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Cloning and expression of genes encoding divergent 4-coumarate : CoA ligase in poplar Cukovic, Daniela 1999

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CLONING AND EXPRESSION OF GENES ENCODING DIVERGENT 4-COUMARATE : CoA LIGASE IN POPLAR By DANIELA CUKOVIC B.Sc,  University of Sarajevo, 1991  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In T H E FACULTY OF GRADUATE STUDIES (Department of Botany) We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH COLUMBIA August 1999 © Daniela Cukovic, 1999  ln  presenting  degree freely  at  this  the  thesis  University  in" partial of  available for reference  copying  of  department publication  this or of  thesis by  this  for  his thesis  or  British and  her  Columbia,  I agree  study.  I further  Columbia  the  requirements that the  agree  may be  representatives.  for financial  Department  DE-6 (2/88)  of  scholarly purposes  permission.  The University of British Vancouver, Canada  fulfilment  gain shall  It not  is  that  Library  an  advanced  shall  make it  permission for  granted  by  understood be  for  allowed  the  extensive  head  that without  of  my  copying  or  my written  Abstract The enzyme 4-coumarate:Coenzyme A ligase catalyzes the third and final step in the general phenylpropanoid metabolic pathway, providing activated thioester substrates for different phenylpropanoid products. To clone putative divergent members o f the poplar 4CL gene family, degenerate primers directed to a part o f the first exon, containing a putative A M P - b i n d i n g motif, were used. Three new 4 C L - l i k e classes were distinguished among 72 cloned P C R amplification products. The new 4 C L - l i k e sequence classes, arbitrarily named 4CL6, 4CL10 and 4CL14, shared 6 1 % to 96% deduced amino acid sequence identity over the 600 bp region compared. A full-length c D N A clone oi4CL6  was isolated by screening a  xylem c D N A library with the P C R fragment. Isolation o f a full-length c D N A clone o f 4CL10 involved both screening a young leaf c D N A library, and 5' and 3' R A C E .  Sequence  analysis showed that the most divergent member o f the poplar 4CL gene family is 4CL10 (61% -65% amino acid identity with others), while 4CL6 is moderately divergent to previously isolated members. R N A blot analysis showed that the 4CL6 gene is expressed exclusively in xylem, green stem and root, while 4CL10 expression is restricted to young leaf, old leaf and root. Recombinant enzymes corresponding to the 4CL6 and 4CL10 genes were expressed using a baculovirus system. The recombinant 4 C L 6 was partially purified by F P L C , and showed a substrate utilization profile (4-coumaric acid > ferulic acid > caffeic acid > cinnamic acid, and no activity toward sinapic acid) similar to the profiles o f the previously studied native and recombinant 4 C L proteins.  ii  Table of Contents  Abstract  ii  Table of Contents  iii  List of Figures and Tables  v  List of Abbreviations  vii  Acknowledgements  ix  CHAPTER I  Introduction  1  1.1  General Phenylpropanoid Metabolism  2  1.2  4-Coumarate:CoA Ligase  5  1.3  4CL Gene Families  7  1.4  Phenylpropanoid Metabolism i n Poplar  9  1.5  Poplar 4CL Gene Family  11  1.6  Lignin  12  CHAPTER II  Materials and Methods  16  2.1  Plant Material  16  2.2  Preparation o f Genomic D N A for P C R Analysis  16  2.3  P C R Search o f Poplar Genomic D N A  16  2.4  Screening c D N A Library  17  2.5  Rapid Amplification o f 5 ' - c D N A and 3 ' - c D N A Ends  18  2.6  D N A Sequencing and Sequence Analysis  19  2.7  R N A Extraction and Northern Blot Analysis  19  2.8  Generating Recombinant 4 C L 6 Baculovirus Particles  20  2.9  Expression and Purification o f Recombinant 4 C L  21  2.10  4 C L Enzyme Assays  21  2.11  C H A P T E R III  S D S - P A G E and Immunoblots  Results Cloning Divergent 4CL Genes  23  3.1.1  P C R Based Search  23  3.1.2  Cloning Full-Length c D N A Clones  29  3.1.3  Sequence Analysis  33  3.2  Analysis o f Divergent 4CL Genes Expression  39  3.3  Characterization o f Recombinant 4 C L Proteins  41  3.3.1  Expression and Purification o f Recombinant 4 C L 6  3.3.2  41  Substrate Utilization Profile o f Recombinant 4 C L 6 Protein  Bibliography  23  3.1  and 4 C L 1 0 Proteins  CHAPTER IV  22  Discussion  45 46  56  List of Figures and Tables  Figure 1 The reactions of general phenylpropanoid metabolism Figure 2  3  Schematic diagram of the biosynthesis o f flavonoid and lignin precursors ... 4  Figure 3 Generic 4CL gene and degenerate primers  24  Figure 4 P C R amplification o f 4 C L gene fragments from P. trichocarpa  using different  degenerate primers  26  Figure 5 Restriction map o f the P C R amplified D N A fragments (-600 bp) o f poplar 4CL genes  27  Figure 6 Alignment of deduced amino acid sequences o f P C R fragments o f poplar 4CL genes  :  28  Figure 7 Nucleotide sequence o f 4CL6 c D N A clone  30  Figure 8 The template and primers i n R A C E reactions and amplification o f the full-length c D N A  32  Figure 9 Nucleotide sequence o f 4CL10 c D N A clone Figure 10 Comparison o f deduced amino acid sequence of4CL6  33 and 4CL10 c D N A  clones  34  Figure 11 Alignment of deduced amino acid sequences o f poplar 4CL c D N A clones  37  Figure 12 Alignment of deduced amino acid sequences o f poplar 4CL6 and 4CL10 c D N A clones and other ATP-dependent enzymes Figure 13 Northern-blot analysis of4CL6  38  and 4CL10 m R N A levels  40  V  Figure 14 Partial purification o f recombinant 4 C L 6 protein b y F P L C on an High-Q ion exchange column  42  Figure 15 S D S - P A G E and immunoblot analysis o f recombinant 4 C L 6 protein  43  Figure 16 S D S - P A G E analysis o f recombinant 4 C L 1 0 protein  44  Figure 17 Substrate-utilization profile o f recombinant 4 C L 6  45  Table I  29  Putative 4CL gene fragments amplified by P C R  Table II Comparison o f 4 C L 6 and 4 C L 1 0 predicted amino acid sequences to each other and to other poplar 4 C L sequences  35  Table III Comparison o f 4 C L 6 and 4 C L 1 0 predicted amino acid sequences to each other and to other 4 C L sequences  36  VI  List of Abbreviations  A c M - N P V Autographa californica nuclear polyhedrosis virus A M P adenosine monophosphate A P I adaptor primer A T P adenosine triphosphate B S A bovine serum albumin C A D cinnamyl alcohol dehydrogenase C C o 3 H 4-coumaryl:CoA 3-hydroxylase C C o O M T caffeoyl:CoA 0-methyltransferase C C R cinnamoyl C o A reductase C 3 H 4-coumarate 3-hydroxylase C 4 H cinnamate-4-hydroxylase 4 C L 4-coumarate:CoA ligase C H S chalcone synthase C o A S H coenzyme A C O M T catechol O-methyltransferase c D N A complementary D N A ds c D N A double stranded c D N A F 5 H ferulate 5-hydroxylase F P L C fast protein liquid chromatography G S P gene-specific primer H I 1 poplar hybrid {Populus trichocarpa X Populus deltoides)  vii  M O I multiplicity o f infection N G S P nested gene-specific primer d N T P deoxynucleotide triphosphate P A L phenylalanine ammonia lyase P C R polymerase chain reaction pfu plaque forming unit PPi  pyrophosphate  R A C E rapid amplification o f c D N A ends R A P D randomly amplified polymorphic D N A R F L P restriction fragment length polymorphism S D S - P A G E sodium dodecyl sulfate polyacrylamide gel electrophoresis Sf9 cells insect cells o f Spodoptera furgipedra, line 9  viii  Acknowledgement  I would like to thank Dr. Carl Douglas for giving me the opportunity o f doing a master o f science degree under his supervision, for his guidance i n developing this thesis research, and for his financial assistance that he provided over the course o f three summers. I am greatly indebted to Dr. Brian Ellis and Dr. Beverley Green for their suggestions and guidance i n developing this thesis research. I would like to thank Dr. Elizabeth Molitor for introducing me to the lab, and thanks to the Douglas, Ellis, Haughn and Kunst lab members for a co-operative and fun environment. M y special gratitude goes to Amrita Singh for providing degenerate primers and sharing P C R experience, to Dr. Sandra A l l i n a and Dr. Cindy Shippam-Brett for their assistance with the baculovirus vector expression system, and to Janis Pereira for her assistance with the F P L C work.  ix  CHAPTER I Introduction  In plants, the phenylpropanoid pathway is responsible for the formation o f several classes o f chemical compounds including coumarins, flavonoids, stilbenes, suberin, lignin and other cell-wall associated phenolics. The functions o f phenylpropanoid derivatives are as diverse as their structural variations. Phenylpropanoids serve as flower pigments (anthocyanins), antimicrobial compounds (isoflavans, isoflavonoids, stilbenes, psoralens, coumarins and flavonols), U V protectants (anthocyanins, flavones, psoralens and isoflavonoids) and signal molecules in symbiotic interactions (flavonoids). Phenylpropanoids with a complex heteropolymer structure, lignin and suberin, function as water-impermeable diffusion barriers in dermal tissues and vascular tissue; they are also induced in response to wounding (Hahlbrock and Scheel 1989; D i x o n and Paiva 1995). Phenylpropanoid metabolism can be divided into a general pathway, required for the synthesis o f all phenylpropanoid metabolites, and specific branch pathways, which lead to synthesis o f specific phenolic end products (Douglas et al., 1992). The biosynthesis o f phenylpropanoid compounds is developmentally activated i n specific tissues and cell types, but can be induced b y various biotic and abiotic stresses. The evolutionary development o f phenylpropanoid metabolism must have been a critical juncture in the transition o f plants from an aquatic to a terrestrial environment, since it provided vascular plants with ability to transport water and nutrients and to cope with new abiotic and biotic stresses (Davin and Lewis, 1992).  1.1 General Phenylpropanoid Metabolism The central pathway o f phenylpropanoid metabolism consists o f three core reactions (Figure 1). The aromatic amino acid phenylalanine is deaminated by the enzyme phenylalanine ammonia lyase ( P A L ) to produce cinnamic acid. The next enzyme i n the pathway, cinnamate-4-hydroxylase (C4H), uses O2 and N A D P F f to hydroxylate the 6membered aromatic ring o f cinnamic acid at the para-position, making 4-coumarate. The enzyme 4-coumarate: C o A ligase ( 4 C L ) catalyzes the last step i n the general phenylpropanoid pathway, formation o f Coenzyme A esters ofp-coumaric acid and other hydroxy- or methoxy- derivatives o f cinnamic acid, such as caffeic acid, ferulic acid, and sinapic acid (Figure 2). Co A esters o f hydroxycinnamic acids serve as substrates for specific branch pathways. Thus, coumaryl-CoA is a substrate for the enzyme chalcone synthase (CHS), which catalyzes the branch point step i n the biosynthesis o f flavonoid compounds and coumaryl-CoA, feruloyl C o A and sinapyl C o A are reduced by cinnamoyl C o A reductase ( C C R ) , and directed to the biosynthesis o f lignin monomers. Hydroxylation and methylation o f the phenylpropanoid aromatic ring, important i n the generation o f different monolignols, can occur at the level o f free acids, or at the level o f the corresponding CoA-esters (Figure 2). The traditional view o f the pathway has been that 4coumaric acid is hydroxylated at the 3-position by 4-coumarate 3-hydroxylase (C3H). Enzymes that carry out this hydroxylation reaction i n vitro have been detected, but little is known about their properties or physiological role (reviewed b y Whetten and Sederoff, 1995).  2  (ISOFLAVONOIDS  SOLUBLE ESTERS  COUMARINS  A  1  1  i  I  I  GENERAL PHENYLPROPANOID COOH  L-Phenylalanine  COOH  Cinnamic acid  METABOLISM  CQOH  OH  OH  4-Coumaric acid  4-Coumaroyl-CoA  I  • WALL-BOUND PHENOLICS  LIGNIN  | SUBERIN  STILBENES  Figure 1. The reactions o f general phenylpropanoid matabolism. Dashed arrows indicate branch pathways emanating from the general pathway. P A L , phenylalanine ammonia-lyase; C 4 H , cinnamate 4-hydroxylase; 4 C L , 4-coumarate:CoA ligase (Hahlbrock and Scheel, 1989).  3  COOH  COOH  COOH  COOH  COOH  r  4-Coumaric add  Cafleie add  J4CL |4CL  J4CL I4CL  OCHj H 0 ^ O 3 C H , HaCO'V^OCHs OH Farufic add 5-Hydroxyfenjllc add Slnaptcadd  J4CL 4CL  | |4CL  |4CL  VcoA  OCH3 H 0 ' " ^ O 3 C H  3  H3CO'  OH ~ ~ "™ O H " " " ~ OH 4-CouniaroyCCcA CafleoytCoA FetutoytCoA S-HydroxyfecutoytCoA SlnapoytCoA  CHS/  Naringenin  Flavonoids  y  5H  CCR  CCR  CCR  CAO  CAO  CAD  CHjOH  CH OH  CH OH  2  COb OH Conifaryl alcohol  J  0  0  1  3  I  CCR  CAO  CH OH  2  2  HO'X.'CCHa H CO OH OH 5-Hydroxyferulyl Sinapyl alcohol alcohol 3  Ugnin  Figure 2. Schematic diagram o f the biosynthesis o f flavonoid- and lignin- precursors. The identification o f novel enzymes suggests that the biosynthesis o f C o A esters from 4coumarate may proceed through a number o f enzymatic steps potentially resulting i n a metabolic grid rather than a linear pathway. C 3 H , 4-coumarate 3-hydroxylase; C O M T , caffeic acid/5-hydroxyferulic acid O-methyltransferase; F 5 H , ferulate 5-hydroxylase; C C o 3 H , 4-coumaroyl:CoA 3-hydroxylase; C C o O M T , caffeoyhCoA O-methyltransferase; ?, uncharacterized metabolic step; C H S , chalcone synthase; C H I , chalcone isomerase; C C R , cinnamoyl-CoA reductase; C A D , cinnamyl alcohol dehydrogenase. Dashed arrows represent enzymatic steps, which have not been clearly demonstrated (Whetten and Sederoff, 1995).  4  A n alternative to hydroxylation o f free 4-coumarate has been supported b y discovery o f a 4-coumaroyl/caffeoyl-CoA hydroxylase (CCo3H) i n an anthocyanin mutant o f Silene dioica, but the potential involvement o f C C o 3 H i n monolignol biosynthesis has not yet been adequately tested (reviewed by Whetten and Sederoff, 1995). The 3- and 5-methylation o f the aromatic ring o f free acids is catalyzed by catechol Omethyltransferase ( C O M T ) , which i n angiosperms utilize both caffeic acid and 5hydroxyferulic acid (reviewed by Whetten and Sederoff, 1995). Y e et al. (1994) showed that a distinct O M T enzyme, caffeoyl C o A 3-O-methyltransferase ( C C o O M T ) , acts at the level o f the caffeoyl-CoA ester and 5-hydroxyferuloyl-CoA during monolignol biosynthesis (Figure 2). This portion o f the monolignol biosynthesis pathway appears to be more o f a network or grid than a linear pathway (Whetten et al., 1998). Hydroxylation o f ferulate to 5-hydroxyferulate, thought to be catalyzed by ferulate 5hydroxylase (F5H), appears to be a key step in sinapyl alcohol and syringyl lignin biosynthesis. A mutant (fah-I) in Arabidopsis  shows a lack o f sinapate-derived residues i n  lignin (Chappie et ah, 1992). Meyer et al. (1998) showed that overexpression o f the F 5 H gene from the C a M V 3 5 S , or AtC4H promoter leads to biosynthesis o f a large amount o f syringyl lignin. Characterization o f the catalytic properties o f recombinant F 5 H protein has recently revealed that F 5 H has the best activity toward coniferyl aldehyde rather than free ferulic acid or feruloykCoA (Chappie, C C , unpublished data).  1.2 4-Coumarate:CoA Ligase The last step o f general phenylpropanoid metabolism is the activation o f cinnamic acids to form C o A thioesters. This reaction is catalyzed by 4-coumarate:CoA ligase  5  (E.C. the group o f acid-thiol ligases) and proceeds v i a a two-step process involving an acyl adenylate intermediates, i n analogy to the activation o f fatty acids with A T P and C o A S H according to the following equations (Gross, 1985): Mg  2 +  4CL + hydoxycinnamic acid + ATP -> 4CL • hydroxycinnamyl-AMP + PPi 4CL • hydroxycinnamyl-AMP + CoASH -* 4CL + cinnamoyl-CoA + ATP 4 C L activity requires the presence o f A T P as a cosubstrate M g  2 +  as a cofactor, and C o A S H  (Gross, 1985). Knobloch and Hahlbrock (1977) showed a sigmoidal dependence o f parsley 4 C L activity on A T P and C o A S H , indicating an alio steric character for this enzyme. During the 1970's and the early 1980's classical biochemical techniques were used to isolate and purify different C o A ligase isoforms from various plant species, such as Forsythia, soybean, Petunia, parsley, pea, carrot, spruce, Erythrina crista-galli, poplar and maize (Gross and Zenk, 1974: Knobloch and Hahlbrock, 1975; Ranjeva et al., 1976; Knobloch and Hahlbrock, 1977; Heinzmann et al, 1977; Wallis and Rhodes, 1977; Luderitz et al, 1982; Kutsuki et al, 1982; Grand et al, 1983; Vincent and Nicholson, 1987). The enzyme is monomeric and molecular weights o f 40 k D a {Erythrina crista-galli), 55 k D a (soybean, Forsythia), 61 k D a (parsley) and 75 k D a (pea) were estimated. Physically distinct 4 C L isoforms have been reported from soybean, Petunia, pea, poplar, carrot and mesocotyl o f maize, (Knobloch and Hahlbrock, 1975; Ranjeva et al, 1976; Wallis and Rhodes, 1977; Grand et al, 1983; Heinzmann et al, 1977; Vincent and Nicholson, 1987). A l l 4 C L isoforms examined to date have the highest activity toward 4coumaric acid. However, i n soybean, Petunia, and poplar, partially purified 4 C L isoforms exhibit different substrate specificity toward substituted cinnamic acids (Knobloch and Hahlbrock, 1975; Ranjeva et al, 1976; Grand et al, 1983). It has been hypothesized that  6  4 C L , with its ability to utilize a number o f related substrates, could control the partitioning o f carbon into different branch-pathways through the activity o f distinct 4CL-isoforms (Knobloch and Hahlbrock, 1975; Grand et al, 1983). For example, i n Petunia leaves one isoenzyme preferentially utilizes caffeic acid (caffeate: C o A ligase), leading to the suggestion that it was involved in caffeic acid ester formation. The second isoenzyme (ferulate: C o A ligase) could be involved in guaiacyl lignin biosynthesis, and the third enzyme, which utilized sinapic acid, could take part i n the formation o f syringyl lignin (Ranjeva et al, 1976). In contrast, a single 4 C L form was purified from Forsythia, Erythrina crista-galli, maize (leaves) and loblolly pine (Gross and Zenk, 1974; Luderitz et al., 1982; Hipskind et al, 1993; V o o et al, 1995), suggesting that 4 C L does not participate i n the metabolic channeling o f phenylpropanoid derivatives i n these species.  1.3 4CL Gene Families More detailed knowledge about 4 C L isoforms was provided b y cloning o f 4 C L genes during the 1980's and 1990's. 4 C L is encoded b y multiple divergent genes i n some plants like rice, soybean, poplar, Lithospermum erythrorhizon, tobacco and aspen (Zhao et al, 1990; Uhlmann and Ebel, 1993; A l l i n a and Douglas, 1994; Y a z a k i et al, 1995; Lee and Douglas, 1996; H u et al 1998); by very similar duplicated genes as i n the case o f parsley, potato and loblolly pine (Lozoya et al, 1988; Becker-Andre et al, 1991; Zhang and Chiang 1997 ); and apparently by a single-gene i n Vanilla planifolia (Brodelius and X u e , 1997). Interestingly, Arabidopsis 4 C L was previously assumed to be encoded b y a single-copy gene  7  (Lee et al, 1995), but two divergent 4CL classes have since been cloned i n Arabidopsis by Ehitingefa/.(1999). Sequence comparisons between deduced amino acid sequences o f 4CL c D N A s o f potato, parsley, soybean, rice and tobacco, and other cloned 4CL genes (Becker-Andre et al., 1991; Uhlman and Ebel, 1993; Lee and Douglas, 1996) indicate that the 4 C L proteins contain a highly conserved seven amino acid motif " G E I C I R G " clustered around a conserved cysteine residue. This motif is conserved i n several apparently unrelated enzymes dependent on A T P , such as luciferase from firefly (deWet et al., 1987; Schroder, 1989); tyrocidin synthetase A and tyrocidin synthetase from Bacillus brevis, and may be associated with catalytic activity (Backer-Andre et al, 1991). A second conserved motif has been proposed (Bairoch, 1991) as a signature for a putative A M P - b i n d i n g domain that is common to a number o f prokaryotic and eukaryotic ATP-dependent enzymes. In addition, the predicted amino acid sequences o f 4 C L proteins from the plants listed above each contain a total o f six conserved cysteine residues. Genomic clones o f 4CL genes from rice, parsley and potato show the presence o f five exons (the first exon is longest) and four introns (Zhao et al, 1990; Lozoya et al, 1988; Becker-Andre et al., 1991). Zhang and Chiang (1997) reported that two loblolly pine 4CL genes have three introns, whereas Arabidopsis 4CL genomic sequences have four introns at the conserved positions, and one o f them contains three additional introns (Ehlting et al. 1999). 4CL gene expression, like that o f many o f the phenylpropanoid genes, is regulated developmentally and is also activated by external stimuli such as pathogen infection, elicitor treatment, wounding, and U V - l i g h t irradiation (Douglas et al, 1987; Schmelzer et al, 1989;  8  W u and Hahlbrock, 1992). In tobacco flowers, in situ hybridization shows that endogenous tobacco 4CL transcripts and those o f an introduced parsley 4CL1 gene accumulate in a celltype specific manner, and that the patterns o f accumulation are generally consistent with the sites o f phenylpropanoid natural-product accumulation (Reinold et al., 1993). A s well, 4CL expression i n tobacco is activated by wounding, light, and methyl jasmonate treatment (Douglas et al., 1991; Ellard-Ivey and Douglas, 1996). The 4CL genes from parsley and soybean are differentially regulated. 4CL2 from parsley is preferentially expressed in the flowering stem and is light inducible, whereas 4CL1 is wound inducible in roots (Lois and Hahlbrock, 1992). In soybean, 4CL16 is inducible by fungal infection whereas 4CL14 is not (Uhlmann and Ebel, 1993).  1.4 Phenylpropanoid Metabolism in Poplar Phenylpropanoid metabolism plays an important role i n the growth and development o f woody plants, since an important component o f wood is lignin. For example, between 2 1 % to 23 % o f P. trichocarpa wood dry weight is lignin (Swan and Kellogg, 1986). M a n y derivatives o f phenolic compounds have been isolate from the bark o f P.  trichocarpa  such as salicin, trichocarpin, salireposide, salicyl alcohol, cinnamic acid, 4-coumaric acid and others (Pearl and Darling, 1968). B u d exudate o f P. deltoides is predominantly composed o f flavanones, chalcones and ester o f flavanones together with the flavone galangin, the flavanon pinocembrin and the flavanonol pinobanksin (Greenaway et al., 1990).  In contrast, the major phenolic compounds i n bud exudate o f P . trichocarpa  are  dihydrochalcone, benzyl salicylate, cinnamic acid, and minor amount o f flavanones, chalcones and flavones (English et al, 1991).  9  Shain and M i l l e r (1982) reported that pinocembrin (5,7-dihydroxyflavanone), as a major component o f poplar bud exudate is active against a fungal pathogen Melampsora medusae. Thus, a sufficient amount of pinocembrin is present on the surface o f young, expanding leaves of P. deltoides contributing to their resistance to M. medusae. A s leaves age, however, the concentration o f pinocembrin is depleted as a result o f weathering, leaves expansion and insufficient replenishment, and leaves become more susceptible to the fungal infection. Induction o f defense response i n poplar is associated with induction of phenylpropanoid metabolism. Hybrid poplar suspension-cultured cells treated with elicitor showed a coordinated and transient increases in extractable P A L and 4 C L enzyme activity at about 7 h after elicitation (Moniz de Sa et al, 1992). These increases were proceeded by an accumulation in extractable cell wall-bound phenolic compounds. Despite o f the economic importance o f wood, little is known about the genetic control o f wood formation. Poplar is an useful organism as a pulp source for paper and as a model organism for the study of physiology, genetics and molecular biology o f a woody perennial. Poplar is easily propagated vegetatively, transformed by Agrobacterium tumefaciens and regenerated as a transgenic tree (Laple et al, 1992; Kajita et al, 1994). Furthermore, a three generation pedigree is available for genetic analysis, derived from a cross between Populus deltoides and P. trichocarpa. A Populus genome linkage map was constructed using R A P D and R F L P markers i n this pedigree (Bradshaw et al, 1994). Several phenylpropanoid genes have been characterized i n the genus Populus (poplars and aspens). P A L is encoded by a small gene family, two members o f which (PALI and PALI) are highly expressed in young leaves and stems, where large amounts o f soluble  10  phenylpropanoid products accumulate i n addition to lignin (Subramaniam et al, 1993). Two more divergent members o f the Populus gene family have recently been identified, which, i n contrast to PALI and PAL2, are strongly expressed i n older stems i n which secondary xylem is undergoing differentiation (Osakabe et al., 1995). This differential expression indicates that specific PAL gene family members could be used to direct phenylalanine into secondary metabolism i n different tissues. The studies b y Grand et al. (1983) strongly suggest the possible existence o f multiple 4 C L isoenzymes i n poplar. Three partially purified 4 C L isoenzymes were obtained from a poplar "euramericana" hybrid stems by chromatofocusing, using 4-coumaric acid as a substrate to measure enzyme activity. The molecular mass o f 41 k D a is similar to the value for 4 C L from Erythrina crista-galli (40 kDa), but lower then values determined for other plants (Grand et al., 1983). The partially purified poplar 4 C L isoenzymes described in that study showed differences i n substrate specificity. 4 C L 1 reacts with 4-coumarate, 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. The 4 C L activity was mainly localized i n lignified tissues, such as xylem and sclerenchyma (phloem fiber cells) (Grand et al, 1983).  1.5 P o p l a r 4CL Gene F a m i l y Recent characterization o f poplar 4 C L isoenzymes and members o f the 4CL gene family has been done i n a poplar hybrid, clone H I 1-11, derived from a cross between Populus trichocarpa and Populus deltoides. Two classes o f 4CL c D N A clones (4CL9 and 4CL216), that do not cross hybridize at high stringency, were isolated b y screening an H I 1 young leaf c D N A library with a parsley 4CL c D N A probe (Douglas et al, 1992; A l l i n a and  11  Douglas, 1994; A l l i n a et al, 1998). The clones showed about 86% identity at the nucleotide and amino acid levels. Genomic Southern blots revealed restriction fragments that strongly cross-hybridize under high stringency conditions specifically either with 4CL216 and 4CL9. However, several weakly hybridizing bands were visible, especially after low stringency hybridization, suggesting that the 4CL gene family might include a divergent gene(s) that weakly hybridize with 4CL9 and 4CL216. The results o f northern analysis indicated that the 4CL9 gene is expressed strongly i n young leaf and weakly i n old leaf, green stem and xylem, while the 4CL216 gene shows strong expression i n all those tissues. Neither the 4CL9 or the 4CL216 genes is expressed i n elicitor-treated suspension tissue culture cells. Expression o f recombinant 4 C L proteins from these c D N A clones showed that they have identical substrate utilization profiles (Allina et al., 1998). Furthermore, 4 C L forms from poplar hybrid H I 1-11, xylem, elicited cell culture, and young leaf were separated b y F P L C ion column exchange, indicating the presence o f 3-4 putative 4 C L isoforms. However, the different forms appear to have identical substrate utilization profiles (Allina et al., 1998). These results do not support previous evidence that three catalytically distinct 4 C L forms exist i n poplar.  1.6 Lignin Lignin (Latin: lignum - wood) is a three-dimensional heteropolymer, resulting from the dehydrogenative polymerization o f three different hydroxycinnamyl alcohols (monolignols): p-coumaryl, coniferyl and sinapyl alcohol, which differ i n the extent o f methylation. These monolignols become cross-linked b y about 20 types o f intersubunit linkages (Chen, 1991).  12  L i g n i n accumulates in the secondary wall and the middle lamellae o f specialized cells such as tracheids, vessel elements, xylem and phloem fibers, and sclereids. When the lignin deposition is complete, the protoplast degenerates leaving a dead cell protected against collapse b y its strengthened walls (Boudet et al, 1995). L i g n i n provides rigidity and hydrophobicity to the cell wall, which is particularly important for cells which conduct water. A l s o , stress-induced lignin deposition provides a mechanism for sealing off sites o f pathogen infection and wounding (Dixon and Paiva, 1995). The monomelic composition o f lignins is one o f the most important characteristics o f lignins from both a taxonomic and an industrial point o f view (Boudet, 1998). The chemical complexity o f lignin has increased from pteridophytes and gymnosperm lignins to the angiosperms. Grasses have lignin with the most complex composition (Boudet et ah, 1995). In conifers, lignin is typically composed only o f coniferyl alcohol (guaiacyl or G units), although, in compression wood, lignin is polymerized from coniferyl and a small portion o f p-coumaryl alcohol ( H units). This composition makes compression wood lignin less methylated and more difficult to hydrolyze (Higuchi, 1985). The typical lignin o f angiosperms is composed o f a mixture o f guaiacyl and syringyl units. The presence o f the more highly methylated syringyl units increases the ability o f hardwood lignin to be hydrolyzed (Sederoff et al, 1994). In monocotyledon plants, lignins contain all three monolignols, but they also have ester- and ether- linked hydroxycinnamic acids, which could be derived from L-tyrosine (Higuchi, 1985; Lewis and Yamamoto, 1990). Lignin composition and intermolecular linkages vary according to cell type, stage o f tissue development and individual cell wall layers (Davin and Lewis, 1992). For example, guaiacyl subunits predominate i n the xylem o f vascular bundles o f Arabidopsis stems, while  13  adjacent, heavily lignified sclerenchyma cells contain syringyl units (Chappie, et al. 1992; Meyer, etal. 1998). Monolignols are derived from C o A esters o f hydroxycinnamic acids v i a a two-step reduction process catalyzed by cinnamyl-CoA reductase ( C C R ) and cinnamyl alcohol dehydrogenase ( C A D ) (Griesbach, 1981). Once made, the lignin monomers may be glycosylated at the 4-hydroxy position and then transported to the site o f lignin deposition. The mechanism o f monolignol transport from cytoplasm to the cell wall is not well understood. In conifers it has been hypothesized that monolignol glucosides are stored in the vacuole, transported from symplast to apoplast, and then de-glycosylated (Whetten and Sederoff, 1995). The polymerization o f monolignols to make lignin is believed to be catalyzed by some combination o f peroxidase and laccase (reviewed by Campbell and Sedoroff, 1996; Whetten etal,  1998).  Proposed mechanisms that control lignin content and composition are (i) substrate specificity o f different isoforms o f monolignol biosynthetic enzymes (e.g. C O M T , 4 C L , C A D ) and (ii) transcriptional regulation o f genes encoding isoforms o f monomer specific pathways (e.g. P A L , 4 C L , C A D ) (Higuchi, 1985; Sederoff et al, 1994; Campbell and Sederoff, 1996). The existence o f 4 C L isoforms in some plants with different substrate utilization profiles has led to the proposal that such isoforms may help control carbon flow into different lignin monomer (Knobloch and Hahlbrock, 1975; Ranjeva et al, 1976; Grand etal, 1983). Characterization o f lignin content and composition i n mutants o f maize, sorghum, and Arabidopsis,  and i n transgenic tobacco, poplar and Arabidopsis  with suppressed enzyme  activities, has led to increased understanding o f the monolignol biosynthesis pathway, and o f  14  the mechanisms by which lignin composition is regulated in different plants. For example, lignin content remained mostly constant i n transgenic plants with suppressed O M T activity in tobacco and poplar (Atanassova et al., 1995, van Doorsselaere et al., 1995), suppressed C A D activity i n tobacco and poplar (Halpin et ah, 1994; Hibino et al. 1995; Baucher et al., 1996), suppressed 4 C L activity i n Arabidopsis (Lee et al., 1997), and ectopic F 5 H activity in Arabidopsis (Meyer et al., 1998). In contrast, changes i n lignin composition (increases or decreases i n the S : G ratio) have been observed i n many transgenic plants with altered enzyme activities generated by suppression o f P A L , 4 C L , C O M T and C C R expression or ectopic F 5 H expression (reviewed by Whetten et al., 1998). Therefore, the amount o f lignin in a plant and a given tissue can be maintained at a relatively constant level even when an enzyme normally involved i n the synthesis o f lignin precursors is suppressed or overexpressed. This response reflects the importance o f lignin to vascular function and mechanical support i n land plants, and indicates a high degree o f plasticity i n lignin biosynthesis. When this project was initiated, the nature o f the 4CL gene family i n poplar and other plants was poorly understood. While different isoforms had been described in many plants, the extent to which the enzymatic properties o f these isoforms differed was unknown. Furthermore, it was possible that genes encoding highly divergent isoforms might not have been detected due to lack o f cross-hybridization to cloned genes. The purpose o f m y project was to test the hypothesis that the poplar 4CL gene family contains, besides the already isolated genes, other genes that encode divergent 4 C L enzymes. It was further hypothesized that such divergent genes have different expression patterns, and that the corresponding isoenzymes have distinct substrate specificities.  15  CHAPTER II Materials and Methods 2.1 Plant Material Clonally propagated individuals of Populus trichocarpa 93-968 and poplar hybrid (P. trichocarpa Torr. & Gray X P. deltoides Marsh) H I 1 were used for organ and tissue isolation. Y o u n g leaves (0.5-2 cm in length), old (fully expanded) leaves and green (nonwoody) stems were harvested from H I 1 plants maintained i n growth chambers at 23°C in a 16-h light/8-h dark regime. Harvested material was immediately frozen i n liquid N2 and stored at - 8 0 ° C until use. Secondary xylem was isolated from field-grown trees as described previously (Allina et al, 1998). The suspension cultures o f H I 1 cells and elicitor treatments were prepared as described by M o n i z de Sa et al. (1992).  2.2.  Preparation of Genomic DNA for PCR Analysis Genomic D N A , used i n P C R reactions, was isolated from fresh leaves o f  chamber-grown P. trichocarpa cuttings by a C T A B method as described by Roger and Bendich(1988).  2.3.  PCR Search of Poplar Genomic DNA Poplar (P. trichocarpa) genomic D N A (-100 ng) was used as a template for P C R  amplification with 20 pmol o f each degenerate primer, 200 u M d N T P s , 1.5 m M M g C k and 2.5.units o f Taq D N A polymerase ( G i b c o - B R L ) i n 50 (j.1 reaction. Conditions for P C R runs with primer combinations 3 & 6 and 3 & 8 were 9 4 ° C / 1 0 min, 35 cycles o f 94°C/ 50 sec, 55°C/50 sec, 72°C/70 sec, and 72°C/10 min. Primer combinations 3 & 2 and 3 & 4 were  16  used under c o n d i t i o n s : ' 9 4 ° C / 1 0 min, 3 cycles o f 94°C/ 50 sec, 50°C/50 sec, 72°C/70 sec, 30 cycles o f the same conditions as previously described. P C R reactions without the template, or reactions with one primer and the template were negative controls for each P C R run. P C R fragments were digested with Bam H I and Xbal, and subcloned into a pBluScript plasmid.  2.4 Screening c D N A L i b r a r y About 4 x 10 recombinant phage from a poplar xylem c D N A library i n A.ZAPII 5  (prepared b y Y u j i Tsutsumi) were screened using the 600 bp P C R fragments oi4CL6, 4CL10 and 4CL14 as described by Sambrook et al. (1989). Probes were radioactively labelled using the Random Primer D N A Labelling System ( G i b c o - B R L ) according to the manufacturer's specifications. Hybridized filters were washed at high stringency (0.2 X S S C , 0.1% S D S , 65°C) for 1 h. After purification o f positive plaques, a single positive clone obtained was subjected to i n vivo excision to yield pBluescript phagemid with helper phage strain E x Assist (Stratagene). The clone was double digested with^Tjal a n d r o l , and separated on 1% agarose gel, followed by partial sequencing o f the 5' and 3' ends o f the clone. It was shown that the clone was a full-length (~ 2 kb) c D N A o f 4CL6. The same approach was applied i n screening a A,ZAPII (Stratagene) H I 1 young-leaf c D N A library (Subramaniam et al., 1993) with the 600 bp 4CL10 P C R fragment. Eight positive plaques were purified and further characterized. A l l clones contained an incomplete (~ 1000 bp) c D N A fragment o f 4CL10.  17  2.5 R a p i d A m p l i f i c a t i o n of 5 ' - c D N A and 3 ' - c D N A E n d s Total R N A was isolated from young leaves o f hybrid poplar H I 1 by the method o f Hughes and Galau (1988) from young leaves. Purification o f poly A + R N A from total R N A was based on a protocol using dynabeads (Dynal). A library o f uncloned, adaptor ligated double strand c D N A was generated using Marathon c D N A amplification kit (Clontech Laboratories, Inc.) and polyA+ R N A from young leaves. Taq polymerase with high fidelity (Boehringer) and the adaptor ligated c D N A , as a template, were used in P C R o f 5 ' R A C E and 3' R A C E . The first P C R o f 3 ' R A C E was performed with the adaptor primer A P I (5' - C C A T C C T A A T A C G A C T C A C T A T A G G G C - 3') and a gene specific primer 4CL10.2 at the position 950 (5' - A G A A C C C A A T G G T G G C G A A C T T C G A C - 3'). To confirm that the amplified region was derived from the desired target, the second P C R o f 3 ' R A C E was performed with A P I and a nested gene-specific primer, 4CL10.4 at the position 1067 (5' - G G G A C A G G G T T A T G G G A T G A C A G A G - 3'). Conditions for both P C R runs were 94°C/1 min, 30 cycles o f 94-°C/ 30 sec, 65°C/30 sec, 68°C/2 min. A n antisense gene-specific primer (GSP), 4CL10.7 at the position 1125 (5' - A G G C T A A G C A C A T T G A T A G C A C T G G C - 3'), and A P I were used in P C R o f 5 ' R A C E under these conditions: 94°C/1 min, 30 cycles o f 94 °C /30 sec, 62°C/30 sec, 68°C/3 min. Primers 4CL10.0 at the position 1 (5' - A T G A T G T C C G T G G C C A C G G T T G A G -  3') and 4CL10.9 at  the position (5' - G G G C A C A A T G A G T G A A G A C A A C A C A - 3') were used to amplify fulllength c D N A o f 4CL10 (conditions: 94°C/1 min, 30 cycles o f 94 °C /30 sec, 60°C/30 sec, 68°C/3 min). The amplified D N A fragments were cloned into the pCR2.1 plasmid vector using the TA-cloning kit (Invitrogen).  2.6.  DNA Sequencing and Sequence Analysis P C R fragments and c D N A clones were sequenced by the University o f British  Columbia Nucleic Acid-Protein Service Unit using the P R I S M Ready Reaction DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems) and D N A sequencer 373 (Applied Biosystems). Sequencing was carried out on D N A isolated either b y a modified mini alkaline/lysis/PEG precipitation procedure (suggested by the N A P S ) or by a Qiagen plasmid kit. D N A and predicted amino acid sequences were analyzed using University o f Wisconsin Genetics Computer Group (Medison) software. Sequenced were searched against the GenBank non-redundant protein and E S T database (National Center o f Biotechnology Information) using B L A S T X . Multiple sequence alignment was obtained using Clustal-W program (Baylor College o f Medicine) and B O X S H A D E . Predicted molecular weight o f deduced amino acid sequences was calculated by software Compute p I / M w Tool (http://www.expasy.ch/tools/pi_tool.html).  2.7 RNA Extraction and Northern Blot Analysis Total R N A was isolated by the method o f Hughes and Galau (1988) from young leaves, old leaves, green stem and root o f poplar hybrid clone H I 1. A trizol reagent ( G i b c o - B R L ) was used to extract total R N A from secondary xylem and the suspension cultures o f H I 1 cells, according to the manufacturer's recommendations. R N A (10 yig per lane) was denatured by 50% formamide and 2.2 M formaldehyde at 65°C for 15 min.; separated by 2.2 M formaldehyde and 1.2% agarose gel electrophoresis; rinsed with water for 45 min; stained with 0.5 u,g/ml and blotted onto H y b o n d - N nylon membrane (Amersham). Probes generated from 4CL6 and 4CL10 c D N A clones (1.9 kb), were radioactively labelled using  19  the Random Primer D N A Labelling System ( G i b c o - B R L ) according to the manufacturer's specifications. Prehybridization and hybridization were performed at 65°C for 16 h i n 7% SDS sodium phosphate buffer (Church and Gilbert, 1984). The membranes were washed at high stringency (0.2 X S S C , 0.1% S D S , 65°C) for 1 h, and autoradiographed for 2 days (the blot probed with 4CL6) or 5 days (the blot probed with 4CL10). To demonstrate evenness o f loading between lanes, the blots were stripped and rehybridized with a probe for a pea r R N A (Jorgensen et al, 1982).  2.8 Generating Recombinant 4 C L 6 B a c u l o v i r u s Particles To subclone the full-length c D N A clones o f 4CL6 and 4CL10 into p V L 1 3 9 2 , a Baculovirus transfer vector, p V L 1 3 9 2 , was digested by Sma I and Not I. The 4CL6 c D N A was cut out from a BlueScript plasmid using Xhol and Not I, and a blunt end was created at the Xhol site by filling i n with Klenow polymerase, while the 4CL10 c D N A was cut out from p C R 2.1 plasmid ( T A plasmid, Invitrogene) using Kpn I and Not I, and a blunt end was created at the Kpn I site. Recombinant plasmids: pVL1392::4CZ,<5 and pVL\392::4CL10,  created after ligation, contained flanking sequences, which were  homologues to the Baculovirus genome. To transfer the genes into genome of Autographa californica nuclear polyhedrosis virus ( A c M - N P V ) , 2 \ig o f highly purified D N A o f the p V L l 392:\4CL6 construct and 0.2 \ig o f A c M - N P V (BaculoGold D N A , Pharmingen) were co-transfected into Spodoptera furgipedra, Sf9 insect cells (American type Culture collection, Accession Number C R L 1711). Recombination took place within insect cells between the homologous regions i n the transfer vector and the BaculoGold D N A . Recombinant baculovirus particles were plaque-  20  purified (Summers and Smith, 1987). Based on an immunoblot and 4CL-enzyme essay, a single recombinant 4 C L 6 baculovirus plaque was chosen for production o f high-titre virus stock. The viral titre (2.45 X I 0 pfu/ml) was calculated by end point dilution 8  (Summers and Smith, 1987). Expression and activity o f the recombinant 4 C L 6 protein was tested over time (every 12 h/3 days). A multiplicity o f infection ( M O I ) o f 1.5 and 36-h infection time was selected based on 4 C L enzyme assay as the best condition for production o f the recombinant 4 C L 6 protein.  2.9 Expression a n d Purification of Recombinant 4 C L 50 x 10 Sf9 cells were infected with the recombinant 4 C L 6 baculovirus at M O I 1.5 6  and harvested 36 hours after infection. Cells were centrifuged at 1000 xg, for 5 minutes at 4°C. The cell pellet was washed twice with Dulbecco's Phosphate Buffered Saline (Sigma), resuspended i n 4 m l 5 0 m M Tris, p H 7.8, and lysed i n a 15 m l Weaton Homogenizer. Cellular debris was removed through centrifugation at 15,000 xg, for 10 minutes, then the supernatant was filtered through a 0.22 p.m filter. The supernatant was loaded onto a H i g h Q column (Bio-Rad) and subjected to anion-exchange F P L C as described b y A l l i n a et al. (1998). Fractions were collected (1 ml), glycerol was added to 30% (v/v) and they were stored at - 2 0 ° C . Protein content in crude extract and F P L C fractions was quantified by the Bradford (1976) method using B S A as a standard and the B i o - R a d Protein Assay K i t .  2.10 4 C L E n z y m e Assays 4 C L activity was measured at room temperature spectrophotometrically as described by Knobloch and Hahlbrock (1977) using 5 m M A T P , 5 m M M g C l  2 )  470 m M Tris, p H 7.8,  21  0.33 m M C o A , and 0.2 m M cinnamic acid derivatives as substrates. The p H o f the reaction mixture was adjusted to p H 7.8 with 4 M K O H (0.5 m l per 10 ml). The change i n absorbance o f the reaction mixtures was monitored at wavelengths o f 311, 333, 346, 345, or 352 nm, according to the absorption maxima for cinnamoyl:CoA, 4-coumaroyl:CoA, caffeoyhCoA, feruloyl:CoA, and sinapoykCoA, respectively (Stockigt and Zenk, 1975).  2.11 S D S - P A G E and Immunoblots Protein samples were electrophoresed in 10% SDS-polyacrylamide separating gels as described by Laemmli (1970). The proteins were blotted onto nitrocellulose Hybond-C (Amersham), blocked with 5% (w/v) nonfat powdered milk, probed with antisera raised against the recombinant 4 C L 9 at a 1:5000 dilution (Allina et al. 1998), and reacted with goat anti-rabbit I g G conjugated to alkaline phosphatase (Pharmacia) at 1:5000 dilution. Alkaline phosphatase activity was visualized using nitroblue tetrazolium and 5-bromo-4-chloro-3indolyl phosphatase as the substrates ( G i b c o - B R L ) .  22  CHAPTER III Results 3.1 Cloning Divergent 4CL Genes 3.1.1  PCR-Based Search Two 4CL c D N A clones, 4CL216 and 4CL9, were previously isolated from a H I 1  young leaf c D N A library, and shown to be derived from two poplar 4CL gene family members, 4CL1 and 4CL2 (Allina et al., 1998). The identity between the predicted amino acid sequences o f these two clones is 86%. To clone other members from the 4CL gene family i n poplar, a P C R strategy was employed. Genomic D N A o f Populus trichocarpa, clone 93-968, was used as a P C R template for amplification o f 4CL genes using sets o f degenerate 4CL-gene-specific primers designed by Amrita Singh. These primers had been designed based on amino acid sequences conserved between all predicted 4 C L proteins from several plants. The primers were directed to a part o f the first exon containing a conserved putative A M P - b i n d i n g motif (Figure 3). Forward primers 1 and 3 correspond to the nucleotide sequence o f seven amino acids conserved near the N-terminus o f 4 C L proteins. This site contains consensus arginine, serine, and leucine residues, which have high codon degeneracy (six codons each). In order to reduce the degeneracy o f primer 3, two codons for arginine ( C G G , C G A ) and two codons for leucine ( U U G , U U A ) were selected as the codons o f preference i n known 4CL genes. A l s o , a deoxyinosine was incorporated at the third position o f the serine codon, further decreasing the complexity o f primer 3. Primer 1 differs from the primer 3 only i n the presence o f deoxyinosine at the third position o f the leucine codon (Figure 3).  23  n nn n  5'  KGVMLT  RSKLPDI  6  2  8  4 P(L/M)FHI(Y/F)(S/A)  primer 1  5' C G GGA T C C G(A/G) CIA A(A/G)T TIC CIG A(CT)A T 3' Bam HI  primer 3 primer 2  5' C G GGA T C C G(A/G) CIA A(A/G)T T(A/G)C CIG A(C/T)A T 3' Bam HI 5' GCTCTAGA GA(G/A) TA(AGT) AT(A/G)TG(A/G) AAI AG(A/G)GG 3'  primer 4  5' GC TCTAGA GA(GA)TA(AGT) AT(G/A) TG(A/G) AA(T/C) AA(A/G) G G 3'  primer 6  5' GCT CTA G A G TIA GCA T(C/A)A CIC C(C/T)T T T 3'  primer 8  5' G C T CTA G A G T(T/A)A ACA T(C/A)A CIC C(C/T)T T 3' Xba I  Xbal Xbal Xbal  Figure 3. Generic 4CL gene and degenerate primers. A generic 4CL gene has five exons and four introns. Primers 1 and 3 are forward degenerate primers corresponding to the sequence which encodes seven conserved amino acids ( R S K L P D I ) at the 5' end o f the first exon. Primers 2,4, 6 and 8 are reverse oligodeoxynucleotides directed to the conserved sequences encoding amino acids K G V M L T and P ( L / M ) F H ( Y / F ) ( S / A ) , respectively. Each primer incorporates a restriction enzyme recognition site to facilitate P C R product cloning into a pBlueScript vector.  24  Reverse primers 6 and 8 were specific to the codons o f six conserved amino acids located next to the putative A M P - b i n d i n g motif. Another set o f reverse primers, 2 and 4, were directed to a region downstream from the putative A M P - b i n d i n g motif (Figure 3). Alternative conserved amino acids o f this region are found i n different 4 C L proteins, such as leucine/methionine, tyrosine/phenylalanine and serine/alanine. Thus, primers 2 and 4 recognized the same region, but they accounted for codons o f the different amino acids. A l l primers incorporated restriction enzyme recognition sites at their 5' ends, to facilitate P C R product cloning into the pBlueScript K S + plasmid. P C R reactions using combinations o f forward (1,3) and reverse (2,4,6,8) primers were expected to generate amplified products about 600-750 bp i n length. P C R reactions using the combination o f primer 1 with any reverse primer, and genomic D N A as a target, yielded either attenuated amplification or no amplification product (Figure 4 A : lane 3, and 4 B : lane3). Therefore, all P C R reactions were carried out using primer 3 as the forward primer. Figures 4 A and 4 B show that primer sets 3+6 and 3+8 amplified a fragment o f ~ 600 bp, as predicted, without background bands. In contrast, primer sets 3+2 and 3+4 amplified fragments o f the predicted size (-750 bp), plus several fragments o f different size (Figures 4 C and 4D). A l s o , P C R negative controls with the template and single primers indicated that primer 4 alone generated non-specific products. Potential contamination o f primers with D N A from other sources was excluded, because neither o f the primer sets i n negative controls without the template amplified any fragment. After subcloning the amplification products into BamHl / Xbal-digested pBlueScript K S + plasmid, 72 clones were obtained. Each clone was fingerprinted b y restriction enzyme analysis using Eco R I , Eco R V , Hind III, Sacl and Stul (Figure 5).  25  \C N C  C  VC SC  SC  SC  D  Figure 4. P C R amplification of 4 C L gene fragments from P. tichocarpa using different degenerate primers. A , amplification with primer sets 1-6 and 3-6; lanes 1 and 2 with c D N A of 4 C L 9 as a template; lanes 3-6 genomic D N A as template; line 7- 8 with no template. B, amplification with primers 1-8 and 3-8; lanes 1 and 2 with c D N A ; lanes 3-7 with genomic D N A ; lanes 8 and 9 without template. C, amplification with primer set 3-2; lane 1 with c D N A , lane 2and 3 with genomic D N A ; lane 4 primers 3 and 2 with no template. D, amplification with primer set 3-4; lane 1 with c D N A ; lane 2 and 3 with genomic D N A ; lane 4 genomic D N A and primer 4 (the second stock); lane 5 with no template.  26  Clones were placed into classes based on their restriction fragment length polymorphism ( R F L P ) . These fell into 4CL9-\ike, 4CL216-X\ks, and classes containing potentially new 4 C L sequences.  4CL9 Eco RV Hind ill  '  5  I I  fcoRV  I Sacl  4CL216  J  EcoRV  EcoRI  L_  4CL6 EcoRV  Eco Rl  J  I  I  Hind III 4CL10 EcoRI  5'  I  EcoR II  I  4CL14 EcoRI  Hind\\\  I  Stu I  I  L_  Pst\  Figure 5. Restriction map o f the P C R amplified D N A fragments (-600 bp) o f poplar 4CL genes.  27  S e q u e n c e a n a l y s i s s h o w e d that a l l c l o n e s f r o m the p r e d i c t e d a m p l i f i e d r e g i o n o f the  4CL9  4CL9  R F L P class contained the  gene, but neither s e q u e n c e f r o m the p u t a t i v e  R F L P class m a t c h e d the c o r r e s p o n d i n g a m p l i f i e d r e g i o n o f the  4CL216 g e n e .  Furthermore,  s e q u e n c e a n a l y s i s s h o w e d that three o f the eight R F L P classes r e p r e s e n t e d n e w sequences:  4CL6, 4CL10 a n d 4CL14.  T h e r e m a i n i n g five classes contained  4CL216  4CL-\ike  non-4CL  s e q u e n c e s . B E S T F I T a n d G A P s e q u e n c e a n a l y s i s r e v e a l e d t h a t t h e 4CL6, 4CL10 a n d 4CL14 c l a s s e s s h a r e b e t w e e n 6 5 % a n d 7 3 % n u c l e o t i d e i d e n t i t y w i t h t h e p r e v i o u s t w o p o p l a r 4CL cDNAs  (4CL216 & 4CL9). 4CL6  and  4CL14  share 8 9 % i d e n t i c a l nucleotide sequence, and  9 0 % identical amino acid sequence, respectively (Figure 6). Since  4CL6  and  4CL10  showed  r e l a t i v e l y h i g h d i v e r g e n c e f r o m e a c h o t h e r ( 6 1 % i d e n t i t y at t h e p r e d i c t e d a m i n o a c i d l e v e l ) , P C R clones f r o m these classes w e r e chosen for c l o n i n g full length c D N A clones.  ******* 4CL216 4CL9 4CL6 4CL14 4CL10 4CL216 4CL9 4CL6 4CL14 4CL10 4CL216 4CL9 4CL6 4CL14 4CL10  120 I 120 '  4CL216 4CL9 4CL6 4CL14 4CL10  Figure 6.  A l i g n m e n t o f deduced a m i n o acid sequences o f P C R fragments o f poplar  4CL  genes. A s t e r i s k s indicate c o n s e r v e d a m i n o acids used for d e s i g n i n g degenerate primers.  28  T a b l e I. Putative 4 C L gene fragments amplified by P C R . Primer set 3x6 3x8 3x2 3x4  4CL14  4CL6  4CL10  11  1 6  -  -  8  -  4CL9  10  -  Non specific 7 5 6 18  Total 19 21 14 18  % Non-specific 37 24 42 100  The efficiency o f different primer sets i n amplification o f 4 C L fragments is summarized i n Table I. Thus, primer sets 3+6 and 3+8 successfully amplified two new classes, 4CL6 and 4CL10, as well as the known 4CL9 class. Primer set 3+2 was biased toward amplification o f the new class 4CL14, while primer set 3+4 amplified only nonspecific fragments.  3.1.2 C l o n i n g F u l l - L e n g t h c D N A Clones A c D N A library from xylem R N A o f hybrid poplar clone H I 1-11 was screened using a mixture o f the P C R fragments 4CL6 and 4CL10 as a probe. Three positive clones out o f approximately 400,000 pfu screened were detected in the first plaque hybridization experiment. After four rounds o f plaque purification, the screening resulted i n purification of a single 1.88 kb c D N A clone corresponding to the 4CL6 P C R fragment. Neither 4CL10 nor 4CL14 were found i n this library. The resulting 4CL6 c D N A was sequenced and found to contain an open reading frame o f 1647 bp, which is predicted to encode a protein o f 548 amino acids with a molecular weight o f 59.2 k D a . There was a 6- bp 5'-untranslated region, a 227 bp 3'-noncoding region, and a poly (A) tail (Figure 7).  29  1 51 101 151 2 01 251 3 01 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 12 01 1251 13 01 1351 14 01 1451 1501 1551 1601 1651 17 01 1751 18 01 1851  CCCGCAATGG AAAATTACCA TTCTTGAAAA GCAAACGGAG AGTTGCTTCT TGCTCTTCCT TCACACAGAG AGAGCTAGCA AGGCTTGTTA AAGGTCATGT GCTAACACAG ATGATGTTGT GGGGTCATGC AGATGGAGAC GTGTGTTGCC GGGCTGAGAG TACTCTGCTG TTCCACCTGT GACTTGTCTT GGAACTTGAA AGGGATATGG TTTGCCAAGG CAGGAATGCA GGAGGAACCA GGATATCTTA ATGGTTGCAC TCATCGTTGA GCTCCTGCTG TGCTGCTGTA CATTTGTAGT CAGTATATTT CTTCATTGAA ATCTGAGAGA TAACCCTCTG TGTAGTGTTG GAACATTACT GGGAAAACAT TTTGGTTTTT  ACGCCATAAT GACATCTACA CTTGTCTAAA ATGTCTACAC GGTCTTAACA ACCAAGTTCA GTGCCATTGT AAACATGCCA CTACGACAAG GCGTAGACTC GCTGACGAAA AGCATTGCCT TAACGCACAA AATCCTAACC TATGTTCCAT TTGGTGCCTC GGATTGATTG GATGTTGGCA CTTTGAGGAT GATACTGTCA AATGACCGAG AACCATTTGA GAAATGAAGA GCCTGGTGAG ATGACCCTGA ACAGGCGATA CAGATTGAAG AACTCGAAGC GTAGGAATGA GAAATCAGAA CAAAACAGGT GCAATTCCCA AACGTTGCCA TCTTATTTCT AATCAAGCGT GTTCTTGTTA AAGTTCTCCT TTAAAAAAAA  GAATTCACAA TCCCGAAAAA TATTCATCAA CTATGCTGAC AGATTGGTAT CCTGAATTCG CACCGCTGCC AGCCTCCAAG GTTAAAGATT TGCCCCAGAT ATGAAGTGCC TATTCATCAG AGGGCTAATA TGTATTTTCA ATCTATGCTC GATTTTGATA AGAAGTACAA ATTGCTAAGT GATAAAATCT GAGCTAAGTT GCAGGACCTG CATAAAACCA TTGTTGACCC ATCTGCATCC GGCAACCTCA TCGGCTACAT GAATTGATCA TTTGTTACTA AAGATGAGGA AAGTCTCAGG GATATTCTAC AGGCGCCATC GGCATATAAC TTAATACTTG GCTTGGAAAA TACAAGCTCT GTCGCCATAT AAAAAAAAAA  GAAGAATTCA CCTTCCTCTG AACCTTGCCT GTTGAGCTCA TCAACAAGGT TGCTTGCTTT AATCCTTTCT AACAAAGCTT TTGCACGAGA GGGTGCTTGC CCAGGTCGAC GGACTACAGG ACCAGTGTGG CAGTGAAGAT TGAATTCAAT ATGCCAAAGT GGTATCTATA CACCTGATTT GGAGGGGCTC TCCTCAGGCC TTCTAGCAAT GGTGCATGTG AGAAACAGGG GGGGTGATCA AGAACAATAG TGATGACGAT AATATAAAGG GCCCATCCAC TGCAGGAGAA CCACCGAAGA AAGAGAATAA AGGCAAAATC TGAAGACGTT CGAAAATGCC GACACGTTAC TTAATGTTGC GGAGTAATTC  TCTTTCGCTC CATTCATACG GATAAATGGC CAGCAAGAAG GACGTGATCA CCTAGGCGCT CCACCCCTGC TTGATAACAC AAGTGATGTT ACTTTTCAGA TTTAGTCCTG GTTACCAAAA CTCAACAAGT GTGATTTTGT AATGCTTTGT TTGATATTGG GCACCAGTTG TGACAAGCAC CATTGGGCAA AGACTTGGTC GTGCTTGGCA GGACTGTCGT GCCTCTCTAC GATCATGAAA ACAAAGAAGG GATGAGCTTT GTTTCAGGTT AGATATCCGA GTTCCTGTTG TGAAATTAAG AACGAGTTTT CTTAGGAAGA ACTGAACATT AATGAATCAT CAAACGTTAA TTTTGTACTT AATTGAATAT  Figure 7. Nucleotide sequence o f the 4CL6 c D N A clone. The sequence includes an open reading frame o f 1647 nucleotides, plus untranslated 5' and 3' end sequences. The putative initiation and termination codons are underlined.  30  Since no 4CL10 clones were identified i n the xylem library, a young leaf c D N A library from H I 1-11 (Subramaniam et al, 1993) was screened with the 4CL10 P C R fragment. Several positive clones were plaque purified, but all were only ~1000-bp i n size. Sequencing analysis showed that these clones contained a 4 C L - l i k e coding region corresponding to the 4CL10 sequence, but that they lacked the 5' and 3' ends o f the c D N A . In order to obtain a c D N A with the full 4 C L 1 0 coding region, R A C E technology was employed. Gene specific primers (GSPs) were designed based on the sequence o f the partial c D N A 4CL10 clones. A pool o f adaptor-ligated c D N A s was made from H I 1 young leaf R N A , and 4CL10 G S P s and adaptor primers were used i n 5 ' R A C E and 3 ' R A C E P C R to amplify the missing ends o f 4CL10 c D N A (Figure 8). The R A C E products were cloned into T A vector pCR2.1 and sequenced. The 3 ' R A C E P C R amplified a 850-bp region including the 3'end o f the predicted 4CL10 coding, a 3' noncoding region, and a poly ( A ) tail. The 5' R A C E P C R amplified a 1050-bp region corresponding to the predicted 5' end o f the 4CL10 c D N A , but the fragment did not contain a predicted A T G start codon. A full length c D N A clone o f the 4CL10 gene was obtained by P C R amplification using the primer-ligated leaf c D N A pool and another set o f 4CL10-specific primers. A 5' primer was designed based on the published sequence o f the 5' end o f the aspen Pt4CL2 gene, since that gene is very similar to m y 4 C L 1 0 sequence. A 3' primer was designed based on the 3' untranslated portion o f the 3 ' R A C E fragment o f 4CL10 c D N A . The amplification product was cloned and sequenced. This 4CL10 c D N A was 1917 bp long, and contained an open reading frame of 1740 bp, predicted to encode a protein o f 579 amino acids (63.1 k D a , predicted molecular weight), including the putative initiation and termination codons (Figure 9).  Adaptor-ligated ds cDNA NNA« NNT<  5'RACE  API,  4^L10-  7  3'RACE 5'  —  •  —  •  •  '  N  N  A  —  —  ;  3  P  1  4CL10^ NGSPJ ••^NT-  Generation of the full-length cDNA of 4CL10 4CL10.9  NNA  3  '  NNT——— — ;  5'  4CL10.0 • Figure 8. The template and primers used i n R A C E reactions and amplification o f the full-length c D N A . Double-stranded c D N A (ds c D N A ) was generated from young leaf poly A + R N A b y reverse transcription and second-strand synthesis, and it was ligated to the adaptor. A P 1 = adaptor primer 1; gene-specific primers = 4CL10.7, 4CL10.2, 4CL10.0, and 4CL10.9. N G S P 1 , nested gene- specific primer. The specificity o f R A C E reactions is greatly enhanced by absence o f an A P I binding site on the adaptor-ligated c D N A s . This site is created on the c D N A o f interest by extension from the inner, gene-specific primer during the first R A C E cycle. The amine group on the c D N A adaptor blocks extension o f the 3'end o f the adaptor-ligated ds c D N A , and thus prevents formation o f an A P I binding site on the general population o f c D N A s .  32  1 51 101 151 2 01 251 3 01 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 12 01 1251 13 01 13 51 14 01 1451 15 01 1551 1601 1651 17 01 1751 18 01 1851 1901  ATGATGTCCG ACAAAACCAA CAAAACTACC TGCTTTGAAA TTCCACGGGA AGGTCGCTGC ATGACCCTGC TTCCATGATT GTGAAATATT CAGTCTCAAC CCAAAAACCG AGAACTGTCT CCAACAGTTT AGGGACAACA TAACAAGTGT AAACAAGATG ATTGAACAGC TAATGCAAAA AATGTTTCGG GAACCCAATG CAGGGGCTGC GTTCCACAGG AGTGCTATCA CTGGATCATG CCTGAGACCG CCGGGGATCC CAAACATCAT GTCGACGACG AAAATTCAAA TAAACCACCC TATGGAAACA GTTTGTGGTC ACTACATTGC TTCGTTCATT CCTCAGAGCC GTTAAATCTG ATTCTAAGAA TGTCCTAGAA CTTCACTCAT  TGGCCACGGT AACGCACCAT AGATATAACC ACCTCTCTGA AAAACCTACT TGGGTTATCC TCCAAAACTG GGTGCAGTCA CAAGCAATTC ATGTGAACAA GAGGAAGATT ACATTTCAAT CAATCCATCC GGGCTCCCAA GGCTCAACAA ATGTCGTTTT GTGTTGTTAT GTTTGAGATC TTGCGGCTGT GTGGCGAACT GCCACTGGGG CCATCCTGGG ATGTGCTTAG TGGAACAGTG GTAGCTCTCT CAAATCATGA AGACGTTGAG ACGACGAGAT GGCTTCCAGG TTCAATTGCG ACAGGCAAAA CGCTCAAATG AAAGCAGGTG CTATTCCCAA AAGCTTGCCA CATTTATTAT AAGACCTCAG TTCATTCCGT TGTGCCC  TGAGCCCCCG CCTCTCATGA ATCTCGAACC TTTCTCAGAT CTTTTGCCGA AATTTGGGCA CCCAGAATTC CCACCACTGT TCTGCTTCTC GCTAAGAGAT TCATAGTAAT GTGCTTGTTG GGATGATCCT AAGGAGTGAT GTTGATGGAG ATGCGTTTTA GCTCGTTGAG GGATCACTGC GGTGCCACCA TCGACTTGAG AAGGAGCTCG ACAGGGTTAT CCTTCTCAAA GTTAGAAACG TGGTCGCAAC AAGGATATTT GGTTGGCTCC TTTCATTGTT TGCCGCCAGC GATGCGGCTG AGACGAGGTT ATCTTGACCT GTGTTCTACA ATCGGCTTCT CAGCCACCAC TTTGTGTTGT AGCCAAGCTT TAAATCTGCA  AAACCGGAAC TCTCCCCCCC AACTGATCAT ATTTTCAGAT A C C T C C C T C T GCACGCATAC AGGCCATGCT TGATTTCAGG AACTCACCTA ATATCTCGAA TCAAGAAAGG CGATGTAATC G T C T T C T C C T TCATGGGTGC GAACCCTTTC TACACTCCAG GTGCGAAACT GATTATCACC AGTGATTACC ATGAAAACAA CACCATTGAT GACCCACCAG AGGCTAACGA GAGTGAAATG GTGGCATTAC C A T T C T C T T C ACTGACTCAC AAGAGCTTGA AGATCCCAAA T T T A T A C T T G CCTTTGTTTC ACATCTTTTC AGCCGGTTCT GCTGTACTTT TAGAGCTCAT TCAGAAACAC CTGGTGCTGG CGTTGGCCAA TTCGATCAGG GTAGTCCTCT AGGAGGCCCT CAGGAGCAGG GGGATGACAG AGGCGGGGCC GCAACCTTTA CCCACCAAGT CAGAGCTCAA GGTCATTGAC CAACCTGGTG AAATCTGCAT GAATGACGCG GAAGCCACGG ACACTGGAGA TATAGGTTAT GATAGAGTGA AGGAAATCAT GGAGCTTGAG GCTCTCCTTG TTGTTCCCCG AGATAACTTG GCTGGTGAAG TTCCTGTCGC TAATGAAGAG GCTGTAAAAG AGAAACTGCA CAAGGTGTTC GGAAAGATTC TAAGAAAAGA CATGTCCTAG A T T T C A T T A C CTTTCACTCG CTGTGGAAAG GCCACAGCCA CCACCATGCA T T T A T A T T A T TTTGTGTTGT  F i g u r e 9. Nucleotide sequence o f the 4CL10 c D N A clone. The sequence includes an open reading frame o f 1740 nucleotides, and an untranslated 3'end sequence. The putative initiation and termination codons are underlined.  3.1.3 Sequence Analysis The deduced amino acid sequences o f the new 4CL c D N A s , designated 4CL6 and 4CL10, were compared to each other using the G A P and B E S T F I T functions o f the G C G sequence analysis software (Figure 10).  33  4CL6  MDAIMNSQEEFIFRSKLPDIYIPKNLPLHSY 31  4CL10 MMSVATVEPPKPELSPPQNQNAPSSHETDHIFRSKLPDITISNHLPLHAY  50  32 VLENLSKYSSKPCLINGANGDVYTYADVELTARRVASGLNKIGIQQGDVI 81 51 CFENLSDFSDRPCLISGSTGKTYSFAETHLISRKVAAGLSNLGIKKGDVI 100 82 MLFLPSSPEFVLAFLGASHRGAIVTAANPFSTPAELAKHAKPPRTKLLIT  131  101  MTLLQNCPEFVFSFMGASMIGAVTTTVNPFYTPGEIFKQFSASRAKLIIT 150  132  QACYYDKVKDF  ARESDVKVMCVDSAPDGCLHFSELTQADENEV  174  151  QSQHVNKLRDSDYHENNQKPEEDFIVITIDDPPENCLHFNVLVEANESEM  2 00  175  PQVDFSPDDWALPySSGTTGLPK3VMLTHKGLITSVAQQVDGDNPNLYF  224  2 01 PTVSIHPDDPVALP SSGTTGLPK3VILTHKSLITSVAQQVDGEIPNLYL  25 0  225  274  HSEDVILCVLPMFHIYALNSIMLCGLRVGASILIMPKFDIGTLLGLIEKY  : | | : | | l l | : | | | : - l l h = ll  II l-- = h l  Ihll-ll  11 = 1 =  251  K Q D D W L C V L P L F H I F S L N S V L L C S L R A G S A V L L M Q K F E I G S L L E L I Q K H 3 00  275  KVSIAPWPPVMLAIAKSPDFDKHDLSSLRMIKSGGAPLGKELEDTVRAK  I hi 301  |||..||::  -  M M - : || I I I I I M I :  324  -  NVSVAAWPPLVLALAKNPMVANFDLSSIRWLSGAAPLGKELEEALRSR  350  32 5 FPQARLGQGYGMTEAGPVLAMCLAFAKEPFDIKPGACGTWRNAEMKIVD  3 74  351 VPQAILGQGYGMTBASPVLSMCLAFSKQPLPTKSGSCGTWRNAELKVID 4 00 II 3 75 PETGASLRRNQPjGEICIRGDQIMKGYLNDPEATSRTIDKEGWLHTGDIGY 424 401 PETGSSLGRNQPJ3EICIRG BQIMKGYLNDAEATANIIDVEGWLHTGDIGY  450  425  IDDDDELFIVDRLKELIKYKGFQVAPAELEALLLAHPQISDAAWGMKDE 474  451  VDDDDEIFIVDRVKEIIKFKGFQVPPAELEALLVNHPSIADAAWPQKDE 500  475  DAGE VP VAF W K S E KS QATED EIKQ Y I S KQ V I F Y KRIKRVF F I E A I P KAP 524  501  VAGEVPVAFWRSNDLDLNEEAVKDYIAKQWFYKKLHKVFFVHSIPKSA  525  S G K I L R K N L R E T L P G I * . . . . 541  551  SGKILRKDLRAKLATATTMS* 571  55 0  Figure 10. Comparison of the deduced amino acid sequences of the 4CL6 and 4CL10 cDNA clones. Identical residues are denoted by | and similar residues by : Conserved regions I (AMP-binding motif) and II (putative catalytic site) are boxed. Asterisks indicate conserved cysteine residues. Bold letters indicate conserved amino acids used for designing degenerate primers.  34  Although the predicted amino acid sequences of 4CL6 and 4CL10 showed only 61 % identity, they have significant similarity throughout their complete lengths. Both proteins contain conserved amino acid motifs at the positions observed in all other 4CL proteins. Motif I (Box I, Figure 10) has been suggested to form part of the AMP-binding domain (Schroder, 1989; Bairoch, 1991), while the Box II amino acid motif GEICIRG (Figure 10) has been proposed to be associated with stability and catalytic activity of 4CL and related enzymes (Becker-Andre et al., 1991).  II Comparison of 4CL6 and 4CL10 predicted amino acid sequences to each other and to other poplar 4CL cDNA sequences. 4CL10 cDNA 4CL216 4CL6 4CL9 % identity % identity % identity % identity  T a b l e  4CL9  *  86  74  64  4CL6  74  74  *  61  4CL10  64  65  61  *  Table II shows a comparison of the predicted amino acid sequences of the four known poplar 4CL genes (4CL9 and 4CL216 previously cloned, and 4CL6 and 4CL10 from this study). The most divergent 4CL gene within the poplar 4CL gene family is 4CL10. This gene shows less identity (61% to 65%) to the three other poplar 4CL genes than to certain 4CL genes from other plants such as aspen Pt4CL2 (96%), Lithospermum  4CL2 (76%) and  soybean 4CL2 (78%) (Table III). The 4CL6 gene shows moderate divergence from other poplar 4CL genes (74%), and higher identity to aspen Pt4CLl (94%), soybean 4CL1 (80%) and tobacco Nt4CLl (78%).  35  Table III Comparison of 4CL6 and 4CL10 predicted amino acid sequences to each other and to other 4CL sequences. cDNA 4CL10 Ref. 4CL6 % identity % identity * 61 4CL6 * 61 4CL10 poplar 4CL1 74 65 Allina & Douglas (1997) 74 64 Allina & Douglas (1997) poplar 4CL2 94 60 Hue?al. (1998) aspen 4CL1 aspen 4CL2 60 96 Hue?al. (1998) 77 67 Lee & Douglas (1996) tobacco M4CL19 78 65 Lee & Douglas (1996) tobacco Nt4CLl Yazakief al. (1995) Lithospermum 4CL1 73 60 Yazakief al. (1995) Lithospermum 4CL2 60 76 70 60 Lee et al. (1995) Arabidopsis 4CL1 64 Uhlmann & Ebel (1993) soybean 4CL1 80 66 78 Uhlmann&Ebel(1993) soybean 4CL2  Alignment of deduced amino acid sequences of the cDNA clones for the four poplar 4CL gene (Figure 11) revealed that the most pronounced similarity between all four predicted amino acid sequences occurs within the central and C-terminal part of the proteins. Based on the sequence alignments, unique sequence features of the predicted 4CL10 relative to the other 4CL proteins are a 19 amino-acid N-terminal extension and two insertions of 4-5 amino acids each near the N-terminus. Similar N-terminal extensions and insertions are present in Lithospermum 4CL2, aspen Pt4CL2 andrice4CL2. The BLAST search for proteins with significant similarity to 4CL at the amino acid sequence level,, identified several non-4CL proteins, including luciferin-4-monooxygenase and long-chainfatty-acid-CoA ligase. These proteins share many conserved amino acids throughout their entire length with 4CL6 and 4CL10, particularly in the AMP-binding and the GEICIRG motifs (Figure 12).  36  4CL9 4CL216 4CL6 4CL10  1 OgNKg 1 JSKNS 1 BUBIM'SO! 1 MMSVATVEPPKPELSPPQNQNBPSSHE  4CL9  42  MPCL  4CL216  42  N P C L IN(  4CL6  42  S P C L INI  4CL10  61  S P C L 1 sj  4CL9 4CL216 4CL6 4CL10  YCFEgLS YCFENLSM. Y__ENLSK YCFENLS  lit  I  SS  :>Ss_  102 102 102 121  4CL9  TSVAQQ  4CL216  TSVAQQ  4CL6  TSVAQQ  4CL10  TSVAQQ  I 4CL9 4CL216  216 216  4CL6  ********** SLMDLVQKY  GENPNLYFHEGDVILCVLPLFHIYSLNSV_LCGLRGGSAILVMQKFD  GENPNLYFHEBDVILCVLPLFHIYSLNSVLLCGLRRGSAILLMQKFETSTLMEL^QKY G_MPNLYFH^DVILCVLP|FHIYALNSIMLCGLRRGASIL:I:M_KFDIBTLL_LIE  4CL10 4CL9 4CL216 4CL6 4CL10 4CL9 4CL216 4CL6 4CL10  II  *******  4CL9  396  QIMKGYLNDPEATERTIDKTJGWLHTGDIGYIDH|DELFIVDRLKELIKYKGFQVAPAELE  4CL216  396  QIMKGYLNDPEATERT§D_DGWLHTGDIGYID_DDELFIVDRLKELIKYKGFQVAPAELE  4CL6  395  QIMKGYLNDPEATHRTIDKJEGWLHTGDIGYIDDDDELFIVDRLKELIKYKGFQVAPAELE  4CL10  421  OIMKGYLNDBEATSMID^GWLHTGDIGYTODDDETFIVDRVKE'IIKFKGFOVHPAELE  4CL9 4CL216 4CL6 4CL10 4CL9 4CL216 4CL6 4CL10  Figure 11. Alignment of deduced amino acid sequences of poplar 4CL cDNA clones. Black shade indicates identical amino acids; grey shade indicates similar amino acids. Conserved regions I (AMP-binding motif) and II (putative catalytic site) are indicates by asterisks.  37  4 CLIO 4CL6 luciferase fadD-5  1 1 1 1  MMSVATVEPPKPEMPPffiSQNAPSgHgTgl ffiDAIMffi @Q@-|F|_ laBlEM ILIGPPPYYJJLEEGJSAGEC MBFTEDI  4CL10 4CL6 luciferase  61 42 3 9 TLAYTDVHjjE  fadD-5  22 [JEF0BYPDRD  4CL10 121 4CL6 102 l u c i f e r a s e 99 fadD-5 82 4CL10  170  --PE  4CL6 145 ES l u c i f e r a s e l 4 3 --LPI fadD-5 142 ERFP@I 4CL10  217 E  4CL6  191  luciferasel98 fadD-5  198  I  4CL10 277 4CL6 251 luciferase256 fadD-5 254 4CL10  337 I  4CL6 311 luciferase316 fadD-5 314 4CL10 397 4CL6 371 luciferase371 fadD-5 372 4CL10 457 4CL6 431 luciferase431 fadD-5 432 4CL10 517 4CL6 482 luciferase482 fadD-5 483  jDEVAGEVPK jDEj^AGEVpR  d  DEVAGgfjPS DEJ^GEIp iRKfflLR^LgJT IRK'LREHLBSH  BRKIKEILI  Figure 12. Alignment o f deduced amino acid sequences o f poplar 4CL6 and 4CL10 c D N A clones and other ATP-dependant enzymes. Luciferase= luciferin-4-monooxygenase from Photuris pennsylvanica (D25416.1); fadD-5= long-chain-fatty-acid-CoA ligase (AE001034). Conserved regions I ( A M P - b i n d i n g motif) and II (putative catalytic site) are indicated by asterisks. Black shade indicates identical amino acids; grey shade indicates similar amino acids.  38  3.2 Expression of Divergent 4CL Genes  To study the regulation of4CL6 and 4CL10 gene expression, total RNA from different tissues of poplar clone HI 1-11 (old leaf, young leaf, green stem, xylem, elicitor-treated and untreated poplar suspension-culture cells) was blotted and hybridized with probes prepared from the 4CL6 or 4CL10 cDNA clones. The level of 4CL6 mRNA accumulation was high in xylem, green stem and root, and quite low in old leaf (Figure 13 A). No expression of 4CL6 was detected in young leaf, or in control or elicitor-treated suspension cultures. A very different expression pattern was observed for 4CL10 (Figure 13B). 4CL10 transcripts were detected in old and young leaves as well as in root tissue, but not in green stem and xylem. The blot hybridized with the 4CL10 probe was exposed twice as long as the blot hybridized with the 4CL6 probe. Thus, the level of 4CL10 mRNA accumulation in old leaf and root appears to be lower than that of 4CL6, and the overall expression level of 4CL10 is quite low.  39  £  83  S3  cu  =  cU  3  >.  4CL6  rRNA  B  -ta»  5  g  cu  —  O  X  *©  j,  +  Cd  W  ""5  1.9 kb  • i t  HI*! 1.9 kb  4CL10  f__L  rRNA  F i g u r e 13.  Northern-blot analysis o f  4CL6  and  m R N A levels. T o t a l R N A (10  fi  F r o m different tissues a n d o r g a n s w a s o n d u p l i c a t e d f o r m a l d e h y d e a g a r o s e g e l s , b l o t t e d to n y l o n m e m b r a n e s , h y b r i d i z e d to either  4CL6 o r 4CL10 c D N A  p r o b e s , a n d w a s h e d at h i g h  stringency ( 0 . 2 X S S C , 0 . 1 % S D S , 65°C). E v e n loading was s h o w n b y stripping blots and rehybridizing with a pea r R N A  probe. E L + , elicitor-treated tissue culture cells: E L I - ,  untreated control cells.  40  g)  3.3 Characterization of Recombinant 4CL Proteins 3.3.1 Expression and Purification of Recombinant 4CL6 and 4CL10 Proteins To test whether the novel 4CL genes from poplar actually encode 4 C L enzymes and whether the enzymes have catalytic properties distinct from previously characterized poplar 4 C L enzymes (Allina et al., 1998), the 4CL6 and ACL 10 c D N A s were expressed as recombinant proteins using a baculovirus vector expression system. Recombinant baculovirus particles expressing 4 C L 6 were generated as a result o f homologous recombination between pVL1392::4CZ<5 and A c N P V viral D N A i n Sf9 insect cells. Identification and purification o f the recombinant 4 C L 6 baculovirus particles were done as described i n Materials and Methods, while a similar procedure was used to transfer 4CLIO from p\L\392::4CL10  into A c N P V viral D N A in the lab o f D a v i d Theilman, Pacific A g r i -  Food Research Center, Summerland, B C . To generate the recombinant 4 C L 6 protein, 50 x 10 Sf9 cells were infected with 6  recombinant 4 C L 6 baculovirus particles from a high titer stock for 36 hours. The 4 C L 6 protein was partially purified from the crude protein extract by fast-protein liquid chromatography using an ion-exchange High-Q column. The recombinant 4 C L 6 protein eluted as a single peak o f 4 C L activity, assayed using 4-coumaric acid as a substrate (Figure 14). The most active fractions o f 4 C L 6 had specific activities between 498 and 597 pkat/p-g protein.  41  g  CM  o co  o *t  © m  o <o  0  Fraction  Figure 14. Partial purification of recombinant protein 4CL6 by FPLC on a High-Q anion exchange column using KC1 gradient. 4CL activity was assayed using 4-coumarate as a substrate.  To assess the purity of FPLC-purified 4CL, two peak FPLC fractions were separated by SDS-PAGE, in parallel with crude extracts from uninfected Sf9 and infected Sf9 cells. This analysis showed that FPLC resulted in an efficient enrichment of the presumed recombinant 4CL6 protein, whose migration corresponded to the predicted molecular mass of 60 kDa (Figure 15A). A parallel immunoblot showed that poplar 4CL-specific polyclonal antibodies (arised against recombinant 4CL9, Allina et al., 1998) cross-reacted with the recombinant 4CL6 (Figure 15B), confirming the identity of the recombinant protein as 4CL.  42  4CL6  49  —  33  —  28  —  F i g u r e 15. S D S - P A G E and immunoblot analysis o f recombinant 4 C L 6 protein. S D S - P A G E gel (A) and an immunoblot o f a parallel gel (B) reacted with antiserum specific to recombinant 4 C L 9 protein, mw, molecular weight standard, wt Sf9, uninfected insect cells, crude extracts o f Sf9 cells infected with 4 C L 6 and 4 C L 9 baculovirus constructs, 4 C L 6 FPLC-purified from Sf9 infected cells.  43  A s shown on Figure 16, eight 4CL10-expressing baculovirus independent stocks, express recombinant proteins o f the size predicted for 4 C L 1 0 protein. The protein has a higher molecular weight than the recombinant 4 C L 6 and 4 C L 9 proteins, as expected from the predicted amino acid sequence. Crude extracts o f Sf9 cells infected with the 4 C L 1 0 baculovirus stocks showed 5-10 fold less 4 C L activity toward 4-coumaric acid than crude extract o f Sf9 infected with 4 C L 6 baculovirus particles.  4CL10  F i g u r e 16. S D S - P A G E analysis o f recombinant 4 C L 1 0 protein. S D S - P A G E gel stained in Coomassie brilliant blue. M W , molecular weight standard, wt Sf9, uninfected insect cells, crude extracts o f Sf9 cells infected with 4 C L 1 0 (1-6 & 8 plaques) and 4 C L 6 baculovirus constructs.  44  3.3.2 Substrate Utilization Profile of Recombinant 4CL6 Protein The ability to use different hydroxycinnamic acids as substrates was tested for the 4 C L 6 protein. Enzyme assays were carried out using 0.2 m M concentrations o f each substrate. Figure 17 shows that ths partially purified recombinant 4 C L 6 protein showed a strong preference for 4-coumaric acid and decreasing activities toward ferulic acid, caffeic acid, and cinnamic acid. N o activity toward sinapic acid was detected. Activity toward 5hydroxyferulic acid was not tested because 5-hydroxyferulic acid was available i n insufficient amount.  120 , 100 •  80-  I  60.  *  40  4  200  coumarate  cinnamate  caffeate  ferulate  sinapate  Substrat  Figure 17. Substrate-utilization profile o f recombinant 4 C L 6 . Recombinant 4 C L 6 enzyme activity was measured using FPLC-purified 4 C L 6 and 0.2 m M concentrations o f hydroxycinnamic acids. Results are averages o f three trials: error bars represent S D values. Asterisks indicate the absence o f detectable activity. Results are reported as a precentage o f the activity against 4-coumaric acid, which was 480.7 pkat/mg.  45  CHAPTER IV Discussion  The results o f this work confirm to the existence o f at least four 4 C L isoenzymes in poplar. This conclusion is based on isolation o f two full-size 4CL c D N A clones in addition to those previously described (Allina et al, 1998), and is consistent with multiple 4 C L proteins detected b y F P L C chromatography (Allina et al, 1998). The use o f degenerate P C R primers that target conserved sequences i n 4CL genes allowed the efficient identification o f divergent 4CL genes. Thus, while previous screening of a poplar young leaf c D N A library resulted i n identification o f two 4CL c D N A clones with 86% identity at the predicted amino acid level (Allina et al, 1998), m y work showed that the poplar 4CL gene family i n fact contains three new 4CL classes (4CL6, 4CL10 and 4CL14) i n addition to the previously studied genes. These were distinguished among 72 clones characterized from the products o f P C R amplification of poplar genomic D N A . P C R techniques have been successfully applied to isolate a divergent Pt4CL2 gene from aspen (Hu et al, 1998). Degenerate P C R primers that target evolutionarily conserved sequences in PAL genes also revealed the presence o f at least eight pal loci i n the jack pine genome (Butland et al, 1998). In contrast, the previous study on the PAL gene family i n loblolly pine had reported that this pine genome contains asingle PAL gene (Whetten and Sederoff, 1992). Thus, it is clear that this P C R strategy can potentially identify phenylpropanoid gene family members that might be difficult to detect b y hybridization. The degenerate primers used in this work performed with different efficiency i n amplifying 4CL gene fragments from P. trichocarpa. Thus, primer sets 3+6 and 3+8  46  amplified fragments of three poplar 4CL genes, 4CL6, 4CL10 and 4CL9 (Table I). Relatively low frequency of nonspecific sequences amplified by these primer sets (24% and 37%) was consistent with results obtained in other studies with PCR-based cloning of different genes. For example, Sells and Chernoff (1995) found that each set of degenerate primers specific for tyrosine phosphatase genes, amplified aboutlO nonspecific sequences out of 30 characterized sequences. In contrast, the 4CL primer set 3+2 amplified only the 4CL14 class. Similar results with degenerate primers were found by Butland et al. (1998), where PAL primers P7-P6 amplified only pal 5gene, while P5-P2 primers amplified four pal sequence classes. 4CL primer set 3+ 4 amplified only nonspecific sequences, suggesting that reverse primer 4 annealed inappropriately due to self-annealing or formation of hairpin structures within the oligonucleotide pool. Moreover, the low efficiency of reverse primers 2 and 4 could be related to the high degeneracy of their sequences, necessary because of the alternative conserved amino acids found between different 4CL genes in this target region. Reverse primers 2 and 4 are also longer (20+8 nt) than the reverse primers 6 and 8 (17+8 nt). Wilks et al. (1989) reported that long degenerate primers (20-30 nt) are less efficient than short (14-20 nt) degenerate primers. Therefore, this work showed that the consensus amino acids RSKLPDI and KGVMLT art; a better choice for designing degenerate primers specific for 4CL than the region wilh the alternating amino acids P(L/M)FHI(Y/F)(S/A). The possibility that additional poplar 4CL genes remain to be isolated appears small due to the exhaustive PCR-based search of poplar genomic DNA and the screening of two cDNA libraries. Furthermore, a poplar database of expressed sequence tags (ESTs) from cambium and xylem (http://www.biochem.kth.se/PopulusDB, Sterky et al., 1998) revealed three ESTs from cambium, which show 90-96% identity to the 4CL6 cDNA clone.  47  The three E S T s represent different parts o f the same gene, which supports the result o f this work that 4 C L 6 is the major 4 C L isoform expressed i n the wood-forming tissues. Neither o f the degenerate primer sets amplified any sequence that corresponded to the 4CL216 gene. A comparison o f the P C R primer sequences with the actual nucleotide sequences o f the four poplar 4CL genes indicates that mispairing between the primer 3 and 4CL216 could explain the absence o f 4CL216 fragments after P C R amplification (Figure 18). This indicates that the P C R primers used in this work were biased toward certain 4CL genes, making it possible that some 4CL genes remain to be isolated. A  # o f mismatches 4CL9  3'TCC  A G G TTT GAG GGA CTG TAG 5'  Primer 3  5'CGG  T C I A A A T T G C C I GAC A T  4CL216  3'TCC  A G G TTT GAG GGA CTA TAG 5'  4  4CL6  3'GCG  A G T TTT A A T GGT CTG TAG 5'  0  Primer 3  5'CGG  T C I AAA TTA C C I GAC AT  4CL10  3'TCT  A G T TTT GAT GGT C T A TAT 5'  Conserved amino acids  B  R  S  K  L  P  D  3'  5'AAA  GGT GTC  Primer 8  3'TTT  C C I C A C (A) T A C A A A T G A G 5 '  4CL216  5'AAA GGT GTC  ATG TTG ACT C 3'  2  ATG CTA ACG C 3'  2  5'AAA  GGG GTC  Primer 6  3'TTT  C C I C A C (A) T A C G A I T G A G 5 '  4CL10  5'AAA  GGT GTG  G  V  2  ATGTTGACT C 3'  4CL6  K  2  I  4CL9  Conserved amino acids  3  3'  A T A CTG A C T C 3'  I  L  1  T  Figure 18. Degenerate primers and corresponding sequences i n poplar 4 C L genes. A , comparison o f the forward primer 3 with the actual nucleotide sequences; B , Comparison o f the reverse primers 6 and 8 with the actual nucleotide sequences o f poplar 4CL genes; bold letters indicate mismatches.  A l l four poplar 4 C L c D N A s sncode predicted polypeptides with a highly conserved region designated as a putative A M P - b i n d i n g motif (region I i n Figure 11), a seven-amino-  48  acid motif containing one cysteine residue (region II in Figure 11) representing a putative catalytic site, and six cysteine residues, suggesting a conserved tertiary structure. These 4CL landmarks have been found in deduced amino acid sequences of all other 4CL predicted products such as those from parsley, potato, soybean, Lithospermum, tobacco and aspen (Lozoya et al., 1988; Backer-Andre et al, 1991; Uhlmann and Ebel, 1993; Yazaki et al, 1995; Lee and Douglas, 1996; Hu et al. 1998). Similarity between the predicted amino acid sequences of poplar 4CL proteins is especially high in the central and C-terminal parts of the proteins. In these regions there are long stretches of essentially identical amino acid residues. These amino acid residues are likely important for enzyme structure and function. However, to date there is no direct experimental evidence to support the hypothetical function assignments of motifs I and II. Site-directed mutagenesis of the cysteine residue in the region II (potato 4CL), carried out in order to test the hypothesis that this cysteine residue is associated with catalytic activity, failed because of instability of the mutated protein in E. coli cells (Becker-Anclere et al, 1991). In addition, the lowest homology among poplar 4CLs is detected in the N-terminal sequences. Therefore the N-terminal region might be involved in phenolic substrate binding specificity (Hu et al, 1998). Different luciferin-4-monooxygenase (firefly luciferase) and long-chain-fatty-acidCoA ligases genes showed similarity to 4CL6, 4CL10 and other 4CLs (Figure 12). These proteins share many conserved amino acids throughout their entire lengths, particularly in the AMP-binding (I) and the GEICLRG (II) motifs (overall 34-36% identity). Although luciferase, long-chain-fatty-acid-Co A ligase, and 4CL catalyse different metabolic reactions, all three enzymes are ATP-dependent, catalyze a two-step reaction which includes an adenylate intermediate compound, and have a hydrophobic substrate (luciferin, a fatty acid,  49  a hydroxycinnamic acid) and a pokr cosubstrate (O2 for luciferase, CoASH for both ligases) (de Wet, 1986; Gross, 1985). The low similarity between these enzymes in their N-terminal regions could be due to their different substrate specificities, while more conserved amino acids in the central and C-terminal parts are probably involved in the conserved function of ATP binding and hydrolysis. It is note worthy that Gross and Zenk (1966) reported that a fairly non-specific acyl-CoA ligase from beef liver mitochondria was able to activate cinnamic acids at low efficiency. Comparison of deduced amino acid sequences of 4CL6 and 4CL10 with aspen 4CLs showed that aspen Pt4CLl and poplar 4CL6 proteins are likely the products of orthologues genes (94% amino acid identity), a:; are the aspen Pt4CL2 and poplar 4CL10 proteins (96% identity). These two pairs of orthologues genes are very distantly related based on an analysis of phylogenetic relationships of 4CL genes (Ehlting et al., 1999). Thus, 4CL6 and aspen Pt4CLl belong to the class I. while the 4CL10 and Pt4CL2 orthologous belong to a distinct class II. 4CL isoforms encoded by genes isolated from parsley, tobacco, potato and poplar [4CL9 and 4CL216) also belong to class I, and they are more closely related to each other than to 4CL isoforms in class II. The deduced amino acid sequence of4CL10 is 61-65% identical to the other three poplar 4CLs. This sequence shows' more identity to Lithospermum 4CL2, soybean 4CL2, Arabidopsis 4CL3 and aspen Pt4CL2 (78-96%, Table II), all of class II, then to the other poplar 4CL genes. The N-terminal part of poplar 4CL10 and other class II4CL proteins differ from other dicot 4CLs in having a terminal extension of 19^24 amino acids and in a two short insertions. Furthermore, genomic clones showed that soybean 4CL2 and Arabidopsis 4CL3 genes (class II genes) have 6 introns, in contrast to genomic clones of  50  4 C L s from class I (Lozoya et al, 1988; Beecker-Andre et al, 1991). H u et al (1998) reported that the promoter region o f aspen Pt4CL2 has none o f the boxes consistently found in all known plant P A L and 4 C L gene promoters. Future work on 4CL gene families from different plants w i l l clarify whether other plants like parsley, potato and tobacco also have 4 C L isoforms from the class II. One likely possibility is that divergent class II isoforms exist i n these plants, but that they remain to be isolated, perhaps due to their divergent sequences (Ehlting et al, 1999). Clear differences in expression patterns have been described that distinguish class I and class II 4 C L isoforms i n aspen, Arabidopsis, and soybean (Hu et al, 1998; Ehlting et al, 1999; Uhlmann and Ebel, 1993). M y work showed that 4CL6 and 4CL10 also have distinct expression patterns, and that these patterns differ from those o f the previously isolated 4CL9 and 4CL216 genes (Allina et al, 1998). 4CL6 is strongly expressed i n xylem, green stem and root, but the expression was not detected i n young leaves, while 4CL10 is expressed only in leaves and root. In contrast, 4CL9 m R N A is most abundant i n young leaf, and 4CL216 is preferentially expressed in old leaf (Allina et al, 1998). Based on these results, the function o f the 4 C L 6 isoform (class I) appears to be related to lignin biosynthesis, and the 4CL.10 isoform (class II) appears to play role i n the biosynthesis o f nonlignin-related phenylpropanoids (e.g. flavonoids and phenylpropanoid esters), that accumulate i n poplar leaves. A similar difference i n the expression patterns o f aspen Pt4CLl and Pt4CL2 has been observed (Hu et al, 1998.). A l s o , heterologous Pt 4CL1 and Pt4CL2 promoter-GUS fusion analyze indicated that Pt 4CL1 (the orthologues to 4CL6) gene expression is xylem-specific and Pt4CL2 (the orthologues to 4CL10) is epidermisspecific (Hu et al, 1998). In Arabidopsis, the most divergent (class II) gene, 4CL3, was  51  expressed to relatively high levels in flowers, but not in lignified tissue such as bolting stem, while the 4CL1 and 4CL2 (class I) are specific for bolting stem and seedling roots. Similarly, the class II 4CL isoformfromsoybean (4CL16) is specifically induced by pathogen infection, after which soybean isoflavanoid phytoalexins accumulate (Uhlmann and Ebel, 1993). This led Ehlting ex al, (1999) to propose that a primary function of the class II4CL isoforms is to channel activated 4-coumarate to chalcone synthase and subsequently to different branch pathways of flavonoid secondary metabolism. On the other hand, a role in lignin biosynthesis has been postulated for the class 14CL isoforms, based on the high expression level in lignifying tissues of 4CL genesfromparsley, tobacco, aspen Pt4CLl and poplar 4CL6, described in this work (Hauffe at al., 1991; Lee and Douglas, 1996; Kuet al, 1998). Neither the previously studied genes nor 4CL6 was activated in response to elicitor treatment, although the same RNA samples hybridized to a poplar PAL probe showed that expression of this gene was strongly stimulated by the elicitor treatment (data not shown). Earlier studies using a heterologous 4CL probe indicated that 4CL expression is elicitoractivated in hybrid poplar suspension-cultured cells, followed by a 10- to 20- fold increase in extractable PAL and 4CL enzyme activities (Moniz de Sa et al, 1992). Furthermore, accumulation of a single 4CL isofoi.m in the elicited suspension culture was detected by partial purification with FPLC (Allina et al, 1998). Therefore, a good candidate for an elicitor-induced poplar 4CL gene is 4CL10, and its expression in elicitor-treated suspensioncultured cells should be tested. To further investigate the regulation of tissue-specific and cell-specific expression of poplar 4CL genes, in situ hybridization could be done using leaf and root sections. That  52  approach could show cell-type specific differences in the expression patterns. For example, 4CL6 and 4CL10 are expressed in root, but their mRNAs might be localised in different tissues of root (epidermis vs. vascular tissue). Also, RFLP analysis of RT-PCR products from different tissues would give better insight into tissue-specific expression of the poplar 4CL genes, and clarify differences between 4CL216 and 4CL9 expression patterns, since they have the highest sequence identity and some cross-hybridization between them was reported (Allina et al, 1998). The baculovirus-expressed protein encoded by 4CL6 shows a strong preference for 4coumaric acid as a substrate and decreasing activities toward ferulic acid, caffeic acid and cinnamic acid. This is consistent with previous results (Allina et al. 1998) indicating that all native isoforms of hybrid poplar had similar substrate utilization profiles, similar to those of recombinant proteins 4CL9 and 4CL216 proteins. Thus, the increase in divergence between the poplar 4CL deduced amino acid sequences (75% identity between 4CL6 and 4CL9; 86% identity between 4CL9 and 4CL216) does not result in a change in the substrate specificities of these isoforms. Since analysis of the substrate specificity of recombinant 4CL10 protein and the kinetic analysis of both recombinant proteins (4CL6 and 4CL10) have not yet been completed, it remains a possibility ihat 4CL10 could have a distinct substrate-utilization profile. In addition, Hu et al. (1998) reported that the major differences between aspen recombinant Pt4CLl (orthologues to 4CL6) and Pt4CL2 (orthologues to 4CL10) were that Pt4CL2 is not active toward 5-hydroxyferulic acid, and that the amount of total specific activity of Pt4CL2 is strikingly lower than that of Pt4CLl. To date, no 4CL proteins have been found to accept substrates other than cinnamic acids. This fact, together with their pronounced preference for hydroxylated cinnamic acids,  53  has led to proposing the systematic name "hydroxycinnamate:CoA ligase (AMP)" (Gross and Zenk, 1974). Alternatively, the name for this enzyme can refer to the best substrate. Since, 4-coumaric acid is the best substrate for the recombinant 4CL9, 4CL216 and 4CL6, the name "4-coumarate:CoA ligase'' is appropriate for these isoforms. The lack of any detectable conversion of sinapic acid by recombinant 4CL6 is consistent with results obtained both with poplar native 4CLs and recombinant 4CL9 and 4CL216 (Allina et al, 1998). Moreover, none of the recombinant 4CL enzymes from tobacco, soybean ox Arabidopsis showed activity toward sinapic acid. The occurrence of sinapic acid: CoA ligase has been reported only for some native 4CL isoformsfromsoybean, Petunia and poplar (Knobloch and Hahlbrock, 1975; Ranjeva et ah, 1976; Grand et al., 1983). Therefore, a number of studies indicate that sinapic acid cannot be efficiently activated to the CoA thioester by most 4CLs, and that the biosynthesis of sinapyl alcohol and syringyl lignin may occur via an alternative pathway. Thus, Higuchi (1985) postulated a biosynthetic pathway of sinapyl alcohol through 5-hydroxy-coniferaldehyde, followed by methylation by an OMT (0-methyltransferee) and reduction by a CAD. Also, Ye and Varner (1995) suggested that 5-hycroxyferuloyl-CoA might be methylated by CCoAOMT (caffeoyl-coenzyme A-3-0-methyli:ransferase) to produce sinapyl-CoA. The genes encoding COMT and CCoAOMT in aspen have been cloned (Bugos et al., 1991; Meng and Campbell, 1995). Meng and Campbell (1998) found that aspen recombinant COMT utilizes free hydroxycinnamic acid, while aspen CCoOMT has activity toward CoA ester substrates with a preference for caffeoyl-CoA. These authors hypothesised that CCoAOMT is likely to be responsible for biosynthesis of lignin precursors in the guaiacyl pathway, while COMT is more likely to be involved in the syringyl pathway. In support of this, COMT-down -  54  regulated poplar trees displayed unchanged lignin content, but the proportion o f G units and resistant biphenyl structures was dramatically enhanced (Lapierre et al. 1999). Because down-regulation o f 4 C L activity in Arabidopsis leads to a decrease i n G residues, but no change i n S residues, Lee et al. (1997) proposed that S-lignin might be synthesized by a 4CL-independent pathway. The absence o f activity toward sinapic acid and absence o f catalytically distinct 4 C L isoforms i n poplar and other plants (Lozoya et al., 1988; V o o et al., 1995; Lee and Douglas, 1996), makes it unlikely that 4 C L controls partitioning o f carbon into guiacyl and syringyl lignin, or into other phenylpropanoid end products.  However, 4 C L is an end point enzyme  between general phenylpropanoid metabolism and branching pathways, and could have an impact on carbon flow from phenylalanine amino acid into monolignols. Thus, the lignin content was decreased to 50% i n 4 C L down-regulated transgenic tobacco (Kajita et al, 1996), and somewhat reduced i n 4 C L down-regulated Arabidopsis (Lee et al., 1997). Antisense suppression o f Pt4CLl i n transgenic aspen showed up to a 4 5 % reduction o f lignin content and increased i n cellulose content (Hu et al, 1999). 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