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Biochemical and molecular analyses of entry-point enzymes of phenylpropanoid metabolism in poplar (Populus… Ro, Dae-Kyun 2002

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Biochemical and Molecular Analyses of Entry-point Enzymes of Phenylpropanoid Metabolism in Poplar (Populus trichocarpa x Populus deltoides) by Dae-Kyun Ro B.Sc, Korea University, 1995 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 2002 © Dae-Kyun Ro, 2002 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Bptah The University of British Columbia Vancouver, Canada Date Apr,[ 2? , looZ. DE-6 (2/88) Abstract Cinnamate-4-hydroxylase (C4H) and its redox partner NADPHcytochrome P450 reductase (CPR), together with phenylalanine ammonia-lyase (PAL), play central roles at the gateway into plant phenylpropanoid metabolism. C4H catalyzes conversion of cinnamate to p-coumarate and has been proposed to anchor a multienzyme complex (MEC) on the endoplasmic reticulum (ER), recruiting PAL and potentially other soluble enzymes. The formation of p-coumarate is a key step in commitment of large amounts of carbon to lignin and soluble phenylpropanoid biosynthesis, especially in woody plants. In this thesis, catalytic and structural roles of poplar (Populus trichocarpa x P. deltoides) C4H and C P R were investigated. Clones of three isoforms of C P R were isolated from poplar xylem and young leaf cDNA libraries. Two of these cDNA clones and a previously cloned C4H cDNA were expressed in yeast. Efficient conversion of cinnamate to p-coumarate by yeast microsomes containing C4H confirmed the authenticity of the C4H cDNA, while co-expression of C4H and CPR substantiated the bona fide CPR activity of the two divergent CPRs . To examine whether the C4H and C P R are localized to the ER, C-terminal green fluorescent protein tagged versions of C4H and the two C P R s were expressed in Arabidopsis. Confocal microscopy analysis of Arabidopsis seedlings demonstrated predominant localization of the chimeric proteins on ER. To test the MEC model, an engineered yeast strain expressing PAL, C4H, and CPR was generated, in which phenylpropanoid product formation and metabolite channeling between enzymes could be investigated. Quantitative measurements showed that the triple-gene expresser synthesized a striking amount of p-coumarate, while PAL-alone and C4H-inhibited triple expressers could not efficiently convert phenylalanine (Phe) to cinnamate. When 3 H-Phe and 1 4C-cinnamate were simultaneously fed to the triple expresser, endogenously synthesized 3H-cinnamate was not preferred by C4H over 1 4C-cinnamate. Therefore, the observed efficient carbon flow from Phe to p-coumarate via the reaction catalyzed by PAL and C4H does not appear to require channeling through a MEC in yeast. Analysis of the biochemical properties of the entry point reactions and enzymes suggested instead that kinetic and thermodynamic coupling of PAL and C4H is sufficient to drive carbon-flux from primary metabolism to the phenylpropanoid pathway. i i Table of Contents Abstract ii Table of Contents iii List of Tables vi List of Figures vii List of Abbreviations x Acknowledgement xii CHAPTER 1 INTRODUCTION 1.1 PHENYLPROPANOID METABOLISM 1.2 PHENYLALANINE AMMONIA-LYASE 1.3 CLNNAMATE 4-HYDROXYLASE AND CYTOCHROME P450 REDUCTASE 1.4 PLANT CYTOCHROME P450 ENZYMES 1.5 RECENT ADVANCES IN LIGNIN BIOSYNTHESIS 1 1.6 COORDINATED REGULATION OF PHENYLPROPANOID GENE EXPRESSION 1 1.7 PHENYLPROPANOID MULTIENZYME COMPLEXES 1 1.8 PHENYLPROPANOID METABOLISM IN POPLAR AND THESIS OBJECTIVES 2 CHAPTER 2 MATERIALS AND METHODS 2 2.1 GENERAL NUCLEIC ACID METHODS 2 2.1.1 Plasmid DNA preparation and DNA sequencing 2 2.1.2 Genomic DNA and total RNA isolation 2 2.1.3 Isolation of C P R c D N A clones 2 2.1.4 Isolation of a PAL cDNA 2 2.1.5 Northern blot analysis 2 2.1.6 Reverse transcriptase PCR (RT-PCR) expression analysis 2 2.2 CLONING PROCEDURES FOR GENE EXPRESSION 2 2.1.1 Host strains, plasmid, and phage 2 2.2.2 Cloning C4H and C4H fusion derivatives in pYeDP60 vector 2 2.2.3 Construction of C4H::GFP fusion in the pBinl9 vector 2 2.2.4 Construction of CPR: :GFP fusions in the pBin19 vector 3 2.2.5 Construction of C4H, CPR, and CPR: :GFP in the pESC-Leu Vector 3 2.2.6 Cloning of PAL genes in the pESC-His vector 2.3 P L A N T G R O W T H C O N D I T I O N S A N D T R A N S F O R M A T I O N 2.4 F L U O R E S C E N C E A N D C O N F O C A L M I C R O S C O P Y A N A L Y S I S 2.5 Y E A S T M E T H O D S 2.5.1 Yeast culture media 2.5.2 Yeast transformation 2.5.3 Yeast cell cultures and gene induction 2.6 Y E A S T A N D P L A N T M I C R O S O M A L P R O T E I N P R E P A R A T I O N 2.7 E N Z Y M E A S S A Y S 2.8 Y E A S T IN VIVO E N Z Y M E A S S A Y S 2.9 C A R B O N M O N O X I D E D I F F E R E N T I A L A B S O R P T I O N S P E C T R O S C O P Y 2.10 S D S - P A G E A N D I M M U N O B L O T S 2.11 A N A L Y S I S O F PAL- O R T R I P L E G E N E (PAL, C4H, A N D C P R ) - E X P R E S S I N G Y E A S T S T R A I N S 39 2.12 3 H - P H E N Y L A L A N I N E F E E D I N G A S S A Y 2.13 D O U B L E - L A B E L I N G A S S A Y 2.14 A N A L Y S I S O F M I X E D P A L - A L O N E A N D C 4 H / C P R - A L O N E E X P R E S S I N G Y E A S T S T R A I N S . . CHAPTER 3 FUNCTIONAL EXPRESSION AND SUBCELLULAR LOCALIZATION OF POPLAR CINNAMATE 4-HYDROXYLASE 3.1 I N T R O D U C T I O N 3.2 R E S U L T S 3.2.1 Expression of recombinant poplar C4H in yeast 3.2.2 Poplar C4H cDNA encodes bona fide cinnamate 4-hydroxylase activity 3.2.3 Poplar C4H is localized to ER in planta 3.2.4 Functional expression of C4H : :GFP fusion protein 3.3 D I S C U S S I O N CHAPTER 4 CLONING, FUNCTIONAL EXPRESSION, AND SUBCELLULAR LOCALIZATION OF POPLAR CYTOCHROME P450 REDUCTASE 4.1 I N T R O D U C T I O N 4.2 R E S U L T S 4.2.1 Poplar genome contains at least three CPR genes 4.2.2 Functional expression of CPRs in yeast 4.2.3 Characterization of a dual expression in yeast 74 4.2.4 Two distinct CPR isoforms are localized to the ER 78 4.2.5 CPR::GFP fusion enzymes retain catalytic activity in support of C4H activity 81 4.2.6 CPR expression patterns in poplar 83 4.2.7 CPR enzyme activity is increased in poplar cultured cells by elicitor treatment... 86 4.3 DISCUSSION 87 CHAPTER 5 RECONSTITUTION OF PHENYLPROPANOID METABOLISM IN YEAST 94 5.1 INTRODUCTION 94 5.2 RESULTS 96 5.2.1 Cloning of a PAL cDNAfrom xylem 96 5.2.2 Differential expression of two PAL genes 97 5.2.3 Simultaneous expression of PAL, C4H, and CPR in yeast 97 5.2.4 Analysis of phenolic products in transformed yeast 101 5.2.5 Production of cinnamate and p-coumarate in transformed yeast fed various concentrations of phenylalanine 103 5.2.6 Testing for modified PAL activity in triple expressing yeast strains 103 5.2.7 Detailed analysis of Phe metabolism in yeast strains 109 5.2.8 Investigation of potential MEC-associated metabolic channeling in yeast 113 5.2.9 Time course for p-coumarate production in triple expressing strains 115 5.2.10 Metabolic coupling of PAL and C4H in mixed culture of strains expressing PAL-alone and C4H/CPR 116 5.3 DISCUSSION 119 CHAPTER 6 CONCLUSIONS AND FUTURE PERSPECTIVES 131 REFERENCES 134 v List of Tables Table 1 Cytochrome P450 content from microsomes prepared from C4H- or C4H::FLAG-expressing yeast strains 48 Table 2 C4H activity in microsome preparations from C4H-, C4H::FLAG-, and C4H::GFP-expressing yeast strains 50 Table 3 Characterization of the C4H::GFP- or GFP-transformed Arabidopsis lines. :: 52 Table 4 Catalytic activities of the C4H and CPR recombinant enzymes from the microsomal fractions prepared from C4H- or C4H/CPR-expressing yeast strains 74 Table 5 In vivo conversion of 3H-phenylalanine to related phenolic metabolites in transgenic yeast strains 112 Table 6 Ratio of incorporation of 3H-phe and 14C-cinnamate into the products, p-coumarate or styrene, in the T2 and T4 yeast strains expressing PAL, C4H, and CPR 115 vi List of Figures Figure 1.1 Various phenylpropanoid compounds and their derivatives which derived from the reactions of all or part of core phenylpropanoid enzymes 2 Figure 1.2 Proposed lignin biosynthetic pathway in plant 13 Figure 2.1 A cloning scheme of C4H::GFP fusion construct in pBin19 binary vector. 29 Figure 3.1 Deduced amino acid sequence of the poplar C4H-550 cDNA and comparison to other plant C4H amino acid sequences 44 Figure 3.2 A schematic diagram of C4H- or C4H::FLAG-construct in pYeDP60 expression vector 46 Figure 3.3 SDS-PAGE and immuno-blot analysis of microsomes from C4H- and C4H::FLAG-transformed yeast 47 Figure 3.4 Carbon monoxide-induced differential absorption spectra analysis of microsomal proteins from C4H- or C4H::FLAG-expressing yeast strains. 49 Figure 3.5 Diagnostic HPLC analysis for identification of p-coumarate converted from trans-cinnamate by the enzyme, cinnamate 4-hydroxylase 51 Figure 3.6 Subcellular localization of hybrid poplar C4H::GFP in transgenic Arabidopsis seedlings 54 Figure 3.7 An enhanced confocal microscopy image for ER-localization of C4H::GFP protein on Arabidopsis cotyledon epidermal cells 55 Figure 3.8 Images Arabidopsis guard cells transformed by C4H::GFP fusion construct observed by concofocal or fluorescent microscopy 56 Figure 3.9 Immuno-blot and carbon monoxide-induced differential absorption spectra analysis of microsomes from C4H::GFP-transformed yeast 58 Figure 3.10 In vivo conversion of cinnamate to p-coumarate by transgenic yeast 59 Figure 4.1 Nucleotide sequence alignment of partial CPR1 cDNA clones 67 Figure 4.2 Restriction enzyme maps of three poplar CPR isoforms and nucleotide sequence alignment of full or partial CPR3 cDNA clones 69 Figure 4.3 Alignment of deduced amino acid sequence of poplar and Arabidopsis CPR isoforms 70 Figure 4.4 Representation of the cloning and expression strategy for C4H and CPR1/2 in the yeast pESC dual expression vector 72 Figure 4.5 In vivo production of p-coumarate from yeast strains expressing C4H-only, C4H and CPR1, and C4H and CPR2 Vr 73 Figure 4.6 SDS-PAGE fractionation of microsomal proteins from vector-only, C4H-only, C4H/CPR1, and C4H/CPR2 transformed yeast strains 75 Figure 4.7 Immuno-blot and RNA-blot analysis of the vector-, C4H-, C4H/CPR1-, and C4H/CPR2-transformed yeast strains 77 Figure 4.8 Comparison of the N-terminal amino acids comparison of poplar CPR, immunoblot analysis of the CPR::GFP proteins from transformed Arabidopsis, and localization of CPR::GFP in Arabidopsis seedlings by confocal microscopy 80 Figure 4.9 Relative in vivo C4H activities in the presence of GFP-fused CPRs, immunoblot analysis of the C4H and GFP-fused CPR recombinant proteins, in vitro C4H assay, and cytochrome c reduction assay 82 Figure 4.10 Specificities of primers for the three CPR-isoforms, and reverse-transcriptase PCR amplification of specific CPR isoforms from poplar tissues and cell culture 85 Figure 4.11 Cytochrome c reduction by microsomal fractions prepared from the cultured poplar cells with (EL+) or without (EL-) elicitor treatment 86 Figure 5.1 Comparison of restriction enzyme maps of PAL18 and PAL7 cDNAs.... 96 Figure 5.2 Expression of two PAL genes in poplar organs and tissue culture cells. 98 Figure 5.3 Phenylpropanoid protein amounts and PAL activity in transgenic yeast strains 100 Figure 5.4 HPLC-fractionation of phenolic metabolites produced by vector control, PAL-alone, and triple expressing yeast strains 102 Figure 5.5 In vivo production of cinnamate and p-coumarate by genetically engineered yeast strains incubated with different concentrations of Phe. '...104 Figure 5.6 Production of cinnamate by PAL-alone expressing strains PAL2 and PAL4 in the presence of varying concentrations of p-coumarate 105 Figure 5.7 Determination of the minimal concentration of piperonylic acid for C4H-inhibition in vivo 107 Figure 5.8 Production of cinnamate in the presence or absence of 10 \xM piperonylic acid in PAL only-expressing yeast strains 107 Figure 5.9 Production of cinnamate and p-coumarate by PAL-alone or triple expressing yeast strains treated with piperonylic acid (PA) 108 Figure 5.10 Phenylalanine metabolism in yeast triple expressing strains (transformed by PAL, C4H, and CPR), showing reconstruction of the entry point into phenylpropanoid metabolism in this host 110 Figure 5.11 Illustration of the double labeling experiment used to analyze a potential multi-enzyme complex (MEC) involving PAL and C4H/CPR 114 Figure 5.12 In vivo production of p-coumarate and cinnamate from triple expressing yeast strains T2 and T4 (transformed with PAL, C4H, and CPR) 117 Figure 5.13 Cinnamate and p-coumarate accumulation in mixed cultures of PAL-alone and C4H/CPR expressing strains and triple expressing strains 118 Figure 5.14 The conversion of tetralin (1,2,3,4-tetrahydronaphtalene) to 1-teteralol (1,2,3,4-tetrahydro-1 -naphtholl) catalyzed by camphor 5-monooxygenase 125 Figure 5.15 Model for the kinetic and thermodynamic coupling between PAL and C4H expressed in yeast 126 Figure 5.16 Biosynthetic pathways to DIMBOA and tryptophan in maize 129 List of Abbreviations A adenine bp base pair C4H cinnamate 4-hydroxylase C cytosine CaMV35S cauliflower mosaic virus 35S promoter cDNA complementary deoxyribonucleic acid CPR cytochrome P450 reductase DAPI 4',6-diamino-phenylindole DIOC 6 dihexaoxacarbocyanine iodide DNA deoxyribonucleic acid EDTA ethylene diamine tetra acetic acid ER endoplasmic reticulum EST expressed sequence tag FAD flavin adenine dinucleotide FITC fluorescein isothiocyanate FMN flavin mononucleotide G guanine GFP green fuorescent protein h hour His histidine HPLC high performance liquid chromatography Kan kanamycin kb kilobase kD kilodalton K m Michaelis-Menten constant LB Luria-Bertani Leu leucine MES 2(N-morpholino)ethanesulfonic acid min minute mRNA messenger ribonucleic acid NADH nicotinamide adenine dinucleotide NADPH nicotinamide adenine dinucleotide phosphate OD optical density ORF open reading frame P450 cytochrome P450 monooxygenase PAGE polyacrylamide gel electrophoresis PAL phenylalanine ammonia lyase PCR polymerase chain reaction Phe phenylalanine pl isoelectric point PMSF phenylmethylsulfonyl fluoride PVDF polyvinylidene difluoride RNA ribonucelic acid rpm rotation per minute s second SD standard deviation SDS sodium dodecyl sulphate SE standard error SSC standard saline citrate T thymine TBS tris buffered saline Tris tris(hydroxymethyl)-aminomethane UV ultraviolet w/v weight per volume w/w weight per weight Acknowledgements The work described here could not have been achieved without the abundant endeavor, encouragement and support from my mentors, friends, and families. In this thesis, I did my best and I don't think I could have done a better job if a second chance were given to me, as I would not be able to meet such a helpful people again listed below. My supervisor, Dr. Carl Douglas, is the most significant contributor in transforming an erroneous fresh graduate from Korea to a competent scientist through his enormous patience and teaching skills. His guidance in research and writing improved my research and this thesis remarkably. My committee members, Drs. Ljerka Kunst, Brian Ellis, Lacey Samuels, and Dave Theilmann, continually supported me to complete my degree. Their diverse expertise in biochemistry, cell biology, and genetics strengthened and broadened my research capability. My sincere gratitude goes to all present and past Douglas lab members who have advised, encouraged, and cheered me in and out of the lab since I started my graduate study in Vancouver. Qing, Jurgen, Lee, Bahram, Tamara, Ben, Shana, Clarice, and AN, you are awesome! I really enjoyed working with you. In particular, many conversations with Jurgen in recent two years made my research attitude different. I thank Daniela, Diana, and Monica for your help and encouragement in my early days at UBC. Dr. Neil Towers, Kevin, and Fiona kindly supported my HPLC-analysis with valuable discussions, and Dr. Elaine Humphrey patiently taught me confocal microscopy. I am also indebted to all members of Kunst and Haughn lab who helped me technically in the lab and socially outside of lab. I also appreciate my Korean friends, Eun-Sook, Sung-Mee, Sang-Won, Woo-Jin, Jin-Young, Kwang-Su, Seoung-Hwan, Jina, Hyuk-Chan, Jin-Sul, Hang-Sik, who organized and provided me with a warm Korean community at UBC. My mother, father, sister Kyung-Hee, and brother Hyun-Kyun closely contacted and spiritually supported me from Korea. I sincerely thank for their pouring affection and their confidence in my study and career. My wife, Eun-Joo Gina, whom I met here in the Biological Sciences building during both of our graduate studies, is indeed a much bigger present to me than any other achievements in my life. Also, I thank all the members of Gina's family for their support. My appreciation to these people goes beyond the acknowledgement I express here. To my parent Moo-Nam Ro and Moon-Ja Ho Chapter 1 Introduction 1.1 Phenylpropanoid metabolism Phenylpropanoid metabolism in plants is responsible for the synthesis of a diverse array of phenylpropanoid products in specific cells and tissues at various developmental stages. The phenylpropanoid compounds deposited in different tissues function as pigments, UV-protective chemicals, inter-species signaling molecules, antimicrobial agents, and structural components (Hahlbrock and Scheel, 1989). Genetic and molecular analyses of mutants defective in phenylpropanoid regulatory or structural genes (e.g., Jin et al., 2000; Landry et al., 1995), phenotypes of transgenic plants with altered expression of key phenylpropanoid structural or regulatory genes (e.g., Elkind ef al., 1990; Tamagnone ef al., 1998) and transgenic plants engineered with novel phenolic pathways (Hain et al., 1993; Mayer et al., 2001) further corroborate the functional significance of phenylpropanoid compounds in plants. In many cases, these studies suggest yet uncharacterized roles of phenylpropanoid derivatives in plant growth and development (Elkind ef al. , 1990; Mo ef al. , 1992; Mayer ef al., 2001; Tamagnone ef al., 1998). In addition, perception of environmental stresses such as pathogen infection, UV-irradiation, and wounding modulates the expression of phenylpropanoid genes (Dixon and Paiva, 1995). These stress signals direct rapid and transient changes in carbon flow into specific phenylpropanoid metabolites necessary for defense against stress. Carbon flux into the phenylpropanoid pathway from primary metabolism is mediated by the catalytic reactions of three gateway enzymes of "general phenylpropanoid metabolism" depicted in Figure 1.1. The first enzyme, phenylalanine ammonia-lyase (PAL), catalyzes the conversion of phenylalanine to frans-cinnamic acid. Trans-cinnamic acid is hydroxylated at the para-position by a cytochrome P450 monooxygenase enzyme (P450), cinnamate 4-hydroxylase (C4H), in conjunction with NADPH:cytochrome P450 reductase (CPR). The enzyme 4-coumarate:CoA ligase (4CL) generates CoA esters of p-coumarate and its derivatives. These high free energy thioesters are used as intermediates for the biosynthesis of many phenylpropanoid compounds. Because the coordinated reactions of these three enzymes channel carbon from phenylalanine into many branch pathways for specific phenylpropanoids or phenylpropanoid derivatives, this part of the phenylpropanoid pathway is referred to as "general (core) phenylpropanoid pathway". 1 Figure 1.1 Various phenylpropanoid compounds and their derivatives derived from the reactions of all or part of core phenylpropanoid enzymes. Modified from Hahlbrock and Scheel (1989). Arrows leading from the box depicting general phenylpropanoid metabolism indicate the central position of this pathway in supplying precursors for specific branch pathways. In many cases, 4-coumaroyl-CoA or other hydroxycinnamoyl-CoAs are used as substrates for these pathways, but in other cases hyroxycinnamoyl-glucose esters, /?-coumaric acid, or even cinnamic acid (for salicylic acid biosynthesis), may be used as substrates. 2 The product of the PAL and C4H reactions, p-coumaric acid, is a direct carbon skeleton for a large number of C6-C3 phenylpropanoid derivatives in plants (Figure 1.1). Functionally important examples of such derivatives are free phenolic acids, or their conjugates, and esters, such as chlorogenic acid. Plants are constantly exposed to ultraviolet B (UV-B)-containing sunlight that can cause DNA damage and oxidative stress. The phenolic esters and conjugates together with flavonoid compounds act as efficient sunscreens and antioxidant chemicals in epidermal cell layers of leaves (Landry ef al., 1995; Li et al., 1993). The functional roles of conjugated phenolic acids are evident from the Arabidopsis fahl mutant defective in sinapylmalate biosynthesis, which is even more sensitive to the UV-B damage than the flavonoid defective mutant tt3, (Landry etal., 1995). Lignin is composed of monomeric lignin subunits that are also derived from the C6-C3 carbon structure (see more details in section 1.5). It is a key structural component of the secondary cell walls of tracheary elements and fibres in xylem and other tissues, which provide these cells with mechanical strength and hydrophobicity. As well, lignin synthesis is induced in response to environmental stresses such as wounding and pathogenic infection especially in monocots. After wounding, the accumulation of lignin may seal off the wound sites to protect the plants from the loss of water and from pathogenic infection. When challenged by a pathogen, the incorporation of lignin into the cell wall is thought to fortify the cell wall and create physical barriers against further infection. Evolution of the lignin biosynthetic pathway is thought to have been a major adaptation that allowed vascular plants to successfully colonize the terrestrial environment (Whetten and Sederoff, 1995). Although the genes and enzymes involved in coumarin and furanocoumarin biosynthesis are not well characterized, it is believed that coumarin is formed by both ortho-hydroxylation of p-coumarate and cis-trans isomerization of its side chain. Formation of the furan-ring of furanocoumarin is catalyzed by three enzymatic reactions including a prenylation reaction (reviewed in Hahlbrock and Scheel, 1989). Several (furano)coumarins such as marmesin, psoralen, umbelliferone, bergapten, and isopimpinellin were found after fungal elicitor treatment in parsley cell cultures (Tietjen ef al., 1983), and these chemicals are known to have antimicrobial activities (Beier and Oertli, 1983). Thus, the (furano)coumarins are believed to function as phytoalexins in parsley and related plant species. Since benzoic acid and its derivatives are C6-C1 compounds, they are not strictly phenylpropanoids. However, they appear to be derived from cinnamic acid (Coquoz ef al., 1998) by a chain shortening reaction, and thus can be considered phenylpropanoid 3 derivatives. Salicylic acid (SA; 2-hydroxy benzoic acid) is an important C6-C1 phenolic compound, probably made by two pathways, one via cinnamate and benzoic acid and another via chorismate in a reaction catalyzed by isochorismate synthase (Wildermuth et al., 2001). SA is an essential signaling molecule required to activate systemic acquired resistance (SAR) and important plant defense responses (Ryals era/., 1995). In the pathway via cinnamate and benzoic acid, the C3 side chain of cinnamate may be shortened by P-oxidation of cinnamoyl-CoA, or a non-oxidative pathway may be used to produce benzoic acid. The last stage of SA biosynthesis in tobacco is reported to involve the release of free benzoic acid from a large pool of preformed conjugated benzoic acid (Yalpani ef al., 1993), followed by the ortr/o-hydroxylation of free benzoic acid by benzoic acid 2-hydroxylase (Leon ef al., 1995). However, in direct contrast to this view, in the same tobacco plant and cell culture, a large pool of conjugated benzoic acid was not detected, and free benzoic acid was not efficiently incorporated into the SA (Chong et al., 2001). Instead of free benzoic acid, the glucose ester of benzoic acid was proposed to be an intermediate for SA biosynthesis. The C15 compound chalcone, the precursor for all flavonoids, is formed by the catalytic reaction of chalcone synthase (CHS), which condenses one p-coumaroyl-CoA and three malonyl-CoA acetate units. Chalcone is rapidly isomerized to yield the naringenin (flavanone) by chalcone isomerase. A large number of structurally and functionally diverse flavonoids are derived from naringenin. For example, chalcone and flavanone derivatives from legume root exudates can activate the nod genes of N2-fixing Rhizobium bacteria and are believed to function as plant-microbe interacting signals for symbiosis between legumes and Rhizobia (Recourt et al., 1991; Zaat ef al., 1987). The condensation of cinnamoyl-CoA with three malonyl-CoA units catalyzed by CHS produces B-ring deoxyflavanones that may be phytoalexins in some plants such as old man cactus (Liu et al., 1995). A variety of modified flavanones are believed to function as UV-protectants and antibiotics (Hahlbrock and Scheel, 1989). Stilbene synthase (STS) belongs to the same family of polyketide synthases as CHS, but it catalyzes an additional decarboxylation reaction, resulting in a C14 carbon structure. Stilbenes with fungicidal effect are found in several unrelated plants such as peanut, grapevine, and pine, and tobacco expressing a grapevine S7"S gene showed enhanced resistance against the pathogen Botrytis cinerea (Hain et al., 1993). The isoflavonoids and anthocyanins constitute two important subclasses of flavonoids. The first reaction into the isoflavonoid pathway is catalyzed by a membrane-bound P450 enzyme, 2-hydroxy isoflavone synthase (2-HIS) or also known as isoflavanone 4 synthase (IFS). This enzyme catalyzes both the migration of B-ring and the hydroxylation of the 2-position of the flavanone to produce 2-hydroxyisoflavanone, to which enzymatic or spontaneous dehydration occurs to yield the isoflavone carbon skeleton (Kochs and Grisebach, 1986). The isoflavonoids (e.g. pterocarpans, isoflavans, and prenylated isoflavonoids) are exclusively distributed in leguminous plants where they are synthesized de novo after pathogenic attack and act as phytoalexins (Dixon ef ai, 1995; Smith and Banks, 1986). Some isoflavonoids such as rotenoids and coumestans are insect feeding deterrents (Dewick, 1993). The key enzymes for anthocyanin biosynthesis are flavanone 3-hydroxylase (F3H), a dioxygenase (Britsch ef a/., 1992), and two subsequent P450 enzymes, flavonoid 3'-hydroxylase (F3'H) and/or flavonoid 3'5'-hydroxylase (F3'5'H) (Brugliera ef a/., 1999; Holton ef a/., 1993). Together, these enzymes direct carbon flow to the dihydroflavonols that can be further used as substrates for biosynthesis of various anthocyanins. Accumulation of anthocyanins in floral petals is important for attraction of insect pollinators, and anthocyanin pigments are common in seeds of many plants. 1.2 Phenylalanine ammonia-lyase The first enzyme in the phenylpropanoid pathway, PAL (EC, catalyzes non-oxidative elimination of ammonia from phenylalanine to yield frans-cinnamic acid, a key starting substrate for subsequent phenylpropanoid metabolism. PAL is a tetrameric enzyme, consisting of approximately 77 kD monomers (Bolwell ef a/., 1985). Normal Michaelis-Menten kinetics with various K m values were documented in native and recombinant PAL. Four PAL isoforms with K m values of 77, 122, 256, and 302 \M were purified from cell cultures of bean (Bolwell et al., 1985). Three PAL isoforms were purified from alfalfa with K m values of 40, 70, 110 each (Jorrin and Dixon, 1990), and one PAL with K m of 27 was isolated from Pinus taeda (Whetton and Sederoff, 1992). Recombinant poplar PAL showed a K m of 450 (McKegney ef al., 1996), while four recombinant parsley PAL isoforms showed K m values ranging from 15 to 25 \xM (Appert ef al., 1994). In bean and alfalfa, the biological significance of PAL isoforms with such substantial differences in kinetic properties is not known. Although negative cooperativity has been detected in native PAL enzymes, co-purification of multiple PAL isoforms with different kinetic properties is believed to have caused misinterpretation of the data (Bolwell etal., 1985; Jorrin and Dixon, 1990). PAL can deaminate tyrosine in vitro but only at very low affinities as approximately indicated by high 5 K m values (2.6 - 7.8 mM in recombinant parsley PAL isoforms), and thus tyrosine is not regarded as an in vivo substrate (Appert et al., 1994). The PAL enzymes characterized show unusual optimal pH ranges from 8.5 to 9.5. It has long been believed that dehydroalanine from serine residue in PAL is directly involved in catalysis; however, the crystal structure of the related enzyme histidine ammonia lyase (HAL) showed that autocatalytic cyclization of three key alanine, serine, and glycine residues, forms the reactive electrophile, 4-methylidene-imidazole-5-one (Schwede ef al., 1999). The same cyclic modification is expected to occur in PAL. Since PAL connects primary to secondary metabolism, a regulatory role for PAL in controlling carbon flow into the phenylpropanoid metabolism has been extensively investigated. The product of PAL, rrans-cinnamic acid, is proposed to be an endogenous PAL modulator in plant cells at the level of transcription and enzyme activity (Bolwell et al., 1988). To test this hypothesis, internal concentrations of cinnamate were varied by the application of exogenous cinnamate, or by the inhibition of either PAL activity or the activity of C4H, the enzyme after PAL that uses cinnamate as a substrate. Inhibition of PAL or C4H is expected to lead to a decrease or increase of the internal cinnamate concentration, respectively. Application of the PAL-specific inhibitor L-oc-aminooxy-phenylpropionic acid (AOPP) to bean cell cultures led to the superinduction of PAL transcription and increased PAL activity, while administration of exogenous cinnamate resulted in loss of PAL transcriptional induction and extractable PAL activity (Amrhein and Gerhardt, 1979; Bolwell et al., 1988; Mavandad et al., 1990). In cinnamate feeding assays, transcripts for the constitutive H1 and elicitor-inducible glucanase genes were not affected by exogenous cinnamic acid. Thus, these data may indicate that plant cells can sense the endogenous concentration of cinnamate to modulate PAL transcription and PAL activity. However, the interpretation of these experiments is still debated since non-physiological levels of cinnamate (100 pM - 1 mM) were applied, and a non-specific inhibitory effect of cinnamic acid on general protein synthesis has been reported (Walter and Hahlbrock, 1984). To elevate endogenous levels of cinnamic acid by inhibition of C4H, a P450 monooxygenase, anaerobic conditions or the P450 inhibitor tetcyclasis were used in early experiments. When a Jerusalem artichoke cell culture was placed under anaerobic conditions, extractable PAL activity rapidly decreased but recovered after aeration. Co-application of a protein synthesis inhibitor abolished the rapid decrease of PAL activity (Durst, 1976), indicating some de novo synthesized proteineous factors are actively involved 6 in the inactivation of PAL. Tetcyclasis treatment of elicited alfalfa cell cultures significantly delayed PAL induction and reduced maximal PAL activity (Orr ef al., 1993). The effect of C4H inhibition on PAL activity has been recently reevaluated using C4H-suppressed transgenic tobacco lines or a C4H-specific inhibitor. In C4H-suppressed tobacco plants, extractable cinnamate amounts from stems decreased with reduced C4H activity, which was interpreted as being due to feedback inhibition of PAL in vivo by elevation of cinnamate levels above a certain threshold concentration, caused by C4H inhibition (Blount ef al., 2000). In contrast to this, addition of elicitor and the C4H-specific inhibitor piperonylic acid to tobacco cells resulted in a 20-fold increase in the cinnamate concentration above control cells without piperonylic acid (Schalk etal., 1998). Induction of PAL activity by elicitor treatment is transient, and degradation of PAL is required to reestablish physiologically normal conditions in plant cells after elicitor-treatment. It is not clear whether cinnamic acid is directly involved in regulating the PAL degradation pathway; however, non-dialyzable factors that facilitate loss of PAL activity only in the presence of cinnamic acid, have been reported in elicitor-treated bean cell cultures (Bolwell ef al., 1986). The cAMP-dependent phosphorylation of PAL during the course of PAL degradation has been suggested in elicited bean cell cultures (Bolwell ef al., 1991). In elicitated bean cultures, a 55 kD protein kinase that phosphorylates a specific threonine residue of recombinant poplar PAL and or a synthetic peptide containing this residue, was partially purified (Allwood ef al., 1999). The phosphorylated PAL showed a reduced Vmax as predicted if the phosphorylation is a part of the PAL degradation process. Screening of several Arabidopsis calcium dependent protein kinases (CDPK) for their abilities to phosphorylate recombinant PAL and the synthetic peptide fragment from PAL identified one Arabidopsis CDPK specific for PAL phosphorylation (Cheng ef al., 2001). Due to the lack of genetic evidence for the functional significance of PAL phosphorylation and a corresponding signal transduction in plants, it is uncertain yet whether PAL phosphorylation is an essential component of complex regulatory circuit for PAL degradation. Nonetheless, the biochemical data strongly suggest that posttranslational modification of PAL is likely to be embedded in the network of elicitor-induced stress responses in plants. 1.3 Cinnamate 4-hydroxylase and cytochrome P450 reductase The enzyme C4H (EC is a member of the CYP73 subfamily within a large cytochrome P450 monooxygenase superfamily (Teutsch etal., 1990). C4H catalyzes the first oxidative reaction in phenylpropanoid metabolism, converting frans-cinnamic acid to p-7 coumaric acid (Russell, 1971). This reaction is not self-sufficient and requires another enzyme, NADPH:cytochrome P450 reductase (CPR; EC as an electron provider. C P R is unique in that it contains both FAD and FMN as prosthetic groups. A pair of electrons from NADPH is transferred to FAD, then to FMN, and ultimately to molecular oxygen in the active site of C4H. The activated oxygen molecule is then used for the monooxygenase reaction in C4H; one oxygen atom forms a water molecule, and the other contributes to the hydroxyl group of p-coumarate. Both C4H and CPR are membrane-bound enzymes, and they are likely to transiently associate with each other for electron transfer mainly by ionic interaction using their charged amino acids (Wang ef al., 1997). Due to the labile and membrane-bound nature of C4H proteins, cloning C4H genes by classical protein purification approaches followed by immunoscreening of cDNA libraries by antibodies has been difficult. However, isolation of the first C4H cDNA from Jerusalem artichoke (Teutsch et al., 1993) has enabled heterologous screening approaches and, to date, at least 20 orthologues of C4H have been cloned from different plant sources. These generally share a high degree of identity in their deduced amino acid sequence (>85%); however, it was recently shown that some plants possess divergent C4H genes encoding a second isoform which show only -60% identity to other C4Hs (Betz et al., 2001; Nedelkina et al., 1999; Potter ef al., 1995). Recombinant and native C4H enzymes display very high affinities for cinnamic acid, with K m values of < 5u.M and high turnover numbers (Chappie, 1998). Regulation of C4H expression has been investigated in various plants and cell-culture systems. Transcriptional regulation by internal and external stimuli seems to be a major mechanism for control of C4H, as it is for PAL and 4CL. Rapid up-regulation of C4H transcripts by light, wounding, elicitors, pathogens, or by internal developmental cues, has been observed in many plants (Batard et al., 2000; Fahrendorf and Dixon, 1993; Frank ef al., 1996; Hotze ef al., 1995; Koopmann ef al., 1999; Mizutani ef al., 1997; Schopfer ef al., 1998). Detection of a biphasic induction mode by wounding or elicitor has been interpreted to indicate that multiple signaling routes may exist for C4H transcriptional activation (Batard ef al., 2000; Schopfer ef al., 1998), but frans-acting factors leading specifically to the activation of C4H expression have yet to be characterized. Since C4H promoter regions share common c/s-elements with those of PAL and 4CL (Bell-Lelong ef al., 1997; Mizutani ef al., 1997), it is generally assumed that C4H should be under similar regulatory control. This would be consistent with reports of tissue- and cell-type specific co-accumulation of the relevant gene products (RNA and protein) (Koopmann ef al., 1999). 8 Plant P450s are generally considered to be localized to the ER as are most animal P450s, but it has also been reported that P450s are found in the plasma membrane (Kjellbom ef al., 1985) or in.the provacuole (Madyastha ef al., 1977), and the N-terminal sequences from CYP74 and CYP79B2 most closely resemble chloroplast targeting transit peptides (Hull ef al., 2000; Song ef al., 1993). Thus, it is likely that the subcellular locations of a given P450 in plants will depend on the specific protein. In the case of C4H, subcellular fractionations of pea seedlings and of Jerusalem artichoke tubers demonstrated that the enzyme was ER-localized (Benveniste etal., 1977), whereas subcellular fractionations of sweet potato roots found most of the C4H in unidentified organelles, perhaps provacuoles (Fujita and Asahi, 1985). In French bean, immunolocalization placed C4H in both the Golgi apparatus as well as the ER (Smith et al., 1994). C P R proteins and corresponding cDNAs have been isolated from Vigna radiata and Catharanthus roseus, where single isoforms of CPR are reported (Meijer ef al., 1993; Shet ef al., 1993). However, three distinct CPR proteins were found from the microsomal fraction of Helianthus tuberosus (Benveniste ef al., 1991), and cDNAs encoding two distinct types of C P R have been isolated from Arabidopsis (Mizutani and Ohta, 1998; Urban et al., 1997) and parsley (Koopmann and Hahlbrock, 1997). These two distinct isoforms of C P R did not display differences in their abilities to interact with C4H, but they showed differential expression patterns; one isoform was constitutively expressed and the other was inducible by external stress such as wounding and light. However, it is not clear whether the two isoforms prefer a distinct group of P450s as substrates, and whether they are differentially localized to different subcellular compartments. 1.4 Plant cytochrome P450 enzymes Cytochrome P450 enzymes were first detected from animal liver extracts as membrane-bound and heme-containing enzymes that show a characteristic absorption at 450 nm after binding to carbon monoxide in the dithionite-reduced form (Omura and Sato, 1964). Other heme-binding enzymes do not show this absorption peak, and this unique spectral property of P450s is attributed to the use of cystainyl thiolate as the fifth ligand of the heme plane at P450 active sites. The presence of a heme binding motif in a predicted amino acid sequence and the characteristic absorption spectrum with a maximum at 450 nm in the recombinant enzyme provide definite evidence that a cloned gene encodes a member of the cytochrome P450 family of enzymes. 9 In plants, cytochrome P450 enzymes are involved in the biosynthesis of extremely diverse metabolites (e.g. fatty acids, phenylpropanoids, alkaloids, terpenoids, and hormones) and in the processes of herbicide or pesticide detoxification (Bolwell et al., 1994; Chappie, 1998). Completion of the Arabidopsis genome sequence reveals that there are 273 P450 genes, falling into 45 distinct gene families ( Thus, in the model plant Arabidopsis, approximately 1.2% of the protein-coding genes belong to cytochrome P450 super-family. The numbers of P450s in the completed genomes from other organisms are 2 for Saccharomyces cerevisiae, 3 for Saccharomyces pombe, 56 for human, 86 for Drosophila melanogaster, and 56 for Caenorhabditis elegans (Nelson, 1999; The small number of P450s in yeast indicates that P450 enzymes do not play key roles in primary metabolism required for a basic unicellular physiology. Functional characterization of human P450s has shown that some P450s are involved in steroid hormone biosynthesis, but their major functions are to detoxify numerous environmental xenobiotics and phytochemicals. In contrast, functional characterization of a limited number of plant P450s has revealed that plant P450s have functions in the biosynthesis of the tremendous diversity of plant secondary metabolites, many of which are correlated with chemical defenses against animals, insects, and microbes. The functional comparison of animal and plant P450s suggests that there has been extensive chemical warfare between animals and plants through million years of evolution. Interestingly, both toxin-synthesizing and detoxifying reactions employ P450 enzymes. The large number of plant P450s exemplified by Arabidopsis indicates that plants are under strong pressure to evolve novel P450 genes. P450 gene duplications are evident from the presence of many loci with clustered, similar P450 genes in the Arabidopsis genome (Paquette ef al., 2000; This may suggest the on-going evolution of new arrays of P450s in plants by gene duplication. Due to the enormous sequence diversity of P450s in different organisms, classifications of P450 enzymes are strictly based on primary sequence comparison. P450s with less than 40% amino acid identity to other known P450s are accepted as members of a new P450 family, while those with less than 55% identity create a new subfamily of the closest P450 family. Based on this nomenclature, the standard P450 gene annotation for Arabidopsis C4H is CYP73A5. CYP represents cytochrome P450, and 73 and A indicate gene family and subfamily names, respectively. The last number (5) simply indicates an annotation number assigned to a specific P450 gene that belongs to the CYP73A group, in 10 order of gene discovery in different species. When the P450 nomenclature system was established in the early 1980s, designated ranges of numbers were assigned to each kingdom so that the P450 family numbers CYP70 to CYP99 were allocated to plant P450s. However, due to the explosion of plant P450 sequences from genomic data, new plant P450 family numbers started being assigned from CYP701. A phylogenetic analysis of Arabidopsis P450s shows that two distinct groups of P450s are encoded in the Arabidopsis genome. The majority of Arabidopsis P450 genes (153/246) used for phylogenetic analysis are part of a single lineage. The presence of one highly conserved intron in this group (Paquette ef al., 2000) suggests that they evolved from a common ancestor. This group of the phylogenetically related genes is plant-specific and is named the A-type family of P450s (Durst and Nelson, 1995). However, the remaining P450 genes (93/246) form several different clades with different numbers of introns. P450s within this class are collectively designated "non-A" P450s. Several families within this non-A group of P450s catalyze reactions similar to those of P450-mediated steroid biosynthesis in animals. Thus, it is likely that non-A P450s share a common origin with animal and fungal P450s, and these P450s are not specific to plants. An important task in P450 research is to identify biochemical functions of the enzymes encoded by this large number of P450 genes in Arabidopsis and other plant species. Predicting candidate substrates for P450s based on phylogenetic analysis (relatedness to enzymes with known functions) is possible but certainly limited to the P450s whose gene families are closely related to already characterized P450 families. Circumventing these difficulties, many plant P450 functions have been identified by screening of mutant lines defective in a specific metabolic step. Sets of important P450s involved in gibberellin (GA) and brassinosteroid biosynthesis were identified from mutant lines (Bishop ef al., 1999; Choe ef al., 2001; Helliwell ef al., 2001a; Helliwell ef al., 1998). It is interesting to note that, in the complex pathways of gibberellin biosynthesis, two multifunctional P450s (CYP701A and CPY88A) catalyze the conversion from enf-kaurene to G A i 2 , which involves six distinct biochemical reactions (Helliwell etal., 2001a; Helliwell et al., 1999). Mutation analysis of P450s in Arabidopsis sometimes suggested the presence of unknown plant growth substances that are directly synthesized or regulated by P450 mediated reactions in specific organs. The Rotundifolia3 (Rot3) mutant of Arabidopsis has a lesion in the CYP90C gene and is defective in the polar elongation of leaf cells (Kim ef al., 1998). The overexpression of this gene in Arabidopsis causes significantly elongated leaves 11 (Kim ef al., 1999). Although CYP90C encodes a sequence similar to P450s required for brassinosteroid biosynthesis, external application of brassinolide did not rescue the mutant phenotype, suggesting that CPY90C is involved in the biosynthesis of a so far undiscovered hormone. The activation of CYP78A9 by a 35S-enhancer results in elongated and wider fruit in Arabidopsis (Ito and Meyerowitz, 2000). Expression of this gene is very specific to fruit, and no known hormone has been reported to cause this phenotype. This was interpreted such that an unknown growth substance regulating the shape and size of the seed pod is likely to be synthesized by CYP78A9 in Arabidopsis. The combinations of biochemical and genetic approaches are expected to unravel the functional identities of many P450 genes from diverse plant sources. Sequence information from a large number of functionally characterized P450 genes in the future will greatly facilitate the engineering of novel P450 enzymes for research and industrial applications. 1.5 Recent advances in lignin biosynthesis Lignin is a complex heterogeneous polymer formed by oxidative linkages of three types of lignin monomers (monolignols); 4-coumaryl, coniferyl, and sinapyl alcohol which constitute p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) moieties of lignin, respectively (Figure 1.2). The composition of the three monomers in lignin varies between species, at different developmental stages or in response to mechanical stress within a plant, and even at subcellular level (Lewis and Yamamoto, 1990). However, it is not known how these compositional variations are controlled in various plants in different situations. Because the area of lignin research is very broad, I will only discuss the recent progress in lignin biosynthesis within angiosperms, focusing on the key enzymes that are proposed to control guaiacyl and syringyl lignin composition. Since expensive and polluting chemical processes are required to remove the undesirable lignin component during the pulping process, alteration of its content and composition has long been the subject of extensive research. Suppression of the key lignin biosynthetic genes has shown that it is feasible to substantially reduce lignin content without severe physiological defects in several cases (for review, see Dixon ef al., 2001). In particular, the reduction of 4CL activity down to 10% of wild-type levels by antisense expression in transgenic aspen results in a 55% decrease in lignin content with a concomitant increase in cellulose content of up to 15% (Hu ef al., 1999). This illustrates the potential of genetic engineering of woody plants to create beneficial traits. 12 COOH H-lignin G-lignin S-lignin Figure 1.2 Proposed lignin biosynthetic pathway in plants. This lignin biosynthetic pathway was constructed based on recent mutant and enzyme charac-terizations in Arabidopsis and poplar by several groups (Meyer et al, 1996; Meyer et al, 1998; Humphreys et al, 1999; L i et al, 2000; Guo et al, 2001; L i et al, 2001; Schoch et al, 2001; Franke et al, 2002). Grey arrows and metabolites indicate a previous lignin biosynthetic path-way utilizing free phenolic acid forms. Empty-arrows indicate cytochrome P450-mediated reactions, and black-lined box indicates proposed syringyl specific pathway (Li et al, 2001). 13 However, suppression of core phenylpropanoid genes has led to unexpected and confusing results in terms of the compositional alteration of lignin. A decrease of 4CL activity to 8% of wild-type levels in Arabidopsis significantly decreases G units but does not affect S units (Lee ef al., 1997), whereas the same approach in tobacco resulted in a decrease of S units (Kajita ef al., 1996). Tobacco plants with down-regulated PAL or C4H activities differ in their lignin composition in that P^\L-suppressed lines have an increased S/G ratio, while C4H-suppressed ones have a decreased S/G ratio. This was interpreted as showing that the biosynthetic fate of carbon destined for guaiacyl or syringyl lignin is determined at the beginning of phenylpropanoid pathway, potentially by multienzyme complexes involving PAL and C4H (Sewalt ef al., 1997). It has been believed that a series of hydroxylations and methylations occurs on p-coumarate and its derivatives to synthesize various hydroxy/methoxy cinnamic acids, which can be used for substrates for the synthesis of different monolignols. However, this classical model for lignin biosynthetic pathway is greatly challenged by discoveries of new P450 enzymes in recent years. It has been shown that the enzymes encoded by two key Arabidopsis P450 genes in lignin biosynthesis, previously named p-coumaric acid 3-hydroxylase (C3H) and ferulic acid 5-hydroxylase (F5H), cannot use free phenolic acids as substrates (Humphreys ef al., 1999; Schoch et al., 2001). Arabidopsis mutants for the P450 gene F5H are unable to synthesize sinapic acid-derived metabolites including syringyl units in lignin (Chappie ef al., 1992; Meyer ef al., 1996), whereas the overexpression of F5H in Arabidopsis, tobacco, and aspen leads to accumulation of lignin with increased syringyl lignin content (Franke ef al., 2000; Meyer ef al., 1998). This genetic evidence indicates that F5H participates in the biosynthesis of the syringyl units of lignin in angiosperms. However, the recombinant F5H enzyme from Arabidopsis cannot convert ferulic acid to 5-hydroxyferulic acid but efficiently catalyzes conversion of coniferaldehyde and coniferyl alcohol to 5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol, respectively (see Figure 1.2; Humphreys ef al., 1999). Further confirming these data, the recombinant F5H enzyme from aspen also shows high substrate specificity for coniferaldehyde but not for coniferyl alcohol (Osakabe ef al., 1999). Therefore, the 5-hydroxylation of the phenolic ring is most likely to occur at the level of aldehyde or alcohol in Arabidopsis and aldehyde in aspen. This unexpected catalytic behavior raises a question about the biosynthetic route for sinapic acid which is used as a precursor for compounds such as sinapoyl malate in Arabidopsis (Figure 1.2). 14 Recent homology based screening of P450s from the Arabidopsis genome database led to the isolation of a C3H cDNA clone. However, the recombinant C3H enzyme has little or no activity towards free p-coumarate and/or its conjugates, but surprisingly specifically converts 5-O-shikimate and 5-O-D-quinate esters of p-coumarate to caffeoyl shikimic acid and chlorogenic acid, respectively (Schoch ef al., 2001). Members of the same cytochrome P450 gene family (CYP98A) are also found as ESTs in poplar xylem (Sterky ef al., 1998). Antibodies specific to C3H located a high accumulation of this protein in differentiating vascular tissue (Schoch ef al., 2001). The Arabidopsis mutant ref8, lacking both G and S lignin as well as sinapoylmalate, was revealed to encode defective C3H gene, which confirms the biological function of this P450 enzyme in vivo (Franke ef al., 2002). The functional identification of these new P450 genes indicates that free phenolic acids other than p-coumarate may be not true intermediates for lignin biosynthesis in plants, and that p-coumaric acid may be the only in situ substrate for 4CL. Once the hydroxylation of the phenolic ring at 3- and/or 5-position occurs, O-methylation of hydroxyl groups is catalyzed by O-methyltransferases using S-adenosyl-L-methionine as a methyl donor. In angiosperms, COMT was originally proposed to be a bifunctional enzyme catalyzing O-methylation of both caffeic acid and 5-hydroxyferulic acid to ferulic acid and sinapic acid, respectively (Davin and Lewis, 1992). Similarly, O-methyltransferase activities at the level of CoA-conjugated phenolic acids (CCoAOMT) have also been shown to be important in monolignol biosynthesis (Pakusch etal., 1989; Ye et al., 1994). However, due to the unexpected catalytic activities of the P450 reactions in lignin biosynthetic pathway, the authentic biochemical identity of the O-methyltransferase reaction catalyzed by COMT has been questioned and recently reevaluated in aspen and alfalfa (Guo etal., 2001; Li etal., 2000). One obvious substrate for COMT is clearly 5-hydroxyconiferaldehyde, a product of F5H reaction (see Figure 1.2). A kinetic analysis of the aspen COMT recombinant enzyme shows that 5-hydroxyconiferaldehyde is preferred over caffeic acid and 5-hydroxyferulic acid (31 and 5 times higher V m a x / K m values, respectively) as a substrate for COMT (Li ef al., 2000). An interesting finding is that 5-hydroxyconiferaldehyde acts as an inhibitor for the O-methylation of 5-hydroxyferulic acid, but free phenolic acids are not inhibitors for the O-methylation of 5-hydroxyconiferaldehyde by COMT. This suggests that O-methylation does not occur at the level of free phenolic acids when a pool of 5-hydroxyconiferaldehyde is present in situ. This COMT enzyme kinetic data are further supported in vivo by the down-regulation of COMT in alfalfa. These plants have lignin with reduced guaiacyl subunits and a 15 complete lack of syringyl lignin (Guo et al., 2001). In these plants, the lack of accumulation of either caffeic acid or caffeoyl glucose and the concomitant incorporation of novel 5-hydroxyguaiacyl unit into lignin also support the finding that caffeic acid is not a true substrate in vivo for COMT in alfalfa. This in vivo alteration of lignin composition provides conclusive evidence for a F5H and COMT-coupled pathway for syringyl lignin biosynthesis at the level of coniferaldehyde (Figure 1.2). In the last step of monolignol biosynthesis, cinnamyl alcohol dehydrogenase (CAD) catalyzes the conversion of coniferaldehyde and sinapaldehyde to coniferyl alcohol and sinapyl alcohol. Gymnosperm CAD activity is specific to coniferaldehyde (Galliano ef al., 1993; O'Malley et al., 1992), while angiosperm CAD is known to catalyze dehydrogenation of both coniferaldehyde and sinapaldehyde (Grima-Pettenati et al., 1994). Supporting this idea, recombinant protein encoded by a Eucalyptus gunnii CAD gene can use both of the substrates equally well (Grima-Pettenati ef al., 1993). However, a decreased S/G ratio mainly due to reduced S lignin has been reported in C/\D-suppressed alfalfa plants, indicating that sinapaldehyde specific CAD enzymes may exist in angiosperms (Baucher ef al., 1999). A divergent form of CAD in aspen, which shares -50% amino acid identity to the previous PtCAD gene and shows substrate preference to sinapaldehyde, was recently identified and named as sinapyl alcohol dehydrogenase (PtSAD) (Li ef al., 2001). The catalytic efficiency of SAD towards sinapaldehyde is 60-fold higher than that towards coniferaldehyde. Immunolocalization studies show that PtCAD is exclusively localized to guaiacyl-rich tracheary elements in the xylem, while PtSAD is abundant in the phloem fiber cells enriched in syringyl lignin. Therefore, angiosperms appear to have evolved a specific set of genes, consisting of F5H, COMT, and SAD that can coordinately redirect metabolic flux from coniferaldehyde to synapyl alcohol. 1.6 Coordinated regulation of phenylpropanoid gene expression Expression of genes in the phenylpropanoid pathway is generally controlled at the transcriptional level (Chappell and Hahlbrock, 1984). External stimuli such as pathogen infection and irradiation with UV light lead to the rapid transcriptional induction of genes in the general phenylpropanoid pathway and branch pathways required for the synthesis of particular stress-related phenylpropanoid compounds. Since individual plant families deploy different arrays of antimicrobial phytoalexins and other defensive phenylpropanoid metabolites, distinct sets of phenylpropanoid genes are induced in different plants. In many members of Leguminoseae in which isoflavonoids serve as phytoalexins, elicitor treatment 16 causes a massive induction of PAL, 4CL, CHS, and CHI transcripts (Ebel and Grisebach, 1988; Lawton and Lamb, 1987; Mehdy and Lamb, 1987). However, in poplar, parsley, and Arabidopsis, CHS transcripts are not induced by elicitor treatment, most likely due to their lack of flavonoid-derived phytolexins (Dong et al., 1991; Kuhn ef al., 1984; Ro ef al., 2001). Instead, in elicitor treated parsley cells, genes encoding SAM:bergaptol O-methyltransferase together with general phenylpropanoid genes are transcriptionally activated to synthesize furanocoumarin phytoalexins (Hahlbrock and Scheel, 1989). On the other hand, CHS and general phenylpropanoid genes in many plants are strongly and coordinately induced by UV light, consistent with the UV-protective role of flavonoid compounds in many plants (Hahlbrock and Scheel, 1989). The tight coordinated expression of genes that eventually lead to the production of confined functional phenylpropanoids implies that the pathway-specific regulatory mechanisms are present that control expression of phenylpropanoid genes. PAL and 4CL enzymes are always encoded by gene families in plants. In some cases, specific genes encoding PAL and 4CL isoforms are specifically induced by different signals or are expressed in specific tissues. For example, parsley PAL4 is not induced by UV light, while PALI, 2, and 3 are rapidly induced, and this PAL isoform is activated by wounding stress only in root but not in leaf tissue (Logemann ef al., 1995a). Arabidopsis 4CL3 does not respond to elicitor-treatment but is strongly induced by UV irradiation (Ehlting ef al., 1999). As an example of differential developmental regulation of phenylpropanoid gene family members, genes encoding two distinct poplar PAL and 4CL isoforms are differentially expressed in young leaf or developing secondary xylem (Cukovic ef al., 2001, Chapter5; Hu etal., 1998; Osakabe etal., 1995). Genes encoding enzymes in primary metabolic pathway are dramatically up-regulated by elicitor or wound treatment in coordination with phenylpropanoid genes (Batz ef al., 1998; Gorlach et al., 1995; Lee ef al., 1997), while cell cycle regulatory genes are significantly repressed (Logemann et al., 1995b). To a lesser extent, UV-light treatment also causes an induction of genes in primary metabolism (Logemann ef al., 2000). These data indicate that primary metabolism is also extensively altered by elicitor or UV-light stress in plants. The coordinated regulation of primary and secondary metabolism gene expression is apparently essential to supply phenylalanine as a substrate for phenylpropanoid metabolism. The importance of phenylalanine supply from primary metabolism is shown by the observation that small changes in the activity of plastid transketolase cause dramatic effects on carbon flux into the phenylpropanoid pathway (Henkes ef al., 2001). 17 Numerous studies on the phenylpropanoid gene expression make it evident that signal transduction pathways activated by external and internal signals play key roles in regulating the phenylpropanoid metabolism. Extensive biochemical, molecular, genetic, and genomic approaches have been performed to identify transcription factors and their binding sites within promoters of phenylpropanoid genes that regulate expression of these genes. Fusions of PAL, C4H, 4CL, and CHS promoters to the GUS reporter gene dictate cell- and tissue-type specific and stress-inducible expression in GUS reporter gene in transgenic plants, showing that these promoters contain the information necessary to control their developmentally regulated expression (Bell-Lelong ef al., 1997; Hauffe ef al., 1991; Liang ef al., 1989; Schmid ef al., 1990). Combinations of in vivo footprinting and promoter mutation analyses have identified segments of promoter regions where putative transcription factors that control expression of these genes bind (Hatton ef al., 1995; Hauffe ef al., 1993; van der Meer et al., 1990; Wingender et al., 1990). The parsley 4CL promoter is one of the most well characterized phenylpropanoid gene promoters and contains a unique 16-bp c/'s-element in the FP56 (footprint 5 and 6) element, as well as adenine/cytosine-rich 'P box' (Hauffe et al., 1993; Hauffe ef al., 1991; Neustaedter ef al., 1999) required for developmentally regulated expression. Several proteins directly or indirectly interact with the short FP56 c/s-segment, forming a protein-DNA complex which appears to determine important cell- and tissue-type specific expression in plants (Neustaedter ef al., 1999). Transcription factors of the MYB class are particularly interesting with respect to their roles in controlling phenylpropanoid gene expression. The first plant MYB regulatory gene was discovered in Zea mays, based on a mutation defective in anthocyanin pigment biosynthesis (Paz-Ares ef al., 1987). MYB genes controlling phenylpropanoid gene expression have subsequently been documented in many plants. For example, ectopic expression of two Antirrhinum majus MYB genes (AmMYN308 and AmMYB330) in tobacco results in repression of C4H, 4CL, and CAD gene expression, and in reduced phenolic acid and lignin biosynthesis, perhaps due to their dominant, negative effect on endogenous tobacco MYB transcription factors (Tamagnone et al., 1998). Overexpression oi Arabidopsis AtMyb4 specifically represses C4H expression, while not affecting other phenylpropanoid genes, and a AtMyb4 mutant exhibits derepressed C4H expression, resulting in enhanced sinapyl ester accumulation (Jin ef al., 2000). However, other transcription factors in addition to those of the MYB class are also important in regulation of phenylpropanoid gene expression. Two parsley transcriptional factors, a basic leucine zipper (bZIP) transcription factor and PcMyb l , bind to the two core 18 c/s-elements of CHS promoter, designated A C E C H S and MRECHS, which are necessary and sufficient for UV-light induction (Feldbrugge ef al., 1994; Feldbrugge ef al., 1997). When the putative tobacco transcription factor Ntliml (member of the LIM protein family that contains a zinc finger motif) was down-regulated by anti-sense in transgenic tobacco, the lignin biosynthetic genes PAL, 4CL, and CAD were repressed, resulting in reduced lignin content (Kawaoka ef al., 2000), suggesting that this transcription factor plays a key role in the coordinated developmental regulation of these genes. Alteration of phenylpropanoid gene expression and phenylpropanoid product accumulation by MYB and other transcription factors shows that specific phenylpropanoid pathways can be controlled by manipulating key transcription factors. However, fine-tuning of phenylpropanoid metabolism, in response to environmental and developmental cues, may not be ruled by a single regulatory factor but controlled by complex DNA-protein and protein-protein interactions involving multiple proteins including MYB transcription factors. 1.7 Phenylpropanoid multienzyme complexes It has been proposed that the sequential enzymes of metabolic pathways are not randomly distributed in cells, but instead are highly organized in the form of multienzyme complexes (MEC), or metabolons (Hrazdina and Jensen, 1992; Srere, 1987). Harsh homogenization of cellular contents, a prerequisite for an in vitro enzyme assay or purification, might destroy such cellular complexes maintained by weak protein-protein associations. MECs might be the most vulnerable in vivo structures disturbed by standard protein purification procedures. The physicochemical environment of intact cells is quite different from that of in vitro enzyme assays. Proteins are much more concentrated in the intracellular and intraorganelle environment (up to 370 mg/ml), and the pool size of substrates is generally low (Mathews, 1993). In contrast, total protein concentrations in in vitro systems, are enormously diluted, and enzyme assays are carried out at substrate saturation. In particular, plant cells, where vacuoles sometimes constitute 90% of the total cellular volume, might strongly constrain the cytosolic space. In such conditions, there may not be enough free water for the random diffusion of enzymes and substrates, conditions on which theoretical enzyme kinetics are based (Jarvis etal., 1990). Introduction of the concept of "MEC and metabolite channeling" into metabolism can rationalize the discrepancies between the limited cellular water potential and the high efficiency of metabolic reactions in situ. Formation of MECs may be promoted by specific surface interactions between enzymes that carry out consecutive reactions, leading to an 19 increase in catalytic efficiency through metabolite channeling because of decreased diffusion times or direct transfer of intermediates. Therefore, cells might be composed of mosaics of metabolic enzyme complexes and substrates involved in sequential metabolic pathways. Enzymes may have evolved not only their catalytic sites, but also their surfaces to facilitate protein-protein interactions among the consecutive enzymes (Edwards, 1996). There are several experimental data supporting the existence of MECs in phenylpropanoid metabolism in plants. On the basis of double radioactive-labeling assays to assess metabolic channeling, the possibility that PAL and C4H from cucumber and green algae are assembled in a MEC was originally reported (Czichi and Kindl, 1975; Czichi and Kindl, 1975; Czichi and Kindl, 1977). When 3H-phenylalanine and 1 4C-cinnamate were simultaneously added as substrates to microsomal fractions isolated by sucrose density gradient centrifugation, 3H-labeled cinnamate derived from 3H-phenylalanine was preferentially used as a substrate for C4H over exogenously provided 1 4C-cinnamic acid. This result lead to the idea of a tight coupling of the two enzymes on the cytoplasmic face of the ER membrane, resulting in a direct metabolite transfer of the cinnamate intermediate. In support of this hypothesis, enzyme activities for core phenylpropanoid and flavonoid biosynthetic reactions are reported to be associated with the ER (Hrazdina and Wagner, 1985). In particular, PAL, C4H and CHS enzymatic activities co-purified in the ER fraction from gently homogenized buckwheat hypocotyl extracts. To obtain direct evidence for the ER-localization of CHS, immunohistochemical investigations were performed, which were interpreted as showing that significant amounts of CHS are associated with the cytoplasmic face of the ER (Hrazdina et al., 1987). These biochemical and immunohistochemical analyses suggest that the C4H anchors otherwise soluble PAL and possibly other phenylpropanoid enzymes on the ER surface to organize a MEC. Investigation of possible MECs in phenylpropanoid metabolism has been further pursued in recent years using various molecular and biochemical techniques. Direct protein-protein interactions among enzymes in the flavonoid pathway (e.g. CHS, chalcone isomerase, dihydroflavonol 4-reductase, and flavanone 3-hydroxylase) were demonstrated by yeast two hybrid assays (for soluble enzymes), affinity chromatography, and co-immunoprecipitation (Burbulis and Winkel-Shirley, 1999). Since the protein-interactions among flavonoid enzymes in yeast two hybrid assays were detectable only with certain combinations of fusion proteins, the macromolecular protein complex, if present in plants, is likely to be of globular organization, rather than in a consecutive linear form. The proposed model for a MEC in flavonoid metabolism is that the P450 enzyme flavonoid 3'-hydroxylase 20 (F3'H) serves to anchor cytosolic enzymes on the cytoplasmic face of the ER membrane. F3'H is encoded by a single-copy gene in Arabidopsis, and an Arabidopsis mutant (tt7) with a cytosolic domain-truncated F3'H has been isolated (Saslowsky and Winkel-Shirley, 2001). The Arabidopsis tt7 mutant was used to test the hypothesis that F3'H anchors soluble flavonoid enzymes on the ER membrane. However, localization patterns of CHS and CHI in the tt7 mutant background were not affected compared to the patterns in wild type plants (Saslowsky and Winkel-Shirley, 2001), suggesting that other proteins must anchor the putative complex. Within the isoflavonoid biosynthetic pathway, in vivo assays have recently shown that alfalfa isoflavone O-methyltransferase (IOMT) relocates from the cytoplasm to endomembranes following elicitor-treatment (Liu and Dixon, 2001). This conditional ER localization of IOMT seems to facilitate the formation of a M E C containing 2-hydroxyisoflavone synthase (2-HIS), which ensures regio-specific methylation on the 4'-position of the isoflavonoid B-ring. This coupled reaction of 2-HIS and IOMT, by not allowing leakage of the unstable intermediate, 2,4',7-trihydroxyisoflavanone, for synthesis of the isoflavonoid isoformononetin, guarantees the biosynthesis of formononectin, which is then used as a substrate for synthesis of medicarpin, a phytoalexin in alfalfa and other legumes. Recent investigations have also focused on the potential MEC at the entry point to phenylpropanoid metabolism hypothesized to contain PAL and C4H. In some cases, indirect support for such a MEC has been obtained from the phenotypes of transgenic plants with down-regulated enzyme activities. Downregulation of PAL or C4H activity in tobacco by introduction of heterologous antisense or sense PAL and C4H genes decreases total lignin content. Interestingly, the ratio of S/G lignin subunits in PAL-suppressed tobacco plants is increased relative to wild-type, while that of C4H-suppressed tobacco plants is decreased (Sewalt ef al., 1997). This unpredicted alteration of S/G ratios was interpreted to indicate that particular isoforms of PAL or C4H may be involved in different MECs that channel carbon flux specifically into guaiacyl or syringyl lignin subunits. In tobacco stem tissue and cultured cells, the existence of a potential MEC containing PAL and C4H was reevaluated in vivo and in vitro by isotope dilution and the double-labeling assay used 20 years earlier (Rasmussen and Dixon, 1999). Using an in vivo assay, these authors showed that 3H-labeled phenylalanine is efficiently channeled to p-coumarate, and that the intermediate 3H-labeled cinnamate does not equilibrate with exogenously fed cinnamate, consistent with the existence of a MEC containing PAL and C4H that efficiently channels the cinnamate to C4H. Interestingly, parallel immunoblot 21 analysis performed to determine the subcellular localization of PAL showed that tobacco PAL1 is localized in both soluble and microsomal fractions, while PAL2 is only found in the soluble fraction, suggesting that different PAL isoforms may differentially associate with the putative MEC. 1.8 Phenylpropanoid metabolism in poplar and thesis objectives Populus species (poplars, cottonwoods, and aspens) provide models for molecular and genetic studies of tree biology because of their small genomes, ease of vegetative propagation, developed transformation systems, and genetic resources (Sterky et al., 1998, and references therein), and an EST genome project has been initiated in Populus (Sterky ef al., 1998). Of particular interest in woody plants is phenylpropanoid metabolism which channels a large amount of carbon into lignin biosynthesis. When this project was initiated, core phenylpropanoid genes encoding PAL and 4CL had been cloned and characterized from several Populus species (Allina ef al., 1998; Hu ef al., 1998; Osakabe ef al., 1995; Subramaniam ef al., 1993). However, important redox enzymes, C4H and CPR, had not been characterized in poplar. Considering the key biochemical reaction of C4H and its potential structural role in MECs, it is essential to fully characterize C4H together with its redox partner CPR. Thus, the objectives of the first part of this thesis (Chapters 3 and 4) are to functionally characterize C4H and CPR and to determine their subcellular locations. Despite strong arguments for the functional significance of potential MECs at the early stages of phenylpropanoid metabolism for channeling of carbon from primary metabolism into phenylpropanoid products, there is currently no definite proof to substantiate the physical existence or physiological roles of such proposed MECs in plants. A complete set of cDNAs for entry point enzymes (e.g. PAL, C4H, and CPR) was obtained from previous work and from this project (Chapter 3 and 4). In order to investigate how these enzymes interact biochemically and physically to channel carbon from primary to phenylpropanoid pathway, I reconstructed the entry point enzymes in Saccharomyces cerevisiae. In Chapter 5, using this novel system, I tested the hypotheses that PAL and C4H can efficiently redirect carbon from primary metabolism to phenylpropanoid pathway in a heterologous host, and that MEC plays an important role in directing intermediate channeling between PAL and C4H. 22 Chapter 2 Materials and Methods 2.1 General nucleic acid methods 2.1.1 Plasmid DNA preparation and DNA sequencing Plasmid DNA was prepared by alkaline lysis according to standard methods (Sambrook et al., 1989), or by use of Qiagen spin Miniprep- or Maxiprep-kits, following the manufacturer's protocol (Qiagen). DNA sequencing was carried out by automated Prism Cycle Sequencing at the University of British Columbia Nucleic Acid and Protein Service unit. 2.1.2 Genomic DNA and total RNA isolation For isolation of poplar genomic DNA, one gram of frozen poplar (P. trichocarpa X P. deltoides clone H11-11) young leaf was ground to a fine powder in liquid nitrogen, and the Nucleon PhytoPure beads (Amersham-Pharmacia) were used to retrieve DNA, according to the manufacturer's instructions. For isolation of poplar total RNA from young leaves, old leaves, and green stem, the modified Nucleon PhytoPure method (Kiefer et al., 2000) was used, except that approximately 300 mg starting material was used, and a repeated Nucleon PhytoPure (50 uL) wash-step was incorporated. Total RNA was isolated from poplar frozen xylem or from cultured cells using Trizol reagent (Gibco-BRL) according to the manufacturer's instructions. For total RNA isolation from yeast, cell pellets from 50 mL cultures of WAT11 yeast strain after 15 h galactose-induction were ground in liquid nitrogen, and extracted with the Trizol reagent. 2.1.3 Isolation of CPR cDNA clones A cDNA library from hybrid poplar H11-11 young leaf or xylem mRNAs was previously constructed in A.ZAPII (Subramaniam et al., 1993; Y. Tsutsumi, Shizuoka University, Japan and C.Douglas, unpublished) according to the manufacturer's instructions (Stratagene). A mixture of two Arabidopsis thaliana CPR cDNA clones (AR1 and AR2; Mizutani ef al., 1998) was radiolabeled by a random priming kit (Gibco-BRL), and these probes were used to screen for CPR cDNA clones in the xylem cDNA library (105 pfu total). Hybridization of the probes was performed for 16 h at 45°C in buffer containing 1% BSA, 7% SDS, 50 mM sodium phosphate (pH 7.5), and 1 mM EDTA. A final wash was performed for 20 min at 23 45°C in 2 X S S C with 0.1% SDS. Three 1.8-kb clones isolated were confirmed to encode an identical 5'-deleted partial CPR sequence that was very similar to known plant CPRs. One of these clones (CPR181) was used to re-screen the H11-11 young leaf cDNA library for a full-length clone, using the following high-stringency conditions: hybridization, 16 h at 65°C in the same buffer as above; final wash, 20 min at 65°C in 0.2 X S S C with 0.1% SDS. Among thirty positive clones, the longest clone isolated and sequenced was a 1.4-kb 3'-deleted clone (CPR122). To obtain a full-length CPR cDNA, a forward (5'-GCTAGTGACCACGTTT-CAAGACAAC-3 ' ) and a reverse (5 ' -AGCTTTAATTCTACCTTGACACCTGG-3') primer were designed from the 3'- and 5'- untranslated regions of CPR122 and CPR181 partial cDNA clones, respectively. These primers were used to amplify a full-length C P R cDNA from a cDNA pool of H11-11 xylem by PCR in a 50uJ reaction mixture containing 20 pmol each primer, 2.5 units Pfu polymerase (Stratagene), 100 ng xylem cDNA template, 0.2 mM dNTP, and 2 mM M g 2 + . The PCR conditions used were: one cycle of 3 min at 94°C, 25 cycles of 1 min at 94°C, 1 min at 62°C, and 3 min at 72°C. The PCR-product was incubated with 5 units of Taq polymerase (Gibco-BRL) at 72 °C for 10 min to add overhang adenine nucleotides. The 2.5-kb PCR products were purified from the gel, and cloned into a pCR2.1 vector using a PCR TA-cloning kit (Invitrogen), according to manufacturer's instructions. This CPR clone was named CPR1 . Other CPR isoforms were recovered from the poplar young leaf cDNA library, using an AR2 coding region as a hybridization probe under low stringency conditions, as described above. As a result, thirty-eight positive clones were identified, which were classified into two groups based on restriction fragment analyses using Hindlll and Pvull. The longest clones from each group were completely sequenced, and named CPR2 and CPR3. The GenBank accession number for CPR1 is AF302496, for the CPR2 is AF302497, and for CPR3 is AF302498. 2.1.4 Isolation of a PAL cDNA Two forward primers encompassing the immediate 5' start codon of the P.kitakamiensis (Osakabe ef al., 1995) PALg2a or PALg2b genes were designed: for PALg2a, 5'-GGCATGGCGGCAGACTCACTAA-3 ' , and for PALg2b, 5'-AAAATGGAATTTTGTCAAGAT-TCACG-3 ' . PALg2a and PALg2b share identical sequences around their stop codons, and a single reverse primer was designed, 5 ' -AGCTTAGCAAATAGGAAGAGGAGC-3 ' . Using each of the forward primers together with the reverse primer, two independent PCR-amplifications were performed to clone PALg2a- or PALg2b-\\ke cDNAs from a pool of H11-24 11 xylem cDNA using the PWO polymerase (Roche Molecular Biochemicals). The PCR products were incubated at 72 °C for 10 min with 1 unit of Taq polymerase (Gibco-BRL) to add adenine overhangs, and ligated into a pCR2.1 vector using a PCR TA cloning kit (Invitrogen). Ten independent clones derived from each primer set were subjected to EcoRI, Pvull, and Hindi 11 digestions. These restriction enzymes are predicted to distinguish the two PAL isoforms, based on the restriction enzyme map of PALg2a and PALg2b sequences. However, all clones from both PCR reactions showed identical PALg2b-\'\We digestion patterns. A search for the P/\L£[2a-like digestion patterns from eighteen more independent clones only revealed P/4Lg2b-like patterns. Subsequent comparison of the 5' regions of PALg2a and PALg2b showed that the PALg2b contains a sequence identical to the PALg2a forward primer binding site 76-bp downstream of its start codon, and thus the primer intended to amplify a P/\Lg2a-like gene seemed to amplify instead a 76-bp shorter fragment from a P/ALg2b-like gene represented in the poplar xylem cDNA pool. One of the poplar P/4Lb2b-like cDNA clones, designated PAL18, was fully sequenced and the corresponding gene named PAL4 to distinguish it from previously characterized poplar clone H11 genes PAL1, 2,and 3. 2.1.5 Northern blot analysis Ten pg of total RNA was resolved on 1.5% formaldehyde agarose gels. Gels were post-stained in 0.5 pg/mL ethidium bromide for 1 h, and the fluorescent signals were photographed under the UV trans-illuminator to confirm the integrity of ribosomal RNA. The gel was equilibrated in a TE buffer (pH 7.5) containing 10 mM Tris and 1 mM EDTA. A capillary blotting apparatus was assembled in 10 X SSC buffer (1.5 M NaCl and 0.3 M Na 3-citrate) to transfer RNA onto Hybond XL membranes (Amersham-Pharmacia), according to standard methods (Sambrook et al., 1989). After disassembly, the blot was washed in 2 X S S C , dried on a piece of 3M Whatman paper for 1 h, and baked at 80 °C for 1 h. Radioactive probes were prepared using appropriate templates using a random priming kit (Gibco-BRL). Probe hybridization was performed overnight at 65°C in a buffer containing 1% BSA, 7% SDS, 50 mM sodium phosphate (pH 7.5), and 1 mM EDTA. Membranes were washed twice for 20 min at 65°C in 2 X SSC with 0.1% SDS. The final wash was performed at 65°C in 0.2 X S S C and 0.1% SDS for 1 h. Radioactive signals were detected using a phosphorimager screen and a Molecular Dynamics "Storm" phosphorimager. If necessary, removal of the probes from the blot was achieved by pouring a boiling solution of 0.1 X SSC 25 and 0.1% SDS onto the blot. After the solution cooled to room temperature, the blot was washed briefly once in 2 X S S C and stored in dry conditions at 4 °C before further use. 2.1.6 Reverse transcriptase PCR (RT-PCR) expression analysis Total RNA and genomic DNA was prepared as described in 2.1.2 from poplar tissues and cell cultures. Gene-specific primers used to amplify a cDNA fragment of PAL1/2 were: a forward primer, 5 ' -GTTGCATCCATTGCTGGTCATGATAC-3 ' and a reverse primer, 5'-GAATCCAGATTCAATGCCAGCTGCTT-3 ' ; for CPR1, a forward primer, 5 ' -CCTAGCGAGG-CAGATAGACTCAAGT-3 ' and a reverse primer, 5 ' -TAGTTCATATCTCTGCTGCTCTATC-3' ; for CPR 2, a forward primer, 5 ' -TCATTATGATTGGCCCTGGAACTGGT-3 ' and a reverse primer, 5 ' -CAAGGCTTCAACGGAGTTAACTTTTG-3' , for CPR3, the same forward primer as for the CPR2 and a reverse primer, 5 ' -GGCTTCGGTATTTATAGAGTAAACTTT-3 ' . The specificities of the CPR primers were verified using 100 ng poplar genomic DNA or 1 ng PAL and C P R cDNA as templates and Taq polymerase (Gibco-BRL) with 30 cycles of PCR under the following conditions: 30 s denaturation at 94 °C, 30 s annealing at 62 °C, and 1 min polymerization at 72 °C. QIAGEN OneStep RT-PCR reagent was used to amplify specific cDNA fragments using 100 ng of total RNA from young leaves after optimization of cycle numbers for each gene such that product accumulation was in the exponential range. Using gene-specific primers, 543-bp PAL2, 669-bp CPR1, 516-bp CPR2, and 513-bp CPR3 fragments were amplified as estimated by agarose gel electrophoresis with 100-bp molecular markers (Gibco-BRL). For expression analysis, RT -PCR was performed according to manufacturer's instructions in 25 fxl final volume. The specific PCR conditions used were as follows: 30 s denaturation at 94 °C, 30 s annealing at 62 °C, and 30 s polymerization at 72 °C for 20 cycles (P.4L and CPR3) or 25 cycles (CPR1 and CPR2). The RT-PCR products were resolved on a 1.5% agarose gel (4 mm thickness), and stained for 30 min by Sybr Green I (Molecular Probes). A Storm Phosphorimager (Amersham-Pharmacia) was used to detect fluorescent signals on the gel. 2.2 Cloning procedures for gene expression 26 2.1.1 Host strains, plasmid, and phage E. coli Genotype Reference D H 5 a X L 1-Blue I N V a F ' supE44, AlacU169 (phi 80 lacZ A M 15), hsdR17, recAl , endAl , gyrA96, thi-1, relAl recAl , endAl , gyrA96, thi-1, hsdR17, supE44, re lAl , lac, [F', proAB + , lacl q, lacZ AM15::Tnl0 (tetr)] F'endAl , recAl , hsdR17, supE44, gyrAl , re lAl , phi 80, lacZAM15 A(lac ZYA-argF) Hanahan(1983) Stratagene Invitrogen Yeast Genotype Reference YPH499 W A T 11 M A T a , ura3-52, lys2-801, ade2-101, trpl-A63, his3-A200, Ieu2-Al M A T a , ura3-l, ade2-l, his3-l 1,-15, leu2-3, canR, cyr", Gall0-CYC1::ATR1 Sikorski and Hieter(1989) Pompon et al. (1996) Agrobacterium Genotype Reference GV3101 with pMP90-virulence plasmid, C59C1, R h * Van Labereke et al. (1974) Phage Construct Reference A.ZAPII pBluescriptSK-, A m p R , colElori, cl857, nin5 Short et al. (1988) Plasmid * Construct Reference pBluescriptSK pCR2.1 pBinl9 p R T l O l pSL1180 pYeDP60 p E S C - L E U pESC-HIS flori, colElori, LacZ, M C S , A m p R flori, colElori, LacZ, M C S , Kan R , A m p R Left border, LacZ, M C S , n p t l l , right border, RK2ori, K a n R colElori, P-CaMV35S, M C S , T-CaMV, A m p R flori, colElori, M C S , A m p R colElori, P - G a l l O : : C Y C l , M C S , T-PGK, URA3, A D E 2 , A m p R 2 a. ori, flori, P - G a l l , T-Cyc, P-GallO, T - A d h l , M C S , L E U 2 , A m p R 2 u. ori, flori, P - G a l l , T-Cyc, P-GallO, T - A d h l , M C S , H I S 3 , A m p R Alting-Mees et al. (1992) Invitrogen Bevan(1984) T6pfer(1987) Brosius (1989) Urban et al. (1994) Stratagene Stratagene Abbreviation: MCS, multiple cloning site; P, promoter; T, terminator 2.2.2 Cloning C4H and C4H fusion derivatives in pYeDP60 vector The pYeDP60 expression vector and strain WAT11 (Urban et al., 1994; Urban et al., 1997) were used to express C4H and its fusion derivatives in yeast. The Pfu-polymerase (Stratagene) was used for PCR in this experiment and hereafter. To create the C4H constructs in pYeDP60, the complete C4H coding sequence from C4H-550 was amplified by 27 PCR using two gene-specific primers, each with a BamHI site (underlined): forward primer, 5 ' - A C A G G A T C C A T C A T G G A T C T C C T T C T C C T G G A - 3 ' and reverse primer 5'-A C A G G A T C C T T A A A A G G A C C T T G G C T T T G C A A C - 3 ' . The PCR products were digested with BamHI, and ligated into the corresponding site of pYeDP60. The correct orientation and fidelity of this and all other constructs hereafter described were confirmed by DNA sequencing. To create a C-terminal fusion of the FLAG epitope to C4H, the complete C4H coding sequence was PCR-amplified using a set of gene-specific primers each containing a N o t l s i t e ( u n d e r l i n e d ) : f o r w a r d p r i m e r 5 ' -A T A A G A C T G C G G C C G C A T C A T G G A T C T C C T T C T C C T G - 3 ' and reverse primer 5'-A A G T A G T A G C G G C C G C A A A G G A C C T T G G C T T T G C A A C A A T A G - 3 ' . The resulting PCR products were digested with Notl, and cloned into the Notl site of pESC vector (Stratagene) to produce a C4H-FLAG in-frame fusion. The entire C4H::FLAG fusion construct was re-amplified by PCR using another set of specific primers containing a BamHI site: the same f o r w a r d C 4 H - s p e c i f i c pr imer , and r e v e r s e pr imer , 5'-ACAGGATCCTCAGATCTTATCGTCATCATCCTT-3 ' . The PCR products were digested with BamHI and cloned into the BamHI site of pYeDP60. For the C4H::GFP fusion, the same forward C4H-specific primer was used together with the reverse primer, 5'-TATGATGGATCCTTATTTGTATAGTTCATCCATGCCATGT-3 ' . to amplify the C4H-GFP fusion construct in the pSL1180 vector described below. The resulting PCR products were digested with BamHI and cloned into the BamHI site of pYeDP60. 2.2.3 Construction of C4H::GFP fusion in the pBin19 vector The CD3-327 clone which contains a plant expression cassette for the red-shift soluble modified GFP (rs-smGFP; GenBank number, U70495) (Davis and Vierstra, 1998) was obtained from the Arabidopsis Biological Resource Center. The cloning procedure is represented in Figure 2.1. To generate a C4H::GFP fusion construct for expression in plants, the coding sequence of GFP was PCR-amplified using Pfu polymerase (Stratagene) and the gene-specific oligonucleotide, 5 ' -CAGCTCTAGAATGAGTAAAGGAGAAGAACTTT-3' together with a T7 vector primer. The amplified GFP coding sequence was digested with Xbal and Sacl and the resulting fragment was inserted into the corresponding sites of the pSL1180 vector (Pharmacia Biotech). The inserted GFP coding region was sequenced to verify the accuracy of the PCR amplification. 28 A. Fusion Gene Preparation B. Vector Preparation f T EcoRI Sad Kpnl Smal BamHI Xbal Sail Pst1 Sphl Hindlll Apal Xbal C4H S a c ^ ^ II I—| GFP *| II pBS SKII Xbal pUC18 \ Apal J " C4H Xbal - u Xbal I H GFP Sacl ~*Hi pBS KSII pSL1180 Sail 1 L i _ Apal Xbal Sacl C4H GFP * Spel -Lii C4H::GFP fusion gene in pSL1180 II LB MCS NPT-II II pBin19 i R.E. digestion 5'-end fill-in by Klenow Blunt-end ligation GAATTTCGAC _ t l c . . u i n r t „ , CTTAAAGCTG P s t l S P h l H l n d l " II LB MCS NPT-II II Modified pBin19 1 Expression cassette insetion from pRT101 pBin19/pRT101 C. C4H::GFP fusion gene in pBin19/pRT101 Hindlll Apal 1 4 * \ ^CaMV-Pro C4H GFP * Xbal Sacl Hindlll Term NPT-II RB Figure 2.1 A cloning scheme for the C4H::GFP fusion construct in the pBinl9 binary vector. 29 In parallel, the poplar C4H cDNA clone C4H-550 was amplified by PCR using a C4H-specific oligonucleotide, 5 ' - C A G C T C T A G A A A A G G A C C T T G G C T T T G C - 3 ' and the M13 reverse primer, specific to pBluescriptll SK. The PCR product was digested with Apal and Xbal and the resulting fragment was inserted into the corresponding sites of pBluescriptll KS. To avoid possible PCR errors, the Apal and EcoRI fragment from this PCR-modified C4H clone was replaced by corresponding fragment from the original C4H cDNA, and the remaining 3' end was sequenced. The entire C4H coding sequence, flanked by Apal and Xbal restriction enzyme sites, was then inserted into the Apal/ Xbal sites of pSL1180, containing the GFP coding region between the Xbal and Sacl sites. This resulted in an in-frame fusion of C4H and GFP in the pSL1180 vector. The pBin19 vector (Bevan, 1984) was modified to contain a plant expression cassette as follows: A fragment of the pBin19 multi-cloning site was removed by digestion with EcoRI and Sail. Both sites were made blunt by filling-in with Klenow fragment, and the plasmid was re-ligated. A Hindlll fragment from the pRT101 vector (Topfer et al., 1987), containing a plant expression cassette consisting of the CaMV 35S promoter and terminator flanking a multiple cloning site, was inserted into the Hindlll site of the modified pBin19 vector to construct a Bin19/pRT101 binary vector. Finally, a Sail/ Spel- digested 2.3kb fragment containing the C4H::GFP fusion coding region was placed in the Xhol/ Xbal- sites of Bin19/pRT101 between the promoter and terminator. Independently, a binary vector with a cassette expressing GFP alone was constructed by inserting the Hindlll/ EcoRI fragment from the CD3-327 clone, which includes the 35S CaMV promoter, GFP, and Nos terminator, into the same sites in the pBin19 vector (Bin19/GFP). Agrobacterium strain GV3101 was transformed either with the binary vector harboring the 35S-C4H::GFP fusion construct, or with the 35S-GFP construct, by electroporation. Transformants were selected on LB medium containing 25 mg L"1 gentamycin, 25mg L"1 rifampicin, and 100 mg L"1 kanamycin, and the presence of the binary vector was verified by PCR. 2.2.4 Construction of CPR::GFP fusions in the pBin19 vector A C4H::GFP fusion construct had previously been generated in the pSL1180 vector (see 2.2.2). To make a series of CPR::GFP fusion constructs, the C4H portion was removed from C4H::GFP in the pSL1180 vector by Apal and Xbal digestion. The CPR coding fragments for CPR1 and two versions of CPR2 (CPR21 and CPR22) were amplified by PWO polymerase (Roche Molecular Biochemicals). Forward and reverse primers with Apal and Xbal restriction sites used in the PCR were: for CPR1, 5 ' -TACAGCGGGCCCAGGATGAG-30 T T C A G G T G G T T C A A A T - 3 ' and, 5 ' -CAGCTCTAGACCAGACATCTCTTAGATACC-3 ' : for CPR21, 5 ' -TACAGCGGGCCCAACATGCAATCATCAAGCAGCTCG-3 ' and 5'-CAGCTCTA-GACCATACATCACGCAGATACCT-3 ' : for CPR22, 5'- TACAGCGGGCCCAACATGAAAGT-GTCACCACTTGAACTT-3 ' and the same reverse primer used for the CPR21 amplification. The amplified PCR-fragments were digested by Apal and Xbal, followed by the ligation into the corresponding Apal and Xbal sites of pSL1180-GFP. These ligations resulted in the construction of in-frame CPR::GFP gene fusions in the pSL1180 vector. All three constructs, CPR1::GFP, CPR21::GFP, and CPR22::GFP, were sequenced to verify the fidelity of the PCR-products. The three constructs were digested by Sail and Spel. These fragments were inserted into the Xhol and Xbal site of the previously generated Bin19/pRT101 vector, which placed the fusion constructs appropriately behind the CaMV 35S promoter in the pBin19 vector background. These three constructs were independently transformed into Agrobacterium GV3101 strains by electroporation, and the transformants were screened as described above. 2.2.5 Construction of C4H, CPR, and CPR::GFP in the pESC-Leu Vector Coding regions for C4H, CPR1, and CPR2 were amplified by PCR using the PWO DNA polymerase. Gene-specific primers with Notl, Sail, or Apal restriction sites used were: for the C4H coding region, a forward primer, 5 ' -ATAAGACTGCGGCCGCATCATGGATCTCCTTC-TCCTG-3' and a reverse primer, 5 ' -AAGTAGTAGCGGCCGCAAAGGACCTTGGCTTTGCA-ACAATAG-3'; for the CPR1 coding region, a forward primer, 5 ' -TACAGCGGGCCCAGGAT-GAGTTCAGGTGGTTCAAAT-3' and a reverse primer, 5 ' -TACAGCGTCGACCCAGACATC-TCTTAGATACCGTCC-3' ; for the CPR2, a forward primer, 5 ' -TACAGCGGGCCCAACATG-CAATCATCAAGCAGCTCG-3 ' and a reverse primer, 5 ' -TACAGCGTCGACCCATACACGC-AGATACCTGC-3 ' . The pESC-Leu yeast dual expression vector (Stratagene) was used to clone and express these genes. The PCR fragment for C4H was digested with Notl and inserted into the corresponding site behind the Gal-10 promoter in frame with the FLAG epitope in the correct orientation. The PCR fragments for CPR1 or CPR2 were digested by Sail and Apal and inserted into the corresponding sites behind the Gal-1 promoter in frame with the c-Myc epitope. Thus, three different forms of the pESC-Leu expression vector were constructed: C4H alone, C4H and CPR1, or C4H and CPR2. A series of CPR::GFP genes were also constructed in a pESC dual expression vector along with C4H. The CPR1::GFP fusion gene previously constructed in the pBin19 31 vector was used as a template in a PCR amplification. The coding sequence for CPR1::GFP was amplified using PWO polymerase, a forward primer 5 ' -TACAGCGGGCCCAGGATGAG-TTCAGGTGGTTCAAAT-3' , and a reverse primer, 5 ' -TACAGCGTCGACTTATTTGTATAGT-TCATCCATGCC-3 ' . The PCR-products were digested by Apal and Sail, and inserted in to the Apal and Xhol sites of the pESC-Leu vector in which the C4H had previously been cloned. For yeast expression of the GFP-fusion of CPR21 and CPR22, DNA fragments containing a CPR21::GFP or CPR22::GFP coding sequence were isolated by Apal/ Xhol digestion from the pSL1180-based CPR: :GFP constructs. These fragments were directionally cloned into the Apal/ Xhol sites of a pESC-Leu vector in which the C4H had previously been cloned. 2.2.6 Cloning of PAL genes in the pESC-His vector Coding sequences for PAL2 and PAL4 were PCR-amplified from PAL18 and PAL7 cDNAs, respectively. For the PAL2 ORF, a forward primer 5'-CAGCTCTAGAAAAATGGAATTTTGT-CAAGATTCACG-3' , and a reverse primer, 5 ' -CAGCTCTAGAAGCTTAGCAAATAGGAAGA-GGAGC-3' were used, and for the PAL4 ORF, a forward primer, 5'-CAGCTCTAGAAAAAT-GGAGACAGTCACCAAGAAT-3' , and a reverse primer, 5 ' -CAGCTCTAGACACTTAACAGA-TAGGAAGAGGGGA-3 ' , were used. The amplified fragments were digested with Xbal and inserted into the Spel site of the pESC-HIS vector in the correct orientation. 2.3 Plant growth conditions and transformation Arabidopsis thaliana ecotype Columbia was germinated and grown on AT medium (Somerville and Ogren, 1982) for one week at 20 °C under constant light, and the seedlings were then transferred to soil (Redi-Earth, W.R. Grace & Co., Ontario, Ca) under the same growth conditions. Plants were grown until abundant immature floral clusters had formed, and then transformed by the floral dip method (Clough and Bent, 1998) using 0.05% Silwet L-77 (Lehle Seeds, TX, USA). Primary transformants (Ti) were selected by screening on AT media containing 40 mg L"1 kanamycin. Ti lines were selfed and the resulting seeds were screened by germination on kanamycin-AT media to select T 2 lines. Homozygous Arabidopsis lines were established based on T 2 segregation patterns for the kanamycin resistant phenotype of C4H::GFP-transformed Arabidopsis. 32 2.4 Fluorescence and confocal microscopy analysis The C4H::GFP-transformed Arabidopsis seeds were germinated on AT-medium containing 40-50 mg"1 kanamycin at 20°C under constant light. Intact five to seven days-old, T 2 or T 3 transformed Arabidopsis plants were mounted under a cover glass with a drop of distilled water. The seedlings were observed using a MRC-600 confocal laser scanning microscope (Bio-Rad Laboratories, Hercules) using the blue high sensitivity (BHS) filter block. Excitation was provided by the 488-nm line of an argon laser, and the laser was reduced to 10 % for optical sectioning at 0.2-pm intervals. For staining nuclei of Arabidopsis seedlings, whole seedlings were dipped in a 50 pM DAPI (4',6-diaminophenylindole) solution for 30 min, and fluorescence was detected by fluorescence microscopy through a DAPI filter set. To detect fluorescent signals in CPR::GFP-transformed Arabidopsis lines, at least 80 independently transformed seedlings (3 to 5 days-old) were pre-screened by fluorescence microscopy using a light microscope equipped with a GFP-filter set, and the strongest fluorescent lines were further analyzed by a Radiance confocal microscope (Bio-Rad Laboratories). Seedling samples were prepared as before, and a line of 488 nm argon laser (10-20%) was used as an excitation light source. Green and red fluorescent signals from GFP and chloroplasts were separately collected through the HQ515/30 and E600LP barrier filters, respectively. NIH Image software was used to process the confocal microscopy data. Five to ten sections at 0.2 pm intervals were overlapped to enhance the fluorescence signals. Collected images were further processed by the NIH Image or Photoshop 5.0 software (Adobe Systems). 2.5 Yeast methods 2.5.1 Yeast culture media Untransformed WAT11 and YPH499 strains were maintained in YPAD medium containing 0.08 g adenine hemisulfate salt, 10 g yeast extract, 10 g bactopeptone, and 20 g dextrose per litre. SGI and Y P E G madia were used for gene induction in the WAT11 strains. SGI medium contains 20 g glucose (Difco), 1 g bactocasaminoacid (Difco), 6.7 g yeast nitrogen base without amino acids (Difco), and 40 mg DL-tryptophan (Sigma) per one litre. Y P E G medium contains 5 g dextrose, 10 g yeast extract, 10 g bactopeptone (Difco), and 3% (v/v) ethanol. A concentrated galactose stock solution (20%, w/v) was used for induction, and 20 g agar per liter was added for solid medium. 33 Synthetic minimal medium supplemented by 2% dextrose (SD) or 2% galactose (SG) were used for the YPH499 strain. One litre of amino acid dropout synthetic minimal medium contains 6.7 g yeast nitrogen base without amino acids, 20 g dextrose or galactose, and 1.3 g of appropriate amino acid dropout powder. Leucine dropout synthetic minimal medium (SD-dL or SG-dL) was used for the YPH499 yeast strain transformed with the pESC-LEU plasmid, containing C4H and CPR. Histidine and phenylalanine dropout synthetic minimal medium (SD-dHP or SG-dHP) was used for the YPH499 strain transformed by the pESC-HIS plasmid, containing PAL2 or PAL4. The double-transformed (pESC-LEU and pESC-HIS) YPH499 strain expressing triple genes (PAL2/4, C4H, and CPR) was cultured and induced in the histidine, leucine, and phenylalanine dropout synthetic minimal media (SD-dHLP or SG-dHLP). 0.5 or 5 mM phenylalanine was added to the media in in vivo assays, when necessary. 2.5.2 Yeast transformation The WAT11 strain was cultured in 2 mL YPAD medium overnight at 28 °C. The whole culture was diluted in 50 mL pre-warmed YPAD medium and cultured at 28 °C for 4-5 h. The 50 mL cultures were centrifuged at 4,000 x g, the cells washed once with 25 mL sterile water, and centrifuged again. Cell pellets were resuspended in 1 mL of 100 mM LiAc, transferred to 1.5 mL microfuge tube, and centrifuged for 15 s at top speed. Pelleted cells were resuspended in 400 uL 100 mM LiAc. The cell suspension was vortexed and 50 uL samples were transferred to labelled microfuge tubes. Yeast cells were centrifuged and the supernatant was removed. To the microfuge tubes containing yeast cells, 240 uL PEG (50%, w/v), 36 uL 1.0 M LiAc, 50 uL single strand DNA, 30 uL sterile water, and 4 uL (0.5 -2 pg) plasmid DNA were added and cells were vigorously vortexed for 1 min to completely mix the components. These mixtures were incubated for 30 min at 30 °C and heat-shocked in a water bath for 30 min. Cells were centrifuged at 6,000 rpm for 15 s and resuspended in 1 mL sterile water. Cell suspensions (50 uL) were plated on suitable selection plates, i.e. SGI for WAT11 strains and appropriate amino acid dropout plate for YPH499 strains. The PAL, C4H, and CPR expressers were generated by co-transformation of pESC-LEU and pESC-HIS vector on leucine and histidine dropout minimal plates. Glycerol stocks of 4 to 5 single colonies were prepared and stored at -80 °C. 34 2.5.3 Yeast cell cultures and gene induction For C4H gene induction in WAT11 strains (Chapter 3), a colony from a SGI selection plate was cultured in 2 mL SGI liquid culture overnight. This starting culture was added to 250 mL SGI media and cultured for 12 to 18 h at 28 °C in an incubator rotating at 150 rpm. This cell suspension was centrifuged at 5,000 x g for 10 min, resuspended in 250 mL YPGE culture media, and further cultured for 36 to 48 h. For gene induction, 25 mL of 20% (w/v) galactose was added to the culture which was then incubated for 12 to 20 h. For C4H and CPR gene induction in the YPH499 strain (Chapter 4), the transformed yeast strains were sub-cultured in 3 mL SD medium overnight and 250 mL SD-dL medium was inoculated by a 1:100 dilution. Yeast cells were cultured at 28 °C on a shaker at 150 rpm for 12 to 15 h until cultures reached mid-log phase (3-5 x 107 cells per mL). The culture medium was changed to fresh SG-dL medium to initiate gene induction, and the yeast cells were cultured for 15 to 20 h until late-log phase (9-12 x 107 cells per mL). 2.6 Yeast and plant microsomal protein preparation Microsome preparation from yeast was performed as described (Pompon et al., 1996) except that ultracentrifugation at 100,000 x g was performed for 60 min. Solutions used were: TE, 50 mM Tris-HCl (pH 7.4) and 1 mM EDTA; TEK, 0.1 M KCI in TE; TES, 1.5 M sorbitol, 20 mM Tris-MES (pH 6.3), 2 mM EDTA; TESB, 0.6M sorbitol in TE; TEG, 20% (v/v) glycerol in TE . Briefly, induced yeast cells were centrifuged at 5,000 rpm for 10 min, resuspended in 10 mL TEK, and left at room temperature for 5 min. Cells were centrifuged and resuspended in 2.5 mL of TESB. Glass beads (425-600 \xm; Sigma) were added until skimming the top of the cell suspension. Cell walls were mechanically disrupted by hand shaking for 5 min in the cold room. The crude bead-yeast extract was extracted three times with 5 mL of ice-cold TESB, and the 15 mL pooled cell extract was centrifuged at 12,000 x g for 10 min. The supernatants were subjected to ultracentrifugation at 100,000 x g for 1 h. The microsomes were suspended in 200 - 400 u l cold TEG, and stored at -80 °C before use. For Arabidopsis microsomal protein preparation, approximately 200 two-week old Arabidopsis seedlings transformed with different GFP-fusion constructs, or wild type control seedlings, were ground in liquid nitrogen. The resulting fine powders were resuspended in a buffer containing 50 mM Tris (pH 7.5), 1 mM PMSF, 5 mM MgCI2, one tablet of protease inhibitor mixture (Roche Molecular Biochemicals), and 14 mM 3-mercaptoethanol. The 35 suspension was centrifuged at 13,000 x g for 5 min at 4 °C and was centrifugated at 100,000 x g for 30 min at 4 °C in a bench-top ultracentrifuge. The pellets were redissolved in 50 uL T E G . Microsomal proteins (20 uq) were fractionated on a 7.5% polyacrylamide gel and blotted onto a PVDF membrane for western blot analysis. 2.7 Enzyme assays C4H enzyme assays were initiated by adding NADPH to a final concentration of 0.5 mM to a reaction mixture (600 pi total volume) containing 100 mM sodium phosphate buffer (pH 7.4), 0.1 mM cinnamic acid, and 30-50 ug microsomal protein. The C4H reaction showed a linear production of p-coumarate for at least 10 min. After incubation for 10 min at 30 °C, the reaction was stopped by adding 40 pi 6M HCI, and the reaction mixture was extracted twice into 600 pi ethylacetate, followed by evaporation of the organic phase under vacuum. Reaction products were analyzed and quantified by an HPLC system equipped with a Waters 996 photodiode array detector. The residue was dissolved in 800 pi HPLC-grade methanol, and samples (40 pi) and standards were separated on 3.9 x 300 mm (10 urn) pBondapak™ C18 column (Waters, MA). Solvents used were A: 0.85 % aqueous phosphoric acid (85%, w/v) and B: acetonitrile (HPLC grade). A linear gradient of 80% solvent A, 20% solvent B to 48% solvent A, 52% solvent B was used at a flow-rate of 0.8 mL min"1 for 30 min. HPLC peaks specific to p-coumarate were identified by migration of standards and diagnostic UV absorption spectra, and the peak area was used to quantify the products. Cytochrome c (bovine heart; Sigma) was used as an artificial electron acceptor to measure cytochrome c reduction activity of CPR. The rate of reduction was calculated by differential absorption coefficiency of 21 mM"1cm"1 at 550 nm. The reaction was initiated by addition of 0.1 mM NADPH or NADH to a final volume of 1 ml reaction solution containing 100 mM sodium phosphate buffer (pH 7.4), 0.05 mM cytochrome c, 1 mM KCN, and 3-6 ug microsomal protein. For the reference sample, buffer solution was added instead of NADPH. Formation of reduced cytochome c was measured for 90 s using a Shimazu 1601 Biospec spectrophotometer at room temperature. For PAL assays, 50 mL cell suspensions of PAL-alone or triple expressing yeast strains were cultured and induced (16 h) as described previously. Cells were centrifuged at 4,000 x g for 10 min, washed with 10 mL of distilled water, and re-centrifuged. For enzymatic lysis of cell walls, yeast cells were resuspended in a solution containing 1 M sorbitol, 0.1 M 36 EDTA, 0.1% (3-mercaptoethanol, and 15 mg of lyticase (Sigma). Cell suspensions were incubated at 28 °C for 30 min with a gentle shaking. The resulting yeast protoplasts were centrifuged for 3 min at 4,000 x g, and the cells were ruptured by addition of 1 mL ice-cold extracting buffer containing 50 mM Tris (pH 7.4) and 14 mM (3-mercaptoethanol for 10 min in ice with periodic vortexing. The yeast extracts were centrifuged in 14,000 x g for 10 min at 4 °C. Aliquots (100 uL) of supernatants containing 60 to 300 u,g total protein were used for the PAL enzyme assays, carried out for 30 min at 30 °C in 0.1 M H 3 B0 3 /KOH (pH 8.8) buffer solution containing 3 mM phenylalanine in 170 | iL total volume. The reactions were terminated by addition of 40 u.L 4 M H 2 S 0 4 , and the reaction solutions were extracted twice with 300 uL ethylacetate. The reaction products were dissolved in methanol after the organic phase had been evaporated under vacuum. The presence of cinnamic acid was identified and quantified using the same HPLC conditions and an authentic cinnamate standard used for the C4H assays. 2.8 Yeast in vivo enzyme assays The number of yeast cells, estimated by the spectrophotometric optical density at 600 nm (OD6oo)> was used to normalize the enzyme activity data obtained from in vivo enzyme assays. An OD6oo of 1 was determined to be equvalent to 3.0 x 107 cells per mL, when OD 6 0 o value from the Shimadzu Biospec 1601 was calibrated by comparing haemocytometeric cell-counting to the OD6oo value, using YPH499 yeast strain (2.94 x107 and 3.15 x 107 cells per mL per 1 OD6oo in two independent countings). For the in vivo enzyme assays shown in Figure 3.10, a yeast suspension culture (10 mL, ~ 3.8 x 108 cells mL"1) induced for 12 h by 2% galactose was pelleted by centrifugation at 4,000 x g for 5 min, and resuspended in 30 mL TE buffer (Tris-HCl [pH 7.4], 1 mM EDTA) supplemented with 0.2 mM cinnamate. The yeast cells were then incubated at 28 °C with gentle shaking. A series of 1-mL samples was collected at different time points and pelleted by centrifugation at 14,000 x g for 1 min. The level of p-coumarate product in the supernatants were quantified by HPLC analysis as described in section 2.7. For the in vivo enzyme assay shown in Figures 4.5 and 4.9, 10 mL late-log phase yeast cells was harvested at 1,000 x g after galactose induction (in 2-3 h time intervals for Figure 4.5) and resuspended in 5 mL of Tris-EDTA buffer (pH 7.5) with 0.2 mM cinnamate. Transformed yeast strains were incubated with cinnamate at 28 °C for 20 min in a 150 rpm shaker. Cells were centrifuged at 4,000 x g and the supernatant was analyzed by a HPLC using the same conditions used for C4H assays. 37 2.9 Carbon monoxide differential absorption spectroscopy Total cytochrome P450 content in protein preparations was determined by obtaining reduced carbon monoxide differential spectra as described before by using a differential absorption coefficient of 91 mM' 1cm" 1 (Omura and Sato, 1964). Microsomal protein was diluted in buffer containing 100 mM sodium phosphate (pH 7.4), 0.1 mM EDTA and 30% glycerol, and divided equally between two 1-mL cuvettes. A few grains of sodium dithionite were added to both cuvettes, and the baseline was recorded in a dual beam UV-visible spectrophotometer (Biospec-1601; Shimadzu). The contents of the sample cuvette were then gently bubbled with a carbon monoxide stream for 1 min, and the resulting differential spectrum was recorded between 400 nm and 500 nm. 2.10 SDS-PAGE and immunoblots Protein concentrations were quantified by the Bradford method using BSA as a standard. Microsomal protein samples (2 - 5 u.g for yeast and 20 \xg for Arabidopsis) were separated on 7.5% / 10% polyacrylamide gels, and either stained with Coomassie Blue or transferred to PVDF membrane (Amersham-Pharmacia) for immunoblot analysis. Protein-containing membranes were incubated with 1 x TBS blocking buffer (10 times dilution from 10 X TBS containing 24.2 g Tris base and 80 g NaCI per liter, pH 7.6) supplemented with 5% (w/v) non-fat dried milk and 0.05% (v/v) Tween 20. Primary antibodies used, depending on the target proteins, were anti-FLAG (Stratagene), anti-cMyc (gift from Dr. M. Gold, Department of Microbiology and immunology, University of British Columbia), anti-GFP (Sigma) monoclonal antibodies, or anti-PAL polyclonal antibody (McKegney ef al., 1996). The amino acid sequences specific (epitope) for FLAG and cMyc monoclonal antibodies are DYKDDDDK and EQKLISEEDL, respectively. Primary antibodies at 1:2,500 (monoclonal antibodies) or 1:5,000 (anti-PAL antibody) dilution in a blocking buffer were reacted with antigens on the membrane for 1 h at room temperature. Horseradish peroxidase (HRP)-conjugated anti-mouse IgG or anti-rabbit IgG antibody (Amersham-Pharmacia) were used as secondary antibodies at 1:2500 dilutions for 1 h at room temperature. Immuno-detection was performed using the ECL system according to the manufacturer's recommendations (Amersham-Pharmacia). For dual detection of C4H and C P R in Figure 4.7A, sequential immunodetection was employed. Duplicate blots were prepared and reacted with FLAG or cMyc primary antibodies independently for 30 min, followed by secondary antibody binding 38 and ECL-based signal detection in standard conditions. Each primary antibody identified a protein band of the predicted size, and no cross-reaction to other proteins was detected. The two blots were then washed with 20 mL of 1 X TBS-T buffer (1 X TBS supplemented with 0.05% [v/v] Tween 20) for 1 h, and reacted again with the reciprocal primary antibody for 1 h (i.e. the blot originally incubated with the FLAG antibody was reacted subsequently with the cMyc antibody, and vice versa). These membranes were then incubated with HRP-anti-mouse IgG secondary antibody at a 1: 5000 dilution for 1h, and signals were detected as before. The dual (FLAG and cMyc) and single signals (FLAG or cMyc) detected in the duplicated blots during this experiment showed very similar patterns. 2.11 Analysis of PAL- or triple gene (PAL, C4H, and CPf?)-expressing yeast strains Empty vectors (pESC-Leu and pESC-His), PAL (PAL2 or PAL4 in pESC-His), and triple genes (P>4L2 or PAL4 in pESC-His together with C4H and CPR in pESC-Leu) were transformed into YPH499 strains as described in 2.5.2. Abbreviations of synthetic dropout media are given in 2.5.1. PAL2- or P>A/.4-transformed yeast strains were pre-cultured in the SD-dHP medium and induced in SG-dHP medium, while T2, T4, or vector-transformed yeast strains were pre-cultured in SD-dHLP medium and induced in SG-dHLP medium. For standard in vivo assays, the medium was supplemented with 500 uM phenylalanine. Yeast starting cultures (2 mL) were prepared overnight by stab inoculation of appropriate yeast strains. Start culture (500 u.L) was added to 50 mL dropout minimal medium supplemented by 2% dextrose, and cultured at 28 °C with 150 rpm shaking for 15 h. Yeast cells were centrifuged at 4,000 x g for 5 min and resuspended in the appropriate dropout minimal medium supplemented by 2% galactose to induce transgene expression for 15 h to 20 h. After the gene induction, yeast cells were centrifuged at 4,000 x g for 5 min and washed once with 20 mL distilled water. Cells were harvested by centrifugation at 4,000 x g for 5 min, and the cell pellet was resuspended in the appropriate amino-acid dropout minimal medum containing 500 u,M phenylalanine and 2% galactose. Phenylalanine (0 or 5 mM) was added for the experiments shown in Figure 5.5. To analyze the feedback effect of p-coumarate, various concentrations of p-coumarate (0 -100 u-M) were added to the culture medium of PAL2 and P>4L4-alone-expressing strains (Figure 5.6). To determine the minimum effective piperonylic acid concentration (PA), concentrations of PA from 0 to 100 u,M were added to the culture medum (Figure 5.7). PA (10 u,M) was added to medium in experiments shown in Figures 5.8 and 5.9. 39 After the appropriate culturing time, yeast cells were centrifuged, and cell pellets and culture medium were separately extracted by organic solvents. The culture medium was acidified by addition of 500 uL 4M H 2 S 0 4 and extracted twice with 20 mL ethylether followed by removal of the ether from the organic phase in a stream of nitrogen gas. Small volumes (2 to 3 mL) of methanol were added at the later stages of evaporation to prevent loss of the sample. The final volume was adjusted to 2 mL by methanol and 20 uf_ was used for HPLC analysis. To extract phenolics from yeast cells, the harvested yeast cells were ground twice in 3 mL cold acetone or extracted twice with 2 mL chloroform. The cell debris and organic solvent were partitioned by centrifugation at 4,000 x g for 5 min. The organic phases were pooled and evaporated under nitrogen gas with methanol addition at later stages of evaporation. The final volume was adjusted to 1 mL and 40 uL of the sample was analyzed by HPLC. When culture medium and cells were separated separately, most of the cinnamate and p-coumarate were recovered from the culture medium, and in repeated experiments low levels (< 10% of total) of these compounds were found in cell extracts prepared either by acetone or chloroform extraction. In all quantitative HPLC analyses except for the experiments shown in Figures 5.12 and 5.13 (where only culture media were analyzed), the organic extracts from media and cells were pooled together for HPLC analysis. For the experiment shown in Figure 5.12, 15 h-induced yeast cells were diluted in 0.5 mM phenylalanine-containing minimal medium (50 mL) to a concentration of 3 x 108 cells per mL. Two-mL aliquots of cultures were sampled every 2 h for 10 h. Cells were removed by centrifugation at 6,000 x g for 2 min, and the supernatants were filtered through a 0.45 pm pore-sized mini-filter (Gelman). The presence of p-coumarate and cinnamate was measured by HPLC analysis. The solvent conditions for reverse phase HPLC (section 2.7) were changed from 80% phosphoric acid (0.85%, v/v) and 20% acetonitrile to 40% phosphoric acid to 60% acetonitrile for 50 min at a constant flow-rate of 0.8 mL min"1. 2.12 3H-Phenylalanine feeding assay The yeast vector control, PAL-only, and triple expressing strains were pre-cultured and induced (16 h) in standard conditions. The cell densities after induction varied between 7.2 x 10 7and 9.0 x 107 cells mL'1, but each batch of the transformed yeast strains was adjusted to a density of 6.0 x 107 cells mL"1 in 25 mL of SG-dHP medium (for PAL2/4 expresser) or SG-dHLP medium (for vector control and triple expresser). Two 25-mL batches were prepared 40 each for the T2 and T4 strains, and the C4H inhibitor piperonylic acid was added to one of the batches for each strain at a final concentration of 10 pM. A final concentration of 500 pM [2,6-ring-3H] phenylalanine (56.9 u.Ci / pmol) was added to the 25 mL culture. These yeast strains were then cultured in standard conditions for 4 h. The accumulated phenolic metabolites from the medium and the cells were extracted as described in 2.10. The extracted metabolites were fractionated on a reverse phase HPLC column (section 2.7) in a gradient of 0.85% phosphoric acid with an increasing proportion of acetonitrile [0 to 30 min, 5% (v/v) acetonitrile; 30 to 40 min, 5 to 15% acetonitrile; 40 to 65 min, 15 to 56% acetonitrile] at a constant flow rate of 1.0 mL min"1. Fractions (500 uL) were collected between 23 to 65 min, and the radioactivity of each fraction was measured by scintillation counting in a LS6000 liquid scintillation counter (Beckman). Major 3H-labelled metabolites detected were: phenylethanol, 33.0 min; phenylacetic acid, 39.5 min; p-coumarate, 45.2 min; cinnamate, 54.5 min; phenylpyruvate, 55.9; styrene, 63.1. In T2 and T4 triple expressers, two unknown 3H-labeled metabolites, each constituting -0.5% of total 3 H activity initially added, were eluted at approximately 43 and 53 min. In all yeast strains including vector-transformed control, unidentified minor 3H-labelled metabolites (- 0.2% of total 3 H activity) were eluted at 27 and 50 min. 2.13 Double-labeling assay T2 and T4 yeast strains were induced by addition of 2% galactose for 15 h in standard conditions. These induced cells were resuspended in 50 mL fresh SG-dHLP medum supplemented with 500 pM [2,6-ring-3H] phenylalanine (56.9 uCi / umol) together with 100 pM (4.8 uCi / pmol) or 10 pM (47.7 pCi / umol) 1 4C-cinnamate. These yeast cell batches were cultured for 30 min at 28 °C. Culture medium and cells were extracted as described in 2.11, and the radioactive metabolites were separated by the HPLC, using the program described in 2.12. Fractions (200 uL) were collected that corresponded to the elution times of p-coumarate, cinnamate, and styrene. The 3 H and 1 4 C radioactivities in the fractions were measured by scintillation counting in a LS6000 liquid scintillation counter (Beckman) with automatic quench compensation for 3 H and 1 4 C dual label counting. 2.14 Analysis of mixed PAL-alone and C4H/CPR-alone expressing yeast strains P/4/.-alone and C4H/CPR expressers were independently cultured, induced for 15 to 18 h, and the cells washed with distilled water as described in 2.11. Approximately 3 x 109 cells 41 each for the PAL-alone or C4H/CPR expresser were mixed in 80 mL of S G medium containing 500 u.M Phe. The T2 or T4 strains were also induced, washed, and cultured in the same conditions as a reference, but half the number of total cells (3x 10 9 cells) were resuspended in 80 mL S G medium (500 uJVI Phe) so that the two systems had similar numbers of P>A/.-expressing yeast cells. One mL aliquots of culture medium were collected in a time course, and cells were removed by centrifugation at 6,000 x g for 2 min. Supernatants were filtered through a 0.45 \xm filter and the quantity of cinnamate and p-coumarate was measured by HPLC fractionation as described in 2.11. 42 Chapter 3 Functional expression and subcellular localization of poplar cinnamate 4-hydroxylase 3.1 Introduction The enzyme cinnamate 4-hydroxylase (C4H) catalyzes the formation of p-coumarate using molecular oxygen and electrons from NADPH. Most C4H activity has been recovered from microsomal fractions (Benveniste ef al., 1977; Benveniste ef al., 1978), whereas its metabolically adjacent enzymes, PAL and 4CL, are considered operationally cytosolic enzymes. Additionally, it has been proposed that C4H functions in direct contact with PAL on the cytosolic side of the ER as part of a multiprotein complex, implying a potentially important structural role of C4H (Rasmussen and Dixon, 1999; Winkel-Shirley, 1999). As a part of our effort to understand lignin biosynthesis from the model organism hybrid poplar {Populus trichocarpa x P. deltoides), the biochemical properties and subcellular localization of poplar C4H were investigated in this study. When this project was initiated, a putative C4H full-length cDNA (C4H-550 cDNA clone; GenBank accession number, AF302495) had been isolated from a hybrid poplar young leaf cDNA library (Ro ef al., 2001). In northern blot analysis, high C4H expression was detected in xylem tissue as well as young leaves and green stems. Also, an elicitor derived from Fusarium oxysporum strongly induced C4H expression in cultured poplar-cells. These results are consistent with a central role of C4H in the biosynthesis of lignin and other defense-related phytochemicals in hybrid poplar. The deduced amino acid from the C4H-550 clone revealed more than 85% identity to other C4Hs from different plant species. All characteristic features for a cytochrome P450 (P450) superfamily enzyme were identified in the primary amino acid sequence, including an N-terminal hydrophobic membrane-anchoring domain, a proline-rich region, several positively charged amino acids, and a strictly conserved heme-binding motif for plant-specific P450s (PFG[ASV]GRRXC[PAV]G) (Durst and Nelson, 1995; Figure 3.1). The sequence information strongly suggested that the cDNA encoded a cytochrome P450 enzyme, most likely C4H. An attempt was made to express this cDNA in a baculovirus/Sf9 cell expression system (J. Norton, C. Douglas, and B. Ellis, unpublished). However, the recombinant proteins recovered were barely active, had no cytochrome P450 specific absorption spectrum, and were targeted mainly to a 10,000 x g mitochondrial fraction, although protein-sequencing confirmed that the correct protein was being expressed. 43 1 -I + I + + ++ + 50 MDLLLLE KTLLGSFVAI LVAILVSKLR GKRFKL PPGP IPVPVFGNWL M FV A L I T I T l KL S IAV V IL TVI KL I I EITVAS G A l AV VG FI K S KL LA I M K-L F - - A SKL- -K--KIffPGp| P-FGNWL 51 100 QVGDDLNHRN LTDLAKKFGD IFLLRMGQRN LVWSSPDLS KEVLHTQGVE Q V Y L M E A D V Y L T G Y E K A QVGDDLN-RN L-D-AKK-G- -F-L-MGQRN LVWSSP-L- K-VLHTQGVE 101 150 FGSRTRNWF DIFTGKGQDM VFTVYGEHWR KMRRIMTVPF FTNKWQQYR FGSRTRKWF DIFTGKGQDM VFTVY-EHWR KMRRIMTVPF FTNKWQQ-R 151 200 YGWEEEAAQV VEDVKKNPEA ATNGIVLRRR LQLMMYNNMY RIMFDRRFES F D R A N L E F S DS K K F FA D LGRA D I R S T I V I M --WE-E V - D - K - - P - - -T-GIV-R-R LQL-MYN-M- R-MFDRRFES 201 250 EDDPLFNKLK ALNGERSRLA QSFDYNYGDF IPILRPFLRG YLKICQEVKE V L E HF L I D LR E D D LR E V K -DDPLF--LK ALNGERSRLA QSF-Y--GDF IP-LRPFLRG YLK-C K-251 -^-300 RRLQLFKDYF VDERKKLAST KNMSNEGLKC AIDHILDAQK KGEINEDNVL K K E I SVD NS IE Q IA K QI S PTGS E EQ S L I TGSKVT D FE EE -R--LFK-YF -DERK S- KC AIDHI--A-- KGEINEDNVL 301 A YIVENINVAA IETTLWSIEW GIAELVNHPE IQKKLRHELD TLLGPGHQIT N V A V C S N V V V M L I Q I A AVI R VPL YIVENINVAA IETTLWS-EW G-AE-VN-PE IQ-K-R-ELD G-G B C 351 • 400 EPDTYKLPYL NAVIKETLRL RMAIPLLVPH MNLHDAKLGG FDIPAESKIL VQ Q Y A Y LH Q V A Y T Q V H Q Y EPD--KLPYL -AV-KETLR- -MAIPLLVPH MNLH-AKL-G -DIPAESKIL ^ ^ D 401 • 450 VNAWWLANNP AHWKNPEEFR PERFLEEEAK VEANGNDFRY LPFGVGRRSC NK D S K I NS K F SH V F EW EK I G . I S F VNAW-LANNP - -W--P-EF- PERF--EE-- -EA-GNDF-- -PFGVGRRSC 500 PGIILALPIL GITLGRLVQN FELLPPPGQS KIDTAEKGGQ FSLHILKHST VI Q I M V S N I ALA V VT N V PGIILALPIL GR-VQN FELLPPPGQS K-D--EKGGQ FSL-IL-HS-501 IVAKPRSF POPLAR C L PARSLEY M NC ARABIDOPSIS V IA PINE -V-KP IDENTITY Figure 3.1 Deduced amino acid sequence of the poplar C4H-550 cDNA and comparison to other plant C4H amino acid sequences. The full amino acid sequence of poplar C4H-550 is given, and only the amino acids that differ from those in poplar are shown in the parsley (Q43033), Arabidopsis (AAB58355), and pine (AAD23378) sequences. The PILEUP and PRETTY options from Wisconsin Package (version 9.1, Genetics Computer Group, Madison, WI) were used to align and process the sequences. Charged amino acids conserved in the N-terminus are indicated by +/- signs. The proline-rich and heme-binding domains are highlighted by a black-lined box and a gray box, respectively. Membrane-anchoring region is indicated by black brackets. The four conserved domains typical of eukaryotic P450s (Kalb and Loper, 1988) are indicated by arrow lines labeled A to D. 44 Possibly, the production of a large quantity of this recombinant protein in infected dying insect cells leads to mistargeting and thus poor activity of this protein. In this study, as an alternative expression system, the C4H cDNA was expressed in yeast, since yeast contains basic eukaryotic endomembranes suitable for the expression of membrane-proteins. Besides its catalytic role in phenylpropanoid metabolism, C4H has been proposed to anchor a hypothetical multienzyme complex (MEC) on the ER, as noted above. The subcellular location of C4H has been studied by various methods, and other organelles in addition to ER, such as the Golgi and perhaps provacuoles, have also been reported as sites of C4H accumulation (Fujita and Asahi, 1985; Smith ef a/., 1994). However, invasive sample preparation, cross-reactivity of antibodies, and contamination between subcellular fractions may have confounded these studies and made it difficult to accurately determine the site of protein localization by these approaches. Considering the postulated structural role of C4H to scaffold the MEC, it is important to determine unambiguously its subcellular localization. The green fluorescence protein (GFP) had emerged as a powerful tool for protein localization studies (Kohler, 1998) when I began this project. With the aid of the GFP-tagging method and confocal microscopy, the subcellular location of C4H was analyzed in vivo in real time. 3.2 Results 3.2.1 Expression of recombinant poplar C4H in yeast The pYeDP60 vector and WAT11 yeast strain developed by Urban et al. (1997) were used to express the cloned C4H cDNA. The vector pYeDP60 allows growth in rich medium necessary for production of high levels of protein products, while allowing use of two independent nutritional selection markers, URA3 and ADE2, on pYeDP60 for plasmid selection (Figure 3.2). Transformed yeast strains were cultured in Ura" minimal medium until saturation, during which time the URA3 served as a selection marker. The Ura"-selected cells were then transferred to a rich medium, where the ADE2 was used as a second selection marker because adenine naturally becomes a limiting factor at a high cell density. In the WAT11 yeast strain, the yeast endogenous cytochrome P450 oxidoreductase (CPR) gene is replaced by the Arabidopsis CPR1 gene (ATR1) under the control of a GAL10-CYC1 hybrid promoter, allowing coupling of recombinant plant P450s such as C4H with a plant CPR. 45 C4H-FLAG C4H |j2>AAG LVSM D YKDDDDKI | U URA3 U ADE2 Yeast 2u origin U Figure 3.2 A schematic diagram of C4H- or C4H::FLAG-construct in pYeDP60 expression vector. Letters beside the C4H-FLAG indicate the amino acid sequence introduced during sequence manipulation. The amino acids for FLAG-epitope are shown in italic letters. The coding sequences for C4H were amplified by PCR, and inserted behind the Gal10-CYC1 promoter in pYePD60 vector (Figure 3.2). To facilitate the immuno-detection of recombinant proteins by antibodies, an eight amino acid FLAG-epitope was added in-frame to the C-terminus of C4H. The two expression constructs and a void pYeDP60 vector were independently transformed into yeast strain WAT11. To examine the profiles of the expressed proteins, microsomal proteins from the vector-, C4H-, or C4H::FLAG-expressing yeast strains were resolved by SDS-PAGE following galactose induction. Coomassie Blue staining of the fractionated microsomal proteins revealed intense protein bands corresponding to the predicted positions, which were absent from the microsomal preparations of vector-transformed yeast (Figure 3.3A). Although the difference of molecular weight between the two proteins estimated from the gel appeared to be slightly bigger than that calculated for C4H and C4H::FLAG (-1.7 kD), seven charged amino acids in FLAG-epitope were likely to interfere SDS-binding to this region, resulting in a slower migration of C4H::FLAG than C4H. The immuno-blot analysis using monoclonal anti-FLAG antibodies confirmed the identity of the novel -60 kD protein from C4H::FLAG expressing yeast as C4H (Figure 3.3B). 46 < B < kD 116_ 97-84-66-55-45-36-29-O J - X > O O Figure 3.3 S D S - P A G E and immuno-blot analysis of microsomes from C 4 H - and C4H::FLAG-transformed yeast. A , 2 ug of microsomal proteins were resolved by a 10% polyacrylamide gel and stained by Coomassie Blue. The positions for recombinant proteins are indicated by asterisks beside the bands. B , Immuno-blot analysis of control (vector only) and experimental (C4H::FLAG-transformed) yeast strains. Microsomal protein prepara-tions (2 |.tg) were separated by 10% S D S - P A G E , and transferred to a P V D F membrane. The blot was incubated with mouse monoclonal an t i -FLAG antibody, and signals detected by E C L chemiluminescence. Authentic cytochrome P450 enzymes should present a characteristic 450 nm peak upon carbon monooxide binding (Omura and Sato, 1964). In order to evaluate if protein-folding and heme-incorporation of the recombinant C4H and C4H::FLAG proteins occurred correctly, spectra of reduced microsomes after the carbon monoxide binding were recorded by differential absorption spectroscopy. Microsomal proteins from yeast transformed with either the C4H or the C4H::FLAG construct showed typical absorption peaks at 450 nm after galactose induction, while a vector-transformed strain showed no such 450 nm peak (Fig. 3.4). Based on the height of their 450 nm peaks, C4H and C4H::FLAG-expressing yeast routinely yielded from 90 to 440 pmol of spectrally active P450 enzyme per mg microsomal protein. Total P450 content varied depending on culturing time in glucose pre-culture and galactose induction (Table 1). Table 1 Cytochrome P450 content from microsomes prepared from C4H- or C4H::FLAG-expressing yeast strains. Experiment Culturing Time (hour) P450 content3 Number Pre-culture (Glu) Induction (Gal) (pmol P450 mg"1 microsomal protein) 1 48 17 290 - 440 2 36 20 220 - 280 3 36 12 90-160 a Two culture-batches each for C4H- or C4H::FLAG-transformed yeast strains were prepared for each experiment. The maximum and minimum P450 amounts recovered are indicated. 48 0.01 d) o c co n >_ o in n < 400 W a v e l e n g t h ( n m ) 500 Figure 3.4 Carbon monoxide-induced differential absorption spectra analysis of microsomal proteins from C4H- or C4H::FLAG-expressing yeast strains. Carbon monoxide-induced differential absorption spectra recorded from dithionite-reduced microsomes of C4H- or C4H::FLAG-transformed yeast. Microsomes from vector-transformed yeast yielded the spectrum shown by the dashed line, and spectrum 1 and 2 were recorded from C4H (0.46 mg/mL) and C4H::FLAG (0.30 mg/mL) containing microsomes, respectively. 3.2.2 Poplar C4H cDNA encodes bona fide cinnamate 4-hydroxylase activity The biochemical identity of the protein encoded by the C4H-550 cDNA was substantiated by enzyme assays using frans-cinnamic acid as a substrate, aided by a diagnostic HPLC-analysis. The microsomal fraction from vector transformed yeast could not convert cinnamic acid to any other phenolic compound (Figure 3.5 A). Conversion of cinnamate to a more hydrophilic product, eluting -11 min earlier than cinnamic acid, was efficiently catalyzed (Table 2) by microsomes from the yeast strains transformed with either C4H or C4H::FLAG construct. The new products were confirmed to possess an identical retention time and absorption spectra to the authentic p-coumarate standard (Figure 3.5 A and B). Specific activities for yeast expressed C4H were measured from microsomes containing either C4H or C4H::FLAG. Both enzymes showed high conversion rates, and there was no significant difference between recombinant C4H and C4H::FLAG proteins (Table 2). These data confirmed that the poplar cDNA encodes an authentic, functional C4H enzyme. Table 2 C4H activity in microsome preparations from C4H-, C4H::FLAG-, and C4H::GFP-expressing yeast strains Construct Specific activity' nmol p-coumarate min"1 nmol P450"1 C4H 274 ± 56 C4H::FLAG 315 ±22 C4H::GFP 148 ±43 nmol p-coumarate mm mg 29 ± 7 39 ± 13 6 ± 2 microsomal protein a mean ± SD. Each value was calculated by three measurements each from two independent yeast transformants. 50 A B Figure 3.5 Diagnostic HPLC analysis for identification of p-coumarate converted from rrans-cinnamate by cinnamate 4-hydroxylase. A . HPLC fractionation of C4H enzyme assay products from microsome of C4H- and C4H::FLAG-transformed yeast strains showed the appearance of novel peak at about the same time as the p-coumarate standard. A l l HPLC chromatograms were the same scale, and the data were extracted at 312 nm. 2 mM p-coumarate was used for standard. B. Spectrum of the novel product was identical to that of p-coumarate standard, maximum peak at 312 nm and second maximum at 226.7 nm. Spectra for substrate, cinnamic acid, is given in the top panal. 51 3.2.3 Poplar C4H is localized to ER in planta A C4H: :GFP fusion gene was placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in the pBin19 vector (Figure 2.1). As a control, the GFP gene was also fused to the same promoter and in the same vector. Following >4grobacfer/i//77-mediated transformation of Arabidopsis, seven C4H: :GFP- and three GFP-transformed Arabidopsis lines were selected for further analysis. Based on the T 2 segregation patterns for kanamycin resistance, all seven C4H::GFP-transformed lines appeared to contain a single insertion, while one of the GFP-transformed lines contained more than two insertions (Table 3). Table 3 Characterization of the C4H::GFP- or GFP-transformed Arabidopsis lines. Construct Arabdopsis T 2 generation Estimated insert GFP lines Kan R e s : K a n S e n number fluorescence level C4H::GFP 4 32: 19 1 + 9 38: 20 1 ++ 19 30: 11 1 ++ 21 18: 12 1 -25 17: 7 1 +++ 28 43: 18 1 ++ 33 14: 3 1 + GFP 1 54: 2 >2 + 2 21: 7 1 ++ 5 N.D a. N.D. +++ a Not determined. Only a few Arabidopsis seedlings were able to germinate and grow. Homozygous lines for the seven C4H::GFP transformed Arabidopsis were generated on the basis of a T 3 segregation ratio for the kanamycin resistant phenotype. One of the lines (C4H::GFP no. 21) presented GFP signals in T 2 generations but apparently lost its fluorescence in the T 3 generation most likely due to the co-suppression of the C4H gene. Though the no. 28 line showed very high GFP signals in the T 2 generation, its fluorescence was significantly reduced in T 3 generation. On the other hand, line no. 25 showed an intermediate level of GFP signals in T 2, but showed higher GFP signals in the T 3 generation. Epidermal cells and guard cells from living transgenic and control seedlings from the T 2 and T 3 generations were examined in vivo by confocal laser scanning microscopy. A series of 0.2 pm optical sections was used to localize fluorescent signals, which were peripherally compressed due to turgor pressure from the large central vacuole. Strong fluorescent signals started to be visualized just beneath the epidermal cell walls down to 0.2 pm - 0.6 pm depth. There was virtually no GFP fluorescence beyond this depth, where large vacuoles are usually located. 52 Non-transformed Arabidopsis seedlings showed weak autofluorescence in hypocotyl epidermal cell walls and cell walls around the stomatal pore of guard cells, but no fluorescence was associated with other cell organelles (Figure 3.6 A, D, and G). When GFP alone was expressed in transgenic Arabidopsis, green fluorescence was observed throughout the cytoplasm, and cell organelles appeared as dark zones against this background (Figure 3.6 B, E, and H). On the other hand, Arabidopsis seedlings transgenic for C4H::GFP presented unique fluorescent patterns in all independent transgenic lines examined. Epidermal cells displayed confined fluorescence patterns that localized to a distinctive reticular network in both hypocotyl and cotyledon epidermal cells (Figure 3.6 C and F, Figure 3.7). The fluorescent structures showed typical three-way junctions with -120 degree angles between the branches, identical to the patterns generated by the ER marker DiOC 6 in onion cells (Knebel et al., 1990). This pattern has also been observed in tobacco leaf epidermal cells expressing an ER-targeted chimeric GFP-KDEL fusion protein from a viral vector (Boevink et al., 1996). The congruence of the C4H::GFP fluorescence pattern with that expected for the ER suggests that C4H::GFP is in fact localized to the ER. Dynamic unidirectional ER streaming was observed in cotyledon epidermal cells (Figure 3.6 F, arrow). No pattern of green fluorescence was associated with other cell organelles such as mitochondria, the Golgi apparatus, or chloroplasts. Despite autofluorescence, fluorescence associated with the cell wall was negligible in comparison to that seen in the ER, and no fluorescence was observed in the apoplastic space. In addition to the reticulate ER pattern of fluorescence, a number of elongated fluorescent organelles were often observed in the hypocotyl epidermal cells, and less frequently in the cotyledon epidermis (Figure 3.6 C, arrow-heads). They were highly mobile when closely located to the streaming ER (data not shown), but otherwise static on the ER. The uppermost optical sections of guard cells also presented typical ER-like patterns (Figure 3.6 I), but an unusual pattern of fluorescence was observed in inner sections (Figure 3.8 A). Here, fluorescence was detected around the nuclear envelope, which forms a continuous membrane system with the ER, and large uniformly fluorescent bodies were frequently observed. Absence of red fluorescence and 4',6-diaminophenylindole (DAPI) staining clearly distinguished these bodies from chloroplasts and nuclei, respectively (Figure 3.8 B), but their identity remains to be clarified. 53 W i l d T y p e G F P C 4 H - G F P F i g u r e 3.6 Subce l lu lar l o c a l i z a t i o n o f h y b r i d poplar C4H : : G F P i n transgenic Arabidopsis seedlings. A - C , H y p o c o t y l epidermal ce l ls ; D - F , coty ledon epidermal cel ls; G - I , guard cel ls f r o m 5-day o l d seedlings were examined by confocal microscopy . A , D , and G , non-transformed Arabidopsis; B , E , and H , 35S-GFP transformed Arabidopsis; C , F , and I, 35S-C4H::GFP transformed Arabidopsis. A r r o w - h e a d s indicate di lated E R patterns i n C , and a l ine o f E R - s t r e a m i n g is s h o w n by the arrow i n F . B a r = 10 p m . 54 Figure 3.7 An enhanced confocal microscopy image for ER-localization of C4H : :GFP protein on cotyledon epidermal cells. Twenty optical sections (0.2 um thickness) were overlapped to increase green fluorescent signals. 55 G F P D A P I M e r g e B • Figure 3.8 Images of guard cells in Arabidopsis transformed by a C4H::GFP fusion con-struct observed by confocal (A) or fluorescent microscopy (B). A , An optical section of inner-sides of Arabidopsis guard cell shows fluorescence in an unidentified organelle; arrow indicates the peri-nuclear membrane, and the arrow-head indicates a strongly fluorescent organelle. B , The C4H::GFP transformed Arabidopsis seedlings were stained by 50 mg/mL DAPI, and observed by fluorescence microscopy by a GFP-filter set or a DAPI-filter set. Arrow indicates the unknown organelle, and arrow-head indicates an nucleus. 56 3.2.4 Functional expression of C4H::GFP fusion protein Although GFP fusions are considered an ideal method to determine subcellular location of proteins in non-invasive in vivo conditions, this 26-kD G F P protein can potentially cause problems in the overall protein folding of fusion proteins. It should be noted that in plants, the subcellular sites for the degradation of mis-folded proteins are not clear yet, and this is still an active area of investigation. Thus, confirming the biological activity of GFP-fused proteins is necessary to validate GFP-based localization studies, and exclude the possibility of mis-targeting of mis-folded proteins. For this purpose, I expressed the C4H::GFP construct in yeast to determine whether the fusion protein is folded correctly and therefore retains its catalytic activity. The fusion protein coding region was amplified by PCR and inserted into the pYeDP60 vector expression cassette. In immuno-blots of microsomal proteins from control or C4H: :GFP expressing yeast strains probed with anti-GFP antibodies, a protein of the predicted size for C4H::GFP was expressed specifically in the C4H::GFP-expressing yeast strain (Figure 3.9 A). A microsomal protein preparation from a C4H::GFP-expressing yeast strain was used to record reduced CO differential spectra. Figure 3.9B shows that a typical P450 peak was observed after a 20-hour galactose induction but that it was accompanied by a strong peak at 420 nm, which has been attributed to denatured and inactivated forms of P450 (Yu et al., 1995, and references therein). By contrast, C4H or C4H: :FLAG microsomal fractions prepared in parallel displayed only minor P420 peaks, indicating that the high P420 content in C4H: :GFP was not caused by inappropriate microsomal preparation (data not shown). Extended heterologous P450 expression has been reported to result in accumulation of P420 at the expense of the P450 (Chen ef al., 1996). I therefore examined the effect of using a shorter induction time for C4H::GFP-expression, but after 12 hour induction, a high P420 content was again observed in the microsomal preparation (Figure 3.9 B). Most importantly, despite the dominance of the P420 fraction in C4H: :GFP expressing yeast, sufficient amounts of the P450, ranging from 40 to 60 pmol per mg microsomal protein, were recovered from microsomal preparations to permit comparisons of enzyme activity. These microsomal fractions were able to efficiently convert cinnamic acid to p-coumaric acid in in vitro enzyme assays, but did so with about two-fold lower specific activities than C4H or C4H::FLAG-expressing strains, when normalized on the basis of spectrally active P450 (Table 2). However, the specific activity of C4H::GFP was much lower when normalized to total microsomal protein amount (Table 2). 57 A B Figure 3.9 Immuno-blot and carbon monoxide-induced differential absorption spectra analysis of microsomes from C4H::GFP-transformed yeast. A , Immunoblot analysis of control (vector only) and experimental (C4H::GFP-transformed) yeast strains. Microsomal protein preparations (2 pg) were separat-ed by SDS-PAGE, and transferred to a PVDF membrane. The blot was incubated with mouse monoclonal anti-GFP antibody, and signals detected by chemiluminescence. B, Carbon monoxide-induced differential absorption spectra recorded from reduced microsomes of C4H-GFP transformed yeast. Microsomes from 20 h or 12 h galactose-induced C4H::GFP-transformed yeast, adjusted to a final assay concentration of 1.0 or 1.1 mg/mL, were used to record spectrum 1 or 2, respectively. The spectrum for microsomes from vector transformed yeast is shown by the dashed line. 58 In order to exclude the possibility of preferential denaturation and inactivation of C4H: :GFP during the process of microsomal preparation, enzyme assays for C4H activity were performed in living yeast cells expressing C4H, C4H::FLAG or C4H::GFP. Cinnamic acid fed to all three yeast strains was converted to p-coumarate in a linear time-dependant manner (Figure 3.10). A vector-only control strain did not produce any p-coumarate (data not shown). As observed in vitro, no significant difference was observed in the catalytic activities of the C4H and C4H::FLAG constructs, while the C4H: :GFP construct converted cinnamate to p-coumarate about two-fold less efficiently. Taken together, these in vitro and in vivo data confirm that C4H::GFP proteins retain enzymatic activity and that the general protein-folding and heme-binding to the C4H: :GFP apoprotein proceeds correctly in a significant fraction of the expressed proteins. 20 40 60 80 Time (min) Figure 3.10 In vivo conversion of cinnamate to p-coumarate by transgenic yeast. The accumulation of p-coumarate in C 4 H - , C4H : :FLAG- , and C4H::GFP-transformed yeast cultures was measured over the times given. Similar results were obtained when the experiment was repeated using independent yeast transformants. 59 3.3 Discussion Although poplar C4H genomic clones and the sequence of a poplar C4H cDNA have been described (Ge and Chiang, 1996; Kawai et al., 1996), C4H genes and proteins have not been further characterized in this genus. In this work, the functional identity of the protein encoded by the poplar C4H-550 cDNA was confirmed as an authentic C4H by heterologous expression in yeast. In addition, predominant ER-localization of C4H was proved by the GFP-fusion in Arabidopsis, and this result was further validated by functional expression of C4H::GFP enzyme in yeast. The amino acid sequence identity of poplar C4H is more than 85% to other well-characterized C4Hs (Ro et al., 2001). However, a highly divergent C4H cDNA (63% amino acid identity to poplar C4H) was identified from French bean, distinct from a previously characterized C4H enzyme (Nedelkina et al., 1999). This C4H cDNA encoded bona fide C4H activity and is proposed to participate in lignin biosynthesis based on its induction pattern in cultured cells undergoing xylogenesis (Nedelkina et al.', 1999). Maize and orange (Citrus sinensis) are also reported to possess divergent C4Hs that are phylogenetically closer to the French bean C4H than other C4Hs, though their biochemical and physiological identities remain to be characterized (Betz ef al., 2001; Potter et al., 1995). In contrast, Arabidopsis C4H has been reported to be encoded by a single gene (Bell-Lelong ef al., 1997; Mizutani et al., 1997), and a search for "C4H-like gene" from a complete Arabidopsis genome sequence did not find any similar gene. Thus, in Arabidopsis, a single C4H form appears to participate in the biosynthesis of a wide array of phenylpropanoid natural products, including both lignin and flavonoids. It is an interesting question why some plants maintain two classes of C4H, while the others can survive with only one type. It is possible that poplar contains one or more copies of the second divergent C4H type enzyme, but this was not investigated. Our interest in C4H came from its central role in lignin biosynthesis in woody plants. Several lines of evidence suggest that the C4H encoded by C4H-550 or very similar genes participates in lignin biosynthesis in the developing xylem of hybrid poplar: 1) In northern blot analysis, the C4H-550 probe detects abundant C4H transcripts in xylem tissue (Ro et al., 2001); 2) I was able to identify sequences for six independent putative C4H clones through a database search of poplar xylem and cambium ESTs (Sterky ef al., 1998), and these were all >95% identical to each other and to C4H-550 in regions where their sequences overlapped. This level of sequence divergence is consistent with the presence of three C4H genes with greater than 90% sequence identity in Populus kitakamiensis (Kawai 60 ef al., 1996); 3) Comparison of the other two partial poplar C4H clones similar to C4H-550 revealed that they are very similar to each other (>90%) in the sequenced regions, but they do not encode identical sequences. These data suggest that C4H genes with sequences significantly divergent from C4H-550 are not highly expressed in poplar xylem. Thus, there appear to be multiple, but very similar, C4H genes in poplar, possibly having arisen from recent gene duplications in this genus. Though the primary sequence of poplar C4H showed high similarity to C4Hs from other plants, it is not safe to assign a catalytic function to cloned P450 genes solely based on sequence similarity. Not only are the sequences generally closely related, but P450s are very plastic in their catalytic behavior, and their substrate utilization profiles can be changed by a single amino acid substitution (Iwasaki et al., 1994; Lindberg and Negishi, 1989). Thus, expression in a heterologous host is an essential step in functionally identifying a newly cloned P450 gene. Efficient expression of C4H, C4H::FLAG, and C4H:GFP in yeast allowed me to measure the spectral properties and catalytic activity of C4H. All three recombinant enzymes presented a typical 450 nm peak by CO differential absorption spectroscopy and catalyzed conversion of cinnamate to p-coumarate with a high turn-over rate. The specific activities (-300 nmol min*1nmol P450"1) of C4H or C4H::FLAG reported here are comparable to those from Arabidopsis and parsley C4H (Koopmann ef al., 1999; Mizutani ef al., 1997). It should be noted that this value is one or two orders of magnitude higher than those of most animal P450s involved in detoxification of xenobiotics (Iwasaki ef al., 1994; Lasker ef al., 1998; Yamazaki ef al., 1999). C4H belongs to the A-group of P450s that appear to have evolved from a common ancestor after divergence of plant and animal lineage (Durst and Nelson, 1995). From a biochemical view point, it is apparent that the diverse A-group P450s specific to plants, have more specific and efficient catalytic functions than animal P450s. Though C4H::FLAG showed similar spectral and catalytic behavior as the untagged C4H, C4H::GFP showed different CO-spectra and reduced catalytic activity. The high content of P420 species detected from microsomes containing C4H::GFP appears to be derived from inactivated P450 enzymes. This result is not unexpected since the heme-binding region is located close to the C-terminal end of C4H adjacent to the GFP-tag (Figure 3.1). Formation of P420 species is not necessarily a result of extensive protein-unfolding but can arise from subtle structural changes or modification of the active site that distorts correct heme-binding to apoprotein (Yu ef al., 1995, and references therein). The GFP domain could potentially constrain the active site and make the heme-binding more susceptible to conformational change, thus leading to the high P420 content of C4H::GFP. On the other 61 hand, the reduced specific activity of C4H::GFP in vitro (Table I) may be caused by the reduced accessibility of the fusion enzyme to CPR, resulting from the interference of the C-terminal 26 kD GFP polypeptide with the C4H-CPR interaction. However, retention of significant activity by the fusion enzyme in vitro and in vivo suggests that proper C4H-CPR interactions can occur in yeast and are also likely to occur in transformed Arabidopsis. In vivo assay of C4H::GFP activity and in vitro assays based on spectrally active C4H::GFP were always more reproducible and had higher activities than the specific activity of in vitro assays based on total protein (Table I and Figure 3.10). This result may suggest that C4H::GFP enzymes are more susceptible to inactivation during or after microsomal preparation. In this aspect, an in vivo assay is a convenient and reliable tool to deal with labile enzymes such as P450s. Expression of the poplar C4H::GFP in transgenic Arabidopsis allowed its sub-cellular location to be determined in vivo. A series of optical sections of epidermal cells using confocal microscopy identified characteristic reticulated ER-pattern, demonstrating that C4H was strictly retained on the cortical ER. No fluorescence was detected in the Golgi apparatus, mitochondria, chloroplasts, or other cellular organelles, although these have been easily identified by GFP-tags in other studies (Kohler, 1998). Immunohistochemical studies in French bean suggested that C4H is abundant in the Golgi of that species (Smith ef al., 1994). However, I found no evidence for fluorescent signals in the mobile, punctuate Golgi organelles visualized by GFP tagging in living tobacco cells (Boevink et al., 1998; Nebenfuhr ef al., 1999) or numerous speck-like Golgi detected by an immunolocalization method in fixed Arabidopsis cells (Wee et al., 1998). Strict ER-retention of C4H demonstrates that the ER is the subcellular site where C4H initiates phenylpropanoid metabolism following the PAL reaction. Two other key P450s, C3H and F5H, are also known to participate in the biosynthesis of monolignols (Humphreys et al., 1999; Schoch et al., 2001), together with other soluble enzymes. It will be challenging but interesting to investigate whether these P450s are required to organize the proposed MECs on ER. Inappropriate retention of proteins in the ER can result from quality-control mechanisms that prevent defective proteins from leaving the ER (Hurtley ef al., 1989; Lodish, 1988). The P420 species in yeast microsomes suggests the possible presence of defective C4H::GFP proteins in Arabidopsis. However, this explanation for C4H localization to the ER, and their apparent absence from the Golgi, can be excluded since a significant fraction of C4H::GFP in yeast microsomes retained normal catalytic and spectral properties. 62 This suggests that functional C4H::GFP should be accessible to the machinery for potential trafficking outside the ER, and argues against the exclusive artifactual trapping of defective C4H::GFP fusion proteins in the ER. In animal systems, P450 2C1/2 and 2E1 are the most studied P450s with respect to their subcellular localization. These two closely related P450s seem to be retained on ER by two different mechanisms; the P450 2C1/2 is static on ER by direct retention conferred by the primary N-terminal sequence (Szczesna-Skorupa ef al., 2000), while the P450 2E1 is retained on ER by retrieval from the pre-Golgi compartment, mediated by an uncharacterized retrograde pathway (Szczesna-Skorupa and Kemper, 2000). Although the presence of an efficient retrograde pathway was recently shown in plants, it is not clear how much the retrograde pathway contributes to the ER-retention of ER-soluble or -integral proteins in plants (Pagny ef al., 2000). Considering the lack of knowledge in this area, it cannot be completely ruled out that C4H shuttles between the ER and the pre-Golgi apparatus. Thus, I might not have been able to detect GFP-signals in pre-Golgi compartment because of the rapid retrieval of C4H to the ER. Immuno-detection of the Golgi specific oligosaccharide fucose-moiety from purified C4H (Smith ef al., 1994) is in agreement with this possibility. Besides ER, 5-10 um-length elongated mobile organelles were frequently detected. Similar organelles have been previously described in Arabidopsis transgenics for an ER-targeted GFP fusion protein (Cutler ef al., 2000, supplemental data; Kohler, 1998; Ridge et al., 1999), but were not observed in other plants such as tobacco (Boevink ef al., 1996, 1998). Gunning (1998) noticed that these subcellular structures closely resemble the dilated ER cisternae characteristic of the Brassicaceae, and thus suggested that they are localized, continuous-ER subdomains in Arabidopsis. However, recent immuno-electron microscopy suggested that the organelles are resident within the lumen of the ER (Hawes ef al., 2001). Further investigations are required to understand the potential biological implication of apparent genus-specific organelles and to explain the extraordinary mobility of the organelles with and/or within ER, independent of the classical cytoplasmic stream. In addition to the ER-localized fluorescent signals in epidermal cells, I frequently found strong fluorescent signals within unidentified organelles in the guard cells of cotyledons in Arabidopsis lines expressing C4H::GFP. To my surprise, a series of optical sections of the organelles revealed that the intense fluorescence was uniformly present inside the organelle. It is possible that these are lytic vacuoles, to which some fraction of malfolded C4H: :GFP is transported for degradation, but lytic vacuole-targeted GFPs are 63 rapidly degraded so that only non- or very weak-fluorescent vacuoles have been observed (Di Sansebastiano et al., 2001). Furthermore, these fluorescent, round organelles were often located next to a non-fluorescent large central vacuole that is usually considered as the lytic vacuole. It is more likely that the unknown organelle is a protein storage vacuole (PSVs) (personal communication with Dr. John C. Rogers, Washington State University). Supporting this interpretation, direct protein-movement from ER to the PSV, bypassing the Golgi apparatus, has been demonstrated (Hara-Nishimura ef al., 1998; Jiang and Rogers, 1998). The dense GFP signals inside the organelles are good agreement with the finding that integral membrane proteins are localized in the lumen of PSV as an internalized, crystalloid membrane structure (Jiang ef al., 2000). A few future experiments could be designed to prove the identity of these organelles. Because of their large size and intense fluorescence, it should be feasible to isolate the intact organelles by classical density gradient centrifugation traced by their fluorescence signals. Marker protein profiles from the purified organelle could reveal its identity. Also, co-localization of the GFP-signal with an other PSV-specific antibody, such as anti a-tonoplast intrinsic protein antibody, could directly test if the organelle is indeed the PSV. However, it is most likely that C4H::GFP localization to these organelles represents artifactual mislocalization, due to overexpression of the fusion protein by the CaMV 35S promoter. The mechanism of C4H: :GFP protein localization to the organelle remains to be solved, but this fusion protein could provide an interesting tool to understand ER-PSV protein trafficking. 64 Chapter 4 Cloning, Functional Expression, and Subcellular Localization of Poplar Cytochrome P450 Reductase 4.1 Introduction Three P450 enzymes are known to play essential roles in defining the physical and chemical properties of monolignols (i.e., p-coumaroyl, coniferyl, and sinapyl alcohols) by catalyzing the 3-, 4-, and/or 5-hydroxylation of cinnamate or its derivatives. Phenylalanine ammonia lyase (PAL) and cinnamate-4-hydroxylase (C4H, CYP73A) together commit carbon flow into the phenylpropanoid pathway from primary metabolism by formation of cinnamate and 4-hydroxylation of cinnamate to p-coumarate, respectively, p-coumarate is a direct precursor for p-coumaroyl alcohol biosynthesis in monocots and specialized cell types such as those in compression wood (Lewis and Yamamoto, 1990). Genetic and biochemical evidence in Arabidopsis shows that 3-hydroxylation of the phenolic ring is catalyzed by the CYP98A3, 5-0-(4-coumaroyl)-shikimate 3-hydroxylase (C3H), and that this is required for coniferyl alcohol and guaiacyl lignin synthesis (Franke etal., 2002; Schoch et al., 2001). Additional 5-hydroxylation is catalyzed by CYP84A1 , coniferaldehyde 5-hydroxylase, which is responsible for the formation of sinapylalcohol and syringyl lignin (Humphreys ef al., 1999). In animal and plant systems, the oxidative reaction of P450s is strictly dependent on NADPH:cytochrome P450 reductase (CPR) which provides electrons from NADPH to a wide range of P450s. Therefore, in developing xylem, combined redox reactions of C4H, C3H, and coniferaldehyde 5-hydroxylase with C P R drive a large carbon flux into the lignin biosynthetic pathway. A single gene and enzyme for CPR was reported from Vigna radiata and Catharanthus roseus (Meijer ef al., 1993; Shet ef al., 1993), while Arabidopsis and parsley genomes apparently encode two divergent C P R isoforms which are differentially regulated by external stresses (Koopmann and Hahlbrock, 1997; Mizutani and Ohta, 1998; Urban ef al., 1997). Despite CPR's central role in oxidative reactions of lignin biosynthesis and the biosynthesis of numerous other secondary metabolites, C P R has not been characterized in the genus Populus. Of particular interest in poplar is the possible presence of a C P R isoform correlated with secondary xylem development. As well, cloning of poplar C P R cDNAs would allow reconstitution of the initial steps of phenylpropanoid metabolism, using enzymes entirely from poplar (see Chapter 5). In this study, poplar CPR cDNAs were 65 isolated, and their biochemical properties, subcellular fates, and expression patterns were examined using the previously characterized C4H as reference for a CPR redox partner. 4.2 Results 4.2.1 Poplar genome contains at least three CPR genes Two divergent Arabidopsis CPR cDNAs (encoding AR1 and AR2; Mizutani and Ohta, 1998), were used to isolate poplar C P R cDNAs from a xylem cDNA library by hybridization. As a result, three ~1.8kb partial cDNA clones were isolated from 100,000 pfu, and partial sequence analysis showed that these clones were identical and encoded sequences very similar to other plant CPRs. Two more rounds of screening for a full-length CPR cDNA clone from the xylem cDNA library were unsuccessful. Subsequently, a poplar young leaf cDNA library was subjected to screening for a full-length cDNA using this partial poplar cDNA clone (CPR181) as a probe. Several candidate full-length cDNAs were retrieved with the aid of PCR-based screening, using a reverse CPR181-specific primer specific to the 5'-end of this clone together with a vector T3 primer. All independent clones purified lacked at least a 750-bp 3'-portion, due to truncation at an internal Xhol site. However, sequence analysis of the 5'-portion of the clones showed that two 1.4 kb clones (CPR122 and CPR161) encoded putative ATG start codons and shared a 624-bp region identical to CPR181 (Figure 4.1). Further sequence comparison revealed that CPR122 and CPR181 were completely identical in their overlapping region, indicating that CPR122 and CPR181 were derived from the same gene. On the other hand, the CPR161 and its related clone CPR131 were identical to each other in their sequenced regions but showed 98.9% (477/482) identity to both the CPR122 and CPR181 clones. The position and nature of nucleotide changes between CPR122/181 and CPR131/161 were identical (Figure 4.1). This fact suggested that the few nucleotide differences were not caused by cloning or sequencing artifacts but that the differences were indeed derived from the poplar genome. Thus, the gene represented by CPR161/131 may be allelic to that represented by CPR122/181. Using two gene specific primers from 5'- or 3'-untranslated regions of CPR122 and CPR181, respectively, a 2.5-kb C P R cDNA was isolated from a pool of xylem cDNAs by a PCR-amplification. This clone, designated as C P R 1 , showed 100% sequence identity to both CPR122 and CPR181. 66 Poplar CPR1 clones 181 122 161 131 624 bp A B * Xhol 122 161 131 122 161 131 1 1 1 61 61 61 ,CGTCGTTTGCTGCTCTAGTTGGATTGMJGGTGCTTGTATTGAAGAGATCGTCCGATCGG RCGTCGTTTGCTGCTCTAGTTGGATTGQ J GGTGCTTGTATTGAAGAGATCGTCCGATCGG CGTCGTTTGCTGCTCTAGTTGGATTG!HGGTGCTTGTATTGAAGAGATCGTCCGATCGG GCAAAGACGTCAAGCCGTTGGTGGTTCCTAAG7CACT GCAAAGACGTCAAGCC3TTGGTGGTTCCTAAGTCACT GCAAAGACGTCAAGCC3TTGGTGGTTCCTAAGTCACT B 181 122 131 161 TATGAAACTGGAGACCATTTGGGGGTGTATGCTGAG TATGAAACTGGAGACCATTTGGGGGTGTATGCTGAG TATGAAACTGGAGACCATTTGGGGGTGTATGCTGAG TATGAAACTGGAGACCATTTGGGGGTGTATGCTGAG 181 122 131 161 61 61 61 g ATAGTGATGAAACTGTTGAAGAAGCAGGGAAGTTGCTAGATAAACCTTTAGATTTGTTG .ATAGTGATGAAACTGTTGAAGAAGCAGGGAAGTTGCTAGATAAACCTTTAGATTTGTTG .ATAGTGATGAAACTGTTGAAGAAGCAGGGAAGTTGCTAGATAAACCTTTAGATTTGTTG ATAGTGATGAAACTGTTGAAGAAGCAGGGAAGTTGCTAGATAAACCTTTAGATTTGTTG 181 122 131 161 121 121 121 121 TTTTCTATTCATGCTGATAATGAGGATGGCACAGCTATTGGAAGCTCATTGCCGCCTCCT TTTTCTATTCATGCTGATAATGAGGATGGCACAGCTATTGGAAGCTCATTGCCGCCTCCT TTTTCTATTCATGCTGATAATGAGGATGGCACAGCTATTGGAAGCTCATTGCCGCCTCCT TTTCTATTCATGCTGATAATGAGGATGGCACAGCTATTGGAAGCTCATTGCCGCCTCCT 181 122 131 161 181 181 181 181 TTCCCAGGTCCCTGCACACTTCACACTGCATTGGCATGCTATGCAGATCTCTTGA3CCCT TTCCCAGGTCCCTGCACACTTCACACTGCATTGGCATGCTATGCAGATCTCTTGA3CCCT TTCCCAGGTCCCTGCACACTTCACACTGCATTGGCATGCTATGCAGATCTCTTGA3CCCT ~ TCCCAGGTCCCTGCACACTTCACACTGCATTGGCATGCTATGCAGATCTCTTGAGCCCT CCTAAAAAGGCTGCTTTGCTTGCGTTGGCTGCTCATGCCAGTGAACCTAGCGAGGCAGAT CCTAAAAAGGCTGCTTTGCTTGCSTTGGCTGCTCATGCCAGTGAACCTAGCGAGGCAGAT C C T A A A A A G G C T O C T T T G C T T G C g T T G G C T G C T C A T G C C A G T G A A C C T A G C G A G G C A G A T CCTAAAAAGGCTGCTTTGCTTGC3TTGGCTGCTCATGCCAGTGAACCTAGCGAGGCAGAT 181 122 131 161 301 301 301 301 GACTCAAGTTTTTATCATCACCGCAAGGAAAGAATGAATACTCTCACTGGGTCATGGCA iGACTCAAGTTTTTATCATCACCGCAAGGAAAGAATGAATACTCTCACTGGGTCATGGCA L G A C T C A A G T T T T T A T C A T C A C C G C A A G G A A A G A A T G A A T A C T C T C A C T G G G T C A T G G C A GACTCAAGTTTTTATCATCACCGCAAGGAAAGAATGAATACTCTCACTGGGTCATGGCA 181 122 131 161 361 n 361 361 361 E GTCAGAGAAGTCTTCTCGAG GTCAGAGAAGTCTTCTCGAG GTCAGAGAAGTCTTCTCGAG GTCAGAGAAGTCTTCTCGAG Xhol Figure 4.1 Nucleotide sequence alignment of partial CPR1 cDNA clones. Gray-colored boxes show the sequenced regions used for the alignment. A 624-bp overlapping region of 3' and 5' partial clones is indicated by the two-arrowed brackets. Start and stop codons are shown by arrows and an asterisk, respectively. Note the consistent nucleotide changes in clones 131 and 161 compared to clones 122 and 181. 67 An in-frame stop codon was identified at 9-bp upstream of the first ATG located in a suitable sequence context for a translation initiation in plants (aggATGA). No other in-frame ATGs were found within the next 300 bp. This clone contained a 2,076 bp ORF, with 83-bp and 333-bp 5- and 3'-untranslated regions, respectively. The ORF encodes a polypeptide of 692 amino acids with a predicted molecular mass of 76,726 D and a pi of 5.18. The CPR1 sequence was most closely related to the Arabidopsis AR1 C P R gene, suggesting that other C P R isoforms more closely related to Arabidopsis AR2 could be present in poplar. Probing the young leaf cDNA library with the AR2 cDNA at low stringency allowed me to collect a set of C P R cDNA clones that were classified into two groups by restriction enzyme digestion analysis (CPR2 and CPR3, Figure 4.2-A). Sequencing a few clones from each group revealed two more full-length cDNAs (CPR216 and CPR351) that were -90% identical to each other in their coding regions and -80% identical in their 3'-untranslated regions. Two more partial clones from the CPR216 group were identical to the CPR216 in the sequenced regions. However, as was seen in the CPR1 sequence analysis, the partial nucleotide alignment of five cDNA clones from the CPR351 group revealed that there were also two very similar but distinct sub-groups within this group, 97.8% identical to each other (Figure 4.2-B). These two very similar classes of clones are likely to represent two alleles. Assuming the presence of a distinct allele for CPR351, CPR216 is not allelic to CPR351 in the diploid poplar genome. The two CPR genes represented by CPR216 and CPR351 were designated as CPR2 and CPR3, respectively. The CPR2 and CPR3 cDNAs possess stop codons 9- and 18-bp upstream of the first ATG codons, respectively, indicating that these are full-length cDNAs. Two more putative ATG codons for CPR2 and one more ATG codon for CPR3 are located downstream of the first start codon. However, the nucleotides neighboring these ATGs were deviated from the dicot start codon consensus, a(a/c)aAUGG, particularly with the occupancy of a pyrimidine at the important - 3 position (with A in ATG as +1) (Joshi et al., 1997). The flanking sequences of the first ATGs in both CPRs generally matched the consensus for initiation codons with an adenine at -3 positions (aacATGC for CPR2 and aacATGG for CPR3). Thus, the first ATGs were assumed to encode the translation start codons in both CPR2 and CPR3. The CPR2 cDNA contains a 26-bp 5'-untranslated region, a 2136-bp ORF, and a 325-bp 3'-untranslated region. The CPR3 cDNA contained a 117-bp 5'-untranslated region, a 2136-bp ORF, and a 351-bp 3'-untranslated region. Both the CPR2 and CPR3 encode 712-amino acids in ORFs with predicted molecular masses of 78,693 and 78,489 D and pis of 5.21, and 5.18, respectively. They showed 91% amino acid identity to each other. 68 A 200 bp CPR1 CPR2 EV X P El H S H B ^ I * l I EV P El Pv S EV ' l l l l * pCR2.1 pBluescript SK CPR3 x Pv J L El Pv I I S EV T B B H pBluescript SK B C T T T T T C G G C A C C " A A A C C G G T A C p J G C A G A A G G A T T T G C T A A G G C T C T A G C T G A G G A G G C C T T T T T C G G C AC C C; A A A C C G G T A c J j G C A G A A G G A T T T G C T A A G G C T C T A G C T G A G G A G G C C T T T T T C GGCAC C CAAACCGGTACGGCAGAAGGATTTGC TAAGGCTCTAGC T G A G G A G G C C T T T T T C O G C A C C S A A A C C G G T A C S G C A G A A G G A T T T G C T A A G G C T C T A G C T G A G G A G G C C T T T T T C G G C A C C C A A A C C G G T A C G G C A G A A G G A T T T G C T ^ G G f l J g B P J J B B J J g B B P AAAAGCTCGGTATSACAAGGCTACATTTAAAACTGTTGATGJTGGATGATTA AAAAGCTCGGTATSACAAGGCTACATTTAAAACTGTTGATJSTGGATGATTA AAAAGCTCGGTATSACAAGGCTACATTTAAAACTGTTGATGTGGATGATTAL AAAAGCTCGGTATSACAAGGCTACATTTAAAACTGTTGATQTGGATGAT'TAI CJ3CGGGTG, PSCGGGTG. 5 3 C G G G 7 G . 5 GCGGGTG, 3 1 5 1 3 1 7 1 3 5 1 1 3 1 1 1 3 6 1 1 3 1 5 6 1 3 1 7 6 1 3 5 1 6 1 3 1 1 6 1 3 6 1 T G A T G A T G A A T A C 3 A A G A G A A A T T G A A G A A A G A G G A T C T G G T T A T T T T C T T C T T G G C T G A T G A T G A A T A C 3 A A G A G A A A T T G A A G A A A G A G G A T C T G G T T A T T T T C T T C T T G G C T G A T G A T C A A T A C 5 A A G A G A A A T T G A A G A A A G A G G A T C T G G T T A T T 7 T C T T C T T G G C T G A T G A T C A A T A C 3 A A G A G A A A T T G A A G A A A G A G G A T C T G G T T A T T T T C T T C T T G G C 3 1 5 121 3 1 7 121 3 5 1 121 3 1 1 121 3 6 1 Figure 4.2 Restriction enzyme maps of three poplar CPR isoforms (A) and nucleotide sequence alignment of full or partial CPR3 cDNA clones (B). The CPR1 cDNA is a PCR product amplified by primers designed from 3' and 5' untranslated region of partial CPR1 clones, 181 and 122. The CPR2 cDNA is from the clone 216, and CPR3 is from the clone 351. Arrows indicate translation initiation codons, and asterisks indicate translation stop codons. The CPR cDNAs are cloned in the vectors listed at the side. Abbreviations of restric-tion enzyme sites are: B, BamHI; EI, EcoRI; EV, EcoRV; H, Hindll l ; P, PstI; Pv, PvuH; S, Sad; X , Xhol . 69 M H H rt H 0. « 0. 04 K U 04 O O ft a u A a u p. < m a. 4 l H I 4 M O. M Pt p. M U f t U U P . o u a fto g i i e g i i a. <t B- a < M i H K OS r» ft K 0. 0. X O 0. U O 0. a u a a u 0 u 0 0 u A < a, o. 4 91 ft A A 9t v n o o o n n n n n H n m H H K at IN o. M o. a M U Pi U U 0< a u a a u O U O O i J 0< 4 Pi tk < t t n r l r l i H m m «• » Pi • P> Pi OS U Pi O U PH a u a a u O U O O t i ft, <« 0 . ft, < i n m Ch cn co PS H M OS oi ft £ ft, £4 K U Pi U U Pi a u a a u O U O O i ) ft «c ft ft « n n ^ n 1*1 ooisnn u> w ip m v *« « m as H p; a s « 0 . a ft ft « U Pi U U PI a u a a u 0 u o 0 u Pi « ft Pi < M S •si s o S o Q S . U = Be a cs 1 i 3 4> P • = *W C/2 ° - 4 <U u ^ I jo 6, c <s £ 3 o J? ' c ^ c/a i * O T 3 C U <D *•> S.a 86 C 7 3 ^ O <^ rt § O cs .s — PH — S rt 7 3 .S o 3 3 o 7 3 C 7 3 D t*- i 7 3 O ?3 ON CQ ON 1 i-T s & s & 7 3 d 3 7 3 7 3 C c S E X> C 8 3 ca &o CA CA 7 3 7 3 .22 0 t>a S 3 ,ca 3 \ B L>13 CA 1 2 M t-, s £ 2 o „ to rt c 7 3 c u a eg g xi K W) <U D w 2 70 The deduced CPR polypeptides possess all functional domains characteristic for the CPR, such as FMN-, FAD-, NADPH-, cytochrome c-, and P450-binding sites (Figure 4.3). N-terminal domains of C P R s contain an approximately 20 amino acid hydrophobic patch required for their membrane-anchoring. Although poplar CPR1 and CPR2/3 share all functionally important domains, their N-terminal ends do not show any homology to each other. The N-terminal end of CPR2/3 is extended 20 amino acid longer than CPR1 and shows high amino acid identity to Arabidopsis AR2 isoform, while CPR1 is more closely related to Arabidopsis AR1 (Figure 4.3). A database search for C P R s revealed that CPRs from some plants (tobacco, Pisum sativum, Eschscholzia californica, and Catharanthus roseus) possess similarly extended N-terminal ends and have higher overall amino acid identity to CPR2/3 than CPR1, whereas CPRs from Vicia sativa and Vigna radiata are more similar to poplar CPR1 in their N-terminal structure and amino acid homology than CPR2/3 (data not shown). Overall deduced amino acid identities of C P R s within each group are always higher than that between groups. Therefore, two distinct C P R isoforms are distributed in plants, and ancient angiosperm plants appear to have evolved two C P R isoforms. 4.2.2 Functional expression of CPRs in yeast CPR1 and CPR2 were expressed in a yeast system together with C4H to ascertain the CPR catalytic activity encoded in the cloned cDNAs. The CPR3 was not examined in the heterologous expression study because of its high sequence identity to CPR2. It is known that endogenous C P R is a limiting factor in yeast when foreign P450s were over-expressed (Urban et al., 1994). Therefore, the enhanced P450 activities achieved by the CPR co-expression in yeast can be considered solid evidence for the biochemical identities of cloned CPR cDNAs. An independent biochemical assay, for CPR catalytic activity in vitro, uses its conventional artificial substrate, cytochrome c. For this purpose, a galatose-inducible yeast dual expression vector pESC-Leu was employed for simultaneous expression of C4H and CPR (Figure 4.4). The coding sequence for the C4H was inserted into the multiple cloning site behind the Gal-10 promoter in-frame with the FLAG-epitope. To the same vector, CPR1 or CPR2 were inserted behind the Gal-1 promoter in-frame with the c-Myc epitope. As a result, three pESC expression vectors were manipulated to harbor C4H alone, C4H/CPR1, or C4H/CPR2, and then three yeast strains were generated by independent transformation of these constructs. 71 An in vivo enzyme assay provides a convenient tool to evaluate P450 enzyme activities such as those of C4H, and the optimal harvesting conditions. Many P450 enzymes have been characterized in vivo by measuring the conversion of substrate in living yeast cells (Urban et al., 1994; Schoendorf, 2001). A series of time course enzyme assays was performed in yeast cells engineered to express three different sets of recombinant enzymes: C4H only, C4H and CPR1, or C4H and CPR2. Figure 4.5 shows that the catalytic activity of C4H was significantly higher when co-expressed with poplar CPR1 or CPR2 than when C4H was expressed alone, indicative of increased CPR activity in yeast system. Strains with a combination of C4H with CPR2 showed 2-3 times higher conversion rate of cinnamate to p-coumarate than those with C4H and CPR1. The maximum level of p-coumarate was produced at a cell density of approximately ~108 cells per mL. CPR1 |cMyc~| OR I F L A G | C4H pESC-Leu 7758 bp CPR2 |cMvcl \ / — T-ADH MCS1 P-GAL10 - P-GAL1 MCS2 T-CYC1 -Figure 4.4 Representation of the cloning and expression strategy for C4H and CPR1/2 in the yeast pESC dual expression vector. Abbreviations: T-ADH and T-CYC1; transcription termination sequences for alcohol dehydrogenase and cyclin 1 gene, respectively; MCS, multiple clon-ing site; P-GAL1 and P-GAL10, promoters for GAL1 and G A L 10 gene, respectively. 72 3 6 9 12 15 Cell Density ( x107 cells mL"1 ) Figure 4.5 In vivo production of p-coumarate from yeast strains expressing C4H-on\y, C4H and CPR1, and C4H and CPR2. Each yeast strain was pre-cultured in glucose for 12 h, and the trans-genes were induced by 2% galactose for 12 h. Formation of p-coumarate from cinnamate was quantified in aliquots harvested at 2-3 hour intervals after induction. Yeast microsomal fractions were prepared from cells at the optimal harvesting conditions to verify the CPR activities in vitro. Using microsomal preparations, recombinant C P R activities were evaluated either by measuring the formation of p-coumarate from cinnamate or by measuring the reduction of cytochrome c. In vitro C4H assays using the microsomes gave very similar data to in vivo estimations of C4H activity (Table 4). The microsomal fractions prepared from yeast expressing C4H/CPR1 and C4H/CPR2 showed significantly higher activities in cytochrome c reduction than C4H alone. The microsomal fractions from CPR1- and CPf?2-expressing yeast showed - 6 and ~3 fold higher cytochrome c reduction activities, respectively, than yeast transformed with C4H alone. The reduction of cytochrome c by CPR1 and CPR2 was dependent on NADPH, but not on NADH, as is known for other CPRs (Koopmann and Hahlbrock, 1997; Mizutani and Ohta, 1998). The yeast strain with C4H paired to CPR2 showed -2.5 times higher conversion rate of cinnamate to p-coumarate than the strain with C4H paired to the CPR1. Therefore, based on the in vivo and in vitro enzyme assays, I conclude that the CPR1 and CPR2 cDNAs encode functional, authentic C P R enzymes that properly interact with C4H and reduce cytochrome c in a NADPH-dependent manner. 73 Table 4 Catalytic activities of C4H and CPR recombinant enzymes from the microsomal fractions prepared from C4H- or C4H/CPR-expressing yeast strains. Vector CPR Act 3 construct C4H Act 3 (Relative Act) b NADH NADPH (Relative Act) C4H/ - 0.9 ±0.1 (1.0 ±0.1) 528 ±28 344 ± 25 (N.A.)C C4H/CPR1 2.8 ±0.2 (9.7 ± 1.3) 504 ± 39 1848 ±95 (1.0 ±0.1) C4H/CPR2 7.4 ±0.3 (14.3 ±1.3) 437 ± 30 1096 ±60 (2.4 ±0.1) a. Specific activities are given by nmol p-coumarate min"1 mg"1 for the C4H assay and nmol cyt c reduction min"1 mg"1 for the C P R assay. Each value is a mean ± SE from four independent microsomal preparations. b. For relative activities, the C4H or C P R specific activities are normalized by arbitrary units derived from the densitometric quantification of the C4H or C P R signals detected by ECL immuno-blot analysis. The C4H activity from the C4H-only expressing yeast or the CPR1 activity from CPR1/C4H-expressing yeast were used as a reference to calculate relative activities. c. N.A. : not applicable 4.2.3 Characterization of a dual expression in yeast Of particular biochemical interest is whether one of the poplar CPR isoforms preferentially interacts with C4H. It was clear from the in vivo and in vitro assays that both the CPR1 and CPR2 enhance C4H activity. However, in both cases, C4H activity was consistently higher when associated with CPR2 than with CPR1, despite a lower rate of NADPH-cytochrome c reduction in CPR2-containing microsomes. These results led me to further investigate whether the higher C4H activity is related to a relatively higher affinity of C4H towards the CPR2 isoform or simply a higher level of C4H protein in the C4H/CPR strains. As shown in Chapter 3, total P450 content in microsomes can be quantified by CO differential absorption spectroscopy. However, the quantification of C4H by this method did not indicate the presence of any detectable P450s in yeast microsomes from C4H or C4H/CPR expressing strains. I presumed that the amount of recombinant C4H in these strains was likely to be below the detection limit of the spectroscopic method (<10 pmol/ mg microsomal protein). In agreement with this conclusion, the presence of C4H was not detectable by S D S - P A G E analysis after Coomassie Blue staining, while new bands for CPR1 and CPR2 recombinant proteins with predicted molecular masses (78.4 kD and 80.4 kD after cMyc epitope fusion) were detected on the gel (Figure 4.6). Thus, the absolute amount of C P R appeared to be much higher than that of C4H in this expression system and C4H may be a limiting component in this dual expression system where C P R is highly expressed. 74 K D 116 -9 7 -8 4 " 6 6 -6 5 -4 5 -3 6 -26 -o > T- CN DC CC CL OL £ $ o o Figure 4.6 S D S - P A G E fractionation of microsomal proteins from vector-only, C4H-only, C 4 H / C P R 1 , and C 4 H / C P R 2 transformed yeast strains. 2 ug microsomal proteins were resolved by 10% polyaer\lumide gel and stained by Coomassie blue. Asterisks indicated putative recombinant CPR, and a arrow indicates the predicted position for recombinant C 4 H (-60 kD). The expression level of C4H in differently transformed yeast strains was compared by semi-quantitative immuno-blot analysis and also by northern blot analysis. In the immunoblot analysis, the same microsomal proteins previously used for the enzyme assays were applied, and the FLAG and c-Myc monoclonal antibodies recognized specific recombinant proteins of predicted molecular weights (Figure 4.7-A). C4H transcript levels were estimated in yeast strains transformed by different vectors (Figure 4.7-B). Unexpectedly, although the expression of C4H in all cases was driven by the same GaUO promoter in an identical vector background, the relative amount of C4H transcript was affected by the presence of C P R s and type of C P R . Yeast expressing C4H alone accumulated significantly higher levels of C4H transcript and protein than the C P R co-expressing yeast lines. Based on immuno-detectable protein level, the relative C4H activity in yeast expressing C4H alone was 10% or less than that in the C4H/CPR dual expressors (Table 4). However, within the dual expressors, the amount of C4H transcript and protein was generally proportional to the C4H activities previously measured by in vivo and in vitro enzyme assays. Thus, considering the C4H protein amount, there were only negligible differences in the relative C4H activities between yeast strains expressing C4H/CPR1 and C4H/CPR2. In addition, when normalized to C4H transcript levels, almost identical C4H relative activities were estimated for C4H/CPR1 and C4H/CPR2 expressors. Thus, the enhanced C4H activity in the yeast strain co-expressing CPR2 was a result of an elevated level of C4H transcript and protein and thus not a result from enhanced ability of CPR2 to support C4H activity. 76 A • kD 1 103" 76-I C4H/ - C4H/ CPR1 C4H/ CPR2 CPR2 M CPR1 < C4H 49" Figurc 4.7 Immuno-blot (A) and RNA-blo t (B) analysis of the vector-, C 4 H - , C 4 H / C P R 1 - , and C4H/CPR2-transt'ormed yeast strains. For immunoblol analysis, 2 ug of microsomal proteins were fractionated by the 109? polyaciylamidc gel and trans-ferred to the P V D F membrane. C P R 1/2 and C 4 H recombinant proteins were immuno-reacted on the same blot by anti-cMyc antibody and an t i -FLAG antibtxl). Specificities of antibcxlics were verified individually on separate blots prior to this immunoblol. For RNA-blo t analysis, 10 pg of total R N A was fractionated on a 1.59? formaldehyde agar-ose gel, transferred to a nylon membrane, and hybridized to a poplar C 4 H probe. 77 4.2.4 Two distinct CPR isoforms are localized to the ER Beginning from the FMN-binding site, three poplar CPR isoforms share high homologies to each other in all functionally important domains (Figure 4.3). However, the CPR1 and CPR2/3 cDNAs encode two entirely different N-termini, each of which has higher identity to its apparent isoform-specific orthologue in Arabidopsis than to its other isoform in poplar. This could suggest a potentially important role of the inter-genus conserved N-terminal sequences in determining different subcellular localization of CPR1 and CPR2/3. Figure 4.8-A illustrates the primary amino acid sequences of the C P R N-termini - non-conserved serine/threonine-rich regions, a 20 amino acid hydrophobic region, a partially conserved (35% identity between CPR1 and CPR2/3) charged amino acid-rich region, and the highly conserved FMN-binding site. The N-termini of CPR1 and CPR2 contain 30% (8/27) and 25% (12/49) serine/threonine, respectively. Although there is no strictly conserved amino acid motif for a chloroplast-targeting, a high composition of serine/threonine in the N-terminus is considered as an indication for chloroplast localization (von Heijne et al., 1989). Applying the C P R amino acid sequence to the program ChloroP ( for the in silico prediction of chloroplast localization did not provide concrete answers. Scores of the chloroplast targeting for CPR isoforms were 0.513 for CPR1 , 0.484 for CPR2, 0.479 for CPR3, where > 0.5 value is considered as chloroplast-localization. In order to determine the subcellular localization of CPRs , I fused GFP to the C-terminus of CPR1 and to the C-termini of two versions of CPR2 isoforms and expressed them in Arabidopsis under the control of the 35S-CaMV promoter. CPR2 and CPR3 contain very similar N-termini, and therefore only full-length CPR2 (CPR21) and its truncated version (CPR22) were used for this targeting study. CPR22 was truncated such that its second methionine is the translation start codon, resulting in deletion of five serines. To meet the nucleotide preference for the translation initiation (Joshi ef al., 1997), the cytosine at - 3 position in CPR22 was replaced with an adenine during PCR amplification. A total of >80 independently transformed T1 and T2 Arabidopsis seedlings for each construct were pre-screened for the presence of green fluorescence by fluorescence microscopy. Most of the kanamycin resistant plants presented weak GFP signals associated with the peri-nuclear membrane in the guard cells but showed no fluorescence in the epidermal cells (data not shown). Several Arabidopsis lines containing each construct showed detectable green fluorescence in the epidermis and some mesophyll cells. An immunoblot analysis using the microsomal proteins from these Arabidopsis seedlings 78 demonstrated that intact fusion-proteins were expressed in transformed Arabidopsis lines (Figure 4.8 B). Although the difference in mobility between CPR21 and CPR22 was difficult to distinguish, this subtle difference could be detected when the same genes were re-cloned and expressed abundantly in yeast (Figure 4.9 B). Confocal microscopy showed that all three different GFP-fused C P R s were strictly localized to the ER membrane in the epidermal cells (Figure 4.8 C, D, and E). The GFP-local izat ion patterns observed here were identical to those observed for C4H: :GFP (Chapter 3). As observed in the Arabidopsis lines expressing the C 4 H : : G F P and other ER-targeted G F P s , the mobile, elongated organelles were also detected in the epidermal cells from all three C P R : : G F P expressing Arabidopsis (Figure 4.8 C-E , arrow heads). Some mesophyll cells from hypocotyls had detectable GFP-s igna ls , and reticulated ER-like patterns were observed along with red autofluorescence from the chloroplasts (Figure 4.8 F, G. and H). These two signals did not overlap with each other, confirming that the C P R s were not localized to chloroplasts. Figure 4.8 Comparison of the N-terminal amino acids of poplar CPR (A), immunoblot analysis of the CPR::GFP proteins from transformed Arabidopsis (B), and localization of CPR::GFP in Arabidopsis seedlings by confocal microscopy (C-H). In A , green letters indicate serine or threonine, and asterisks indicate conserved amino acids in all three CPRs. Hydrophobic domains are shown by an underline, and an arrow-head points to the first amino acid for CPR22. Conserved charged amino acids are shown as + or - signs. (B) microsomal proteins from transformed or non-transformed control (WT) Arabidopsis plants were resolved in a 7.5% polyacrylamide gel, transferred to a PVDF membrane, and immuno-reacted with anti-GFP-antibody. The ECL-plus detection system was used to visualize signals. (C-H), GFP-signals were detected in epidermal cells from cotyledon (C) or hypocotyl (D and E). Auto-fluorescence from chlorophyll and GFP-signals were separately collected through red- and green-channels, respectively, by confocal microscopy and merged by Photoshop software (F, G, and H). White arrows indicate elongated organelles associated with the ER. Bar =10 pm. 79 Poplar Cytochrome P450 Reductase CPR22 CPRl ^ MSSGGSNLARFVQ VLGI FGD L D WVIITTSFAA CPR21 MQ. -. MKV PLELMQAIIKGKVDP7NV E GG AAEMA LIRENREFVIILTTSIAV CPR3 MES SS SSIKVSPLDLMQAIIKGKVDPANV E GG VAEVA LILENREFVMILTTSIAV + CPRl LVGLWLVLKRSSDRSKDVKPLWPKSLSIKDEEDESEALGGKT KVTIFYGTQTGTAEGF 98 CPR22 LIGYWLLIWRRSSGYQKPKVPVPPKPLIVKDLEPEVDD—GKK KVTIFFGTQTGTAEGF 120 CPR 3 LIGCVWLIWRRSSGYQRPKVPVPPKPLIVKDLEPEVDD—GKKKVTIFFGTOTGTAF.GF1 120 3 8 60 60 * * ***** ********** FMN-binding B GFP-fusion I 1 r N N i a: cr Df C L C L C L o o o o 80 4.2.5 CPR::GFP fusion enzymes retain catalytic activity in support of C4H activity To exclude potential localization artifacts caused by inactive G F P fusions, functional verification of the G F P fusion proteins is necessary. For this purpose, the C P R : : G F P constructs were recloned into a pESC yeast expression vector already containing the C4H by restriction enzyme digestion (CPR21: :GFP and CPR22: :GFP) or PCR amplification (CPR1::GFP). In vivo analysis was used to measure C4H activities in the absence or presence of the CPR::GFP-fusion protein. Yeast transformed with the CPR: :GFP constructs catalyzed the C4H reaction at a significantly higher rate than the control where only C4H was expressed, though their C4H-supporting activities were lower in comparison to the native, non-GFP-fused CPRs (Figure 4.9 A). To validate this observation, the presence and relative amount of the CPR: :GFP and C4H protein were estimated by an immunoblot using anti-GFP or anti-FLAG antibodies (Figure 4.9 B). As previously observed, the highest amount of C4H was detected in yeast expressing C4H alone, although this strain had the lowest C4H activity. The lower C4H activity supported by C P R 1 : : G F P relative to CPR21: :GFP and CPR22: :GFP correlated with a lower C4H protein level in this strain. These data demonstrate that C P R : : G F P enzymes are correctly folded, and that the GFP fusions at the C-termini did not significantly interfere with interaction with C4H in vivo. Microsomal proteins prepared for immunoblot analysis were further used for in vitro enzyme assays of C P R activity. The patterns for the p-coumarate formation closely resembled those from in vivo feeding assay, i.e. the C P R : : G F P proteins increased C4H activity in all cases, with CPR21: :GFP and CPR22: :GFP showing higher activities (Figure 4.9 C). Interestingly, despite the fact that the in vitro assay data showed there were catalytically active CPR: :GFPs in the microsomes, the same microsomal fractions were not able to reduce cytochrome c (Figure 4.9 D). Thus, the CPR: :GFPs appeared to preferentially interact with C4H, but not with cytochrome c. 81 B C4H CPRGFP C4H GFP fusion + + + + - CPR1 CPR21 CPR22 2500 E 2000 •| 1500 § 1000 c o •c 500 • NADPH • NADH rl ri rl rl o o 3 o 33 O o u 33 o 33 N> N> O Figure 4.9 Relative in vivo C4H activities in the presence of GFP-fused CPRs (A), immunoblot analysis of the C4H and GFP-fused CPR recombinant proteins (B), in vitro C4H assay (C) and cytochrome c reduction assay (D). A, The C4H-only expressed yeast was used as a negative control (- control) and set as a relative value 1 (5.4 ±1.1 pmol p-coumarate min"! 10^ cell -!). The relative C4H activities from the yeast strains expressing C4H together with various CPR constructs are shown. Data represent mean ± SE from three independent experiments. B, 2 pg of microsomal proteins were fractionated on 7.5% SDS-PAGE, and protein blots were prepared on PVDF membranes. C4H or CPR::GFP recombinant proteins were detected by the ECL method using anti-FLAG or anti-GFP antibodies, respectively. C and D, C4H activity (C) or cytochrome c reduction (D) in microsomes from the yeast strains expressing CPR::GFP fusions together with C4H or C4H alone (- control). D, reduction of cytochrome c by CPR1 was used as a positive con-trol (+ control), and a strain expressing C4H alone as a negative control (- control) Data represent mean ± SE (n >3). Data in D was reproducible in an independent experiment. 82 4.2.6 CPR expression patterns in poplar The mRNA accumulation patterns of poplar C P R s in various tissues and in poplar cultured cells with or without elicitor-treatment were examined by a semi-quantitative reverse transcriptase (RT)-PCR analysis. This analysis employed C P R isoform-specific primers and 18S-specific primers as controls for RNA amount. PCR cycle number was optimized for each gene to ensure amplification endpoints were in the logarithmic phase. Amplified 18S fragments interfered with the RT-PCR of the CPR2 and CPR3, and these two genes were amplified without 18S internal control. CPR7-specific primers were designed in the coding region of CPR1. For CPR2 and CPR3, a common forward primer in the 3'-coding region of CPR2/3 and distinct reverse primers each for CPR2 or CPR3 in the 3'-untranslated regions were designed. When tested using CPR cDNA clones as templates, these gene-specific primer sets were able to distinguish different CPR cDNAs (Figure 4.10 A). However, using poplar genomic DNA as templates, the CPR1 -specific primers did not amplify any PCR-product, while the CPR2 and CPR3 primers showed ~700-bp longer PCR-products than those calculated from the corresponding CPR cDNAs. These data suggest that the CPR1 primer set may span an exon-intron junction or flank a very long intron (resulting in no PCR-products). The longer genomic fragments amplified by CPPR2/3 primer sets likely arose due to the presence of introns in poplar CPRs as shown in Arabidopsis CPR (17 introns for each Arabidopsis isoform). Therefore, the primer sets for each CPR isoform were not only gene specific, but also distinguished CPR cDNA amplicons from those of genomic DNA. We have previously documented the expression pattern of the poplar PAL2 gene (PAL7 cDNA) by northern-blot analysis (Subramaniam et al., 1993). Thus, the reliability of the RT-PCR analysis was first evaluated using this gene as a control. Relative levels of the predicted PAL2 RT-PCR products using P/\/.2-specific primers and RNA from different sources closely matched the expression pattern of this gene estimated by northern blot analysis (i.e. a highest expression in young leaf and a strong induction by elicitor). RT-PCR analyses of CPR transcript levels using the same total RNA preparations showed that CPR1 transcripts were ubiquitously present in all tissues and cells with highest expression in young leaves and lowest expression in mature leaves when normalized to 18S amplification. (Figure 4.1 OB). The highest amount of CPR2 transcript was detected in young leaf, and its expression in cultured poplar cells was barely detectable. The CPR3 gene appeared to have the strongest expression level among the CPR genes because with the same amount of total RNA it required about five fewer PCR-cycles than CPR1 and CPR2. We have previously shown a strong induction of genes involved in phenylpropanoid metabolism, such 83 as PAL, C4H, and 4CL, from the poplar cell-cultures treated with an elicitor derived from Fusarium oxysporum (Moniz de Sa et al., 1992; Ro et al., 2001). However, I did not find a significant induction of any CPR isoform by the same elicitor-treatment, while the transcripts of control PAL2 were induced more than 100-fold. 84 Primers CPR1 CPR2 I 1 I CPR3 1 I 1 Template G 1 2 3 G 1 2 3 G 1 2 3 B CD _ i co CD I c o E CD 00 c CD CD o £ • _j UJ + _ l UJ PAL2 I-CPR1 CPR2 CPR3 Figure 4.10 Specificities of primers for the three CPR-isoforms (A) and reverse-transcriptase P C R amplification of specific C P R isoforms from poplar tissues and cell culture (B). A , 100 ng of poplar genomic D N A (G) or 1 ng of CPR1 (1), C P R 2 (2), and CPR3 (3) c D N A s were P C R amplified using gene-specific primers B , 100 ng of total R N A isolated from poplar tissues or cell cultures was used for R T - P C R after the optimization of P C R cycle number (20 cycles for P A L 2 and C P R 3 ; 25 cycles for CPR1 and CPR2). The arrow-head indicated specific R T - P C R products, and the arrow indicates the 18S internal control amplified by 18S primers with competitors (Ambion). A l l four R T - P C R products were amplified i rom the same R N A batch, and similar expression patterns were obtained in an independent experiment. 4.2.7 CPR enzyme activity is increased in poplar cultured cells by elicitor treatment In cultured poplar cells, none of the poplar CPR genes were significantly induced by elicitor-treatment. The major secondary metabolites synthesized de novo upon elicitor-treatment in poplar are not well characterized, however these are known to include caffeic acid derivatives that require at least two P450-catalyzed reactions for their biosynthesis (P. Spencer and C. Douglas, unpublished). Thus, it is unexpected that none of the CPR genes was coordinately activated with phenylpropanoid defense-related genes. I further examined CPR induction at the protein level. CPR enzyme assays based on cytochrome c reduction were performed on microsomal fractions prepared from the same elicitor treated and non-treated control cells that were used for RT-PCR analysis. Microsomes from the elicitor-treated samples had about 4 times higher activity than those from non-treated control cells (Figure 4.11). Therefore, although I could not detect transcriptional up-regulation of the three poplar CPR genes, CPR enzyme activity was induced by elicitor in cultured poplar cells. c o *3 o _ a T •o c £ I " £ o E e 10, 8 6 4 2 0 EL- EL+ Figure 4.11 Cytochrome c reduction by microsomal fractions pre-pared from the cultured poplar cells with (EL+) or without (EL-) elic-itor treatment. Microsomal fractions were prepared from elicitor-treated or control cells and used for cytochrome c reduction assays. Data are mean ± SE from three independent measurements. 86 4.3 Discussion Plant CPR genes have been characterized in Arabidopsis (Mizutani and Ohta, 1998; Urban et al., 1997), parsley (Koopmann and Hahlbrock, 1997), poppy (Rosco et al., 1997), and Douglas fir (Tranbarger et al., 2000). However, CPR genes have not been characterized in hybrid poplar, an economically important woody plant. In this chapter, C P R cDNAs from hybrid poplar were isolated and characterized with respect to the biochemical functions of recombinant enzymes, subcellular localization, and expression patterns. As is the case for other poplar phenylpropanoid genes such as PAL, 4CL and C4H (Allina et al., 1998; Cukovic et al., 2001; Ro et al., 2001; Subramaniam ef al., 1993), C P R is encoded by a small gene family in poplar. Among the three CPR cDNAs studied here, CPR1 and CPR2/3 displayed highly divergent sequences, while the CPR2 and CPR3 showed 92% amino acid identity to each other. In addition, partial sequencing of several additional CPR cDNA clones (Figures 4.1 and 4.2) indicated the presence of two putative alleles each for CPR 1 and CPR3. The cDNA libraries in this study were constructed from a hybrid poplar (Populus trichocarpa x P. deltoides), and thus complete heterozygosity is expected in its genome and cDNA libraries. Considering that two probable CPR3 alleles were found in the cDNA population, CPR2 and CPR3 appear to be two different genes, likely to have arisen from a recent gene duplication. CPR2 and CPR3 did not show identical expression patterns in the RT-PCR analysis (see below for details), further supporting the idea that they were not allelic to each other. Duplicated, very similar genes have often been identified in core phenylpropanoid metabolism of poplar, apart from highly divergent genes. Two very similar PAL genes (PALg2a and PALg2b) were clustered in Populus kitakamiensis genome (Osakabe ef al., 1995), and three genomic clones containing C4H genes with > 96% nucleotide homology were identified in the same poplar species (Kawai et al., 1996). Two closely related PAL genes (PAL1 and PAL2) distantly related to the PALg2a and PALg2b were characterized in hybrid poplar, (Gray-Mitsumune et al., 1999; Subramaniam ef al., 1993). Finally, two similar, recently diverged 4CL genes (Ptd4CL1 and Ptd4CL2) encode enzymes that are identical in their substrate use preference but differ in their gene expression patterns (Allina et al., 1998; Cukovic et al., 2001). Thus, recent duplications of genes encoding enzymes in core phenylpropanoid metabolism seem to be common in Populus. The identification of two divergent classes of CPR genes (CPR1 and CPR2/3) shows that poplar contains at least two distantly related CPR genes, as other plants (Koopmann and Hahlbrock, 1997; Mizutani and Ohta, 1998; Urban et al., 1997). In contrast, evidence for 87 a single C P R in animal systems was shown (Porter ef a/., 1990; Simmons ef al., 1985). Multiple plant C P R isoforms have,also been inferred by western-blot analysis after biochemical purification of C P R proteins from the microsomal fraction of Jerusalem artichoke tubers (Benveniste et al., 1991). In Arabidopsis, besides the two classes of characterized CPR genes (Mizutani and Ohta, 1998), a representative of a potential third CPR gene class was deposited in the GenBank database (accession number AAF02110), though its authenticity as CPR needs to be proved. The presence of multiple CPRs is however not unexpected in plants, considering the diversity of P450s that have physiological requirements for CPRs, and the wide range of primary and secondary metabolites essential for plant growth and development that are synthesized through P450-mediated reactions (Chappie, 1998). Despite the diversity of biochemical reactions they catalyze, 62% of Arabidopsis P450s (153/246) subjected to phylogenetic analysis clustered within the same lineage group of so called plant specific "A-type P450s" (Durst and Nelson, 1995; All the P450s belonging to this A-type are presumed to have evolved after divergence of plant and animal/fungal lineages. Along the same line, it is reasonable that plants may have evolved CPRs with specific functions to cope with various reducing demands upon diverse and unique plant P450s that are apparently absent in animals. We measured the CPR-supported C4H activities after a dual expression in yeast. Although different levels of recombinant proteins that accumulated complicated analysis, the data here in general showed that both C P R isoforms could equally support the best characterized A-type P450 enzyme, C4H. This is in agreement with findings reported from other plants (Mizutani and Ohta, 1998; Urban ef al., 1997). However, other cloned P450s from either other A-type or non-A type P450s (e.g., CYP51; obtusifoliol 14 oc-demethylase) (Bak ef al., 1997; Kushiro ef al., 2001) should be applied to the experimental reconstitution system with different CPR isoforms to determine if there is any specificity in P450 enzyme-CPR isoform interaction. Sequence comparison of the CPRs from poplar and Arabidopsis presented striking homologies in the N-termini within the same CPR group. The overall length of N-termini and even many amino acids were conserved in each class of CPR in an isoform-specific manner (Figure 4.3). Thus, this conserved primary sequence may suggest functional relevance of this N-terminal domain maintained through evolution in distantly related species, Arabidopsis and poplar. One plausible explanation for this is that the different N-termini contain information for the different subcellular destination of each C P R isoform'. In 88 particular, poplar CPR2/3 and its orthologue Arabidopsis CPR2 possess an extended N-terminus with high serine/threonine content, generally believed to be required for chloroplast targeting (von Heijne ef a/., 1989). However, due to the lack of absolutely conserved amino acids for chloroplast-targeting, the assessment of chloroplast-localization requires experimental evidence such as an in vitro chloroplast import assay or in vivo localization of a GFP-fusion. The GFP-localization experiments for CPR here showed that both classes of C P R isoforms are localized and retained on the ER in the same localization pattern as the C4H: :GFP, and that neither was localized to chloroplasts. In addition, the CPR2 isoform engineered to utilize the second Met, in which the serine-rich region was deleted, was also localized to the ER, suggesting that that serine-rich region does not function significantly in subcellular targeting, and that minor N-terminal modification does not interfere with localization of CPR. Therefore, the conserved N-terminal regions do not direct differential subcellular localization of CPRs, and both types of CPRs are targeted and retained on ER in the plants. About 20 Arabidopsis P450 genes were identified that encode enzymes with high serine/threonine content at their N-termini (Watson ef al., 2001). Of these genes, enf-kaurene oxidase, the P450 enzyme involved in gibberellin biosynthesis, and a P450 enzyme with unknown function, CYP86B1 have recently proved to be localized to the outer membrane of the chloroplast without processing their leader sequences (Helliwell ef al., 2001b; Watson et al., 2001). CYP79B2/3, proposed to be a key enzyme for indole-3-acetic acid biosynthesis, was also predicted to be targeted to the chloroplast where its substrate tryptophan is synthesized (Hull ef al., 2000). It is evident that the chloroplast serves as an organelle for P450-mediated metabolism. Therefore, electron donors for P450s should be present in the chloroplast, although the absence of CPR: :GFP targeting to the chloroplast in this study is not consistent with roles of CPRs as electron donors for P450 reactions in that organelle. Thus, it is questionable whether plant CPRs function as electron donors for P450-catalyzed reactions in chloroplasts or other plastids. Plastids may employ a distinct electron donor for P450s as the animal mitochondria do. Several animal P450s are competent to function in mitochondria, and in these cases, a specific mitochondrial electron donor system comprised of two proteins, adrenodoxin and adrenodoxin reductase, supplies electrons from NADPH to the mitochondrial P450s (Bernhardt, 1996). Chloroplast thylakoid membranes also contain similar proteins, ferredoxin and ferredoxin reductase, which are encoded in the nucleus and serve as electron carriers in photosynthetic electron transport chain to produce NADPH. However, by a reverse 89 reaction, these proteins can also serve as electron donors from NADPH for various enzymes, such as glutamate synthase and nitrate reductase (Hirasawa et al., 1991; Privalle et al., 1985). Plant ferredoxin and ferredoxin reductase could serve as electron donors for bacterial and eukaryote P450s in reconstituted systems (Dong et al., 1996; Xiang ef al., 2000; Yamazaki et al., 1995), and a translational fusion protein composed of the rat P4501A1, chloroplast ferredoxin, and ferredoxin reductase, supports P450 1A1 catalytic activity (Lacour and Ohkawa, 1999). Thus, the chloroplast ferredoxin and ferredoxin reductase can substitute for C P R in vitro, though the relevance of these interactions for P450s in the chloroplast outer membrane is not clear. Due to the difficulty of isolation, the protein profiles of chloroplast envelope (outer)membranes are not well characterized, and there is currently no evidence for the biological function of the ferredoxin redox system in the envelope membrane. For these reasons, it is premature to suggest that ferredoxin and its reductase serve as the redox partner for the chloroplastic P450s. However, it should be noted that purified chloroplast envelope membranes catalyze an efficient desaturase reaction in a ferredoxin-dependent manner, indicating that important redox reactions are present in the choroplast envelope membrane (Schmidt and Heinz, 1990). Several ferredoxin- and ferredoxin reductase-like genes were found in a Arabidopsis database (TAIR accession no. for ferredoxin-like genes: At1g60950, At1g10960, At2g27510, At5g10000, At4g14890; for ferredoxin reductase-like genes: At1g30510, At4g05390, At5g66190, At1g20020). Functional characterization of these genes and subcellular/suborganellar localization of the encoded proteins in relation to the chloroplastic P450s will test whether the ferredoxin reducing system is involved in P450-mediated reactions in chloroplasts and other plastids. Though it is a prevailing dogma in cell biology that the primary sequence of a protein determines the subcellular destination of that protein, dual targeting of an identical animal P450 enzyme to two different subcellular organelles (ER and mitochondria) depending on its phosphorylation status has been reported (Anandatheerthavarada ef al., 1999; Robin et al., 2001). In this respect, it cannot be completely ruled out that a small fraction of CPR can gain competence to be localized to the chloroplast at a level undetectable by the confocal microscopy method used in this study, but at a sufficient level to support chloroplast P450 reactions. As was shown in the C4H: :GFP expression studies in Chapter 3, fluorescent, elongated, mobile organelles along the ER were observed in the epidermal layers of Arabidopsis expressing the various C P R : : G F P constructs (Figure 4.8). Based on the 90 accumulation of cysteine proteinases and a vacuolar processing enzyme in these organelles, Hayashi ef al. (2001) have recently defined these organelles as novel proteinase-storing ER-bodies. However, Golgi-targeted GFP and a secretory form of GFP are also transiently retained in these elongated organelles before being sorted to the destined organelles (Hawes ef al., 2001). In addition, it is obvious from my work that these organelles can harbor CPR and the P450 enzyme C4H. Thus, the organelles are more likely to be an integral part of the ER where all ER-proteins are localized, rather than a specialized organelle only for a specific set of enzymes. Most C4H: :GFP transformed lines showed an easily detectable level of green fluorescent signals by confocal microscopy analysis. However, CPR::GFP-transformed Arabidopsis lines exhibiting an adequate level of fluorescence were not easily identified, though the same vector background was employed in both constructs. More than 80 independent C P R : : G F P transformants were screened, but the fluorescence level from the selected highest expressors was still lower than that from weak C4H: :GFP expressors. I speculate that CPR structure may interfere with the GFP-conformation required for optimal fluorescence. The C P R : : G F P localization results were further substantiated by the ability of C P R : : G F P to support C4H activity in yeast, indicating that the GFP domain of the fusion enzyme does not significantly interfere with C P R interaction with C4H in living cells and microsomes, and the correct protein folding and orientation of C P R on the ER. In contrast, the C P R : : G F P fusion enzyme was incapable of supporting reduction of cytochrome c in microsomes. A reverse situation, in which the soluble CPR released from ER by protease-treatment retained its catalytic activity to reduce cytochrome c but not P450s has been reported (Lu ef al., 1969; Phillips and Langdon, 1962; Williams and Kamin, 1962). Experimental data such as the inhibitory effect of cytochrome c on P450 activity (Gillette ef al., 1957), cross-linking studies (Nisimoto, 1986), and site directed mutagenesis (Shen and Kasper, 1995), together with protein structural analysis (Wang ef al., 1997) consistently indicate that both cytochrome c and P450 proteins interact with a cytosolic CPR domain in close proximity to the C P R electron exit site at the FMN-binding domain. Bulky P450s should require a larger contact area for CPR than the small cytochrome c molecule, which at first sight would seem to cause more potential steric hindrance of C4H to CPR: :GFP than cytochrome c to C P R : : G F P . However, the observed preferential reduction of C4H by C P R : : G F P may be due to the fact that cytochrome c interacts with C P R from the soluble side in an in vitro assay, whereas C4H interacts with the CPR in a tight membrane-bound 91 form through the ER. In this case, cytochrome c interaction with C P R : : G F P could be preferentially disrupted by the bulky GFP domain extending into the cytoplasm; however, the membrane domains of P450 and CPR may significantly stabilize the P450-CPR association either by direct N-terminal hydrophobic interactions or by conferring the favorable structural conformations for P450-CPR interactions. It is generally accepted that electrostatic interactions make a major contribution to P450-CPR interactions (Cvrk and Strobel, 2001; Shen and Kasper, 1995), but the N-terminal domain seems to play a role in CPR-P450 interactions. RT-PCR analysis detected all three CPR transcripts in several poplar tissues, but young leaves always showed higher levels of CPR transcript accumulation. All three CPR transcripts were present in xylem, and a xylem-specific C P R gene was not found, suggesting that there is not a CPR enzyme specific to lignin-biosynthesis in xylem. CPR2 showed similar but slightly different expression patterns from CPR1 and CPR3. Accumulation of CPR2 transcripts was more predominant in young leaf, and CPR2 expression was low in both elicitor-treated and non-treated poplar cell cultures. Although speculative, this suggests a specific role for CPR2 in poplar young leaves in which other phenylpropanoid genes are also highly expressed (Subramaniam et al., 1993; Hu et al., 1998; Cukovic et al., 2001). The confined expression of poplar PAL and 4CL genes encoding specific isoforms in the epidermal or subepidermal cells of young leaf has been shown by in situ hybridization or promoter-GUS analysis (Gray-Mitsumune ef al., 1999; Hu et al., 1998; Subramaniam et al., 1993). Poplar buds and young leaves are rich in phenolic compounds, including flavonoids and hydroxycinnamic acid conjugates and esters (English et al., 1991; Pearl and Darling, 1971), whose biosynthesis requires P450 enzymes. In situ hybridization and promoter-GUS fusions could determine the precise cell-type specific expression of CPR2 relative to CPR1 and CPR3 in poplar. There was no significant induction of any of the three CPR genes by an elicitor, prepared from the cell wall fragments from Fusarium oxysporum. However, a significant increase of C P R enzyme activities was observed in the same elicitor-treated cell cultures (Figure 4.11). We have optimized the harvesting times following elicitor treatment on the basis of maximal PAL and 4CL enzyme activity (Moniz de Sa ef al., 1992), which are not necessarily optimal for CPR induction. Thus, it is possible that the induction mode of CPR is different from other phenylpropanoid genes such that transient CPR induction precedes that of PAL and 4CL. However, even assuming that the significantly increased C P R enzyme activities in cells in which CPR transcripts were not induced suggests that posttranslational 92 regulation such as CPR protein stability may play an important role to coincide CPR activity to other enzymes in phenylpropanoid metabolism. Similar posttranslational modulation of CPR activity was also observed previously in parsley (Koopmann and Hahlbrock, 1997) and Douglas fir (Tranbarger ef al., 2000). However, it is also possible that other unidentified elicitor responsive CPR genes are present in poplar. The dual expression vector employed allowed convenient co-expression of C4H and C P R in a single yeast strain. The Gal l and GaMO promoters controlling C4H and C P R expression are strictly regulated by glucose/galactose at the level of transcription and the promoters comparably induce reporter genes (information supplied by manufacturer). Thus, we expected that similar levels of both recombinant proteins would accumulate. This was expected to simplify in vivo evaluation of differential ability of C P R to support C4H activity without membrane reconstitution in vitro using individually prepared recombinant proteins. However, we unexpectedly found unequal amounts of recombinant C4H and C P R in different strains (Figure 4.7). The significantly decreased C4H recombinant protein levels relative to C P R in C4H/CPR dual expressing strains could result from C4H codons unfavorable for use in yeast. Codon use in both CPR1 and CPR2 mRNAs does not severely deviate from that in yeast, but the predicted C4H mRNA contains a few unfavorable codons for yeast expression such as CTC (Leu) and A G G (Arg). We recovered >100 pmol C4H per mg protein from yeast grown in rich medium (Chapter 3). However in the minimal medium conditions used for dual expression, rare tRNAs could be limiting factors for efficient translation of C4H, especially in combination with C P R . Batard ef al. (2000) showed that codon usage is a serious problem for yeast expression of monocot P450s with a high proportion of rare yeast codons. In addition, expression of different combinations of C4H and CPR resulted in different levels of C4H transcripts. We do not have a clear explanation for this variation, but other unknown variations in transcription seem to be introduced by dual expression of C4H and CPR. 93 Chapter 5 Reconstitution of phenylpropanoid metabolism in yeast 5.1 Introduction Phenylalanine (Phe) is used as one of the building blocks for protein synthesis, and is generally accepted as an end product of primary metabolism. However, starting from Phe, a variety of phenylpropanoid metabolites are synthesized in plants by way of phenylpropanoid metabolism. The first committed step in the phenylpropanoid pathway, the production of frans-cinnamate from Phe, is catalyzed by phenylalanine ammonia-lyase (PAL). PAL is considered to be a key enzyme, whose appearance may have allowed successful adaptation of plants to land by providing a pathway for the biosynthesis of key phenylpropanoid products from primary metabolism. For example, the most abundant phenylpropanoid product, lignin, provides plants with the rigid, hydrophobic secondary cell walls of tracheary elements and fibres within vascular bundles, while other products such as flavonoids function as efficient UV-absorbing sun-screens. Both lignin and phenolic sun-screens are likely to have played important roles in allowing vascular plants to become dominant terresterial colonizers (Whetten and Sederoff, 1995). Since PAL resides at a metabolically important position, linking a secondary pathway to primary metabolism, the regulation of overall flux into phenylpropanoid metabolism has long been suggested to be modulated by PAL as a rate-limiting enzyme (Hahlbrock et al., 1976). How this regulation is accomplished, however, is not completely clear. Feedback inhibitory regulation of PAL by its own product, frans-cinnamate, has been demonstrated in vitro (Appert et al., 1994; Havir and Hanson, 1968; Jorrin and Dixon, 1990), and proposed to modify transcription of the PAL gene in vivo (Bolwell et al., 1988; Mavandad ef al., 1990). It has also been suggested that PAL isoforms could form hypothetical multienzyme complexes (MECs) that would include downstream enzymes such as C4H. These complexes could synthesize different phenolic products, or different lignin precursors (for details, see Chapter 1). It has been shown that a specific PAL isoform (tobacco PAL1) is physically associated with both isolated microsomes and the cytoplasmic fraction in fractionated tobacco cell extracts, consistent with the model that PAL may associate with MECs in an isoform-specific manner (Rasmussen and Dixon, 1999). In our laboratory, poplar cDNAs encoding the entry point enzymes (PAL, C4H, CPR, and 4CL) of the phenylpropanoid pathway have been cloned and characterized previously (Allina ef al., 1998; Cukovic ef al., 2001; Gray-Mitsumune et al., 1999; Ro ef al., 2001; 94 Subramaniam ef al., 1993; Chapters 3 and 4 of this thesis). While PAL is operationally a cytosolic enzyme, the redox enzymes C4H and CPR have been shown in previous work to be ER-bound (Ro ef al., 2001; Chapters 3 and 4 of this thesis), where they could, in principle, help anchor a MEC that includes PAL. While the poplar C4H, CPR, and 4CL gene families in the P. trichocarpa X P. detloides hybrid used in our studies have been well characterized (Allina ef al., 1998; Cukovic ef al., 2001; Ro ef al., 2001; Chapters 3 and 4 of this thesis), the nature of the PAL gene family, and the expression of different PAL genes and isoforms in this genotype remains uncertain. In previous studies in our lab, a poplar cDNA, PAL7, was isolated that was specific to poplar PAL2 and closely related to PALL PALI/2 were shown to be highly expressed in epidermal cells of young leaves and stems, but poorly expressed in developing secondary xylem of poplar (Gray-Mitsumune et al., 1999; Subramaniam ef al., 1993). The isoforms encoded by PAL1/2 are thus most closely associated with biosynthesis of flavonoids and other phenylpropanoid derivatives. There is strong evidence supporting the presence of one or more additional PAL genes associated with lignin biosynthesis in this genotype. In northern blot analysis, a genomic fragment derived from a putative divergent PAL gene, named PAL3, detects a higher level of PAL transcripts in developing xylem than does PALI/2 (Ro ef al., 2001). More direct evidence comes from a report that Populus kitakamiensis contains two 96% identical PAL genes (PALg2a and PALg2b) whose expression appears to correlate with secondary xylem development in poplar (Osakabe et al., 1995). However, a cDNA for the presumed orthologue of PALg2a or PALg2b has not been characterized in P. trichocarpa X P. deltoides. In this chapter, I describe the cloning of a cDNA for a divergent, xylem-expressed PAL gene that completes the set of entry-point phenylpropanoid genes from poplar. I then describe the introduction of the entry-point enzymes, PAL, C4H, and CPR, into the yeast Saccharomyces cerevisiae in different combinations in an attempt to reconstitute the entry-point of phenylpropanoid metabolism in a phenylpropanoid-free eukaryotic cell. Using simple and novel analytical tools, I have used this system to explore how the entry-point enzymes of phenylpropanoid metabolism redirect the carbon flow from primary to secondary metabolism. Specifically, I test the hypotheses that PAL is a key enzyme controlling carbon flux into phenylpropanoid metabolism, that physical association between PAL and C4H as part of a MEC is important for efficient carbon flux into phenylpropanoid metabolism from primary metabolism, and that pathway intermediates (cinnamate and p-coumarate) help control enzyme activities and phenylpropanoid carbon flux. If evidence for a MEC containing 95 PAL and C4H were obtained, the yeast system would provide an ideal genetic background to investigate its functional properties in more detail, especially with respect to biosynthesis of lignin precursors. 5.2 Results 5.2.1 Cloning of a PAL cDNA from xylem It has been shown that PAL isoforms may differentially participate in MEC formation, (Rasmussen and Dixon, 1999). Since we had not yet isolated a poplar PAL gene whose expression was highly correlated with lignin biosynthesis, it was necessary to do this before proceeding with the planned phenylpropanoid reconstitution experiments in yeast. Since the P. trichocarpa X P. detloides PAL7 cDNA (encoded by PAL2) and its orthologue PALgl in P. kitakamiensis display substantial nucleotide sequence identity (94%), direct PCR-based cloning of a PAL cDNA from a P. trichocarpa X P. detloides clone H11-11 xylem cDNA pool was attempted, based on the reported sequences of the PALg2a and PALg2b genes. Following PCR with primers specific to these genes, ten PALg2b-\\ke clones with identical restriction enzyme maps were isolated, but P/4Lg2a-like clones could not be identified. The detailed restriction enzyme map of one of these clones (PAL18 cDNA) showed that it encoded sequences quite different from that of PAL7 cDNA (Figure 5.1). 100 bp i Hill SI i i El i * PAL18 cDNA Hill El SI i Hill El i i * PAL7 cDNA Figure 5.1 Comparison of restriction enzyme maps of PAL18 and PAL7 cDNAs. EI, HIII, SI, and XI indicate EcoRI, Hindlll, Sacl, and Xhol, respectively. 96 Sequencing of the PAL 18 cDNA confirmed that it contained a 2133-bp ORF (77% identical to the PAL7 cDNA) encoding 711 amino acids and a predicted molecular mass of 77,795 D with a pi of 5.75. The deduced amino acid sequence shared 96% (684/711) and 94% (645/686) identity to PALg2b and PALg2a, respectively, but 85% (608/711) identity to the poplar PAL7 cDNA. The PAL18 clone also possessed nucleotide sequences distinct from that previous PAL3 gene fragment. In order to distinguish this clone from the previously identified PAL clones, the corresponding gene for PAL18 cDNA was named PAL4. 5.2.2 Differential expression of two PAL genes Northern blot analysis was performed to investigate steady state PAL4 mRNA accumulation relative to PAL2 mRNA using the corresponding cDNAs as probes (Figure 5.2). Predominant expression of the PAL4 gene was detected in secondary xylem and to a lesser extent in green stems. However, very weak hybridization signals were found with RNA from young leaf tissue, and virtually no transcript was detected in mature leaf. In addition, PAL4 expression was undetectable in cultured poplar cells but strongly induced by elicitor-treatment. These expression patterns suggest that the expression of the PAL4 gene is developmentally regulated in association with secondary xylem formation and is stress-induced by elicitor treatment. To corroborate this finding, the same blot was re-probed with the PAL7 cDNA. As predicted, the expression pattern of PAL1/2 detected by this probe was very similar to that previously reported (Subramaniam et al., 1993), i.e. abundant transcripts in young leaf and elicitor-treated cell culture but little in xylem tissue. Therefore, the PAL4 gene clearly showed a predominant expression pattern in secondary xylem, while PAL2 is mainly expressed in young leaves. 5.2.3 Simultaneous expression of PAL, C4H, and CPR in yeast The cDNAs for these two differentially expressed PAL genes were used, together with those for C4H and C P R genes, to reconstruct the entry-point of the phenylpropanoid pathway in yeast cells. It has been shown that poplar C4H and CPR2 are expressed both in young leaf and xylem (Ro ef al., 2001; Chapter 4). Based on these expression patterns, I conjectured that the combined catalytic reactions of PAL2, C4H, and CPR2 could be involved in the biosynthesis of UV-protective phenolics and flavonoids in young leaves and stems, while PAL4, C4H, and CPR2 could be required for lignin biosynthesis in developing xylem. 97 Figure 5.2. Expression of two PAL genes in poplar organs and tissue culture cells. Total RNA was isolated from the poplar organs indicated or from tissue culture cells treated w ith (EL+) or without (EL-) an elicitor. 10 ug of total RNA was resolved on 10% formaldahvde agarose gels, transferred to nylon membranes, and hybridized with poplar a PAL18 cDNA probe for PAL4 or a' PAL7 cDNA probe for PAL2. Hybridization of stripped blots to an rRNA probe as a loading control is shown. Epitope-tagged versions of the C4H and CPR2 ORFs had been previously cloned in the pESC-LEU vector (Chapter 4). Thus, the ORF of the PAL7 cDNA (PAL2) or PAL 18 cDNA (PAL4) were cloned into the pESC-HIS vector under the control of the Gal-10 promoter. Single transformation of yeast with pESC-HIS containing P>4L2 or PAL4, and co-transformation with this PAL-containing vector, together with pESC-LEU containing both C4H and CPR2, resulted in generation of four different yeast strains. Two single transgene strains would express PAL2 or PAL4 alone, and two triple transgene strains would express PAL2/C4H/CPR2 or PAL4/C4H/CPR2. In addition, empty vectors of pESC-LEU and pESC-HIS were co-transformed to generate a vector-transformed control strain. In the following description, the two single P/\L-expressers are referred to as PAL2 or PAL4, and the two triple expressers are called T2 (PAL2/C4H/CPR2) or T4 (PAL4/C4H/CPR2). Expression of the three genes in yeast was verified by immunoblot analysis. The presence of C4H and C P R in microsomes from strains T2 and T4 was shown using anti-FLAG and anti-cMyc antibodies specific to the epitope-tagged C4H and CPR, respectively (Figure 5.3A). To detect recombinant PAL enzymes, a polyclonal PAL antibody previously raised against recombinant PAL2 protein (McKegney et al., 1996) was used in an immunoblot of cytosolic proteins. This antibody recognized strong protein bands for PAL2 recombinant protein in both single and triple expressers (Figure 5.3B). Although deduced amino acid sequences from PAL2 and PAL4 appear similar enough to provide many common epitopes, the PAL4 recombinant protein was not detected in either single or triple expressers using this antibody. Repeated experiments, and prolonged exposure times with higher concentrations of the antibody, still could not detect the PAL4 recombinant protein (data not shown). As an alternative method of confirming functional expression, PAL enzyme assays were performed using cytosolic fractions from transformed yeast strains. The cytosolic fractions of all four yeast strains expressing PAL, including PAL4 and T4, efficiently catalyzed the deamination of Phe to produce authentic frans-cinnamate as judged by diagnostic HPLC analysis (data not shown). Therefore, although the anti-PAL2 antibody failed to recognize PAL4 recombinant enzyme, it is clear that PAL4 enzyme was present in PAL4 and T4 yeast strains. Under Phe-saturated assay conditions, the PAL2 recombinant enzyme displayed higher catalytic activity than PAL4 (Figure 5.3 B), but within strains expressing the same PAL isoform, there were no significant differences in PAL activities between single and triple expressers. 99 O CM o —I —I 0) < < > 0. Q . CM CPR C4H B KD 1 0 3 -76 H 4 9 -33-100 80 g 60 ~ r o ro Q . • PAL-alone • Triple expressor PAL4 PAL2 Figure 5.3 Phenyl propanoic! protein amounts and P A L activity in transgenic yeast strains. (A) Immunoblot analysis of microsomal proteins from the yeast strains indicated, react-ed with a cMyc monoclonal antibody to detect CPR and a F L A G monoclonal antibody to detect C4H. (B) Immunoblot analysis of total protein from the yeast strains indicated, reacted with a polyclonal anti-PAL2 antibody. (C) P A L enzyme activity in PAL-alone or triple expressing yeast strains. Microsomal proteins (2 ug; A) or total proteins (5 ug; B) were resolved by L09E SDS-PAGE, transferred to PVDF membranes, and immuno-dctectcd by chemiluminescence. P A L enzyme assays were performed using soluble cytoplasmic fractions as described in "Materials and Methods". Values arc means ± SD of triple measurements each from two independent experiments. 100 5.2.4 Analysis of phenolic products in transformed yeast Having confirmed that comparable amounts of recombinant proteins were present in the four transformed yeast strains, the efficiency with which these strains re-direct Phe to phenylpropanoid metabolites was analyzed. PAL, C4H, and C P R expression was induced for 15 - 20 hours, after which the induced yeast cells were cultured in fresh media containing Phe (0.5 mM) for 2 hours. The culture media and cell-pellet were subsequently extracted with organic solvent, and the extracts were fractionated by HPLC. When the extract from vector-transformed control cells was analyzed by HPLC, a few small peaks were identified at 290 nm, but no other major peaks were detected (Figure 5.4). One of these small peaks appeared to be phenylpyruvate, based on its comparison to the authentic standard. Phe is synthesized from phenylpyruvate by a reversible reaction catalyzed by a transaminase (Urrestarazu et al., 1998), and Phe could therefore be readily converted back to phenylpyruvate in these cells. A rapid increase in the content of phenylpyruvate in cell cultures fed with high Phe concentrations was in fact detected (data not shown). HPLC analysis of culture media from PAL2- or PAL4- transformed yeast strains showed an additional small peak. Its retention time and spectrum were identical to those of authentic cinnamate (Figure 5.4). In contrast to the low levels of cinnamic acid produced in the PAL2 and PAL4 strains, strikingly large amounts of p-coumarate were accumulated in cultures of the triple expressers T2 and T4, accompanied by a small amount of cinnamate (Figure 5.4). These results were consistently observed in repeated experiments. Thus, the simultaneous expression of PAL, C4H, and C P R in yeast cells resulted in efficient production of p-coumarate, whereas PAL-alone expressers seemed to convert Phe to cinnamate with much lower efficiency. In these and subsequent experiments, neither cinnamate nor p-coumarate were consistently detected in yeast cells that had been separated from medium by centrifugation and extracted with acetone or chloroform, probably due to their low quantities within cells (data not shown). Although cinnamate was sometimes detected in cells, the amount was much smaller than that recovered from media. The maximum amount of cinnamate recovered from cell pellets was less than 1/10 of that recovered from media. Thus, the bulk of the cinnamate and p-coumarate was being secreted into the culture media. 101 A B Figure 5.4 HPLC-fractionation of phenolic metabolites produced by vector control, PAL-alone, and triple expressing yeast strains. (A) HPLC chromatograms of extracts from different yeast strains or standards, with absorbance at 290 nm in arbitrary units. Insets show peaks eluting between 30 and 40 minutes in an expanded scale. (B) U V absorption spectra of peaks eluting at positions corresponding to p-coumarate (PCA), cinnamate (CA), and phenylpyruvate (PPA) standards, with spectra of the authentic standards below. UV-spectra of PAL2 and T2 are the same as those of PAL4 and T4, and thus they are not shown in the figure. Yeast strains were grown to mid-log phase in glucose media, transferred to inductive galactose media for 18 hour. After 2 h incubation in fresh media supplemented with 500 pM Phe, media and cells were extracted by ether and acetone, respectively, and both fractions were pooled together for HPLC analysis. A l l chromatograms are at the same scale except that with standards. Retention times of peaks are given in minutes. 102 5.2.5 Production of cinnamate and p-coumarate in transformed yeast fed various concentrations of phenylalanine The yeast strain YPH499 used in these studies is prototrophic for Phe and in the absence of Phe added to the media, protein sythesis will be supported by an intracellular Phe pool. To examine whether triple-gene expressing yeast strains, T2 and T4, could produce p-coumarate from endogenous Phe, the strains were cultured in minimal media lacking Phe following galactose-induced PAL, CPR, and C4H expression. For comparison, yeast cells from the same induced cultures were also cultured in media supplemented with 0.5 mM or 5 mM Phe. Some obvious conclusions can be drawn from the results of these experiments, which are summarized in Figure 5.5. First, even in the absence of added Phe, significant amounts of p-coumarate were synthesized by the T2 and T4 strains, although cinnamate was not detectable in any yeast strain under these conditions. Thus, PAL and C4H in the triple expressing strains appear to act in a tightly coupled fashion, and together they efficiently redirect carbon from a pool of endogenous Phe to the secondary product p-coumarate in this host. Strains expressing PAL alone, on the other hand, do not have this capability. Second, with increasing Phe concentrations in the medium, the amounts of both cinnamate and p-coumarate produced by the triple expressers increased. However, in PAL-alone expressers, the amounts of cinnamate increased from undetectable levels to levels only slightly lower than the p-coumarate levels at 5 mM Phe. These data suggest that Phe availability (concentration) is a limiting factor in vivo for cinnamate production by strains expressing PAL alone, but that triple expressers are able to catalyze the conversion of Phe to p-coumarate very efficiently even at the low Phe concentrations produced endogenously by yeast. 5.2.6 Testing for modified PAL activity in triple expressing yeast strains From the data in sections 5.2.4 and 5.2.5, it was clear that efficient carbon-flow from Phe into cinnamate or p-coumarate via PAL only occurred in triple expressers, but not in strains expressing PAL alone. Several working hypotheses can be made to explain this apparently enhanced carbon-flux in triple expressers. Simplest model can be that C4H modifies the equilibrium of PAL reaction such that the formation of p-coumarate (i.e., elimination of cinnamate) can act as an overall reaction-driver in triple expresser. Alternatively, considering that both kinds of strains expressed similar levels of PAL protein, one plausible idea to 103 explain this is that the kinetic properties of PAL are somehow enhanced when PAL was expressed together with C 4 H and CPR, resulting in an increased affinity of the enzyme for Phe and/or a higher turn-over number. Feedback inhibition of PAL activity by cinnamate is also possible as indicated in section 1.2. Finally, feedback stimulation of PAL activity by p-coumarate could occur in the triple expressers since p-coumarate is present in triple expressing strains but not in PAL-alone expressers. These hypotheses will be tested in this and the following sections or will be discussed (for the model for cinnamate feedback inhibition) in section 5.3. 2000 1600 o 3 "g 1200 a o "5 c Q> JC a o E c 300 400 • /7-coumarate o cinnamate I I Strain P2 P4 T2 T4 I I Phe n I P2 P4 T2 T4 I I 0.5 mM P2 P4 T2 T4 I I 5 mM Figure 5.5 In vivo production of cinnamate and p-coumarate by genetically engineered yeast strains incubated with different concentrations of Phe. Cells induced for 18-20 h were resuspended in fresh media containing 0, 0.5, or 5 mM Phe for 2 h. P2 and P4 indicate PAL2- or PAL4-expressing yeast strains, and T2 and T4 indicate PAL2, C4H, and CPR2, or PAL4, C4H, and CPR2 triple expressing yeast strains, respectively. Levels of p-coumarate and cinnamate in media and cells were determined by HPLC. Data are mean ± SE from at least three independent cultures. 104 In order to examine the last possibility first, PAL-alone expressers induced for PAL expression were cultured in media containing various concentrations of p-coumarate ranging from 0 to 100 pM. No apparent enhancement of cinnamate accumulation was found in either strain PAL2 or PAL4 at any concentration tested (Figure 5.6). It is, of course, possible that p-coumarate could not reach the inside of cells. However, blocking C4H activity by use of a C4H inhibitor at 1 hour post-culture (thus, ensuring that significant amounts of intracellular p-coumarate were present) did not result in a high rate of cinnamate production (see below, Figure 5.7). p-Coumarate therefore does not appear to feedback activate PAL enzyme activity, and this is not a likely explanation for the efficiency of carbon flux into p-coumarate in the triple expressers. 1 10 p-coumarate (uM) 100 Figure 5.6 Production of cinnamate by PAL-alone expressing strains PAL2 and PAL4 in the presence of varying concentrations of p-coumarate. PAL2 and PAL4 strains were induced for 18-20 h and resuspended in fresh media containing 500 pM Phe and the indicated concentrations of p-coumarate. Cinnamate levels in cells and media were assayed by HPLC. Another possibility for the apparent enhancement of PAL kinetic properties in triple expressers would be an allosteric change in PAL brought about by physical interaction between PAL and C4H/CPR in a MEC within these yeast strains. If this hypothesis were correct, inhibition of C4H activity in triple expressers might be predicted to result in a large accumulation of cinnamate, equivalent to the levels of p-coumarate found in control strains. An underlying assumption of this prediction is that the hypothetical PAL-C4H interaction is not disturbed by C4H-inhibition. 105 For the C4H-inhibition assay, the C4H-specific inhibitor piperonylic acid (PA) was employed. PA has been reported to inhibit C4H activity with a K,of 17pM (Schalk et al., 1998). To determine the minimum concentration of PA required for the complete inhibition of C4H in vivo in my system, a series of PA concentrations (10 - 500 pM) was added to the medium containing the induced triple expressing strain T2, and product formation was measured by HPLC. It was determined that 10 pM PA almost completely inhibited C4H activity in vivo (Figure 5.7), as measured by the ability of the strain to produce p-coumarate. At 10 pM PA, the PAL activities from the PAL-alone expressers P2 and P4 were measured in a time course to examine whether PA interfered with PAL activities. As shown in Figure 5.8, no inhibition of PAL activity by 10 pM PA was observed. Thus, 10 uJVI PA was able to completely inhibit C4H activity but did not interfere with PAL activity in vivo. The conditions established for C4H-inhibition were then used to block C4H activity in triple expressers. Galactose-induced cultures of the triple expressers T2 and T4 were resuspended in fresh culture media supplemented with 500 pM Phe, and PA (10 pM) was added either at the beginning of the culture period, or one hour after the culture started. The production of phenylpropanoid compounds over time was measured by HPLC. The accumulation of phenylpropanoids in PAL-alone expressers was measured in parallel to the triple expressers treated with PA. When PA was added at the beginning of the culture period, p-coumarate was not detected and the kinetics of cinnamate accumulation closely simulated those seen in PAL-alone expressers (Figure 5.9). Although an increase in cinnamate production was detected in strain T2, the final production of cinnamate was remarkedly lower than the accumulation of p-coumarate reproducibly observed in this strain in the absence of PA (Figure 5.5 and 5.9). When PA was added one hour after culture, the production of p-coumarate abruptly ceased, and small increases in the rates of cinnamate production were detected in both triple expressers. However, these increases were not comparable to the rates of p-coumarate production observed in the first 60 min in the absence of inhibitor. Thus, the inhibition of C4H activity in triple expressers did not result in a rapid accumulation of cinnamate but instead closely mimicked accumulation patterns of product accumulation in PAL-alone expressers. This suggests that PAL activity is not significantly altered by the physical presence of C4H and CPR in strains T2 and T4, and that this is not an explanation for the apparently enhanced channeling of carbon into phenylpropanoid metabolism in these strains. 106 0 2 0 0 0 1 E S 1500 •4-1 (0 1 1000 ? 500 0 10 50 100 Piperonylic acid concentration (u.M) Figure 5.7 Determination of the minimal concentration of piperonylic acid for C4H-inhibition in vivo. Piperonylic acid at the given concentrations and 500 uM Phe were added to media containing induced triple expressing yeast stains. Accumulation of p-coumarate was measured by HPLC after 3.5 h culture. 100 E 7 5 CD C C 50 "o " 5 25 E c 100 PAL2 S 5 30 s 8 75 50 • no PA | 2 5 ° 1 0 \iM PA | 60 min 90 120 120 Figure 5.8 Production of cinnamate in the presence or absence of 10 uM piperonylic acid in PAL only-expressing yeast strains. 10 uM of piperonylic acid and 500 uM Phe were added to the media containing PAL2- or PAL4-expressing yeast strains, and accumulation of cinnamate was measured by HPLC over time. Similar results were obtained in two additional independent experiments. 107 Time (min) Figure 5.9 Production of cinnamate and p-coumarate by PAL-alone or triple expressing yeast strains treated with piperonylic acid (PA). Induced yeast strains were resuspended in fresh media containing 500 p M Phe, and 10 pM piperonylic acid was added at the times indicated by arrows. Cinnamate and p-coumarate from media and cells were extracted and quantified by HPLC. Data are mean ± SE from three independent cultures. 108 5.2.7 Detailed analysis of Phe metabolism in yeast strains To exclude the possibility that cinnamate does not accumulate to high levels in the PAL2 and PAL4 strains because of further metabolism of cinnamate into derivatives that could not be detected by UV absorbance, and to better characterize Phe-derived carbon flux in PAL-alone and triple expressers, a sensitive radioactive-tracking method was used. For radio-tracer assays, 1 4C-Phe was added to fresh medium containing induced yeast cells, and the resulting metabolites were analyzed by thin layer chromatography (TLC). In the course of developing methods to analyze metabolites by TLC, it was noticed that in addition to heavy labelling of p-coumarate in the triple expressers, several other radiolabeled metabolites were produced from 1 4C-Phe in vector-control, PAL-alone, and triple expressers. Attempts to completely separate cinnamate and p-coumarate from other unknown metabolites by TLC, failed despite using several different mobile phases (data not shown). Nevertheless, these TLC analyses indicated that several metabolites were being synthesized from Phe in both the control and transformed yeast strains. It has been documented that Saccharomyces cerevisiae can use Phe as a sole nitrogen source (Cooper, 1981). In this pathway, Phe is converted to phenylpyruvate by a transaminase, a subsequent decarboxylation reaction produces phenylacetaldehyde, and a reduction reaction then converts phenylacetaldehyde to phenylethanol (Webb and Ingraham, 1963). Other phenolics, such as phenylacetate and phenyllactate, could also be synthesized from the intermediates in the Phe catabolic pathway. As far as cinnamate and p-coumarate are concerned, Saccharomyces cerevisiae is also reported to metabolize various (hydroxy)cinnamic acids (Clausen et al., 1994; Goodey and Tubb, 1982). According to these reports, cinnamate and p-coumarate can be decarboxylated to styrene and 4-vinylphenol by a single phenylacrylic acid decarboxylase (PAD) with similar efficiency both in vivo and in vitro. These potential pathways and metabolites, which could explain the multiple Phe-derived phenolics I observed, are illustrated in Figure 5.10. The reported presence of PAD activity in yeast is particularly relevant, since this indicates that cinnamate and p-coumarate could be further metabolized. Further metabolism of either compound would affect HPLC-based evaluation of carbon flux in transformed yeast strains, which made it essential to re-evaluate the metabolic flux of Phe in control and transformed yeast by the more comprehensive radioactive feeding assay. A vector-transformed control strain, PAL-alone expressers, and triple expressers with or without 10 uM PA were examined in 3H-Phe radioactive feeding experiments. Labeled metabolites extracted from both cells and culture media from 3H-Phe-fed yeast cultures were 109 Exogenous Phe Feeding phenylacetate Phe Biosynthesis phenylacetate Figure 5.10 Phenylalanine metabolism in yeast triple expressing strains (transformed by PAL, C4H, and CPR), showing reconstruction of the entry point into phenylpropanoid metabolism in this host. Endogenous yeast reactions involved in phenylalanine (Phe) catabolism are shaded in gray. The new pathway generated by introduction of PAL, C4H, and CPR genes is boxed by black-line, and endogenous pathways that metabolize cinnamate and p-coumarate are circled. ADH, alcohol dehydrogenase; DC, decarboxylase; PAD, phenylacrylic acid decarboxylase; TA (transaminase). Width of arrows is proportional to carbon flux, and potentially reversible reactions are indicated by double arrows. Question marks indicate potential but uncharacterized reactions or products. See text for further explanation. 110 fractionated by HPLC, and the radioactivity associated with each fraction across the elution profile was quantified. 3H-Phe was independently measured by HPLC fractionation of the aqueous medium fraction remaining after ether-extraction. Several authentic standards for the metabolites described above were chromatographed in this elution system to verify complete separation of these compounds, but 4-vinylphenol was not commercially available. Styrene exhibited a characteristic UV-absorbance spectrum around a maximum of 245 nm which, together with the retention time of the standard, guided the identification of the radioactive fractions corresponding to this compound. However, phenylalanine, phenylethanol, and phenylacetate only showed non-diagnostic absorbance peaks at around 200 nm, and their identification as labeled metabolites was solely based on comparison of their retention times to the authentic standards. By comparing radioactive profiles from vector-control, PAL-alone, or triple expressing strains to each other, it could be determined whether synthesis of the 3H-metabolites required PAL or C4H reactions. Special attention was given to the metabolites unique to PAL-alone and triple expressing strains. The results of a set of metabolic profiling experiments are shown in Table 5. Induced yeast strains were cultured in the presence of 500 pM 3H-Phe at a specific activity of 56.9 pCi/pmole, and 3H-labeled metabolites were identified as described above. In good agreement with previous data, a small amount of 3H-cinnamate (0.5-1.6% of added label) was only found in PAL-alone and triple expressing strains, and a large amount of 3H-p-coumarate (-8.9 - 10.2% of added label) was found only in the triple expressers (Table 5). However, a few additional radioactive metabolites could be identified in these yeast strains. In fact, 3H-styrene was detected in all four PAL-containing yeast strains (~1% of added label) but not in the vector-control, indicating that cinnamate is further metabolized in yeast probably by the endogenous enzyme PAD. Due to the lack of an authentic chemical standard, 4-vinylphenol, formed from p-coumarate by the same PAD, could not be definitively identified in triple expressers. However, two additional small radioactive peaks specific to triple expressers were identified, each with -0.5% of the added label. These two chemicals may have been 4-vinylphenol and its metabolic derivatives, or other metabolites from p-coumarate. The low levels of 3H-styrene and putative 3H-4-vinylphenol in PAL-only expressers. and triple expressers, relative to 3H-p-coumarate levels in triple expressers, suggests that metabolism of cinnamate and p-coumarate to these compounds is not a major metabolic route in these strains. I l l Table 5 In vivo conversion of  3H-phenylalanine to related phenolic metabolites in transgenic yeast strains. Strains 10u.M PA a PET PAC pCA tCA PPA Styrene PHE Vector - 7.5b 2.4 0 0 1.2 0 62.8 PAL2 - 2.9 2.2 0 1.6 0.4 1.0 60.1 T2 + 3.6 2.3 0.2 1.2 0.3 1.4 56.4 T2 - 2.9 2.3 10.2 0.6 0.8 1.0 50.5 PAL4 - 3.0 2.1 0 0.6 0.7 1.1 61.3 T4 + 4.8 2.4 0 0.8 0.6 1.4 59.5 T4 - 3.3 2.2 8.9 0.5 0.8 0.8 51.7 a Abbreviations: PA, piperonylic acid; PET, phenylethanol; PAC, phenylacetate; pCA, p-coumarate; tCA, frans-cinnamate; PPA, phenylpyruvate; PHE phenylalanine b Numbers represent the percentage of 3 H recovered from cells and media by HPLC fractionation, out of initial 3H-Phe feeding. Radioactive metabolites common to all the yeast strains used should represent metabolites synthesized from Phe by endogenous yeast enzymes. A major Phe-derived metabolite appeared to be phenylethanol, a known end-product of the Phe catabolic pathway. Phenylethanol accumulated in the vector-control at a level about twice that in the other strains. Radioactive metabolites with retention times similar to phenylpyruvate and phenylacetate were also detected. A metabolic intermediate for phenylethanol, phenylacetaldehyde, was expected to be present but would have eluted before the first fraction (23 min) in the HPLC program and could not be analyzed further. However, the total radioactivity eluted before 23 min was insignificant (<1%), and thus this chemical constitutes only a minor portion of the Phe-derived metabolites. Barely detectable levels of radioactivity were eluted at around 27 and 50 min, each with less than 0.2% radioactivity, in all yeast strains. The first metabolite could be phenyllactate, based on the elution time reletive to the standard, but the identity of the second one remains to be clarified. Approximately 30% of the radioactivity from 3 H-Phe supplied to the yeast cells was not recovered and was presumed to be incorporated into proteins. An important finding from this experiment was that Phe utilization was significantly accelerated only in triple expressers relative to PAL-alone expressers, or to triple expressers treated with piperonylic acid. About 50 - 60% of the Phe label was recovered as unmetabolized Phe in all strains, but the amount of Phe metabolized varied according to the strain. The residual 3 H-Phe recovered from triple expressing strains T2 and T4 was lowest (about 50% of added label). The difference between this level and that found in PAL-only expressing strains, in the PA-treated T2 and T4, or in the vector control strain was roughly comparable to the amount of 3H-p-coumarate that accumulated in the triple expressers. In 112 both triple expressers, when C4H was inhibited by PA, small increases were detected not only in cinnamate and its metabolite styrene, but also in phenylethanol, the final product of an independent metabolic pathway that shares Phe as an initial substrate. These data show that efficient conversion of cinnamate to p-coumarate by C4H acts as a strong driver for carbon-flux through the engineered plant secondary pathway in yeast. Yeast can metabolize cinnamate, and probably p-coumarate, but the endogenous enzyme catalyzing these reactions (PAD) cannot drive the reaction to styrene or 4-vinylphenol as efficiently as C4H does to p-coumarate. 5.2.8 Investigation of potential MEC-associated metabolic channeling in yeast A primary goal of this reconstitution analysis was to investigate the nature of a potential MEC containing PAL and C4H, in a simplified heterologous host system. One explanation for the very efficient production of p-coumarate by yeast cells expressing PAL, C4H, and CPR is that direct transfer of the pathway intermediate, cinnamate, from PAL to C4H increases flux towards p-coumarate via a tightly channeled MEC without requiring kinetic alteration of PAL activity. One reliable and frequently used biochemical approach to demonstrate metabolic channeling and the possible existence of a MEC is to examine whether endogenously formed intermediates from the first enzyme in a pathway are preferentially used as substrates for a second enzyme, relative to those introduced externally (Czichi and Kindl, 1977; Rasmussen and Dixon, 1999). Using differential radioactive labeling (e.g.3H and 1 4C) of the substrate and intermediate, it can be determined which proprotion of final products is derived from endogenously produced intermediate and which is formed from externally introduced intermediate. With regards to the yeast strains I constructed, the experimental logic is illustrated in Figure 5.11. If 3H-Phe and 14C-cinnamate were simultaneously fed to the triple expressing strains T2 and T4, these two radiochemicals would be readily incorporated into p-coumarate by way of the PAL and C4H reactions and also, though to a much lower extent amount, incorporated into styrene by the PAL and PAD reactions. Assuming that endogenous PAD and the introduced PAL do not form a MEC, the ratio of 3 H - to 14C-styrene can be used as a reliable internal control for the case of two physically separated enzymes in a linear pathway. If PAL and C4H were organized in a functional MEC in yeast cells, significant amounts of cinnamate endogenously made by PAL will be directly and preferentially transferred to C4H, and thus the ratio of 3 H- to 1 4 C in p-coumarate should be notably higher 113 than that in styrene. In the absence of a MEC involving PAL and C4H, the ratio of 3 H - to 1 4 C in p-coumarate should be similar to that in styrene. 1 4C-CA 3H-Phe Figure 5.11 Illustration of the double labeling experiment used to'analyze a potential multi-enzyme complex (MEC) involving PAL and C4H/CPR. Width of arrows is proportional to carbon flux. The box represents a yeast cell fed with 3 H -Phe and 14C-cinnamate. C4H and CRP are shown anchored to the cytoplasmic face of the ER. The light double arrow with a question mark denotes the hypothesized physical association between PAL and C4H/CPR. Abbreviation: CA, cinnamate; PCA, p-coumarate; PAD, phenylacrylic acid decarboxylase. First, 100 uM 1 4C-cinnamate (4.8 uCi/ nmol) was added to culture media containing induced triple expressing strains T2 and T4, together with 500 \xM 3 H-Phe (57.8 uCi/ umol). When the 3 H / 1 4 C ratios were measured from HPLC fractions containing target phenolics, much larger amounts of 1 4C-cinnamate than 3H-Phe were incorporated into p-coumarate and styrene, resulting in very low 3 H / 1 4 C ratios. 100 uM cinnamate appeared to essentially block the incorporation of 3H-cinnamate into p-coumarate and styrene. Although the cinnamate concentration was excessive in this case, it should be noted that the 3 H / 1 4 C ratio of p-coumarate formed in both triple expressers was not higher than styrene, but apparently lower (Table 6). 114 In a second trial, a more physiological concentration of cinnamate (10 pM; 47.7 uCi/ umol), together with the same amount of Phe (500 pM) used previously, was supplied to the induced triple expressing strains. In this experiment, substantial amounts of 3 H were partitioned to p-coumarate and styrene, allowing more reliable calculation for 3 H / 1 4 C ratios. However, as observed in the first experiment, the 3 H / 1 4 C ratios of p-coumarate did not exceed that of styrene in either triple expressing strain T2 or T4 (Table 6). In fact, the 3 H / 1 4 C ratios of p-coumarate were again lower than those of styrene. Since most of the initial 1 4 C -cinnamate was utilized and some 3H-cinnamate accumulated, the 3 H / 1 4 C ratios of cinnamate were significantly increased relative to the experiment using 100 pM cinnamate. These results were confirmed in an independent experiment using the same Phe and cinnamate concentrations. The low ratio of 3H-p-coumarate/ 1 4C-p-coumarate relative to the 3 H / 1 4 C ratio for styrene indicates that 3H-cinnamate endogenously synthesized from PAL was not preferentially used by C4H in the yeast strains. This is not consistent with a model for direct metabolite channeling between PAL and C4H in these strains, and suggests that PAL and C4H are not part of a MEC in vivo in yeast. Since preferential metabolite-transfer was not detected, it can be concluded that a MEC is not required for the observed carbon flux from Phe to p-coumarate in the triple expressing strains. Further, since two different PAL isoforms were used, it also appears that a specific PAL isoform does not preferentially associate with C4H in a MEC in this heterologous host. Table 6 Ratio of incorporation of 3H-Phe and 14C-cinnamate into p-coumarate or styrene, in Products 100 pM 1 4C-cinnamate a 10 pM 1 4C-cinnamate b T2 T4 T2 T4 3 H / 1 4 C a H / 1 4 C 3 H / 1 4 C C a H / 1 4 C c Cinnamate 0.04 0.06 4.70,4.13 2.84, 2.38 p-coumarate 0.02 0.03 0.58, 0.50 0.47, 0.54 Styrene 0.14 0.13 1.37, 1.71 1.68, 1.55 cinnamate to induced strains and cultured for 30 min. b 500 u.M 3H-Phe (57.8 uCi/ umol) were added together with 10 pM 1 4C-cinnamate (47.7 pCi/ umol) to induced strains and cultured for 30 min. 0 Data from two independent experiments are given. 5.2.9 Time course for p-coumarate production in triple expressing strains If C4H commits carbon flow to p-coumarate by a highly favorable forward reaction, p-coumarate is expected to accumulate at a high concentration after prolonged culture times. 115 To determine how much p-coumarate the engineered strains T2 and T4 could produce over time, and whether this flux would be affected by end-product inhibition, the flux from phenylalanine to p-coumarate was monitored over an extended time (10 hours) in 500 uM Phe-supplemented medium. As shown in Figure 5.12, within the experimental time, the production of p-coumarate remained linear. The final concentration of p-coumarate in the medium after 10 hours culture was -120 uJVI for strain T2 and -150 u.M for strain T4 in 50-ml_ cultures. By extrapolation of these data, the rates of p-coumarate production were 49 mg L"1 day"1 for strain T2 and 57 mg L"1 day'1 for strain T4. The linear production of p-coumarate by these triple expressers shows that C4H catalyzes an irreversible forward reaction and that there is no feedback control by p-coumarate on the flux from Phe through the entry-point enzymes of the phenylpropanoid pathway in vivo, at least in the yeast. 5.2.10 Metabolic coupling of PAL and C4H in mixed culture of strains expressing PAL-alone and C4H/CPR Radiotracer studies with differently labeled substrates showed that physical contact or proximity between PAL and C4H are not required for highly efficient Phe conversion to p-coumarate in the reconstituted yeast system. If a MEC is not required for high metabolic flux into phenylpropanoid metabolism in yeast, and simply the biochemical properties of the system dictate the carbon flux, I hypothesized that it might not even be necessary to express the three genes (PAL, C4H, and CPR) in a single cell to achieve efficient conversion of Phe to p-coumarate. In order to test whether similarly efficient conversion could occur even with physically separated PAL and C4H/CPR enzymes, independently induced PAL-alone strains, PAL2 or PAL4, and a C4H/CPR dual expressing strain (Chapter 4) were mixed together after induction (M2 and M4, Figure 5.12A), and cinnamate and p-coumarate accumulation was measured. In parallel, product accumulation by pure cultures of triple expressing strains T2 and T4 was measured as control. The accumulation of cinnamate and p-coumarate in mixed cultures of either PAL expressing yeast strain with the C4H/CPR strain mimicked that of triple expressing strains T2 and T4, although the amount of p-coumarate accumulated in the mixed cultures was about half of that in the triple expressers (Figure 5.13). Cinnamate appears to freely exit and enter the cells, since conversion of Phe to p-coumarate remained tightly coupled in the mixed cultures. The yeast strains used are of the same mating type, and thus the formation of diploid cells could be excluded. These data, together with the previous double-labeling 116 assays, show that biochemical coupling of PAL and C4H is sufficient for redirecting metabolic flux from Phe to p-coumarate without requiring recruitment of PAL and C4H into a MEC in engineered yeast. T2 H o E E o T4 <u _ <2 a> 1 II 8000 6000 4000 2000 A p-coumarate A cinnamate 2 4 6 Culture time (hour) 8000 6000 4000 2000 Culture time (hour) Figure 5.12 In vivo production of p-coumarate and cinnamate from triple expressing yeast strains T2 and T4 (transformed with PAL, C4H, and CPR). Yeast strains were induced for 15 h with galactose, re-suspended in fresh galactose-containing media supplemented with 500 uM Phe. Culture media was sampled every 2 h. Cinnamate and p-coumarate in culture media were quantified HPLC. Data points are the average of two measurements. 117 Time (min) Figure 5.13 Cinnamate and p-coumarate accumulation in mixed cultures of PAL-alone and C4H/CPR expressing strains (A) and triple expressing strains (B). Equal numbers (3x 109 cells) of induced PAL-alone expressing strains PAL2 or PAL4 and an induced C4H/CPR expressing strain were mixed to generate M2 and M4 mixed cultures, and the mixed cultures were incubated with 500 pM Phe (A). Alternatively, 3x 109 cells of induced triple expressing strain T2 or T4 were incubated with 500 pM Phe (B). Cinnamate (CA) and p-coumarate (PCA) amounts in culture media at different time points were quantified by HPLC. Data points in (A) are the means from two independent experiments; data points in (B) are measurements from a single experiment. Note that (A) and (B) have different scales. 118 5.3 Discussion Carbon flux into the phenylpropanoid pathway in plants requires efficient metabolic redirection from a Phe pool. Lignin is the second most abundant bio-polymer after cellulose, and 15 to 36% of assimilated carbon is directed to the lignin biosynthesis in woody plants (Sarkanen and Hergert, 1971). In vegetative tissues, soluble flavonoid or phenylpropanoid derivatives such as sinapoyl malate in Arabidopsis, or rutin and chlorogenic acid in tobacco, are known to accumulate in abundance (Landry ef al., 1995; Lorenzen et al., 1996; Mayer et al., 2001). Thus, PAL and subsequent enzymes in the phenylpropanoid pathway can direct significant carbon into many different phenylpropanoid metabolic end-products, as dictated by developmental or environmental signals. In this study, I have constructed yeast strains with metabolic gateways for two representatives of poplar phenylpropanoid pathways. Based on the compartmentalized expression of the poplar PALM2 and PAL4 genes observed by northern-blot analysis (Figure 5.2), and the expression of C4H and CPR2 in all organs (Ro et al., 2001; Chapters 3 and 4), the yeast strain T2 (expressing PAL2, C4H, and C P R 2 cDNAs) represents a surrogate of the entry-point into flavonoid and other soluble phenylpropanoid biosynthesis. On the other hand, strain T4 (expressing PAL4, C4H, and CPR2 cDNAs ) represents the entry-point into monolignol biosynthesis. Although PAL is the first committed step to both lignin and soluble phenylpropanoid metabolic pathways, its role in controlling carbon flux to lignin or chlorogenic acid appears to be tissue-dependent. In transgenic tobacco lines displaying different levels of PAL activity, a small reduction in PAL activity caused a proportional decrease in chlorogenic acid accumulation in the leaf tissue. However, more than 80% reduction in PAL activity was required to effect any decrease in lignin content (Bate et al., 1994). At the subcellular level, a certain fraction of a specific PAL isoform in tobacco is proposed to be associated with the ER (Rasmussen and Dixon, 1999). Thus, different PAL isoforms may be situated in different biochemical and cellular environments in plants, making it necessary to reconstruct the initial steps of the phenylpropanoid pathway using PAL cDNAs encoding distinct isoforms. Excessive expression of introduced genes is not necessary to couple a plant secondary pathway with the primary metabolism of a heterologous host, as I attempted. In general, the level of heterologous gene expression in yeast does not overwhelm expression of endogenous genes (Romanos ef al., 1992). Thus, moderate levels of transgene expression and the eukaryotic cellular structure of yeast make it an ideal host for such reconstruction studies, particularly for ER-bound enzymes such as C4H and CPR. In PAL-119 transformed yeast strains, the enzyme activities of PAL2 and PAL4 (25 - 100 pkatal mg"1) were similar to those measured from elicitor-induced tobacco, pine, or poplar cell cultures (20-60 pkatal mg"1) (Campbell and Ellis, 1991; Moniz de Sa etal., 1992; Rasmussen and Dixon, 1999), suggesting that this level of expression represents a physiologically relevant commitment to phenylpropanoid metabolism. The situation with respect to the heterologous C4H activity is somewhat different. In induced cell cultures of alfalfa and Catharanthus roseus, C4H activities of 240 and 1800 pmol min"1 mg"1, respectively, have been reported (Fahrendorf and Dixon, 1993; Vetter et al., 1992), while avocado fruit and wound-induced Jerusalem artichoke tuber possessed 174 and 2000 pmol min"1 mg" 1 of C4H activity, respectively (Benveniste et al., 1977; Bozak ef al., 1992). While C4H and C P R enzyme assays were not repeated in the triple expressing strains T2 and T4, these strains are expected to contain levels of these proteins similar to those in a C4H/CPR2 dual expresser in which C4H activity was 7,400 pmol min"1 mg"1 (Chapter 4). Based on these data, C4H activities in the triple expressing strains are likely several-fold higher than those measured from plant tissues or cell culture. Thus, under the conditions used to induce transgene expression (15 to 20 hours in the presence of galactose), the PAL and C4H enzyme activities in yeast were similar to, or somewhat higher than, induced plant cell cultures, but did not entirely diverge from in planta condition. An initial goal of this research was the investigation of a possible formation of a MEC between PAL and C4H, and its functional significance in a heterologous background. Direct channeling of cinnamate from PAL to C4H has been demonstrated in isolated microsomes from some plants (Czichi and Kindl, 1975; Czichi and Kindl, 1977; Rasmussen and Dixon, 1999) and recently in cultured tobacco cells in vivo (Rasmussen and Dixon, 1999). In my analysis, the possibility of metabolite channeling between recombinant PAL and C4H in an in vivo heterologous host was assessed by simultaneous feeding of 3H-labeled Phe and Re-labeled cinnamate. However, these assays provided no evidence for a functional PAL/C4H MEC in yeast. Previously reported double-labeling assays have depended entirely on the calculated ratios of 3 H - and 14C-radioactivities in pools of intermediate and a product (coupling factor) (Rasmussen and Dixon, 1999, and references therein). This is justifiable in a homogeneous (non-compartmentalized) system, but in the present study, it was difficult to reliably estimate the size of intracellular pools of cinnamate and p-coumarate since they were rapidly secreted into the culture medium. Instead, the formation of styrene from cinnamate by endogenous PAD activity in yeast was used as an internal control for a non-MEC-derived 120 product, to which the incorporation ratio of 3 H to 1 4 C in p-coumarate could be compared. This method is virtually independent of most potential experimental artifacts since the two metabolites (p-coumarate and styrene) being compared were derived from the same cell population, and they were simultaneously processed through the same sample preparation and fractionation processes. In all three independent double-labeling experiments, the 3 H / 1 4 C ratios of p-coumarate were never higher than those of styrene (Table 6). Strains expressing either PAL isoform generated very similar 3 H / 1 4 C ratios in p-coumarate, which further indicates that isoform-specific channeling between PAL and C4H is not occurring in this heterologous host. These results strongly argue against the concept that a MEC readily forms between PAL and C4H when they co-occur in one cell. Nevertheless an explanation is needed for the very efficient flow of carbon from Phe to p-coumarate that occurs in this reconstituted system. Interestingly, the ratio of 3H-styrene / 1 4C-styrene was consistently higher than that of p-coumarate. These higher 3 H / 1 4 C ratios in styrene are not necessarily a reflection of PAL/PAD metabolic coupling. PAD is known to be a cytoplasmic enzyme (Clausen et al., 1994), and the higher 3 H / 1 4 C ratios in styrene could reflect the fact that "cytosol to cytosol transfer" (PAL to PAD) of endogenously produced 3H-cinnamate will be topological^ more efficient than "cytosol to ER transfer" (PAL to C4H), and incorporation of an excess amount of exogenously added 1 4C-cinnamate is equally efficient by PAD and C4H. Although the theoretical ratio of 3 H - styrene / 1 4 C - styrene should be < 1 if a non-MEC associated reaction sequence links PAL and PAD, the 3 H / 1 4 C ratios of styrene were slightly higher than 1 when 10 pM 1 4C-cinnamate was fed. It is possible that 3H-cinnamate that accumulates after prolonged incubation starts to be used more efficiently than 1 4 C -cinnamate by PAD and C4H as the cells move into a 1 4C-cinnamate-depleted condition, resulting in an increase of 3 H / 1 4 C ratios in both styrene (more than 1) and p-coumarate. If this interpretation is correct, reducing the experimental time of incubation with labeled substrates or increasing the 1 4C-cinnamate concentration (as shown in the experiment in which 100 pM cinnamate was fed) would be predicted to concomitantly decrease the 3 H / 1 4 C ratios in both p-coumarate and styrene. The double labeling experiments in this study only show that PAL and C4H do not directly interact or are not closely associated on the ER when expressed in yeast. It is possible that plant-specific factors are required to modify one or both enzymes, or to provide a protein-protein interaction scaffold, before a functional MEC can be formed. This 121 possibility is supported by a recent finding that GFP-tagged isoflavone O-methyltransferase (IOMT), an enzyme normally localized in the cytosol in alfalfa, moves to the ER upon elicitation (Liu and Dixon, 2001). Phosphorylation of PAL by a calmodulin-like domain protein kinase has been recently demonstrated (Allwood ef a/., 1999; Cheng ef a/., 2001), and it would be interesting to investigate whether this modification is correlated with a conditional ER-localization of PAL as shown in alfalfa IOMT. My experiments consistently demonstrated that Phe is an excellent precursor for p-coumarate in triple expressing yeast strains but not for cinnamate in PAL-alone expressers. This can be interpreted to mean that efficient carbon flux into phenylpropanoid metabolism from Phe requires both PAL and C4H, and that PAL alone is unable to efficiently channel carbon away from primary metabolism into phenylpropanoid metabolism. However, several other factors could influence the apparent low level of carbon-flux into cinnamate in PAL-alone expressing yeast strains. First, it is possible that PAL-alone and triple expressers were physiologically different during the gene induction period because they possess different sets of transgenes and produce different metabolites. A particular effort was taken to minimize this problem by pre-culturing yeast cells in glucose to suppress the transgene expression until mid-log phase and by inducing transgenes for less than 20 hours in all experiments performed. Also, as an independent approach, C4H activity was inhibited by PA immediately before or during the Phe-feeding experiment so that the cell-culture conditions were identical except for the a few hour's experimental time. Flux from Phe through PAL was significantly reduced by C4H inhibition as shown in both non-radioactive and radioactive feeding assays. In addition, a modest level of cinnamate and styrene production was associated with both the triple expressing strains as well as the PAL-alone expressers. Therefore, potential negative effecters are present in PAL-alone expressers, but do not appear to be the major cause of the reduced Phe-flux. Second, it is possible that endogenously made cinnamate is metabolized to other compounds in the PAL-alone expressers. The 3H-Phe feeding assays showed that the yeast strain used in this study can catalyze decarboxylation of cinnamate, and possibly p-coumarate, perhaps by the reported endogenous enzyme PAD (Clausen ef al., 1994; Goodey and Tubb, 1982). In various yeast and bacterial strains, PAD is known to catalyze the decarboxylation reaction of (hydroxy)cinnamate derivatives (Barthelmebs et al., 2000; Clausen ef al., 1994). Therefore, a certain fraction of the cinnamate and p-coumarate produced endogenously in transformed yeast strains will be converted to their vinyl derivatives. In addition, the presence of unidentified products specific to triple expressers 122 indicates that p-coumarate or its vinyl derivative can be further metabolized to other compounds in yeast. However, it is unlikely that PAD specifically and actively reroutes a significant amount of Phe to styrene or that its activity could account for the apparent low level of cinnamate accumulation in the PAL-only expressing strains, relative to p-coumarate accumulation in triple-gene expressing strains. This enzyme can convert both p-coumarate and cinnamate with similar efficiencies (Clausen et al., 1994; Goodey and Tubb, 1982), which suggests that cinnamate and p-coumarate accumulation should be equally affected. In fact, in 3 H-Phe feeding experiments, the amount of 3H-styrene in PAL-alone expressers and the two additional unknown 3H-compounds in triple expressers accounted for only about 1% of the total radioactivity, suggesting this is a pathway of minor significance with regards to carbon flux, in either kind of yeast strain. It is possible that the metabolic flux into styrene is underestimated because styrene may be further metabolized rapidly or could be lost due to its volatile nature. However, the significantly higher amount of unmetabolized 3H-Phe that remained in PAL-alone strains and C4H-inhibited triple expressing strains argues against these possibilities (Table 5). Based on this data, a reasonable interpretation is that PAL-alone expressers cannot efficiently produce cinnamate, and that rapid metabolism of cinnamate to styrene and other compounds does not account for the low amounts of cinnamate produced in these strains. A plausible explanation for the reduced carbon-flux in the absence of C4H could be feedback inhibition of PAL by cinnamate. Intracellular cinnamate pools in PAL-alone and C4H-inhibited expressers might rapidly inhibit PAL activity, but in triple expressers this metabolic inhibition could be released by efficient conversion of cinnamate to p-coumarate. In plants, cinnamate has been proposed to modulate PAL transcription and, potentially, PAL enzyme stability at the post-translational level by yet uncharacterized mechanisms (Bolwell et al., 1986; Bolwell ef al., 1988; Mavandad ef al., 1990). However, these regulatory networks would presumably not be operating in yeast. It has been routinely reported that PAL can be feedback-inhibited by its product, cinnamate, in in vitro assays using purified native enzymes or recombinant enzymes (Appert ef al., 1994; Havir and Hanson, 1968; Jorrin and Dixon, 1990). In C4H-suppressed tobacco, reduced C4H activity was correlated with a decrease in intracellular cinnamate, suggesting a feedback inhibition (i.e., auto-regulation) of PAL at a certain threshold level of endogenous cinnamate (Blount ef al., 2000). However, the role of cinnamate in regulating PAL activity in vivo is still unclear. With the poplar recombinant PAL2 enzyme, the Kj for cinnamate inhibition was determined to be -700 uM (McKegney ef al., 1996), a level unlikely to be reached in any of 123 the engineered yeast strains, making the in vivo importance of feedback inhibition questionable. In principle, feedback inhibition might be accessed in vivo experimentally in yeast by feeding varying concentrations of cinnamate and measuring conversion of 3 H-Phe to 3H-cinnamate. However, the results of this experiment could be uninformative because, according to the feedback inhibition model, the PAL-alone expressers are always in a PAL-suppressed condition even before feeding cinnamate. In an in vivo assay using cultured tobacco cells, feedback inhibition of PAL by cinnamate was not detected at concentrations of up to 100 u.M exogenously fed cinnamate (Rasmussen and Dixon, 1999). My data show that increasing the external Phe concentration to 5 mM in PAL-alone expressing strains resulted in an enhanced accumulation of cinnamate (Figure 5.5), which is not consistent with an explanation based on the robust inhibitory effect of cinnamate on PAL activity. Finally, the difference in carbon flux in PAL-only expressers and triple-expressing strains may simply demonstrate that PAL-alone or C4H-inhibited triple expressers have a thermodynamically unfavorable metabolic pathway for Phe metabolism. Consistent with this hypothesis, PAL enzyme is known to catalyze a reversible reaction, a fact which has been exploited for commercial synthesis of L-phenylalanine from cinnamate and ammonia (Tewari, 1990). By contrast, the reaction mediated by P450 is highly irreversible in nature, with a large free energy change (Grayson et al., 1996). There are convincing experimental and theoretical data showing that PAL catalyzes a reverse reaction both in vitro and in vivo (Havir and Hanson, 1968; Tewari et al., 1987; Yamada et al., 1981). The equilibrium constant for the conversion of Phe to cinnamate and ammonia varies between 1.5 to 4.5 M depending on physical conditions (Tewari ef al., 1987). On the other hand, the equilibrium constant for a typical aromatic P450 reaction, the conversion of tetralin to 1-tetralol by camphor 5-monooxygenase has been estimated to be ~4 x 10 6 5 (Figure 5.14) (Grayson et al., 1996). Thus, P450-reactions are essentially irreversible and can be expected to proceed until substrate is almost completely consumed. This will be particularly true for C4H and other plant P450s that possess very low K m values (see below). This thermodynamic comparison of the PAL and P450 reactions, together with experimental evidence from the engineered yeast strains, indicates that C4H is a major driving force in directing carbon source from Phe to p-coumarate at the entry-point to phenylpropanoid metabolism. The thermodynamic parameters only reveal the final equilibrium state of the reactions but cannot elucidate their kinetic efficiency. For the tightly coupled reactions shown in Figures 5.9, 5.12 and 5.13, the kinetic properties of PAL and C4H are expected to 124 play an important role. The K m value for recombinant PAL2 is approximately 450 uM (McKegney et al., 1996), while that of native or recombinant C4H from Jerusalem artichoke (Helianthus tuberosus) is 2.6 u.M or 3.9 uM, respectively (Gabriac et al., 1991; Urban ef al., 1994). On the other hand, turn-over numbers reported for PAL are 2.6 s"1 or 22 s"1 (Appert ef al., 1994; Hanson and Havir, 1972) and that for C4H has been calculated to be 5 s"1 (Chapter 3; -300 nmol min"1 nmol P450"1), indicating that the two enzymes possess comparable catalytic rates. These kinetic properties of PAL and C4H predict that C4H will efficiently carry out catalysis even at very low substrate concentrations, whereas PAL requires a much higher concentration of its substrate Phe in order to attain equivalent activity. Camphor 5-monooxygenase Figure 5.14 The conversion of tetralin (1,2,3,4-tetrahydronaphtalene) to 1-teteralol (1,2,3,4-tetrahydro-l-naphtholl) catalyzed by camphor 5-monooxygenase. Thus, PAL-alone and C4H-inhibited triple expressers are equipped with a thermodynamically unfavorable pathway, while triple expressers possess a high throughput metabolic pathway, leading to the synthesis of p-coumarate (Figure 5.15). A simple solution to overcome these unfavorable thermodynamics is to either rapidly remove the accumulated PAL product (cinnamate), or to supply a high concentration of the PAL substrate (Phe). In triple expressers, the intracellular cinnamate level should be very low due to the highly efficient irreversible reaction catalyzed by C4H. This allows an accelerated carbon flow from Phe to cinnamate and then to p-coumarate, an effect that would be most pronounced at low Phe concentration (Figure 5.5, Figure 5.10, and Table 5). In the absence of C4H, a high 125 concentration of Phe can provide an alternative drive for cinnamate formation, as seen in PAL-alone expressing strains grown in supplemented Phe (5 mM) (Figure 5.5). These are straightforward thermodynamic and biochemical explanation for the control of carbon flux through the entry point of phenylpropanoid metabolism that avoid any need to invoke the participation of a MEC. Kinetic and Thermodynamic Coupling K' = 2 . 5 M eq b Overall Equil. Constant K ' e q = 4 x 1065 a X b = 1 Q 6 6 PAL PHE N H 3 CA C4H CPR pCA °K m = 450 uM d K m = 2-4 uM Figure 5.15 Model for the kinetic and thermodynamic coupling between PAL and C4H expressed in yeast a, The value of the apparent equilibrium constant K ' e q for PAL reaction at 298 K and pH 7 (Tewari et al., 1987; personal communication with R. N . Goldberg), b, Inferred value from similar type of P450 reaction (D. A. Grayson et al., 1996; Fig-ure 5.14). c, The value measured from the recombinant PAL2 enzyme expressed by baculovirus/Sf9 system (McKegney et al., 1996). d, Inferred range of K m val-ues from native and recombinant Jerusalem artichoke C4H (Gabriac et al., 1991; Urban et al., 1994). Abbreviation: PHE, phenylalanine; CA, cinnamic acid; pCA, p-coumaric acid It is now believed that there is no single rate-limiting enzyme governing the overall flux through a given metabolic pathway, and that the control points for metabolism are distributed to multiple enzymes by flux-control coefficients (Fell, 1998 and references therein). However, recent evidence shows that C4H is an enzyme with a high flux-control coefficient for sinapoyl malate synthesis in Arabidopsis. The AtMYB4 transcription factor was characterized as a specific negative regulator of C4H in phenylpropanoid metabolism, and the enhanced C4H expression in an Arabidopsis AtMYB4 mutant resulted in a 6-fold increase in sinapoyl malate, an UV-absorbing phenylpropanoid (Jin et al., 2000). 126 The model for control of carbon flux into phenylpropanoid metabolism by PAL and C4H can be more broadly applied to plant secondary metabolism. As the name "cytochrome P450 monooxygenase" implies, in P450-mediated reactions, molecular oxygen is cleaved, and the individual oxygen atoms are inserted into the substrate and H 2 0 . In the course of the reaction, a major contribution to the large negative free energy change is derived from H 2 0 formation (Grayson ef al., 1996). Thus, considering the strong thermodynamic drive embedded in the nature of P450 reaction, together with high specificity constants (V m a x /K m , relative to animal P450s), of plant P450 enzymes for their specific substrates, one simple prediction is that P450s involved in plant secondary metabolism should be tightly regulated, especially those that utilize primary metabolites as substrates. The presence of P450s in a specific cell or tissue type, together with their substrates, is expected to result in the efficient accumulation of particular products. In recent years, a number of novel P450 genes and P450-mediated secondary pathways have been functionally characterized, but there is no thermodynamic interpretation of the role of P450s in these metabolic pathways in the literature. One example of how the principles of my thermodynamic model for the control of carbon flux by PAL and C4H may be applied to other pathways of secondary metabolism occurs in maize. The cyclic hydroxamic acid, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), is involved in chemical defense in maize, where it is synthesized by reactions catalyzed by at least four consecutive P450 enzymes (Figure 5.16) (Frey et al., 1997). This pathway uses indole-3-glycerol phosphate as its first substrate, a compound that is also required in the tryptophan biosynthetic pathway. However, DIMBOA accumulates in maize seedlings to levels 10 to 20-fold higher than tryptophan (Frey ef al., 1997). The first enzyme of the DIMBOA pathway is the enzyme Bx1 (cloned from maize benzoxazinless 1 mutant), which links primary metabolism to the DIMBOA pathway. Interestingly, this enzyme catalyzes a reaction with an apparent equilibrium constant of 1.8 x 10"3, which greatly favors the reverse reaction (Kishore etal., 1998). Therefore, the subsequent P450 reactions must play essential roles in driving the overall flux towards DIMBOA synthesis. Similarly, tryptophan synthase a (TBA), an enzyme homologous to Bx1 but involved in the parallel tryptophan biosynthetic pathway, catalyzes the same unfavorable reaction. However, the overall pathway is strongly driven toward tryptophan synthesis because the subsequent reaction catalyzed by tryptophan synthase (3 (TSB) strongly favors the forward reaction (K e q = 1.1 X 10 1 1) (Kishore et al., 1998). To further reinforce the forward reaction, TBA forms a tight enzyme complex with the subsequent enzyme TSB in bacteria (Hyde et al., 1988) and 127 also probably in plants (Radwanski et al., 1995). This observation indicates that efficient biochemical (kinetic and thermodynamic) coupling does not necessarily exclude MECs, and that a metabolic flux may be synergistically further enhanced by use of a MEC. In the case of DIMBOA biosynthesis, however the primary driving force appears to be the thermodynamics of the P450 catalyzed reactions later in the pathway. Successful manipulation of yeast metabolism by introduction of PAL plus its P450 partner enzyme, C4H, indicates that redirection of carbon flux into reconstructed target pathways in heterologous hosts is a promising future area in biotechnology, particularly if the pathway involves one or more plant P450 enzymes. Despite the short history of plant P450 gene discovery, there have already been some successful examples of plant P450-mediated metabolic engineering in plants. For example, metabolic patterns in the model plant Arabidopsis have been modified by introduction of sorghum and soybean P450s (Bak et al., 2000; Jung et al., 2000; Petersen et al., 2001). The most dramatic case is the genetic engineering of Arabidopsis for dhurrin (para-hydroxy-S-mandelonitrile (3-glucoside) production. Dhurrin is a cyanogenic glycoside synthesized in soybean tissues via a pathway that shares some mechanistic features with glucosinolate biosynthesis in the crucifers. Introduction of two multifunctional microsomal cytochromes P450 (CYP79A1 and CYP71E1) and a soluble UDPG-glucosyltransferase from soybean allowed tyrosine to be diverted to cyanogenic glycoside synthesis in Arabidopsis, which was accompanied by an increase in pest resistance in the transgenic plants (Tattersall et al., 2001). Thus, efficient kinetic properties that have arisen through evolution, together with the innate thermodynamic properties of plant P450s, may explain why plants have evolved an unusually wide range of P450 genes, most of which are likely to have roles in supporting the rich palette of specialized metabolisms found in plants. 128 lndole-3-glycerol phosphate Indole ^ BX2 (P450) H lndolin-2-one ^ BX3(P450) •OH 3-hydroxyindolin-2-one BX4 (P450) 2-hydroxy-benzoxazin-3-one BX5 (P450) Indole TSB Tryptophan N H 2 CHgO, O ^ ,OH T P450? OH N I OH DIBOA DIMBOA Figure 5.16 Biosynthetic pathways to DIMBOA and tryptophan in maize Genes in the DIMBOA pathway (BX1 to BX5) were cloned from maize benzoxazinless mutants (bxl and bx3) or from a X genomic clone containing BX2 to BX5 genes. Adopted fromFrey etal. (1997). Abbreviation: BX, Benzoxazinless; 2,4-dihydroxy-l,4-benzoxazin-3-one, DIBOA; 2,4-dihydroxy-7-methoxy-l,4-benzoxazin-3-one, DIMBOA; TSA, tryptophan synthase a; TSB, tryptophan synthase p. 129 The arguments presented here for thermodynamic and kinetic flux control of the coupled PAL and C4H reaction are likely to be most relevant in a heterologous host where the metabolic flux leads to a dead-end. However, the situation will usually be more complicated in plants for the following reasons. 1) Cinnamate is not always solely committed to p-coumarate biosynthesis. Following pathogen attack or other stresses, cinnamate is also used as a precursor for the important defense-related molecule, salicylic acid, which is made via benzoic acid or its conjugates (Chong et al., 2001; Yalpani ef al., 1993). 2) Most of the free cinnamate in the cytoplasm, if not correctly compartmentalized, is converted to cinnamate glucose ester as part of a general detoxification mechanism (Anterola et al., 1999). 3) Ammonium released by the PAL catalyzed reaction is rapidly incorporated into a nitrogen recycling pathway that returnes nitrogen to the Phe biosynthesis process (van Heerden etal., 1996). These reports show that the products of PAL reaction, cinnamate and ammonia, are efficiently linked to other metabolic pathways in plants. Nonetheless, it is clear from this study that biochemical coupling of PAL and C4H is sufficient to efficiently redirect and commit carbon from the primary metabolic product Phe into phenylpropanoid metabolism without requiring supramolecular organization within a MEC. Control of steady state carbon flux by this mechanism could be predominant in cells and tissues specialized for formation of large amounts of phenypropanoid products such as xylem, or leaf epidermal cells. Involvement of MEC formation, if it occurs at all, may be restricted to cells producing a more diversified patten of phenylpropanoid metabolites that require additional regulatory mechanisms to partition the fluxes. 130 Chapter 6 Conclusions and Future Perspectives In this thesis, authentic C4H and CPR enzyme activities encoded by cloned poplar cDNAs were substantiated by expression in yeast. The experimental data presented in Chapter 5 further suggest that C4H and C P R , as well as PAL the first committed enzyme of phenylpropanoid metabolism, are required to efficiently commit the flow of carbon into the phenylpropanoid pathway in experimental yeast strains. A major conclusion is thus that both PAL and C4H are essential for effective re-direction of primary metabolism into the phenylpropanoid pathway. Apparently, PAL and C4H are not spatially organized in yeast in a manner that channels their intermediate frans-cinnamate between the two catalysts, as judged by the double-labeling assay, but rather they are kinetically linked and in this way fulfill their key function of re-routing carbon flux to the phenylpropanoid pathway. Formation of a MEC may provide several biological advantages in plant secondary metabolism, such as efficient intermediate channeling for high throughput pathways (e.g. lignin), sequestering of cytotoxic intermediates in a micro-environment, and potential regulation of flux into specific pathways. However, co-expression of PAL, C4H and C P R in yeast without formation of a MEC has been demonstrated to be sufficient for efficient, and sustained metabolic flux into the engineered yeast phenylpropanoid pathway and maintenance of low levels of the cinnamate intermediate. This capability results from the kinetic and thermodynamic coupling of the reactions catalyzed by these entry point enzymes. Although three isoforms of poplar C P R were isolated and two of the divergent classes were investigated in depth, there is no indication that they are not functionally redundant. In subcellular localization studies, GFP-tagged C4H, CPR1 , and CPR2 were all demonstrated to be predominantly targeted to, and retained on, the ER despite the chloroplast targeting-like motif in CPR2 . This confirms that the ER is a key subcellular structure for P450-mediated reactions and is consistent with the postulated anchoring role for C4H in the proposed MEC model. Failure to find intermediate channeling in the reconstituted yeast system implies that the formation of a MEC in plants, if present, may require plant-specific components not found in yeast. As an alternative to the biochemical approach used here to try to detect a MEC containing PAL and C4H, physical approaches could also provide evidence concerning the existence of a potential MEC on the ER. In fact, I initiated experiments to determine the subcellular location of PAL in Arabidopsis plants. In parallel to the GFP-fusion to C4H, I constructed GFP-PAL2 and blue fluorescent protein (BFP)-PAL2 fusions under the control 131 of the CaMV-35S promoter and transformed them into Arabidopsis. These transformants were to be used to examine the in vivo localization of PAL and the possibility of potential fluorescence resonance energy transfer (FRET) between BFP-PAL and C4H: :GFP. However, I was not able to identify Arabidopsis lines expressing detectable levels of PAL-G F P and PAL-BFP signals using either fluorescence microscopy or confocal microscopy analysis, probably due to the low abundance of the chimeric proteins in transformed Arabidopsis lines (data not shown). It may be useful in the future to use particle bombardment method to transiently express the GFP-fused PAL at higher levels in wild-type Arabidopsis or in the 35S-C4H Arabidopsis lines where a high amount of the putative C4H anchor is present. On the other hand, BFP appears to be a poor reporter for plant system due to the high autofluorescence in plant cells under UV excitation, and the weak fluorescence of BFP. Red fluorescent protein (RFP) has recently been used in plants (Jach ef al., 2001), and a RFP-PAL fusion could be transiently co-expressed in C4H: :GFP transformed Arabidopsis for co-localization studies. If such a simple co-localization analysis showed substantial ER-localization of PAL, FRET assays could be performed to try to detect direct protein-protein interactions. Alternatively, chemically labeled fluorescent PAL protein, compatible for the FRET assay with G F P , could be injected into cells of C4H: :GFP expressing Arabidopsis to detect close proximity of PAL and C4H in plant epidermal cells, where chloroplasts are lacking. Since recombinant proteins for PAL, C4H, and CPR, and their antibodies (specific to the tagged epitopes for C4H and CPR) are available from this study, other classical biochemical methods such as affinity chromatography and immuno-coprecipitation could be attempted in vitro. Direct detection of PAL in the ER-membrane by subcellular fractionation or in the vincinity of ER by immuno-cytological analysis could also provide clues for the MEC that involves PAL in plant system. In the triple expressing strain, the joint action of PAL and C4H in driving carbon flux into the phenylpropanoid pathway was remarkable, although I detected no alteration in the kinetic properties of PAL brought about by potential interaction with C4H in this strain. High levels of p-coumarate were measured in the triple expresser in both unlabeled and 3 H -labeled Phe feeding assays, but equivalent levels of cinnamate were absent in the PAL-alone expresser and C4H-inhibited triple expressing strains. The proposed model for the tight biochemical and thermodynamic coupling of PAL and C4H at the gateway into the phenylpropanoid pathway fits well with this experimental data, and could account for the massive carbon-flux into lignin biosynthesis in woody plants. Similar coupling of entry-point 132 enzymes with P450 enzymes in other pathways of secondary metabolism also appears to occur, as discussed in Chapter 5.3. With regard to the proposed thermodynamic explanation, however, this thesis has not provided any direct evidence that the reverse PAL reaction (i.e.,the synthesis of Phe from cinnamate and ammonia) occurs in vivo in the PAL-only expressing yeast strain. This reverse-reaction would be predicted to occur, based on the PAL equilibrium constant, and would help account for the low amount of cinnamate produced in this strain. To confirm that this is happening, and to determine its scale, radiolabeled cinnamate could be fed to the PAL-only expressing strain together with ammonium salt to determine how much radiolabeled Phe is synthesized by the PAL reverse reaction. However, Phe has several metabolic fates including protein synthesis, which could complicate interpretation of such data. Alternatively, a genetic approach could be applied to investigate the efficiency of the PAL reverse reaction. Two Phe auxotropic mutants have been reported in Saccharomyces cerevisiae: the pha2 mutant is defective in prephenate dehydratase specific to Phe synthesis (Lingens ef al., 1966), while the aro8 and aro9 double mutant is defective in aromatic amino acid transferase I and II, resulting in auxotropy for both Phe and tyrosine (Urrestarazu ef al., 1998). Therefore, PAL could be transformed into these mutants to test whether the P/4L-transformed Phe-auxotropic mutant can grow in the absence of Phe when cinnamate and ammonia are provided. The evolution of different plant secondary metabolic pathways appears to have involved repeated use of similar strategies, such as selection of P450 enzymes with new activities, to generate new arrays of phytochemicals. The kinetic and thermodynamic properties of P450 enzymes, in turn, may have enabled plants to recruit new gateway enzymes that may not provide efficient catalysis themselves. This coupling could thus expand the repertoire of biosynthetic possibilities still further. As discussed in section 5.3, the DIMBOA biosynthetic pathway is another candidate pathway that appears to fit this model. Plant P450 research still in its infancy, and a large number of P450 genes in Arabidopsis and other plants are awaiting functional identification. 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