco-stem extracts (see section 4.2). Also, unmodified recombinant poplar-4CL protein produced in an eukaryotic (baculovirus) expression-system also used cinnamate as a substrate, an activity not found in poplar extracts (A. Pri-Hadash, S. Allina, B. Ellis, and C. Douglas, unpublished). Results shown in Figures 4.7, 4.8 and 4.9 suggest that the discrepancy between the recombinant-4CL and endogenous-4CL in their abilites to use cinnamate as a substrate may be attributed to the presence of a 4CL-modifying factor(s) present in tobacco extracts. The factor(s) in tobacco-stem extracts specifically affects the ability of recombinant-4CL to use cinnamate as a substrate, reducing it by five to ten-fold (Figure 4.7), but has no effect on activity towards other substrates (Figure 4.8). Thus, we hypothesize that the  in vivo  enzymatic properties of 4CL1 and 4 C L 2 in tobacco are  partially determined post-translationally by interaction with factor(s), absent in the E. coli  host used to produce the recombinant proteins. What is the nature of the 4CL-modifying activity in tobacco stems? We have  accumulated evidence that the 4CL-modifying activity is present in high quantities in tissues which have high 4 C L activity (Figure 4.9 and 4.10). Thus the presence of the modifier correlates well with the predicted location where its function would be most required. Studies with alkaline phosphatase and NaF, a phosphatase inhibitor, suggest that the 4CL-modifying activity may be a protein kinase (Figure 4.11). At least three mechanisms can be postulated by which 4 C L activity could be post93  translationally modified by a putative kinase. The most simple explanation, is that 4 C L is covalently altered by phosphorylation and that this renders the enzyme inactive towards cinnamate as a substrate. This model (Figure 4.13, A) is supported by the fact that there are putative phosphorylation-sites in the predicted 4CL1 and 4 C L 2 amino acid sequences (Figure 4.12); however, using P - y - A T P , we have not been able to 32  clearly demonstrate phosphorylation of the recombinant-4CL proteins (not shown). Reversible phosphorylation-dephosphorylation as method of regulating enzyme activity has been extensively characterized in animal systems (Cohen, 1980) and has been demonstrated in plants (Ranjeva and Boudet, 1987). In particular, phosphorylation-dephosphorylation of nitrate reductase (Kaiser and Huber, 1994) and phosphoeno/pyruvate carboxlase (Jiao and Chollet, 1991) has been well characterized and putative kinases which mediate this modification have been identified and partially purified (Bachmann et al., 1995; Wang and Chollet, 1993b; Bakrim era/., 1992). Light-mediated phosphorylation of spinach nitrate reductase at serine-543 (Bachmann et al., 1996) leads to inactivation of the enzyme and thereby serves as an "on/off" switch. In contrast, phosphorylation of a serine residue (Bakrim et al., 1992) at the far N-terminus of the phosphoeno/pyruvate carboxlase leads to changes in enzyme kinetics. The phosphorylated form of phosphoeno/pyruvate carboxlase is less sensitive to feedback-inhibition by L-malate and exhibits higher enzyme activities at a suboptimal pH range (Nimmo etal., 1987; Bakrim etal., 1992). P A L has been shown to be phosphorylated but this was associated with enzyme degradation and not changes in enzyme activity (Bolwell, 1992). A second method in which 4 C L may be regulated by a kinase is by the interaction of 4 C L with another protein which is phosphorylated (Figure 4.13, B). In such a model, when crude tobacco-extracts are added to recombinant 4CL, the interacting-protein (phosphorylated) interacts with the recombinant 4 C L and alters its abiity to use cinnamate as a substrate. In the presence of alkaline phosphatase, the phosphate group is removed from the 4CL-interactor resulting in complex dissociating 94  and the re-acquisition of recombinant-4CL activity towards cinnamate as a substrate. This model is supported by the fact that immunoprecipitation experiments suggest that enzymes of phenylpropanoid biosynthesis pathways may form multienzyme complexes  in vivo  (Hradzina and Wagner, 1985; Deshpande  et al.,  1993). However, if  this model is true, the putative interactors must be present in the tobacco-stem extracts in great excess. This is because in the co-incubation experiments described above, the concentration of recombinant 4 C L is approximately 50-75 times greater than that of endogenous tobacco-4CL protein (based on enzyme activity). Although possible, it is unlikely that tobacco-stem extracts would contain 4CL-interactors that are 50-75 fold in excess compared to the levels of endogenous tobacco-4CL. A third possibility is that 4 C L is regulated by a cascade of events which includes a phosphorylation step (Figure 4.13, C). It is unlikely that this model is accurate since the results presented in this section suggest that recombinant-4CL activity is modified by reversible phosphorylation-dephosphorylation. The reversibility of a signal cascade, as presented in Figure 4.13, C is difficult to envision. The potential regulation of 4 C L substrate specificity by phosphorylation would be more meaningful if the occurance and the biological significance of 4 C L activity towards cinnamate as a substrate was understood. We have not identified a developmental stage, a physiological state, or an experimental treatment where endogenous tobacco 4 C L uses cinnamate as a substrate. In the presence of gibberellic acid, the 4 C L activity found in carrot cell cultures does not use cinnamate as a substrate. However, carrot cell cultures grown in medium lacking gibberellic acid accumulate large amounts of anthocyanins and contain a 4 C L activity which uses cinnamate as a substrate (Heinzmann et al., 1977). It is not clear whether this difference in substrate specificity is due to post-translational modification, as might be the case of the tobacco 4 C L described here, or due to the presence of divergent 4CL gene-products. Nevertheless, these results suggest that, in carrot cell cultures, 4 C L activity toward cinnamate as a substrate is induceable and may be important in 95  phenylpropanoid metabolism. If tobacco 4 C L uses cinnamate as a substrate  in  planta,  what kinds of products can be made from cinnamoyhCoA? The 4 C L from Cephalocereus  senilis  (old-man-cactus; Liu et  al.,  1993) uses cinnamate as a  substrate probably for the formation of B-ring deoxy flavonoids such as pinocembrin (a flavanone), cephalocerone (an aurone), and baicalein (a flavone). (Shain and Miller, 1982) and cephalocerone (Pare  etal.,  Pinocembrin  1991) have antimicrobial  activities. Although similar compounds have not been identified in tobacco, pinocembrin has been found in Nierembergia  hippomanica,  a member of the  Solanaceae family (Pomilio and Gros, 1979), suggesting that such B-ring deoxy flavonoids may exist in tobacco. Salicylic acid is made from cinnamate (Yalpani et al., 1993) and it has been hypothesized that the conversion of C6-C3 to C6-C1 may involve a mechanism analogous to "B oxidation of fatty acids" (Lewis, 1993; Loscher and Heide, 1994). If this suggestion is true, then cinnamoyhCoA would be a prime candidate as the precursor to salicylic acid. The biosynthesis of salicylic acid is still under debate but results suggest that salicylic acid biosynthesis occurs through a "non oxidative" route thereby by-passing the 4CL-catalyzed step (French Yazaki  etal.,  1991; Schnitzler  etal.,  1992).  96  etal.,  1976;  + Tobacco Extract • + Alkaline Phosphatase  B + Tobacco Extract •  i  PCX  + Alkaline Phosphatase  C  + Tobacco Extract A  A-po,  O 97  Figure 4.13: Three models describing the involvement of phosphorylation-dephosphorylation in the regulation of 4CL. A: 4CL is modified by phosphorylation. B: 4CL interacts with another protein which is phosphorylated. C: 4CL substrate specificity is regulated by a signal cascade which includes a phosphorylation step. White globular structures represent recombinant-4CL proteins which use cinnamate as a substrate. Grey structures represent modified recombinant-4CL proteins which do not use cinnamate as a substrate. Black structures represent protein(s) which interact with 4CL. PO4 represents phosphate group; its location on the proteins is schematic. A and A-PO4 represent components of a signal cascade.  Chapter 5 4CL-Suppressed Transgenic  Plants  5.1 Introduction 5.2 4CL-Suppressed Tobacco Plants 5.2.1 Generation of 4CL-Suppressed Tobacco Plants 5.2.2 Discussion 5.3 4CL-Suppressed Arabidopsis Plants 5.3.1 Generation of 4C/_-Suppressed Arabidopsis Plants 5.3.2 Characteristics of 4CL-Suppressed Lines 5.3.3 Wound-Activated Gene Expression in 4CL-Suppressed Lines 5.3.4 Discussion  5.1  Introduction One of the ways in establishing the developmental significance or physiological  role of a given gene is to examine the consequences of its absence. Since mutants lacking 4 C L activity have not been identified, we used sense suppression and antisense R N A to down regulate 4CL expression. The mechanism by which sense suppression functions is still unclear (Jorgensen, 1995); however, it has been successfully used to down-regulate, most notably, the expression of CHS in petunia (Napoli etal., 1990; van der Krol etal., 1990) and PAL in tobacco (Elkind etal., 1990). A major disadvantage of sense suppression is that it can be genetically-unstable giving "revertants" in subsequent generations and, sometimes, within sectors of the primary transformant (Jorgensen, 1995). Antisense R N A has been widely used to down-regulate genes in primary and secondary metabolism. For example, genes encoding ACC-oxidase (ethylene-forming enzyme; Hamilton etal., 1990), D A H P 98  (Jones  etal.,  1995), C O M T (Ni  1995; Van Doorsselaere  etal.,  etal.,  1994; Dwivedi  etal.,  1995), and C A D (Halpin  1994; Atanassova  etal.,  etal.,  1994) have been  successfully down-regulated by antisense RNA. However, antisense R N A also has limitations (reviewed in Lee and Douglas, 1996a), one of them being that it rarely produces transgenics completely suppressed at the target-gene. The physiological role of 4 C L in tobacco and Arabidopsis  plants was examined  by generating transgenic plants suppressed in 4CL expression. In 1992, when this work was initiated, the 4CL c D N A clones from tobacco and Arabidopsis  had not been  isolated; thus antisense R N A and sense suppression were attempted in tobacco using the c D N A encoding 4 C L from potato (Becker-Andre et al., 1991). By 1993, the Arabidopsis  4CL  c D N A had been cloned and well characterized (Lee  et al.,  1995a)  and the endogenous 4CL-gene family in tobacco was better understood (Lee and Douglas, 1996b). Since 4 C L appeared to be encoded by a single gene in Arabidopsis,  as opposed to tobacco where 4 C L is encoded by a diverse gene-family  of 4 or more members, and because  Arabidopsis  is a good model-system for  molecular and genetic analysis (Estelle and Somerville, 1986; Meyerowitz, 1987), we chose to concentrate our studies on the Arabidopsis  system. Furthermore, since  sense suppression has been shown to be unstable (Bates  etal.,  1994; Jorgensen,  1995), we limited our analysis to the antisense-suppressed plants. This decision was appropriate especially since  Arabidopsis  has a short generation-time and it is  inevitable that our research would span over a few Arabidopsis  generations.  This chapter is a summary of the work done in tobacco and Arabidopsis  using  antisense-RNA suppression and sense suppression. Some sections (tobacco and sense-suppressed transgenic lines) contain results laying the groundwork which will serve as a foundation for further analysis by other researchers. The antisense 4CL Arabidopsis  transgenic-plants are analyzed in detail and are the main topic of  discussion in this chapter. The questions we would like to address using the antisense 4CL plants are as follows. First, what are the phenotypic consequences of 99  blocking 4CL expression? Second, how does blocking 4CL expression affect the biosynthesis of phenylpropanoid-products? And lastly, if we are successful in blocking carbon flow into phenylpropanoid biosynthesis, what is the affect of blocking 4CL expression on stress-activated gene expression? 5.2 4CL-Suppressed Tobacco Plants 5.2.1  Generation of 4CL-Suppressed Tobacco Plants The potato-4CL cDNA was subcloned behind the 35S promoter in sense or  antisense orientation (Figure 5.1) and transformed into tobacco using  Agrobacterium-  mediated transformation. The regenerated plants (TO-generation) were allowed to set seed (T1-generation); however, approximately 9 % (8 out of 93) of the primary transformants were sterile so that propagation of these transgenic lines and analysis of the progeny was not possible (Table 5.1). Half of these sterile transgenic-tobacco plants (4 out of 8) appeared to have filaments that projected far beyond (exserted) the stigma such that the pollen-bearing anthers were removed from the stigma. Attempts to pollinate the stigma by manually placing pollen from the same plant onto the stigma were not successful. To ensure that the T1-generations contained the transgene and to assess the copy-number of the transgenes in each plant, the seeds from the T1-plants were screened for the presence of the  nptll  selectable-marker; namely, the seeds were  surface-sterilized, plated on M S plates supplemented with kanamycin and scored for their ability to grow in the presence of kanamycin (Table 5.2). Results demonstrate that most of the transgenic tobaccos contained one locus of the transgene (KanR/KanS ratio of -3:1). Some transgenic lines contained 2 (KanR/KanS ratio of -15:1) or more transgene loci. Approximately 8 % (7 out of 85) of the seedlots were nonviable, and of the seeds that were viable, approximately 1 5 % (12 of 78) had particularly low germination frequencies or produced seedlings with unusual characteristics such as short roots or chlorotic, yellow leaves (Table 5.1 and 5.2). 1 0 0  These seedlings, however, were not correlated with low levels of 4 C L proteins as determined by western blot analysis (not shown). Using the antibody raised against the parsley 4 C L to detect tobacco 4CL, T1 transgenic-tobacco seedlings were screened by western blot analysis to determine which transgenic lines had decreased levels of 4 C L protein. Seedlots were surfacesterilized, germinated on MS plates supplemented with kanamycin and 17-day old kanamycin-resistant seedlings were pooled for protein extraction. A representative western blot is shown in Figure 5.2. Many of the transgenic tobacco lines had lower levels of 4 C L protein as compared to the wild-type plants. To ensure that these results were accurate, screening by western blot analysis was performed twice on all -80 transgenic lines and duplicate gels were electrophoresed and stained with Coomassie Blue to ensure eveness of protein-loading (not shown). The protein extracts used in the western blot screening represent a pool from seedlings of homozygous and heterozygous genotype. Individuals homozygous for the transgene were identified by examining the T2-generation of selected lines. Transgenic lines which consistently showed lower levels of 4 C L compared to that in wild-type seedlings were allowed to self and set seed (T2-generation). The T2-seeds were plated on M S media supplemented with kanamycin and seedlots which had 1 0 0 % kanamycin-resistant seedlings, and were thus likely homozygous for the antisense transgene, were stored for future analysis (Table 5.3). The 4CL-protein levels and 4 C L enzyme activities of these putative homozygous lines have not been determined due to time constraints. Transgenic tobacco plants with lower 4CL-protein levels, as demonstrated by western blot analysis, appeared relatively normal in size and growth habits compared to that of wild-type SR1 tobacco plants. The number of flowers and the color of the petals in the transgenic plants were similar to that of the wild-type tobacco plants. One exception is that transgenic-tobacco plants with lower 4CL-protein levels appeared to have curved leaf-margins (repand) whereas wild-type tobacco leaf-blades tend to be planar. One transgenic line, containing 1.6-kb of the potato 4CL c D N A in sense 101  orientation (Eco-Kpn Sense, transgenic line #9.1), had severely curved leaf-margins and the intervening tissue between the secondary veins were buckled (Figure 5.3).  Construct Designation  Size of St4CL Orientation of St4CL cDNA cDNA (kb)  Eco Anti  Antisense  Xba-Kpn Anti  Antisense  Eco Sense  Sense  Eco-Kpn Sense  Sense  Schematic Diagram  35S  1.6  — 35S 1.6  —  anti St4CL  -term  anti St4CL  term  -  35S  St4CL  term —  St4CL  ~^)-|term —  Figure 5.1: Summary of potato-4CL cDNA constructs. The potato 4CL cDNA (St4CL) was subcloned in sense and antisense orientation behind the CaMV 35S promoter (35S). The DNA constructs included the 35S polyadenylation/termination sequences (term). Designation of the DNA-constructs stem from the restriction enzymes used during the subcloning process (see section 2.10 for details). Table 5.1:  Characteristics of primary transgenic-tobacco plants and their seeds.  Construct Designation  # Primary Transformants  # Fertile Transformants  # Transformants with Non-viable Seeds  # Unusual Seedlots*  Eco Anti Xba-Kpn Anti Eco Sense Eco-Kpn Sense  17 29 19 28  15 27 18 25  1 3 0 3  2 5 2 3  TOTAL  93  85  7  12  * Unusual seedlots are ones which had low germination frequencies or produced seedlings with short roots or chlorotic leaves.  102  Table 5.2: The proportion of kanamycin-resistant (KanR) to kanamycin-sensitive (KanS) T1 tobaccoseedlings. Transgenic Line  1 2 3 4 6 8 9 10 11 12 14 15 16 17  Transgenic Line  1 2 3 4 5 6 8 9 10 11 13 14 15 16 17 18 20 21 22 24 26 27 28 29 °  Eco Anti  #KanR plants  26 34 35 21 41 17 28 24 28 22 28 18 50 37  # KanS plants  5 7 2 11 16 12 10 6 13 7 0 10 0 10  Xba-Kpn Anti  #KanR plants  53 16 39 56 14 54 27 28 48 54 49 0 38 22 45 41 19 32 41 29 0 30 38 44  # KanS plants  9 13 18 4 6 0 15 3 5 7 5 62 12 18 4 9 17 2 3 12 45 13 9 6  KanR:KanS ratio  Transgenic Line  5.2 4.8 17.5 1.9 2.5° 1.4 2.8 4 2.1 3.1  1 2 3 4 5 6 7 9 10 11 12 13 14 15 16 17 18 19  oo°  1.8 CO  3.7  KanR:KanS ratio  Transgenic Line  5.8 1.2* 2.2 14 2.3*  1 2 3 4 5 6 7 8 9 10 11 13 14 18 20 21 22 23 24 26 27 28  OO  1.8 9.3 9.6 7.7 9.8 0 3.2 1.2 11.3 4.6t 11 16* 14 2.4 0 2.3 4.2* 7.3  ^Seedlings with short roots. Seedlings with chlorotic, yellow leaves. * Seedlots with low germination frequency.  103  Eco Sense  # KanR plants  33 38 47 31 0 41 47 40 37 42 53 28 38 60 39 53 24 24  # KanS plants  10 18 0 14 40 21 1 8 20 15 0 16 14 4 14 0 18 5  Eco-Kpn Sense  # KanR plants  64 61 21 55 42 57 49 58 58 26 23 37 45 39 55 48 24 39 31 39 59 38  # KanS plants  0 20 21 14 17 6 17 21 7 14 7 18 14 11 4 20 16 25 7 19 15 0  KanR:KanS ratio  3.3 2.1 oo  *  2.2 0 1.9 47 5 1.9 2.8 oo  1.8 2.7 15 2.8f oo  1.3 4.8  KanR:KanS ratio oo  3° 1* 4 2.5 9.5 2.9 2.8 8.3 1.9* 3.3 2 32 3.5 13.8 2.4 1.5 1.6 4.4 2 4 CO  1  2oo  2  3 4  6  7 9 1 0 wt  69 — • 46 — 30 —  Construct Designation  Transgenic Lines with Lower 4CL Protein  Putative Homozygous Individuals (100% KanR T2 Seedlings)  Eco Anti  9 10 1 1 16  9.2 10.4 11.1 16.8  Xba-Kpn Anti  1 15 17 29  1.3 1.6 15.2 15.6 17.2 17.3 -  1 7 10 14 15  1.1 1.2 7.2 10.7 10.8 14.4 14.6 15.2 15.8  Eco Sense  Eco-Kpn Sense  2 9 10 18  2.2 10.4 18.6  9.3 9.6 10.5 10.7 11.2 11.7 16.9  1.8 15.8 17.4 17.5  9.8  17.7  14.7  2.3  2.4  10.5 18.7  10.7 18.8  104  9.7 10.8 11.8  2.5  2.8  Figure 5.2: An example of a western blot used to screen transgenic tobacco plants. Protein extracts are from pools of T1-tobacco plants transgenic for the 2-kb potato 4CL cDNA subcloned in sense orientation behind the CaMV 35S promoter (Eco Sense). Numbers above the figure refer to the transgenic line whereas wt refers to proteins from wild-type SR1 seedling. Molecular mass standards (in kDa) are on the left.  Table 5.3: Summary of 4CL-suppressed transgenictobacco lines. Transgenic lines with lower 4CL-protein levels as demonstrated by western blot analysis are listed together with putative homozygous individuals within each transgenic line.  Figure 5.3: An extreme case of altered leaf morphology in 4CLsuppressed transgenic tobacco. Transgenic tobacco "Eco-Kpn Sense 9.1" is heterozygous for the 1.6-kb potato-4CZ. cDNA, subcloned behind the CaMV 35S promoter, transgene. Note the curving of the leafmargin and the buckling of the tissue between the secondary veins.  5.2.2  Discussion Approximately 9 % of the primary transformants were sterile and have since  died, making further examination of them impossible. Whether the sterility was caused by low 4 C L activity is unknown; however, it is conceivable that suppression of 4CL may cause sterility since flavonols, downstream-products of the 4CL-catalyzed reaction, have been shown to be important in some plants for pollen development and pollen-tube growth (Yistra et al., 1992; 1994). Sense-suppressed PAL tobacco-plants had abnormal flowers containing stamens with short filaments as compared to those of wild-type tobacco plants (Elkind et al., 1990). This is in contrast to the 4CLsuppressed tobacco plants reported here which have unusually long filaments. How the down-regulation of 4CL (or PAL) leads to structural differences in the stamen is not immediately obvious and remains to be resolved in the future. Sense-suppressed PAL plants had white or faded-pink flowers, an observation not paralleled here in the 4C/_-supressed tobacco plants. This latter result was unexpected since anthocyanins are downstream-products of the 4CL-catalyzed reaction so that one would predict that a block in 4 C L should cause a block in anthocyanin accumulation. Without direct measurement of the residual 4 C L enzyme activity remaining in the transgenic 105  tobaccos, it is difficult to speculate on the mechanism in which anthocyanins are made in these plants. It should be noted that antisense-4CL  Arabidopsis  plants with - 8 %  residual 4CL activity accumulated anthocyanins in a manner analogous to wild-type Arabidopsis  plants (see section 5.3.2).  The cause of the unusual leaf morphology in antisense-4CL tobacco plants is unknown. However, one possibility is that the lower levels of 4CL activity may result in lower levels of accumulated lignin and that this may cause an abnormal rate of cell expansion between the highly lignified trachery elements in the vasculature as compared to the parenchyma-cells in the intervening tissue. Sense suppression of  PAL  in tobacco (Elkind et al., 1990) also produced transgenic plants with altered leaf  morphology; however, the leaves were described as "spoon-like" and "epinastic" in nature. Sense-suppressed PAL plants accumulated lower levels of lignin compared to wild-type plants (Elkind era/., 1990; Bate et al., 1994) thereby supplying a possible link between the biochemical lesions with the observed morphology. The antisenseDAHP  potato plants were reported to have structural differences in stem-diameter,  stem-length, and growth habits but these results are puzzling since the levels of residual D A H P synthase activity did not correlate with the levels of lignin (Jones et al., 1995). However, in other studies, transgenic tobacco (Ni potato (Jones  etal.,  1995; Y a o  etal.,  etal.,  1995), and transgenic  1994), transgenic  Arabidopsis  (see section  5.3.2) plants had lower levels of lignin but did not exhibit differences in leaf- or stemmorphology. The effect of antisense 4CL on transgenic tobacco needs to be investigated further; however,  I  have chosen to concentrate on the Arabidopsis  results of these studies are described below.  106  system and the  5.3 4CL-Suppressed 5.3.1  Arabidopsis  Plants  Generation of 4CL-Suppressed  Arabidopsis  Plants  In order to generate Arabidopsis plants with suppressed 4CL levels, approximately 1.6-kb of the Arabidopsis 4CL cDNA (Lee et al., 1995a) was subcloned behind the parsley-4CL7 promoter or the 35S promoter in sense or antisense orientation (Figure 5.4) and transformed into Arabidopsis using Agrobacteriummediated root-transformation. Two Arabidopsis accessions were used; the R L D ecotype, which has been reported to be easier to transform and regenerate (Dr. L. Kunst, personal communication), and the Columbia ecotype, which is one of the more commonly studied varieties of Arabidopsis. A s predicted, the RLD variety of Arabidopsis was more efficiently transformed and regenerated such that 27 antisense 4CL transgenic RLD-lines were obtained whereas only 8 antisense-4CL transgenic Columbia-lines were obtained (Table 5.4). The primary transformants (TO) were allowed to self and set seed while in tissue culture (see section 2.11). The T1-seeds were selected for the presence of the nptll transgene and then allowed to self and set seed. The T2-generation was screened to isolate seedlots with 1 0 0 % kanamycinresistance seedlings and these putative homozygous lines were used for western blot analysis. Protein extracts from wild-type and antisense-4CL transgenic Arabidopsis stems were used to screen for plants with decreased levels of 4 C L protein, using an antibody raised against the parsley 4CL. Screening by western blot analysis was performed twice to ensure their accuracy and duplicate gels were electrophoresed and stained with Coomassie Blue to ensure eveness of protein-loading (not shown). More than 5 0 % of the transgenic RLD-lines had lowered 4CL-protein levels and the efficacy of the parsley 4CL1 promoter (8 suppressed lines out of 12) was comparable to that of the 35S promoter (9 suppressed lines out of 15) given the small sample-size. Although only a few transgenic Columbia-lines were obtained, many of them had lower levels of 4 C L protein and both the parsley-4CL 1 promoter (4 suppressed lines out of 6) and the 35S promoter (2 suppressed lines out of 2) were effective in causing 107  antisense RNA suppression. An example of a western blot is shown in Figure 5.5. Arabidopsis lines transformed with the sense constructs were not examined but are stored as T2-seeds. Construct Designation  Schematic Diagram Pc4CL-P  P c 4 C L Anti 35S Anti  35S  Pc4CL Sense 35S  —  K  Pc4CL-P  Sense  35S -  anti At4CL antiAt4CL  -term — - term  At4CL  term  At4CL  term —  Figure 5.4: Summary of Arabidopsis -4CL cDNA constructs. The Arabidopsis 4CL cDNA (At4CL) was subcloned in sense and antisense orientation behind the CaMV 35S promoter (35S) or the parsley-4Ct 7 promoter (Pc4CL-P). The DNA constructs included the 35S polyadenylation/termination sequences (term).  Table 5.4: Summary of transgenic Arabidopsis plants # Primary generated. Transformants  Construct Designation  Ecotype  P c 4 C L Anti 35S Anti  RLD RLD  12 15  P c 4 C L Sense 35S Sense  RLD RLD  22 21  Columbia Columbia  6 2  P c 4 C L Anti 35S Anti  kDa  PC4CL-P wt  35S  1.7 3.6 4.1 5.86.5 1.3 wt  108  Figure 5.5: An example of a western blot used to screen transgenic Arabidopsis plants. Protein extracts are from stems of Arabidopsis plants (Columbia ecotype) transgenic for the Arabidopsis 4CL cDNA subcloned in the antisense orientation behind the parsley-4CL7 promoter (PC4CL-P) or the CaMV 35S promoter (35S). Numbers above the figure refer to the transgenic line whereas wt refers to proteins isolated from the stems of wildtype plants. Molecular mass standards (in kDa) are on the left.  5.3.2  Characteristics of 4CL-Suppressed  Lines  Based on western blot analysis, four antisense-4CL  lines with  Arabidopsis  lowest levels of 4 C L protein were selected for detailed analysis. The four transgenic Arabidopsis-\ines  appeared normal with similar size and growth habits as that of wild-  type plants of the same variety (Figure 5.6) and genomic Southern blot analysis indicated that the transgene was stably integrated in these  lines (Figure  Arabidopsis  5.7). Despite their normal appearance, these plants exhibited significant differences at the biochemical level. 4 C L enzyme assays using  Arabidopsis-sXem  extracts showed  that the transgenic lines had 1 5 % (Pc4CL Anti, RLD ecotype, homozygous line 11.3), 5 0 % (35S Anti, RLD ecotype, homozygous line 11.1), 2 0 % (Pc4CL Anti, Columbia ecotype, homozygous line 1.7) and 8 % (35S Anti, Columbia ecotype, homozygous line 1.3) residual 4CL-enzyme activity compared to that of wild-type plants of the same variety (Figure 5.8). For simplicity, these transgenic lines were designated RLD:Pc4CL, RLD:35S, C O L : P c 4 C L , and C O L 3 5 S respectively. Lignin is one of the major downstream-products of the 4CL-catalyzed reaction. To examine the affect on lignin accumulation in the 4CL-suppressed  Arabidopsis  lines, lignin was extracted from the bolting stems using the thioglycolic acid method. Figure 5.9 show that the lignin levels, as expressed per mg of fresh weight, were reduced in the antisense lines. Similar results were obtained when the lignin content was expressed per mg dry (alcohol-insoluble residue) weight (not shown).  The  degree of lignin reduction corresponded well with the levels of residual 4CL-enzyme activity found in the transgenic plants (compare Figures 5.8 and 5.9). Although the lignin content followed the same pattern as the levels of residual 4CL-enzyme activity, only the plants with less than 2 0 % residual 4CL-activity had lignin levels that were significantly less than that found in wild-type plants. When the lignin content is plotted as a function of 4 C L enzyme activity (Figure 5.10), it becomes clear that lignin levels begin to drop precipitously when 4 C L activity is below 2 0 % of that found in wild-type plants. Extrapolation of the curve in Figure 5.10 could provide a method for predicting 109  the levels of lignin that will be accumulated in plants with a given level of 4 C L activity. For example, plants with 5 % residual 4 C L enzyme activity would be predicted to have - 7 5 % decrease in lignin content.  Figure 5.6: Phenotype of antisense-4CZ. suppressed transgenic Arabidopsis. Arabidopsis plants, Columbia ecotype, were transformed with the Arabidopsis 4CL cDNA subcloned in antisense orientation behind the parsley-4CL7 promoter or the CaMV 35S promoter. Plants are: A and D, COL:Pc4CL; B and E, COL35S; and C and F, wild-type.  110  PC4CL XBH  kb 10.2 6.1 " 4.0  35S  wt  XBH XBH  Pc4CL  wt  XBH XBH  -*  m  35S  XBH  ~*  *m  2.0 1.0 0.5  COL  RLD  Figure 5.7: Genomic Southern blot analysis of transgenic Arabidopsis. Genomic DNA (10 ug) from RLD (RLD) and Columbia (COL) Arabidopsis plants were digested with different restriction enzymes, transferred onto nylon membranes and used in Southern blot analysis. Plants were either untransformed (wt) or transformed with the Arabidopsis 4CL cDNA subcloned in antisense orientation behind the parsley-4CZ-7 promoter (Pc4CL), or behind the CaMV 35S promoter (35S). The blot was probed with the 2-kb Arabidopsis 4CL cDNA and asterisks indicate hybridization-signals arising from the endogenous Arabidopsis 4CL gene. Xba\ (X) cuts the transgene to release a fragment containing the promoter and the antisense 4CL cDNA. BarrfrU (B) cuts the transgene once at a location 5' to the promoter. HindiW (H) cuts the DNA construct to release the entire transgene containing the promoter, the antisense 4CL and the termination sequence. These restriction enzymes do not cut within the Arabidopsis 4CL cDNA. Figure 5.8: 4CL enzyme activity in transgenic and wild-type Arabidopsis. Proteins from the bolting stem of Arabidopsis plants were used for 4CL enzyme assays. Activity is expressed as a percentage of the activity in untransformed (wt) RLD or Columbia plants. Results are averaged from three determinations and error bars represent standard deviations.  Pc4CL 35S  E  wt  Pc4CL 35S  wt  Figure 5.9: Lignin content in transgenic and wild-type Arabidopsis. Lignin was extracted from the bolting stem of Arabidopsis plants using the thioglycolic acid method and lignin amount is expressed as the optical density at 280 nm. Plants were either wild-type (wt) or transformed with the antisense 4CL construct as indicated. Results are averaged from three determinations and error bars represent standard deviations.  10  Q  PC4CL 35S  wt  Pc4CL 35S  wt  11 1  Figure 5.10: Lignin content plotted as a function of 4CL enzyme activity. The results from Figures 5.8 and 5.9 are summarized in a single plot showing the relationship between lignin accumulation and 4CL enzyme activity. Results are expressed as a percentage of the lignin content and the 4CL enzyme activity in wild-type plants. Open circles represent data from the RLD ecotype of Arabidopsis and closed circles represent data from the Columbia ecotype of Arabidopsis. Data points represent the average of three determination and the error bars represent standard deviation in lignin determination.  125  25 0  0  20  40  60  80  100  120  % 4CL Enzyme Activity  Another major phenylpropanoid-product thought to be derived from cinnamoyhCoA derivatives are the anthocyanins. To examine if a block in 4 C L would affect endogenous anthocyanin levels, wild-type and antisense-4CL  Arabidopsis  seedlings were visually scored to characterize pigment accumulation. Anthocyanins have been shown to accumulate in the hypocotyls and the rim of cotyledons during early development of Arabidopsis Arabidopsis  seedlings (Kubasek era/., 1992). Antisense-4CL  seedlings appeared to accumulate purple pigments in the same manner  as wild-type seedlings (not shown). There was no observable difference in pigmentation intensity nor timing or location of pigment accumulation. This result was unexpected since anthocyanins are downstream-products of the 4CL-catalyzed reaction and one would predict that a decrease in 4 C L function might lead to a decrease in end-products. Flavonoids have been shown to accumulate in the petals and the seed coat of Arabidopsis  (Shirley  et al.,  1995) and one way to examine  flavonoid content is by fluorescence microscopy. Flavonoids absorb UV-light so that tissues which contain flavonoids do not fluoresce under UV-excitation. In contrast, tissue which lack flavonoids fluoresce blue under UV-light (Shirley era/., 1995). When examined under fluorescent microscopy, there were no observable differences in the petals and seeds of wild-type and transgenic  112  Arabidopsis  plants (not shown).  We further characterized the antisense plants by testing for differences in the induced accumulation of anthocyanin. Plants were placed under high-intensity whitelight to elicit environmentally-induced anthocyanin production. After 24h of highintensity white-light treatment, anthocyanins were extracted from the leaves of wildtype and antisense-4C/_  Arabidopsis  plants. A s previously reported (Feinbaum and  Ausubel, 1988), 24 h of high-intensity white-light treatment caused an accumulation of purple pigments in the vegetative tissues (not shown). However, extraction and quantitation of anthocyanins showed that there was no significant difference in anthocyanin content between the light-treated 4CL-suppressed plants and the lighttreated wild-type plants (Figure 5.11). These results indicated that anthocyanins and flavonoids accumulate in antisense 4CL plants to levels analogous to the levels found in wild-type  Arabidopsis  plants.  To examine the expression of 4CL and CHS in antisense-4CL suppressed lines subsequent to high-intensity white-light treatment, northern blot analysis was performed. Total R N A was isolated from leaves of antisense-4CL suppressed and wild-type plants before and after high-intensity white-light treatment. Hybridization of the northern blot to a CHS probe showed that CHS steady-state R N A levels increased dramatically after light-treatment in both the antisense and wild-type plants (Figure 5.12A, C H S ) . Re-hybridization of the same blot to a single-stranded 4CL riboprobe showed that the steady-state levels of 4CL transcripts were relatively abundant in the RLD.35S transgenic and in wild-type plants. The 4CL m R N A levels were less abundant in the R L D : P c 4 C L and C O L : P c 4 C L transgenic lines and was almost undetectable in the COL.35S transgenic line (Figure 5.12A, 4CL). Furthermore, 4CL transcript levels in the wild-type and the transgenic lines did not increase after highintensity white-light treatment (Figure 5.12A, 4CL). In agreement, 4 C L protein levels  113  as demonstrated by western analysis was highest in wild-type and RLD.35S plants; lower in R L D : P c 4 C L and C O L : P c 4 C L plants; and very low in the C O L : 3 5 S plants (Figure 5.12B). The levels of 4 C L protein did not increase significantly after 24 hours of high-intensity white-light stress (Figure 5.12B). These results show that: 1) highintensity white-light caused an increase in CHS m R N A levels but no apparent changes in the levels of 4CL m R N A or 4 C L protein; and 2) the levels of 4CL m R N A and 4 C L protein in the antisense-suppressed leaves parallel the levels of residual 4 C L enzyme activity (Figure 5.8) and 4 C L protein found in the stems. Figure 5.11: Anthocyanin levels in transgenic and wild-type Arabidopsis after 24 hour high-intensity white-light treatment. Anthocyanin levels are expressed as a percentage of the levels in wild-type plants (WT). Transgenic lines are as indicated. Results are averaged from three determinations and error bars represent standard deviations.  250  Pc4CL 35S WT  RLD PC4CL 35S wt - + - + --»  Pc4CL 35S WT Figure 5.12: Steady-state levels of CHS RNA, 4CL RNA, and 4CL protein in highintensity white-light treated transgenic and COL wild-type Arabidopsis leaves. (A) Total Pc4CL 35S wt RNA was isolated from leaves which were untreated (-) or had been exposed to high- + - + - + intensity white-light for 24 hours (+). RNA 4CL (10 ug) was separated on formaldehyde gels, transferred onto nylon membranes CHS and hybridized sequentially to the Arabidopsis 4CL (single-stranded probe) rRNA and CHS probes. Hybridization to a rRNA probe demonstrated evenness of loading. Blots were washed at high stringency. (B) Western blot analysis was performed using anti 4CL 25 jag of protein from the tissues described above. The blot was reacted with a polyclonal antibody raised against the parsley 4CL (anti 4CL).  BUR B  114  5.3.3  Wound-Activated  Gene Expression in 4CL-Suppressed  Lines  Many of the phenylpropanoid genes are coordinately regulated so that during pathogen attack and environmental stress, the genes encoding enzymes in the phenylpropanoid-biosynthetic pathways are sequentially or simultaneously induced (Ragg  etal.,  1981; Fritzemeier  etal.,  1987; Lois era/., 1989). More recently, genes  encoding enzymes in the shikimic acid pathway (Henstrand et al., 1992; Gorlach et al., 1995) and the oxidative pentose phosphate pathway (Fahrendorf  etal.,  1995) have  also been shown to be activated in cell cultures by elicitor treatment and light treatment. The transcriptional activation of genes in these pathways likely have a role in the biosynthesis of phenylalanine or the conversion of this amino acid into a variety of phenylpropanoid-products. W e used wounding as a representative stress-stimulus to determine if genes involved in primary and secondary metabolism are coordinately regulated in Arabidopsis. before in  An extensive study of this nature has not been reported  Arabidopsis.  Mature, fully-expanded leaves were wounded and, after 0, 0.5, 1, 2, 4, and 6 h, were harvested for R N A extraction. Total R N A was isolated for northern blot analysis (Figure 5.13). The R N A samples were electrophoresed in triplicate blots and probed with genes encoding enzymes in glycolysis (hexokinase,  HEXO;  phosphofructo  kinase, PFK), the oxidative pentose phosphate pathway (glucose 6-phosphate dehydrogenase,  G6PDH;  6-phosphogluconate dehydrogenase,  6PGDH),  the shikimic  acid pathway (3-deoxy-D-arab/no-heptulosonate 7-phosphate synthase, DHS1; 5Eno/pyruvylshikimate-3-phosphate synthase,  the general phenylpropanoid  EPSPS),  pathway (phenylalanine ammonia-lyase, PAL; cinnamate 4-hydroxylase, C4H; 4coumarate:CoA ligase, 4CL), and the lignin-biosynthetic pathway (cinnamyl alcohol dehydrogenase, levels for 6PGDH,  CAD). DHS1,  In wild-type Columbia EPSPS,  PAL,  C4H  Arabidopsis  and  4CL  plants, steady-state m R N A  increased subsequent to  wounding and reached maximal levels 1 to 2 hours after wounding (Figure 5.13). Wound-induced accumulation of  6PGDH,  DHS1,  115  EPSPS,  PAL,  and C4H R N A was  also observed in the 4CL-suppressed Columbia lines, C O L : P c 4 C L and C O L 3 5 S , despite the significantly lower levels of 4CL transcripts, 4 C L protein and 4 C L enzyme activity in these lines (Figures 5.13, 5.12, and 5.8). Taking into account minor variations in RNA-loading in each lane, as determined by hybridization to a rRNA probe, the wound-induced activation of all of these genes (with the exception of 4CL itself) occurred with the same timing and intensity in leaves from the antisense-4CL suppressed plants as compared to those from wild-type plants. Identical results were obtained with the wounded leaves of wild-type and antisense-4CL suppressed RLDlines of Arabidopsis  (not shown). Hybridization signals were extremely weak, or  undetectable when  HEXO,  PFK,  G6PDH, and CAD probes were used (not shown).  Pc4CL 0  0.5  1  2  •  4  35S 6  0  0.5  1  2  m 0  wt 4  6  •  *  0  0.5  1 2  4  6  probe 6PGDH DHS1  if  -ti -  mm)  EPSPS  f•  PAL C4H 4CL  IIIIII L L i i r a IIITTTl  rRNA  Figure 5.13: Wound-induced RNA accumulation in wild-type and antisense-4CL suppressed Arabidopsis leaves. Columbia Arabidopsis plants were wild-type or were transformed with the antisense 4CL construct as indicated. Total RNA was isolated from mature, fully-expaned leaves which were wounded for 0, 0.5, 1, 2, 4 and 6 h. RNA samples (10 ug) were electrophoreses in triplicate, in formaldehyde gels, transferred to nylon membranes and hybridized to the Arabidopsis-genes encoding 6-phosphogluconate dehydrogenase (6PGDH), 3-deoxy-D-ara/b/'no-heptulosonate 7-phosphate synthase (DHS1), 5-eno/pyruvylshikimate-3-phosphate synthase (EPSPS), phenylalanine ammonialyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:CoA ligase (4CL, single-stranded probe). Hybridization of the blots to a rRNA probe demonstrated evenness of RNA-loading; the blot used to probe PAL, C4H and 4CL transcripts is shown as an example (rRNA). Blots were washed at high stringency.  116  5.3.4  Discussion Using antisense-RNA technology, we have generated  Arabidopsis  plants with  suppressed 4 C L activity such that one transgenic line, COL:35S, had 4 C L enzyme activity as low as 8 % compared to that of wild-type plants. The parsley-4CL antibody recognized the Arabidopsis  4 C L and screening of the transgenic lines by western  analysis was an effective method in detecting 4 C L suppressed lines. The lowered levels of 4 C L enzyme activity in the transgenic lines were paralleled by the lower levels of 4 C L protein, as demonstrated by western blot analysis, and the lower levels of steady-state 4CL R N A levels. Both the 35S promoter and the parsley-4CL7 promoter were capable of suppressing 4CL. This was not unexpected since promoter-  GUS  fusion analysis has shown the parsley-4CL7 promoter to direct proper  developmental- and wound- induced  GUS  expression in Arabidopsis  (Lee  et al.,  1995a). The level of 4CL suppression observed here is comparable to the level of suppression reported in the PAL sense-suppressed tobacco lines which showed 9 0 % (Maher  etal.,  1994) to 9 5 % (Elkind  etal.,  1990) lower levels of P A L activity. Similarly,  antisense- CAD tobacco plants had a 9 3 % decrease in C A D activity (Halpin et al., 1994). Some antisense  COMT  (caffeic acid 3-O-methyltransferase) tobacco plants  had activities as low as 2 % of that found in the wild-type counterparts (Atanassova et  al., 1995) whereas in transgenic poplar, residual C O M T activity was - 5 % compared to wild-type plants (Van Doorsselaere  etal.,  1995). A transgenic line with no 4 C L activity  was not found and it is not clear whether this was because antisense R N A rarely causes 1 0 0 % suppression, or whether a plant completely lacking 4 C L activity is nonviable. The C O L : 3 5 S transgenic-line had approximately 5 0 % lower levels of lignin but did not show any differences in morphology or growth habit. Lignin levels have also been decreased in transgenic potato plants over-expressing the tryptophan decarboxylase gene (Yao C O M T activity (Ni  etal.,  etal.,  1995) and transgenic tobacco plants suppressed in  1994) with no reported differences in plant morphology. In 117  contrast, a subset of the P>4L-suppressed tobacco plants (Elkind et al., 1990) and the antisense-D/lHPpotato plants (Jones  1995) exhibited differences in the growth  etal.,  of the leaves and stems, respectively. Whether decreasing lignin levels results in abnormal plant anatomy probably depends on the percentage of lignin that has been reduced. Results reported here and in the literature (Ni era/., 1994; Yao et al., 1995) suggests that a decrease in lignin content of 5 0 % or less allows the plants to maintain their structural integrity and wild-type appearance. Abnormal leaves and stunted growth of P>4/_-suppressed tobacco plants were reported to have as little as 1 0 % lignin compared to wild-type tobacco-stems (Bates  etal.,  1994) suggesting that highly  reduced lignin levels result in irregular phenotypes. Jones et al. (1995) reported anomalous stem-growth in potato plants which had 3 5 % to 7 5 % of the lignin levels found in untransformed plants; however, the lignin content in these plants did not correlate with the levels of D A H P synthase activity, making it difficult to understand the relationship between the residual enzyme activity, the lignin content, and the resulting phenotype. Lignin content was only significantly different from that of wild-type plants when the 4 C L enzyme activity was lowered >80%. This suggests that  in vivo  levels of 4 C L  activity are in excess and only when the 4 C L activity has been suppressed by > 8 0 % does the enzymatic reaction restrict carbon flow into lignin formation. Similar results were reported with P A L where the enzyme only becomes rate-limiting in lignin biosynthesis when P A L activity is decreased by 7 5 % - 8 0 % compared to wild-type levels (Bates et al., 1994). In light of these experiments it is likely that, under normal laboratory conditions (uninduced and non-stressed), the general phenylpropanoid enzymes are in excess compared to that that is required for lignin biosynthesis. There have been a number of biotechnological attempts to decrease the quantity or quality of lignin for the purpose of increasing the cattle digestibility of forage crops, or to decrease the cost and environmental impact in the pulp and paper industry (Halpin  etal.,  1994; Ni  etal.,  1994; Dwivedi 118  etal.,  1994; Atanassova  etal.,  1995; Van  Doorsselaere et al., 1995). Transgenic plants containing antisense genes encoding C O M T (Dwivedi  etal.,  and C A D (Halpin  1994; Atanassova  etal.,  1995, Van Doorsselaere  etal.,  1995)  1994) did not exhibit quantitative differences in lignin  etal.,  accumulation; however, the monomeric composition was altered. Tobacco plants with low levels of C O M T had a lower ratio of syringyl/guiacyl (S:G) subunits and this qualitative change was observed only when C O M T activity was decreased by > 8 0 % compared to wild-type levels (Atanassova  etal.,  1995; Van Doorsselaere era/., 1995).  C/AD-suppressed tobacco plants accumulated hydroxy- and methoxycinnamylaldehyde monomers which caused the xylem to appear red-brown in color and this phenotype was observed when the C A D activity was decreased by > 8 4 % compared to that of wild-type plants (Halpin era/., 1994). Genetic mutants with defects in the corresponding genes show comparable phenotypes. The mutant of maize has a defective an altered S:G ratio (Vignols  COMT  etal,  brown  midrib  (bm3)  gene and the lignin in these mutants also has  1995). The  bmro  mutant of sorghum has a lower  level of C A D activity, it deposits aldehyde lignin monomers, and it has a red-brown color (Pillonel era/., 1991). The  fahl  {sin1) mutant of Arabidopsis  has a defective  ferulate 5-hydroxylase (F5H) gene and it lacked sinapate-derivatives, including syringyl lignin (Chappie  etal.,  1992). It is interesting to note that low levels of D A H P ,  P A L and 4 C L caused a decrease in lignin quantity whereas low levels of F5H, C O M T , and C A D resulted in changes in lignin quality but no differences in lignin quantity. Taken together, it is tempting to suggest that manipulation of genes early in the phenylpropanoid pathway may regulate carbon flux into lignin biosynthesis whereas manipulation of genes downsteam in the lignin-biosynthesis pathway modulate the kinds of lignin-subunits formed. This observation should be considered with caution since in one report, antisense  COMT  plants had lower levels of lignin (Ni  and the bm3 mutant of maize, which has a defective of lignin (Grand  etal.,  1985). The bm6mutant of  COMT  Sorghum  et al.,  1994)  gene also has lower levels (Pillonel  etal.,  1991) has  lower C O M T and C A D activities and it has a changed lignin composition as well as a  119  1 5 % - 2 5 % decrease in lignin content. Transgenic tobacco plants over-expressing tryptophan decarboxylase had a preferential decrease in syringyl subunits as compared to the guaiacyl subunits in addition to an overall lower lignin content (Yao et  al., 1995). In this manuscript, suppression of 4CL has caused a decrease in lignin content; however an unexpected affect on lignin quality may also have occurred. Studies to determine whether the 4CL suppressed  Arabidopsis  plants have altered  lignin composition in addition to lower lignin quantity are in progress. Preliminary results using Maule staining suggest that the levels of syringyl lignin is unchanged in antisense  4CL  and wild-type  Arabidopsis  plants (C.J. Douglas, unpublished).  Under the experimental conditions described here, a significant block in 4 C L function does not block anthocyanin accumulation during high-intensity white-light stress or during early  development. These results are in contrast with  Arabidopsis  P/AL-suppressed tobacco plants which have lower levels of pigments in the flowers and in the leaves (Elkind  etal.,  1990; Bates  etal.,  1994). Similarly, chemical inhibitors  of P A L cause lower levels of anthocyanin accumulation in the flowers, seedling, and hypocotyls of a number of plants (Amrhein and Hollander, 1979; Laber era/., 1986). High-intensity white-light treatment caused an increase in steady-state levels of CHS mRNA, but no significant increase in 4CL transcripts or 4 C L proteins (Figure 5.12). This latter result is surprizing since PAL and 4CL have been reported to be lightresponsive. However, careful examination of the literature reveals that, where induction has been demonstrated, PAL and 4CL transcripts are induced by white-light if the plants were dark-adapted prior to light-treatment (Liang et al., 1989; Ohl et al., 1990; Kubasek et al., 1992; Wu and Hahlbrock, 1992) or if the white-light was supplemented with UV-light (Lois  etal.,  1989; Wu and Hahlbrock, 1992). The results  presented here suggests that steady-state 4CL R N A levels do not increase under the high-intensity white-light treatment given (-900 uE s- nr ) when the plants were 1  2  grown and maintained under normal levels of continuous light (-120 jxE s- nrr ) prior 1  2  to treatment. The possible induction of 4CL R N A accumulation in dark-adapted or UV120  light treated  Arabidopsis  plants was not investigated. Thus, while we cannot yet  conclude that 4CL is not light-activated under some conditions, it is not high-intensity white light-induced in the way that CHS is (Figure 5.12; Feinbaum and Ausubel, 1988). In this respect, we have uncovered a differential response between 4CL and  CHS  towards high-intensity white-light treatment and this suggests that CHS and 4CL  are regulated by different mechanisms. The fact that high-intensity white-light resulted in: 1) no increase in 4CL transcripts, 2) no increase in 4 C L protein, 3) dramatic increase in CHS transcripts, and 4) anthocyanin accumulation, suggests that the residual 4 C L enzyme activity (>8%) in the antisense plants is sufficient to supply 4-coumaroyl:CoA esters for anthocyanin biosynthesis. Another possibility is that anthocyanins that are observed subsequent to high-intensity white-light stress are not synthesized  de novo,  but are converted from  pre-existing precursors. In such a situation, the low levels of 4 C L in the antisense plants may not be an impediment as long as the plants have had sufficient time to accumulate a sizable metabolic-pool. This metabolic pool may serve as a buffer such that changes in 4 C L activity do not greatly affect anthocyanin production. The dramatic increase in steady-state levels of CHS suggest that the activity of C H S is probably involved in the de  novo  biosynthesis of at least a portion of the observed  anthocyanins. It is possible that C H S is a very efficient enzyme such that the 4coumaroykCoA esters made are immediately drawn into the biosynthesis of flavonoids. During wounding, steady-state m R N A levels of  6PGDH,  DHS1,  EPSPS,  PAL,  C4H and 4CL increased to maximal levels 1 to 2 hours post-wounding. Although there has been some evidence that the genes encoding these enzymes are upregulated coordinately, these reports are derived from different plants under different stimuli (Chappell and Hahlbrock, 1984; Davis and Ausubel, 1989; Fahrendorf era/., 1995; Gorlach  et al.,  1995). In Arabidopsis,  this is the first report demonstrating the  coordinate regulation of genes encoding enzymes in the oxidative pentose phosphate 121  pathway, the shikimic acid pathway and the general phenylpropanoid pathway.  In the  4CL suppressed plants, the genes examined here were also coordinately regulated and responded to wounding irrespective of the significantly lower levels (>8%) of endogenous 4 C L activity. This suggests that gene activation is due to early events in the wound-signal and not a consequence of metabolic-flux through 4 C L . A similar conclusion was reported with elicitor-induced gene expression in the presence of chemical inhibitors of P A L (Gorlach  etal.,  1995; Fahrendorf  et al.,  1995). Conserved  c/'s-elements like the P-, A-, and L- boxes and putative Myb-binding sequences have been identified in the promoters of genes involved in phenylpropanoid metabolism thereby potentially providing a mechanism for the coordinate, yet independent, upregulation of gene expression (Lois era/., 1989; Grotewald  etal.,  1994; Sablowski  et  al., 1994; Logemann era/., 1995). Whether these c/'s-elements are found in the promoters of all the wound-induced genes described here is a question yet to be answered. Using antisense-RNA technology, transgenic  Arabidopsis  lines with suppressed  4 C L activity have been generated. The regulation of phenylpropanoid metabolism and the role of 4 C L in carbon flow into phenylpropanoid-product formation can be characterized using these 4CL-suppressed lines. Further and more detailed analysis of the biochemical- and stress-related phenotypes of these lines is beyond the scope of this thesis. Collaborative efforts with other laboratories have been initiated to examine the lignin, phenolic ester, and flavonoid composition of the wild-type and transgenic  Arabidopsis  and transgenic  lines. A s well, the response to pathogen attack in the wild-type  Arabidopsis  lines is under investigation.  122  Chapter 6 Conclusions and Future Directions  The work presented here represents a broad examination of 4 C L from two model plants. At the level of the DNA, it was shown that 4 C L was encoded by a genefamily in tobacco and by a single gene in Arabidopsis  (chapter 3). This difference in  genome-organization is interesting in that it shows that one copy of 4CL per haploid genome is sufficient; however in plants like tobacco, multiple copies of 4CL exist. Because 4 C L is encoded by a single gene in Arabidopsis, identify genetic mutants of Arabidopsis Arabidopsis  it may be possible to  that are defective at the 4CL locus. The  research community has made available T-DNA tagged lines which may  aid in the identification of 4CL null-mutants (McKinney era/., 1995), a phenotype that was not achieved in the antisense-RNA suppressed lines reported here. Sequencing of the entire  Arabidopsis  genome and anonymous c D N A clones (expressed sequence  tags, E S T s ; Newman et al., 1994) will eventually result in the theoretical availability of all  Arabidopsis  genes and cDNAs.  Using northern blot analysis, 4CL expression was examined at the level of the R N A (chapter 3). In tobacco, 4 C L is encoded by at least 2 classes of genes; however, members of the classes did not appear to be differentially expressed. The two divergent 4CL genes were both expressed in tobacco and were both detected in the progenitor species suggesting that multiple-copies of 4CL exist and that it may even confer an evolutionary advantage. The expression pattern of 4CL in tobacco and Arabidopsis  was comparable to the expression of other phenylpropanoid genes and  for the most part, can be correlated with the location where phenylpropanoid-products would be expected to accumulate. Of particular interest is the high levels of 4CL R N A transcripts in the unpigmented portion of the petals of tobacco flowers. This result is in 123  contrast to the many transgenic studies where phenylpropanoid promoters directed  GUS  expression in the pigmented portion of the petals and no G U S staining in the  white part of tobacco petals (Bevan Hauffe  et al.,  1989; Liang  etal.,  etal.,  1989; Schmid  1991). However, in agreement with the results found here,  etal.,  1990,  in situ  hybridization (Reinold era/., 1993) clearly show accumulation of 4CL transcripts in the white part of the flower. One area of further investigation would be to elucidate the function of 4 C L and P A L in the white portion of tobacco petals. Recently, flavonoidspecific staining has shown high levels of flavonoids in the white part of tobacco petals (Reinold, 1995) suggesting that 4CL and PAL may be expressed in this tissue for flavonoid biosynthesis. The characteristics of the 4 C L proteins were examined by expressing the two divergent tobacco  4CL  c D N A s in E.  coli  (chapter 4). The two recombinant-4CL  proteins had almost identical enzyme activities and did not use sinapate as a substrate. This latter result was particularly enigmatic since crude tobacco-stem extracts contained 4 C L activities which use sinapate as a substrate and results in the literature demonstrated high levels of syringyl lignin in tobacco stems (Halpin era/., 1994). W e found evidence of other divergent 4CL-genes (genomic Southern blot analysis and Genbank sequences) and this represents an area of further investigation. Cloning and characterization of these highly divergent tobacco-4CL genes  (4CL3  class genes) may provide evidence of differential expression of the tobacco 4CL genes and more importantly, expression of class 3 4CL c D N A s may generate 4 C L isoenzymes which use sinapate as a substrate. If a sinapate-specific 4 C L isoenzyme was identified, the gene(s) encoding for the isoenzyme may be used to engineer plants with higher levels of syringyl lignin, a biotechnologically desirable trait. Expression of the  4CL  c D N A s in E.  coli  allowed us to identify a novel activity  against the 4 C L which may involve the post-translational modification of 4CL. The putative post-translational modification has been characterized sufficiently to suggest  124  that a kinase is involved in determining the substrate specificity of tobacco 4 C L . Further studies might include: 1. Determine if 4 C L itself is phosphorylated or if another protein is phosphorylated resulting in altered 4 C L substrate-utilization. 2. Purify the putative kinase and clone the corresponding gene using generated antibodies or synthesized oligonucleotides derived from protein microsequencing data. 3. Determine when and where endogenous tobacco 4 C L utilizes cinnamate as a sustrate and link the biochemistry with the physiology. Namely, what products are made from cinnamoyhCoA esters and how do these downstream products relate to the biology of the plant? Using antisense RNA, we have generated, for the first time ever, transgenic Arabidopsis  lines with severely suppressed 4 C L levels and we have begun  characterizing these transgenic lines. A particularly unique result is the apparently normal accumulation of anthocyanins in antisense-4CL suppressed plants. Downregulation of PAL by sense suppression in tobacco (Elkind et al., 1990) and chemical inhibitors of P A L activity (Amrhein and Hollander, 1979) resulted in a decrease in anthocyanin accumulation.  DFR  Arabidopsis  mutants with defective  CHS  (tt4), CHI (tt5),  or  (tt3, ttg) fail to accumulate anthocyanidins in the seeds, the seedlings and the  leaves (Shirley et al., 1995). The results presented here, show for the first time that a decrease in 4 C L enzyme activity by >90% did not decrease anthocyanin accumulation. Further studies are required before we can understand the basis of this unexpected result. It is possible that alternative pathways exist for the biosynthesis of anthocyanins that by-pass the 4CL-catalyzed reaction. Antisense suppression of COMT  did not decrease lignin levels and this provided support that an alternative  pathway, involving C C o A O M T , in monolignol biosynthesis existed (Atanassova era/., 1995; Van Doorsselaere  etal.,  1995). Another possibility is that the 8 % residual 4 C L  enzyme activity in the antisense 4CL suppressed lines may be sufficient to allow for 125  wild-type levels of anthocyanins to be made. One way to resolve this would be to identify genetic mutants of 4CL which completely lack 4 C L activity. Since 4 C L is encoded by a single gene in Arabidopsis,  such a mutant can, in theory, be isolated.  However, since 4 C L is important in lignin biosynthesis (Figure 5.10), it is conceivable that such a mutation may be lethal. Another area which deserves analysis is plant-pathogen interactions. PALsuppressed tobacco plants have been shown to be more susceptible to pathogen invasion possibly because of diminished levels of pre-existing chlorogenic acid (Maher  et al.,  1994). Is this phenomenon paralleled in the antisense-4C/_  Arabidopsis  plants? The antisense-4CL plants described here are distinct from the PALsuppressed plants since furanocoumarins, phenolic acids, and other phenylpropanoid derivatives are/may be made after the PAL-catalyzed step but before the 4CLcatalyzed step (see section 1.3.3) and the response of antisense-4CL plants to pathogens may be different from the response in P>4L-suppressed plants. 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