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Towards genetic modification of the lignin biosynthetic pathway in interior spruce (Picea glauca x engelmanni… Gray-Mitsumune, Madoka 2000

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TOWARDS GENETIC MODIFICATION OF L I G N I N BIOSYNTHETIC PATHWAY I N INTERIOR SPRUCE {Picea glauca x engelmanni c o m p l e x ) By MADOKA GRAY-MITSUMUNE B . S c , Ehime U n i v e r s i t y , J a p a n , 1990 M . S c , Ehime U n i v e r s i t y , J a p a n , 1992 M . S c , The U n i v e r s i t y o f New B r u n s w i c k , 1995 A THESIS SUBMITTED I N PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY I n THE DEPARTMENT OF PLANT SCIENCES THE FACULTY OF GRADUATE STUDIES We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA December 2000 © . M a d o k a G r a y - M i t s u m u n e , ,2000 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o lumbia, I a gree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head of my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada Date D e c , 2.Q , zzjpv-Q 11 Abstract Although the lignin biosynthetic pathway has been altered successfully in angiosperm species via genetic engineering approach, this has not yet been achieved in gymnosperm species. Therefore, the goal of my thesis research was to apply the transgenic approach to economically important interior spruce (Picea glauca x engelmanni complex). In the first half of my thesis, the poplar PAL2-GUS fusion gene was introduced into hybrid poplar (Populus tremula x P. alba) and interior spruce in order to to evaluate the potential use of this promoter for directing xylem-specific gene expression. In transgenic poplar, the poplar PAL2 promoter directed the expression of the GUS gene in the tissues associated with synthesis of phenolic compounds (epidermal/subepidermal cells) and in the tissues associated with lignin synthesis (xylem and phloem cells). In contrast, in transgenic spruce, the activity of the poplar PAL2 promoter was detected only in the tissues associated with lignin and suberin synthesis. The differences in the activity of the poplar PAL2 promoter between the two hosts suggest that the gene regulation system that leads to the synthesis of essential structural components such as lignin and suberin is more likely to be conserved than that leading to the synthesis of specialized phenolic compounds. The second half of this thesis investigated a contribution of coniferin (3-glucosidase (CG) in lignin synthesis in spruce. C G is believed to be involved in the last steps of the lignin biosynthetic pathway by releasing the lignin precursor, coniferyl alcohol, from its glucosides form, coniferin, before polymerization in the cell wall. A n antisense construct against the CG gene was prepared using the lodgepole pine CG c D N A sequence and was introduced into interior spruce. Among 45 antisense lines examined, no transgenic lines contained reduced levels of endogenous CG mRNA levels. The failure of the antisense CG gene to cause inhibitory effects on the endogenous CG expression levels in transgenic spruce i i i could be attributed to low expression of the antisense gene, or to insufficient sequence homology between the antisense and target CG sequences. To my knowledge, this is the first study to employ an antisense approach in a gymnosperm species. IV T A B L E OF CONTENTS Abstract ii Table of Contents iv List of Abbreviations vii List of Tables ix List of Figures x Acknowledgements xii Chapter 1 Lignin Biotechnology 1 1.1 Introduction 1 1.2 Lignin biosynthetic pathway 3 1.2.1 Monolignol biosynthesis 3 1.2.2 Storage and transport of monolignols 6 1.2.3 Oxidative polymerisation of monolignols 9 1.3 Genetic engineering approach to modify lignin biosynthesis 10 1.3.1 Gene manipulation to control lignin content 10 1.3.2 Gene manipulation to control lignin monomer composition . 17 1.4 Natural and artificial mutations in the lignin biosynthetic pathway 21 1.4.1 Brown-midrib mutants 21 1.4.2 Arabidopsis mutants 23 1.5 Proposal of thesis research - application of lignin biotechnology to conifers 24 Chapter 2 Developmental regulation of the poplar PAL2-GUS fusion gene in transgenic poplar 28 2.1 Introduction 28 2.2 Materials and Methods 30 2.2.1 Agrobacterium-msdiated transformation of hybrid poplar. 30 2.2.1.1 Plant material and growth condition 30 2.2.1.2 Agrobacterium-mediated transformation . . . 30 2.2.2 GUS assays 32 2.2.2.1 Histochemical GUS staining 32 2.2.2.2 Fluorometric GUS assay 32 2.2.3 Lignin staining 33 2.2.4 Genomic D N A extraction 33 2.2.5 PCR analysis of putative transformants 34 2.3 Results 34 2.3.1 Generation of transgenic poplar 34 2.3.2 Expression of the PAL2-GUS fusion in different organs . . 37 2.3.3 Expression of the PAL2-GUS fusion in different cell types 42 2.4 Discussion 46 Chapter 3 Generation of Transgenic Spruce 50 3.1 Introduction 50 3.2 Materials and Methods 51 V 3.2.1 Overview of transformation process 51 3.2.2 Tissue culture conditions 52 3.2.3 Transformation vectors 53 3.2.4 Particle bombardment 54 3.2.5 Transient expression assays 57 3.2.6 Selection and screening of transformed cell lines 57 3.2.7 Screening for GUS activity 60 3.2.8 Extraction of spruce genomic D N A 60 3.2.9 PCR analysis of putative transformants 61 3.3 Results 62 3.3.1 Comparison of D N A delivery efficiencies 62 3.3.2 Kanamycin selection of transformed tissues 65 3.3.3 GUS screening of putative transformants 68 3.3.4 PCR analysis 69 3.4 Discussion 72 Chapter 4 Vascular-specific expression of the poplar PAL2-GUS fusion gene in transgenic spruce 78 4.1 Introduction 78 4.2 Materials and Methods 80 4.2.1 Generation of transgenic spruce 80 4.2.2 GUS assays 80 4.3 Results: 80 4.4 Discussion 90 Chapter 5 Effects of an antisense coniferin P-glucosidase (CG) gene in transgenic spruce 94 5.1 Introduction 94 5.2 Materials and Methods 96 5.2.1 P C R cloning of the spruce coniferin P-glucosidase (CG) gene . . . 96 5.2.2 R N A extraction 98 5.2.3 RT-PCR analysis 99 5.2.4 Construction of transformation vectors 102 5.2 5 Generation of transgenic spruce 107 5.2.6 Protein extraction 107 5.2.7 Coniferin glucosidase assay 107 5.2.8 Western blotting 108 5.2.9 Statistical analysis 109 5.3 Results 109 5.3.1 Structure and expression of the spruce CG gene 109 5.3.1.1 P C R cloning 109 5.3.1.2 Intron structure of spruce CG genes 113 5.3.1.3 Sequence homology of CG genes 114 5.3.1.4 Expression of the spruce CG genes during seedling development 120 5.3.2 Effects of the antisense CG mRNA on development of transgenic spruce 125 VI 5.3.2.1 Generation and growth of transgenic spruce 12 5 5.3.2.2 C G enzyme activity and C G protein levels in transgenic spruce 133 5.3.2.3 Detection of sense and antisense CG mRNA in transgenic spruce 137 5.4 Discussion 145 5.4.1 Structure and expression of CG genes in spruce 145 5.4.2 Effect of an antisense CG gene in transgenic spruce 150 Chapter 6 Towards genetic engineering of the lignin biosynthetic pathway in conifers (Concluding remarks and future considerations) 154 References 158 List of Abbreviations 4CL 4-coumarate:coenzyme A ligase A B A abscisic acid A M V alfalfa mosaic virus B A P 6-benzylaminopurine BCIP 5-bromo-4-chloro-3-indolyl phosphate B S A bovine serum albumin C3H coumarate 3-hydroxylase C4H cinnamate 4-hydroxylase C A D cinnamyl alcohol dehydrogenase C a M V cauliflower mosaic virus CAPS 3-[cyclohexylamino]-l-propanesulfonic acid C C o A O M T caffeoyl-coenzyme A O-methyltransferase C C R cinnamoyl coenzyme A reductase C G coniferin P-glucosidase C O M T caffeate/5-hydroxyferulate O-methyltransferase C T A B hexyadecyltrimethyl ammonium bromide DTT dithiothreitol E D T A ethylenediaminotetraacetic acid ER endoplasmic reticulum F5H ferulate 5-hydroxylase G guaiacyl GUS P-glucuronidase H hydroxyphenyl M E S 2-[N-morpholino] ethanesulfonic acid M - M L V Molony-murine leukemia virus M U 4-methylumbelliferone M U G 4-metylumbelliferyl P-D-glucuronide N A A naphthalic acid N B T nitroblue tetrazolium NPTII neomycin phosphotransferase II gene V l l l P A G E polyacrylamide gel electrophoresis P A L phenylalanine ammonia-lyase PCR polymerase chain reaction P E G polyethylene glycol PIG particle inflow gun PO peroxidase PVP polyvinylpyrrolidone PVPP polyvinylpolypyrrolidone RT-PCR reverse transcription-polymerase chain reaction S syringyl SDS sodium dodecyl sulphate X-gluc 5-bromo-4-chloro-3-indol-l-glucuronide IX List of Tables Table 1.1 Lignin modification in transgenic plants 11 Table 1.2 Examples of natural and artificial mutants of lignin synthesis 24 Table 1.3 Cloning of lignin synthesis genes from conifers 26 Table 1.4 Production of transgenic conifers 27 Table 2.1 Media for poplar micropropagation 31 Table 2.2 Primers used for PCR screening 34 Table 2.3 Comparison of different explants for ability to produce transformed shoots . . . 37 Table 3.1 Media for spruce micropropagation 53 Table 3.2 Plasmids used for spruce transformation 54 Table 3.3 Primers used for PCR screening 61 Table 3.4 Effect of kanamycin selection on wild-type spruce cells 67 Table 3.5 Kanamycin selection of transformed cells 68 Table 3.6 Effect of initial selection on transformation efficiency 68 Table 3.7 Summary of spruce transformation 71 Table 3.8 Summary of PCR analyses 71 Table 5.1 Primers for P C R amplification of the CG gene 98 Table 5.2 Primers used for RT-PCR analysis 101 Table 5.3 Pairwise comparison of three CG genes 117 Table 5.4 Selected list of genes that are homologous to ScgA and ScgB 118 Table 5.5 Comparison of sense and antisense CG mRNA levels in transgenic spruce . . . 145 X List of Figures Figure 1.1 Structures of the three monolignols and their resulting residues in the lignin polymer 2 Figure 1.2 Outline of monolignol biosynthesis 4 Figure 1.3 Synthesis and hydrolysis of monolignol glucosides 8 Figure 1.4 Monolignol glucosides may serve as intracellular transport forms of monolignols 8 Figure 2.1 Generation and growth of transgenic poplar 36 Figure 2.2 Expression of the poplar PAL2-GUS fusion gene in in W/ro-grown transgenic poplar 39 Figure 2.3 Expression of the PAL2-GUS fusion gene in soil-grown transgenic poplar.... 40 Figure 2.4 Expression of the poplar PAL2-GUS fusion gene in greenhouse-grown transgenic poplar 42 Figure 2.5 Histochemical activity of PAL2-GUS in transgenic poplar 44 Figure 3.1 Transformation process 52 Figure 3.2 Schematic diagrams of two particle bombardment devices 56 Figure 3.3 Selection of transformed cells 59 Figure 3.4 Generation of transgenic spruce 62 Figure 3.5 Comparison of two particle bombardment devices 64 Figure 3.6 Effect of rupture disc pressures on D N A delivery efficiency 64 Figure 3.7 Example of PCR analyses 70 Figure 4.1 Expression of the poplar PAL2-GUS fusion gene in different tissues of transgenic spruce 82 Figure 4.2 Histochemical assay for expression of the PAL2-GUS fusion gene in micropropagated transgenic spruce 86 Figure 4.3 Histochemical assay for expression of the PAL2-GUS fusion gene in soil-grown transgenic spruce plants 88 Figure 5.1 Primer binding sites 98 Figure 5.2 Primers used for detection of antisense CG mRNA 101 Figure 5.3 Strategy for making PCR competitors 102 Figure 5.4 Generation of a full-length CG c D N A fragment 103 Figure 5.5 Construction of p B A C G 104 Figure 5.6 Construction of p B A C G G U S 106 Figure 5.7 Structure of p B A C G G U S 106 Figure 5.8 PCR amplification of spruce CG sequences I l l Figure 5.9 Intron structure of spruce CG sequences 114 Figure 5.10 Alignment of deduced amino acid sequences of the spruce and pine CG genes 116 Figure 5.11 Phylogenetic tree of (3-glucosidase genes 119 Figure 5.12 RT-PCR analysis of the CG gene expression during germination of spruce ... 121 Figure 5.13 Xylem development of spruce during germination 123 Figure 5.14 Growth of transgenic spruce plants 127 Figure 5.15 Height comparison of transgenic spruce plants 129 Figure 5.16 Lignin staining of transgenic spruce plants 131 Figure 5.17 Western blotting analysis of protein extracts from embryogenic tissues of transgenic spruce 135 Figure 5.18 Levels of the sense CG mRNA in transgenic spruce as detected by RT-PCR ..139 Figure 5.19 Levels of the antisense CG m R N A in transgenic spruce as detected by RT-P C R 141 Figure 5.20 R N A quantification by competitive RT-PCR 143 X l l Acknowledgements During the six years of my Ph.D. program, I was fortunate to have a supervising committee made up of superb scientists, each of whom provided me with their different expertises. I would like to thank Dr. John Carlson for being my academic supervisor for the first three years of my study, and for exposing me to the world of forest genetics. I am grateful to Dr. Brian Ell is for being my academic supervisor for the last three years of my study, and for introducing me to biochemical approaches to study plants. I would like to thank Dr. Dave El l i s at B C Research Inc. (BCRI) for being my industrial supervisor and for teaching me the skil l and knowledge of genetic transformation. I am indebted to Dr. Carl Douglas for providing me with many critical suggestions to my research and for exposing me to plant molecular biology. I would like to thank Dr. Rob Guy for providing me with insights as a tree plant physiologist. The last year of my study has been a struggle between thesis writing and my new job as a postdoctoral fellow. I would like to thank my postdoc supervisors Drs. Bjorn Sundberg and Ewa Mellerowicz for understanding the need to complete my thesis writing. M y thesis research would not have been possible without technical assistance from many people. I acknowledge M s Margarita Gilbert for her technical supports and advice in generating transgenic spruce. M y acknowledgements also go to B C R I technicians, M s Michelle Parsons, M r . Tei Kyung and M s Mir ian K i m for their assistance with tissue culture work and for showing me an excellent example of teamwork. I thank Green Timbers Nursery for taking care of transgenic spruce trees, and M r . Ron Rollo at the U B C Botanical Garden Nursery and Dr. David Kaplan at the U B C Horticultural Greenhouse for taking care of transgenic poplar trees. I thank M r . Victor Luk for his advices in tissue culture and X l l l culture and transformation procedures of Populus species. I am grateful to M s Stefanie Butland for her assistance and advices in molecular biological procedures. The proposal for my thesis research was based upon the knowledge obtained through thesis works of former graduate students at U B C . I am particularly thankful to Dr. Elizabeth Moli tor for her work on poplar PAL promoters and for providing me poplar PAL-GUS fusion gene constructs, and also to Dr . Palifha Dharmawardhana for his work on coniferin P-glucosidase (CG) and for providing me with a lodgepole pine CG c D N A clone. Many discussions with these Drs. were invaluable to my thesis research. M y life as a Ph.D. student was enriched by everyday interactions with people in the laboratories o f Drs John Carlson and Brian El l i s . M y special appreciation goes to Dr. Amrita Kumar, whose inquisitive mind had cultivated my critical thinking towards scientific research. I was fortunate to work with M s Stefanie Butland, whose positive attitude towards science also inspired me with much excitation. I also enjoyed discussion with Dr. Lacey Samuels, whose enthusiasm towards her research was infectious to my own. It was a pleasure to work with the technicians at Dr. John Carlson's laboratory, M s Claudia Hentschel, M s Lijuan Sun, M r . Daryl Louie, M s L i z a De Castro and M s Shirley Wong, whom I continued to see at the Friday lunch rituals long after Dr. Carlson's departure. I would like to thank B C Science Council for financial support to my Ph.D. study through G R E A T Awards and B C Research Inc. for being my industrial collaborator and for granting me the access to their research facilities. I would like to thank my parents K e n and Sumiko Mitsumune for their trust and moral support throughout the long years o f my school education. A t last and not the least, I would like to express my deepest gratitude to my family Darrin and Zack. M y school years would not have been such positive experience i f in the xiv absence of Damn 's friendship and encouragement. It was Darrin who suggested that I pursue a Ph.D. degree. He has always given me an extra push to overcome my obstacles. Thanks to Darrin, my English skil l has improved significantly to the point that I would win awards for oral presentations. Thanks to him, I had no concern over my son's care while I was busy with school work. Also, I would like to thank my son Zack for being there to cheer me up when I came home exhausted after a long day of work, and for respecting my work when I was at home writing my thesis. In closing, I am grateful to the fact that I have this many people to acknowledge and share the pleasure of completing Ph.D. degree. After all, what would be the meanings of achievements i f there were nobody to share with? Chapter 1 Lignin biotechnology l 1.1 Introduction Lignins are phenolic polymers that constitute a major portion of wood. During xylogenesis, lignins are deposited in the secondary cell walls to form a matrix surrounding the cellulose/hemicellulose microfibril network. The physicochemical properties of lignins give the plant cell wall important characteristics. Lignified cell walls are hydrophobic, which may support water conductivity by xylem cells and prevent water loss from the plant body. Lignification also gives mechanical strength to the cell wall by reinforcing the cellulose microfibril network and strengthening the bonding between microfibrils. These properties probably played crucial roles in the evolutionary adaptation of plants as they migrated from an aquatic environment to a land environment millions of years ago. Lignins may also be important for plant survival under stress conditions. For example, upon pathogen attack, lignin deposition is induced at the site of challenge, forming a physical barrier against pathogen penetration (Dean and Kuc, 1987). Similarly, lignins are deposited around wound sites to provide a sealant. Lignins are formed by oxidative polymerization of three hydroxycinnamyl alcohols (monolignols), p-coumaryl, coniferyl and sinapyl alcohols, which give rise to the hydroxyphenyl (H), guaiacyl (G) and syringyl (S) residues of lignin, respectively (Figure 1.1). The three monolignols differ in the degree of O-methylation at the aromatic ring. Lignins are highly heterogeneous in nature, with the exact polymer compositions depending on species, cell types and different parts of the cell wall. For example, gymnosperm lignins consist mainly of G units, whereas angiosperm lignins consist of both G and S units (Terashima et al, 1993). Vessels of angiosperm species are rich in G units, 2 whereas fibres of the same plant are rich in G and S units (Chappie et ah, 1992; Terashima et ah, 1993). Within the same fibre cells of an angiosperm plant, lignins in the middle lamella consist predominantly of G units, whereas a majority of monomer units in the secondary cell wall are S type (Terashima et ah, 1993). Variation is also observed in lignin content of woody tissues among different species. In general, angiosperm woods (hardwoods) contain less lignin ( 1 8 - 2 5 % of wood dry weight) than gymnosperm woods (softwoods, 25 - 35 % of wood dry weight) (Biermann, 1996). C H 2 O H C H 2 O H C H 2 O H ^ t f ^ O C H 3 H a t t ^ Y ^ O C H , O H O H O H 4-coumaryl alcohol conlferyl alcohol slnapyl alcohol ^ polymerization | polymerization ^ polymerization - l i g n i n — — l i g n i n — — l i g n i n — ^ ^ ^ O C H 3 H 3 C O ^ y ^ < O H numbering convention O C H 3 6-R O -R O - R hydroxyphenyl residue gualacyl residue syrlngyl residue Figure 1.1 Structures of the three monolignols and their resulting residues in the lignin polymer (modified from Whetten et ah, 1998). The quantity and quality of lignins in plants are key factors in some commercial uses of plant materials. In pulp and papermaking, lignins are unwanted components and must be removed by harsh chemical treatments, which are energy-consuming and hazardous to the environment (Boudet and Grima-Pettenati, 1996). Forage crops with high lignin content are difficult for animals to digest, and thus have a lowered nutritional value as animal feed (Sewalt et ah, 1997a). Therefore, the focus of lignin biotechnology has been to reduce lignin content or to modify lignin monomer composition, for easier removal by 3 chemical treatments or digestion. During the last decade, extensive research efforts have been devoted to modifying the lignin biosynthetic pathway via genetic engineering. This chapter reviews our current understanding of the lignin biosynthetic pathway and the related progress in lignin biotechnology. 1.2 Lignin biosynthetic pathway Lignin biosynthesis proceeds in three steps: a) monolignol biosynthesis, b) storage and transport of monolignols, and c) oxidative polymerisation of monolignols. The pathway has been reviewed extensively in the last five years (Boudet et al, 1995; Boudet and Grima-Pettenati, 1996; Whetten and Sederoff, 1995; Campbell and Sederoff, 1996; Douglas, 1996; Whetten et al., 1998; Grima-Pettenati and Goffner, 1999; Boudet, 2000). The following is a brief overview of the lignin biosynthetic pathway. 1.2.1 Monolignol biosynthesis A n outline of monolignol biosynthesis is shown in Figure 1.2. The initial steps of monolignol biosynthesis follow the general phenylpropanoid pathway. Phenylalanine ammonia-lyase (PAL) catalyses the first step in phenylpropanoid metabolism: the deamination of L-phenylalanine to form ?ra«s-cinnamic acid. The /rara-cinnamic acid is hydroxylated at the 4-position of the aromatic ring by cytochrome P450-dependent cinnamic acid 4-hydroxylase (C4H) to form p-coumaric acid. At this stage, the aromatic ring of />-coumaric acid may be modified to form various hydroxycinnamic acids (caffeic acid, ferulic acid, 5-hydroxyferulic acid and sinapic acid). Alternatively, the ring modification may also occur after modification of the side chain acid group (see below). Hydroxycinnamic acids are activated to form CoA thioesters (p-coumaroyl-CoA, caffeoyl-4-C O O H PAL COOH N H 2 6 ^ * J L-phenylolanfnc COOH C 3 H CCoA3H O H p-coumaryl alcohol H (hydroxyphenyl) COOH C O M T CCoAOMT COOH J-COOH J A s C O M T A J L — • * > Y O C H 3 C H 3 0 V ^ o c H a O H O H I * * T COSCaA J * COSC6A CCoAOMT J L — * • [ J L OCH3 C H 3 0 O H O H COMT H O ' ^ N f ^ C < H 3 C H 3 O ' OCH3 OH O H 5 - O H contferaldehyde sinapaldehyde O H conlferyl alcohol 6 (Guaiacyl) C O M T H O " y " 0 C H 3 C H 3 0 ' f 0CH3 O H O H 5 - O H conlferyl alcohol slnapyl alcohol 5 - O H 6 S (5-hydroxygufliacyl) (Sinapyl) Figure 1.2 Outline of monolignol biosynthesis (modified from Grima-Pettenati and Goffner, 1999). P A L , Phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; C3H, coumarate 3-hydroxylase; COMT, caffeate/5-hydroxyferulate 0-methyltransferase; CCoAOMT, caffeoyl-coenzyme A (9-methyltransferase; F5H, ferulate 5-hydroxylase; 4CL, 4-coumarate:coenzyme A ligase; CCR, cinnamoyl coenzyme A reductase; C A D , cinnamyl alcohol dehydrogenase. 5 CoA, feruloyl-CoA, 5-hydroxyferuloyl-CoA, sinapoyl-CoA) by the enzyme 4-coumarate: CoA ligase (4CL). The reactions involved up to this point are common to the synthesis of a wide variety of phenylpropanoids, including lignin, suberin, flavonoids, isoflavonoids, coumarins and stilbenes (Dixon and Paiva, 1995). The first committed step towards monolignol synthesis is catalyzed by cinnamoyl-CoA reductase (CCR), which reduces lignin CoA-thioesters to aldehydes (p-coumaraldehyde, coniferaldehyde, 5-hydroxyconiferaldehyde, sinapaldehyde). These aldehydes are subsequently reduced by cinnamyl alcohol dehydrogenase (CAD) to form monolignols. Understanding how the aromatic rings of hydroxycinnamic acids are modified (hydroxylated or methylated) has been an important issue for lignin biotechnology. The efficiency of lignin removal during pulping is influenced by lignin monomer compositions because methylation at the 3- and 5-positions prevents monolignols from maximizing covalent crosslinks through these ring positions (Boudet et al, 1995). Thus, lignins rich in S units are more susceptible to chemical degradation than lignins made up primarily of G and H units (Chiang et al, 1988). Originally, it was proposed that the ring modification reactions occur mainly at the level of hydroxycinnamic acids (Higuchi, 1985). That is, p-coumarate is hydroxylated at the 3-position by coumarate 3-hydroxylase (C3H), which is followed by methylation of the 3-hydroxyl group by bi-functional caffeate/5-hydroxyferulate O-methyltransferase (COMT) to form ferulate. The subsequent conversion of ferulate to sinapate involves hydroxylation at the 5-position by ferulate 5-hydroxylase (F5H), followed by 5-O-methylation by C O M T . This view has been extensively revised in recent years. The first challenge to the traditional view was the isolation of an enzyme that methylates CoA-thioesters of hydroxycinnamate (caffeoyl-CoA O-methyltransferase, 6 C C o A O M T ) , suggesting that 3-O-methylation could occur at the CoA thioester level (Ye et al, 1994). The second challenge was the finding that purified 4CL proteins from angiosperm plants were generally unable to utilize sinapate as a substrate (Lee and Douglas, 1996; Allina et al, 1998; Ehlting et al, 1999) despite the presence of S lignin in these plants. This indicated that the G to S conversion might not occur at the level of hydroxycinnamic acids. Third, feeding of angiosperm plants with deuterium-labelled coniferyl alcohol resulted in labelling in S lignin as well as G lignin, suggesting that the G to S conversion might take place at the level of the monolignol (Chen et al, 1999; Matsui et al, 2000). Finally, two independent studies have shown that the cytochrome P450-dependent monooxygenase that was initially characterized as F5H (Meyer et al, 1996) preferentially utilized coniferyl aldehyde and coniferyl alcohol over ferulic acid, and the resulting 5-hydroxylated aldehyde and alcohol were efficiently methylated by C O M T (Osakabe et al, 1999; Humphreys et al, 1999). These observations clearly indicate that the ring modification of cinnamic acids can also occur at the level of the CoA-thioesters, aldehydes and alcohols. 1.2.2 Storage and transport of monolignols Monolignols must be transported from their site of synthesis, the cytoplasm, to the site of polymerization, the cell walls. Mechanisms of monolignol transport are not well understood. Developing xylem cells contain many electron-dense vesicles derived from the Golgi apparatus (L. Samuels unpublished data). These vesicles may contain cell wall components and precursors and may subsequently release their contents to the cell wall via vesicle-plasmalemma fusions (Terashima et al, 1993). It is possible that the vesicle contents include monolignols. 7 A potential problem underlying monolignol storage and transport is that monolignols are chemically reactive compounds and their accumulation in plant cells is likely to be toxic. In gymnosperms, the 4-O-P-glucosides of monolignols (p-coumaryl alcohol glucoside, coniferin and syringin) accumulate in developing xylem and have been postulated to represent the stabilized storage and transport forms of monolignols (Freudenberg, 1965; Terazawa and Miyake, 1984; Savidge, 1988). Reactions involved in synthesis and hydrolysis of monolignol glucosides are shown in Figure 1.3. Monolignols are glucosylated by a specific UDP-glucose-utilizing glucosyltransferase, and the resulting glucosides can be hydrolysed by a specific glucosidase (coniferin (3-glucosidase, CG) to release the aglycone. Glucosylation presumably occurs in the cytoplasm, since that is where monolignol glucosides appear to accumulate (Leinhos and Savidge, 1992). Hydrolysis, on the other hand, occurs in the cell wall, as suggested by the apoplastic localization of C G (L. Samuels, unpublished). The released monolignols can then be oxidatively polymerized within the wall matrix to form lignin (Figure 1.4). Although the involvement of monolignol glucosides in lignin biosynthesis is well supported in gymnosperm species, the data from studies in angiosperm species are rather puzzling. Coniferin and syringin have been detected in the cambium sap of only two families (Magnoliaceae and Oleaceae) out of nine angiosperm families studied (Terazawa et al, 1984), and the accumulation pattern of syringin in Magnolia did not correlate well with lignification either spatially and temporally. First, syringin accumulates much more in the phloem side of the cambium (inner bark) rather than in the xylem (Terazawa and Miyake, 1984). Second, its accumulation does not increase during the active growing season (Terazawa and Miyake, 1984). The inability to detect monolignol glucosides in many angiosperm tissues could be the result of rapid turnover rates. Alternatively, 8 angiosperm species may have evolved a different, glucoside-independent pathway for storage and transport of monolignols, and the glucosides detected in angiosperm tissues may be involved in biosynthesis of phenolic compounds other than lignin. Coniferin p-glucosidase (CG) Figure 1.3 Synthesis and hydrolysis of monolignol glucosides Coniferin Cytosol © p e r o x i d a s e l a c c a s e L i g n i n Coniferin p-glucosidase Cell Wall Figure 1.4 Monolignol glucosides may serve as intracellular transport forms of monolignols 1.2.3 Oxidative polymerisation of monolignols Lignins are assembled by the coupling of radicals produced by enzymatic oxidations of monolignols. Two types of enzymes, H202-dependent (peroxidase, PO) or H202-independent (laccase-like) oxidases, have been implicated in catalyzing the oxidation process. Both enzymes are present in developing xylem cells and their tissue distribution exhibits a good correlation with lignification in each case (Sterjiades et al., 1993). There has been heated debate over which enzyme is the main participant of lignin biosynthesis. One of the difficulties in studying these enzymes is that they exhibit a broad range of substrate specificity and thus it is difficult to correlate in vitro enzyme activities with enzyme functions in planta (Boudet, 2000). Another difficulty is that both enzymes exist in many different isoforms, some derived from different genes and some derived from the same gene but differing in the carbohydrate moiety (Lagrimini et al, 1990; Boudet, 2000). This complicates identification of those isoforms specifically involved in lignification. Antisense PO or laccase genes have been introduced into transgenic plants (Lagrimini et al, 1997a; Boudet, 2000; Table 1.1). However, the down-regulation of neither PO nor laccase resulted in a reduction in lignin content (Lagrimini et al, 1997a; Boudet, 2000). This likely reflects the fact that both peroxidase and laccase genes exist as large gene families with high potential for functional redundancy (LaFayette et al, 1999; Ranocha et al, 1999; Quiroga et al, 2000). 10 1.3 Genetic engineering approach to modify lignin biosynthesis 1.3.1 Gene manipulation to control lignin content Table 1.1 summarises the lignin modification studies that have been carried out by creating transgenic plants. To date, only angiosperm species have been used in these lignin modification studies. Reductions in lignin content have been achieved by sense- or antisense-suppression of PAL, C4H, 4CL, CCR, COMT and CCoAOMT. The first attempt to modify lignin content was made by Elkind et al, who introduced a bean PAL2 sense construct into tobacco (1990). Introduction of the transgene resulted in severe inhibition of both endogenous and introduced PAL gene expression, a phenomenon commonly known as co-suppression. The inhibition not only resulted in a reduction of lignin content but also caused pleiotropic effects on plant development, including altered leaf shape and texture, stunted growth, reduced pollen viability, and altered flower morphology and pigmentation (Elkind et al., 1990). The effects were presumed to be due to a general reduction in synthesis of phenylpropanoid metabolites. Interestingly, successive selfing of these same sense-PAL transgenic lines resulted in progressive recovery of PAL gene activity, and even PAL gene overexpression, which allowed Bate et al. to create transgenic tobacco plants with a wide range of P A L activity (0.2 - 200 % of wild-type; Bate et al, 1994). Quantitative analyses of these transgenic plants showed that P A L activity only becomes a limiting factor in lignin biosynthesis when the activity is 3- to 4-fold below wild-type levels, whereas the accumulation of a soluble phenolic, chlorogenic acid, is directly correlated with P A L activity (Bate et al, 1994; Howies et al, 1996). 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CD £ X O CD ^ o t— ^ 5 o H o H X 3" CD a, is « 3 5 a, 5 cy CD a. i J3 o f— o H T 3 C CO a , o 3 o a , S 0 o o o c0 O 8 a 13 <U 3 C e o C "3. u 'S c« e cs B O u E •3 o SJD 3 S3 H Reference Meyer etal., 1998; Marita et al, 1999 etal, 1990; , 1991, etal, 1993; etal, 1997b etal, 1997a Boudet, 2000 Tamagnome et al., 1998 Reference Meyer etal., 1998; Marita et al, 1999 Lagrimini Lagrimini Lagrimini Lagrimini Lagrimini Boudet, 2000 Tamagnome et al., 1998 Other characteristics wilting upon matuarity, increased browning upon wounding, altered IAA metabolism, shorter plants altered IAA metabolism? Taller plants. increased soluble phenolics, normal growth white lesions in leaves, altered flower morphology, reduced soluble phenolics, stunted growth Lignin composition G type lignin changed to S/G type lignin unchanged reduction in S units in young stem, reduction in G units in mature stem Lignin content (% of wild-type) 1 2-fold increase in pith tissue unchanged unchanged 6s-ro OO Enzyme activity (% of wild-type) restored F5H activity 10-fold increase 0s-V reduced mRNA reduced activities of 4CL, C4H and CAD Transgenic plant Arabidopsis fahl mutant (deficient in F5H) Tobacco, tomato Tobacco Populus Tobacco Transgene sense Arabidopsis F5H sense tobacco PO antisense tobacco PO antisense poplar laccase genes sense Anthirrhunum AmMYB308 or AmMYB330 14 antisense alfalfa C4H constructs. A reduction in lignin content was observed in C4H-suppressed plants as well (Sewalt et al, 1997). Suppression of the 4CL gene by introduction of either antisense or sense 4CL constructs resulted in a 30 - 50 % reduction of lignin content in tobacco (Kajita et al, 1996), Arabidopsis (Lee et al, 1997), and Populus tremuloides (Hu et al, 1999). The 4CL suppression caused different effects on development in the three plant species. 4CL-suppressed Arabidopsis plants grew at a normal rate and were morphologically indistinguishable from wild-type plants (Lee et al, 1997). Tobacco antisense 4CL plants were similar to wild-type plants except that the xylem tissues were unevenly pigmented (Kajita et al, 1996). The degree of brown coloration was correlated with 4CL suppression in the tissue (Kajita et al, 1996). In contrast to the rather minor effects on development in Arabidopsis and tobacco, 4CL-suppression resulted in dramatic growth enhancement in transgenic trembling aspen (Hu et al, 1999). The growth enhancement was reflected in an increase in stem growth, leaf area and root growth, but it is unlikely that these changes were caused by changes in lignin content alone. Since 4CL is involved in the syntheses of many phenolic compounds, the growth enhancement may result from indirect effects on the metabolism of other phenylpropanoids. 4CL genes exist in small gene families. A phylogenetic study of 4CL genes has shown that they form two major clusters, class I and class II, which are evolutionarily and functionally distinct from each other (Ehlting et al, 1999). The class I 4CL genes have been associated with lignifying tissues in aspen (Hu et al, 1998) and Arabidopsis (Lee et al, 1995), while the class II 4CL genes have been proposed to be involved in the syntheses of other phenolic compounds (Hu et al, 1998; Ehlting et al, 1999). In a situation where the class I 4CL genes have been specifically suppressed, the pool of cinnamic acid 15 substrates may become more readily available to the class II 4CL. Since different plant species contain a different set of phenolic compounds, this type of indirect effect could explain divergent phenotypes of 4CZ,-suppressed plants. Although phenolic compounds are not generally considered to be a determining factor for plant growth, a link between levels of soluble phenolics and plant growth has been suggested in a recent study by Tamagnome et al. (1998), in which the disappearance of soluble phenolics caused by ectopic expression of two Anthirrhinum MYB genes (AmMYB308 and AmMYB330) resulted in severe growth inhibition in transgenic tobacco plants. It wil l be interesting to compare levels of phenolic compounds between wild-type and 4CZ,-suppressed plants in order to understand secondary effects of 4CL suppression. Antisense suppression of CCR, the first committed enzyme in lignin synthesis, caused a 60 % reduction in C C R enzyme activity and resulted in a 50 % reduction in lignin content (Piquemal et al, 1998). The inhibition also caused pleiotropic effects on plant development, including abnormal leaf morphology, reduced growth and collapsed vessels. It is not clear whether all of these developmental changes are solely caused by lignin reduction, since lignin reduction via introduction of antisense 4CL (Kajita et al, 1996; Lee et al, 1997; Hu et al, 1999) or CCoAOMT (Zhong et al, 1998) did not result in overall growth inhibition. One study reported a slight reduction (20 %) of lignin content in COMT-suppressed tobacco plants (Ni et al, 1994), whereas four other studies reported no lignin reduction in COMT-suppressed transgenic plants (Dwivedi et al, 1994; Atanassova et al, 1995; Van Doorsselaere et al, 1995; Tsai et al, 1998). This difference may be related to the different transgenes that were used. 16 A strong reduction in lignin content resulted from antisense inhibition of the CCoAOMT gene in transgenic tobacco (Zhong et al, 1998). Inhibition of CCoAOMT caused up to a 50 % reduction in lignin content, consistent with an important role for this enzyme in monolignol biosynthesis. The simultaneous inhibition of CCoAOMT and COMT in double transgenic plants resulted in even more reduction in lignin content (up to 66 % reduction). This suggests that, in antisense CCoAOMT plants, the alternative methylation pathway via C O M T is compensating the C C o A O M T inhibition. As was seen in lignin-reduced CCR antisense plants, many vessels in CCoAOMT antisense plants were collapsed. Interestingly, this damage to the vascular system had no observable effect on the overall growth or leaf morphology of the transgenic plants (Zhong et al, 1998). Attempts to reduce lignin content through either PO- or laccase-suppression have not been successful (Lagrimini et al, 1997a; Boudet, 2000). This could be due to functional redundancy in these oxidative enzymes, since multiple isoforms exist in plants (Boudet, 2000). Interestingly, overexpression of PO in transgenic tobacco resulted in a marked increase in pith lignin (Lagrimini, 1991). This outcome contrasts with the results of overexpression of other lignin synthesis genes {PAL, C4H, CAD and COMT), where no changes of lignin content were observed (Sewalt et al, 1997b; Baucher et al, 1996; Atanassova et al, 1995). PO-overexpressing plants also exhibited an unexpected wilting phenotype when they reached maturity (Lagrimini et al, 1990). The wilting phenotype was later found to be due, not to the change in lignin synthesis, but rather to altered IAA metabolism in the roots (Lagrimini et al, 1997b). Lignin modification has also been achieved by introduction of regulatory genes. The promoter regions of many phenylpropanoid genes contain one or more binding sites (AC-like elements) for M Y B transcription factors (Douglas, 1996). The interactions 17 between these upstream cz's-elements and M Y B transcription factors are thought to be important in coordinating the expression of different phenylpropanoid structural genes. Overexpression of two Anthirrhinum MYB genes (AmMYB308 and AmMYB330) in tobacco simultaneously inhibited expression of at least four phenylpropanoid genes (4CL, C4H, CAD and chalcone synthase; Tamagnome et al., 1998). This resulted in a 17 % reduction in lignin content and in a severe reduction in soluble phenolic contents. The M Y B -overexpressing plants exhibited abnormal plant development, including stunted growth, altered flower morphology, reduced flower pigmentation and white lesions on their leaves (Tamagnome et al., 1998). 1.3.2 Gene manipulation to control lignin monomer composition Although the obvious targets for altering lignin monomer composition would be the biosynthetic steps responsible for aromatic ring modifications (F5H, COMT and CCoAOMT), modification of expression of other genes (PAL, C4H, 4CL, CCR and CAD) has also been found to result in unexpected changes in lignin monomer composition (Table 1.1). The changes in monomer compositions in PAL- and C^/f-suppressed plants are perhaps the most intriguing, since these enzymes act on substrates that are common to all three monomer biosynthetic pathways (Figure 1.2). Sewalt et al. reported an increase in S/G ratio in /MZ-suppressed plants and a decrease in S/G ratio in C4//-suppressed plants (1997b). As a possible explanation of these results, the authors hypothesized that P A L and C4H might normally be organized into enzyme complexes or metabolic channels, which would be uniquely associated with branch-specific enzymes later in the pathway. Such complexes could lead L-phenylalanine or cinnamate specifically into the production of G or 18 S units (Sewalt et al, 1997b). The existence of such enzyme complexes has also been suggested by apparent co-localization of P A L and C4H activities in the microsomal fraction of cell extracts, and by preferential conversion from L-phenylalanine to p-coumarate (Rasmussen and Dixon, 1999 and references therein). However, before this hypothesis can be accepted, it is important to establish the physical existence of these proposed complexes and their association with the downstream enzymes. The lignin monomer compositions of 4CZ-suppressed plants are surprisingly divergent. Kajita et al. reported an increase in H units and a decrease in both S and G units in 4CL-suppressed tobacco plants (1996, 1997). The ratio of S to G units was also reduced in these plants. 4CL-suppressed Arabidopsis plants, on the other hand, exhibited no increase in H units but contained elevated levels of S units and reduced level of G units, resulting in an increase in the S/G ratio (Lee et al, 1997). In contrast to these examples, no changes in lignin monomer compositions were observed in 4CL-suppressed aspen (Hu et al, 1999). 4CL-suppressed tobacco and aspen also contained elevated levels of hydroxycinnamic acids in the non-lignin cell wall fraction as a result of 4CL inhibition (Kajita et al, 1997; Hu et al, 1999). Genetic suppression of the two O-methyltransferase genes, COMT and CCoAOMT, yielded opposite effects on the S/G ratio in the transgenic plants. Suppression of COMT caused a reduction in the S/G ratio and accumulation of novel 5-hydroxyconiferyl alcohol residues (Dwivedi et al, 1994; Atanassova et al, 1995; Van Doorsselaere et al, 1995; Tsai et al, 1998). This is consistent with the idea that G to S conversion occurs at either the aldehyde or alcohol levels, but not at the acid level (Osakabe et al, 1999; Humphreys et al, 1999) and, therefore, C C o A O M T is unable to compensate for the lack of C O M T activity. Transgenic reduction in the C C o A O M T activity yielded a small increase in the S/G ratio 19 but the simultaneous inhibition of COMT and CCoAOMT resulted in a reduction in the S/G ratio (Zhong et al, 1998). This is again consistent with the idea that the action of C O M T is located downstream of C C o A O M T . Another point of interest in these results is the lack of detectable H units in COMT- and CCoAOMT-suppressed plants. This suggests that the lack of H units in wild-type tobacco may be due to the absence of a pathway for conversion of /?-coumaroyl CoA to p-coumaryl alcohol (Zhong et al, 1998). In contrast to the dramatic impact of COMT suppression, overexpression of COMT did not cause any changes in monomer composition, suggesting that this enzyme is not rate-limiting for the G to S conversion and that the activities of other enzymes such as F5H may control the final ratios (Atanassova et al, 1995). The Arabidopsis mutant fahl is deficient in S-type lignin, and thus accumulates a gymnosperm-type lignin instead of the normal angiosperm-type lignin (Chappie et al, 1992). The FAH1 locus has been cloned and shown to code for a cytochrome P450-dependent monooxygenase, which initially was deduced to have F5H activity (Meyer et al., 1996). More recently, the recombinant enzyme has been shown to catalyze the hydroxylation of coniferyl aldehyde and coniferyl alcohol much more efficiently than the hydroxylation of ferulate (Osakabe et al, 1999; Humphreys et al, 1999). Nevertheless, it is clear that this enzyme plays a critical role in the synthesis of S-type lignin in angiosperms. In support of this, overexpression of a F5H gene in the fahl mutant under control of either the CaMV35S promoter or the Arabidopsis C4H promoter restored synthesis of S-type lignin (Meyer et al, 1998). This confirmed that the presence of F5H is a limiting factor for G to S conversion in Arabidopsis. It would be interesting to see i f overexpression of this gene promotes S-type lignin synthesis in conifers. 20 A n unexpected degree of plasticity in lignin biosynthesis was revealed by analysis of the lignin phenotype of C4Z)-suppressed plants. C A D is responsible for the last step in the lignin biosynthetic process, namely conversion of hydroxycinnamyl aldehydes to monolignols (Figure 1.2). Although an anticipated phenotypic outcome of the down-regulation of CAD was a reduction in lignin content, in fact, no reduction in lignin content was observed in C4D-suppressed plants (Halpin et al, 1994; Higuchi et al., 1994; Baucher et al, 1996). It appears that in order to compensate for the lack of monolignols, CAD-antisense plants incorporated hydroxycinnamyl aldehydes into lignin (Ralph et al, 1998). C4Z)-suppressed plants exhibited normal plant development except that the xylem tissues showed red coloration (Halpin et al, 1994; Higuchi et al, 1994; Baucher et al, 1996). An important property of CAD antisense plants is that this new aldehyde-rich lignin is more susceptible to chemical degradation than normal lignin, which, therefore, reduces the cost of pulping wood (Lapierre et al, 1999). Incorporation of aldehyde units into lignin is also associated with a reduction in C O M T activity in COMT co-suppressed aspen plants (Tsai et al, 1998), although this is less easily explained. Unusual lignin has also been observed in CC/?-suppressed plants. This was first suggested by the orange-brown coloration of the xylem tissue (Piquemal et al, 1998). An N M R analysis had shown that these plants contained an elevated amount of tyramine ferulate (Ralph et al, 1998). The authors suggested that this new unit was incorporated into lignin in compensation for the restricted supply of monolignols. There is a general trend towards increasing S/G ratio when lignin content is reduced (e.g. PAL-, 4CL- and CQ?-suppressed plants). This could be because there is preferential carbon flow towards the synthesis of S-type lignin when monolignol supply is limited. 21 1.4 Natural and artificial mutations in the lignin biosynthetic pathway 1.4.1 Brown-midrib mutants Screening of natural and artificial mutants offers an alternative method for lignin modification. It is a valuable approach for species in which transformation protocols are not established. Several natural mutants of lignin biosynthesis have been isolated in maize, sorghum and pearl millet, as well as loblolly pine. Brown-midrib (bm) mutants (bml, bm2, bm3 and bm4) of maize are characterized by reddish-brown pigments in leaf midribs and stem sclerenchyma (Table 1.2; Cherney et al, 1991). These mutants have attracted considerable interest over the past 30 years because they provide superior characteristics for maize as a forage crop. They exhibit increased digestibility due to lowered lignin content and modified lignin composition (Cherney et al, 1991). Similar mutants have been isolated in sorghum and pearl millet as well (Cherney et al, 1991). The BM1 and BM3 genes have recently been cloned and were shown to encode CAD (Halpin et al, 1998) and COMT (Vignols et al, 1995), respectively. It is interesting that the bm mutants display a similar but distinct phenotype from CAD- or COMT-suppressed dicotyledonous transgenic plants. COMT suppression in both bm3 and transgenic plants resulted in a decrease in S units and the appearance of 5-hydroxyguaiacyl units (Boudet et al, 1995; Atanassova et al, 1995; Van Doorsselaere et al, 1996; Tsai et al, 1998). Unlike the bm3 mutation, however, red-brown xylem and lowered lignin content are not always associated with COMT suppression in transgenic plants (Table 1.1). G4Z)-suppressed transgenic plants more closely resemble bm mutants than do COMT-suppressed transgenic plants. G4Z)-suppressed transgenic plants accumulated lignin containing cinnamyl aldehydes (Halpin et al, 1994; Higuchi et al, 1994; Baucher et al, 1996), and an increase in cinnamyl aldehyde units was also suggested in bml mutants 22 (Halpin et al, 1998). Red-brown coloration of xylem was observed in both bml plants and C4D-suppressed transgenic plants (Cherny et al, 1991; Halpin et al, 1994; Higuchi et al, 1994; Baucher et al, 1996). However, a reduction in lignin content was only observed in bml mutants, not in transgenic plants. These differences may represent differences between monocots and dicots. Grass lignins are distinct from dicot lignins in that grass lignins contain a substantial fraction of H units, whereas H units have not been detected in dicot lignins (Boudet et al, 1995). Coumaric and ferulic acids are found bound to lignin via labile ester and/or ether bonds in grasses, but not in dicots (Boudet et al, 1995). Since the difference in lignin structure in these two phyletic groups predicts different control points in lignin biosynthesis, gene modification would likely cause somewhat different effects on lignin biosynthesis. A CAD mutant {cad-nl) has been identified in loblolly pine, which also exhibited a bm-hke phenotype (MacKay et al, 1997). The mutant is characterized by red-brown xylem and reduced lignin content. Detailed analyses of the cad-nl mutant revealed the presence of unusual monomer units derived from dihydroconiferyl alcohol, as well as increased amounts of coniferaldehyde units (Ralph et al, 1997). Since synthesis of dihydroconiferyl alcohol cannot be explained by the conventional lignin biosynthetic pathway, this suggests the existence of an alternative pathway in these plants. Surprisingly, the cad-nl allele was also associated with enhanced radial growth, resulting in a 14 % increase in de-barked volume (Wu et al, 1999). The growth enhancement co-segregated with the cad-nl allele, suggesting that the cad mutation itself may somehow be responsible for the changes in stem growth (Wu et al, 1999). It would be interesting i f indeed the growth enhancement were caused by the cad mutation. However, it would be difficult to confirm this linkage by segregation analysis alone. It would require generating G4Z)-suppressed transgenic plants 23 to see i f it causes the similar phenotype as cad-nl, or complementation of the cad-nl mutation by ectopic expression of the wild-type CAD gene. Nevertheless, the cad-nl mutant is economically valuable because of its enhanced growth and the susceptibility of its wood to pulping treatments (Wu et al, 1999; MacKay et al, 1999). 1.4.2 Arabidopsis mutants Studies of Arabidopsis mutants have provided a powerful tool in modern plant biology, and lignin research is no exception. A n excellent example is the identification of the fahl mutant, which eventually led to cloning of the F5H gene (See above). A number of other mutants also hold promise for providing insight into lignin biosynthesis / development of secondary cell walls (for example, Table 1.2; reviewed in Fagard et al, 2000). The ifll mutation blocks the formation of lignified interfascicular fibres in Arabidopsis stems (Zhong et al, 1997). One interesting aspect of this mutant phenotype is its inability to stand upright, which demonstrates the importance of fibres in providing mechanical support to plants. The elpl mutation causes lignin deposition in the pith, a tissue in which there are normally no lignified cell walls (Zhong et al, 2000). This ectopic lignification is not accompanied by secondary cell wall thickening and, therefore, resembles wound-induced lignin deposition. The authors suggested that the ELP1 gene might encode a repressor for wound-inducible lignification under normal conditions (Zhong et al, 2000). It will be interesting to establish the identity and function of the ELP1 gene. 24 Table 1.2 Examples of natural and artificial mutants of lignin synthesis Species Mutation Description Reference Maize Brown-midrib 1 {bml) Mutation in the CAD gene, brown pigments in leaf midribs and stems sclerenchyma, reduced lignin content, increased digestibility, increase in cinnamyl aldehyde units Halpin etal., 1998 Maize Brown-midrib3 (bm3) Mutation in the COMT gene, brown pigments in leaf midribs and stems sclerenchyma, reduced lignin content, reduced S units, increased 5-OH G units, increased digestibility Vignols et al, 1995 Loblolly pine cad-nl Deficient in CAD, phenotype similar to brown-midrib mutants, brown xylem, reduced lignin content, increased aldehyde units, incorporation of dihydroconiferyl alcohol into lignin, increased chemical extractability, enhanced growth MacKay et al., 1997; Ralph et al, 1997; W u et al, 1999 Arabidopsis fahl Mutation in the F5H gene, deficient in S-type lignin M e y e r s al, 1996 Arabidopsis W Mutation in homeodomain-leucine zipper protein, lack of lignified interfascicular fibres, altered xylem differentiation, unable to stand upright Zhong etal, 1997; Zhong and Ye, 1999 Arabidopsis elpl Ectopic lignin deposition in the pith without secondary cell wall thickening, similar to wound-induced lignin, rich in S units Zhong et al, 2000 1.5 Proposal of thesis research - application of lignin biotechnology to conifers In contrast to the considerable progress made towards genetic engineering of lignin in angiosperm species, the progress in gymnosperm species has been very limited. The impact of lignin modification would potentially be far more economically significant in gymnosperm wood (softwood) than angiosperm wood (hardwood), because gymnosperms contain more lignin than angiosperms (Biemann, 1996) and because gymnosperm lignin mainly consists of G units, which are the most difficult to extract (Boudet et al, 1995). In order to attempt genetic engineering of lignin in gymnosperms, three requirements have to be met: isolation of conifer lignin biosynthesis genes, development of effective transformation vector systems and development of efficient transformation protocols. 25 When this thesis work was initiated in 1995, only a few lignin biosynthesis genes had been cloned and characterized in conifers (Table 1.3) and very few studies had reported successful recovery of transgenic conifers (Table 1.4). There also were major uncertainties about which kind of constructs would work best for conifer transformation. Therefore, the two main goals of my thesis research were 1) to evaluate the potential use of the poplar PAL2 promoter for directing xylem-specific gene expression in conifers, and 2) to study the effect of an antisense CG gene on lignin formation in transgenic conifer plants. The poplar PAL2-GUS fusion gene had previously been introduced into tobacco and was shown to express specifically in xylem tissues (Molitor, 1995). To establish whether this held true for woody perennials as well, I introduced the poplar PAL2-GUS fusion gene into hybrid poplar {Populus tremula x P. alba) and studied its expression pattern. These results were compared with the behaviour of the same construct when it was introduced into an embryogenic line of interior spruce (Picea glauca x engelmanni complex). Chapter 3 of this thesis describes the process of generating transgenic spruce and Chapter 4 describes the expression analysis of the poplar PAL2 promoter in the resulting transgenic plants. Chapter 5 describes my attempt to modify the lignin biosynthetic pathway in spruce via introduction of an antisense CG gene. A CG c D N A sequence from lodgepole pine had previously been isolated and characterized (Dharmawardhana et al., 1999). I used this sequence to construct an antisense CG gene, and then introduced this construct into spruce. In order to analyse the effect of the antisense CG transgene, it was necessary to clone the spruce CG genes. Therefore, the first half of Chapter 5 describes the cloning and expression analysis of spruce CG genes, and the second half describes my analysis of 26 transgenic spruce containing the antisense CG gene. To my knowledge, this is the first study to employ an antisense approach to metabolic engineering in a gymnosperm species. Table 1.3 Cloning of lignin synthesis genes from conifers (GenBank search was performed on July 13, 2000) Gene Species (accession number) Reference Pinus taeda Whetten and Sederoff, 1992 Pinus banksiana (AF013485, AF013484, AF013483, AF013482, AFO13481) Butlande/a/. , 1998 PAL Pinus taeda (U39792) Zhang X H and Chiang V L , unpublished Picea sithensis (Y13581) Woodward N G , Woodward S, Kervinen T M A and Karjalainen R, unpublished Pinus monticola (AFO 19965) Baker S M and White E E , unpublished C4H Pinus taeda (AF096998) Anterola A M , Jeon JH, Davin L B and Lewis N G , unpublished Pinus taeda (U12013, U12012) Voo etal, 1995 4CL Pinus taeda (U39405, U39404) Zhang and Chiang, 1997 Pinus armandi (AF144503, AF144502, AF144501), Picea smithiana (AF144504), Pinus banksiana (AF144500, A F 144499) Wang et al, 2000 COMT Pinus taeda (\J3930\) L i etal, 1997 Pinus radiata (AF119225) Moyle etal, 1999 CCoAOMT Pinus taeda (AF098159, AF036095) L i etal, 1999 Pinus taeda O'Mal ley etal, 1992 CAD Picea abies (AJ001926, AJ001925, AJ001924) Schubert R, unpublished Pinus radiata (AF060491) Moyle etal, 1998 CG Pinus contorta (AF072736) Dharmawardhana et al, 1999 27 Table 1.4 Production of transgenic conifers Species Method Tissues used for transformation Transformation efficiency Reference Picea glauca Particle bombardment Mature somatic embryos - Ellis et al, 1993 Picea glauca Particle bombardment Mature somatic embryos 2.2 % of bombarded mature somatic embryos Bommineni et al, 1993 Larix decidua Agrobacterium Hypocotyls of seedlings 0.2 % of inoculated seedlings Shin etal, 1994 Picea mariana Particle bombardment Mature somatic embryos 0.3 % of bombarded mature somatic embryos Chareste?a/., 1996 Pinus radiata Particle bombardment Embryonal masses 0.5 transgenic lines per 200 mg bombarded embryonal masses W a l t e r s al, 1997 Larix kaempferi x L. decidua Agrobacterium Embryonal masses 1 - 2 lines out of 50 co-cultivated embryonal masses Levee etal, 1997 Picea abies Agrobacterium Embryonal masses 0.01-0.24 per number of cells showing transient expression Wenck etal, 1999 Pinus strobus Agrobacterium Embryonal masses 4 lines per gram co-cultivated embryonal masses LevieetaL, 1999 28 Chapter 2 Developmental regulation of the poplar PAL2-GUS fusion gene in transgenic poplar 2.1 Introduction Phenylalanine ammonia-lyase (PAL) catalyses the first step in phenylpropanoid metabolism: the deamination of L-phenylalanine to form trans-cirmamic acid. Among many natural products derived from cinnamic acid are lignin, flavonoids, coumarins and phenolic esters. These compounds confer a wide variety of functions necessary for plant survival, such as structural support, U V protection, defence against pathogens and pests, and deterrence of insect feeding. PAL gene expression is controlled in plants by developmental and environmental signals. PAL m R N A accumulation has been reported in cells associated with lignification (xylem cells and phloem fibres of vascular tissue) and with flavonoid synthesis (epidermal/subepidermal cells of leaves and petals) (Shmelzer et al, 1989; Wu and Hahlbrock, 1992; Subramaniam et al, 1993). It is also stimulated by environmental signals such as U V light, wounding, and pathogen attack (Schmelzer et al., 1989; Lois et al, 1989; Ohl et al, 1990; Osakabe et al. 1995). The control of PAL expression occurs mainly at the transcriptional level. PAL promoters fused to the GUS reporter gene have been introduced into both homologous and heterologous hosts. The developmental and environmental regulation of GUS fusion genes correlates with phenylpropanoid accumulation (Bevan et al, 1989; Liang et al, 1989; Ohl et al, 1990; Shufflebottom et al, 1993; Kawamata et al, 1997). Two genomic clones of PAL genes, PALI and PAL2, have been isolated from hybrid poplar Populus trichocarpa x P. deltoides using a PAL c D N A sequence obtained from a young leaf library (Subramaniam et al, 1993). In situ R N A hybridization of 29 PALI/2 revealed high levels of expression in subepidermal cells of young stems and leaves, and lower levels of expression in vascular tissues. High expression of PALI/2 in subepidermal cells correlated with high levels of synthesis of phenolic compounds in young leaves and stems, which may serve as deterrent for insect feeding (English et al, 1991). Expression in vascular tissues was correlated with lignification in xylem cells and phloem fibre cells (Subramaniam et al, 1993). Promoter regions of the PAL 1 and PAL 2 genes were 75 % identical and contained two copies of A C elements (Molitor 1995). A C elements are cw-acting elements conserved in the promoters of PAL and other phenylpropanoid genes (Hauffe et al, 1991; Loake et al, 1992; Yamada et al, 1994; Grotewold et al, 1994; Logemann et al, 1995; Hatton et al, 1995; Seki et al, 1996; Feldbriigge et al, 1997). Fusion genes have been constructed to express GUS under control of PALI and PAL2 promoters, and those have been introduced into tobacco (Molitor 1995). PAL1-GUS and PAL2-GUS fusion genes directed similar patterns of expression in transgenic tobacco. GUS activity was detected in developing primary xylem of leaves, stems and other organs, and in secondary xylem of stems. However, contrary to the in situ R N A hybridization results obtained earlier in poplar (Subramaniam et al, 1993), no promoter activity was detected in subepidermal cells of young leaves and stems in transgenic tobacco (Molitor 1995). This discrepancy might result from critical cw-acting elements of PALI and PAL2 being located outside of the isolated promoter regions. Alternatively, it is possible that certain PALI/2 cw-acting elements do not function properly in the heterologous tobacco host. In order to examine the latter possibility, it was necessary to study expression of PAL-GUS fusion genes in a homologous host. In this chapter, the poplar PAL2-GUS fusion gene was introduced into a 30 hybrid poplar and developmental regulation directed by the poplar PAL2 promoter was studied in the transgenic plants. 2.2 Materials and Methods 2.2.1 Agrobacterium-mediated transformation of hybrid poplar 2.2.1.1 Plant material and growth condition Two hybrid poplar lines Populus trichocarpa x P. deltoides, clone 53-246 (Subramaniam et al, 1993) and Populus tremula xP. alba, INRA clone 717-1B4 (Leple et al, 1992) were maintained in vitro in Magenta boxes containing R I M medium (Table 2.1) at 25 °C. They were incubated under a 16 h light/8 h dark photoperiod unless otherwise mentioned. Plants were subcultured by transferring shoot apices to fresh medium every fourth week. 2.2.1.2 Agrobacterium-mediated transformation Agrobacterium-mediated transformation was carried out using a modification of the method of Leple et al. (1992). Leaf explants ( 1 - 2 cm2) or petiole pieces (1 cm) were incubated on SIMlmedium (Table 2.1) for two days at 25 °C. Agrobacterium tumefaciens strain LBA4404 containing a transformation vector was grown in L B medium overnight at 30 °C in a rotary shaker at 200 rpm. The cultured cells were collected by centrifugation at 2000 g for 5 min in a tabletop centrifuge. The bacterial pellet was resuspended in W P M medium and diluted to OD6oo=0.3 (5.7 x 108 c.f.u.). Explants were placed in the bacterial suspension and incubated for 24 h at 25 °C on a rotary shaker at 60 rpm. They were then blotted dry on sterile filter paper and incubated on SIM1 for two days at 25 °C. Explants were covered with bacterial colonies by the end of this co-cultivation. Explants were 31 washed three times 5 min each with 1 g/1 carbenicillin in W P M , blotted dry onto a sterile filter paper and incubated on SIM1 containing 500 mg/1 carbenicillin for two weeks at 25 °C in the dark. Table 2.1 Media for poplar micropropagation SIM1 SIM2 RIM W P M salt W P M salt Vi W P M salt 0.5 % agar 0.5 % agar 0.5 % agar 3 % sucrose 3 % sucrose 1.5 % sucrose 200 mg/1 L-glutamine 200 mg/1 L-glutamine 200 mg/1 L-glutamine 1 u M B A P 0.1 u M thidiazuron pH5.7 0.5 u M N A A pH5.7 pH5.7 For selection, explants were transferred to SIM1 containing 500 mg/1 carbenicillin and 100 mg/1 kanamycin and incubated for two weeks at 25 °C. For shoot induction, explants were transferred to SEVI2 containing 500 mg/1 carbenicillin and 100 mg/1 kanamycin (SIM2 selection medium) and incubated at 25 °C. The explants were transferred to fresh SIM2 selection medium every fourth week. For root induction, shoots formed during incubation on SIM2 selection medium were removed, transferred onto RIM medium (Table 2.1) containing 500 mg/1 carbenicillin and 100 mg/1 kanamycin, and incubated at 25 °C. Putative transgenic lines were screened for GUS activity by staining leaflets in X-Gluc GUS reaction mixture as described below. Presence of the transgene in GUS-positive lines was confirmed by PCR analysis as described below. GUS-positive 32 lines were maintained on R I M medium as described above, and transferred to soil (Sun Shine mix, Sun Gro Horticulture) when they were about 5 cm in height. They were incubated in a growth chamber at 22 °C for two months and then transferred to a plastic tunnel greenhouse in May 1996 and grown under ambient light at the U B C Botanical Garden Nursery. They were watered every other day and given fertiliser (Nurseryland 20-20-20, Greenleaf products Inc., Burnaby, B.C.) every second week. 2.2.2 GUS assays 2.2.2.1 Histochemical GUS staining Histochemical staining for GUS activity was performed as described by Jefferson (1987). For localization of GUS-active cells, leaves and stems were hand-sectioned (about 0.5 mm thick) and fixed for 30 min in 0.5 % paraformaldehyde in 0.1 M sodium phosphate (pH 7.0). Fixed sections were rinsed in 0.1 M sodium phosphate (pH 7.0) and incubated in the GUS reaction mixture (1.5 m M 5-bromo-4-chloro-3-indol-l-glucuronide (X-Gluc), 10 m M N a 2 E D T A , 0.1 % Triton X-100 in 0.1 M sodium phosphate pH 7.0) at 37 °C for 1 to 16 hours. They were rinsed once in 0.1 M sodium phosphate (pH 7.0) and incubated in a graded series of ethanol (30-100 %, v/v) to extract chlorophyll. After chlorophyll was removed, ethanol was replaced stepwise with 50 % glycerol. The sections were then mounted on glass slides for observation in a light microscope. The fixation step was omitted for GUS screening of putative transformants. 2.2.2.2 Fluorometric GUS assay Tissues were homogenized in extraction buffer (10 m M P-mercaptoethanol, 10 m M N a 2 E D T A , 0.1 % (v/v) Triton X-100, 50 m M sodium phosphate buffer, pH 7.0) with a 33 pinch of sand, followed by centrifugation at 8,000 g at 4 °C for 10 min. A n aliquot of the supernatant was added to the GUS reaction mixture (1 m M 4-methylumbelliferyl glucuronide (MUG), 20 % (v/v) methanol in extraction buffer) and incubated at 37 °C. The GUS reaction was stopped by adding 0.2 M Na 2 C03. Enzymatic conversion of M U G to 4-methylumbelliferone (4-MU) was measured using a fluorometer (365 nm excitation and 455 nm emission). The GUS activity was expressed as the concentration of 4 - M U released per minute per milligram of protein in the cell extract. The protein concentration was determined according to the method of Bradford, using a Protein Assay Kit (Bio-Rad, Mississauga, Ontario) and bovine serum albumin as a standard. 2.2.3 Lignin staining Stems and leaves of transgenic poplar were hand-sectioned and fixed as described above. Fixed tissue sections were stained for lignin using phloroglucinol/HCl (5% in an ethanol/HCl mix 9:1). 2.2.4 Genomic D N A extraction Leaves of in vitro-grown poplar were ground to powder in liquid nitrogen using a mortar and pestle. Seven volumes of poplar D N A extraction buffer (100 m M Tris-HCl, 20 m M E D T A , 2 % C T A B , 1.4 M NaCl, 1 % (w/v) PVP and 0.2 % (v/v) (3-mercaptoethanol; pH 8.0) were added to the powder, mixed well and incubated at 60 °C for 30 min. The extract was incubated with 10 (J.g/ml of DNase-free RNase A at 37 °C for 30 min, extracted twice with chloroform/isoamyl alcohol 24:1 and D N A was precipitated using two-third volume of isopropanol. The D N A precipitate was collected by centrifugation at 17,000 g for 5 min, rinsed with 70 % ethanol, dried briefly, dissolved in TE buffer (10 m M Tris-HCl 34 and 1 m M E D T A ; pH 8.0) and incubated at 65 °C for 30 min. D N A concentration was determined by absorbance at 260 nm, diluted to give a concentration of 0.25 ug/p.1, and kept at 4 °C. Undiluted D N A stock solution was kept at -20 °C. 2.2.5 PCR analysis of putative transformants Two primer sets used in detection of transgenes are listed in Table 2.2. The PCR reaction contained 250 ng genomic D N A , 400 n M each of forward and reverse primers, 200uM dNTP, 10 m M Tris-HCl pH 8.3, 1.5 m M M g C l 2 , 50 m M KC1 and 0.025 U/ul Taq D N A polymerase (Boehringer Mannheim). The PCR reaction was run in a Perkin Elmer 9600 thermal cycler. The thermal cycling regime was as follows: 1 cycle of 94 °C/5 min; 30 cycles of 94 °C/1 min, 55 °C/1 min, 72 °C/1 min; and 1 cycle of 72 °C/10 min. The amplification products were analysed by 1 % agarose gel electrophoresis. Table 2.2 Primers used for PCR screening Target Primer Sequence Product gene size NPTII npt4 npt6 C A A G A T G G A T T G C A C G C A G G T T C T C G A A T C G G G A G C G G C G A T A C C G T A A A 0.74 kb GUS G N L G N R T C T C T T T G A T G T G C T G T G C C A A A T T C G A G C T C G G T A G C A A 0.5 kb 2.3 Results 2.3.1 Generation of transgenic poplar Explants from two hybrid poplar lines Populus trichocarpa xP. deltoides, clone 53-246 (Subramaniam et al., 1993) and Populus tremula x P. alba, INRA clone 717-1B4 35 (Leple et al, 1992) were infected with Agrobacterium tumefaciens carrying a vector containing the poplar PAL2-GUS fusion gene. The P. trichocarpa x P. deltoides hybrid was chosen because the poplar PAL2 gene was originally isolated from this hybrid (Subramaniam et al, 1993). The Populus tremula x P. alba hybrid was chosen as it was highly amenable to Agrobacterium-mediated transformation (Leple et al, 1992). Transformed cells were selected for kanamycin resistance on shoot induction medium containing B A P and N A A (SIM1 in Table 2.1). Adventitious shoots were readily formed from wounded sites on leaf veins, petioles and stems of both lines as early as two weeks after co-cultivation. The adventitious shoots continued growing on kanamycin medium for at least a month. However, they eventually stopped growing and turned white on kanamycin medium. None of these initial shoots were GUS-positive, indicating that they were "escapes". Explants stopped producing new shoots about 2 - 4 weeks post-infection. At this point, explants were transferred to shoot induction medium containing thidiazuron (SIM2 in Table 2.1). New shoots, some white and some green (Figure 2.1 A), were generated after a few weeks of incubation on SIM2. About half of the green shoots from clone 717-IB possessed GUS activity whereas no GUS-positive shoots were obtained from clone 53-246. Figure 2.IB shows an example of GUS staining. Among the green shoots obtained from clone 717-IB, true transformants and "escapes" were indistinguishable for several weeks. The highest transformation rate (39 %) was obtained from petiole explants (Table 2.3). A total of 24 GUS-positive 717-1B lines were obtained, all of which rooted on root induction medium (RIM) containing kanamycin. The presence of the GUS fusion gene in the poplar genome was confirmed by PCR, using primers specific for the GUS sequence. GUS sequence was amplified from all of the GUS-positive lines, whereas no amplification was detected in wild-type control lines (Figure 2.1C). G U S - ^ C. PCR analysis of transgenic poplar Figure 2.1 Generation and growth of transgenic poplar 37 Table 2.3 Comparison of different explants for ability to produce transformed shoots Explants # of explants producing GUS-positive shoots after 5 months # of explants tested transformation efficiency* (%) Clone 717-IB Leaf 4 38 11 Petiole 9 23 39 Stem 2 33 6 Root 0 42 0 * Number of explants producing GUS-positive shoots / number of total explants. 2.3.2 Expression of the PAL2-GUS fusion in different organs Transgenic poplars grown in vitro were analysed for GUS activity when they were 5-7 cm tall. Young leaves (ca. 2 cm in length) of twenty-four independent transgenic lines exhibited varying degrees of GUS activity, ranging from 0.16 to 26.24 nmole per minute per mg protein (Figure 2.2A). GUS activities in different organs were examined in five transgenic lines. A l l five lines exhibited a similar expression pattern; highest in roots, followed by stems and leaves (Figure 2.2B). Six lines were randomly selected and transferred to soil. GUS activities were assayed in these lines two months after transfer to soil, at which time the plants were ca. 40 cm tall (Figure 2.ID). GUS activity in stem and leaf samples was expressed relative to the activity in the upper one cm of stem tissue from each line. This facilitated comparison of the relative GUS expression levels at different developmental stages using data from all six lines. Similar to in vitro grown plants, GUS activity in all lines was highest in roots (relative value of 1.72, not shown in the figure), followed by stems and leaves (Figure 2.3). Figure 2.3 shows changes in expression levels in the aerial tissues according to 38 developmental age. GUS activity was highest in immature leaves and stems within 5 cm of the shoot apex and consistently decreased as leaves and stems matured. These values were several-fold lower in fully expanded leaves (20 cm from the apex) and in stems undergoing significant amounts of secondary growth (30-40 cm from the apex). A similar trend was found in 4 month-old plants that had been grown in a plastic tunnel greenhouse for two months (Figure 2.4). These plants had grown over 1 meter by this point (Figure 2.IE). Again, GUS activity in the aerial tissues was highest in immature leaves and decreased as leaves matured. Mature leaves retained relatively high activity (40 % of that in immature leaves) compared to those in growth chamber-grown plants (20 % of that in immature leaves, Figure 2.3). This difference could reflect differences in leaf morphology, growth response or growth conditions such as light, humidity and temperature. For example, mature leaves of greenhouse-grown plants were smaller and stiffer than those of growth chamber grown plants, probably because they were exposed to stronger light and low humidity conditions. Figure 2.2 Expression of the poplar PAL2-GUS fusion gene in in vitro-grown transgenic poplar Transgenic poplar plants were harvested when they were 5-7 cm tall. Samples were extracted and assayed for GUS activity. A . Variation in GUS activity among transgenic lines. Leaves (1-3 cm in length) from individual transformants indicated were assayed for GUS activity. GUS activity in line 6.1 (26.24 nmole MU/min/mg protein) is indicated at the top of the bar. B. GUS activity in different organs of selected transgenic lines. Activity was measured in roots (open bars), stems (grey bars) and leaves (black bars). 40 Figure 2.3 Expression of the PAL2-GUS fusion gene in soil-grown transgenic poplar. Leaves and stem segments at different distances from the apex of transgenic lines 1.1, 1.2, 4, 5.1, 6.2, and 8.2 (ca 40 cm in height, centre photograph) were harvested and assayed for GUS activity. GUS activities in stems (A) and leaves (B) are shown relative to activity in the highest expressing sample, the youngest stem segment (<lcm from the apex). Values are the mean of six transgenic lines ± SE. Letters in A and B refer to locations from which stem and leaf tissue was taken for histochemical assay of GUS activity and refer to panels shown in Figure 2.5. (wo) x a d e UJOJJ eoue is iQ 42 R e l a t i v e G U S a c t i v i t y 0 0.5 1 0 E L e a f o 01 40 r u c ra to 60 -Figure 2.4 Expression of the poplar PAL2-GVS fusion gene in greenhouse-grown transgenic poplar. Transgenic poplar lines were transferred to a plastic tunnel greenhouse two months after transfer to soil. Leaves of transgenic lines, 1.1, 2, 4, 6.2, 8.2 and neg3 (1-1.5m in height) were harvested four months after transfer to soil. Leaves at different distances from the apex were harvested and assayed for GUS activity. GUS activity is shown relative to activity in the youngest leaf tissue (<lcm from the apex). Values are the mean of six transgenic lines±SE. 2.3.3 Expression of the PAL2-GUS fusion in different cell types As shown in Figure 2.IB, GUS staining in leaves of in vitro-gwwn plants was strong in vascular tissues, which is consistent with the role of P A L in lignin biosynthesis. For further investigation of tissue-specific patterns of expression directed by the poplar PAL2 promoter, tissue sections were made from the 2 month-old growth chamber-grown plants and assayed for GUS activity. The sites of sampling for histochemical assays are indicated on Figure 2.3, and representative results from line 9.2 are shown in Figure 2.5. Similar staining patterns were observed in lines 1.1 and 5.1. Cross-sections through the youngest leaves at the shoot apex revealed very high GUS activity of the PAL2 promoter in the epidermal and/or subepidermal layer of the leaf midrib, as well as in the developing leaf 43 blade (Figure 2.5A). Vascular tissues were not apparent in stained sections at this stage. Expanding young leaves one cm from the shoot apex showed distinct histochemical staining in epidermal/subepidermal cells of the midrib, strong staining in the developing xylem and phloem of the midrib, and strong staining in the developing leaf blade (Figure 2.5B). Weaker staining was detected in parenchyma cells in the midrib. The first fully expanded young leaves (20 cm from the apex) exhibited a staining pattern similar to that seen in young leaves, except that staining was generally weaker and restricted to vascular tissues and epidermal/subepidermal cells (data not shown). In petioles, strong GUS activity was observed in epidermal/subepidermal cells and was associated with the developing xylem vessels and with phloem fibres (Figure 2.5D). Histochemical staining of similar petiole sections with phloroglucinol to show sites of lignin deposition indicated that the xylem vessels were heavily lignified, while large amounts of lignin were not yet detectable in the phloem fibres (Figure 2.5C). Phloroglucinol-stained stem sections taken 1, 5, 10, and 30 cm from the apex are shown in Figure 2.5E, G, I, K , and similar sections stained for GUS activity are shown in Figure 2.5F, H , J, L . In stems one cm from the apex, primary xylem was just beginning to form in vascular bundles, which could be recognised by the presence of scattered lignified vessels (Figure 2.5E). At this stage, strong GUS activity was detected in primary xylem and phloem and in the ring of interfascicular cambium (Figure 2.5F). Staining was also seen in epidermal/subepidermal cells. Cross-sections of stems at 5 cm from the apex were characterized by distinct bundles of developing, lignified primary xylem and a ring of differentiating phloem fibre cells (Figure 2.5G). In these sections, GUS activity was specific to vascular tissues, with little or no activity in epidermal/subepidermal cells. The most intense staining was observed in cells adjacent to phloem fibres, the interfascicular 44 cambium, and in parenchyma cells near primary xylem vessels. Stems at 10 cm from the apex were characterized by well-developed secondary xylem and lignified phloem fibres (Figure 2.51). Strong GUS activity was seen in phloem and phloem fibre cells (Figure 2.5J). A weak activity was seen in parenchyma cells of primary xylem. Very weak or no activity was detected in ray parenchyma cells of the secondary xylem and in the cambium. At 30 cm from the apex, secondary xylem occupied the major part of the stem and secondary xylem and phloem fibres cells were highly lignified (Figure 2.5K). GUS activity at this point was weak and only a faint staining was detected; histochemically detectable GUS activity was present in parenchyma cells of primary xylem vessels and in phloem fibre cells (Figure 2.5L). Figure 2.5 Histochemical activity of PAL2-GUS in transgenic poplar. Cross-section from different tissues of 2 month-old soil-grown transgenic poplars were taken from the locations indicated in Figure 2.3 (letters A - L). A , B , D, F, H , J and L , sections stained for GUS activity. C, E, G, I and K , sections were stained for lignin using phloroglucinol. A . A cross section through a young leaf 0.5 cm from apex. B. Cross section of an expanding young leaf 1cm from apex. C, D. Cross sections of petioles of an expanding leaf. (Next page) E, F. Cross sections of stems 1 cm from apex. G, H . Cross sections of stems 5 cm from apex. I, J. Cross section of stems 10 cm from apex. K , L. Cross section of stems 30 cm from apex. e, epidermal/subepidermal cells; ic, interfascicular cambium; pf, phloem fibres; px, primary xylem cells. Bars = 200 um. 46 2.4 Discussion This study investigated the spatial and temporal expression pattern conferred by the poplar PAL2-GUS fusion gene in a homologous host, poplar. GUS activity was highest in roots, followed by stems and leaves in both in vz'/ro-grown.and soil-grown plants (Figure 2.2 and 2.3) and was higher in young stems and leaves close to the shoot apex than in older stems and leaves (Figure 2.3). The expression pattern was consistent with the pattern of PAL 1/2 m R N A accumulation reported previously in hybrid poplar P. trichocarpa x P. deltoides using northern analysis (Subramaniam et al, 1993; Gray-Mitsumune et al, 1999). High expression in roots has also been reported with expression of GUS fusion genes under control of other phenylpropanoid gene promoters (Ohl et al, 1990; Schmid et al, 1990; Feuillet et al, 1995). This may be associated with synthesis of structural components such as lignin and suberin and synthesis of phytoalexins against soil-borne pathogens. High expression of the PAL2 promoter in young tissues is consistent with production of a range of phenolic compounds in exudates of poplar leaf buds (English et al, 1991). The cell-type specific expression of the GUS fusion gene was similar to that observed using in situ R N A hybridization (Subramaniam et al, 1993). High GUS activity was first observed in epidermal/subepidermal layer cells of very young leaves and then in developing vascular tissues of young leaves and stems. As stems matured, GUS activity in non-vascular tissues disappeared and the activity was restricted to phloem cells and primary xylem cells. Very weak or no GUS activity was detected in ray parenchyma cells of secondary xylem and in the cambium (Figure 2.5). Lack of strong GUS activity in secondary xylem is also consistent with in situ hybridization results (Subramaniam et al, 1993). However it is notably different from the expression patterns conferred by bean PAL2-GUS (Bevan et al, 1989), parsley 4CL1-GUS (Hauffe et al, 1991) and Eucalyptus 47 CAD-GUS (Feuillet et al, 1995) in transgenic plants, where strong GUS activity was detected in ray parenchyma cells of the secondary xylem. Many genes involved in phenylpropanoid metabolism exist as gene families. Expression of each family member may differ greatly both in spatial patterns and in response to different environmental stimuli to confer different physiological functions. For example, the bean PAL2 promoter directed strong expression of GUS in developing xylem and exhibited only a limited wounding response, whereas another member of this gene family (PAL3) directed no expression in xylem but exhibited an extensive wounding response (Shufflebottom et al 1992). More recently, expression of two Populus tremuloides 4CL genes, Pt4CLl and Pt4CL2, was found to be compartmentalized into two different tissues, xylem tissues and epidermal cells, respectively (Hu et al, 1998). Lack of strong expression of poplar PALI/2 in secondary xylem may imply the existence of an additional PAL gene(s) in poplar that could confer expression in these tissues. Four PAL genes, palgl, palg2a, palg2b, and palg4, have been isolated from Populus sieboldii x P. grandidentata hybrid P. kitakamiensis (Osakabe et al, 1995a; Osakabe et al, 1995b). Palgl m R N A was abundant in young stems and leaves whereas palg2a was more highly expressed in mature stems undergoing secondary growth (Osakabe et al, 1995a). The coding region of poplar PALI72 is 92 % identical to palgl, consistent with their similar patterns of expression (Subramaniam et al, 1993; Osakabe et al, 1994a). The palg2a/b-like sequence has recently been isolated from P. trichocarpa x P. deltoides using a PCR approach (C. Hutcheon and C. Douglas, unpublished data). This may represent an orthologue of palg2a/b in this hybrid that is more specific to tissues undergoing secondary growth. 48 M y results show that the poplar PAL2 promoter contained the information required for correct regulation of expression in poplar. The expression pattern of the poplar PAL2-G US in a homologous host correlated closely with the data obtained by northern blotting and in situ hybridization. The expression patterns differed from that seen in a heterologous host in that no expression was detected in epidermal/subepidermal cells in the latter, and that PAL-GUS retained activity in ray parenchyma cells of secondary xylem in tobacco (Molitor 1995). The differences in expression specified by the PAL2 promoter in poplar and tobacco may be due to species-specific differences in the distribution and/or activity of transcription factors that regulate this promoter. For example, cellular signals that regulate the PAL2 promoter activity in epidermal/subepidermal cells of young poplar leaves may be absent in similar tobacco cells. Similarly, signals that activate the PAL2 promoter in ray parenchyma cells may be absent in secondary xylem of poplar but present in secondary xylem of tobacco. It is interesting that the poplar PAL promoters retained high activity in vascular tissues in the heterologous host (Molitor 1995), suggesting that transcriptional regulation in vascular tissues is more conserved between the two species. One might speculate that the regulation system that leads to synthesis of essential structural components such as lignin is more likely to be conserved than that leading to synthesis of more specialized phenolic compounds. The poplar PALI and PAL2 promoters both contain copies of A C cw-elements (Gray-Mitsumune et al, 1999). A C elements are conserved in the promoters of PAL and other phenylpropanoid genes (Hauffe et al, 1991; Loake et al, 1992; Yamada et al, 1994; Grotewold et al, 1994; Logemann et al, 1995; Hatton et al, 1995; Seki et al, 1996; Feldbriigge et al, 1997), and they are implicated as critical components for tissue-specific and light/elicitor-induced expression of these genes (Hauffe et al, 1991; de Costa e Silva et 49 al, 1993; Hatton et al, 1995; Seguin et al, 1997; Faktor et al, 1997). Transcription factors that interact with A C cw-elements in vitro and/or in vivo have been isolated, including myb transcription factors (Grotewold et al, 1994; Sablowski et al, 1994; Feldbriigge et al, 1997; Tamagnome et al, 1998) and other transcription factors (de Costa e Silva et al, 1993; Seguin et al, 1997). Expression of poplar PAL2 may be determined by dynamic interactions between different transcription factors as well as by interactions between different cw-elements (Leyva et al, 1992; Loake et al, 1992; Hauffe et al, 1993; Hatton etal, 1995). In summary, the PAL2 promoter isolated from hybrid poplar contained the information required for correct patterns of expression in poplar. The discrepancy between the expression patterns directed by this promoter in tobacco and poplar indicate that there may be species-specific differences in distribution and/or activation of transcription factors in these plants. The poplar PAL2 genes appear to be specific for epidermal/subepidermal cells and developing xylem in young leaves and stems but not for secondary xylem in mature stems. A n additional gene(s) may confer expression of PAL in secondary xylem. Chapter 3 Generation of transgenic spruce 50 3.1 Introduction When this study was initiated in 1995, transformation of coniferous species was not a routine procedure, and very few studies had reported successful recovery of transgenic plants (Ellis et al, 1993; Shin et al., 1994). Initial transformation studies had examined various D N A transfer protocols, including protoplast transformation (Gupta et al, 1988; Bakkaoui et al, 1988; Wilson et al, 1989; Charest et al, 1991), Agrobacterium-mediaXed transformation (Sederoff et al, 1986; Dandeker et al, 1987; Ellis et al, 1989; Huang et al, 1991; Yibrah et al, 1996) and transformation using microprojectile bombardment or the "gene gun" technology (Stomp et al, 1991; Goldfarb et al, 1991; Newton et al, 1992; Charest et al, 1993; Walter et al, 1994). Protoplast transformation had proven inefficient due to technical difficulty in regenerating conifers from protoplasts. Agrobacterium-mediated transformation was promising, but it had yet to be combined with a suitable regeneration system such as somatic embryogenesis. Somatic embryogenesis offers a number of advantages over organogenesis: i) large numbers of clonal lines can easily be obtained, and ii) embryogenic cell lines can be subjected to cryopreservation. Cryopreservation not only serves as a method of storage and reducing unnecessary tissue culture procedures but also serves to reduce somaclonal variation and loss of embryogenic potential due to prolonged exposure to growth regulators (Cyr et al, 1994). Microprojectile bombardment transformation was, at that time, the only method that worked reliably with the conifer somatic embryogenesis system (Robertson et al, 1992; Bommineni et al, 1993; Ellis et al, 1993; Charest et al, 1996; Walter et al, 1998). 51 Agrobacterium-mediated transformation protocols combined with somatic embryogenesis have since been reported (Levee et al, 1997; Levee et al, 1999; Wenck et al, 1999). This chapter describes the process of generating transgenic spruce using microprojectile bombardment. Transformation experiments were performed using two types of plasmids, one designed for studying promoter functions and one designed for antisense inhibition of the coniferin (3-glucosidase (CG) gene. Phenotype analyses of the transgenic plants are described in chapters 4 and 5. Only those transformation experiments using the antisense CG gene are described in detail, since they yielded a large number of independent transgenic lines. 3.2 Materials and methods 3.2.1 Overview of transformation process Figure 3.1 shows the overall process used to generate transgenic spruce, with an approximate time line. Somatic embryos of interior spruce (Picea glauca x engelmanni complex) were bombarded using either a PDS 1000/He™ biolistic gun or a particle inflow gun (PIG; Finer et al, 1992). Transient GUS assays were performed two days after the bombardment to confirm efficient D N A delivery to the target cells. Embryogenic tissues were selectively induced from somatic embryos on medium containing 2 or 5mg/l of kanamycin, and putative transgenic cell lines were screened for GUS activity. PCR analysis was performed to confirm the presence of transgenes in putative transgenic lines. Tissues of transgenic lines were processed for cryopreservation and stored in liquid nitrogen for future use and to minimize loss of embryogenic potential (Cyr et al. 1994). Transgenic plants were recovered via somatic embryogenesis and planted in soil. 52 Transient assay (Ml t t Cryopreservation 1 1 Analysis Selection screening — • Regeneration 1 1 Molecular analysis Screening by phenotype Gene gun Start 6 months 1 year F i g u r e 3.1 T r a n s f o r m a t i o n p r o c e s s 3.2.2 Tissue culture conditions Embryogenic tissue line of interior spruce (Picea glauca x engelmanni complex) clone 11026 was obtained from B.C. Research Inc., Vancouver, British Columbia (Cyr et al, 1994) and grown on maintenance medium (Table 3.1) at 25 °C in the dark. For maturation, actively growing tissues were spread on nylon mesh screens, placed on charcoal medium (Table 3.1) for three days, then transferred to A B A medium (Table 3.1) and incubated for 4 - 6 weeks under dim light. Tissues were transferred to fresh medium every second week. For desiccation treatment, mature somatic embryos at stage 4 - 5 (Ellis et al, 1993) were manually removed from calli, arranged on 53 um nylon mesh screens, placed in a high humidity chamber and incubated at 25 °C in the dark for 3 - 6 weeks. For germination, desiccated embryos were transferred to hormone-free germination medium (GMD medium) and incubated under dim light. After one to two weeks, the radicle tips of germinated "emblings" were inserted into the medium and incubated under 16 h light/ 8 h 53 dark photoperiod. After six weeks on G M D medium, emblings were transplanted to soil and grown in a greenhouse at GreenTimbers Nursery (Surrey, British Columbia) under ambient light. Table 3.1 Media for spruce micropropagation Maintenance medium Charcoal medium A B A medium G M D medium Basal salt ^ L M s a l t * 1 ^ L M s a l t * 1 ^ L M s a l t * 1 ' /zLMsalt* ' Casein hydrolysate lg/1 lg/1 1 g/1 lg/1 L-Glutamine 500 mg/1 500 mg/1 500 mg/1 500 mg/1 Sucrose 1 % 3.4 % 3.4% 2 % Activated charcoal - 0.8 % - -B A 5 u M - - -2,4-D 10 u M - - -± A B A - - 60 u M -IBA - - 1 u M -Agar 0.64 % 0.8 % 0.64 % 0.64 % PH 5.8 5.8 5.8 5.8 *]-Litvay etal., 1985 3.2.3 Transformation vectors The plasmids used for spruce transformation are listed in Table 3.2. A pUC-based plasmid pBI221 containing a fusion of the cauliflower mosaic virus 35S (CaMV35S) promoter and GUS coding gene, derived from pBI121 (Jefferson et al. 1987), was only used for transient expression assays. Plasmid pCMCHOO contained the Nos-NPTII gene 54 and a fusion of the CaMV35S promoter, the alfalfa mosaic virus (AMY) leader sequence and the GUS coding sequence (Ellis et al, 1991). A pUC-derived plasmid, pJDD1.7, contained the CaMV35S-NPTII gene and the poplar PAL2-GUS fusion gene (Molitor 1995). A binary vector, p B A C G G U S , was made for this study and contained the antisense pine coniferin (3-glucosidase gene under control of the CaMV35S promoter, a Nos-NPTII gene and a wheat Em-GUS fusion gene. Construction and structure of p B A C G G U S is described in Chapter 5. Table 3.2 Plasmids used for spruce transformation Plasmid Description Reference pBI221 CaMV35S promoter- GUS Jefferson et al 1987 pCMCHOO CaMV35S promoter-^MK leader-GLtf NOS-NPTII Ellis et al 1991 p J D D I . 7 Poplar PAL2-GUS CaMV35S-NPTII Molitor 1995 p B A C G G U S CaMV35S-mtisense CG Wheat Em-GUS NOS-NPTII Chapter 5 in this study. 3.2.4 Particle bombardment Two devices, a PDS 1000/He™ biolistic gun (BioRad, Mississauga, Ontario), and a particle inflow gun (PIG; Finer et al, 1992; assembled by Dr. Stephanie Mclnnis, B C Research Inc., Vancouver, British Columbia), were used for particle bombardment. Schematic diagrams for the devices are shown in Figure 3.2. Standard settings for PDS 1000/He™ bombardment were a 1300-psi rupture disk and target distance of 5.1 cm. Standard settings for the PIG gun were helium pressure of 80 psi and target distance of 9 55 cm. A baffle of nylon mesh was used to reduce excess airflow to the target for bombardment using the PIG gun. Mature somatic embryos placed in the centre of a petri dish containing agar-solidified A B A medium were used as target tissues. Plasmid D N A was purified using CsCl density gradient centrifugation (as per Sambrook et al., 1989). The purified plasmid D N A was precipitated onto gold particles (spherical 1.5-3.0 um, Aldrich) using a CaCb and spermidine method modified from Klein et al. (1988). Five ul of 1 ug/ul D N A were mixed with 100 ul of 50 m M spermidine. The D N A mixture was added to 10 mg gold particle (spherical 1.5-3.0 um, Aldrich) while vortexing. One hundred ul of 1.0 M CaCb. was added to the mixture, vortexed thoroughly, and incubated at room temperature for 10 min to let the particles settle to the bottom. For PIG bombardment, 140 ul supernatant was removed and the remaining mixture was vortexed thoroughly to make a uniform suspension. Three ul of the suspension was dispensed in the centre of a syringe filter holder (13 mm diameter, V W R ) and used for particle bombardment. For PDS 1000/He™ bombardment, the supernatant was removed completely, and the particles were washed once with 100 % ethanol and resuspended in 80 ul of 100 % ethanol. Three ul of the suspension was dispensed in the centre of a macrocarrier. The particle mixture was dried under a sterile air stream and used immediately for particle bombardment. 56 h e l i u m g a s m i c r o p r o j e c t i l e s s y r i n g e f i l ter h o l d e r • ^ ! ! ! E ^ E E E e | baf f le target t i s s u e A. Particle inflow gun (PIG) h i g h - p r e s s u r e h e l i u m g a s ruptu re d i s k m a c r o c a r r i e r ( p l a s t i c d i sk ) m i c r o p r o j e c t i l e 3 r e t a i n i n g s c r e e n t a r g e t t i s s u e B. PDS 1000/He™ (1) / 1 \ (2) (3) Figure 3.2 Schematic diagrams of two particle bombardment devices. A . Particle inflow gun (PIG). The suspension of microprojectiles is placed in the centre of the syringe filter holder. Sudden release of helium gas directly accelerates the microprojectiles toward the target tissue. B. PDSIOOO/He . (1) Helium gas is retained by rupture disk. Microprojectiles are placed on macrocarrier. (2) Helium gas builds up sufficient pressure to break the rupture disk. (3) Expanding helium gas hits the macrocarrier, launching the microprojectiles into the target tissue. 57 3.2.5 Transient expression assays Transient expression assays were performed using zygotic and somatic embryos of spruce to assess D N A delivery efficiency under different conditions. To obtain zygotic embryos, seeds of white spruce (Picea glauca) were soaked for 5 min in a sterile Petri dish with 95 % (v/v) ethanol containing a few drops of wetting agent (Tween 20, Sigma Chemical). The seeds were then transferred to undiluted commercial hypochlorite bleach (6 % (w/v) hypochlorite, Javex, Bristol-Myers, Toronto, Ontario) containing a few drops of Tween 20 and soaked for 15 min. The surface-sterilised seeds were rinsed three times in sterile distilled water for 10 min each. Sterilised seeds were imbibed in sterile distilled water for 5-8 hours. Zygotic embryos were excised aseptically from imbibed seeds, placed in the centre of half-strength Litvay's medium (1/2 L M ) containing 0.4 % Gelrite, 2 % sucrose and 0.05 % glutamine, and used for bombardment. Somatic embryos were placed in the centre of a petri dish containing A B A medium and used for bombardment. Each petri dish contained 10 zygotic or somatic embryos. Zygotic and somatic embryos were assayed for GUS activity two days after the bombardment, using the X-Gluc staining procedure described below, and the number of blue loci (Figure 3.4A) was counted under a dissecting microscope. 3.2.6 Selection and screening of transformed cell lines A flow chart describing the selection process is shown in Figure 3.3. Somatic embryos without bombardment were used as controls in the selection process. Tissues were incubated at 25 °C in the dark during the entire selection process. After incubation for two days, 25 embryos were stained for GUS activity to confirm D N A delivery to the tissues as described below. The rest were dispersed onto fresh A B A medium to minimise 58 contamination and incubated for five days. For callus induction, they were transferred to maintenance medium and incubated for one week. For kanamycin selection, the tissues were incubated on maintenance medium containing 2 or 5 mg/1 kanamycin (kanamycin medium). From this point, tissues were transferred to fresh medium every two and one half weeks. After calli had been transferred to kanamycin medium two more times (initial selection), they were incubated on medium without kanamycin to recover actively growing embryogenic tissues. Embryogenic tissues were then transferred back to kanamycin medium for evaluation of kanamycin resistance. Cell lines that survived four subsequent transfers on kanamycin medium after the initial kanamycin selection were considered to be kanamycin-resistant lines. Kanamycin-resistant lines were screened for GUS activity, and the presence of transgenes in the putative transformants was confirmed by PCR as described below. Cell lines were cultured on maintenance medium without kanamycin once they were confirmed to be true transformants. Recovery from bombardment Ca l lus initiation Initial selection r r Recovery from initial selection Evaluat ion of kanamycin res istance v. Bombardment ^ 2 days ^ 2 days Fresh A B A medium Transient a s s a y ^ 5 days Maintenance medium ^ 1 week Kanamyc in medium ^ 2.5 w e e k s * 3 t imes Maintenance medium ^ 2.5 w e e k s * 2 times Embryogenic t issues induced? Y e s No Discard Kanamyc in medium ^ 2.5 weeks * 4 times Growth of embryogenic t i ssues? Y e s No i Discard Kanamycin-resistant lines Figure 3.3 Selection of transformed cells 6 0 3.2.7 Screening for GUS activity Tissues of kanamycin-resistant lines were incubated in the GUS reaction mixture (1.5 m M 5-bromo-4-chloro-3-indol-l-glucuronide (X-Gluc), 10 m M N a 2 E D T A , 0.1 % Triton X-100 in 0.1 M sodium phosphate pH 7.0) at 37 °C for overnight. The presence or absence of staining (blue colour development) was scored under a dissecting microscope. 3.2.8 Extraction of spruce genomic D N A Actively growing embryogenic tissues (approx. 100 mg) were mechanically ground in 0.5 ml spruce D N A extraction buffer (50 m M Tris-HCl, 20 m M E D T A , 1 % (w/v) PVP, 0.1 % (w/v) B S A , 0.1 % (w/v) spermine, 0.1 % (w/v) spermidine and 0.1 % (v/v) p-mercaptoethanol; pH 8.0) in a 1.5 ml microcentrifuge tube using a fitted plastic pestle. To the mixture, 122 ul 5 % Sarkosyl was added and incubated at room temperature for 15 min on a shaker. The cell lysate was mixed well with 100 ul 5 M NaCl , then 100 ul 8.6 % C T A B (in 0.7 M NaCl) was mixed in, and incubated at 65 °C for 30 min. The sample was incubated with 2.5 ug/ml DNase-free RNase A at 37 °C for 30 min, extracted twice with chloroform/isoamylalcohol (24:1) and D N A was precipitated using one-fifth volume of 5 M NaCl and two-thirds volume of isopropanol. The D N A precipitate was collected by centrifugation at 17,000 g for 5 min. The pellet was rinsed with 70 % ethanol, dried briefly, and dissolved in TE buffer (10 m M Tris-HCl and 1 m M E D T A ; pH 8.0) by incubating at 65 °C for 30 min. The D N A concentration was determined by measuring its absorbance at 260 nm. D N A samples were diluted to a concentration of 0.25 ug/ul and stored at 4 °C. Undiluted D N A stock solution was kept at -20 °C. 61 3.2.9 PCR analysis of putative transformants The primers used for PCR screening are listed in Table 3.3. Tubulin primers were used as positive controls for the PCR reactions, while the other three primer sets were used for detection of transgenes. Each PCR reaction contained 250 ng genomic D N A , 400 n M each of forward and reverse primers, 200uM dNTP, 10 m M Tris-HCl pH 8.3, 1.5 m M M g C l 2 , 50 m M KC1 and 0.025 U/ul Taq D N A polymerase (Boehringer Mannheim). PCR reactions were performed in a Techne3 Thermal Cycler. The thermal cycling regime was as follows: 1 cycle of 94 °C/5 min; 30 cycles of 94 °C/1 min, 55 °C/1 min, 72 °C/1 min; 1 cycle of 72 °C/10 min. The amplification products were analysed by 1 % agarose gel electrophoresis. Table 3.3 Primers used for PCR screening Target gene Primer Sequence 5' 3' Product size NPTII npt4 npt6 C A A G A T G G A T T G C A C G C A G G T T C T C G A A T C G G G A G C G G C G A T A C C G T A A A 0.74 kb GUS G N L G N R T C T C T T T G A T G T G C T G T G C C A A A T T C G A G C T C G G T A G C A A 0.50 kb antisense CG P C G l a PCG6a A G C T A G G C T G G A C A G G A A C A T C G T C G T G C T G A A G A A A T T G 1.44 kb (3-tubulin tubl tub2 G T G A C T T G A A C C A T C T G A T C T C C C A T G C C T T C C C C G G T A T A C C A 0.53 kb 62 3.3 Results 3.3.1 Comparison of D N A delivery efficiencies Transient assays were performed to compare the efficiencies of D N A delivery under different conditions. Somatic or zygotic embryos of spruce were bombarded with pCMCHOO (CaMV35S-AMV-GUS), pJDD1.7 (poplar PAL2-GUS) or pBI221 {CaMV35S-GUS) and assayed for GUS activity two days after bombardment. Transformed cells, seen as blue loci in Figure 3.4A, were counted under a dissecting microscope. Figure 3.5 shows a comparison of D N A delivery efficiencies using two different particle bombardment devices. Under the conditions tested, the PDS 1000/He™ gave about 3-fold higher efficiency than the PIG, using both plasmids. When different rupture disc pressures were compared in the PDS 1000/He™ system, a rupture disc pressure of 1300 psi gave the highest efficiency (Figure 3.6). For these reasons, most of subsequent transformation experiments were performed using the PDS 1000/He™ with a rupture disc pressure of 1300 psi. Figure 3.4 Generation of transgenic spruce (next page) A . Example of transient GUS expression assay results showing transformed cells as blue loci. B. Induction of embryogenic tissues from transformed tissues. Embryonal masses are seen as translucent projections. C. Actively growing embryogenic tissues. D. Mature somatic embryos just before desiccation treatment. E. Transgenic plants germinated in vitro. G. Transgenic plants grown in soil for six months. 64 16 o E 1 2 1_ 0 Q-O S 8 0 _3 - 0 CD E 3 PIG P D S 1000/He B PIG P D S 1000/He Figure 3.5 Comparison of two particle bombardment devices. D N A delivery efficiency of two particle bombardment devices was compared using two different gene constructs. Somatic embryos of spruce were bombarded with pCMCHOO (A) or pJDD1.7 (B) using either a particle inflow gun (PIG) or PDS 1000/He™. Embryos were stained for GUS activity two days after the bombardment and number of blue loci was counted under a dissecting microscope. Values are the mean of 30 (A) or 80 (B) embryos ± SE. o £" 1 4 i— Q. O o CP i 2 CD E 900 psi 1100 psi 1300 psi Rupture disc pressure Figure 3.6 Effect of rupture disc pressures on DNA delivery efficiency Zygotic embryos of spruce were bombarded with pBI221 using different rupture discs in PDS 1000/He™. Embryos were stained for GUS activity two days after bombardment and blue loci were counted under a dissecting microscope. Values are the mean of 40 embryos ± SE. 65 3.3.2 Kanamycin selection of transformed tissues In order to establish transgenic cell lines, embryogenic tissues were selectively induced on kanamycin medium from embryos bombarded with pJDD1.7 or p B A C G G U S . Detailed analyses were only carried out on transformation experiments using p B A C G G U S . The plasmid p B A C G G U S contained three fusion genes, a NPTII gene under control of the NOS promoter, a GUS gene under control of the wheat Em promoter, and an antisense coniferin (3-glucosidase gene under control of the CaMV35S promoter. Particle bombardment was performed using either the PIG or the PDS 1000/He™ device. Somatic embryos without bombardment were used as controls. Tissues were allowed to recover from the physical stress of the bombardment by incubation on A B A medium for one week and then incubated on maintenance medium without kanamycin (Figure 3.3) for another week. The cotyledons and hypocotyls of the embryos started expanding soon after this transfer, forming callus-like structures. These embryos were transferred to maintenance medium containing 2 or 5 mg/1 of kanamycin (referred to as the K 2 or K5 medium respectively) to apply a selection pressure during induction of embryogenic tissues. Tissues were incubated on this kanamycin selection medium for 7.5 weeks, during which time they were transferred to fresh kanamycin medium every two and a half weeks (the initial selection stage shown in Figure 3.3). In the absence of kanamycin, the calli turned brown within two weeks on the maintenance medium and embryonal masses started appearing as translucent projections (Figure 3.4B). Embryonal masses continued to proliferate and became established as embryogenic tissues (Figure 3.4C). Inclusion of kanamycin in the medium inhibited embryonal mass induction at concentrations as low as 2 mg/1. Embryogenic tissue formation from both control and bombarded tissues was 66 moderately inhibited on the K2 medium. It was completely inhibited on the K5 medium for control tissues, and reduced to 1 % on the K5 medium for bombarded tissues (Tables 3.4 and 3.5). After this initial selection, the embryogenic tissues were transferred to maintenance medium without kanamycin to recover actively proliferating tissues. This step was necessary because many transformed tissues displayed only a weak resistance to kanamycin and continuous incubation on a kanamycin medium would eventually ki l l these weak expressing lines. After this proliferation stage, actively growing embryogenic tissues were returned to a kanamycin medium for evaluation of kanamycin resistance (Tables 3.4 and 3.5). Embryogenic tissues were transferred to fresh K2 medium every two and one half weeks and their growth were scored at each passage. Only minor growth inhibition was observed in control cell lines after the first incubation on K 2 medium (Table 3.4), but the survival rate was reduced to 13 % after the second incubation and stayed around the same level (10 %) after the third incubation. Lines that survived three consecutive incubations on K 2 medium remained resistant to K2 medium. Control embryogenic lines originally induced in K2 medium exhibited a similar response except that more lines remained resistant after the second incubation on K2 medium (Table 3.4). Embryogenic lines derived from bombarded embryos and induced on K 2 medium responded to K 2 medium in a similar manner to control lines (Table 3.5). Thirteen percent of embryogenic tissues remained resistant after the third incubation on K2 medium. About half of these lines proved to be true transformants as demonstrated by the detection of the transgenes in PCR analysis (data not shown). Embryogenic lines induced on K5 medium after the bombardment were markedly more resistant to kanamycin than those induced on K2 medium (Table 3.5). Half of these lines survived through three transfers on K 2 medium 67 and all of these kanamycin-resistant lines were subsequently shown to be true transformants. Table 3.6 summarises the effect of the initial selection process on transformation efficiency. Selection on K5 medium yielded a lower overall transformation efficiency (0.5 %) than selection on K2 medium (1.6 %), probably because many transgenic lines displayed only weak antibiotic resistance that was insufficient to allow growth on the higher kanamycin level. In fact, 42 % of the recovered transgenic lines were unable to survive through two consecutive transfers on K5 medium (data not shown). Table 3.4 Effect of kanamycin selection on wild-type spruce cells Initial selection ' % of embryos forming embryogenic tissue*2 after initial selection Growth of embryogenic tissues on K2 medium after recovery from initial selection 3 1 transfer to K 2 medium 2 transfers to K2 medium 3 transfers to K2 medium Omg/1 kanamycin 94% 71 % 13 % 8 % 2 mg/1 kanamycin 18% 60% 4 0 % 10% 5 mg/1 kanamycin 0 % N / A N / A N / A *1- Initial selection consisted of 7.5 weeks of incubation on medium containing 0, 2, or 5 mg/1 kanamycin (See Figure 3.3). *2- Embryos with proliferating embryonal masses. *3- The K 2 medium is maintenance medium containing 2 mg/1 kanamycin. One transfer represents incubation on kanamycin medium for 2.5 weeks. Values are the number of cell lines exhibiting any signs of growth as a fraction of the total number of embryogenic lines obtained. 68 Table 3.5 Kanamycin selection of transformed cells Initial selection*1 % of embryos with embryogenic tissues 2 after initial selection Growth of embryogenic tissues on the K2 medium after recovery from initial selection 3 1 transfer to K2 medium 2 transfers to K 2 medium 3 transfers to K 2 medium 2 mg/1 kanamycin 42% 63% 25% 13% 5 mg/1 kanamycin 1% 93% 57% 50% *1- Initial selection consisted of 7.5 weeks of incubation on kanamycin medium (See Figure 3.3). *2- Embryos with proliferating embryonal masses. *3- The K 2 medium is maintenance medium containing 2 mg/1 kanamycin. One transfer represents incubation on kanamycin medium for 2.5 weeks. Values are the number of cell lines exhibiting any signs of growth as a fraction of the total number of embryogenic lines obtained. Table 3.6 Effect of initial selection on transformation efficiency Initial selection Number of embryos tested Number of transgenic lines 1 obtained Transformation efficiency 2 2 mg/1 kanamycin 2737 44 1.6% 5 mg/1 kanamycin 2713 13 0.5% *1- Transgenic line was defined as a line in which the presence of the NPTII gene was confirmed by PCR analysis. *2- Number of PCR-positive (NPTII) lines / total number of embryos bombarded. 3.3.3 GUS screening of putative transformants Kanamycin selection of transformed spruce tissues is a slow and labour-intensive process because of the difficulty in distinguishing between escapes and true transformants. Evaluating kanamycin resistance for all lines required five to six months. The response of tissues to kanamycin was influenced by variables such as the size of tissue being 69 transferred, the medium depth in each plate and variation between medium preparations, making evaluation even more challenging. Therefore, GUS staining was performed routinely to identify true transformants at earlier stages of kanamycin selection. Portions of actively growing tissues were stained for GUS activity and the presence of blue staining was scored using a dissecting microscope. The first GUS-positive lines were detected immediately after the initial kanamycin screening (two months after bombardment) and all the GUS-positive lines were detected within four months after bombardment. Inclusion of GUS screening in the selection process also made it possible to rescue transgenic lines that were relatively susceptible to kanamycin and would therefore have been lost during prolonged selection on kanamycin medium. 3.3.4 PCR analysis The presence of a transgene in putative transformants was examined by PCR analysis using primers specific to the NPTII gene. The amplified NPTII gene fragment was detected as a single band in transgenic lines (Figure 3.7). No amplification product was detected in a wild-type control. A summary of the results from four transformation experiments is shown in Table 3.7. On average, only 46 % of kanamycin-resistant lines proved to be PCR-positive for the NPTII gene. In contrast, there was a much stronger correlation between GUS-positive lines and PCR-positive results. Although GUS screening failed to identify a few of the PCR-positive lines, this either resulted from absence of the GUS gene or low expression of the GUS gene in these lines (Table 3.7 and 3.8). In total, 57 transgenic spruce lines were obtained from these transformation experiments. The PDS 1000/He™ system produced higher transformation efficiencies than 7 0 the PIG device (Table 3.7), which is consistent with the results from transient expression assays (Figure 3.5). The overall transformation efficiency was 1 % when calculated on a per embryo basis. This corresponds to an efficiency of 36 % when expressed per bombarded petri dish. To confirm the presence of other transgenes, PCR analysis was performed using primers specific to the GUS gene and to the antisense CG gene, the results of which are summarised in Table 3.8. Forty-six lines contained all three genes, eight lines contained only the NPTII and GUS genes, and three lines contained only the NPTII gene. Tissues of all the transgenic cell lines were processed for cryopreservation for long-term storage. Plants were regenerated from the transgenic lines containing all three genes via somatic embryogenesis (Figure 3.4D and E). A l l 46 of these lines produced mature embryos although three lines exhibited notably lower embryogenic potential. Somatic embryos were germinated in vitro and transferred to soil (Figure 3.4F) for phenotypic analyses as described in Chapters 4 and 5. 8 putative transformants ~ (kanamycin resistant lines) co , v "5. 1 2 3 4 5 6 7 8 9 10 Figure 3.7 Example of PCR analyses. 71 Table 3.7 Summary of spruce transformation Total number of embryos bombarded Kanamycin-resistant lines 1 GUS-positive lines PCR-positive lines Transformation efficiency *2 Experiment 1 (PIG) 889 10 3 3 0.3% Experiment 2 (PDS 1000/He™) 1484 36 5 8 0.5% Experiment 3 (PDS 1000/He™) 1450 39 18 19 1.3% Experiment 4 (PDS 1000/He™) 1618 38 21 27 1.7% Total 5441 123 47 57 1.0% * 1 - Number of lines that survived four consecutive transfers to kanamycin medium after recovery from initial selection (See Figure 3.3). *2- Number of PCR-positive (NPTII) lines / total number of embryos bombarded. Table 3.8 Summary of PCR analyses Genomic D N A was extracted from embryogenic tissues. PCR analysis was performed as described in Materials and Methods using primers specific for wither the NPTII, GUS or antisense CG gene. Transgenes Number of transgenic lines NPTII + GUS+ antisense CG 46 NPTII + GUS only 8 NPTII only 3 Total 57 72 3.4 Discussion Kanamycin selection Of all the procedures used in this study, kanamycin selection of transformed tissues was the most problematic. This is mainly because transformed spruce cells that had acquired the NPTII gene did not display strong kanamycin resistance. To obtain the maximum number of transgenic lines, it was necessary to select tissues using a low concentration (2 mg/1) of kanamycin, which is a sublethal dose for spruce tissues (Table 3.4). This made the selection process slow and labour-intensive, since it produced a large number of escapes. The number of escapes was reduced only when they were incubated on the kanamycin medium for a prolonged period of time (Table 3.4 and 3.5). The selection process was further complicated by the observation that some non-transformed tissues also acquired resistance against this level of kanamycin, while some transgenic lines proved to be susceptible to 2 mg/1 kanamycin. It was, therefore, impossible to distinguish transformed cells based on kanamycin selection alone. Selection using 5 mg/1 kanamycin drastically reduced the number of escapes (Table 3.4 and 3.5) but it also resulted in a three-fold reduction in transformation efficiency (Table3.6). Other workers have also reported weak kanamycin resistance in transgenic spruce tissues carrying the NPTII gene (Ellis et al, 1993; Bommineni et al, 1993; Bommineni et al, 1997). One reason for this may be the high susceptibility of spruce tissues to kanamycin. Kanamycin concentrations as low as 1 mg/1 have been reported to have inhibitory effects on the development of spruce embryos (Ellis et al, 1989), while a concentration of 3 mg/1 completely inhibited growth of embryogenic suspension cultured cells (Bommineni et al, 1997). This is a very low tolerance compared to other plant species. For example, embryogenic tissues of Pinus radiata maintained growth in medium 73 containing 300 mg/1 of kanamycin (Walter et al, 1998), while a kanamycin concentration of 100 mg/1 is routinely used for poplar transformation (Chapter 2 in this study). Another reason for the weak kanamycin resistance in transgenic spruce may be low expression of the NPTII gene in spruce tissues. The NPTII gene in the transformation vector p B A C G G U S is controlled by the NOS promoter (Table 3.2). Although this fusion gene is effective for use in angiosperm species, it may not direct sufficient levels of expression in gymnosperm species. Although no reports have quantified expression of the NOS-NPTII fusion gene in conifers, studies using transient expression assays have found that a CAT or GUS reporter gene fused to the NOS promoter is only weakly expressed in conifer tissues (Charest et al, 1991; Duchesne and Charest, 1992). Use of a stronger promoter, such as the wheat Em promoter or the CaMV35S promoter fused with the AMV enhancer element (Charest et al, 1993) to direct NPTII expression might improve kanamycin resistance in transgenic spruce. It should be noted that recent studies of Agrobacterium-mediated conifer transformation did not encounter problems with kanamycin selection (Levee et al, 1999; Wenck et al, 1999). Levee et al obtained kanamycin-resistant white pine cell lines with a high efficiency even when expression of the NPTII gene was driven by the NOS promoter. A l l of the kanamycin-resistant white pine lines were true transformants, as confirmed by PCR analysis (1999). Does this indicate that different transformation methods lead to different levels of transgene expression? Transformation mediated by Agrobacterium may give a higher transgene expression possibly by yielding to a reduced copy number of transgenes, thus reducing transgene silencing, or by integrating transgenes preferentially into active chromatin. Alternatively, this may simply be a result of a much higher incidence of stable transformation when using Agrobacterium. Higher transformation 74 efficiency in combination with higher selection pressure would facilitate a cleaner selection of high expression lines. Consistent with this idea, I was able to eliminate escapes when I used a selection pressure of 5 mg/1 kanamycin (Table 3.4 and 3.5). GUS screening Since step-wise kanamycin selection was not a reliable way to distinguish transformants from escapes, it was necessary to incorporate additional methods for screening transformants. Routine staining for GUS activity with kanamycin-resistant cell lines made the screening process significantly faster. A l l of the GUS-positive lines were identified within four months after the bombardment, whereas evaluation of kanamycin resistance could not be completed until six months after the bombardment. This also allowed shorter exposure of embryogenic tissues to kanamycin, which is advantageous in maintaining the embryogenic potential of transgenic spruce cells. Prolonged exposure to kanamycin is known to reduce embryogenic potential (Robertson et al., 1992). Most importantly, GUS screening successfully identified a number of transgenic lines that were susceptible to kanamycin. There was a strong correlation between GUS screening results and PCR screening results (Table 3.7). GUS screening is, therefore, a simple, reliable and straightforward method to identify transgenic tissues. One drawback to GUS screening is that it fails to identify lines with low levels of expression. Seven percent of the transgenic lines did not stain for detectable GUS activity as embryogenic tissues but gave positive reactions only when the embryos matured (data not shown). Twelve percent of the transgenic lines did not stain for GUS at all in either tissue types (Table 3.7). This problem is exacerbated i f the GUS gene is fused to a weak promoter. For example, only faint GUS staining was detected in embryogenic tissues 75 transformed with the poplar PAL2-GUS fusion gene (Chapter 4). Since GUS screening does not eliminate non-transformed cells by itself, it needs to be combined with kanamycin selection to enable efficient screening. PCR analysis P C R analysis is the most direct and straightforward way of identifying transgenic tissues. However, it is an inefficient method for screening a large number of cell lines, as it still requires about two weeks of work for screening 100 lines. In comparison, it takes one day to screen the same number of putative transformants via GUS assays. In cases where no other screenable markers are included in transformation vectors, PCR screening is the only method capable of distinguishing transgenic tissues from escapes. It is, therefore, critical to minimize the number of escapes in order to avoid the labour and costs associated with P C R assays. The time and labour required for PCR analysis could be shortened i f it used a dye-based detection system such as TaqMan system (Applied Biosystems), which would eliminate the need to run agarose gels. P C R analysis of the transgenic spruce lines showed that 11 out of 57 transgenic lines did not contain the antisense CG gene (Table 3.8). This is not surprising, as the antisense CG gene was physically distant from the NPTII gene that was under selection (Figure 5.7 in Chapter 5). The transgenic lines lacking the antisense CG gene are still useful, since they can serve as appropriate transgenic controls in phenotype analysis (Chapter 5). Other studies have reported presence of additional PCR amplification products in some transgenic lines (Robertson et al., 1992; Charest et al., 1996). This was interpreted as an indication of the presence of multiple copy transgenes. Such additional bands were not 76 detected in any of the transgenic lines obtained in this study. Nevertheless, copy number variation among the transgenic lines was suggested by variation in the intensity of the amplification product among different lines (Figure 3.7). This point needs to be confirmed using Southern hybridization analysis. Transformation efficiency The overall transformation efficiency in this study was 1.6 % when transgenic lines were selected on K 2 medium and 0.5 % when they were selected on K5 medium. An efficiency of 1.6 % is comparable to the spruce transformation studies reported by Bommineni et al. (1993, 1998), in which they did not use kanamycin selection but used GUS screening as the only method to isolate transgenic lines. This indicates that, by using a low concentration of kanamycin, I was able to rescue transgenic lines that had gained only weak kanamycin resistance. It is difficult to directly compare my results with efficiencies obtained in other transformation studies using embryogenic tissues because results in those studies were expressed as either per gram fresh weight or per plate (bombardment) (Charest et al, 1996; Walter et al, 1998). A recent study has reported Agrobacterium-mediated transformation of white pine with high transformation efficiency (average 2 transgenic lines per plate; Levee et al, 1999). Although this is promising, microprojectile transformation still provides a reliable transformation method for those species where useful levels of transformation have not yet been achieved using Agrobacterium. In summary, I successfully obtained a large number of transgenic spruce lines using microprojectile bombardment. Kanamycin selection was problematic due to the kanamycin 77 susceptibility of transgenic spruce tissues carrying the NPTII gene. Kanamycin screening was therefore complemented with screening using GUS assays and PCR analysis, which identified some transgenic lines that were susceptible to kanamycin. Although the transformation efficiency may not be as high as that mediated by Agrobacterium, microprojection does provide a reliable protocol for conifer transformation. 78 Chapter 4 Vascular-specific expression of the poplar PAL2-GUS fusion gene in transgenic spruce 4.1 Introduction In order to control and predict the effects of genetic modification, it is important to understand when and where transgenes are expressed. This is normally determined by the expression pattern of the promoters used to control transgenes. The CaMV35S promoter and its derivatives are used in the majority of plant genetic engineering studies because they can direct strong expression of transgenes in many species. This trend also applies to use in tree species (Fillati et al, 1988; Ellis et al, 1993; Robinson, et al, 1994; Leple et al, 1995; Wang et al, 1996; Doorsselaere et al, 1995; Nilsson et al, 1996; Tsai et al, 1998; E l Euch et al, 1998). However, as the commercialisation of genetically engineered trees approaches reality, arguments have arisen against the use of the CaMV35S promoter for transgene expression. First, since the CaMV35S promoter directs constitutive expression of transgenes, it may cause unintended effects in other tissues. For example, lignin is believed to play an important role in defence against pathogens and in the healing process after wounding (Dean and Kuc, 1987). If we were to reduce lignin in trees using a constitutive promoter, this might interfere with these stress-response processes, resulting in trees susceptible to pathogens and mechanical stress. It is, therefore, desirable to restrict transgene expression only to targeted tissues. Second, since the CaMV35S promoter is derived from a virus sequence and plants have evolved efficient mechanisms for interfering with viral replication and spread in host tissues, this may be more easily subjected to transgene silencing (Matzke and Matzke, 1995). This could pose a major problem in long-lived plants such as trees, which require long-term stability of transgene expression. Third, the 79 public may be more reluctant to accept commercialization and release of transgenic trees carrying transgenes driven by a virus promoter. There have been heated public debates over the safety of genetically modified organisms (Gavaghan, 1999). Use of a virus gene sequence may further exacerbate public concern about use of the technology. For these reasons, it is important to develop plant gene-derived promoters that direct expression of transgenes only in target tissues (tissue-specific promoter) and/or only in response to appropriate stimuli (inducible promoter). For the purpose of modifying wood properties, it is desirable to express transgenes only in vascular tissues. Vascular-specific expression has been observed for many angiosperm genes, using in situ R N A hybridization or immunolocalization. This includes genes involved in lignin biosynthesis (PAL [Shmelzer et al, 1989; Subramaniam et al, 1993]; 4CL [Shmelzer et al, 1989]; C A D [Hawkins et al, 1997]; C O M T [Ye and Varner, 1995]; C C o A O M T [Ye and Varner, 1995]) and genes coding for cell wall structural proteins (glycine-rich protein [Keller et al, 1989a]; extensin [Ye et al, 1991]; arabinogalactan protein [Shindler et al, 1995]; proline-rich protein [Ye et al, 1991]; tyrosine- and lysine-rich protein [Domingo et al, 1994]). Promoters of these genes have been shown to direct vascular-specific expression in transgenic angiosperm plants (Bevan et al, 1989; Keller et al, 1989b; Hauffe et al, 1991; Intapruk et al, 1994; Feuillet et al, 1995; Capellades et al, 1996). However, identification of vascular-specific genes is far less advanced in gymnosperm species. To date, only a few conifer promoters that fall into the above category have been isolated, notably promoters from arabinogalactan protein genes (Loopstra et al, 1995) and from a CCoAOMT gene (Li et al, 1999) from loblolly pine. Therefore, transgenic research in conifers still largely relies upon use of well-characterized angiosperm promoters. 80 As part of this search for additional promoters that might direct vascular-specific expression in conifers, I have chosen the poplar PAL2 promoter because the expression pattern of this promoter has been well characterized in both transgenic tobacco and poplar (Molitor, 1995; Chapter 2 in this study). It was shown that the PAL2 promoter directs expression specifically in vascular tissues in each of these hosts, indicating that transcriptional regulation in vascular tissues may be conserved between the two species. It was not known, however, whether this conservation also extends to gymnosperm species. The evolutionary lineage of angiosperm and gymnosperm diverged from each other over 130 million years ago. To explore this possibility, the poplar PAL2-GUS fusion gene was introduced into spruce and the expression pattern of the GUS gene directed by the PAL2 promoter was studied in the resulting transgenic plants. 4.2 Materials and Methods 4.2.1 Generation of transgenic spruce Plasmid pJDD1.7 was introduced into embryogenic spruce line 11026 and transgenic plants were regenerated as described in Chapter 3. 4.2.2 GUS assays GUS activity was assayed using histochemical or fluorometric substrates as described in Chapter 2. 4.3 Results A plasmid containing the poplar PAL2-GUS fusion gene (pJDD1.7) was introduced into embryogenic spruce cells in order to study the expression pattern of the poplar PAL2 81 promoter in transgenic spruce. Bombardment of 1046 embryos produced 14 kanamycin-resistant lines, of which eight lines were positive for GUS activity. PCR analysis confirmed the presence of the GUS gene sequence in all of the GUS-positive lines. Out of the eight GUS-positive transgenic lines obtained, six lines retained embryogenic potential. These six GUS-positive transgenic lines were regenerated in vitro and assayed for GUS activity. GUS activity in PAL2-GUS transgenic spruce seedlings was generally weak, ranging from 0.05 to 1.14 nmole M U / minute / mg protein in one month-old germinants (Figure 4.1 A) . GUS activities were measured at three early developmental stages and were expressed relative to the activity in the one month-old germinants. The activity was highest in one month-old germinants, followed by embryogenic tissues and somatic embryos (Figure 4.IB). In order to compare GUS activity among different organs, two month-old germinants were divided into radicles (roots), hypocotyls (stems) and cotyledons (leaves), and each tissue type was assayed separately for GUS activity. GUS activity was found to be highest in radicles, followed by hypocotyls and cotyledons (Figure 4.1C). 82 Figure 4.1 Expression of the poplar PAL2-GUS fusion gene in different tissues of transgenic spruce. A. Variation in GUS activity among transgenic lines. Crude extracts were prepared from one month-old germinants of six transgenic spruce lines (5, 10, 16, 19, 22 and 121) and a wild-type control and assayed for GUS activity. Values shown are the mean of three extractions ± SE. B. Comparison of GUS activity between three developmental stages. Samples were taken from actively growing embryogenic tissues, mature somatic embryos and one month-old germinants. GUS activities were assayed separately in each line. They are shown relative to activity in germinants. Values are the mean from six transgenic lines ± SE. C. Comparison of GUS activity between organs. Radicle, hypocotyl, and cotyledon tissues from two month-old germinants were harvested and assayed for GUS activity. GUS activities are shown relative to activity in radicles. Values shown are the mean of four transgenic lines (10, 16, 19 and 22) ± SE. 83 84 Figure 4.2 shows histochemical GUS staining in in vitro-grown transgenic spruce carrying the poplar PAL2-GUS fusion gene. GUS activity in embryogenic tissues was weak and only faint staining was detected in embryonal masses (Figure 4.2A). Staining for GUS activity in somatic embryos was more readily detected than that in embryogenic tissues or germinants, possibly because of the high protein content and the small cell volumes in embryos. Mature somatic embryos exhibited intense staining at the radicle end (Figure 4.2B). Cross-sections of somatic embryos were also examined, which yielded intense and uniform GUS staining at the radicle end but no staining in the hypocotyl or the cotyledon (data not shown). In one month-old germinants, only two lines (lines 10 and 19) exhibited GUS staining at detectable levels. GUS staining of whole plants was detected in the centre of the hypocotyl and the cotyledon, suggesting expression in vascular tissues (Figure 4.2C). This was confirmed by histochemical staining of tissue cross-sections. Cross-sections of one month-old germinants revealed two to three vascular bundles with lignified xylem cells in the centre of the hypocotyl. GUS activity was associated with the primary xylem cells in this tissue (Figure 4.2D). Cross-sections of cotyledons revealed one centrally located vascular bundle, which was surrounded by endodermal cells. The xylem cells were not yet lignified at this stage (data not shown). In the cotyledon sections, GUS activity was detected only in the vascular tissues (Figure 4.2E). In radicles, GUS activity was observed in both vascular tissues and in epidermal/ectodermal cell layers (Figure 4.2F). GUS activity in epidermal/ectodermal cells may be associated with suberin synthesis in these tissues. Histochemical GUS staining was also performed in cross-sections of one year-old PAL2-GUS transgenic spruce. Transgenic spruce plants had been held in a plastic tunnel 85 greenhouse for seven months and grown through one complete cycle of dormancy. Tissue sections were taken in June, when new shoots were actively growing and the plants were about 20 cm tall. GUS staining in these sections was very faint, indicating that the poplar PAL2 promoter supported only a weak activity in spruce. In stem sections taken near the shoot apex, faint GUS staining was detected in the primary xylem tissues (data not shown). Development of secondary xylem was first observed in cross-sections of stems taken from a point 3 cm below the shoot apex. In these sections, faint GUS staining was detected in xylem ray parenchyma cells and in developing xylem (Figure 4.3A-C). The pattern of GUS staining remained the same in older stems that showed extensive lignification of the xylem (Figure 4.3D). Similar to the GUS expression pattern observed in cotyledons, GUS activity was detected in the vascular tissues at the centre of needles (Figure 4.3F). When whole needles were stained, GUS activity was also detected in the guard cells of the stomata (data not shown), which is consistent with the observation that guard cells are lignified in conifers (Esau, 1977). Cross-sections of expanding roots revealed GUS activity in the primary xylem and in the endodermal cell layer (Figure 4.3G). GUS activity in the endodermis may reflect the demands on phenylpropanoid metabolism associated with synthesis of suberin in the Casparian strip (Esau, 1977). 86 Figure 4.2 Histochemical assay for expression of the PAL2-GUS fusion gene in micropropagated transgenic spruce (next page). GUS activity as detected in - A. Actively growing embryogenic tissue. Arrowhead points to GUS staining in embryonal masses. B. Mature somatic embryos. C-F. One month-old germinants. C. GUS staining of a whole germinant. D. Cross-section of a hypocotyl. E . Cross-section of a cotyledon. F. Cross-section of a radicle. 88 Figure 4.3 Histochemical assay for expression of the PAL2-GUS fusion gene in soil-grown transgenic spruce plants. Germinants of transgenic spruce lines were transferred to soil after six weeks on G M D medium and grown through one complete cycle of dormancy in a plastic tunnel greenhouse. Plants were harvested in June when new shoots were actively growing. Cross sections from newly flushed shoot were stained for GUS activity. A . Cross section of a young stem showing GUS activity in ray parenchyma cells (arrowhead). B. Cross section of a young stem showing staining in developing xylem (asterisk). C. A n enlargement of B, showing staining in developing xylem (arrowhead). D. Cross section of a mature stem showing staining in ray parenchyma cells (arrowhead). E . Cross section of a young needle showing staining in vascular tissues (arrowhead). F. Cross section of a young root showing staining in metaxylem (arrowhead) and in the endodermis (asterisk). 90 4.4 Discussion This chapter reports the first investigation of a vascular-specific promoter in transgenic conifers. The expression pattern of the GUS reporter gene directed by the poplar PAL2 promoter is correlated with sites of lignin/suberin synthesis in transgenic spruce. GUS activity was 5- to 10-fold lower in embryogenic tissues and somatic embryos than in one month-old germinants (Figure 4.IB), which is consistent with the lack of lignification in embryo tissues. When different organs were compared in the transgenic plants, GUS activity was highest in radicles, followed by hypocotyls and cotyledons (Figure 4.1C). This is similar to the expression pattern observed in transgenic poplar, where the highest expression of the same construct was observed in the roots (Chapter 2). Histochemical staining of GUS activity confirmed an association of PAL2 promoter activity with sites of lignin/suberin synthesis. GUS activity was detected in vascular tissues of one month-old germinants and one year-old plants. Expression in non-vascular tissues (guard cells of needle, epidermal/exodermal cells of radicles, endoderm cells of young roots) was also associated with sites of lignin and/or suberin synthesis. The pattern of cell-specific expression of the PAL2-GUS fusion gene in transgenic spruce was strikingly similar to that seen in transgenic tobacco. GUS activity was detected in primary xylem of one month-old germinants (Figure 4.2) and in ray parenchyma cells and developing xylem of stems undergoing secondary growth (Figure 4.3). This suggests that the transcriptional regulation system that controls vascular expression of PAL2 may be conserved between the two evolutionarily distinct vascular plant species, tobacco and spruce. This conservation may reflect a core mechanism that has evolved to construct structural components such as lignin and suberin in vascular plants. Such structures would have been essential for plant survival on land by providing mechanical support and a water 91 conducting system. The presence of AC-like elements in the promoter regions of the pine arabinogalactan protein genes (Loopstra et al, 1995) and the pine CCoAOMT gene (Li et al., 1999) also supports a model of evolutionary conservation between angiosperms and gymnosperms for regulation of genes encoding cell wall-associated functions. It is interesting in this regard that genes for myb-like proteins, which may interact with A C elements, have been isolated from black spruce (R Rutledge, personal communication) and loblolly pine ( M Campbell, personal communication). The activity of the poplar PAL2-GUS fusion gene was generally very weak in the transgenic spruce. When cotyledons of one month-old germinants of transgenic spruce (Figure 4.1) were compared with leaves of in vzYro-grown transgenic poplar (Figure 2.2 in Chapter 2), GUS activity was 30 to 300-fold lower in spruce. Intense staining was detected only in somatic embryos; all the other tissues exhibited very faint GUS staining. The strong GUS staining in embryos was not the result of a high specific activity of GUS, because the quantitative assay with extracts detected relatively low GUS activity in these tissues (Figure 4.1). Instead, the strong staining was probably due to the high protein content and small cell volume in somatic embryos, which created a compartment relatively rich in GUS enzyme. The weak overall expression of PAL2-GUS observed in spruce may result from low transgene expression due to position effects. Position effects are thought to be the variation in transgene expression resulting from variation in structure and activity of the chromatin into which the transgene has been incorporated. This is demonstrated in Figure 4.1 as variation in GUS activity among different transgenic lines, which most likely have different sites of insertion of the transgene. Since a relatively small number (6) of spruce transgenic lines were obtained, it is possible that more strongly expressing lines could be obtained by 92 examining a larger number of transgenic lines. By comparison, 24 transgenic lines were obtained from poplar and their activity ranged from 0.16 to 26.24 nmole M U per minute per mg protein (Chapter 2). Another possible explanation for the weak transgene GUS activity is that some cis-elements of the poplar PAL2 promoter may not function properly in spruce. Although the pattern of expression suggests that the cw-elements that confer vascular-specific expression is conserved, additional enhancer element(s) specific to gymnosperms may be required for optimal expression in spruce. Enhancer element(s) may lie in the promoter regions used, but these elements may not be conserved between angiosperm and gymnosperm. For example, a study of GUS expression directed by the spruce 2S albumin promoter in spruce and tobacco has shown that transcription factors in tobacco recognise cw-elements that control tissue-specific expression of this promoter but do not recognise some of the enhancer elements (Mclnnis 1998). Partial conservation between angiosperm and gymnosperm promoters has also been reported for GUS gene expression directed by photosynthesis gene promoters (Campbell et al, 1994; Kojima et al, 1994; Gray-Mitsumune et al, 1996). Alternatively, additional enhancer elements may lie outside of the promoter regions used in our PAL2-GUS fusion construct. Introns can also affect the expression level of genes. The pine CCoAOMT gene contains an intron in the region upstream of the translation initiation site (Li et al, 1999). Such introns have also been observed in other plant genes (rice actin [McElroy et al, 1990]; maize ubiquitin [Christensen et al, 1992]; Arabidopsis elongation factor 1 [Gidekel et al, 1996]; Arabidopsis enol-acyl carrier protein reductase [de Boer et al, 1999]), and have been shown to be required for strong expression of reporter genes. The presence of introns may enhance gene expression by increasing the rate of transcription (Gidekel et al, 93 1996; Lumbreras et al, 1998) and/or by increasing the stability of m R N A (Koziel et al, 1996; Lumbreras et al, 1998). Enhancer elements have also been detected both in the 3' non-coding region (Larkin et al, 1993; Ruegger et al, 1999) and in the coding region (Douglas et al, 1991) of plant genes. In summary, this study showed that the poplar PAL2 promoter directs vascular-specific expression in transgenic spruce, suggesting that the transcriptional regulation system that controls tissue-specific expression of PAL2 is conserved between gymnosperm and angiosperm species. However, since expression of this promoter was weak, it may be necessary to add gymnosperm enhancer elements to the construct in order to obtain expression levels that would be useful for genetic modification of wood properties. 94 Chapter 5 Effects of an antisense coniferin (3-glucosidase (CG) gene in transgenic spruce 5.1 Introduction Despite the wealth of knowledge accumulated about the lignin biosynthetic pathway in plants, comparatively little is known about storage and transport of the monolignols. The 4-O-P-D-glucosides of monolignols (p-hydroxycinnamyl alcohol glucoside, coniferin and syringin) have been implicated as their storage and transport forms (Freudenberg 1965), an idea that is supported by several observations. First, high levels of coniferin accumulate in the cambium sap of gymnosperm trees during the growing season (Freudenberg and Harkin 1963; Terazawa and Miyake, 1984; Savidge, 1988). Second, exogenously applied radiolabeled monolignol glucosides can be efficiently incorporated into lignin in the stems of both gymnosperm and angiosperm trees (Terashima et al., 1986; Fukushima and Terashima, 1990; Fukushima and Terashima, 1991). Third, seasonal changes in the activity of coniferyl alcohol glucosyltransferase, an enzyme that synthesises monolignol glucosides, coincide with the active growing season (Savidge and Forster, 1998). Finally, histochemical activity staining of coniferin (3-glucosidase, an enzyme that hydrolyses monolignol glucosides, reveals activity only in developing xylem (Dharmawardhana et al, 1995). Glucosides may offer functional advantages since they are chemically more stable than the monolignol aglycones. Once monolignol glucosides are transported to the site of lignification, most likely in the cell wall, they must be hydrolysed to release their aglycones. A (3-glucosidase that exhibits specificity for coniferin and syringin has been purified from lodgepole pine xylem tissue (Dharmawardhana et al., 1995) and its corresponding c D N A sequence has been 95 obtained from a lodgepole pine xylem c D N A library (Dharmawardhana et al, 1999). The protein predicted from the isolated c D N A has a molecular mass of 58 kDa and exhibits high similarity to family 1 (3-glycosyl hydrolases (Dharmawardhana et al, 1999). It also contains a 23 amino acid N-terminal signal peptide for ER targeting. However, since no ER retention signal appears in the sequence, the protein is secreted presumably into the apoplast (Dharmawardhana et al, 1999). By using specific antibodies against pine coniferin (3-glucosidase (CG), it was recently shown that C G was localized in the cell walls of developing xylem (L. Samuels, unpublished). This localization is consistent with a role for C G in lignin deposition. Although these observations are all consistent with the involvement of monolignol glucosides in lignin biosynthesis, none of them provides direct evidence for an obligatory role in that process. To test the hypothesis that formation and hydrolysis of monolignol glucosides is a necessary part of lignin biosynthesis, it would be desirable to block either the synthesis or cleavage of coniferin and evaluate the resulting phenotype. Therefore, this chapter describes an attempt to down-regulate the CG gene via expression of the specific antisense R N A . Availability of the pine CG c D N A allowed me to construct an antisense gene against the CG gene. However, since no transformation protocol was available for lodgepole pine, I introduced this construct into interior spruce (Picea glauca x engelmanni complex). It was also necessary to characterize the CG gene(s) in spruce in order to validate the use of the heterologous antisense sequence, and to enable me to analyze the effects of the antisense CG gene in transgenic plants. Therefore, the first half of this chapter describes the cloning and expression of spruce CG genes, while the second half describes analysis of transgenic spruce plants that carry the antisense CG gene. 96 5.2 Materials and Methods 5.2.1 PCR cloning of spruce coniferin P-glucosidase (CG) genes Genomic D N A was extracted from embryogenic tissues of interior spruce 11026 as described in chapter 3. Three sets of primers (PCGla and PCG2a; PCG3a and PCG4a; PCG5a and PCG6a. See Table 5.1) were designed based on the pine CG c D N A sequence (Dharmawardhana et al, 1999), using web-based Primer3 software at http://www.genome.wi.mit.edu/cgi-bin/primer/primer3 www.cgi (Rozen and Skaletsky, 1998). The position of each primer in the pine CG c D N A sequence is shown in Figure 5.1. Spruce CG fragments were then amplified from interior spruce genomic D N A with these primers using either Taq D N A polymerase (Boehringer Mannheim) or Accurase™ (DNamp Inc.). PCR reactions using Taq D N A polymerase contained template D N A (250 ng genomic D N A or 10 ng plasmid DNA) , 400 n M each forward and reverse primers, 200uM dNTP, 10 m M Tris-HCl pH 8.3, 1.5 m M M g C l 2 , 50 m M KC1 and 0.025 U/ul Taq D N A polymerase (Boehringer Mannheim). The thermal cycling regime was as follows: 1 cycle of 94 °C/5 min; 30 cycles of 94 °C/1 min, 55 °C/1 min, 72 °C/1 min; 1 cycle of 72 °C/10 min. PCR reactions using Accurase™ contained 250 ng spruce genomic D N A , 400 n M each forward and reverse primers, 200uM dNTP, Accurase Long Template Buffer, 1.5 m M MgSCM, and 0.025 U/ul Accurase™. The thermal cycling regime was as follows: 1 cycle of 94 °C/5 min; 10 cycles of 94 °C/1 min, 55 °C/1 min, 68 °C/2 min; 20 cycles of 94 °C/1 min, 55 °C/1 min, 68 °C/2 min plus 5 sec increment per additional cycle; 1 cycle of 72 °C/7 min. The amplification products obtained using primers PCG5a and PCG6a were cloned into a Bluescript SK vector according to the T/A cloning protocol of Holton and Graham (1991). White colonies were screened for the presence of insert by direct PCR. Individual 97 colonies were picked with wooden toothpicks and suspended in 10 p.1 sterile water, boiled for 10 sec and then cooled on ice immediately. One ul of a 1/10 dilution of the boiled extract was used in a 20 u.1 PCR reaction. The presence of insert was confirmed by PCR analysis using primers PCG5a and PCG6a. Plasmid D N A from selected clones was purified using the alkaline lysis/PEG precipitation method (Lis 1980). The D N A sequence of the insert was obtained by A B I AmpliTaq dye termination cycle sequencing according to the manufacture's protocol and analysed on a A B I 373 D N A sequencer (Nucleic Acid and Protein Services unit, University of British Columbia). After manual editing, nucleotide sequences were assembled using web-based C A P Sequence Assembly software at http://gcg.tigem.it/ASSEMBLY/assemble.html (Huang 1996). Intron splice sites were predicted using Arabidopsis splice site prediction software NetGene2 World Wide Web Server at http://www.cbs.dtu.dk/NetPlantGene.html (Hebsgaard et al, 1996). Exons were also predicted by aligning spruce genomic sequences against the lodgepole pine CG cDNA sequence using B L A S T 2 Sequences software at NCBI web server (http://www.ncbi.nlm.nih. gov/gorf/b!2 .html). The amino acid sequence was translated from the predicted c D N A sequence using the Sequence Utilities software on the B C M Search Launcher server (http://dot.imgen.bcm.tmc.edu:9331/seq-util/seq-util.html). Multiple sequence alignment of c D N A and amino acid sequences were performed using the PILEUP program in G C G software (University of Wisconsin). A phylogenetic tree was constructed using P A U P 4.0 software (Swofford 1999; Sinauer Associates, Inc.). 98 Table 5.1 Primers for PCR amplification of the C G gene Primer sequence product size expected from the pine CG c D N A P C G l a A G C T A G GCT G G A C A G G A A C A 0.35 kb PCG2a G G A TCC C A T TTT G C A G A A G A PCG3a C A A TTT CTT C A G C A C G A C G A 0.2 kb PCG4a C T G A A T G G G GTT TTC TGC A T PCG5a TTC GGT G A C C G T GTC A A A T A 1.0 kb PCG6a T C G T C G TGC T G A A G A A A T T G 5' • 1a I I . N E P . . . . . . E N G . . 2a 5a 6a • A 3a 4a • < Primer binding sites • Predicted active site residues • Glycosylation site E l Signal peptide Figure 5.1 Primer binding sites 5.2.2 R N A extraction Precautions were taken as described in Sambrook et al. (1989) to minimise RNase contamination. Plant tissues were ground into powders in liquid nitrogen. Up to 350 mg powdered tissue was homogenized with a fitted pestle in a microcentrifuge tube in 750 ul R N A extraction buffer (150 m M Tris-HCl, 750 m M NaCl, 250 m M L i C l , 1 % P E G 8000, 2 99 % C T A B , 20mM E D T A , 0.3 % lauroyl sarcosine, 1.5 m M aurintricarboxylic acid, 5mM thiourea, pH 8.2). To the extract was added 540 l i l chloroform/isoamylalcohol (24:1) and the resulting mixture was then vortexed. The aqueous layer was separated from the organic layer by centrifugation at 17,000 g for 5 min. R N A was selectively precipitated from the recovered aqueous layer by adding one-third volume of 8M L i C l followed by incubation on ice for at least 2 hrs. The R N A precipitate was collected by centrifugation (17,000 g, 5 min) rinsed with 70 % ethanol, dried briefly, and dissolved in RNase-free water. When complete removal of D N A was necessary, the R N A was treated with RNase-free DNasel for 15 min at 37°C and the reaction was stopped by addition of 1/2 volume of stop buffer (50 m M E D T A , 1.5M sodium acetate, 1 % SDS). To remove DNasel, the R N A was extracted once with an equal volume of phenol/chloroform/isoamylalcohol (25:24:1) and once with an equal volume of chloroform/isoamylalcohol (24:1). Two volumes of 100 % ethanol were added to the final aqueous phase and R N A was precipitated at -20 °C for 20 min. The R N A pellet was collected, washed, dried and dissolved in RNase-free water as described above. R N A samples were stored at -80°C. 5.2.3 RT-PCR analysis The final reverse transcriptase reactions contained 50 m M Tris-HCl (pH 8.3), 75 m M KC1, 3 m M M g C l 2 , 10 m M DTT, 0.5 m M dNTP mix, 0.01 ug/ul random hexamers, 0.5 U/ul RNAguard™ (Amersham Pharmacia Biotech), 10 U/ul M - M L V reverse transcriptase (GibcoBRL) and 0.1 ug/ul R N A . The R N A was first mixed with an appropriate amount of random hexamers and RNase-free water to make a final volume of 12 ul. This mixture was overlaid with 50 ul mineral oil and incubated at 95 °C for 3 min for denaturation, then cooled at 4 °C for 3min. The other components of the reverse 100 transcriptase reaction were added to denatured RNA/primer mixture to make a final volume of 20ul This was then incubated at 37 °C for 60 min for first strand c D N A synthesis. The reaction was stopped by incubating at 90 °C for 5 min and then cooling to 4 °C. One pi of this solution was used to provide c D N A for a 20 u,l PCR reaction. The primers used for RT-PCR are listed in Table 5.2. Primers SCG1 and SCG5 amplify the two spruce CG genes, ScgA and ScgB, but not the pine CG gene. Primers C N L and C N R were designed to specifically amplify the antisense CG, and their target sites within this gene are shown in Figure 5.2. Primers for the histone H3 and (3-tubulin genes were used as amplification controls. Primers HIS1 and HIS2 were designed by Amrita Kumar and targeted conserved regions of the histone H3 gene. The tubl and tub2 primers were obtained from B C Research Inc. and targeted conserved regions of the (3-tubulin gene. The standard P C R reactions contained 1 ul cDNA, 0.4 - 1 . 6 u M left and right primers, 200uM dNTP, 1 x Taq buffer (Boehringer Mannheim) and 0.025 U/ul Taq D N A polymerase (Boehringer Mannheim). The thermal cycling regime was as follows: 1 cycle of 94 °C / 5 min; 34 cycles of 94 °C/30 sec, 56°C/30 sec, 72 °C/lmin; 1 cycle of 72 °C/10 min. The amplification products were separated by 2 % agarose gel electrophoresis, stained with 1 u,g/ml ethidium bromide, viewed under a U V light and photographed using an IS-500 Digital Imaging System (Alpha Innotech Corporation). For quantification, intensity of ethidium bromide staining was analysed using Scion Image software (Scion Corporation). 101 Table 5.2 Primers used for RT-PCR analysis Primer Target gene Sequence 5' 3' Product size SCG1 Spruce CG genes C C C C T A A C A G G A A T T C T G C G 240 bp (cDNA) or 330 bp (competitor) SCG5 A C C A T C G C A G A T T G A A G G A C C N L Antisense CG gene G C T G T G C C G A A C A T G A A A T 329 bp (cDNA) or 235 bp (competitor) C N R A A T GTT T G A A C G A T C G G G G HIS1 Histone H3 A T G GCI CGI A C I A A ( G / A ) CA(G/A) A C I G C 455 bp H1S2 CG(T/G/A) ATI C(T/G)I CGI GCI AG(C/T) TG(T/G/A) A T tubl P-tubulin G T G A C T T G A A C C A T C T G A TCT C 534 bp tub2 C C A TGC CTT C C C C G G T A T A C C A 3 5 S U - C G c D N A N O S • < CNL CNR Figure 5.2 Primers used for detection of antisense CG mRNA For competitive PCR, one ul of an appropriate dilution of competitors, ranging from 10"16 M to 10"14 M , was added to the PCR reaction. For quantification of spruce CG mRNA, a competitor fragment was obtained by PCR amplification of a portion of genomic clone (pSCG18) using the SCG1 and SCG5 primers. This fragment contained an intron and yielded PCR products that were 90 bp larger than those derived from spruce CG cDNA. Since no appropriate competitor was available for antisense CG sequences, a synthetic competitor was generated by adding target primer sequences to an unrelated D N A fragment using P C R (Figure 5.3). The binding sites for C N L and C N R were added to the PCR fragment derived from amplification of ScgA c D N A using SCG1 and SCG5 primers. The resulting fragment contained the C N L and C N R primer binding sites at both ends but 102 the internal region of this fragment did not contain any homologous sequences to the target cDNA. target sequence target sequence ^ PCR amplification Competitor with target primer sequences at the each end Figure 5.3 Strategy for making PCR competitors 5.2.4 Construction of transformation vectors Two plasmids containing partial c D N A sequences of the pine CG gene were obtained from Dr. Palitha Dharmawardhana (1999). Plasmid p F l contained a 0.45 kb fragment of CG c D N A 5' end sequence and plasmid p l A 6 contained 1.8 kb of CG c D N A 3' end sequence with a single nucleotide mutation at the position 438 (A—>G). Full-length CG c D N A clone pCG3 was obtained by fusing these two sequences using a unique restriction site (XmnI) in their overlapping regions (Figure 5.4). Plasmid pF l was digested with restriction enzymes Xba I and Xmn I to obtain a 0.27 kb fragment of CG c D N A 5' end sequence (5'CG). Plasmid p l A 6 was digested with restriction enzymes Xho I and Xmn I to obtain a 1.63 kb fragment of CG c D N A 3' end sequence (3'CG). Plasmid p F l was digested with Xba I and Xho I to obtain a 2.9 kb fragment of linear Bluescript K S vector sequence (BKS). The three fragments, 5 'CG, 3 'CG and B K S , were gel purified using a Qiaex II D N A Purification Kit (Qiagen) and ligated using T4 ligase (GibcoBRL) in a molar ratio of 3:3:2 at 14 °C for 16 hrs. The ligation product was introduced into D H 5 a competent E. coli cells (GibcoBRL) using the heat shock method (Sambrook et al, 1989). 103 White colonies were picked using wooden toothpicks, suspended in 3 ml L B medium and incubated at 37 °C in a rotary shaker (150 rpm) overnight. Plasmid D N A was isolated from actively growing E. coli cells using the mini-prep method of Zhou et al. (1990), and the presence of the desired full c D N A sequence was confirmed by restriction analyses using Xmn I, Kpn I and Dra II. Xba I Xmn I Xmn I I I p F 1 I Xho I ^ p 1 A 6 J<ba^J<mn^ Xho I Full length CG cDNA-p C G 3 Figure 5.4 Generation of a full-length CG cDNA fragment Figure 5.5 presents a schematic diagram of the antisense CG gene construction. Sst I and Xba I restriction sites were added to 5'and 3' ends of CG c D N A sequence using PCR with primers P5 ( A T G A G C T C G G A T T T G G A C C T G A A ) and P6 ( T A T C J A G A C A A T G T T C T T A C C C T G C ) . The thermal cycling regime was as follows: 1 cycle of 94 °C / 5 min; 25 cycles of 94 °C/1 min, 55 °C/2 min, 72 °C/2 min; 1 cycle of 72 °C/10 min. The PCR product was digested with Xba I and Sst I for 24 hrs and the resulting CG c D N A fragment was ligated with the 11 kb fragment of the binary vector pBI121 (Jefferson et al, 1987) obtained by digestion with Xba I and Sst I. This process replaced the GUS coding region of pBI121 and placed the CG c D N A under the control of the CaMV35S promoter in an antisense orientation (pBACG). The ligation product was introduced into E. coli ElectroMAX DH10B™ (GibcoBRL) cells using Cell/Porator 104 (BRL). E. coli strains were screened for the ligated product (pBACG) using plasmid D N A mini-prep procedure (Zhou et al, 1990), followed by restriction analysis using Kpn I. The orientation of the CG sequence in the promoter-terminator cassette was confirmed by restriction analysis using Eco RI and Pst I, and the two ends of the construct were sequenced to confirm the presence of the CG sequence. ^ C G cDNA - »- K X b a I S s t l p C G 3 3 5 S G U S N O S pBI121 | P C R + R E d i g e s t i o n X b a I S s t l X b a I 35S R E d i g e s t i o n S s t I N O S | — . C G c D N A • X b a I S s t l 3 5 S - C G c D N A N O S p B A C G Figure 5.5 Construction of pBACG A GUS fusion gene was added to p B A C G to aid the screening of putative spruce transformants (Figure 5.6). The plasmid pBM113kp (Marcotte et al, 1988) contains the GUS gene under control of the wheat Em promoter. The plasmid was first digested with Eco RI and ligated with an Eco RI - Hind III adapter. For synthesis of the Eco RI - Hind III adapter, two oligonucleotides, A G C T T C C C G G G and A G G G C C C T T A A , were synthesised using an Oligo 1000M D N A synthesizer (Beckman) and their 5' ends were 105 phosphorylated using T4 polynucleotide kinase (New England Biolabs). These oligonucleotides were incubated at 94 °C for 5 min for denaturation. Identical molar amounts of the phosphorylated oligonucleotides were mixed at 94 °C and then annealed by slowly cooling the mixture to room temperature to create the following: Hind III overhangs 5'- A G C T T C C C G G G -3' 3'- A G G G C C C T T A A - 5 ' Eco RI overhangs After the Eco RI-Hind HI adapter was ligated to the ends of the Eco RI digestion fragment of pBM113kp, the fragment was digested with Hind III to isolate the Em-GUS fusion gene (Figure 5.6). This fragment was inserted into the Hind III site between the NPTII and the antisense CG genes of p B A C G to make p B A C G G U S . The presence of the GUS sequence was confirmed by restriction analysis and PCR analysis. Orientation of the Em-GUS fusion gene in relation to the NPTII and antisense CG genes was established by restriction analysis using Eco RI. The functionality of the GUS gene was confirmed by transient expression assay in spruce zygotic embryos using the PDS 1000/He™ device as described in Chapter 3. The complete structure of p B A C G G U S is diagrammed in Figure 5.7. p B M 1 1 3 K p E m p r o G U S 3 5 S te r H i n d H i n d III E c o R l R E d i g e s t i o n + a d a p t e r E c o R l H i n d III NPTII I A n t i C G Hincl III p B A C G G U S Figure 5.6 Construction of pBACGGUS N O S p ro NPTII NosV / i i s t e r / \ ter G U S E m p ro 3 5 S p ro a n t i C G N O S \ te r L B pBACGGUS R B K a n R Or i Figure 5 .7 Structure of pBACGGUS 107 5.2 5 Generation of transgenic spruce Plasmid p B A C G G U S was introduced into embryogenic spruce line 11026 using the microprojectile bombardment apparatus, and transgenic cell lines were established as described in Chapter 3. Transgenic plants were recovered from embryogenic tissues via somatic embryogenesis as described in Chapter 3. 5.2.6 Protein extraction Plant tissues were ground into a powder in liquid nitrogen. The powder was transferred to a 15 ml plastic centrifuge tube, to which cold protein extraction buffer (2 ml per 1 g fresh weight; 50mM M E S , 3mM DTT, pH 6.0) containing insoluble polyvinylpolypyrrolidone (O.lg/ g fresh weight) was added. The contents of the tube were mixed well using a spatula and vortexed briefly before incubation on a shaker at 4 °C for 30 min. The slurry was filtered through three layers of K i m Wipe tissue to remove debris and the resulting filtrate was transferred to an Eppendorf tube. The remaining debris in the filtrate was removed by centrifugation (17,000 g; 4 °C; 10 min). The protein concentration in the supernatant was determined according to the method of Bradford, using a Protein Assay Kit (Bio-Rad, Mississauga, Ontario) and bovine serum albumin as a standard. The protein extracts were used immediately for enzyme assay, while other portions of the extracts were stored at -80°C and used later for SDS-PAGE analysis. 5.2.7 Coniferin glucosidase assay Fifty pi fresh protein extract was mixed with 100 pi reaction mixture (3 m M coniferin in 0.2M M E S , pH 5.5 buffer) and incubated at 30°C for 30 min. The reaction was stopped by basification of the assay mixture with an equal volume of 0.5 M CAPS, pH 108 10.5 to convert the released aglycone to its ionized form. Product yield was measured by determining the absorbance of the released aglycone at 325 nm, using a molar extinction coefficient (e) of 7 mM" 1 x cm"1. 5.2.8 Western blotting Rabbit anti-CG antiserum was obtained from Dr. Palitha Dharmawardhana. The antiserum had been raised against recombinant pine C G protein and purified by affinity chromatography. SDS-PAGE was conducted according to the standard Laemmli (1970) procedure, in 12% polyacrylamide gels. BioRad broad range molecular weight marker was used as molecular weight standards. After SDS-PAGE, proteins were electroblotted onto nitrocellulose membranes. The blots were pre-treated in blocking solution (5 % w/v non-fat milk powder, 100 m M Tris-HCl pH 7.5, 150 m M NaCl, 0.1 % Tween20) for 2 hours, incubated in a 1/300 dilution of primary antibody (rabbit anti-CG antibody) in blocking solution for 1 hour, washed three times (15 min each) in blocking solution, incubated in a 1/2500 dilution of secondary antibody (goat anti-rabbit antibody conjugated with alkaline phosphatase) for 1 hour, washed three times (15 min each) in blocking solution and rinsed briefly in TBS (100 m M Tris-HCl pH 7.5, 150 m M NaCl). Detection of the secondary antibody was performed using a BCIP/NBT detection system (Gibco B R L ) following the manufacturer's instructions. For molecular weight estimation, the molecular weight marker lane was cut off after blotting and stained with Coomassie Brilliant Blue (Sambrook et al, 1989). In addition, a duplicate blot was stained with Coomassie Brilliant Blue to check for equal loading of protein. 109 5.2.9 Statistical analysis When statistical analysis was necessary, an analysis of variance ( A N O V A ) was performed using SigmaStat 2.0 (Jandel Corporation). A multiple comparison test (Dunnett's test) was performed to indicate treatments that are different from the control group. 5.3 Results 5.3.1 Structure and expression of the spruce CG gene 5.3.1.1 PCR cloning In order to confirm the presence of orthologous CG sequences in the spruce genome, P C R was performed using primer sets designed according to the pine CG c D N A sequences. The primers P C G l a and PCG2a were designed to amplify a target region near the N-terminal region of the pine CG c D N A with an expected product size of 0.35 kb (Table 5.1, Figure 5.1), which included a part of the signal peptide sequence (Figure 5.1). PCR amplification using these primers yielded a single band with an approximate size of 0.7 kb (Figure 5.8A). The fragment size was larger than expected, possibly due to introns in the genomic sequence. The primers PCG3a and PCG4a were designed to amplify a 3'end region containing the C-terminus and non-coding sequence of the pine CG cDNA, with an expected size of 0.2 kb (Table 5.1, Figure 5.1). PCR amplification using these primers yielded a single band with an approximate size of 0.2 kb (Figure 5.8A). The primers PCG5a and PCG6a were designed to amplify the region near the C-terminal end of the pine CG coding sequence, with an expected product size of 1.0 kb (Table 5.1, Figure 5.1). This region included the two highly conserved active site residues of family 1 (3-glucosidases (Figure 5.1). PCR amplification using these primers yielded 1 1 0 two faint bands with approximate sizes of 1.4 kb and 2.0 kb (Figure 5.8A, B). Southern hybridization of these bands using a pine CG c D N A probe showed that both PCR fragments were homologous to the pine CG sequence (data not shown). Therefore, the two PCR bands may represent two CG genomic sequences with different intron sizes. PCR amplification was also performed using primers PCG5a and PCG4a, which yielded a single band with an approximate size of 1.6 kb. Since PCG4a should bind approximately 0.2 kb downstream of the PCG6a site, this 1.6 kb fragment was probably derived from the same gene that gave a 1.4 kb fragment after amplification using PCG5a and PCG6a. In order to further characterize spruce CG sequences, the PCR fragments amplified using primers PCG5a and PCG6a were cloned into a Bluescript SK vector using the T/A cloning method (Holton and Graham, 1991). This yielded one 1.4 kb PCR clone and two identical 2.0 kb PCR clones. The genes represented by these fragments were termed ScgA and ScgB, respectively. To obtain more sequence information, the 1.6 kb-PCR fragment amplified using primers PCG5a and PCG4a was cloned into the TOPO cloning vector using the TOPO T A Cloning® kit (Invitrogen). Sequence analysis of two of the resulting clones confirmed that they were derived from ScgA. Figure 5.8 PCR amplification of spruce C G sequences PCR was performed using Taq D N A polymerase (A) or Accurase™ (B) as described in Materials and Methods. The figure is a negative image of the ethidium bromide-stained agarose gel. A. PCR amplification using Taq D N A polymerase. PCR reactions were performed in a Techne3 thermal cycler. Ten ng of the plasmid p B A C G G U S was used as a template in lanes 1 to 3. p B A C G G U S contained the pine c D N A sequence and, therefore, served as positive control. Two hundred and fifty ng of interior spruce genomic D N A was used as a template in lanes 5 to 7. Lane 1: 1 Kb D N A ladders. Lanes 2 and 5: PCR amplification using primers P C G l a and PCG2a. Lanes 3 and 6: PCR amplification using primers PCG3a and PCG4a. Lanes 4 and 7: PCR amplification using primers PCG5a and PCG6a. B. P C R amplification using Accurase™. P C R reactions were performed in a PTC 100 thermal cycler (MJ Research). Lane 1: 1 Kb D N A ladder. Lane 2: PCR amplification using primers PCG5a and PCG6a. Lane 3: PCR amplification using primers PCG5a and PCG4a. 0.5 -B 113 5.3.1.2 Intron structure of spruce CG genes The larger sizes of genomic spruce CG P C R fragments relative to the pine CG c D N A suggested the presence of intron(s) in these spruce sequences (Figure 5.8). In order to determine the intron structure of the spruce CG genes, intron-exon junctions of the ScgA and ScgB were predicted using Arabidopsis splice site prediction software available at NetGene2 World Wide Web Server (http://www.cbs.dtu.dkyservices/NetGene2/) (Hebsgaard et al, 1996). The predicted exons were also confirmed by aligning the genomic ScgA and ScgB sequences against the pine CG c D N A sequence using BLAST2 Software (Tatusova and Madden, 1999) at the N C B I web server (http://www.ncbi.nlm.nih.gov/gorf/bl2.html). Both ScgA and ScgB contained five introns at the same positions (Figure 5.9). The two genes were very similar in their exon regions (See analysis below) but not in their intron regions. The introns of ScgB were larger than those in ScgA, thus contributing to the larger size of the ScgB PCR fragment (Figure 5.8 and 5.9). The fourth intron of ScgB was the largest and spanned 603 bp. A l l of the splice site sequences of ScgA and ScgB fit G T - A G U2-dependent consensus borders, where GT refers to intron residues at the 5' splice site and A G refers to intron residues at the 3' splice site (Simpson and Filipowicz, 1996). 114 5' 3' .NEP. . . • 5a . . .ENG. . •4 6a Lodgepole pine CG cDNA (0.9 kb) ScgA (1.4 kb) ScgB (2.0 kb) E I I e x o n ^ ™ i n t r o n Figure 5 .9 Intron structure of spruce CG sequences 5.3.1.3 Sequence homology of C G genes In order to compare the spruce and pine C G sequences, the exons of ScgA and ScgB were assembled and their deduced amino acid sequences were predicted from the resulting cDNA sequences. Alignment of the deduced amino acid sequences revealed high similarity between spruce and pine CG peptides throughout the sequenced region (Figure 5.10). ScgB was more similar to the pine CG, with amino acid and nucleotide identities of 88 % and 89%, respectively (Table 5.3). The amino acid and nucleotide identities between ScgA and the pine CG were 85 % and 86 %, respectively (Table 5.3). Conserved regions include the sequence motifs NEP and E N G at residues 190-192 and 408-410 of the pine CG, respectively (Figure 5.10). These motifs are conserved among familyl (i-glycosidases and their acidic residues are believed to be essential for enzyme activity (Baird et al, 1990; Withers et al, 1990, Ly and Withers, 1999). Studies using site-directed mutagenesis of Agrobacterium P-glucosidase (Abg) have shown that the Glu residue in the E N G motif is 115 the active-site nucleophile (Withers et al, 1990; Trimbur et al, 1992; Withers et al, 1992). The Glu residue in the NEP motif is proposed to be the acid/catalyst of Abg (Ly and Withers, 1999). The Tyr residue 72 residues downstream of the E N G motif, which assists in hydrolysis of the glycosyl-enzyme intermediate in Abg (Ly and Withers, 1999), is also conserved in spruce CG genes. Other conserved residues in ScgA and ScgB include the two potential N-glycosylation sites (residues Asn223 and Asn447 of the pine CG) (Dharmawardhana et al, 1999) and two cysteine residues (positions 210 and 219 of the pine CG) (Figure 5. 10). A B L A S T search in the ScgA and ScgB confirmed their similarity to other members of the family 1 (3-glycosyl hydrolases. Not surprisingly, ScgA and ScgB score their highest similarity to the lodgepole pine CG sequence. Other genes that exhibit high similarity (up to 45 % of sequence identity) to spruce CG include cyanogenic [3-glucosidase and glucoalkaloid P-glucosidase genes from several plant species, and several putative P-glucosidase genes from Arabidopsis (Table 5.4). A phylogenetic tree of these glucosidase genes was constructed using P A U P software (Figure 5.11). Interestingly, three Arabidopsis genomic sequences (AC004392A, AC004392B and AL161555) formed a cluster with the three CG sequences. ] 16 WATMNEPNLFVPMGYTVGIYPPTRCSAPjggNSAC ITGN 3SSSEPYLAAHHVLLAHASAVE WATMNE PNL FVPLGYTVGIFP PTRCTAPHGNSACITGNSSSSE PYLAAHHVLLAHASAVE WATVNEPNLFVPLGYTVGIFPPTRC0APHANBICMTGN iSSAEPYLAAHHVLLAHASAVE KYREKYQKIQGGGLGGVMSAPWYEPLEDSPEEGSAVDRILSFNLRWFLDPIVFGDYPREM KYREKYQKIQ^GSIGLVMSAPWYEPLEDSPEERSAVDRLGSFNLRWFLDPIVFGDYPREM KYREKYQKIQGGSIGLVTSAPWYEPLEHJSPEERSAVDRILSFNLRWFLDPIVFGDYPBEM ScgA ScgB PCG 1 1 186 ScgA ScgB PCG 61 61 246 ScgA 121 ScgB 121 PCG 306 REEVGSRLPSISSDLSGKLRGSFDYIGINHYTTLYATIJJTPTRSP REJVGSRLPSISJJELSAKLRGSFDYLGINHYTTLYATSTPTRSPD RESLGSRLPSISSELSAKLRGSFDYMGINHYTTLYATSTPBBSPD ScgA ScgB PCG 181 181 366 ERHGVPIGERTGMDGLYMVPgGI J£KIVEYVKgFYgNPAI11TENGYgES ERHGHPIGERTGMDGLYWPHGIQKlVEYVKEglYDNPAI 11TENGYPDS ERHGvHlGERTGMDGLFVVPHGIQKIVEYVKEFYDNpRlII0ENGYPES Etaaataiii NDVRRIRFHGDgLSYLAAAIRNGSDVRGYFVWSLLDNFEWAJJGYTIRFGLYHVDIISDQK NDVRRIRFHGDCLSYLAAAIHNGSDVRGYFVWSLLDNFEWAFGYTIRFGLYHVDlBsDQK NDVRRIRFHGDCLSYLSAAIKNGSDVRGYFVWSLLDNFEWAFGYTIRFGLYHVDglSDQK ScgA 241 ScgB 241 PCG 426 ScgA 301 RYPK LSAQWFT ScgB 301 RYPK LSAQWFT PCG 486 RYPK ScgA 312 IQFI J Q R | DQGSIRSS PCG 497 IQFI .QHB DQGSIRSS B. Figure 5.10 Alignment of deduced amino acid sequences of the spruce and pine CG genes A. Alignment at amino acids 186 - 496 of lodgepole pine CG (PCG, accession number AF072736). Amino acid sequences of ScgA and ScgB were predicted from nucleotide sequences of three ScgA PCR clones and one ScgB PCR clone. Asterisks and black dots indicate conserved active site residues (NEP, E N G , and Tyr 336) and potential N -glycosylation sites, respectively. B. Alignment at C terminal ends (497 - 513) of PCG. C-terminal sequence of ScgA was predicted from a 1.6 kb PCR clone of ScgA. 117 Table 5.3 Pairwise comparison of three C G genes A . Amino acid sequence identity in percentage for ScgA, ScgB and residues 186 - 496 of lodgepole pine CG. Pine CG ScgA ScgB Pine CG ScgA 85 88 89 B. Nucleotide identity in percentage for ScgA, ScgB and pine CG, corresponding to amino acid residues 186 - 496 of the lodgepole pine CG. Pine CG ScgA ScgB Pine CG ScgA 86 89 88 118 Table 5.4 Selected list of genes that are homologous to ScgA and ScgB B L A S T search was performed on Sept 6, 2000. Accession number Organism (common name) Gene product (function) AF072736 Pinus contorta (Lodgepole pine) Coniferin P-glucosidase (lignin synthesis) U50201 Prunus serotina (black cherry) Prunasin hydrolase (cyanogenic P-glucosidase) AF221526 Prunus serotina Prunasin hydrolase (cyanogenic P-glucosidase) U26025 Prunus serotina Amygdalin hydrolase (cyanogenic P-glucosidase) U39228 Prunus avium (sweet cherry) P-glucosidase AF163097 Dalbergia cochinchinensis (Thai rosewood) Dalcochinin P-glucosidase (isoflavonoid metabolism) AF149311 Rauvolfia serpentina Raucaffricine p-glucosidase (alkaloid metabolism) D83177 Costus speciosus Furostanol glycoside P-glucosidase (triterpene metabolism) S35175 Manihot esculenta (cassava) Linamarase (cyanogenic p-glucosidase) AF112888 Catharanthus roses Strictosidine P-glucosidase (alkaloid metabolism) U28047 Oryza sativa (rice) P-glucosidase (unknown function) X56734 Trifolium repens (white clover) P-glucosidase (unknown function) AC004392 (2 genes)*, AC004521 (5 genes)*, AB024024*, AB023032*, AL161555*, AC006053*, AB020749 (2 genes)* Arabidopsis thaliana Putative P-glucosidase (unknown function) *- Genomic sequences. 119 Arabidopsis AC004521B - Arabidopsis AC004521C Arabidopsis AC004521E 100 85 50 changes 100 72 100 94 — Pinus contorta AF072736 ScgB - ScgA - Arabidopsis AC004392A — Arabidopsis AC004392B Arabidopsis AL161555 99 100 I Arabidopsis AB020749A Arabidopsis AB020749B Oryza sativa U28047 70 100 - Arabidopsis AB023032 Arabidopsis AB024024 Arabidopsis AC004521A 100 I— Prunus avium U39228 89 94 1— Prunus serotina U50201 — Prunus serotina AF221526 81 " Prunus serotina U26025 Dalbergia cochinchinensis A F 163097 Rauvolfia serpentina AF149311 Catharanthus roses AF112888 Costus speciosus D83177 — Arabidopsis AC004521D — Manihot esculenta S35175 Trifolium repens X56734 Figure 5.11 Phylogenetic tree of p-glucosidase genes Amino acid sequences of the genes listed in Table 5.4 were truncated to make fragments corresponding to the residues 186-496 of pine CG. Truncated sequences were aligned using the PILEUP program (gap creation penalty: 12; gap extension penalty: 1) of G C G (see Appendix A for the alignment). A phylogenetic tree was constructed using P A U P software. The bootstrap method with heuristic search was used with the following options: 100 replications, random stepwise addition of taxa, tree bisection and reconnection branch swapping. 120 5.3.1.4 Expression of the spruce CG genes during seedling development Changes in the levels of CG expression were studied during spruce seedling development. Primers SCG1 and SCG5 were designed to amplify both ScgA and ScgB sequences and used for RT-PCR analysis. RT-PCR of spruce m R N A using primers SCG1 and SCG5 yielded a single band of 240 bp (Table 5.2, Figure 5.12A). Since the efficiency of c D N A synthesis could vary among different R N A preparations, the histone H3 gene was used as a reference gene for RT-PCR analyses. After first strand c D N A synthesis using random hexamers, PCR amplification was performed using two sets of primers, ones for the CG gene (SCG1 and SCG5) and ones for the histone H3 gene (HIS1 and HIS2). This yielded two PCR products of 240 bp (CG) and 450 bp (histone), respectively (Figure 5.12A). The ratios of intensity of the histone and the CG bands were used to compare expression levels of the CG gene in different tissues (Figure 5.12B). CG m R N A was barely detectable in one week-old seedlings, but increased as the seedlings grew older (Figure 5.12A and B). A similar pattern of CG expression was observed when the p-tubulin gene was used as a reference gene (data not shown). The increase in CG m R N A coincided with the degree of lignification in the spruce seedlings, as detected histochemically. The results of lignin staining of these seedlings are shown in Figure 5.13. One week-old spruce seedlings do not contain any lignified tissues (data not shown). One month-old seedlings contained two to three vascular bundles with lignified xylem tissues -in hypocotyls (Figure 5.13A), similar to those seen in one month-old germinants from somatic embryos (Chapter 4). The beginning of secondary xylem development was detected in hypocotyls of two month-old seedlings (Figure 5.13B) and secondary xylem was well developed by the time seedlings were three months old (Figure 121 5.13C). Epidermal cells of these seedlings also stained positively for lignin (Figure 5.13B and C). Figure 5.12 RT-PCR analysis of the C G gene expression during germination of spruce (next page) White spruce seeds were germinated on wet filter papers at 25 °C in the darkness. They were transferred to soil when they were four weeks old and incubated in a growth chamber at 25°C with a 16h light/8h dark photoperiod. R N A was extracted from actively growing embryogenic tissues (callus); from 1, 2, 4, or 14 week-old spruce seedlings; or from stems of 6 month-old emblings (mature stems). RT-PCR was performed as described in Materials and Methods. PCR reactions were performed using 0.4 u M each of HIS1, HIS2, SCG1 and SCG5 primers. A. Negative image of ethidium bromide-stained agarose gel is shown. The upper (455 bp) and lower bands (240 bp) correspond to the histone H3 and the CG genes, respectively. B. The intensity of the histone and CG bands was quantified using Scion Image software and their ratio is shown in the graph. Values are the mean of four RT-PCR reactions ± SE. 123 Figure 5.13 Xylem development of spruce during germination White spruce seeds were germinated and grown as described in Figure 5.12. Stem sections were taken from one (A), two (B) or three (C) month-old seedlings and stained for lignin using phloroglucinol/HCl (5% in an ethanol/HCl mix 9:1). 12LT 125 5.3.2 Effects of the antisense CG mRNA on development of transgenic spruce 5.3.2.1 Generation and growth of transgenic spruce In order to test potential role of C G in the lignin biosynthetic pathway, a tri-functional plant transformation vector (pBACGGUS) was constructed to direct expression of an antisense version of the CG gene. Construction of p B A C G G U S is described in Materials and Methods. Briefly, the lodgepole pine CG c D N A was fused to the CaMV35S promoter in an antisense orientation by using pine CG to replace the GUS coding region of pBI121 (Jefferson et al, 1987; Figure 5.5). In order to facilitate visual screening of transgenic tissues (Chapter 3), the wheat Em-GUS fusion gene from pBM113kp (Marcotte et al, 1988) was inserted between the NPTII gene and the antisense CG gene (Figure 5.6). The wheat Em promoter directs strong expression of the GUS gene in conifer embryogenic tissues (Duchesne and Charest 1992). The resulting plasmid, p B A C G G U S , thus contained three fusion genes; the antisense coniferin (3-glucosidase gene under control of CaMV35S promoter, the NPTII gene under control of the NOS promoter, and the wheat Em-GUS fusion gene (Figure 5.6). Plasmid p B A C G G U S was introduced into embryogenic spruce line 11026 and transgenic cell lines were established as described in Chapter 3. A total of forty-six transgenic lines were recovered that contained the antisense CG sequence (Table 3.8). Twenty-eight to fifty-six transgenic plants were generated via somatic embryogenesis from each of the lines except the line 5F 23.5. This line exhibited severely reduced embryogenic potential and yielded only three plants from more than 50 grams of embryogenic tissues. By comparison, five grams of embryogenic tissues typically yield more than 100 somatic embryos. The low embryogenic potential could be due to somaclonal variation caused by 126 prolonged exposure to growth regulators and/or kanamycin in the culture medium or due to a mutation caused by insertion of the introduced D N A into the spruce genome. Transgenic plants were transferred to soil in September 1998, January 1999 or March 1999. They were grown in a plastic tunnel greenhouse without supplemental light. Those planted in September 1998 were grown through one cycle of dormancy, while those planted in January 1999 or March 1999 grew continuously through the summer. The height of each transgenic plant was measured in August 1999 (Figure 5.15). Although most lines did not differ significantly from control plants in overall appearance and growth rate, growth rates were significantly reduced in six transgenic lines (G12J 5.6, 30F 23.5, 34C 6.2, 28J 3.4, 10A 3.4, 52A 3.4; Figure 5.14 and 5.15). In particular, line 30F 23.5 exhibited severe growth inhibition, which was apparent both in the roots and shoots (Figure 5.14C). The growth inhibition of line 30F 23.5 became apparent only after the seedlings broke dormancy. This inhibition is also reflected in the results of lignin staining of stem sections (Figure 5.16). First- and second- year growth can be compared in sections taken from stems just below the first branch (Figure 5.16A and B). Notable growth inhibition was seen in second-year growth but not in first-year growth. Sections taken from stems just above the first branch represent only second-year growth (Figure 5.16C and D). These sections revealed a significant reduction in stem diameter and uneven lignin staining in the line30F 23.5. Lignin staining of stem sections revealed unusual xylem development in the line 28J 3.4. Although the older part of xylem appeared to be normal, the new xylem was greatly distorted (Figure 5.16F). Xylem cells were uneven in shape and size, and were not arranged linearly. Some appeared collapsed. The secondary cell walls were thinner and less lignified than in control plants (Figure 5.16G and H). Aside from lines 30F 23.5 and 127 28J 3.4, the other transgenic lines did not display obvious changes in xylem development or a reduction in lignin staining. Stem sections of two other lines are shown in Figure 5.16E and I. Figure 5.14 Growth of transgenic spruce plants (next page) Plants were collected in May 1999 and washed thoroughly with water to remove soil. A - C . Lines planted in September 1998. A . Non-transgenic control. B. Line G12J 5.6. C. Line 30F 23.5. D and E. Lines planted in March 1999. D. Non-transgenic control. E. Line 52A 3.4 Lines planted in March 1999 Figure 5.13 Growth of transgenic spruce plants 129 Figure 5.15 Height comparison of transgenic spruce plants Transgenic spruce lines were transferred to soil in September 1998 (A), January 1999 (B) or March 1999 (C). Height of ten randomly selected plants was measured in August 1999. The values are the mean of ten measurements ± SE. Non-transgenic control plants were also planted on each planting date. The line A 1 K 5.6 is a transgenic control that contained the NPTII and GUS genes but not the antisense CG gene. Transgenic and non-transgenic controls are shown as solid bars. Asterisks indicate the lines that are significantly different from controls at P<0.05. 130 Height (cm) Height (cm) 03 5" 3 ft a • s -131 Figure 5.16 Lignin staining of transgenic spruce plants Hand-sections were made from plants harvested either in July 1999 (A-F) or in September 1999 (G-I). The sections were stained for lignin using phloroglucinol/HCl (5% in an ethanol/HCl mix 9:1). Sections A to E were taken from plants planted in September 1998. For A , B and E, hand-sections were made through stems just below the first branch. These sections contain the previous year's growth as well as the current year's growth. For C and D, sections were made through stems just above the first branch, which only contain the current year's growth. A , C. Non-transgenic control. B, D. Line 30F 23.5. E. Line G12J 5.6. Sections F to I were taken from plants planted in January 1999. F. Line 28J 3.4 harvested in July 1999. G. Non-transgenic control. H . Line 28J 3.4. I. Line 1H 6.2. Bars = 500 um (A - F) or 60 um (G-I). 133 5.3.2.2 C G enzyme activity and C G protein levels in transgenic spruce To examine the effects of the antisense CG gene on C G protein, the activity of the C G enzyme was measured in the transgenic spruce lines. In order to select tissues appropriate for C G assays, preliminary experiments were performed using embryogenic tissues, mature somatic embryos, two month-old germinants and young stems of 6 month-old seedlings. I was unable to assay C G activity in either two month-old germinants or stems of 6 month-old seedlings, even though these tissues exhibited a higher level of CG gene expression (Figure 5.12). This was primarily due to the low level of total protein in these tissues. Less than 1 pg total protein was obtained from 500 mg of ground tissues. The assay was also confounded by phenolic compounds in the extract, which gave a high background in the C G assay solution, making it impossible to detect low levels of enzyme activity. Addition of PVPP to the protein extraction buffer was not sufficient to remove these phenolic compounds. Attempts to detect C G protein in these extracts using western blotting also failed. Surprisingly, although they were not actively lignifying, embryogenic tissues yielded a high level of C G activity (0.58 pKat/u.g protein). Therefore, C G enzyme assays were performed using actively growing embryogenic tissues of 30 antisense C G lines, and 7 transgenic and non-transgenic control lines. Transgenic control lines contained the NPTII and GUS genes but not the antisense CG gene. The coniferin hydrolysing activity detected in embryogenic tissues varied greatly among different cell lines. Enzyme activity ranged from 0.18 to 4.23 pKat/pg protein among antisense CG lines, 0.44 to 1.85 pKat/u.g protein among transgenic control lines and 0.58 nKat/mg protein for the non-transgenic control. Due to the high variation in C G 134 activity among the control lines, none of the antisense CG lines exhibited a significant difference from the transgenic control lines. In order to further examine C G protein levels in embryogenic tissues, protein extracts from selected lines (two high C G activity lines and three low C G activity lines) were analysed by western blotting using antibodies raised against recombinant pine C G protein (Dharmawardhana et al., 1999). Protein extracts from the non-transgenic control exhibited two bands with approximate sizes of 55 and 24 kDa (Figure 5.17). The 55 kDa protein corresponds to the size of mature lodgepole pine C G protein (Dharmawardhana et al, 1999), while the 24 kDa protein may correspond to a cleavage product of the 55 kDa protein. Dharmawardhana et al. reported two cleavage products with sizes of 24 kDa and 28 kDa derived from the 55 kDa C G protein (1995). A 28 kDa protein can also be seen as a weak signal in wild-type embryogenic tissues (Figure 5.17). The 55 and 24 kDa protein bands sometimes appeared as doublets (Figure 5.17). This may be due to variations in posttranscriptional modification of the C G protein, as spruce and pine C G sequences both contain two potential N-glycosylation sites (Figure 5.10). Extracts of the transgenic line 5F 23.5 exhibited stronger doublet signals of both the 24 kDa and 55 kDa proteins on western blots, while the transgenic line 341 6.2 exhibited stronger single band signals at 55 and 24 kDa. On the other hand, the intensity of C G bands in transgenic lines 45D 6.2, 34C 6.2 and 3IB 6.2 did not differ significantly from those in wild-type cells. Overall, there was no correlation between C G protein levels on western blots and coniferin hydrolysing activity. For example, the protein extract of line 341 6.2 exhibited low C G activity although western blotting detected more C G protein in the extract (Figure 5.17). Similarly, although protein extracts of lines 45D 6.2, 34C 6.2, 3 IB 6.2 and wild type contained about same amount of C G protein, C G enzyme activity in 135 these extracts varied from 0.42 to 2.78 pKat/ug protein (Figure 5.17). The lack of correlation between C G protein levels and C G activity, together with the fact that embryogenic cells do not contain lignified cell walls suggest that the C G activity measured in embryogenic tissue extracts may be due to the presence of other glucosidases not necessarily involved in lignin biosynthesis. Since the purpose of this study was to detect C G activity specifically associated with lignification, glucosidase activity in embryogenic tissues was not studied any further. Figure 5.17 Western blotting analysis of protein extracts from embryogenic tissues of transgenic spruce (next page) Proteins were extracted from actively growing embryogenic tissues (10 days after subculture) and C G enzyme assays were performed as described in Materials and Methods. SDS-PAGE and western blotting were performed as described in Materials and Methods. Each lane contained 10 ug of total proteins. Lines 341 6.2 and 45D 6.2 (indicated by asterisk) were transgenic controls and contained only the NPTII and GUS genes but not the antisense CG gene. Lines C G activity pKat/ug protein 55 kDa — • CD C L 5F23.5 341 6.2* 45D 6.2* r N ( ^ 0.38 0.18 0.35 0.38 0.33 0.35 0.42 2.78 28 kDa 24 kDa 0 CL Lines C G activity pKat/ug protein 55 kDa — • 45D 6.2* 34C 6.2 \ ( 1.57 1.13 31B6.2 1.75 2.48 2.28 0.42 \ 0.67 0.22 28 kDa 24 kDa 137 5.3.2.3 Detection of sense and antisense CG m R N A in transgenic spruce Since it was not possible to study C G enzyme activity in spruce seedlings (see above), assessment of CG gene expression in transgenic spruce had to rely upon mRNA quantification. The abundance of sense CG mRNA was compared between transgenic plants using RT-PCR. After first strand c D N A synthesis using random hexamers, PCR was performed using two sets of primers, one set specific for the spruce CG genes (SCG1 and SCG5) and one set specific for the histone H3 gene (HIS1 and HIS2). The ratios of intensity of the specific band to that of the histone band were compared as described above. Control PCR reactions were performed in order to test the specificity of primers SCG1 and SCG5. Primers SCG1 and SCG5 amplified spruce CG sequences but not the pine CG sequence, the sequence from which the antisense CG construct was derived (data not shown). Any CG PCR bands would therefore be derived from the sense spruce CG mRNAs, not from antisense pine CG mRNAs. A comparison of sense CG mRNA levels among different transgenic lines is shown in Figure 5.18. No significant reduction in the levels of endogenous CG m R N A was observed in any of the transgenic lines, including the two lines that exhibited abnormal development (30F 23.5 and 28J 3.4). Levels of antisense mRNA were also compared using the RT-PCR approach described above. Primers C N L and C N R were designed to amplify only the antisense CG transcripts and then used for RT-PCR analysis. Antisense transcript levels varied greatly between transgenic lines (Figure 5.19). The highest level was detected in line 1H 6.2, which showed an antisense CG I histone ratio of 4.82. Other lines exhibited much lower expression levels, with antisense CG I histone ratios of less than 1. No antisense CG bands were detected in three lines (G1A 5.6, G15N 5.6 and 33A 6.2). There was no correlation 138 between antisense and sense CG mRNA levels within individual transgenic lines, nor was there any correlation between antisense mRNA levels and growth response. The failure of the antisense CG construct to inhibit endogenous spruce CG mRNA accumulation could possibly be attributed to several factors, one of which may be low expression of the antisense CG gene. Since the RT-PCR antisense bands were much weaker than the histone bands in most lines, it was necessary to add four times more antisense CG primers than histone primers (see the legend of Figure 5.19). This might reflect weak expression of the antisense CG gene, or it could be due to weak binding of the antisense CG primers to their target sites, since different primers exhibit different affinities for their binding sites. In order to clarify this point and to more rigorously quantify antisense and sense CG m R N A levels, competitive RT-PCR was performed using mRNA extracts from three of the transgenic lines (lines 30F 23.5, G12J 5.6 and 1H 6.2). Artificial D N A competitors were designed to contain the same primer binding sites as the target sequence and these were then added to the PCR reactions. The amount of specific cDNA in the sample can be estimated by establishing the competitor concentration that yields equal amounts of the PCR products from the competitor and the target sequence (Figure 5.20Aand B). The level of antisense CG mRNA was very low in lines 30F 23.5 and G12J 5.6, and was only one-third the level of the sense CG mRNA (Table 5.5). This confirmed the low expression of the antisense CG genes detected earlier in most of the transgenic lines (Figure 5.19). By contrast, line 1H 6.2 exhibited much higher expression of the antisense CG gene than of the endogenous CG gene. As analyzed by competitive PCR, the accumulation of antisense CG m R N A was 17-fold higher than sense CG m R N A (Table 5.5), yet the 139 presence of such an excess amount of antisense m R N A had no apparent effect on the accumulation of sense mRNA. Figure 5.18 Levels of the sense CG mRNA in transgenic spruce as detected by RT-PCR (next page) Transgenic spruce lines were transferred to soil in September 1998 (A), January 1999 (B) or March 1999 (C). Plants were harvested in August 1999 and R N A was extracted from stem samples as described in Materials and Methods. RT-PCR was performed using 0.4 u M each of HIS1, HIS2, SCG1 and SCG5 primers. The SCG1 and SCG5 primer set amplifies the endogenous spruce CG sequence but not the antisense CG sequence. The intensity of the CG band is shown relative to that of the histone band. Values presented are the mean of four RT-PCR reactions ± SE. Non-transgenic control plants were planted on each planting date. The line A 1 K 5.6 was a transgenic control and contained the NPTII and GUS genes but not the antisense CG gene. Transgenic and non-transgenic controls are shown as solid bars. In none of transgenic lines was the level of sense CG m R N A significantly different from that detected in control plants. 140 sense C G / histone o o &v - * s a 5' — s B 65 *% *4 I—» SO SO 3 63 S a 5' 6S "1 O SO sense C G / histone o control a G12J5.6 05 « 15C23.5 g . G15N5.6 re *« S G1A5.6 so 00 sense C G / histone 44I 3.4 control 141 Figure 5.19 Levels of the antisense CG mRNA in transgenic spruce as detected by RT-PCR Transgenic spruce lines were transferred to soil in September 1998 (A), January 1999 (B) or March 1999 (C). Plants planted in September 1998 were grown through one cycle of dormancy in a plastic tunnel greenhouse. Plants were harvested in August 1999 and R N A was extracted from stem samples as described in Materials and Methods. RT-PCR was performed using 0.4 u M each of HIS1 and HIS2 primers, and 1.6 p M each of C N L and C N R primers. Intensity of the antisense CG band is shown relative to that of the histone band. Values are the mean of four RT-PCR reactions ± SE. Asterisks indicate lines with no detectable antisense CG bands. IM-Z Antisense C G / histone GO 65 3 re a 3 B M *<: h- ' 1H6.2 43B 3.4 3A 3.4 48D3.4 HH 21A 3.4 p.^HH 28J 3.4 | ^ H H 34M6.2 38M 6.2 Eyi I 40D 3.4 JUIH 44A3.4 10C 3.4 16E 6.2 41B6.2 44B 6.2 4B 3.4 38B 3.4 10A3.4 23E 6.2 34 J 6.2 38H 6.2 39D 6.2 12D 6.2 310 6.2 34C6.2 39A3.4 42E 3.4 41E6.2 36G 3.4 31B6.2 33A 6.2 H—I Antisense C G / histone 3 re a G12J 5.6 CM 30F23.5 re ro 3 G19Q5.6 3^  re S G19J 5.6 1 00 M 15C 23.5 G1A5.6 G15N 5.6 Antisense C G / histone 3- 48C 3.4 ro a. 52A 3.4 &9 ^ 30B 3.4 441 3.4 42G 3.4 28C 3.4 21C 3.4 143 Figure 5.20 RNA quantification by competitive RT-PCR RT-PCR reactions were performed as described in Materials and Methods. For quantification of antisense CG cDNA, PCR was performed using 0.4 u M each of C N L and C N R primers. One u.1 of an appropriate dilution of competitor was added to 20 pi PCR reaction (See Materials and Methods for description of competitor). A . Negative image of ethidium bromide-stained agarose gel. The upper and lower bands correspond to antisense CG and competitor bands, respectively. B. The intensity of the antisense CG and competitor bands was quantified using Scion Image software. The intensity of competitor was multiplied by the ratio of the fragment size of antisense CG band to that of competitor band. This is to correct for staining the intensity difference related to the size difference. The log of the ratio of antisense CG I competitor was plotted against the log of competitor concentration. The concentration of antisense CG m R N A is determined by the competitor concentration at which equal molar amounts of antisense CG and competitor bands (i.e. log (antisense CG/competitor) = 0) were observed after PCR amplification (shown by an arrow in the graph). 2 concentration of 2 competitor £ antisense C G competitor A . s s s s m \ o r— © o © © x x x x v o en tN oo CN O O O —i u-i <N 00 145 Table 5.5 Comparison of sense and antisense C G mRNA levels in transgenic spruce Competitive PCR was performed as described in Materials and Methods and the amount of specific m R N A was estimated as described in Figure 5.20. Values presented are the mean of three replicate sets of competitive PCR reactions ± SD. There was no statistically significant difference in the sense CG mRNA levels between four lines. Lines antisense CG mRNA (x 10"21 mole/ugRNA) sense CG mRNA (x 10"21 mole/LigRNA) Antisense / sense Control - 14.3 ±2 .5 -G12J5.6 4.0 ± 3 . 0 12.4 ±6 .1 0.32 30F 23.5 5.7 ± 4 . 0 14.5±6.0 0.39 1H6.2 155.6 ±78 .1 9.3 ± 2 . 0 16.70 5.4 Discussion 5.4.1 Structure and expression of CG genes in spruce Structure of spruce CG genes Using a PCR-based approach, I was able to isolate partial genomic sequences of two spruce CG genes, ScgA and ScgB. The sequences of the two genes were similar in exon regions but more divergent in intron regions (data not shown). The longer intron sequence of ScgB resulted in longer PCR amplification products although the positions of introns were conserved between the two (Figures 5.8 and 5.9). It is not surprising to find multiple copies of the CG gene in spruce, since most genes involved in lignin biosynthesis exist as gene families. Unpublished Southern blotting data by Dharmawardhana also suggests the presence of multiple CG genes in the lodgepole pine genome (DP Dharmawardhana personal communication). The functional significance of this redundancy is unknown but the two genes may have slightly different substrate specificity and/or may be expressed differentially. 146 Although both ScgA and ScgB exhibited similarity to other members of family 1 (3-glycosidases, they are much more closely related to the pine CG sequence, forming a tight cluster of three genes (Table 5. 4). Conservation was seen throughout the sequenced regions, including all of the key features in the pine CG (Figure 5.10). The three sequences contain the NEP and E N G motifs that are conserved in all of family 1 (3-glycosidases (Trimbur et al, 1992). Studies using enzyme inhibitors and site-directed mutagenesis have shown that these motifs are located in or near the catalytic site (Baird et al., 1990; Trimbur et al, 1992), and crystal structures of enzyme-glycosyl complexes later confirmed the physical presence of these motifs at the active site (Barrett et al, 1995). It is significant that the sequence conservation between spruce and pine CG extends to the C-terminal region (Figure 5.10), since the C-terminal sequence of (3-glucosidases has recently been shown to be important for determining substrate specificity. Exchanging the C-terminal domain of maize (3-glucosidase Glu l with the C-terminal domain of sorghum dhurrin (3-glucosidase revealed that the substrate specificity of dhurrin (3-glucosidase was controlled by a 47 amino acid C-terminal sequence (Cicek et al, 2000). Also, exchanging the C-terminal 58 amino acid sequence of a prokaryotic (3-glucosidase from Celvibrio gilvus with the C-terminal 60 amino acid sequence of Agrobacterium tumefaciens (3-glucosidase incorporated in the substrate specificity of A. tumefaciens (3-glucosidase into the chimeric enzyme (Singh and Hayashi, 1995). Sequence conservation at the C-termini of the CG genes thus suggests conservation of substrate specificity between these enzymes. However, this point needs to be confirmed experimentally by heterologous expression of full-length spruce CG sequences. Spruce CG sequences exhibited high similarity to family 1 (3-glucosidases from a variety of angiosperm species (Table 5.4). Although the reported substrates of these 147 glucosidases seem diverse, the enzymes do share some similar characteristics. Since the aglycones of target substrates are often cytotoxic, it is important that the glucosides be kept separate from glucosidases capable of hydrolysing them. The glucosides are therefore typically stored in the plant in different cells or different cell compartments from the storage site of the glucosidases. For example, in sorghum the cyanogenic glucoside dhurrin is localized in the vacuole of epidermal cells, whereas its specific glucosidase is localized in neighbouring mesophyll cells (Kojima et al, 1979). In Catharanthus roseus, the glucoalkaloid strictosidine is stored in the vacuole whereas its specific glucosidase is localized in the E R (Geerlings et al, 2000). Upon cell damage, the stored glucosides come in contact with their corresponding glucosidases and are rapidly hydrolysed to release their aglycones, which may serve as defence molecules to deter herbivores or opportunistic pathogen colonization of damaged tissues. Similar to these glucosidase systems, coniferin and C G are localized in different subcellular compartments. Leinhos and Savidge reported that the majority of coniferin is localized in the cytoplasm in developing xylem of Pinus banksiana and P. strobus (1992). C G , on the other hand, appears to be largely localized in the cell wall (L. Samuels, unpublished). If hydrolysis of coniferin occurs in a manner analogous to other (3-glucosidase systems, coniferin would be stored in the cytoplasm until after cell death. Loss of cytoplasm would result in release of coniferin to the apoplast, which would allow delivery of monolignols to the site of lignification. Although this type of passive transport may occur in the last steps of xylogenesis, such a process would not explain how lignification occurs in living xylem cells. It has been suggested that lignin precursors may be transported through Golgi-derived vesicles in living xylem cells (Takabe et al, 1985; Terashima et al, 1993). 148 The Arabidopsis genome contains a number of sequences that exhibit homology to CG (Table 5.4). It is particularly interesting that three Arabidopsis glucosidase sequences (AC004392A, AC004392B and AL161555) appear to be more closely related to CG genes than other (3-glucosidase genes (Figure 5.11). An interesting question is whether these sequences represent functional orthologues of CG in Arabidopsis. If the substrate specificity of C G is controlled by the C-terminus sequence as shown in dhurrin (3-glucosidase (see above), the three Arabidopsis sequences do not appear orthologous to CG. However, it is impossible to answer this question from sequence comparisons alone. It would require studying the substrate specificity of these glucosidases. Some Arabidopsis (3-glucosidase sequences are found in clusters. These include two tandemly situated glucosidase genes in chromosome 1 (AC004392), five genes in chromosome 2 (AC004521) and three genes in chromosome 3 (AL138658). The significance of the gene cluster arrangements is not clear. Since similarities between individual genes within each cluster are not necessarily high (34-74 %), it is unlikely that these represent the product of a recent duplication event. Instead, there may have been selection pressure to keep these sequences together. Such situations may arise i f they are expressed in the same spatial or temporal manner. Alternatively, some of the genes may represent pseudogenes. Although they appear functional in their coding regions, it is possible that they have mutations in the promoter/terminator regions. Another point of interest is that some Arabidopsis [3-glucosidase genomic sequences (AB024024, AB023032, AC006053) do not contain any introns. This is in striking contrast to the genomic spruce CG sequences, the genomic sequence of barley (3-glucosidase BGQ60 (Leah et al, 1995), the genomic sequences of poplar (3-glucosidases (MacDonald, 1999) and the clustered Arabidopsis (3-glucosidases (AC004392, AC004521), which all 149 contain multiple introns. The biological significance of the presence or absence of introns in plant genes is not clear. The existence of these CG homologues in the Arabidopsis genome combined with the array of molecular genetics tools available for this species, offers an excellent opportunity to clarify the role of (3-glucosidases in lignin biosynthesis. Since Arabidopsis can exhibit secondary growth under prolonged short day conditions (Busse and Evert, 1999), this would provide an ideal model system for such a study. Expression of spruce CG genes during early seedling development RT-PCR analysis confirmed the expression of the identified spruce CG genes (Figure 5.12). The observed expression pattern roughly corresponded to the extent of lignification during early seedling development (Figure 5.12 and 5.13). CG m R N A levels started to rise in seedlings as young as four weeks old (Figure 5.12). This is consistent with an earlier finding that coniferin accumulated in spruce seedlings as young as 40 days old (Marcinowski and Grisebach, 1977). Since these seedlings have lignified cell walls only in primary xylem (Figure 5.13), these observations suggest C G may also be involved in lignification of primary xylem. Marcinowski et al. reported that coniferin p-glucosidase activity in spruce {Picea abies) seedlings peaked 7 days after germination and declined afterward (1979). Their finding is puzzling, since lignin is barely detectable in 7-day-old spruce seedlings (data not shown) and lignification continues to become more extensive as the plants grow older (Figure 5.13). One possible explanation for this discrepancy is the presence of other P-glucosidases in spruce seedlings. Many family 1 P-glucosidases exhibit a broad range of substrate specificity. For example, the maize P-glucosidase G l u l hydrolyses a broad 150 spectrum of artificial and natural substrates in addition to its natural substrate, DEVIBOAGlc (Cicek et al, 2000), while coniferin is readily hydrolysed by commercially available almond P-glucosidase. It is therefore possible that coniferin is hydrolysed non-specifically by other glucosidases in extracts from these seedlings. Another possible explanation is that the C G activity detected in young spruce seedlings may be involved in biosynthesis of compounds other than lignin. For example, coniferin has been postulated as a precursor of lignans in Linum flavum (van Uden et al, 1990). Since lignan synthesis has been reported to take place in spruce (Picea glehnii) suspension cultured cells (Nabeta et al, 1994), it is possible that lignan synthesis also occurs in young spruce seedlings. In either case, the reported enzyme activity may not be exclusively associated with lignin biosynthesis. 5.4.2 Effect of an antisense CG gene in transgenic spruce Although an antisense CG gene was successfully introduced into interior spruce, expression of this gene in transgenic spruce plants did not result in a reduction of sense CG transcripts (Figure 5.18 and 5.19). This was unexpected, since most antisense inhibition studies reported a reduction of corresponding sense transcript levels (Rothstein et al, 1987; Halpin et al, 1994; Dwivedi et al, 1994; Atanassova et al, 1995; van Doorsselaere et al, 1995; Yahiaoui et al, 1998; Piquemal et al, 1998; Hu et al, 2000). Although the exact mechanisms of antisense R N A inhibition are not yet established, formation of double stranded R N A (dsRNA) in the cell is believed to play an important role (Mol et al, 1994; Arndt and Rank, 1997). This normally results in a reduction of steady state mRNA levels possibly due to degradation by dsRNA-specific RNase (Mol et al, 1994; Arndt and Rank, 1997), which is mediated through sequence-specific defense mechanisms against viruses 151 (Hamilton and Baulcombe, 1999; Dalmay et al, 2000). Other mechanisms may also operate, since two studies have reported inhibitory effects of antisense genes without apparently affecting sense mRNA levels (Oliver et al, 1993; Temple et al, 1993). In these cases, inhibition at the translational level was suggested. Therefore, it is still possible that expression of the CG gene is being inhibited at the translational level. Unfortunately, this point could not be examined because of lack of enzyme assay systems and inability to detect C G proteins in western blotting using extracts from mature stem tissues. One possible explanation for the failure of antisense inhibition might be the generally weak expression of the antisense gene in spruce. Although the CaMV35S promoter is commonly used in angiosperm species to direct transgene expression, it is now known to be only weakly expressed in conifer tissues (Walter et al, 1994). In fact, all but one spruce line in this study exhibited weaker expression of the antisense CG gene compared to expression of endogenous spruce CG gene (Figure 5.18 and Table 5.5). On the other hand, weak expression cannot be the reason for lack of inhibition in line 1H 6.2 because this line exhibited antisense transcript levels 17-fold higher than endogenous CG transcripts (Table 5.5). Nevertheless, the presence of high levels of antisense CG transcripts had no effect on endogenous CG transcript levels in 1H 6.2 (Table 5.5), nor did it not produce any effects on plant development, since this line was indistinguishable from wild-type plants (Figure 5.15 and 5.16). Another possible explanation for the observed lack of inhibition might be that homology between the antisense gene and the target gene is not high enough to have any inhibitory effects. This did not appear to be a major issue at the time this study was initiated, as heterologous sequences are commonly used to create antisense inhibition in angiosperm species (Van der Krol et al, 1988; M o l et al, 1994; Dwivedi et al, 1994; 152 Higuchi et al, 1994; N i et al, 1994). However, a study of transgenic tobacco transformed with either homologous or heterologous antisense genes has shown that the heterologous sequence is not as effective as the homologous sequence for induction of antisense inhibition (Yahioui et al, 1998). Although the pine and spruce CG genes exhibit 86 - 89 % of nucleotide sequence identity (Table 5.3), this may not have been sufficient for effective antisense inhibition. It has been suggested that double stranded R N A (dsRNA) formed by introduction of antisense R N A are rapidly degraded as a part of defense mechanisms against virus sequences (Hamilton and Baulcombe, 1999; Dalmay et al, 2000). It is possible that gymnosperm species have evolved alternative mechanisms for viral defense and do not employ dsRNA-mediated defense responses, or gymnosperm species may have lower sensitivity to the presence of dsRNA. However, given the broad phylogenetic distribution of this phenomenon, it is unlikely to be absent from gymnpspers. The transgenic plants did not differ phenotypically from wild-type plants except for two lines: line 30F 23.5, which exhibited severe growth retardation (Figure 5.14C), and line 28J 6.2, which exhibited abnormal xylem development (Figure 5.16F). Since there was no general trend in phenotypic changes, these unusual phenotypes might result from somaclonal variation rather than transgene effects. Somaclonal variations are stable phenotypic variations among plants derived from clonal propagation, and are often triggered by prolonged exposure to tissue culture conditions. Several mechanisms have been suggested for somaclonal variation, including a) chromosomal aberrations (polyploidy, translocations, chromosomal breakage), b) activation of transposable elements, c) D N A methylation changes, d) point mutations, and e) epigenetic variation (Veilleux and Johnson, 1998). This phenomenon is problematic for studying effects of transgenes, since 153 the stress of the selection process itself appears to increase the frequency of somaclonal variation (Veilleux and Johnson, 1998). Somaclonal variation cannot be excluded as the source for a novel phenotype unless the phenotypic changes in transgenic plants exhibit a good correlation with gene expression pattern, or unless a clear trend is shown among different transgenic lines. Since neither was the case for the antisense CG transgenic spruce plants, I conclude that the expression of the antisense CG gene is unlikely to have caused the observed changes in xylem development or growth response. The unusual xylem development in line 28J 6.2 is still fascinating even i f it is caused by somaclonal variation. Plants of line 28J 6.2 exhibit only mild growth inhibition despite this xylem abnormality (Figure 5.15). It would be interesting to establish the exact cause of the phenotypic change. In summary, partial sequences of two CG genes were obtained from interior spruce. Their expression pattern correlated with extent of lignification in young spruce seedlings, which supported the role of C G in lignin biosynthesis. However, an attempt to inhibit CG gene expression via antisense inhibition failed, possibly due to low expression of the antisense gene, or due to insufficient sequence homology between the antisense and target CG sequences. To my knowledge, this is the first study to employ an antisense approach in a gymnosperm species. Whether this is a viable approach in conifers remains to be answered in the future. Improvements in antisense gene construction will be necessary for future antisense inhibition studies. 154 Chapter 6 Towards genetic engineering of the lignin biosynthetic pathway in conifers (Concluding remarks and future considerations) Trees and tree products are among the most important natural resources in Canada. There are increasing demands for improved quality and productivity of the wood resource. Genetic engineering provides a powerful tool to improve tree properties. Although the quantity and quality of lignin have already been altered successfully in angiosperm trees (poplar and aspen; Chapter 1), the application of lignin biotechnology to coniferous species poses new and important challenges. This thesis has presented important findings and highlighted some of the difficulties involved in this area of research. In the following, I summarise this work's contribution towards genetic engineering of the lignin biosynthetic pathway in conifers. Expression of the poplar PAL2 promoter - homologous (poplar) vs. heterologous (spruce and tobacco) systems Expression studies of the poplar PAL2 promoter in homologous and heterologous systems revealed that at least one of the transcriptional regulation systems involved in the synthesis of lignin is conserved between two different angiosperm species and between angiosperms and gymnosperms, whereas the regulating system leading toward the synthesis of specialized phenolic compounds is not conserved (Chapter 2 and 4). This evolutionary conservation is an important finding, since it indicates that it will be feasible to use a promoter from a heterologous source to direct vascular specific expression in different taxa. 155 Role ofCG in lignin biosynthesis The presence of the CG sequences in both spruce and pine is consistent with a universal role of C G in conifer lignin biosynthesis. This is the first study to show a correlation of CG m R N A levels with lignification. It would be interesting to study seasonal changes in CG m R N A levels in xylem tissues in order to obtain further evidence for the role of C G in lignin biosynthesis. These correlative studies, however, would not demonstrate an essential role for C G . That test wi l l still require specific inhibition of the CG genes via antisense- or sense-suppression. Although my attempt to inhibit the spruce CG gene via expression of antisense R N A failed, this may still be a viable approach i f suitable gene constructs were to be used (see below). Need for isolating conifer promoter/enhancer elements One of the difficulties faced in this work was weak expression of the promoters used. Weak kanamycin resistance displayed by the transgenic spruce was due to weak activity of the NOS promoter that directed expression of the NPTII gene (Chapter 3). The poplar PAL2-GUS fusion gene was also very weakly expressed in spruce (Chapter 4). The antisense CG gene exhibited weak expression in spruce due to the relative ineffectiveness of the CaMV35S promoter (Chapter 5). Although each of these promoters has been shown to be highly active in angiosperms, this does not appear to be the case for conifers. Conifer transgenic research currently relies upon the use of well-characterized angiosperm promoters because these are readily available and only a few conifer promoter/enhancer elements have been isolated to date. The results of my study show that there is an urgent need to isolate highly active promoter/enhancer elements from conifers to obtain optimal expression of transgenes. 156 Need for cloning genes from homologous system In recent years, considerable effort has been dedicated to gene discovery from different organisms. Conifers are no exception. More than 5000 EST clones have been sequenced from the compression wood library of loblolly pine (Allona et al, 1998). Currently, the EST database (dbEST, http://www.ncbi.nlm.nih.gov/dbEST) contains ten EST library entries for loblolly pine, four for Pinus radiata, one for Norway spruce and one for Sitka spruce. However, these gene discovery efforts are not well coordinated with the availability of transformation systems. For example, no efficient transformation system exists for loblolly pine (Table 1.4), and little effort has been directed towards gene discovery from those species in which transformation protocols are established (Picea glauca, Picea mariana or Larix species). Since heterologous sequences are not as effective as homologous sequences for use in antisense inhibition, it is important to isolate genes from the target species for gene modification. Spruce CG sequences obtained in this study wil l allow the future construction of homologous antisense CG genes to introduce into spruce. Need for conifer systems for rapid measurement of the effectiveness of transgenes The slowness of obtaining transgenic conifer plants is the major disadvantage in this area. At least one year of constant work is necessary before any preliminary analyses are made (Chapter 3). This makes it difficult to assess the effectiveness of different gene constructs. 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Appendix A Sequence alignment of (3-glucosidase genes AC004521B AC004521C AC004521E PCG ScgB ScgA AC004392A AC004392B AL161555 AB020749A AB020749B U2 8 04 7 AB023032 AB024024 AC004521A U39228 U50201 AF221526 U26025 AF163097 AF149311 D83177 AC004521D S35175 X56734 AF112888 WMTLNEP WMTLNEP WMTLNEP wfiJTLNEP WpTLNEP WITLNEP WITLNEP WITLNEP WMTLNEP WITLNEP WITINEPi W M T S N E P TLNEP FT. tfTFS rtSLSTl M I F A V : S A Y V G WJgFSNSI MHlFHiaiaHTYVAsI AC004521B AC004521C AC004521E PCG ScgB ScgA AC004392A AC004392B AL161555 AB020749A AB020749B U28047 AB023032 AB024024 AC004521A U39228 U50201 AF221526 U26025 A F 1 6 3 0 9 7 AF149311 D83177 AC004521D S35175 X56734 AFII2888 AC004521B AC004521C AC004521E PCG ScgB ScgA AC004392A AC004392B AL161555 AB020749A AB020749B U28047 AB023032 AB024024 AC004521A U39228 U50201 AF221526 U26025 AF163097 AF149311 D83177 AC004521D S35175 X56734 AF112888 AC004521B AC004521C AC004521E PCG ScgB ScgA AC004392A AC004392B AL161555 AB020749A AB020749B U2 8 04 7 AB023032 AB024024 AC004521A U39228 U50201 AF221526 U26025 AF163097 AF149311 D83177 AC004521D S35175 X56734 AF112888 H C J H Q W R G K R H 1 H H F E K K C K Y Y I K K F . RIllQGffiDVJMKTNIDGKQ _ PDS RVYJITGERJ5G 'AHIgQA. . D P A R P TAHLgHI. . D H T R P SIKHV..D P T Q STgPLSPDHTQY: S T W T R S P D T T Q N jTRSPDNTQT I Q D C L T S A C N T G H G . I Q D C L I T A C N S G D G A S J K D C L H S V C E P G K G G S R A L S G F G Y A N A L | D . H K I S T T P K D L G E Q Q B W N W T E . B H P T T K P K D L G R Q O B W N M E KG . Q Q L . M Q Q T P T ^ B S A H W Q B T KDVgcSSE. . . N V H L F ^ P C A S KDvHcSSE. . .NVJjLF KDvScSTK...DVKMF I E D Y S I P T P P S YTNNYSVPTPP H D F S N D Y I . . A P P g j T^AgKITSVHA K S D A S T C C P P H N A S T N S S G S N N F I Q H A S V T E D H T P D N ^ E D V . . M F Y A N T N 2EPlgPVDPKFR liNA@. . S H G N A K P | jNADKI. . . PDTPG0E PMTN M V Y J T G E M V Y I T G E : G Y A I K L D : GLAWKLDRgGN H I N V N I H R G B L . . Y F S L Q D D R G K I . " | K C B E R T S E R I B E R T S E L T D \ , E E L T D \ , B E . ^RgNKNIFiKKvfGKEaRffiEPCYG' I | l P I V P I G H P I C - P G R P I G P G E P I G P G V P I G P G V P I G P G V P I G P G V P I G P G V P I G P G V P I G P G V P I G P GV0IGP G V P I G P G V P I G P G V P V G O J I G S I I G P I G I P 1 G P I G  AC004521B AC004521C AC004521E PCG ScgB ScgA AC004392A AC004392B AL161555 AB020749A AB020749B U28047 AB023032 AB024024 AC004521A U39228 U50201 AF221526 U26025 AF163097 AF149311 D83177 AC004521D S35175 X56734 AF112888 190 .QSHPE 197 .HSHPE 196 ..TYPT 201 H 201 H 201 Y 200 T 200 N AC004521B AC004521C AC004521E PCG ScgB ScgA AC004392A AC004392B AL161555 AB020749A AB020749B U2 8 04 7 AB023032 AB024024 AC004521A U39228 U50201 AF221526 U26025 AF163097 AF149311 D83177 AC004521D S35175 X56734 AF112888 IDNDHT KT.FQ 180 AC004521B AC004521C AC004521E PCG ScgB ScgA AC004392A AC004392B AL161555 AB020749A AB020749B U28047 AB023032 AB024024 AC004521A U39228 U50201 AF221526 U2602S AF163097 AF149311 D83177 AC004521D S35175 X56734 AF112888 

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