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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 THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1999 © Daniela Cukovic, 1999 ln presenting this thesis in" partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) 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 of the poplar 4CL gene family, degenerate primers directed to a part of the first exon, containing a putative AMP-bind ing motif, were used. Three new 4CL- l ike classes were distinguished among 72 cloned P C R amplification products. The new 4CL- l ike sequence classes, arbitrarily named 4CL6, 4CL10 and 4CL14, shared 61% 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 of a full-length c D N A clone of 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 of 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 of 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 in 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 of Genomic D N A for P C R Analysis 16 2.3 P C R Search of Poplar Genomic D N A 16 2.4 Screening c D N A Library 17 2.5 Rapid Amplification of 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 of Recombinant 4 C L 21 2.10 4 C L Enzyme Assays 21 2.11 S D S - P A G E and Immunoblots 22 C H A P T E R III Results 23 3.1 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 of Divergent 4CL Genes Expression 39 3.3 Characterization of Recombinant 4 C L Proteins 41 3.3.1 Expression and Purification of Recombinant 4 C L 6 and 4CL10 Proteins 41 3.3.2 Substrate Utilization Profile of Recombinant 4CL6 Protein 45 C H A P T E R I V Discussion 46 Bibl iography 56 List of Figures and Tables Figure 1 The reactions of general phenylpropanoid metabolism 3 Figure 2 Schematic diagram of the biosynthesis of flavonoid and lignin precursors . . . 4 Figure 3 Generic 4CL gene and degenerate primers 24 Figure 4 P C R amplification of 4 C L gene fragments from P. trichocarpa using different degenerate primers 26 Figure 5 Restriction map of the P C R amplified D N A fragments (-600 bp) of poplar 4CL genes 27 Figure 6 Alignment of deduced amino acid sequences of P C R fragments of poplar 4CL genes : 28 Figure 7 Nucleotide sequence of 4CL6 c D N A clone 30 Figure 8 The template and primers in R A C E reactions and amplification of the full-length c D N A 32 Figure 9 Nucleotide sequence of 4CL10 c D N A clone 33 Figure 10 Comparison of deduced amino acid sequence of4CL6 and 4CL10 c D N A clones 34 Figure 11 Alignment of deduced amino acid sequences of poplar 4CL c D N A clones 37 Figure 12 Alignment of deduced amino acid sequences of poplar 4CL6 and 4CL10 c D N A clones and other ATP-dependent enzymes 38 Figure 13 Northern-blot analysis of4CL6 and 4CL10 m R N A levels 40 V Figure 14 Partial purification of recombinant 4 C L 6 protein by F P L C on an High-Q ion exchange column 42 Figure 15 S D S - P A G E and immunoblot analysis of recombinant 4 C L 6 protein 43 Figure 16 S D S - P A G E analysis of recombinant 4CL10 protein 44 Figure 17 Substrate-utilization profile of recombinant 4 C L 6 45 Table I Putative 4CL gene fragments amplified by P C R 29 Table II Comparison of 4 C L 6 and 4CL10 predicted amino acid sequences to each other and to other poplar 4 C L sequences 35 Table III Comparison of 4 C L 6 and 4CL10 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 GSP gene-specific primer H I 1 poplar hybrid {Populus trichocarpa X Populus deltoides) vii M O I multiplicity of infection N G S P nested gene-specific primer dNTP deoxynucleotide triphosphate P A L phenylalanine ammonia lyase P C R polymerase chain reaction pfu plaque forming unit P P i pyrophosphate R A C E rapid amplification of 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 of Spodoptera furgipedra, line 9 viii Acknowledgement I would like to thank Dr. Carl Douglas for giving me the opportunity of doing a master of science degree under his supervision, for his guidance in developing this thesis research, and for his financial assistance that he provided over the course of three summers. I am greatly indebted to Dr. Brian Ell is and Dr. Beverley Green for their suggestions and guidance in developing this thesis research. I would like to thank Dr. Elizabeth Moli tor for introducing me to the lab, and thanks to the Douglas, El l i s , 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 Al l ina 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 of several classes of chemical compounds including coumarins, flavonoids, stilbenes, suberin, lignin and other cell-wall associated phenolics. The functions of 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; Dixon and Paiva 1995). Phenylpropanoid metabolism can be divided into a general pathway, required for the synthesis of all phenylpropanoid metabolites, and specific branch pathways, which lead to synthesis of specific phenolic end products (Douglas et al., 1992). The biosynthesis of phenylpropanoid compounds is developmentally activated in specific tissues and cell types, but can be induced by various biotic and abiotic stresses. The evolutionary development of phenylpropanoid metabolism must have been a critical juncture in the transition of 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 of phenylpropanoid metabolism consists of 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 in the pathway, cinnamate-4-hydroxylase (C4H), uses O2 and N A D P F f to hydroxylate the 6-membered aromatic ring of cinnamic acid at the para-position, making 4-coumarate. The enzyme 4-coumarate: Co A ligase (4CL) catalyzes the last step in the general phenylpropanoid pathway, formation of Coenzyme A esters ofp-coumaric acid and other hydroxy- or methoxy- derivatives of cinnamic acid, such as caffeic acid, ferulic acid, and sinapic acid (Figure 2). Co A esters of 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 in the biosynthesis of flavonoid compounds and coumaryl-CoA, feruloyl C o A and sinapyl C o A are reduced by cinnamoyl C o A reductase (CCR) , and directed to the biosynthesis of lignin monomers. Hydroxylation and methylation of the phenylpropanoid aromatic ring, important in the generation of different monolignols, can occur at the level of free acids, or at the level of the corresponding CoA-esters (Figure 2). The traditional view of the pathway has been that 4-coumaric acid is hydroxylated at the 3-position by 4-coumarate 3-hydroxylase (C3H). Enzymes that carry out this hydroxylation reaction in vitro have been detected, but little is known about their properties or physiological role (reviewed by Whetten and Sederoff, 1995). 2 (ISOFLAVONOIDS COUMARINS SOLUBLE ESTERS 1 A i 1 I I GENERAL PHENYLPROPANOID METABOLISM COOH COOH CQOH OH OH L-Phenylalanine Cinnamic acid 4-Coumaric acid 4-Coumaroyl-CoA I • WALL-BOUND PHENOLICS LIGNIN | SUBERIN STILBENES Figure 1. The reactions of 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 r COOH OCHj H 0 ^ O 3 C H , HaCO'V^OCHs 4-Coumaric add Cafleie add |4CL I4CL J J J4CL | Farufic add 5-Hydroxyfenjllc add  4CL OH Slnaptcadd |4CL VcoA 5H OH ~ ~ "™ O H " " " J ~ OH 0 0 1 3 4-CouniaroyCCcA CafleoytCoA FetutoytCoA S-HydroxyfecutoytCoA SlnapoytCoA OCH3 H 0 ' " ^ O 3 C H 3 H3CO' C H S / y CCR CAO CCR CAO CCR I CAD CCR CAO Naringenin CHjOH CH2OH CH2OH CH2OH COb HO'X.'CCHa H3CO OH OH OH Conifaryl 5-Hydroxyferulyl Sinapyl alcohol alcohol alcohol Flavonoids Ugnin Figure 2. Schematic diagram of the biosynthesis of flavonoid- and lignin- precursors. The identification of novel enzymes suggests that the biosynthesis of C o A esters from 4-coumarate may proceed through a number of enzymatic steps potentially resulting in 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 of free 4-coumarate has been supported by discovery of a 4-coumaroyl/caffeoyl-CoA hydroxylase (CCo3H) in an anthocyanin mutant of Silene dioica, but the potential involvement o f C C o 3 H in monolignol biosynthesis has not yet been adequately tested (reviewed by Whetten and Sederoff, 1995). The 3- and 5-methylation of the aromatic ring of free acids is catalyzed by catechol O-methyltransferase ( C O M T ) , which in angiosperms utilize both caffeic acid and 5-hydroxyferulic 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 of the caffeoyl-CoA ester and 5-hydroxyferuloyl-CoA during monolignol biosynthesis (Figure 2). This portion of the monolignol biosynthesis pathway appears to be more of a network or grid than a linear pathway (Whetten et al., 1998). Hydroxylation of ferulate to 5-hydroxyferulate, thought to be catalyzed by ferulate 5-hydroxylase (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 in lignin (Chappie et ah, 1992). Meyer et al. (1998) showed that overexpression of the F 5 H gene from the C a M V 3 5 S , or AtC4H promoter leads to biosynthesis of a large amount of syringyl lignin. Characterization of the catalytic properties of 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 of general phenylpropanoid metabolism is the activation of cinnamic acids to form C o A thioesters. This reaction is catalyzed by 4-coumarate:CoA ligase 5 (E.C. 6.2.1.12 the group of acid-thiol ligases) and proceeds via a two-step process involving an acyl adenylate intermediates, in analogy to the activation of fatty acids with A T P and C o A S H according to the following equations (Gross, 1985): M g 2 + 4CL + hydoxycinnamic acid + ATP -> 4CL • hydroxycinnamyl-AMP + PPi 4CL • hydroxycinnamyl-AMP + CoASH -* 4CL + cinnamoyl-CoA + ATP 4 C L activity requires the presence of 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 of 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; Wall is 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 of 40 k D a {Erythrina crista-galli), 55 kDa (soybean, Forsythia), 61 k D a (parsley) and 75 kDa (pea) were estimated. Physically distinct 4 C L isoforms have been reported from soybean, Petunia, pea, poplar, carrot and mesocotyl of maize, (Knobloch and Hahlbrock, 1975; Ranjeva et al, 1976; Wall is 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 4-coumaric acid. However, in 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 of related substrates, could control the partitioning of carbon into different branch-pathways through the activity of distinct 4CL-isoforms (Knobloch and Hahlbrock, 1975; Grand et al, 1983). For example, in 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 in the formation of 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; Voo et al, 1995), suggesting that 4 C L does not participate in the metabolic channeling of phenylpropanoid derivatives in these species. 1.3 4CL Gene Families More detailed knowledge about 4 C L isoforms was provided by cloning of 4 C L genes during the 1980's and 1990's. 4 C L is encoded by multiple divergent genes in some plants like rice, soybean, poplar, Lithospermum erythrorhizon, tobacco and aspen (Zhao et al, 1990; Uhlmann and Ebel, 1993; Al l ina and Douglas, 1994; Yazaki et al, 1995; Lee and Douglas, 1996; H u et al 1998); by very similar duplicated genes as in the case of parsley, potato and loblolly pine (Lozoya et al, 1988; Becker-Andre et al, 1991; Zhang and Chiang 1997 ); and apparently by a single-gene in Vanilla planifolia (Brodelius and Xue, 1997). Interestingly, Arabidopsis 4 C L was previously assumed to be encoded by a single-copy gene 7 (Lee et al, 1995), but two divergent 4CL classes have since been cloned in Arabidopsis by Ehitingefa/ .(1999). Sequence comparisons between deduced amino acid sequences of 4CL c D N A s of 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 in 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 AMP-b ind ing domain that is common to a number of prokaryotic and eukaryotic ATP-dependent enzymes. In addition, the predicted amino acid sequences of 4 C L proteins from the plants listed above each contain a total of six conserved cysteine residues. Genomic clones of 4CL genes from rice, parsley and potato show the presence of 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 of them contains three additional introns (Ehlting et al. 1999). 4CL gene expression, like that of many of the phenylpropanoid genes, is regulated developmentally and is also activated by external stimuli such as pathogen infection, elicitor treatment, wounding, and UV-l ight 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 of an introduced parsley 4CL1 gene accumulate in a cell-type specific manner, and that the patterns of accumulation are generally consistent with the sites of phenylpropanoid natural-product accumulation (Reinold et al., 1993). A s well , 4CL expression in 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 in the growth and development of woody plants, since an important component of wood is lignin. For example, between 21% to 23 % of P. trichocarpa wood dry weight is lignin (Swan and Kellogg, 1986). Many derivatives of phenolic compounds have been isolate from the bark of P. trichocarpa such as salicin, trichocarpin, salireposide, salicyl alcohol, cinnamic acid, 4-coumaric acid and others (Pearl and Darling, 1968). Bud exudate of P. deltoides is predominantly composed of flavanones, chalcones and ester of flavanones together with the flavone galangin, the flavanon pinocembrin and the flavanonol pinobanksin (Greenaway et al., 1990). In contrast, the major phenolic compounds in bud exudate of P . trichocarpa are dihydrochalcone, benzyl salicylate, cinnamic acid, and minor amount of flavanones, chalcones and flavones (English et al, 1991). 9 Shain and Mi l l e r (1982) reported that pinocembrin (5,7-dihydroxyflavanone), as a major component of poplar bud exudate is active against a fungal pathogen Melampsora medusae. Thus, a sufficient amount of pinocembrin is present on the surface of young, expanding leaves of P. deltoides contributing to their resistance to M. medusae. A s leaves age, however, the concentration of pinocembrin is depleted as a result of weathering, leaves expansion and insufficient replenishment, and leaves become more susceptible to the fungal infection. Induction of defense response in 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 of the economic importance of wood, little is known about the genetic control of 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 in this pedigree (Bradshaw et al, 1994). Several phenylpropanoid genes have been characterized in the genus Populus (poplars and aspens). P A L is encoded by a small gene family, two members of which (PALI and PALI) are highly expressed in young leaves and stems, where large amounts of soluble 10 phenylpropanoid products accumulate in addition to lignin (Subramaniam et al, 1993). Two more divergent members of the Populus gene family have recently been identified, which, in contrast to PALI and PAL2, are strongly expressed in older stems in 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 in different tissues. The studies by Grand et al. (1983) strongly suggest the possible existence of multiple 4 C L isoenzymes in 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 of 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 in substrate specificity. 4CL1 reacts with 4-coumarate, ferulate, and sinapate as substrates; 4 C L 2 can use 4-coumarate and ferulate as substrates; and 4CL3 can use 4-coumarate and caffeate as substrates. The 4 C L activity was mainly localized in lignified tissues, such as xylem and sclerenchyma (phloem fiber cells) (Grand et al, 1983). 1.5 Poplar 4CL Gene Fami ly Recent characterization of poplar 4 C L isoenzymes and members of the 4CL gene family has been done in a poplar hybrid, clone H I 1-11, derived from a cross between Populus trichocarpa and Populus deltoides. Two classes of 4CL c D N A clones (4CL9 and 4CL216), that do not cross hybridize at high stringency, were isolated by 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 of northern analysis indicated that the 4CL9 gene is expressed strongly in young leaf and weakly in old leaf, green stem and xylem, while the 4CL216 gene shows strong expression in all those tissues. Neither the 4CL9 or the 4CL216 genes is expressed in elicitor-treated suspension tissue culture cells. Expression of recombinant 4 C L proteins from these c D N A clones showed that they have identical substrate utilization profiles (All ina et al., 1998). Furthermore, 4 C L forms from poplar hybrid H I 1-11, xylem, elicited cell culture, and young leaf were separated by F P L C ion column exchange, indicating the presence of 3-4 putative 4 C L isoforms. However, the different forms appear to have identical substrate utilization profiles (All ina et al., 1998). These results do not support previous evidence that three catalytically distinct 4 C L forms exist in poplar. 1.6 Lignin Lignin (Latin: lignum - wood) is a three-dimensional heteropolymer, resulting from the dehydrogenative polymerization of three different hydroxycinnamyl alcohols (monolignols): p-coumaryl, coniferyl and sinapyl alcohol, which differ in the extent o f methylation. These monolignols become cross-linked by about 20 types of intersubunit linkages (Chen, 1991). 12 Lignin accumulates in the secondary wall and the middle lamellae of 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 by its strengthened walls (Boudet et al, 1995). Lignin provides rigidity and hydrophobicity to the cell wall , which is particularly important for cells which conduct water. Also , stress-induced lignin deposition provides a mechanism for sealing off sites of pathogen infection and wounding (Dixon and Paiva, 1995). The monomelic composition of lignins is one of the most important characteristics of lignins from both a taxonomic and an industrial point of view (Boudet, 1998). The chemical complexity of 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 of coniferyl alcohol (guaiacyl or G units), although, in compression wood, lignin is polymerized from coniferyl and a small portion of p-coumaryl alcohol (H units). This composition makes compression wood lignin less methylated and more difficult to hydrolyze (Higuchi, 1985). The typical lignin of angiosperms is composed of a mixture of guaiacyl and syringyl units. The presence of the more highly methylated syringyl units increases the ability of 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 of tissue development and individual cell wal l layers (Davin and Lewis, 1992). For example, guaiacyl subunits predominate in the xylem of vascular bundles of 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 of hydroxycinnamic acids via 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 of lignin deposition. The mechanism of monolignol transport from cytoplasm to the cell wal l 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 of monolignols to make lignin is believed to be catalyzed by some combination of peroxidase and laccase (reviewed by Campbell and Sedoroff, 1996; Whetten etal, 1998). Proposed mechanisms that control lignin content and composition are (i) substrate specificity of different isoforms of monolignol biosynthetic enzymes (e.g. C O M T , 4 C L , C A D ) and (ii) transcriptional regulation of genes encoding isoforms of 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 of 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 of lignin content and composition in mutants of maize, sorghum, and Arabidopsis, and in transgenic tobacco, poplar and Arabidopsis with suppressed enzyme activities, has led to increased understanding of the monolignol biosynthesis pathway, and of 14 the mechanisms by which lignin composition is regulated in different plants. For example, lignin content remained mostly constant in 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 in tobacco and poplar (Halpin et ah, 1994; Hibino et al. 1995; Baucher et al., 1996), suppressed 4 C L activity in Arabidopsis (Lee et al., 1997), and ectopic F 5 H activity in Arabidopsis (Meyer et al., 1998). In contrast, changes in lignin composition (increases or decreases in the S:G ratio) have been observed in many transgenic plants with altered enzyme activities generated by suppression of 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 of lignin in a plant and a given tissue can be maintained at a relatively constant level even when an enzyme normally involved in the synthesis of lignin precursors is suppressed or overexpressed. This response reflects the importance of lignin to vascular function and mechanical support in land plants, and indicates a high degree of plasticity in lignin biosynthesis. When this project was initiated, the nature of the 4CL gene family in poplar and other plants was poorly understood. While different isoforms had been described in many plants, the extent to which the enzymatic properties of these isoforms differed was unknown. Furthermore, it was possible that genes encoding highly divergent isoforms might not have been detected due to lack of cross-hybridization to cloned genes. The purpose of my 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. Young leaves (0.5-2 cm in length), old (fully expanded) leaves and green (nonwoody) stems were harvested from H I 1 plants maintained in growth chambers at 23°C in a 16-h light/8-h dark regime. Harvested material was immediately frozen in liquid N2 and stored at - 8 0 ° C until use. Secondary xylem was isolated from field-grown trees as described previously (All ina et al, 1998). The suspension cultures of H I 1 cells and elicitor treatments were prepared as described by Moniz de Sa et al. (1992). 2.2. Preparation of Genomic DNA for PCR Analysis Genomic D N A , used in P C R reactions, was isolated from fresh leaves of 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 of each degenerate primer, 200 u M dNTPs, 1.5 m M M g C k and 2.5.units of Taq D N A polymerase (Gibco-BRL) in 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 of 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 condi t ions: '94°C/10 min, 3 cycles of 94°C/ 50 sec, 50°C/50 sec, 72°C/70 sec, 30 cycles of 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 HI 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 5 recombinant phage from a poplar xylem c D N A library in A.ZAPII (prepared by Yuj 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 (Gibco-BRL) according to the manufacturer's specifications. Hybridized filters were washed at high stringency (0.2 X SSC, 0.1% SDS, 65°C) for 1 h. After purification of positive plaques, a single positive clone obtained was subjected to in 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 of the 5' and 3' ends of the clone. It was shown that the clone was a full-length (~ 2 kb) c D N A of 4CL6. The same approach was applied in 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 of 4CL10. 1 7 2.5 R a p i d Ampli f ica t ion of 5 ' - c D N A and 3 ' - c D N A Ends Total R N A was isolated from young leaves of hybrid poplar H I 1 by the method of 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 of 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 of 5 ' R A C E and 3' R A C E . The first P C R of 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 of 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 of 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 of 5 ' R A C E under these conditions: 94°C/1 min, 30 cycles of 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 full-length c D N A of 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 of 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 by 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 of Wisconsin Genetics Computer Group (Medison) software. Sequenced were searched against the GenBank non-redundant protein and E S T database (National Center of Biotechnology Information) using B L A S T X . Multiple sequence alignment was obtained using Clustal-W program (Baylor College of Medicine) and B O X S H A D E . Predicted molecular weight of deduced amino acid sequences was calculated by software Compute pI /Mw 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 of Hughes and Galau (1988) from young leaves, old leaves, green stem and root of poplar hybrid clone H I 1. A trizol reagent (Gibco-BRL) was used to extract total R N A from secondary xylem and the suspension cultures of 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 Hybond-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 (Gibco-BRL) according to the manufacturer's specifications. Prehybridization and hybridization were performed at 65°C for 16 h in 7% SDS sodium phosphate buffer (Church and Gilbert, 1984). The membranes were washed at high stringency (0.2 X SSC, 0.1% SDS, 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 of 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 Baculovirus Particles To subclone the full-length c D N A clones of 4CL6 and 4CL10 into pVL1392, a Baculovirus transfer vector, pVL1392, 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 in 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 of 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 in the transfer vector and the BaculoGold D N A . Recombinant baculovirus particles were plaque-2 0 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 of high-titre virus stock. The viral titre (2.45 X I 0 8 pfu/ml) was calculated by end point dilution (Summers and Smith, 1987). Expression and activity of the recombinant 4 C L 6 protein was tested over time (every 12 h/3 days). A multiplicity of infection (MOI) of 1.5 and 36-h infection time was selected based on 4 C L enzyme assay as the best condition for production of the recombinant 4 C L 6 protein. 2.9 Expression and Purification of Recombinant 4 C L 50 x 10 6 Sf9 cells were infected with the recombinant 4 C L 6 baculovirus at M O I 1.5 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 in 4 m l 50mM Tris, p H 7.8, and lysed in 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 High Q column (Bio-Rad) and subjected to anion-exchange F P L C as described by 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 Bio-Rad Protein Assay Ki t . 2.10 4 C L Enzyme 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 of the reaction mixture was adjusted to p H 7.8 with 4 M K O H (0.5 ml per 10 ml). The change in absorbance of 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 (All ina et al. 1998), and reacted with goat anti-rabbit IgG conjugated to alkaline phosphatase (Pharmacia) at 1:5000 dilution. Alkaline phosphatase activity was visualized using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphatase as the substrates (Gibco-BRL) . 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 (All ina et al., 1998). The identity between the predicted amino acid sequences of these two clones is 86%. To clone other members from the 4CL gene family in poplar, a P C R strategy was employed. Genomic D N A of Populus trichocarpa, clone 93-968, was used as a P C R template for amplification of 4CL genes using sets of 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 of the first exon containing a conserved putative AMP-bind ing motif (Figure 3). Forward primers 1 and 3 correspond to the nucleotide sequence of seven amino acids conserved near the N-terminus of 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 of 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 of preference in known 4CL genes. Also , a deoxyinosine was incorporated at the third position of the serine codon, further decreasing the complexity of primer 3. Primer 1 differs from the primer 3 only in the presence of deoxyinosine at the third position of the leucine codon (Figure 3). 23 5' n nn n RSKLPDI 6 8 2 4 KGVMLT P(L/M)FHI(Y/F)(S/A) primer 1 5' CG GGA TCC G(A/G) CIA A(A/G)T TIC CIG A(CT)A T 3' Bam HI primer 3 5' CG GGA TCC G(A/G) CIA A(A/G)T T(A/G)C CIG A(C/T)A T 3' Bam HI primer 2 5' GCTCTAGA GA(G/A) TA(AGT) AT(A/G)TG(A/G) AAI AG(A/G)GG 3' Xbal primer 4 5' GC TCTAGA GA(GA)TA(AGT) AT(G/A) TG(A/G) AA(T/C) AA(A/G) GG 3' Xbal primer 6 5' GCT CTA GAG TIA GCA T(C/A)A CIC C(C/T)T TT 3' Xbal primer 8 5' GCT CTA GAG T(T/A)A ACA T(C/A)A CIC C(C/T)T T 3' Xba I 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 of 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)FH(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 of six conserved amino acids located next to the putative AMP-bind ing motif. Another set of reverse primers, 2 and 4, were directed to a region downstream from the putative AMP-b ind ing motif (Figure 3). Alternative conserved amino acids of this region are found in 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 of 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 of forward (1,3) and reverse (2,4,6,8) primers were expected to generate amplified products about 600-750 bp in length. P C R reactions using the combination of primer 1 with any reverse primer, and genomic D N A as a target, yielded either attenuated amplification or no amplification product (Figure 4A: lane 3, and 4B: lane3). Therefore, all P C R reactions were carried out using primer 3 as the forward primer. Figures 4 A and 4B show that primer sets 3+6 and 3+8 amplified a fragment of ~ 600 bp, as predicted, without background bands. In contrast, primer sets 3+2 and 3+4 amplified fragments of the predicted size (-750 bp), plus several fragments of different size (Figures 4C and 4D). Also , P C R negative controls with the template and single primers indicated that primer 4 alone generated non-specific products. Potential contamination of primers with D N A from other sources was excluded, because neither of the primer sets in negative controls without the template amplified any fragment. After subcloning the amplification products into BamHl / Xbal-digested pBlueScript KS+ plasmid, 72 clones were obtained. Each clone was fingerprinted by restriction enzyme analysis using Eco RI , Eco R V , Hind III, Sacl and Stul (Figure 5). 25 \C NC VC S C S C S C C 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 (RFLP) . These fell into 4CL9-\ike, 4CL216-X\ks, and classes containing potentially new 4 C L sequences. 4CL9 Eco RV I I 5' Hind ill fcoRV I Sacl 4CL216 EcoRV EcoRI J L_ 4CL6 EcoRV Eco Rl J I I Hind III 4CL10 5' EcoRI EcoR II I I 4CL14 Hind\\\ EcoRI I L _ Stu I I Pst\ Figure 5. Restriction map of 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 4CL9 R F L P c l a s s c o n t a i n e d 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 g ene , b u t n e i t h e r s e q u e n c e f r o m the p u t a t i v e 4CL216 R F L P c l a s s 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 . F u r t h e r m o r e , s e q u e n c e a n a l y s i s s h o w e d that t h ree o f the e i g h t R F L P c l a s s e s r e p r e s e n t e d n e w 4CL-\ike s e q u e n c e s : 4CL6, 4CL10 a n d 4CL14. T h e r e m a i n i n g f i v e c l a s s e s c o n t a i n e d 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 that the 4CL6, 4CL10 a n d 4CL14 c l a s s e s sha re 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 the p r e v i o u s t w o p o p l a r 4CL c D N A s (4CL216 & 4CL9). 4CL6 a n d 4CL14 sha re 8 9 % i d e n t i c a l n u c l e o t i d e s e q u e n c e , a n d 9 0 % i d e n t i c a l a m i n o a c i d s e q u e n c e , r e s p e c t i v e l y ( F i g u r e 6 ) . S i n c e 4CL6 a n d 4CL10 s h o w e d 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 the 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 c l o n e s f r o m these c l a s s e s w e r e c h o s e n f o r c l o n i n g f u l l l e n g t h c D N A c l o n e s . ******* 4CL216 4CL9 4CL6 4CL14 4CL10 4CL216 4CL9 4CL6 4CL14 4CL10 4CL216 120 I 4CL9 120 ' 4CL6 4CL14 4CL10 4CL216 4CL9 4CL6 4CL14 4CL10 Figure 6. A l i g n m e n t o f d e d u c e d a m i n o a c i d s e q u e n c e s o f P C R f r a g m e n t s o f p o p l a r 4CL genes . A s t e r i s k s i n d i c a t e c o n s e r v e d a m i n o a c i d s u s e d f o r d e s i g n i n g degene ra t e p r i m e r s . 28 Table I. Putative 4 C L gene fragments amplified by P C R . Primer set 4CL6 4CL10 4CL14 4CL9 Non specific Total % Non-specific 3 x 6 11 1 - 7 19 37 3 x 8 - 6 10 5 21 24 3 x 2 - - 8 6 14 42 3 x 4 - - - 18 18 100 The efficiency of different primer sets in amplification of 4 C L fragments is summarized in 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 of the new class 4CL14, while primer set 3+4 amplified only non-specific fragments. 3.1.2 C lon ing Ful l -Length c D N A Clones A c D N A library from xylem R N A of hybrid poplar clone H I 1-11 was screened using a mixture of the P C R fragments 4CL6 and 4CL10 as a probe. Three positive clones out of approximately 400,000 pfu screened were detected in the first plaque hybridization experiment. After four rounds of plaque purification, the screening resulted in 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 in this library. The resulting 4CL6 c D N A was sequenced and found to contain an open reading frame of 1647 bp, which is predicted to encode a protein of 548 amino acids with a molecular weight of 59.2 kDa. There was a 6- bp 5'-untranslated region, a 227 bp 3'-noncoding region, and a poly (A) tail (Figure 7). 29 1 CCCGCAATGG ACGCCATAAT GAATTCACAA GAAGAATTCA TCTTTCGCTC 51 AAAATTACCA GACATCTACA TCCCGAAAAA CCTTCCTCTG CATTCATACG 101 TTCTTGAAAA CTTGTCTAAA TATTCATCAA AACCTTGCCT GATAAATGGC 151 GCAAACGGAG ATGTCTACAC CTATGCTGAC GTTGAGCTCA CAGCAAGAAG 2 01 AGTTGCTTCT GGTCTTAACA AGATTGGTAT TCAACAAGGT GACGTGATCA 251 TGCTCTTCCT ACCAAGTTCA CCTGAATTCG TGCTTGCTTT CCTAGGCGCT 3 01 TCACACAGAG GTGCCATTGT CACCGCTGCC AATCCTTTCT CCACCCCTGC 351 AGAGCTAGCA AAACATGCCA AGCCTCCAAG AACAAAGCTT TTGATAACAC 401 AGGCTTGTTA CTACGACAAG GTTAAAGATT TTGCACGAGA AAGTGATGTT 451 AAGGTCATGT GCGTAGACTC TGCCCCAGAT GGGTGCTTGC ACTTTTCAGA 501 GCTAACACAG GCTGACGAAA ATGAAGTGCC CCAGGTCGAC TTTAGTCCTG 551 ATGATGTTGT AGCATTGCCT TATTCATCAG GGACTACAGG GTTACCAAAA 601 GGGGTCATGC TAACGCACAA AGGGCTAATA ACCAGTGTGG CTCAACAAGT 651 AGATGGAGAC AATCCTAACC TGTATTTTCA CAGTGAAGAT GTGATTTTGT 701 GTGTGTTGCC TATGTTCCAT ATCTATGCTC TGAATTCAAT AATGCTTTGT 751 GGGCTGAGAG TTGGTGCCTC GATTTTGATA ATGCCAAAGT TTGATATTGG 801 TACTCTGCTG GGATTGATTG AGAAGTACAA GGTATCTATA GCACCAGTTG 851 TTCCACCTGT GATGTTGGCA ATTGCTAAGT CACCTGATTT TGACAAGCAC 901 GACTTGTCTT CTTTGAGGAT GATAAAATCT GGAGGGGCTC CATTGGGCAA 951 GGAACTTGAA GATACTGTCA GAGCTAAGTT TCCTCAGGCC AGACTTGGTC 1001 AGGGATATGG AATGACCGAG GCAGGACCTG TTCTAGCAAT GTGCTTGGCA 1051 TTTGCCAAGG AACCATTTGA CATAAAACCA GGTGCATGTG GGACTGTCGT 1101 CAGGAATGCA GAAATGAAGA TTGTTGACCC AGAAACAGGG GCCTCTCTAC 1151 GGAGGAACCA GCCTGGTGAG ATCTGCATCC GGGGTGATCA GATCATGAAA 12 01 GGATATCTTA ATGACCCTGA GGCAACCTCA AGAACAATAG ACAAAGAAGG 1251 ATGGTTGCAC ACAGGCGATA TCGGCTACAT TGATGACGAT GATGAGCTTT 13 01 TCATCGTTGA CAGATTGAAG GAATTGATCA AATATAAAGG GTTTCAGGTT 1351 GCTCCTGCTG AACTCGAAGC TTTGTTACTA GCCCATCCAC AGATATCCGA 14 01 TGCTGCTGTA GTAGGAATGA AAGATGAGGA TGCAGGAGAA GTTCCTGTTG 1451 CATTTGTAGT GAAATCAGAA AAGTCTCAGG CCACCGAAGA TGAAATTAAG 1501 CAGTATATTT CAAAACAGGT GATATTCTAC AAGAGAATAA AACGAGTTTT 1551 CTTCATTGAA GCAATTCCCA AGGCGCCATC AGGCAAAATC CTTAGGAAGA 1601 ATCTGAGAGA AACGTTGCCA GGCATATAAC TGAAGACGTT ACTGAACATT 1651 TAACCCTCTG TCTTATTTCT TTAATACTTG CGAAAATGCC AATGAATCAT 17 01 TGTAGTGTTG AATCAAGCGT GCTTGGAAAA GACACGTTAC CAAACGTTAA 1751 GAACATTACT GTTCTTGTTA TACAAGCTCT TTAATGTTGC TTTTGTACTT 18 01 GGGAAAACAT AAGTTCTCCT GTCGCCATAT GGAGTAATTC AATTGAATAT 1851 TTTGGTTTTT TTAAAAAAAA AAAAAAAAAA Figure 7. Nucleotide sequence of the 4CL6 c D N A clone. The sequence includes an open reading frame of 1647 nucleotides, plus untranslated 5' and 3' end sequences. The putative initiation and termination codons are underlined. 30 Since no 4CL10 clones were identified in 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 in size. Sequencing analysis showed that these clones contained a 4CL- l ike coding region corresponding to the 4CL10 sequence, but that they lacked the 5' and 3' ends of the c D N A . In order to obtain a c D N A with the full 4CL10 coding region, R A C E technology was employed. Gene specific primers (GSPs) were designed based on the sequence of the partial c D N A 4CL10 clones. A pool of adaptor-ligated c D N A s was made from H I 1 young leaf R N A , and 4CL10 GSPs and adaptor primers were used in 5 ' R A C E and 3 ' R A C E P C R to amplify the missing ends of 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 of 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 of 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 of the 4CL10 gene was obtained by P C R amplification using the primer-ligated leaf c D N A pool and another set of 4CL10-specific primers. A 5' primer was designed based on the published sequence of the 5' end of the aspen Pt4CL2 gene, since that gene is very similar to my 4CL10 sequence. A 3' primer was designed based on the 3' untranslated portion of the 3 ' R A C E fragment of 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 of 579 amino acids (63.1 kDa, predicted molecular weight), including the putative initiation and termination codons (Figure 9). Adaptor-ligated ds cDNA NNA« NNT< 5'RACE A P I , 4 ^ L 1 0 - 7 3'RACE 5' — • — • • ' N N A — — 3 ; P 1 4 C L 1 0 ^ N G S P J • • ^ N T -Generation of the full-length cDNA of 4CL10 4CL10.9 4CL10.0 • NNA 3 ' N N T — — — ; — 5' Figure 8. The template and primers used in R A C E reactions and amplification of 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 by reverse transcription and second-strand synthesis, and it was ligated to the adaptor. AP1= 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 of R A C E reactions is greatly enhanced by absence of 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 of 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 of the 3'end of the adaptor-ligated ds c D N A , and thus prevents formation of an A P I binding site on the general population of c D N A s . 32 1 ATGATGTCCG TGGCCACGGT TGAGCCCCCG AAACCGGAAC TCTCCCCCCC 51 ACAAAACCAA AACGCACCAT CCTCTCATGA AACTGATCAT ATTTTCAGAT 101 CAAAACTACC AGATATAACC ATCTCGAACC ACCTCCCTCT GCACGCATAC 151 TGCTTTGAAA ACCTCTCTGA TTTCTCAGAT AGGCCATGCT TGATTTCAGG 2 01 TTCCACGGGA AAAACCTACT CTTTTGCCGA AACTCACCTA ATATCTCGAA 251 AGGTCGCTGC TGGGTTATCC AATTTGGGCA TCAAGAAAGG CGATGTAATC 3 01 ATGACCCTGC TCCAAAACTG CCCAGAATTC GTCTTCTCCT TCATGGGTGC 351 TTCCATGATT GGTGCAGTCA CCACCACTGT GAACCCTTTC TACACTCCAG 401 GTGAAATATT CAAGCAATTC TCTGCTTCTC GTGCGAAACT GATTATCACC 451 CAGTCTCAAC ATGTGAACAA GCTAAGAGAT AGTGATTACC ATGAAAACAA 501 CCAAAAACCG GAGGAAGATT TCATAGTAAT CACCATTGAT GACCCACCAG 551 AGAACTGTCT ACATTTCAAT GTGCTTGTTG AGGCTAACGA GAGTGAAATG 601 CCAACAGTTT CAATCCATCC GGATGATCCT GTGGCATTAC CATTCTCTTC 651 AGGGACAACA GGGCTCCCAA AAGGAGTGAT ACTGACTCAC AAGAGCTTGA 701 TAACAAGTGT GGCTCAACAA GTTGATGGAG AGATCCCAAA TTTATACTTG 751 AAACAAGATG ATGTCGTTTT ATGCGTTTTA CCTTTGTTTC ACATCTTTTC 801 ATTGAACAGC GTGTTGTTAT GCTCGTTGAG AGCCGGTTCT GCTGTACTTT 851 TAATGCAAAA GTTTGAGATC GGATCACTGC TAGAGCTCAT TCAGAAACAC 901 AATGTTTCGG TTGCGGCTGT GGTGCCACCA CTGGTGCTGG CGTTGGCCAA 951 GAACCCAATG GTGGCGAACT TCGACTTGAG TTCGATCAGG GTAGTCCTCT 1001 CAGGGGCTGC GCCACTGGGG AAGGAGCTCG AGGAGGCCCT CAGGAGCAGG 1051 GTTCCACAGG CCATCCTGGG ACAGGGTTAT GGGATGACAG AGGCGGGGCC 1101 AGTGCTATCA ATGTGCTTAG CCTTCTCAAA GCAACCTTTA CCCACCAAGT 1151 CTGGATCATG TGGAACAGTG GTTAGAAACG CAGAGCTCAA GGTCATTGAC 12 01 CCTGAGACCG GTAGCTCTCT TGGTCGCAAC CAACCTGGTG AAATCTGCAT 1251 CCGGGGATCC CAAATCATGA AAGGATATTT GAATGACGCG GAAGCCACGG 13 01 CAAACATCAT AGACGTTGAG GGTTGGCTCC ACACTGGAGA TATAGGTTAT 13 51 GTCGACGACG ACGACGAGAT TTTCATTGTT GATAGAGTGA AGGAAATCAT 14 01 AAAATTCAAA GGCTTCCAGG TGCCGCCAGC GGAGCTTGAG GCTCTCCTTG 1451 TAAACCACCC TTCAATTGCG GATGCGGCTG TTGTTCCCCG AGATAACTTG 15 01 TATGGAAACA ACAGGCAAAA AGACGAGGTT GCTGGTGAAG TTCCTGTCGC 1551 GTTTGTGGTC CGCTCAAATG ATCTTGACCT TAATGAAGAG GCTGTAAAAG 1601 ACTACATTGC AAAGCAGGTG GTGTTCTACA AGAAACTGCA CAAGGTGTTC 1651 TTCGTTCATT CTATTCCCAA ATCGGCTTCT GGAAAGATTC TAAGAAAAGA 17 01 CCTCAGAGCC AAGCTTGCCA CAGCCACCAC CATGTCCTAG ATTTCATTAC 1751 GTTAAATCTG CATTTATTAT TTTGTGTTGT CTTTCACTCG CTGTGGAAAG 18 01 ATTCTAAGAA AAGACCTCAG AGCCAAGCTT GCCACAGCCA CCACCATGCA 1851 TGTCCTAGAA TTCATTCCGT TAAATCTGCA TTTATATTAT TTTGTGTTGT 1901 CTTCACTCAT TGTGCCC Figure 9. Nucleotide sequence of the 4CL10 c D N A clone. The sequence includes an open reading frame of 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 of 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 of the G C G sequence analysis software (Figure 10). 33 4CL6 MDAIMNSQEEFIFRSKLPDIYIPKNLPLHSY 31 4CL10 MMSVATVEPPKPELSPPQNQNAPSSHETDHIFRSKLPDITISNHLPLHAY 5 0 32 VLENLSKYSSKPCLINGANGDVYTYADVELTARRVASGLNKIGIQQGDVI 81 51 CFENLSDFSDRPCLISGSTGKTYSFAETHLISRKVAAGLSNLGIKKGDVI 100 82 MLFLPSSPEFVLAFLGASHRGAIVTAANPFSTPAELAKHAKPPRTKLLIT 131 101 MTLLQNCPEFVFSFMGASMIGAVTTTVNPFYTPGEIFKQFSASRAKLIIT 150 132 QACYYDKVKDF ARESDVKVMCVDSAPDGCLHFSELTQADENEV 174 151 QSQHVNKLRDSDYHENNQKPEEDFIVITIDDPPENCLHFNVLVEANESEM 2 00 2 01 PTVSIHPDDPVALP 175 PQVDFSPDDWALPySSGTTGLPK3VMLTHKGLITSVAQQVDGDNPNLYF 224 SSGTTGLPK3VILTHKSLITSVAQQVDGEIPNLYL 2 5 0 225 HSEDVILCVLPMFHIYALNSIMLCGLRVGASILIMPKFDIGTLLGLIEKY 274 : | | : | | l l | : | | | : - l l h = l l II l-- = h l I h l l - l l 11 = 1 = 251 KQDDWLCVLPLFHIFSLNSVLLCSLRAGSAVLLMQKFEIGSLLELIQKH 3 00 275 KVSIAPWPPVMLAIAKSPDFDKHDLSSLRMIKSGGAPLGKELEDTVRAK 324 I h i | | | . . | | : : - M M - : || I I I I I M I : -301 NVSVAAWPPLVLALAKNPMVANFDLSSIRWLSGAAPLGKELEEALRSR 350 32 5 FPQARLGQGYGMTEAGPVLAMCLAFAKEPFDIKPGACGTWRNAEMKIVD 3 74 351 VPQAILGQGYGMTBASPVLSMCLAFSKQPLPTKSGSCGTWRNAELKVID 4 00 401 PETGSSLGRNQPJ3EICIRG I I 3 75 PETGASLRRNQPjGEICIRGDQIMKGYLNDPEATSRTIDKEGWLHTGDIGY 424 BQIMKGYLNDAEATANIIDVEGWLHTGDIGY 450 425 IDDDDELFIVDRLKELIKYKGFQVAPAELEALLLAHPQISDAAWGMKDE 474 451 VDDDDEIFIVDRVKEIIKFKGFQVPPAELEALLVNHPSIADAAWPQKDE 500 475 D AGE VP VAF WKS E KS Q ATED EIKQ YIS KQ VIF Y KRIKR VF FIE AIP KAP 524 501 VAGEVPVAFWRSNDLDLNEEAVKDYIAKQWFYKKLHKVFFVHSIPKSA 55 0 525 SGKILRKNLRETLPGI*.... 541 551 SGKILRKDLRAKLATATTMS* 571 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). T a b l e II Comparison of 4CL6 and 4CL10 predicted amino acid sequences to each other and to other poplar 4CL cDNA sequences. cDNA 4CL9 % identity 4CL216 % identity 4CL6 % identity 4CL10 % identity 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 4CL6 % identity 4CL10 % identity Ref. 4CL6 * 61 4CL10 61 * poplar 4CL1 74 65 Allina & Douglas (1997) poplar 4CL2 74 64 Allina & Douglas (1997) aspen 4CL1 94 60 Hue?al. (1998) aspen 4CL2 60 96 Hue?al. (1998) tobacco M4CL19 77 67 Lee & Douglas (1996) tobacco Nt4CLl 78 65 Lee & Douglas (1996) Lithospermum 4CL1 73 60 Yazakief al. (1995) Lithospermum 4CL2 60 76 Yazakief al. (1995) Arabidopsis 4CL1 70 60 Lee et al. (1995) soybean 4CL1 80 64 Uhlmann & Ebel (1993) soybean 4CL2 66 78 Uhlmann&Ebel(1993) 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 and rice 4CL2. 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-chain-fatty-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 1 O g N K g 4CL216 1 JSKNS 4CL6 1 BUBIM'SO! 4CL10 1 MMSVATVEPPKPELSPPQNQNBPSSHE 4CL9 4CL216 4CL6 4CL10 4CL9 4CL216 4CL6 4CL10 4CL9 4CL216 4CL6 4CL10 4CL9 4CL216 4CL6 4CL10 4CL9 4CL216 4CL6 4CL10 4CL9 4CL216 4CL6 4CL10 4CL9 4CL216 4CL6 4CL10 4CL9 4CL216 4CL6 4CL10 Y C F E g L S YCFENLSM. Y__ENLSK YCFENLS lit I SS :>Ss_ 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 102 102 102 121 I ********** 216 216 TSVAQQ TSVAQQ TSVAQQ TSVAQQ SLMDLVQKY G E N P N L Y F H E G D V I L C V L P L F H I Y S L N S V _ L C G L R G G S A I L V M Q K F D G E N P N L Y F H E B D V I L C V L P L F H I Y S L N S V L L C G L R R G S A I L L M Q K F E T S T L M E L ^ Q K Y G _ M P N L Y F H ^ D V I L C V L P | F H I Y A L N S I M L C G L R R G A S I L : I : M _ K F D I B T L L _ L I E 396 396 395 421 I I ******* Q I M K G Y L N D P E A T E R T I D K T J G W L H T G D I G Y I D H | D E L F I V D R L K E L I K Y K G F Q V A P A E L E Q I M K G Y L N D P E A T E R T § D _ D G W L H T G D I G Y I D _ D D E L F I V D R L K E L I K Y K G F Q V A P A E L E Q I M K G Y L N D P E A T H R T I D K J E G W L H T G D I G Y I D D D D E L F I V D R L K E L I K Y K G F Q V A P A E L E O I M K G Y L N D B E A T S M I D ^ G W L H T G D I G Y T O D D D E T F I V D R V K E ' I I K F K G F O V H P A E L E 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 1 MMSVATVEPPKPEMPPffiSQNAPSgHgTgl 4CL6 1 ffiDAIMffi @ Q @ - | F | _ l u c i f e r a s e 1 l a B l E M ILIGPPPYYJJLEEGJ S AGEC fadD-5 1 M B FTEDI 4CL10 61 4CL6 42 l u c i f e r a s e 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 E S l u c i f e r a s e l 4 3 --LPI fadD-5 142 ERFP@I 4CL10 217 E 4CL6 191 l u c i f e r a s e l 9 8 fadD-5 198 I 4CL10 277 4CL6 251 l u c i f e r a s e 2 5 6 fadD-5 254 4CL10 337 I 4CL6 311 l u c i f e r a s e 3 1 6 fadD-5 314 4CL10 397 4CL6 371 l u c i f e r a s e 3 7 1 fadD-5 372 4CL10 457 4CL6 431 l u c i f e r a s e 4 3 1 fadD-5 432 4CL10 517 4CL6 482 l u c i f e r a s e 4 8 2 fadD-5 483 j D E V A G E V P K jDEj^AGEVpR dDEVAGgfjPS D E J ^ G E I p iRKff lLR^LgJT IRK'LREHLBSH BRKIKEILI Figure 12. Alignment of deduced amino acid sequences of 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 (AMP-binding 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 4CL6 rRNA B 4CL10 rRNA 83 S3 cU £ cu -ta» = 5 g *- j , + 3 cu — © ""5 >. O X Cd W • i t 1.9 kb HI*! f__L 1.9 kb Figure 13. N o r t h e r n - b l o t a n a l y s i s o f 4CL6 a n d m R N A l e v e l s . T o t a l R N A ( 1 0 fi g) F r o m d i f f e r e n t t i s s u e s 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 e i t h e r 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 s t r i n g e n c y ( 0 . 2 X S S C , 0 . 1 % S D S , 6 5 ° C ) . E v e n l o a d i n g w a s s h o w n b y s t r i p p i n g b l o t s a n d r e h y b r i d i z i n g w i t h a p e a r R N A p r o b e . E L + , e l i c i t o r - t r e a t e d t i s s u e c u l t u r e c e l l s : E L I - , u n t r e a t e d c o n t r o l c e l l s . 40 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 (All ina 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 of homologous recombination between pVL1392::4CZ<5 and A c N P V viral D N A in Sf9 insect cells. Identification and purification of the recombinant 4 C L 6 baculovirus particles were done as described in 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 of David Theilman, Pacific Agr i -Food Research Center, Summerland, B C . To generate the recombinant 4 C L 6 protein, 50 x 10 6 Sf9 cells were infected with 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 of 4 C L activity, assayed using 4-coumaric acid as a substrate (Figure 14). The most active fractions of 4 C L 6 had specific activities between 498 and 597 pkat/p-g protein. 41 0 g o o © o CM co *t m <o 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 — Figure 15. S D S - P A G E and immunoblot analysis of recombinant 4 C L 6 protein. S D S - P A G E gel (A) and an immunoblot of 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 of Sf9 cells infected with 4 C L 6 and 4 C L 9 baculovirus constructs, 4 C L 6 FPLC-purif ied from Sf9 infected cells. 43 As shown on Figure 16, eight 4CL10-expressing baculovirus independent stocks, express recombinant proteins of the size predicted for 4CL10 protein. The protein has a higher molecular weight than the recombinant 4CL6 and 4 C L 9 proteins, as expected from the predicted amino acid sequence. Crude extracts of Sf9 cells infected with the 4CL10 baculovirus stocks showed 5-10 fold less 4 C L activity toward 4-coumaric acid than crude extract of Sf9 infected with 4CL6 baculovirus particles. 4CL10 Figure 16. S D S - P A G E analysis of recombinant 4CL10 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 of Sf9 cells infected with 4CL10 (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 of 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 5-hydroxyferulic acid was not tested because 5-hydroxyferulic acid was available in insufficient amount. 120 , 100 • 80-I 60. * 40 4 20-0 coumarate cinnamate caffeate Substrat ferulate sinapate Figure 17. Substrate-utilization profile of 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 of hydroxycinnamic acids. Results are averages of three trials: error bars represent SD values. Asterisks indicate the absence of detectable activity. Results are reported as a precentage of the activity against 4-coumaric acid, which was 480.7 pkat/mg. 45 CHAPTER IV Discussion The results of this work confirm to the existence of at least four 4 C L isoenzymes in poplar. This conclusion is based on isolation of two full-size 4CL c D N A clones in addition to those previously described (Al l ina et al, 1998), and is consistent with multiple 4 C L proteins detected by F P L C chromatography (Al l ina et al, 1998). The use of degenerate P C R primers that target conserved sequences in 4CL genes allowed the efficient identification of divergent 4CL genes. Thus, while previous screening of a poplar young leaf c D N A library resulted in identification of two 4CL c D N A clones with 86% identity at the predicted amino acid level (Al l ina et al, 1998), my work showed that the poplar 4CL gene family in fact contains three new 4CL classes (4CL6, 4CL10 and 4CL14) in addition to the previously studied genes. These were distinguished among 72 clones characterized from the products of PCR 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 of at least eight pal loci in the jack pine genome (Butland et al, 1998). In contrast, the previous study on the PAL gene family in 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 by hybridization. The degenerate primers used in this work performed with different efficiency in 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 ESTs represent different parts of the same gene, which supports the result of this work that 4CL6 is the major 4 C L isoform expressed in the wood-forming tissues. Neither of the degenerate primer sets amplified any sequence that corresponded to the 4CL216 gene. A comparison of 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 of 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 # of mismatches 4 C L 9 3 ' T C C A G G T T T G A G G G A C T G T A G 5 ' 3 Primer 3 5 ' C G G T C I A A A T T G C C I G A C A T 3 ' 4 C L 2 1 6 3 ' T C C A G G T T T G A G G G A C T A T A G 5 ' 4 4 C L 6 3 ' G C G A G T T T T A A T G G T C T G T A G 5 ' 0 Primer 3 5 ' C G G T C I A A A T T A C C I G A C A T 3 ' 4 C L 1 0 3 ' T C T A G T T T T G A T G G T C T A T A T 5 ' 2 Conserved amino acids R S K L P D I B 4 C L 9 5 ' A A A G G T G T C A T G T T G A C T C 3 ' 2 Primer 8 3 ' T T T C C I C A C (A) T A C A A A T G A G 5 ' 4CL216 5 ' A A A G G T G T C A T G T T G A C T C 3 ' 2 4 C L 6 5 ' A A A G G G G T C A T G C T A A C G C 3 ' 2 Primer 6 3 ' T T T C C I C A C ( A ) T A C G A I T G A G 5 ' 4 C L 1 0 5 ' A A A G G T G T G A T A C T G A C T C 3 ' 1 Conserved amino acids K G V I L T Figure 18. Degenerate primers and corresponding sequences in poplar 4 C L genes. A , comparison of the forward primer 3 with the actual nucleotide sequences; B , Comparison of the reverse primers 6 and 8 with the actual nucleotide sequences of 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 AMP-bind ing motif (region I in 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-acid-CoA 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 5 0 4CLs from class I (Lozoya et al, 1988; Beecker-Andre et al, 1991). H u et al (1998) reported that the promoter region of 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 in 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 in 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 (All ina et al, 1998). 4CL6 is strongly expressed in xylem, green stem and root, but the expression was not detected in young leaves, while 4CL10 is expressed only in leaves and root. In contrast, 4CL9 m R N A is most abundant in young leaf, and 4CL216 is preferentially expressed in old leaf (All ina et al, 1998). Based on these results, the function of 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 in the biosynthesis of nonlignin-related phenylpropanoids (e.g. flavonoids and phenylpropanoid esters), that accumulate in poplar leaves. A similar difference in the expression patterns of aspen Pt4CLl and Pt4CL2 has been observed (Hu et al, 1998.). Also , 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 epidermis-specific (Hu et al, 1998). In Arabidopsis, the most divergent (class II) gene, 4CL3, was 5 1 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 isoform from soybean (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 genes from parsley, 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 elicitor-activated 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 suspension-cultured 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 4-coumaric 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 isoforms from soybean, 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 of G units and resistant biphenyl structures was dramatically enhanced (Lapierre et al. 1999). Because down-regulation of 4 C L activity in Arabidopsis leads to a decrease in G residues, but no change in S residues, Lee et al. (1997) proposed that S-lignin might be synthesized by a 4CL-independent pathway. The absence of activity toward sinapic acid and absence of catalytically distinct 4 C L isoforms in poplar and other plants (Lozoya et al., 1988; Voo et al., 1995; Lee and Douglas, 1996), makes it unlikely that 4 C L controls partitioning of 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% in 4 C L down-regulated transgenic tobacco (Kajita et al, 1996), and somewhat reduced in 4 C L down-regulated Arabidopsis (Lee et al., 1997). Antisense suppression of Pt4CLl in transgenic aspen showed up to a 45% reduction of lignin content and increased in cellulose content (Hu et al, 1999). 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