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ASSIMILATION OF SYMBIOTICALLY-REDUŒD NITROGEN IN TROPICAL LEGUMES: REGULATION OF PEROXISOME
PROLIFERATION AND UREIDE PRODUCTION
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of
The Ohio State University
by
Tianyun Wu, B.S., M S.
*****
The Ohio State University
1998
Dissertation Committee: Approved by Profesor Desh-Pal S. Verma, Adviser Professor Keith R. Davis Professor Terrence L. Graham Adviser Department of Plant Pathology UMI Number: 9822389
UMI Microform 9822389 Copyright 1998, by UMI Company. Ail rights reserved.
This microform edition is protected against unauthorized copying under Title 17, United States Code.
UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT
Peroxisomes are specialized subcellular organelles existing in all eukaryotic
organisms, which carry out diverse metabolic roles essential for living cells. Plant peroxisomes have long been known to be involved in mobilization of storage oil
reserves, synthesis of ureides and salvage of photorespiratory phosphoglycolate. In root nodules of tropical legumes, the de novo purine biosynthesis pathway is induced with
the onset of Nz-fixation, converting symbiotically reduced nitrogen to ureides. Peroxisomes are proliferated in root nodules in response to synthesized purines during N2-6xation as oxidation of purines requires this organelle.
Using Saccharomyces cerevisiae, we demonstrated that catalase and uricase (two
marker enzymes of peroxisomes) activities were induced by xanthine. This study suggested that xanthine could induce peroxisome proliferation in S. cerevisiae, but uric
acid could only induce uricase activity. A strain of S. cerevisiae was mutagenized with
ethyl methyl sulfonate (EMS). A positive selective procedure was used based on the lethality of H2O2 accumulation in wild-type cells with application of a catalase inhibitor,
3-amino-1,2,4-triazol. Four putative peroxisome biogenesis mutants were isolated which failed to utilize oleic acid as the sole C-source and uric acid as the sole N-source, respectively, and fell into three different complementation groups. Their peroxisomal catalase activities were less induced by oleic acid than that of the wild-type cells. One mutant (Spbl) showed a restrict phenotype of non-utilizing uric acid or oleic acid, and had a single mutation. Spbl showed less induction of peroxisomal enzymes including
u uricase and catalase, which was used for complementation with a soybean nodule cDNA
library constructed in LAMDA MAXI. A cDNA (spbl) encodes a DNA binding protein
which contains a basic region/helix-loop-helix (bHLH) domain was isolated on oleic acid
selective medium. The deduced amino acid of SPB1 contains 150 amino acids and
shows less than 40% homologies with other plant bHLH containing transcription factors,
e.g. phaseolin G-box binding protein, myc-like-R factor and rice transcription activator
Ra. Sequence comparison revealed that SPB 1 likely represents a new member of bHLH protein family in plants, which may be involved in regulation of peroxisome proliferation in plants.
The urate oxidation into allantoin by uricase is carried out within peroxisomes during the oxidative decomposition of purines. In nodule tissue, the major urate oxidation takes place through the xanthine-dehydrogenase-uricase system that is widely distributed in bacteria and animals. Previous studies have suggested that a urate degradation system composed of a peroxidase and a diamine oxidase exists in soybean radicles. The diamine oxidase catalyze the deamination of polyamines into aldehyde, ammonia and hydrogen peroxide. Cadaverine, a diamine, serves as a cofactor in this enzymes system. Therefore, the hydrogen peroxide generated from cadaverine oxidation regulates the peroxidase urate-degrading activity. By functional complementation of a yeast S. cerevisiae mutant Spbl, two soybean cDNA fragments were isolated from a soybean nodule cDNA library which encode a cytochrome P450 and a copper-containing amine oxidase, respectively. The full length clones of these two cDNAs were isolated by using the cDNA fragments as probes. P450 full length cDNA is 1.9-kilobase long and contains an open reading frame encoding a protein having 511 amino acids with a molecular weight of 67 kDa. This P450 was identified as a HiOi-dependent urate- degrading peroxidase (P450W). The full length cDNA of copper-containing amine oxidase is 2,663 base pairs long and encode a protein with 701 amino acid residues. Its
m enzyme activity is highly efficient towards diamines putrecine and cadaverine. Both
proteins contain a typical peroxisomal targeting signal (PSTl) near/at the C-terminus
which strongly suggests that they are peroxisomal enzymes. Both P450W and soybean peroxisomal amine (SPAO) oxidase are expressed in cotyledon, root, leaf and nodule
tissues, but SPAO is expressed in a very low content suggesting that it may be substrate inducible. Amino acid sequence comparison reveals that P450W and SPAO share less
than 40% homology with other known P450s and copper-containing amine oxidases and form a new gene family of each superfamily, respectively. The feature of P450W implies that the mode of action as a peroxidase or a monooxygenase may be regulated by the ratio of O2/H2O2 in the compartment of peroxisomes. This study confirmed the existence of an alternate pathway for urate oxidation, and revealed a new cellular function of peroxisomes. Thus, amine oxidation and urate oxidation can be catalyzed in a diamine oxidase-peroxidase enzyme system in plant peroxisomes. This alternate pathway may contribute to nitrogen supplement and messenger molecule generation during plant growth and play an important role in cell growth, development and protection of plant against stresses.
IV To my parents, and sisters, Tianling and Tianwen,
who love and encourage me. ACKNOWLEDGMENTS
My sincere gratitude is expressed to my great adviser. Dr. Dash-Pai S. Verma. I truly admire him the serious scientific attitude and his deep knowledgeable vision of science. He provided guidance, advise and patience and very generous contributions to both my educational and professional development.
I wish to express great appreciation to my Smdent Advisory Committee member, Drs. K. R. Davis, T. L. Graham and D. Coplin, for their suggestions and comments on this study.
My appreciation is particularly expressed to all persons in my Lab who have contributed to making my graduate work positive and successful. Thank for their friendship and encouragement. I give special thanks to Drs. Zonglie Hong, Chunsheng
Zhang, for their patient advice and help through my research. Sincere appreciation also extended to Drs. Xiangju Gu, Jone Heon Kim and Zhaohua Peng, and Mr. 29iongming Zhang, Sunhun, Kim.
Finally, I thank my friends, my parents and sisters for their enduring faith, fellowship and love.
VI VTTA
December 26, 1964 ...... Bom-Handan, Hebei Province, People's
Republic of China July 1 9 8 ...... B.S. Microbiology, Shandong University, Jinan, Shandong, P. R. China 198 -1988 ...... Research Assistant, Institute of Applied
Atomic Energy, Chinese Academy of
Agricultural Sciences (CAAS) 1989-1991 ...... Research Assistant, Institute of Plant
Protection, CAAS
April 1991- October 1993 ...... M. S. Graduate Fellow, Graduate Research Associate, Department of Plant Pathology, Ohio Agriculture Research and
Development Center (OARDQ, The Ohio State University
October 1993 -March 1994 ...... Research Assistant, Plant Biotechnology Center, The Ohio State University
vii PUBLICATION
1. Boehm, M.J., Wu, T., Stone, A.G., Kraakman, B., Wilson., G. E., Maddon, L.V., and Hoitink, H.J., 1997, Cross-Polarized Magic-Angle Spinning ^^Nuclear Magnetic Resonance Spectroscopic Characterization of Sole Organic Matter Relative to Cultivable Bacterial Species Composition and Sustained Biological Control of Pythium Root Rot. Appl. Environ. Microbiol.Vol. 63:162-168.
2. Verma, D. P.S., Kim., J.H. and Wu, T., 1997, Assimilation of reduced nitrogen in tropical legum nodules regulation of de novo purine biosynthesis and peroxisome proliferation. Biological Fixation of Nitrogen for Ecology and Sustainable Agriculture, Springer-Verlag Berlin Heidelberg, NATO ASI Series, Vol. G 39:205-209.
3. Wu, T., Madden, L. V., and Hoitink, H. A. J. 1993. Relationship between organic Matter development level, microbial species diversity and Pythium root rot suppression. Soil Ecology Society Meeting 1993, An international Conference on the Functional Significance and Regulation of soil Biodiversity, May 3-6, 1993, Michigan State University, East Lansing, MI.
4. Wu, T., and Verma, D. P.S., 1997, Assimilation of symbiotically-reduced nitrogen in tropical legumes: Regulation of induction of de novo purine biosynthesis and peroxisome proliferation. The 11th International Congress on Nitrogen Fixation, July 20-25, 1997, Institute Pastece, Paris, Franc.
Vlll FIELD OF STUDY
Major Field: Plant Pathology
Specializing in Plant Molecular Biology. Studies in : Using Biotechnology Techniques to Study of Plant Peroxisome Biogenesis and Biological Functions, and Soybean Nitrogen
Assimilation. Advisor: Professor Desh-Pal S. Verma
IX TABLE OF CONTENTS
Abstract ...... ü Dedication ...... v Acknowledgments ...... vi V ita ...... vii List of Tables ...... xvii List of Figures ...... xx
Chapter Page
I. Peroxisomes ...... 1
1.1 Introduction ...... 1 1.2 Peroxisomes in human metabolism ...... 2 1.3 Peroxisomes in plant metabolism ...... 3 1.3.1 Glyoxysomes ...... 3 1.3.2 Leaf peroxisomes ...... 4 1.3.3 Transition forms of peroxisomes (unspecialized peroxisomes) ...... 4 1.3.4 Nodule peroxisomes ...... 4 1.4 Yeast as model system to study of peroxisome biogenesis ...... 5 1.5 Peroxisome biogenesis ...... 8 1.5.1 Membrane lipid acquisition ...... 8 1.5.2 Proliferation (duplication) ...... 8 1.5.3 Segregation ...... 9 1.5.4 Protein import ...... 9 1.6 Peroxisome polypeptide synthesis and import ...... 9 1.6.1 Matrix polypeptides ...... 9 1.6.2 Peroxisomal membrane proteins (PMPs) ...... 10 1.7 Peroxisome protein import ...... 13 1.7.1 Peroxisome targeting signals ...... 13 1.7.2 PST receptors ...... 16 1.7.3 A PTS receptor docking protein ...... 16 1.7.4 Other import proteins ...... 17 1.8 Peroxisome proliferation regulation ...... 20 1.9 The role of peroxisome in cellular signaling ...... 21 1.9.1 Extracellular signaling and peroxisome membranes ...... 22 1.9.2 Peroxisomal metabolites as regulatory signals ...... 22 1.9.2.1 Lysophosphatidic acid derivatives as peroxisomal signals ...... 23 1.9.2.2 Hydrogen peroxide and oxygen free radicals ...... 23 1.9.3 Further involvement of the peroxisome in cellular signaling (at the intercellular level of signaling) ...... 26 1.10 Peroxisomes, stresses and active oxygen species in plants ...... 27 1.10.1 Peroxisome and active oxygen species ...... 27 1.10.1.1 Catalase and H2O2 ...... 27 1.10.1.2 Superoxide dismutases (SODs) and superoxide radicals in peroxisomes ...... 28 1.10.3 Oxidative stress ...... 29 1.10.4 Peroxisomes as a source of superoxide and hydrogen peroxide in stressed plants ...... 30 1.11 Plant peroxisomes, purine metabolism and soybean ureides biosynthesis ...... 35 1.11.1 Nodule formation and nitrogen assimilation ...... 35 1. 11.2 Nitrogen assimilation and peroxisome proliferation ...... 37 1.11.2.1 Ammonia assimilation ...... 37 1.11.2.2 de novo purine biosynthesis ...... 37 1.11.2.3 Purine catabolism and ureides biosynthesis ...... 37
XI 1.11.3 The morphology feature of infected and uninfected cells ...... 39 1.11.4 Catabolism of Purines ...... 40 1.11.5 Polyamine catabolism in peroxisomes ...... 43 1.12 Proposed research ...... 43
2. Isolation of peroxisome biogenesis mutants and cloning of soybean genes required for peroxisome biogenesis ...... 45
2.1 Abstract ...... 45 2.2 Introduction ...... 47 2.3 Materials and methods ...... 52 2.3.1 Strains, plasmids, media and culture conditions ...... 52 2.3.2 Mutagenesis and selection of mutants ...... 53 2.3.3 Genetic analysis ...... 53 2.3.3.1 Dissection of asci (tetra dissection) ...... 54 2.3.3.2 Mating type assay ...... 54 2.3.3.3 Reverse mutation determination ...... 54 2.3.4 Cell-free extraction and enzyme assays ...... 55 2.3.4.1 Determination of catalase activity distribution in wildtype and mutant cells ...... 55 2.3.4.2 Biochemical analysis of mutant cells ...... 55 2.3.5 Functional complementation ...... 56 2.3.5.1 Soybean nodule cDNA library ...... 56 2.3.5.2 Yeast cell transformation and positive clone selection ...... 56 2.3.5.3 Yeast plasmid extraction from yeast cells ...... 57 2.4 Results ...... 58 2.4.1 The peroxisome biogenesis mutant failed to grow on the uric acid as sole nitrogen source and oleic acid as sole carbon source ...... 60
XU 2.4.2 Spbl contains a recessive mutation at a single allele ...... 63 2.4.3 Biological analysis of mutants by fractionation of peroxisomal marker enzymes ...... 66 2.4.4 Plant cDNA isolation by functional complementation ...... 68 2.4.4.1 Selection of transformants on the uric acid-containing medium ...... 68 2.4.4.2 Selection of transformants on the oleic acid-containing medium ...... 68 2.5 Discussion ...... 69
3. A putative G-box binding protein with Basic Helix-Loop-Helix may be involved in regulation of peroxisome proliferation in soybean ...... 74
3.1 A bstract ...... 74 3.2 Introduction ...... 75 3.3 Results ...... 78 3.3.1 SPB 1 cDNA isolation through functional complementation ...... 78 3.3.2 SPB 1 is a DNA binding protein containing a bHLH motif ...... 79 3.4 Discussion ...... 83
4. Isolation and characterization of a novel peroxisomal cytochrome P450 cDNA encoding a H2C>2-dependent urate-degrading peroxidase in soybean ...... 88
4.1 Abstract ...... 88 4.2 Introduction ...... 90 4.3 Materials and Methods ...... 93 4.3.1 Strains, plasmids, media and culture conditions ...... 93
xm 4.3.2 Functional complementation ...... 94 4.3.3 Full length cDNA isolation and DNA sequencing ...... 95
4.3.4 Sequence alignment, comparison and phylogenetic trees ...... 95 4.3.5 RNA isolation and Northern hybridization ...... 96 4.3.6 Expression of P450W cDNA in E.coli, protein extraction and SDS-PAGE ...... 96 4.3.7 Cell free firactionation and induction ...... 97 4.3.8 Enzyme assay ...... 97 4.3.9 Determination of H2O2 effect on yeast cell growth ...... 98 4.4 Results ...... 101 4.4.1 Xanthine can act as a peroxisome proliferator to S. cerevisiae ...... 101 4.4.2 Isolation of a cytochrome P450 cDNA by functional complementation ...... 102 4.4.3 P450W is a novel member of the P450 superfamily ...... 105 4.4.4 P450W contains a peroxisomal targeting motif and is a membrane bond protein ...... 108 4.4.5 Expression of P450W correlates with purine metabolism in plants ...... I ll 4.4.6 P450W has peroxidase activity ...... 114 4.4.7 Expression of P450W confers H2O2 tolerance in yeast ...... 115 4.5 Discussion ...... 117 4.5.1 A putative peroxisomal cytochrome P450 isolated from soybean ...... 117 4.5.2 P450 H202-dependent hydroxylation activity possibly is regulated by the O2/H2O2 ratio ...... 119 4.5.3 Catabolism of uric acid through the alternate pathway ...... 120 4.5.4 Xanthine is a peroxisome proliferator ...... 122
XIV 4.5.5 Ureide fonnatioa by diamine-peioxidase enzyme system may be involved in nitrogen supplement during seeding growth or under stress conditions ...... 123
5. Isolation and characterization of a soybean peroxisome amine oxidase cDNA ...... 126
5.1 Introduction ...... 126 5.2 Materials and methods ...... 130 5.2.1 Functional complementation ...... 130 5.2.2 Soybean nodule cDNA library screening and DNA sequencing ...... 130 5.2.3 Sequence alignment, comparison and polygenetic trees ...... 130 5.2.4 RNA isolation and Northern hybridization ...... 131 5.2.5 Expression of soybean peroxisomal (SPAO) cDNA in E.coli...... 131 5.2.6 Enzyme assay ...... 132
5.3 Results ...... 133 5.3.1 Isolation of a soybean peroxisomal amine oxidase (SPAO) via functional complementation ...... 133 5.3.2 Isolation and characterization of SPAO full length cDNA ...... 135 5.3.3 SPAO contains the conserved region for topa quinone cofactor formation ...... 139 5.3.4 A conserved region exists at N-terminus of some amine oxidases ...... 140 5.3.5 A peroxisomal targeting signal SKL/AKL resides at C-terminus of SPAO ...... 141 5.3.6 SPAO is likely inducible in soybean tissues ...... 144 5.3.7 Diamines are highly efGcient substrates of SPAO ...... 146
XV 5.4 Discussion ...... 148 5.5 Perspectives ...... 153
6. General discussion and summary ...... 154
6.1 Xanthine is a peroxisome proliferator ...... 154 6.2 The mutant and its functional complementation ...... 158 6.3 Urate degrading alternative pathway in peroxisomes ...... 158 6.4 The hydroxylation activity of peroxisomal P450W may be regulated by the ratio of O2/H2O2 ...... 159 6.5 A possible linkage of salicylic acid biosynthesis and poly amine oxidation ...... 160
List of Reference ...... 164
XVI LIST OF FIGURES
Figure Page
1.1 Diagram showing location and relative sizes of the known peroxisomal biogenesis proteins ...... 18 1.2 Model of peroxisomal matrix protein import ...... 19 1.3 The successive addition of electrons to oxygen in the production of oxygen free radicals ...... 24 1.4 An illustration of how oxygen free radicals may be generated, detoxified, and act as messengers via their modification of proteins and lipids ...... 25 1.5 A hypothetical scheme of the signal transduction pathway when plants are under a stress situation ...... 34 1.6 Role of peroxisomes in nitrogen assimilation pathway in root nodules of tropical legumes ...... 36 1.7 The major pathway of purine catabolism in eukaryotic cells ...... 42 1.8 The degradation of uric acid to allantoic acid ...... 41
2.1 Peroxisomal hydrogen peroxide metabolism in yeast grown on a fatty acid ...... 49 2.2 The schemes of fatQr acid ^-oxidation and urate oxidation pathways in peroxisomes ...... 50 2.3 The outline of yeast 5. cerevisiae mutagenesis and peroxisome biogenesis mutant isolation and identification ...... 59 2.4 Cell-free extract catalase activities of yeast strains ...... 62
xvu 2.5 The growth ability of on uric acid containing medium of the peroxisome biogenesis mutants generated in this study ...... 64 2.6 Enzyme activities of Spbl mutant cells under different growth conditions ...... 65
2.7 The growth ability of pgroxisome biogenesis mutants pebl, peb2, peb3 and peb4 on the uric acid as the sole nitrogen source ...... 70 2.8 The oleic acid and uric acid non-utilizer could be due to the mutation in the peroxisome proliferator activated receptor ...... 73
3.1 Nucleotide and deduced amino acid sequence of soybean putative G-box binding protein (SPBl) ...... 80 3.2 The hydrophobicity and, acidic and basic amino acid usage profile of SPBl ...... 81 3.3 Comparison of SPB 1 with other bHLH containing transcription regulatory proteins in plants ...... 82 3.4 Hypothetical models of regulation of genes coding for peroxisomal proteins ...... 87
4.1 Induction of yeast peroxisomal enzyme activities by xanthine ...... 99 4.2 Functional complementation of Spbl mutant with pYP450W grown on uric acid medium (NUGY agar) ...... 100 4 .3 Nucleotide sequence of cDNA and deduced amino acid sequence of cytochrome P450W ...... 103 4.4 Comparison of the amino acid sequence of P450 with other plant cytochrome P450s ...... 104 4.5 Amine acid sequence of P450s in the distal helix region ...... 107
x v m 4.6 The phylogenetic trees of P450W with other P450s and the hydrophobicity profile ...... 109 4.7 RNA blot analysis of P450W gene expression in soybean tissues ...... 110 4.8 Peroxidase and uricase activities of P450W expressed in yeast cells ...... 112 4.9 SDS-PAGE analysis of P450W expression in E.coli and the enzyme assays ...... 113 4.10 Spb 1 expression of P450W growth in H202-containing medium ...... 116 5.1 Functional complementation of Spb 1 mutant with p YSPAOi growth on uric acid-amino acid amended m edium ...... 134 5.2 cDNA and deduced amino acid sequence of soybean peroxisomal copper-containing amine oxidase ...... 136 5.3 Comparison of the amino acid sequences of SPAO with other amine oxidases ...... 137 5.4 The phylogenetic trees and sequence distance of SPAO with other amine oxidases ...... 138 5.5 The conserved region at N-terminal regions of several amine oxidases ...... 142 5.6 Comparison of C-terminal region amino acid sequence soybean peroxisomal amine oxidase isomers ...... 143 5.7 RNA blot analysis of P450W gene expression in soybean tissues ...... 145 5.8 The SDS-PAGE of SPAO expression in £. c o lt...... 145 5.9 The hypothetical model of the altemate urate-degrading enzyme system in soybean peroxisomes ...... 152
6.1 Xanthine is a peroxisome proliferator ...... 156 6.2 Functional complementation to the peroxisomal biogenesis mutant ...... 157 6.3 The hypothetical model of peroxisomes in plant defense through a diamine oxidase-peroxidase urate-degrading enzyme system ...... 162
XIX LIST OF TABLES
Table Page
1.1 Genes required for peroxisome biogenesis ...... 7 1.2 Peroxisome membrane polypeptides involved in 02- generation in pea leaf peroxisomes ...... 11 1.3 Leaf peroxisome membrane polypeptides in different plants ...... 12 1.4 Peroxisomal proteins containing S-K-L motif at the C-terminus ...... 15 1.5 The metabolism of activated oxygen in leaf peroxisomes from pea plants subjected to different types of stresses ...... 31 1.6 Peroxisome proliferation in plants ...... 32 2.1 Yeast mutant isolation scheme ...... 61 2.2 The features of the peroxisome biogenesis mutants Spbl and Spb2 ...... 67 5.1 Comparison of the relative catalytic ability of SPAO to the different substrates ...... 147
XX CHAPTERl
PEROXISOMES
1.1 Introduction
Peroxisomes are single membrane bound suborganelles present in all eukaryotic
cells ranging firom yeast to higher plants and mammals. This specialized organelle contains a set of unique enzymes that play diverse metabolic roles. Generally,
peroxisomes are in round or oval shapes, but in certain cells, they can display complex and irregular forms (Gorgas, 1984; Yamamoto, 1987). Removal of hydrogen peroxide and P-oxidation of fatty acids are two basic biochemical functions performed by
peroxisomes of different eukaryotic cells. A broad spectrum of metabolic activities are
found in peroxisomes (Master, 1992; Van den Bosch, 1992). An important aspect of
peroxisomal metabolism is its effect on metabolic pathways in other cell compartments. Peroxisomal metabolism integrates transport systems for peroxisomal substrates,
metabolic pathways shared with other compartments (like chloroplasts and
mitochondria), and indirect regulation of the other metabolic routes by means of the pool size of substrates or products. Therefore, peroxisomes are suggested to play an essential role in the central cellular metabolic signaling (see review. Master, 1996). A remarkable property of peroxisome biogenesis is that these organelles are substrate-inducible. 1.2 Peroxisomes in human metabolism In mammalian, approximate SO peroxisomal enzymes have been isolated, more than 70 per cent of peroxisomal enzymes are localized in the matrix of peroxisomes. A few enzymes are associated with crystalloid matrical inclusions (e.g. uricase). The remainder of the enzymes involved in the activation of fatty acids and the synthesis lipids and cholesterol from famesyl-pyrophosphate is membrane-bound. Of these membrane- bound enzymes, some have their catalytic sites exposed to the matrix, others to the cytosol. Small water-soluble molecules with molecular weights up to at least 800 can freely transport through the purified peroxisomes (Van Veldhoven, 1983; 1987). The non-selective permeability of the peroxisomal membrane is based on the presence of a proteinaceous pore which allow the diffusion of substrates, products and cofactors (Van
Veldhoven, 1987). Labarca, 1986) ), however, some substrate transportation request ATPs (Wolvetang, 1991).
In human, peroxisomal enzymes play an essential role in a number of metabolic pathways, including 1) lipid biosynthesis; 2) saturated and unsaturated fatty acids 13- oxidation ; 3) glyoxylate metabolism; 4) dicarboxylic acid p-oxidation; 5) metabolism of xenobiotic compounds; 6) polyamine oxidation; 7) the oxidative degradation of phytanic acid, purines, D-amino acids and pipecolic acid, and 8) oxygen and reactive oxygen species metabolism. Most of peroxisomal enzymes involved in these pathways have their counterparts in other subcellular compartments like mitochondria, chloroplasts and ER.
The enzyme content of peroxisomes is versatile and may vary from species to species, tissues to tissues.
The importance of peroxisomes in metabolism in human is demonstrated by existence of numerous genetic disorders affecting this organelle (van den Bosch, 1990;
Lazarow, 1994). Many of these disorders are the result of deficiencies in a specific peroxisomal enzymes. Examples of these genetic disorders include, X-linked adrenoleukodystrophy (X-ALD), amyotrophic lateral aclerosis (ALS) and other defects in the biogenesis of the entire organelle, e.g. Zellweger syndrome, a fetal disease of new
home in which peroxisomes are absent or grossly deficient (Lazarow, 1994).
1.3 Peroxisomes in plant metabolism
In plants, peroxisomes have been demonstrated to have a key function in the oxidative photosynthetic carbon cycle of photorespiration, ^-oxidation of fatty acids, and
glyoxylation (Gerhardt, 1986; Huang et al., 1983). The major functions of peroxisomes
in plant and yeast are listed as following: 1) oxidative photosynthetic carbon cycle of photorespiration; 2) fatty acid ^-oxidation; 3) glyoxylate cycle; 4) purine metabolism; 5) oxygen and reactive oxygen species metabolism; 6) methanol metabolism (fimgi, yeast);
7) amine metabolism (yeast) and 8) alkane metabolism (yeast). Four different types of specialized peroxisomes have been classified in plants, they are leaf peroxisomes, root glyoxysomes, transient peroxisomes and root nodule peroxisomes. Each of these peroxisomes possesses a distinct set of enzymes to perform a physiological function specific to each tissue (Tolbert and Essner, 1981; Huang et al., 1983; Schubert, 1986).
1.3.1 Glyoxysomes
Glyoxysomes are specialized plant peroxisomes existing in the storage tissues of oil seeds, which contain enzymes unique to reactions of the glyoxylate cycle and P- oxidation of fatty acids (Olsen and Harada, 1995; Gietl, 1996). These metabolic reactions efficiently convert the seed reserved lipids into sugars which are used for germination and plant growth (Kindi, 1987). The substrates for this conversion are different forms of fatty acids, and the glyoxysomes provide enzymes to oxidize various fatty acids. 1.3.2 Leaf peroxisomes
Leaf peroxisomes are specialized peroxisomes present in phosphosynthetic tissues that carry out the major reactions of photorespiration. In this reaction, glycolate is
converted to glycine, a key step in the photorespiration cycle. Hydroxypyruvate
reductase and glycolate oxidase are two important leaf peroxisomal proteins. A unique
suborganellar compartment likely exists in leaf peroxisomes, which can channel their
metabolites within the remaining structures even lacking an intact membrane boundary (Heupeletal, 1991).
1.3.3 Transition formf of peroxisomes (unspecilized peroxisomes) During plant lifetime, cells undergo many changes in their intracellular structures and biochemical functions. Distinct forms of peroxisomes are observed during the formation and maturation of seeds, in dry seeds, and during mobilization of storage compounds in germinating seeds. Under light induction, leaf peroxisomes appear simultaneously with chloroplasts. Glyoxysomes, transient peroxisomes and leaf peroxisomes coexist during this transition stage (Behrends et al., 1990; Kindle, 1982).
Later, tissues undergo alteration towards senescence and that means reappearance of glyoxysomal properties. Catalase gene expression is switched during transition state.
Thus, the transition state is distinguished by the presence of a number of heteromeric hybrid isoforms of many peroxisomal enzymes (Eising et al., 1990).
1.3.4 Nodule peroxisomes
Another specialized peroxisomes are root nodule peroxisomes existing in legume plants. Peroxisome proliferation in root nodules is induced (Atkins, 1981) during N2- flxation as oxidation of purine requires this organelle in the tropical legume plants. In the uninfected cells, the final step of ureide biosynthesis occurs inside peroxisomes where oxidation of uric acid to allantoin is catalyzed by uricase (Nguyen et al., 1985; Webb and Newcomb, 1987). Peroxisome proliferation is observed in the uninfected cells. Thus, peroxisomes are play an important role in nitrogen assimilation in tropical legumes.
1.4 Yeast as a model system to smdy of peroxisome biogenesis An important breakthrough in research on peroxisome biogenesis is caused by the application of yeast genetics. Yeast is an ideal genetic model to study peroxisome biogenesis due its ability to control the proliferation and enzymatic composition of organelle via growth substrate manipulation (Veenhuis, 1991). In some yeasts including
Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipilytica, peroxisome proliferation is repressed by glucose, derepressed by non-fermentable carbon sources and induced by oleic acid when it is used as the sole carbon source (Veenhuis, 1991). Yeast Hansenula polymorpha or P. pastoris growth on methanol induces a large amounts of peroxisomes. The application of genetics to yeast peroxisome studying results in the availability of peroxisome-deficient mutants.
To date, many peroxisomal mutants have been identified and isolated in difierent yeast mentioned above (Borst, 1989; Erdmann et al., 1989; Van Der Leij, 1992). These mutants have helped to elucidate the possible mechanism and the proteins involved in peroxisome assembly as well as import of peroxisomal proteins. The initially isolated peroxisome mutants were defined as peroxisome assembly (pas) mutants based on the utilization of oleic acid as a sole carbon source. The oleic acid non-utilizer can be classified into two groups ;
1) Eatty Acid Qiridation mutants (FOX) defect in structural genes of )3-oxidation;
2) Eeroxisome Assemblv mutants (PAS) or Peroxisome-deficient mutants (PER) defect in peroxisomal assembly or peroxisome biogenesis. PAS mutants are further classified into three different classes based on the morphology of the peroxisomes: i: Peroxisomes are not detectable;
ii: Peroxisomes present but non-proliferating;
iii: Normal peroxisomes are present but distinct peroxisomal matrix are mislocalized.
So far, more than 20 complementation group have been identified and 21 polypeptides have been detected in S. cerevisiae as constitunents of this organelle (Kunau et al., 1992). Recently, these mutants were originally assigned names such as pas, pay, or peb have been renamed pex as part of an effort to unify peroxisome biogenesis mutant, gene and protein nomenclature (Distel, 1996). Table 1.1 presents most of the genes identified so far that are involved in peroxisome biogenesis in S. cerevisiae, and some of the genes from other yeast in term of representing each model of pex family. In total, over 30 PEX genes have been cloned primarily by functional complementation of pex mutants in different species. Based on these pex genes, a hypothetical model of peroxisome protein import machinery has been postulated (see below). Gene Bonner name Functons Reference
PEXI ScPASI lo fllT k D Erdmann, et ai^ 1991 that ooottms two puiaiive ATPlimdmg nies. PEX2 PpPER6 PIMP of 3S-52kPa with COOH-lmmnal Watetham.e*af., 1996 CgHC^nmoiifl
PEX3 ScPAS3 PIMPofWkDa, penudaamaliniegnl Hbhfeld.e«if.. 1991 membniiepralem. PEX4 SePASZ Ubiqnüm-ooiqiigatmg enQme o f 21-24 icDa Wiebel and Kunau, 1992 located oo cytoaolic fide « penndfaaial
PEXS SePASlO A ld n it Hohfeld.eia(,,1991 the ût^oft matrix pratBÎDS with SKL agnaL PEX6 ScPASS A 127kDa protBiD member o f AAA Aiinly o f Voom-Bioawer.eto/., 1993 AlPasea, cootams ooe AlP-bmdmg i&B in a cooaerved 185 aa domam. PEX7 ScPAS7t Putative PTS2 receptor with WD-40 modf Maizioch, ef of,, 1994 PEBl Various rabceUnlar kxalioa. Zhang and Lazarow. 1995
PEX8 PpPER3 Aoteinof71-81kD a located on matrix site o f Lin, et n/., 1995 peroxisomet» membrane, contains both FTSl and PTS2. PEB9 YIPAÏ2 PIMP o f 42kDa with cysteine-tich region. Eitzen.ernf.. 1995 PEBIO PpPAS? PIMP o f 3 4 ^ kDa with COOH-terminal CaHC* Kalish.et PEX13 Putative PTSl Nuttley.eroi,, 1994 o f 43 kDa with H-terminal SH3 rtaxif ScPASM Kos,erai,1995 transduction route required for relmf of glucose repression. ScPASI9 The transcription bctorA D R l, asapositive Simon, et ni., 1991 activator k r many peroxisomal proteins. Abbreviatioos: Sc, Saccharomyces cerevisiae; Pp. Pichia pastoris; Yl, Yarrowia lipolytica. (This table is modified according to Waietham, and Cr%g, 1997) Table 1.1. Genes required for pa^oxisome biogenesis 1.5 Peroxisome biogenesis Peroxisome was first described as an electron dense subcellular organelle in rat liver. Peroxisome proliferation takes place by growth and division through budding from the pre-existing peroxisomes (Trelease, 1984; Lazarow and fiijiki, 1985; Borst 1989). In rat liver, de novo proliferation of peroxisomes suggests that peroxisome is initiated by membranous attachments onto the surface of pre-existing peroxisomes. The membrane structures may provide the appropriate lipid environment for the incorporation of peroxisomal membrane proteins and subsequently become the sites for import of the newly synthesized matrix proteins (Lueres et al., 1993; Fahimi et al., 1993). Peroxisomal proteins are synthesized on free ribosomes and imported post- translationally firom the cytosol (Lazarow and fiijiki, 1985; Nguyen et al., 1985). These proteins contain the so-called peroxisome targeting sequences (PTS) that are present at the C-termini of the proteins or at the N-terminal regions of the molecules. Thus, peroxisome biogenesis can be subdivided into the following specific categories. 1.5.1 Membrane lipid acquisition The peroxisomal membrane is composed primarily of phosphatidy choline and phosphatidyl ethanolamine (Subramani, 1993). Lacking its own lipid biosynthetic apparatus, peroxisomes likely acquire lipid firom the endoplasmic reticulum (ER) and their site of synthesis. The mechanism is not known yet. 1.5.2 Proliferation (duplication) Peroxisome proliferation occurs by fission or budding firom an pre-existing peroxisome (Subramari, 1993). This fission process is observed only in the cells where peroxisomes undergo rapid proliferation in response to peroxisome-inducing substrates. So far, first two components of the peroxisome proliferation associated machinery have 8 been described: HpPexlOp, a peroxisomal integral-membrane protein from H. polymorpha (Tan, 1995) and ScPexl Ip, a peroxisomal membrane-associated protein from S. cerevisiae (Erdmann and Blobel, 1995; Marshall et al., 1995). S. cerevisiae pexl 1-deleted strain was observed to contain only a single large peroxisome under oleate- induction of peroxisomes. It indicates that ScPexl Ip is an essential for peroxisome proliferation. 1.5.3 Segregation Under glucose repression, yeast typically contains only one or two small peroxisomes per cell (Veenhuis, 1991). A mechanism must exist to maintain such equal segregation of peroxisomes to the daughter cells. As with other organelles, peroxisomal movement and segregation is likely to involve cytoskeletal elements such as microtubules or actin filaments. 1.5.4 Protein import Peroxisomal proteins are known to be synthesized in cytosol on the free ribosomes and posttranslationally imported into peroxisomes (Nguyen et al., 1985; Lazarow and Fujiki, 1985). A targeting signal and an import machinery are required for the import of peroxisomal proteins. A detailed discussion will be found in the following text. 1.6 Peroxisomal polypeptides synthesis and import 1.6.1 Matrix polypeptides Unlike mitochondria and chloroplasts, peroxisomes do not contain its own DNAs. The gene-encoding peroxisomal polypeptides are located in genomic DNAs. All 9 peroxisomal polypeptides, matrix and membrane peptides, studied thus far are synthesized on firee polysomes in cytoplasm and then imported into peroxisomes. Unlike mitochondrion and chloroplast secretary polypeptides, many peroxisomal matrix proteins do not involve their proteolytic processing (Lazarow and Fujiki, 1985; Borst, 1986; Vemer and Schatz, 1988). Previous studies reveal that all polypeptides are synthesized and released in the cytosol from where they enter peroxisomes with quite different import kinetics. Newly synthesized peroxisomal polypeptides are released into cytosol and imported into peroxisomes very quickly. Catalase, Acyl-CoA oxidase and uricase, for example, are typical peroxisome matrix proteins. Most peroxisomal enzymes are inducible in the presence of specific substrates. 1.6.2 Peroxisomal membrane proteins (PMPs) Peroxisomal membrane contains several major integral membrane proteins that likely are synthesized on free polysomes without recognizable presequences, and are post-translationally inserted into the peroxisomal membrane (Lazarow and Fujiki, 1985). Several peroxisomal integral membrane polypeptides have been characterized and purified from rat liver peroxisomes, they have molecular weight of 70, 68, 41, 27, 26 and 22 kDa, respectively (Hashimoto et al., 1986). However, the physiological functions of most integral membrane proteins are unknown. PMP70 is a member of the Mdr (P- glycoprotein) -related ATP-binding protein superfamily (Kamijo, et al., 1990). PMP22 is a type Hlb integral membrane protein with four hydrophobic membrane spanning domains. In Hansenular polymorpha, the constitunent PMPs are 22, 31,42, 49, and 51 kDa (Suiter et al., 1990). In plants, PMPs have been identified mostly in seedling glyoxysomes. Six major PMPs have been observed in the enlarged glyoxysome membrane in cotyledons of cotton seedlings (Chapman and Trelease, 1992). Donaldson's group (Hicks and Donaldson, 10 1982; Donaldson and Fang, 1987; Fang et al., 1987) demonstrated that Cytbg, CytP450, NADH:ferricynaide reductase, NADH:Cyt c reductase, and NADPHrCyt c reductase were present in glyoxysomal and ER membranes of castor bean endosperm, del Rio's group has shown that peroxisome membrane polypeptides are involved in 0%" generation which is due to the Cytbs : cytochrome C reductase electron transport system in pea leaf peroxisomes (1996) (see. Table 1.2). Jiang et al., (1994) demonstrated that there are six prominent nondenatured PMP complexes and 10 SDS-denatured PMPs from the cotyledons of sunflower seedlings in glyoxysomes, transition forms of peroxisomes and leaf-like peroxisomes. Three PMPs (57, 49 and 31) were constituted in all types of peroxisomes. Peroxisome membrane protein contents vary depending on the plant origin. Some proteins are conserved, see Table 1.3. Molecular mass Electron Possible kDa donor identity 61.0 - 7 56.0 - 7 32.0 NADH Ravoprotein ferricyanide reductase activity 29.0 NADPH Cytochrome C reductase activity (?) 18.0 NADH Cytochrome b5 (del Rio et al. 1996) Table 1.2. Peroxisome membrane polypeptides involved in 0%" generation in pea leaf peroxisomes. 11 Plant tissue Molecular mass Reference kDa Pea leaf peroxisome 61.0 del Rio, 1996 56.0 32.0 29.0 18.0 Cotton leaf peroxisome 57.0 Jiang, et al., 1994 49.0 31.0 53.0 28.0 27.5 Table 1.3. Leaf peroxisome membrane polypeptides in different plants. 12 1.7 Peroxisomal protein import Peroxisomal protein import mechanism has been intensively studied recently. Several groups have reconstructed the process in vitro (Subranmani, 1993). These efforts have let to significant advances in our understanding in the import pathway. Most major systems that transport proteins across a membrane share a common transportation pathway in all organisms including bacterial protein exportation, ER, mitochondria, chloroplasts and peroxisomes (see review, Schatz and Dobberstein, 1996). The common features of transport proteins across a membrane consist 1) an amino- terminal transient signal sequence on the transported protein; or a carboxyl-terminal targeting signal residing in peroxisomal proteins; 2) a targeting system on the cis -side of the membrane; 3) a hetero-oligomeric transmembrane channel that is gated both across and within the plant of the membrane; 4) a peripherally attached protein translocation motor that is powered by the hydrolysis of nucleoside triphosphate, and 5) a protein folding system on the trans-side of the membrane (Schatz and Dobberstein, 1996). Until now, peroxisomal protein transportation system was the least studied in comparison to other organelles. With recent peroxisome biogenesis studies and gene isolation, the peroxisome import mechanism is getting to be elucidated. These studies suggest that, the mechanism of peroxisome transportation is very similar to that of other organelles. 1.7.1 Peroxisomal targeting signals Peroxisomal protein import is conserved among yeast, fungi, plants, and animals (Gould, 1989). Polypeptides destined for the peroxisome matrix contain specific molecule determinants (Subramani, 1993) which are recognized post-translationally (Lazarow and Fujiki, 1985). Two evolutionarily conserved peroxisomal targeting signals (PTSs) have been identified. PTSl, used by the majority of peroxisomal polypeptides, consists of the C-terminal tripeptide Ser-Lys-Leu (SKL) or degenerate form of S/A/C/- 13 K/H/R-L defined by Gould et al (1989). This signal appears to be universal and conserved within mammalian, plant and yeast peroxisomes (Gould et al. 1990), it is non- cleavable after protein import. Recent study on the carboxyl terminus of microbodies function as peroxisomal targeting signal in higher plants, reveals that carboxyl-terminal tripeptide sequences of the form C/A/S/P-K/R-I/L/M function as a microbody-targeting signal. Tripeptides with proline as the first amino acid position and isoleucine at the carboxyl terminus show weak targeting efficiencies (Hayashi, 1997). Some peroxisomal proteins with peroxisomal SKL targeting signals are listed in Table 1.4. The second targeting signal, PTS2, resides at the N-terminus, with the consensus sequence Arg-Leu-X5-His/Gln-Leu (RLX5H/QL), which was first identified on rat thiolase (de Hoop, 1992; Swinkles, 1991). PTS2 is less common and less conserved than PTSl (Osumi, 1991; Swinkles, 1991). It can be cleaved in some proteins (rat thiolase, watermelon, malate dehydrogenase) but not in others (yeast thiolase, amine oxidase). Some peroxisomal membrane proteins (PMP) also contain PTSl, But the precise topogenic information of PMP have not been defined (McCammon, 1994). 14 Protein Source Sequence Reference Luciferase Photinus pyralis -KSKL Keller, era/., 1987 Acyl-CoA oxidase Rat -QSKL Miyazawa, era/., 1989 Bifunctional enzyme Rat -GSKL Osumi, et al., 1985 (peroxisomal) Uricase Soybean -NSKL Nguyen, er a/., 1985 Malate qmthase Cucumber -LSKL Smith and Leaver, 198( Citrate synthesis S. cerevisiae -ESKL Lewin, er a/., 1990 (peroxisomal) (According to Roggenkamp, R., 1992) Table 1.4. Peroxisomal proteins containing S-K-L motif at the C-terminus 15 1.7.2 PST receptors The presence of PTSs on peroxisomal proteins implies the existence of receptors that recognize and bind to these sequences as a first step in protein import. Now, both PTS 1 and PTS2 receptor proteins have been identified (Rachubinski and Subramani, 1995). The first PTS 1 receptor candidate was identified as the product of the P. pastoris PEX5 gene, PpPexSp. This protein is predicted to be a 68-kDa polypeptides with seven copies of a motif called the snap helix or tetratricopeptide repeat (TPR) and has high affinity of and specificity to SKL ending peptides in vitro (McCollum et al., 1993; Terlecky et al., 1995). It is mainly localized in cytoplasm. The pex5 mutant are defective in the import of proteins with PTSl but not to the proteins with PTS2. Therefore, PpPex5p imports proteins with this specificity. The candidate of PST2 receptor has been identified as Pex7p in S. cerevisiae based on the phenotype of mutants affected in its gene (Marzioch et al., 1994; Rehling et al., 1996). The mutants faü to import PTS2 enzymes but not PTSl enzymes. ScPex7p is predicted to be a 42-kDa protein with six copies of a motif of approximately 40 residues, called WD repeat, named for the conserved presence of a Trp-Asp (WD) pair at carboxyl terminus of each copy of the motif. It has shown that WD-repeat protein interacts with TPR proteins (Rehling et al., 1996). The localization of PTS2 receptor protein is in cytoplasm or at intraperoxisomal region (Zhang and Lazarow, 1996). 1.7.3 A PTS receptor docking protein A candidate for a PTS receptor docking protein, Pexl3p, has been identified in S. cerevisiae (Ermann and Blobel, 1996). ScPexl3p is a 43-kDa PIMP (Peroxisome Import Protein), which contains a src homology 3 (SH3) domain near its carboxyl terminus that interacts with ScPex5p, the putative PTSl receptor protein. This interaction, in conjunction with its peroxisomal membrane location, is consistent with the 16 notion that Pex 13p functions as a docking protein on the surface of the peroxisome that binds matrix protein receptor complexes at the site of the translocation machinery as a second step in ± e import process. Pex 13p does not interact with PTS2 receptor protein Pex7p. 1.7.4 Other import proteins The remaining biogenesis (import) proteins include two ATP-binding proteins, a ubiquitin-conjugating enzyme, five PIMPs (in addition to the putative docking protein), and a protein located on the inner surface of the peroxisomal membrane. The ATP- binding proteins Pexlp and Pex6p are large (>100 kDa) polypeptides, with potential ATP-binding sites in conserved domains of approximately 185 amino acids, belonging to the AAA family of ATPases (Erdmann et al., 1991; Yahraus et al., 1996). AAA proteins are involved in a wide variety of cell functions, as their name implies (ATPases associated with diverse cellular activities). The peroxisome biogenesis AAA proteins exist in low-abundant. Pex6p is a peroxisomal membrane-associated protein, while in human ortholog is cytoplasmic (Tsukamoto et al., 1995; Yahraus et al., 1996). A hypothetical model of peroxisomal protein import is drawn in Figure 1.1. The proteins involved in peroxisome biogenesis are drawn in the Figure 1.2, which represent the proteins listed in Table 1.1. 17 Cytoplasm Peroxisomal Matrix Figure 1.1. Diagram showing location and relative sizes of the known peroxisomal biogenesis proteins. The numbers of the proteins correspond to those of the genes in Table 1.1 (According to Waterman and Cregg, 1997). 18 / XP.ti.sp TPas»; Cytosol Peroxisomal matrix Figure 1.2. Model of peroxisomal matrix protein import. 1) Nascent peroxisomal protein with PTS 1 recognized by PTS 1 receptor (PexSp) located in the cytoplasm. 2) The PTS I receptor complex interact with PTS receptor docking protein (PexI3p) through PexSp, Pexl3p interaction. 3) protein transportation requires energy. Proteins are then transferred to the translocation apparatus to import into matrix with expense some ATPs (Pexlp and Pex6p are ATPases). 4) Peptides are formed native form through chaperons which may locate in or out of peroxisomes. (According to Water and Cregg, 1997; Schatz and Doblxrstein, 1996). 19 1.8 Peroxisome proliferatioa reguladon As previous described, peroxisomal proteins and peroxisomal compartment of yeast S. cerevisiae can be repressed, derepressed and induced in response to cellular demand or substrates present. Peroxisome proliferation, therefore, has been found to be controlled at the level of transcription and regulated by a multiple transcription factors (Alexandra et al. 1993; Wang, 1994). Oleate induces the transcription of genes involved in peroxisome biogenesis and proliferation of these organelles in 5. cerevisiae. A c/j-acting DNA element has been defined as oleate-response element (ORE) which is a positive regulatory element initially found in the 5' flanking region of the F0X3 gene encoding the peroxisomal enzyme 3- oxoacyl-CoA thiolase (Einerhand et al, 1991). ORE has been found in the 5' flanking region of most the S. cerevisiae genes encoding peroxisomal proteins or enzymes involved the biogenesis of the organelle (e.g. 3-oxoacyl-CoA thiolase , catalase A , PAS3 and PAS 10). DNA band-shift experiments indicated that trans-acting factors bind to this palindrome and DNase-I footprint showed that it covers the entire palindrome. Such oleate-responsive elements are not only present in the 5' flanking regions of genes encoding the proteins involved in peroxisome biogenesis but are also found in the genes encoding the factor(s) binding to the oleate-responsive element(s). The fibrate analogs [such as clofibrate, ciprofibrate and 2,4- dichlorophenoxyacetic acid (2,4-D), an active herbicide] are strong peroxisomal proliferators in animals (Cherkouimalki et al., 1991). A peroxisomal integral membrane protein of 35-kDa acts as a peroxisome assembly factor 1 (PAF-1). The PAF-1 contains a conserved cysteine-rich sequence at the C-terminal region which is exactly the same as that of a novel cysteine-rich RING finger motif family (Tsukamoto et al., 1991, 1994). Chinese hamster ovary cells deficient in peroxisome biogenesis regain peroxisomes after transfection with a cDNA coding for PAF-1 from rat liver (Thieringer and Raetz, 1993). 2 0 In animals, several peroxisome proliferator-activated receptors (PPARs) have been identified as members of the steroid hormone receptor family and peroxisome proliferator-responsive genes contain an upstream regulatory element (Green and Wahli, 1994). The retinoid X receptor forms a heterodiamer with PPAR and binds to the peroxisome proliferator-responsive element. This explains how a number of genes may be affected by PPAR during the nutritional change (Auwerx, 1992). No such receptor has yet been identified in plants. 1.9 The role of peroxisomes in cellular signaling With intensive studies on the peroxisome biogenesis and functions, a new role has been given to the peroxisomes, that is, peroxisomes play an important role in cellular signaling (see review. Masters, 1996). In the past, the peroxisome is considered to play a " garbage" role inside cells and with only a few metabolic activities, such as D-amino acids, L-a-hydroxyl acids and urate oxidation (De Duve, 1966). However, peroxisomes are found not only to play an important role in a broad spectrum of metabolic activities (see the section of peroxisome functions), but also are involved in the biosynthesis of many important cellular constituents like cholesterol, bile acids and other lipids (Masters, 1992, Van Den Bosch, 1992). In addition, peroxisome deficiency causes serious genetic disorders. Taken together, these developments have raised general questions in relation to the role of the peroxisome in cellular signaling. Recent studies, pointed out that peroxisomes have specific signaling role at several different levels of communication (Masters, 1995; Hamilton, 1987; Masters, 1984). A unique feature of peroxisome in its proliferator-induciblity makes it an important play in the cellular-signaling network (Masters, 1996). 21 1.9.1. Extracellular signaling and peroxisome membrane Xenobiotics, clofibrate, for example, acts as a peroxisome proliferator, and induces rat liver peroxisome proliferation, as well as plant pea leaf peroxisome proliferation (Palma, 1991). Peroxisome proliferator action is through the alternation produced as the level of membrane phospholipids in this organelle. Peroxisome proliferators cause an increase of synthesis of phosphatidylcholine is required for the increase in peroxisomal membrane mass, and allows for an associated increase in the production of other membrane components such as phosphatidylethanolamine and phosphatidylserine (Kawashima, 1994). Peroxisome proliferators also can alter the permeability of peroxisomal membranes and result of some regulatory molecules release into cytosol. For instance, catalase can pass selectively through the peroxisomal membrane into the cytoplasm under appropriate physiological conditions (Crane, 1990). Peroxisomal catalase as well as cytosol catalase together may regulating the levels of oxygen firee radicals in the cellular environment and their potential role as secondary messengers (Stadtman, 1986). There are several H2O2 production source outside peroxisomes. It is also expected that permeability influence would be even greater at the level of smaller regulatory molecules (Masters, 1995). 1.9.2 Peroxisomal metabolite as regulatory signals Peroxisome proliferators have been shown to interact with many of the main components of the cellular-signaling matrix e.g., calcium levels, sodium-hydrogen ion exchange, protein kinase C, and proto-oncogenes. Two particular groups of messenger involved in cellular signaling hold special relationships with peroxisomal function, they are those lipidic messengers associated with the hydrolysis of phosphatidylcholine, and those messengers associated with hydrogen peroxide. 2 2 1.9.2.1 Lysophosphatidîc acid derivatives as peroxisomal signals The hydrolysis of phosphatidylcholine by PLA2 is a reaction closely associated with peroxisomal function and typically leads to the production of lysophosphatidic acid and arachidonic acid. Both molecules play an important role in signaling and contribute significantly to the coordination of cellular processes during peroxisomal proliferation and function (Masters, 1996). Lysophosphatidylcholine influences the permeability of the peroxisomal membrane and participates intracellular and extracellular signaling. It particularly influences the action of protein kinase C (PKC) which is a prerequisite for long-term physical responses (Asaoka, 1992). 1.9.2.2 Hydrogen peroxide and oxygen fiee radicals Free radicals arise in tissues by a number of mechanisms, both enzymatic and non-enzymatic, but principally as metabolic by-products of the two main energy-deriving processes in living organisms: photosynthesis and reduction of oxygen to water (Chance, 1979). Although the oxidative metabolism of lipids and carbohydrates is generally contained within organelles such as the peroxisome and mitochondrion, it has been estimate that some 1-2% of the molecular oxygen evades the detoxification processes and escapes as free radicals (Chance, 1979; Stadtman, 1986; Wolff, 1986). If 0% accepts only one electron, the product is superoxide; if it accepts two electrons and hydrogen, the product is hydrogen peroxide; further electron acceptation leads to the hydroxyl radical and subsequently to water (Figure 1.3). Oxygen free radicals are well recognized as playing an important role in many aspects of long-term physiological regulation such as aging and degenerative disease. Also oxygen fiee radicals have been proposed to act in 23 the short term as second messenger, with the oxygen-radical-mediated modulation of biomolecules underlying a general mechanism for regulating signal-transduction pathway (Statman, 1986; 1990) (Figure 1.4). Figure 1.3. The successive addition of electrons to oxygen in the production of oxygen free radicals. 0%-, superoxide; H2O2, hydrogen peroxide; .OH", hydroxyl radical finally to H2O. Oxygen-radical modulation of biomolecules may occur at many levels, particularly effecting proteins and lipids, for example, oxygen free radicals may cause the oxidation of several residues like prolyl, arginyl, lysyl, and histidyl of amino acids. This may lead to a substantial alteration of the normal physiological fimctions of the host proteins, as well as accelerating their degradation and causing a concomitant loss of the ability of the cell to maintain homeostasis under stress conditions. Especially, the effects on a regulatory protein, e.g. PCK, can influence on a diverse cellular processes such as receptor fimction, ion transport, movement and shape changes, metabolism, and DNA transcription (Asaoka, 1992; Kiss, 1994). In the case of lipids, free-radical attack is generally directed at the polyunsaturated fatty-acid components of membranes. Hydrogen-radical attack, for example, may set off a radical chain reaction, as the result, lipid peroxides generation clearly exerts a marked influence on membrane stability and fimction. 24 OXVGBSl IVETABOLiaVI BIOTOXINS CYTOKIINES RADIATION INITIATORS FEIVEDIATORB CATALASE SUPEROXIDE DISMUTASE GLLTTATHION PEROXIDASE Vi MESSENGER EFFECTS REGULATORY PROTEINS METOBOLIC EFFICIBCY MBVBRANES 0R3ANB.LE FUNCTION Figure 1.4. An illustration of how oxygen free radicals may be generated, detoxified, and act as messengers in biological systems (Masters, 1996). 25 Catalase and superoxide dismutase are as the most efficient enzymes to scavenge these oxygen firee radicals. Their combined action converts superoxide and hydrogen peroxide to water and oxygen. O2” + O2" + 2H ^ H2O2 + O2; 2H2O2 ^ 2H2O + O2 Both of these enzymes have been identified within or without peroxisomes. The firee radicals produced by most eukaryotic cells has been recognized to contribute a considerable influence on the functioning of both membranes and signaling networks, and that the peroxisome is intimately involved in the regulation of firee-radical concentrations and hence has the potential for a major influence in cellular signaling (Fig. 1.4). 1.9.3 Further involvement of the peroxisome in cellular signaling At the intercellular level of signaling in mammals, evidence has shown that inter tissue communication related to peroxisome function (Masters, 1984; Masters, 1987). At the level of tissue-tissue communication, one possibility is the existence of a chemical messenger related to peroxisomal metabolism (Masters, 1995; Hamilton, 1987; Masters, 1987). It has been suggested that inter-and intra tissues signaling is related to peroxisome metabolism, implying some peroxisomal metabolites may be the systemic signals. Several lipid compounds and medium-chain fatty acids are already considered as messengers. Regarding the cellular signaling in intracellular terms, one of the notable features of peroxisomal metabolism is the occurrence of many important relationships with other organelles and with the cytoplasm at the enzymatic and metabolic levels. Many activities identified in the peroxisome also appear in sim ilar or identical form in other cellular compartments such as the detoxification of hydrogen peroxide, the oxidation of fatty acids, transaminations, and the synthesis of complex lipids often involve a close 26 coordination of function between two or more compartments (Masters, 1995). Such inter-compartmental cooperation may lead to the interpretation of a significant role for the peroxisome in cellular signaling and the regulation of cellular metabolism. 1.10 Peroxisomes, stresses and active oxygen species in plants 1.10.1 Peroxisome and active oxygen species 1.10.1.1 Catalase and H2O2 Peroxisomes contain catalase and H202-production flavin oxidases as basic enzymatic constituents (Tolbert, 1981, Huang et al., 1983). In plants, H2O2 is produced via two major routes: (a) reduction of molecular oxygen in Mehler reaction and (b) photorespiration. In peroxisomes, H2O2 is generated as a by-product by reducing O2 in the oxidative reactions catalyzing by a series of flavin oxidase, like xanthine oxidase, urate oxidase, acyl-CoA oxidase, and glycolate oxidase. Under normal physiological conditions, H2O2 is decomposed either catalytically or peroxidatically (de Duve and Baudhuin, 1966; Chance, 1979). Generally, H2O2 concentration can be controlled by catalase inside peroxisomes so that it does not reach toxic levels that could bring about oxidative damage inside peroxisomes and surrounding cytoplasm. However, under certain stress conditions, catalase activity can be depressed. Catalase (ECl.11.1.6) is a ubiquitous peroxisomal matrix enzyme. In Plants, all catalases derive from a common ancestral catalase gene (Guan, 1996). Catalase is encoded by more than one gene, e.g. there are three genes in maize that are expressed in a development and tissue specific manner (Skadsen et al., 1990; Redinbaugh et al., 1990). Catalase can also exist in many isoforms, CAT2 through CAT8 have been isolated firom sunflower cotyledons peroxisomes (Grotjoham et al., 1997). A catalase of 59 kDa is identified as the predominant protein component in purified cores firom sunflower cotyledons. A 55-kDa catalase is the major catalase protein in matrix firaction obtained 27 from lysed peroxisomes suggest that two peroxisomal populations of catalase differing in molecular structure and subperoxisomal compartmentations in sunflower cotyledons (Kleff et al, 1997). Cotton seed contains tetrameric isoforms of catalase (Ni et al., 1990). Catalase functions as a sink for H2O2, it is critical for maintaining the redox balance during oxidative stress (Willekens, 1997). Catalase-deficient plants develop necrotic lesions on leaves in elevated light, and become more sensitive to paraquat, salt and ozone (Willekens, 1997). The discovery of salicylic acid binding to catalases have bring much attention to H2O2 which is suggested to act as secondary messenger to activate plant defense response when plant is under pathogen attack (Chen, 1993). A mechanism has been postulated that inhibition of catalase results in elevated level of H2O2 which then activates defense-related genes (Chen, 1993). It is not known whether the inhibition of catalases caused by the stress factors (del Rio, 1992) other than pathogen infection is also due to the salicylic acid inhibition. 1.10.1.2 Superoxide dismutases (SODs) and superoxide radicals in peroxisomes The generation of superoxide radical has been demonstrated in two classes of plant peroxisomes, glyoxysomes and leaf peroxisomes. Superoxide radicals are generated in both soluble and membrane fraction of glyoxysomes of watermelon cotyledon (Sandalio, 1988), and pea leaf peroxisomes (del Rio, 1989). The one from soluble fraction is identified to be produced by xanthine/ xanthine oxidase, and that from membrane fraction is contributed by NADH-dependent enzyme system (review, see del Rio et al., 1992). The NADH-dependent production of O2" by leaf peroxisomal membranes could involve a small electron transport chain similar to that reported in peroxisomal membranes from castor bean endosperm (Fang, 1987). This electron transport chain is composed of flavoprotein NADH; ferricyanide reductase of about 32 28 kDa and cytochrome bg (measured as DANH: cytochrome c reductase activity see Table 1.2) (Fang, 1987; Luster, 1988). A potential source of superoxide anions in peroxisomes is xanthine oxidase, which in certain species is associated with the core of hepatic peroxisomes (AngermuUer, 1987). One of the important features of peroxisomes is that ±ey have an essentially oxidative type metabolism to dispose the oxidative oxygen species. Superoxide dismutases (SODs) (EC 1.15.1.1) are a family of metalloenzymes that catalyze the disproportion of superoxide (0%") radicals, and they play an important role in protecting cells against the toxic effects of superoxide radicals produced in different cellular loci (Halliwell, 1989, Fridovich, 1995). Superoxide dismutases have been known to present in mitochondria, chloroplasts, and the cytosol (Halliwell, 1993). A Mn-SOD was discovered in plant peroxisome by using inmmunoelectron microscopy localization by del Rio and colleagues (Sandalio, 1987a,b), thereafter several plant SODs have been isolated and identified (see Review, del Rio et al., 1992). These metalloenzymes have been found existing in peroxisomes of pea leaves (Sandalio, 1987a, b), watermelon cotyledons (Sandalio, 1987a), carnation petals (Droillard, 1990), cucumber, cotton and sunflower cotyledons (Corpo, 1994) and castor bean endosperm (del Rio, 1995). SOD has been also found in the peroxisomes of human fibroblasts (Keller, 1991), hepatoma cells (Crapo, 1992), and also in rat liver cells (Dhaunsi, 1992; Wanders, 1992). The type of SOD present in peroxisomes varies depending on the tissue origin. 1.10.3 Oxidative stress Oxidative stress refers to the damage caused by superoxide radicals. The unpaired electrons of superoxides react with many cellular molecules, such as DNAs, proteins, membrane lipids and other organic molecules with unsaturated bonds, resulting 29 in a chain processes involving crosslinkage, peroxidation, mutations membrane peroxidation and toxic compound production (Fridovich, 1978; Davies, 1995). Under certain condition, in peroxisomes, O2" radicals could react with H2O2, abundant in catalase-deficient peroxisomes, by a metal-catalyzed site-specific Haber- Weiss reaction (Halliwell and Gutteridge, 1989), and give rise to the vastly more reactive hydroxyl radicals (OH") (Figure 1.4). This strong oxidizing species could severely damage biological membranes, lipids and DNAs (Halliwell and Gutteridge, 1989). This simation could be prevented by the O2" scavenging action of SOD, which in peroxisomes is located at the sites where O2" radicals are produced. Nevertheless, oxygen free radicals also have some useful functions in the metabolism of peroxisomes. These organelles may serve as specific cellular sites for the purpose-oriented production, under certain conditions, of activated oxygen species useful for some cellular metabolic reactions (Halliwell and Gutteridge, 1989; Sandalio, et al., 1988). The peroxisomal superoxide dismutase could modulate the O2" dependent site-specific formation of OH" radicals so that they could be effectively used for reactions which require strong oxidizing agents. However, the oxidative reactions of the peroxisomal metabolism where these highly reactive oxygen radicals could be used beneficially are still unknown. 1.10.4 Peroxisomes as a source of superoxide and hydrogen peroxide in stressed plants The metabohsm of activated oxygen in leaf peroxisomes from pea plants subjected to different types of stress, including salinity, copper excess, xenobiotics such as chlofibrate (ethyl-a-p-chlorophenoxyisobutyrate) and dark incubation has been studied by del Rio's group. The metabolism of activated oxygen in peroxisomes was observed from leaf peroxisomes of pea plants (see review, del Rio et al, 1995). Table 1.5 summarizes the responses caused by these stress factors. 30 Causative agent H2p2-geneiating Mn-SOD Catalase Odier effects Reference enzyme NaCl (salinity) Olycolate oxidase DO effect inhibited H2O2 defusion Corpas, etal., 1987 Cu( heavy metal) increase inhibition H 2O2 increase Palma, et of., 1987 Gofibrate Ayl-CoA oxidase decrease inhibited proxisome proliferation Palma, et 0/., 1991 w Senescence Xanthine oxidase increase inhibited H2Q2 increase Pastori and del Rio, 19 Unte oxidase peroxisome proliferation Table 1.5. The metabolism of activated oxygen in leaf peroxisomes fiom pea plants subjected to different types of stresses. Plant tissue Causation agent Reference Rygrass leaves Isoproturon de Felipe, et al., 1988 Norway Spuce needles Ozone Morte, et ai, 1990 Pea leaf Clofibrate Palma, etal., 1991 Carnation petals Senenscence Droillard and Paulin, 1990 Pea leaves Secenscence Pastori and del Rio, 1994 Soybean Symbiotic nitrogen Nguyen, et al., 1985 root nodule assimilation (Based on del Rio, 1992 with modification) Table 1.6. Peroxisome proliferation in plants. Under stress, a H202-producing enzyme is induced, however, catalase activity is inhibited. The peroxisomal Mn-SOD activity, the NADH-dependent generation of O2" radicals and the lipid peroxidation of peroxisomal membranes behave differently subject to various factors. For example, under salinity conditions, the activity of H2O2- producing enzyme, glycolate oxidase, is induced and catalase is inhibited (Corpas et al., 1987). Incubation of leaves with clofibrate also increases the peroxisomal activity of H202-producing acyl-CoA oxidase and, to a lesser extent, of xanthine oxidase. However, there is a complete loss of catalase activity and a decrease in Mn-SOD. Peroxisomal and mitochondrial populations are induced as the eHect of inhibition of catalase and Mn-SOD (Palma, 1991). During pea leaf senescence, xanthine 32 oxidoreductase activîQr increases, especially the O2" producing xanthine oxidase. The activity of H202-producing Mn-SOD and urate oxidase are also enhanced by senescence. Whereas, catalase activity is severely depressed (Pastori, 1994). The NADH-dependent generation of O2' radicals by peroxisomal membranes and H2O2 concentration in intact peroxisomes increase significantly in senescent leaf peroxisomes. The population of peroxisomes also increases with senescence (Pastori, 1994). A factor common to almost all plant response to the stresses mentioned above is inhibition of catalase activity and an increase in the peroxisomal generation of O2" and/or H2O2 (Table 1.5). As 02" radicals have a short halflife and quickly dismutate into H2O2 and O2 (Halliwell and Gutteridge, 1993), H2O2 finally builds up in the peroxisomes as the stabilized molecules. This appears to indicate that peroxisomes could act as sensitive cellular indicators of plant stress, under toxic situations, releasing H2O2 to the cytosol. Superoxide and H2O2 may act as specific chemical messengers in cellular signal transduction pathways (Figure 1.4) (Saran and Bors, 1989; Schrecket al., 1991; Levine, et al., 1994; Prasad, et al., 1994). Therefore, peroxisomes could have a role in signal transduction processes that lead to specific gene expression. The well-known inducible nature of many peroxisomal enzymes (Tobert, 1981; Huang et al., 1983; Fahimi and Sies, 1987; Van den Bosch et al., 1992; Mannaerts and Van Veldhover, 1993) and the demonstrated proliferation of peroxisomes in plant cells under different stress conditions (Table 1.6) show the role of peroxisomes in plants. 33 STRESSES I HgOg GBERATION I CATALASE INHIBITION 1 H 2 O 2 ACCUMULATION 1 TRANSCRIPTION INDUCTION O F SPECIRC GENES Figure 1.5. A hypothetical scheme of the signal transduction pathway when plant is under a stress situation. 34 1. L1 Plant peroxisomes, purine metabolism and soybean ureide biosynthesis As previous stated, there are four different types of peroxisomes existing in plants, they are root glyoxysomes, leaf peroxisomes, transit peroxisomes and nodule peroxisomes. Each one contains a unique set of enzymes and plays an important role in different tissues during plant development. In this section, I focus on the discussion of the root nodule and its peroxisomes. Previous smdies have been shown that peroxisomes play an essential role in nitrogen assimilation. 1.11.1 Nodule formation and Nitrogen assimilation Nodules are specialized root organs formed in legume plants as result of bacterium-plant symbiotic interaction. Initially, developing nodule is a sink for nitrogen, as nitrogen fixation starts it becomes a source for nitrogen and synthesizes nitrogenous compounds which are subsequently transported to the other parts of the plant. An effective nodule follows normal development and processes nitrogen-fixing activity. Both oxygen (Xue, 1991) and the availability of nitrate or ammonia (McNeil and Lane, 1984; Reynolds, 1990) affect the process of the nitrogen assimilation in nodules. 35 Ammonium N2 N2 fixation Assim nation icterad Ririne Glutam ine Biosynthesis FRPP + Qutamine ■< & pmr^ IMP R astid J INFECTE) CELL7 Purine UNINFECTED CELL Cat aboi ism Hypoxanthlne Xanthine oxidase Xanthine Xanthine oxidase Uric Acid Uricase Allantoic Acid ER Ailanto in V Peroxisome Ureides transport Figure 1.6. Role of peroxisomes in nitrogen assimilation pathway in root nodules of tropical legumes. Peroxisomes are required for the conversion of uric acid to allantoin, which is catalyzed by uricase and takes place in the uninfected cells of root nodules. Gin, glutamine; GS, Glutamine synthetase; PRFF, 5-phosphoribosyl-l- pyrophosphate; IMP, Inosine monophosphate. 36 1.11.2 Nitrogen assimilation and peroxisome proliferation As shown in Figure 1.6, the final products of nitrogen assimilation in tropical legumes are the ureides (allantoin and allantoic acid), which are formed in nodules by oxidative catabolism of de novo synthesized purine nucleotides. The ureides, allantoin and allantoic acid, are the most efficient forms of nitrogen transport compounds in tropical legume plants since they contain a higher molar ratio of nitrogen per carbon atom as compared to amides which is the final product of nitrogen assimilation in temperate legume plants (Schubert, 1986). The tropical legumes such as soybean cowpea and bean, thus are defined as a ureides-transporters due to their primarily transport ureides as the fixed-nitrogen compounds. Three steps are shown to be involved in the ureide biosynthesis in tropical legumes, they are 1) ammonia assimilation, 2) de novo purine biosynthesis, and 3) oxidative purine catabolism. These biochemical processes are carried out in the compartmentalized infected and uninfected cells in nodules. 1.11.2.1 Ammonia assimilation Ammonia assimilation is mediated by glutamine synthetase (GS; EC 6.3.1.2). Glutamine generation is as the result of assimilation of fixed nitrogen. In the infected cells of determinate nodules, the fixed nitrogen is incorporated into the amide position of L-glutamine by the cytosolic GS activity, which is then funneled into the de novo purine biosynthesis pathway after transport into the plastids. The de novo purine synthesis pathway is highly induced in the nodules during nitrogen fixation. 1.11.2.2 de novo purine biosynthesis In ureide-transporting determinate nodules, plastids are the sites for de novo purine biosynthesis where enzymes necessary for catalyzing purine pathway as well as providing carbon to this pathway exist. Glutamine phosphoribosylpyrophosphate 37 amîdotransferase (PRAT) is the first enzyme of the de novo purine biosynthesis pathway, which is the key step for the purine biosynthesis. Purine biosynthesis is induced by glutamine production as nitrogen assimilation and nodule maturation (Kohl, 1988; Kim et al. 1995). Studies of the PRAT promoter region, indicates that the induction of this enzyme is tightly regulated by the availability of glutamine formed by symbiotically reduced nitrogen. Levels of PRAT mRNA in soybean and cowpea nodules are found to increase steadily as the nodule matures (Kim, 1996 ). PRAT gene expression in root elongation zone and root tips is very low, but a high level of expression is detected following the commencement of nitrogen fixation (Kim, 1996). Treatment of uninfected root with L-glutamine induced the PRAT mRNA transcript suggesting that glutamine produced as a result of assimilation of fixed nitrogen is funneled into the de novo purine biosynthesis and controls the expression of this pathway in root nodules. 1.11.2.3 Purine catabolism and ureides biosynthesis The enzymes catalyzing the oxidation of purines are located in the uninfected cells of nodules. Xanthine dehydrogenase (XDH) converts xanthine to uric acid, and greater activity of this enzyme is detected in the cytoplasm of uninfected cells (Datta et al., 1991). The uricase, catalyzing the conversion of uric acid to allantoin is localized in the peroxisomes of uninfected cells (Nguyen et al., 1985; Webb and Newcomb, 1987). Kim (1996) found that uricase expresses in root tips and elongation zone, but the expression level is lower than that of GS in these tissues. Similar to GS, the uricase gene expression is highly induced following the onset of nitrogen fixation (Kim, 1996). However, increased level of uricase gene expression is also detected in 6- and 8-day-old nodules, indicating the developmental control of this gene. The expression of GS, PRAT and uricase genes is higher in fix+ nodules especially in mature nodules than that of the Fix- nodules indicate the role of nitrogen fixation in providing the metabolic substrate for 38 ureide biosynthesis pathway in this organ. His results indicate that the expression of these genes is tightly controlled by the availability of ammonia, glutamine and the nitrogen-fîxation products, respectively. 1.11.3 The compartmentation of metabolite flux between the infected an uninfected cells The key feature of the ureide synthesis in determinate nodules is the compartmentation of metabolite flux between the infected and uninfected cells. The infected cells are responsible for the oxidative catabolite flux between the infected and the uninfected cells. The infected cells that microaerobic carry out nitrogen fixation and assimilation, while the uninfected cells are responsible for the oxidative catabolism of de novo synthesized purines to ureides. In soybean nodules, the uninfected cells occupy 21% of the total volume of the central infected region grown without nitrate (Selker and Newcomb, 1985). The infected cells occupy more space than that of the uninfected cells, however, its number is much lower than that of the uninfected cells. Both cells are spacially organized in a manner that the uninfected tissue generates large surface area to contact with the infected tissue, in such a way, that the fixed nitrogen from the infected cells is more efficiently transported to the uninfected cells (Selker and Newcomb, 1985). Due to the distinct functions of these cells, their subcellular organelles become specialized. In the infected cells, two prominent organelles, mitochondria and plastids, are congregated at the cell periphery, and the numbers of these organelles increase as the infected cells enlarge (Newcomb et al., 1985). Mitochondria in the infected cells have a much larger volume and number per unit cytoplasm than those in the uninfected cells. The plastids have a dense, granular matrix penetrated by a few cistemae and lamellae, their 39 shape are variable and starch granules are usually absent (Newcomb et al., 1985). Small peroxisome-like stmctures are occasionally found in the periphery of the infected cell cytoplasm. In the uninfected cells, on the other hand, the plastids and mitochondria do not congregate along the plasma membrane (Newcomb et al., 1985). The plastids usually contain large starch granules depending on the amount of starch present, and their size varies greatly (Selker and Newcomb, 1985; Newcomb et al., 1985). The differentiation of plastids in nodules are, therefore, cell-specific. Uninfected cells of soybean root nodules are highly vacuolated, and have large peroxisomes as well as a prominent system of tubular endoplasmic reticulum (Newcomb and Tandom, 1981). The enlarged peroxisomes are closely associated with the intercellular free spaces which may enable peroxisomes to take up oxygen more efficiently from the spaces (Newcomb and Tandom, 1981). 1.11.4 Catabolism of Purines The major pathways of purine nucleotide and deoxynucleotide catabolism in animals are diagrammed in Figure 1.7. Other organisms may have some minor differences but all of these pathways leads to uric acid (Voet and Voet, 1990). In humans and other primates, the final product of purine degradation is uric acid, which is excreted in the urine. In all other organisms uric acid is further processed before excretion (Figure 1.8). Mammals oxidize it to their excretory product, allantoin, in a reaction catalyzed by the Cu-containing enzyme urate oxidase which makes up the core of heptatic peroxisomes (Volkl, 1988). A further degradation product, allantoic acid, is excreted by teleost (bony) fish. The excess amino acid nitrogen also can be catabolized into uric acid via purine biosynthesis. 40 6 ? "X x? mb- " 7 ^ , pmia* H<0 HHt Boctoa RaoM-l-P« pho^aoqluB Baow-l-P-»^ Pkxpkqb* Hbon-l-P«^ phoM X/ 1 JL > = ( H « 0ifeasid Figure 1.7. The major pathway of puiine catabolism in eukaiyoüc cells. The various purine nucleotides are all degraded into uric acid via a final step that xanthine is oxidized into uric acid by xanthine oxidase. 41 HN Uric acid 2HzO + Oi Uricase CO2 + H2Q2 A ant cm Allontomase COOH H H Allante! c acid Figure 1.8. The degradation of uric acid to allantoic acid. Uric acid is oxidized by a peroxisomal uricase into allantoin and H2O2 is released as a by-product. Allantoin is further oxidized into allantoic acid by allantoinase in the cytoplasm. 42 1.11.5 Polyamîne catabolism in peroxisomes Polyamines are involved in the regulation of cell growth and differentiation. Peroxisomes contain polyamine oxidase found in animal cells, which convert spermine and N-acetylspermine to spermidine, spermidine and N-acetylspermidine to putrecine (Beard, 1985). Amine oxidation result in H2O2 production which lead to oxidative stress (Gaugas, 1980). Whether polyamine oxidases play an active role in the regulation of cellular polyamine levels is unknown yet (Hayashi et al., 1989). Some yeast like H. polymorpha can use amines as sole nitrogen source, a peroxisomal amine oxidases is induced (Zwart et al., 1980). There is no peroxisomal amine oxidase has been found in plants yet. 1.12 Proposed Research Plant peroxisomes play important and often different roles from their mammalian and yeast counterparts. It has long been known that plant peroxisomes are involved in mobilization of storage oil reserves, synthesis of ureides and salvage of photorespiratory phosphoglycolate (Tolbert, 1981). Recently, plant peroxisomes are discovered to be major sites of formation and detoxification of active oxygen species and have been proposed to play an important role in protecting plants from oxidative stress, which can arise as a result of environmental conditions such as pathogen attack, metal toxicity, or salt stress. The de novo purine biosynthesis pathway is induced with the onset of N2- fixation, converting symbiotically reduced nitrogen to ureides. Peroxisome proliferation in root nodules apparently occurs in response to de novo synthesized purines during N2- fixation as oxidation of purines requires this organelle. Early studies in this lab an antisence cDNA of nodulin-35 encoding a subunit of soybean uricase was introduced into 43 Vigna plants (Lee et ai., 1993). The transgenic vigna plants showed a reduction in the activity of uricase by antisense expression of nodulin-35 and its effect on nitrogen assimilation in root nodules. Uricase catalyzing uric acid into allantoin is processed in peroxisomes of the non-infected cells within the nodules. Up until now, little information is available on peroxisome biogenesis and proliferation in plants. We would like to generate a molecular approach to smdy plant peroxisome biogenesis, as well as determine its new functions. As stated, in tropical legume root nodules, peroxisomes proliferation is highly induced as this organelle is used for nitrogen assimilation (Newcomb and Tandom, 1981; Nguyen, et al, 1985). Nitrogen source, like purines (xanthine or uric acid), could be an inducer of peroxisome proliferation in the uninfected cells of soybean nodules. The primary objective of this research is to determine the ability of xanthine or uric acid to induce peroxisome proliferation when they are used as the sole nitrogen sources. Our long-term goal is the isolate plant peroxisome biogenesis related genes. To achieve this goal, I propose that peroxisome proliferation by a nitrogen source like purines shares a similar or an identical induction pathway with those of fatty acid inducers. To isolate plant genes involved in peroxisome biogenesis or nitrogen metabolism, yeast 5. cerevisiae mutants need to be generated and isolated. To isolate plant genes involved in peroxisome assembly or involved in nitrogen metabolism or any other peroxisomal proteins, my approach would be to functionally complement of yeast mutants with a soybean nodule cDNA library. Through this study, several genes were isolated and function of the genes were determined. The last prospective of this study was to determine and characterize the proteins encoded by the cDNAs isolated from the yeast mutant with the functional complementations. 44 CHAPTER2 ISOLATION OF PEROXISOME BIOGENESIS MUTANTS AND CLONING OF SOYBEAN GENES REQUIRED FOR PEROXISOME BIOGENESIS 2.1 Abstract The goal of this study was to isolate soybean genes involved in peroxisome function and biogenesis. Although several peroxisome biogenesis mutants of S. cerevisiae had been isolated by Veenhuis and colaborators, they were not available at the time we started this project. We attempted to isolate yeast peroxisome biogenesis mutants that are unable to utilizing uric acid as the source of nitrogen. A positive selecdon scheme developed by Van Der Leij (1992) and colaborators was employed in this study. EMS mutagenized cells were screened on a positive selective medium based on the lethality of H2O2 accumulation in wild-type cells with application of a catalase inhibitor, 3-amino- 1,2,4-triazol (3-AT). There were 34 stains isolated according to the phenotype of variously non-utilizing oleic acid as the sole carbon source. To avoid fatty acid P- oxidation deficient mutation, uric acid was used as the sole nitrogen source since urate oxidation is also carried out inside peroxisomes. Among them, four mutants were isolated which failed to utilize oleic acid as the sole carbon source and uric acid as the sole nitrogen source, respectively. They fell into three different complementation groups. Their peroxisomal catalase activities were lower than that of the wild-type cells. One 45 mutant showed a restrict phenotype of non-utilizing uric acid or oleic acid, and had a single mutation, which is defined as a peroxisome biogenesis mutant Spbl. Ftmctional complementation with a soybean nodule cDNA library, resulted three cDNA firagment isolations. They encode a putative transcription factor (G-box binding protein), a cytochrome P450 and a diamine oxidases, respectively. 46 2.2 Introduction Peroxisomal organelles are widely distributed in eukaryotic organisms from fungi to animals and plants. Peroxisomes are essential to cellular functions as described previously. A human genetic disease Zellweger syndrome is the result of lack of functional peroxisomes, causing death of new-born infants. In tropical legumes, symbiotically fixed nitrogen is assimilated via the peroxisomal urate oxidation. Therefore, peroxisome biogenesis and maintenance are of fundamental importance in all eukaryotic cells. The establishment of a genetic system in yeast has provided a ideal tool to study peroxisome biogenesis in higher organisms. A significant progress has been made recently in understanding of peroxisome proliferation in yeast and human. But the study of peroxisome biogenesis in plants is still at the beginning. The objective of this study was to isolate and characterize peroxisome biogenesis mutants of Saccharomyces cerevisiae in order to build up a tool for the isolation plant genes involved in peroxisomal biogenesis and nitrogen metabolism through a functional complementation, and to finally reach the goal of smdying the plant peroxisome biogenesis. Oleic acid has been identified as an inducer of peroxisomal proliferation in 5. cerevisiae, when it is used as a sole carbon source. There are 18 complementation groups of Peroxisomal assembly (PAS) mutants and Peroxisome-deficient (PER) mutants that have been generated based on the phenotype of oleic acid non-utilizing property (Erdmann et al., 1989; Van Der Leij, 1992; Zhang, 1993). Several mutants have been isolated and complemented (Hohfeld et al., 1991; Marzioch, et al., 1994) and corresponding genes isolated. The desired mutant for our study should be defective in utilizing uric acid as the sole nitrogen source as result of the peroxisome biogenesis deficiency. It is known that purine catabolism results in the production of uric acid which is subsequently oxidized 47 into allantoin in plants, yeast and some animals. Thus, peroxisomes play an essential role in purine metabolism, as well as in nitrogen assimilation in soybean. 5. cerevisiae peroxisome assembly mutants were first identified by screening for cells unable to use oleic acid as the sole carbon source (Erdmann et al., 1989; Cregg et al., 1990). Different positive selections have been developed based on the toxicity of H2O2 during the ^-oxidation of fatty acids. Lazarow and coworkers used a catalase- deficient strain which failed to grow on oleic acid due to the toxicity of accumulated H2O2. Peroxisome defective mutants were able to survive on oleic acid in the presence of an alternative carbon source, such as glycerol and maltose (Fujiki, 1989; Zhang et al., 1993a). Several peroxisome clustering mutants were generated. Strategy for isolation of peroxisome biogenesis mutants In S. cerevisiae, peroxisome proliferation is induced when fatty acid is provided as the sole C-source (Veenhuis et al., 1987). Fatty acid is degraded by p-oxidation pathway carried out in the peroxisomes. In fatty acid oxidation, hydrogen peroxide is generated as a by product, which is toxic to cell proteins, lipids, and DNAs. To scavenge H2O2, peroxisomal catalase is induced during peroxisome proliferation. However, H2O2 is accumulated when catalase is inhibited by its inhibitor, 3-AT, and wild-type cells die. If the strain lacks peroxisomes, and the second C-source is present in the medium then the mutants which either lacks of peroxisomes or lacks of P-oxidation can survive (Van Der Leij, 1992, Zhang et al., 1993), see Figure 2.1. 48 CELL DEATH Fatty acyl CoA (n) TOXIC 3-AT ► Fatty acyl CoA catalase T (n-2) catalase A 3-AT H2O +1/2 Og Cytosol Peroxisome Figure 2.1. Peroxisomal hydrogen peroxide metabolism in yeast grown on a fatty acid (Van Der Leij, et al., 1992). 49 Fatty acid ^oxidatioa Urate oxidation Oleic acid Uric acid ^ 0 2 Acyl-CoA ^ 0 2 Uricase oxidase LH2O2 L H2O2 Acyl-CoA catalase A Allantoin catalase A H2O H2O Peroxisome Peroxisome y Oleic acid as sole carbon source Uric acid as sole nitrogen source Figure 2.2. The schemes of fatty acid p-oxidation and urate oxidation pathways in peroxisomes, respectively. Fatty acid ^-oxidation and uric acid oxidation both occur in peroxisomes. If a mutation occurs on the genes involved in peroxisome biogenesis, a defective peroxisome can cause cell deficiency of utilizing oleic acid as the sole carbon source and uric acid as the sole nitrogen source. 50 Xanthine oxidase and uricase, the enzymes of purine metabolism pathway shown in Figure 1.6., have been identified in peroxisomes in yeast, plant and animals. These enzymes can cause H2O2 and O2 ' generation inside peroxisomes (del Rio, 1992). In soybean nodules, nitrogen assimilation induces purine biosynthesis and catabolism. Uricase and peroxisomes in the uninfected cells are induced (Lee, et al., 1993; Kim, 1996). E*urines (including xanthine, hypoxanthine, and uric acid) are suggested to be able to induce nodule peroxisome proliferation. Therefore, when xanthine or uric acid serves as the sole nitrogen source, xanthine oxidase and uricase must be induced as peroxisome proliferation. Since both ^-oxidation and urate-oxidation share the common subcellular compartment, peroxisomes, if peroxisome biogenesis is defective, both pathways could become deficient and the mutant could fail to use oleic acid as the sole carbon source or uric acid as the sole nitrogen source, respectively. This is the strategy we used for isolation of peroxisome biogenesis mutants in this study (Figure 2-2). In this study, we found that uric acid is able to induce uricase in S. cerevisiae when it is provided as a sole nitrogen source. We have isolated about 34 oleic acid non utilizer of S. cerevisiae by using a positive selection procedure of pas mutants developed by Van Der Leij et al. (1992). Among them there were six strains unable to use oleic acid or uric acid, respectively. Within the six strains, one strain was selected and defined as a peroxisome biogenesis mutant (Spbl) which was used to isolate homologous plant genes. Functional complementation with yeast peroxisome biogenesis mutants is a potential way to isolate plant genes related to the peroxisome biogenesis. A soybean nodule cDNA library was constructed in LAMDA MAXI expression vector pYEUra3 that allows expression of soybean genes in yeast cells. This library was used to complement the yeast mutants which fail to use uric acid as the sole nitrogen source. Positive clones that could rescue the mutant were isolated and used for cloning of the 51 corresponding soybean genes. DNA sequencing and expression of these genes in plants were determined by the standard molecular genetic techniques. 2.3 Material and Methods 2.3.1 Strains, media and culture conditions Wildtype yeast strains used in this study were S. cerevisiae DBY1034 (MAT a, lvs2-80l. his4-539. ura3-52) (Seger and Botsein, 1987), and a SS328 (MATa, ade2— 101, his3A200, lys2-801, uraS-52). Minimal media contained 0.67 yeast nitrogen base without amino acids (YNB) (Difco laboratories, Detroit, MI) and amino acid lys (4Gug/ml) his (40ug/ml), ura (20ug/ml) as needed, supplemented with 2% glucose (SG), 2% dextrose (SD), 2% (wt/vol) glycerol plus 0.05% yeast extract (SGY), or 0.1% (vol/vol) oleic acid, 0.5% (wt/vol) Tween-40 and 0.1% yeast extract (SOY). Positive selective medium consisted of 0.67% YNB. 0.5% maltose, 0.06% (wt/vol) lauric acid, 0.2% Tween-40, and 0.1% yeast extract, 25mM 3-AT, and amino acids as required (20pM/ml) as described by Van Der Leij (1992). Rich media are YPD and YEPD as described by Sherman (1991). The xanthine or uric acid containing medium contained 0.17% nitrogen base without amino acid and ammonium sulfate (Difco, Detroit, MI), ImM xanthine (the N source) and 2% of dextrose or 2 mM uric acid (UGY), 2% (wt/vol) galactose used for selecting pYEUraS containing colonies, supplement with amino acids lys 50p,g/ml, his 40p,g/ml, ade 20pg/ml, leu 40p,g/ml, arg 50|i.g/ml, which was modified according to recipe of uracil synthetic medium described in Clontech. 52 2.3.2 Mutagenesis and selection of mutants: The DBY 1304 cell grown on 2% SD were treated with 3% ethyl methyl sulfonate (EMS), and cells with a survival rate between 30%-50% were screened according to the method described by Lawrence (1991). The cultures were allowed to recover from EMS treatment on 2% SD medium for about three generations and subsequently plated on a positive selective plates at a concentration of 10^ cell/per petri dish. After 5 days incubation at 30° C, colonies survive on the positive selection medium were picked double plated onto SOY and SGY plates. Mutants grown on SGY but not on SOY were transferred on uric acid-containing medium with glycerol as C-source. Cells failed to grow on uric acid-containing medium were putative peroxisome biogenesis mutants. 2.3.3 Genetic analysis Mutants (MATa) were backcrossed with wild-type strain SS328 (MAT a). The mating and sporulation procedure were modified according to the described methods (Sherman, 1991). Each haploid mutant strain was spread on a YPD plate at a density sufficient to form a lawn (up to 8 strains per plate), then mixed with about the same amount of wild- type strain SS328, respectively. After overnight growth at 30° C, a loop of cells were picked up and streaked on a histidine drop-out mineral selective medium (SD, 0.67% YNB, 2% dextrose agar medium). An efficient sporulation method was used in this study, which is described as follows. The diploid strain received from the backcross mating, was incubated on a 2x YPD [2% (w/v) Bacto-yeast extract, 4% (w/v) Bacto- peptone, 4% (w/v) dextrose, 2% (w/v) agar] for overnight. A full loop of cells was transferred into a SO ml flask containing 5ml sporulation medium (0.5% sodium acetate, 1% potassium chloride, and 1% glucose, amino acids were required as needed at 30mg/ml). Then the cell culture was incubated at 30° C in a water bath for 2-3 days. Spores were examined under microscope. 53 2.3.3.1 Dissection of asci (tetrad dissection) Spomlated cells were collected by centrifugation. A small amount of cells was resuspended in lyticase (Sigma) containing cell wall digestion solution (lyticase 2000 units/ml, 10% sorbitol-50mM Tris-Q, pH 8.0,4M KCl to a final concentration of ImM, and lOOmM spermidine, pH 7.8 to a final concentration of 5mM). Cells were digested for 10-30 min. at 4° C on ice. After digestion, a 0.2ml aliquot of suspension was streamed on a YPD agar plate. Then spores were dissected with a micromanipulator. About 12 spores were dissected for each strain. After 1-2 days growth on YPD, colonies were transferred by a replica method onto SOY selective plate, his-dropout SD plate, adenine-dropout SD plate to determine the allele segregation. 2.3.3.2 Mating type assay The mating type of spores originated cells were determined by matting type determination assay as described by (1991). BBMB plates contained YEPD with addition of O.IM citrate pH4.5,0.03% (w/v) methylene blue after autoclave. Spread 10^ to 5x103 cells of a pheromone tester strain (YTC 39 RC634, MAT a sstl; YTC 40 RBH-8-2C, MAT a sst2) on the surface of a BBMB plate. 2.3.3.3 Reverse mutation determination Mutant grown on SGY plate washed into a tube with 0.5ml distilled water to about 10? cells/ml, dilute cell suspension into 10?, lO^, 10^ and 10^ cells/ml, respectively, and then an equal aliquot was double plated onto SOY and YPD plates. Plates were incubated at 30° C, 1 day for YPD plates, 4-5 days for SOY plates. If there is any colonies appearing on the SOY plates, they were considered as the reverse recovery mutation. The total number of cells was determined on YPD plates. The recovery efficiency was then determined. 54 2.3.4 Cell-fiæe extraction and enzyme assays 23.4.1 Determination of catalase activity in wildtype and mutant ceils SD overnight cell cultures were washed twice by centrifugation with ice-cold water and then transferred into SOY liquid induction medium. After 16 hr induction, cells were collected by centrifugation and then washed twice with ice cold water and once with ice cold buffer ( EMS-sorbitol buffer). Finally, cells were resuspended in a 400jti of the same buffer, and added a same volume of 0.5-mm-diameter acid-washed glass beads. The mixture was vigorously mixed for 30 sec. by using a bench-top vortexer and held on ice for Imin. Each sample was repeated for four cycles. Unbroken cells were removed by low speed centrifugation at 3,GG0rpm. Extract which remained trapped in the glass beads was recovered by washing the beads with 300p.l of cold buffer and adding this wash to the extract. To remove the large cell debris, extracts were centrifuged at SOOOrpm for 5 min. The supernatant was then transferred and centrifuged at 20,000g for 25 min to separate peroxisomal fraction and soluble fraction according to Liu et al. (1994). The supernatant was transferred to a frresh microcentrifuge tube and the pellet was resuspended. 2.3.4.2 Biochemical analysis of mutant cells The mutant and wild type cells were induced by oleic acid as the sole carbon source and uric acid as the sole nitrogen source, cells grown in SD were used as controls. Cell frree extracts were prepared by following above procedure. After removal of the unbroken cell and cell debris, the cell-free extracts were assayed for enzyme activities. Enzyme activities were measured with a spectrophotometer at room temperature. Catalase activity was determined using H2O2 as a substrate according to the method described by Luck et al, (1963). Uricase activity was determined using uric acid as 55 substrate, and measuring the decrease in absorption at 293 nm according to the method described previously (Suzuki and Verma, 1991). Peroxidase activities were determined at 450nm using o-phenylenediamine as the substrate (Tajima et al., 1985). Protein concentrations were determined using Bio-Rad reagent (Bradford, 1976). 2.3.5 Functional complementation 2.3.5.1 Soybean nodule cDNA library 3-week old soybean nodule mRNA were chosen to construct a yeast cDNA expression library. The library is constructed in a LAMDA MAXI shuttle vector (Clontech) which contains pYEUra3 sequence. Soybean cDNAs were inserted unidirectionally under the control of the GALl-10 promoter. The pYEUra3 sequence containing soybean cDNA inserts can be in vivo excised from LAMDA MAXI and converted into plasmid by a helper phage. A host strain was E. coli XLl-Blue. 2.3.5.2 Yeast cell transformation and positive clone selection Yeast electrocompetent cell preparation and electroporation procedure described by Becker and Guarente (1991) were followed with some modifications. Yeast cells grown on YPD liquid culture for about 18-20 hrs were collected and washed and finally resuspended in ice cold IM sorbitol at a concentration of 10^ i cells/ml. An aliquot (400^il of yeast cell) was transferred to a cold 0.2-cm sterile electroporation cuvette, adding less than lOOng isolated plasmid DNA and incubated for 5 min. Electroporation was carried with a pulse at 2.0 kV, 25pP, 2000, then 1 ml cold sorbitol was immediately added to the cuvette. Aliquots of the transformed suspension was plated by spreading on URA drop-out SO medium containing IM sorbitol, the plasmid-containing colonies were selected for uracil prototropy on the plates. After three days growth, these uracil 56 prototropic cells were transferred onto uric acid containing URA drop-out selective medium by replica technique. 2.3.5.3 Yeast plasmid extraction from yeast cells Positive strains were picked into a 3 ml minimal URA drop-out medium (SG) for overnight shaking. Cells were harvested and then resuspended in Buffer I (SOmM Tris- Cl, pH 8.0; lOmM EDTA in 0.6M sorbitol solution) at volume of 200ml. Add Img/ml lyticase into the cell suspension and let sit on ice for up to 4 hr. Then add the same volume of buffer H (0.2 NaOH, 10% SDS) (200ml) and incubate for 5 min., and then neutralized by adding the same volume of buffer El (3M NaAc, pH 5.2) for 5 min.. After centrifugation for 5 min., the supernatant was transferred into a tube. An equal volume of isopropanol alcohol was added to precipate the DNAs. Then DNAs were collected by centrifugation for 10 min. and washed by 70% alcohol. Finally, DNA was resuspended in TE buffer (10 mM Tris-Cl, pH 8.0 and ImM EDTA). Plasmid was recovered by transforming in to E. coli cells by heat shock method. The plasmids of the positive yeast transformants were isolated and used to transform into E. coli XL-Blue in order to receive a good quality and high amount of plasmid. The plasmid was then used to retransform yeast to verify complementation and the insert was continued by restriction enzyme analysis and sequencing. 57 2.4 Results The positive selection system described by Van Der Leij (1992) based on the potential lethality by H2O2 generated during the peroxisomal ^-oxidation of fatty acids (Figure 2.1) was used. H2O2 is generally rapidly degraded by catalase, however in this method, presence of a catalase inhibitor (3-amino-1,2,4-triazol, 3AT) H2O2 decomposition is prevented. As a result, H2O2 accumulation occurs which is lethal for the cell. Eventually, continually H2O2 accumulation will cause cell death. Only those cells that do not have functional ^-oxidation or a defective in peroxisome assembly are able to survive on the positive selective medium in the presence of a secondary carbon source. Laurie acid and maltose are chosen to be used as the carbon source in the selective medium and 3-AT is supplied as the catalase inhibitor. Laurie acid is the most efficient substrate to induce ^-oxidation and catalase activity (Van Der Leij, 1992). If a single mutation occurs in the genes involved in peroxisome biogenesis, cells will be defective to utilize uric acid as the sole nitrogen source. Therefore, effect on utilizing uric acid indicates the mutation is not due to the defective ^-oxidation system. A schematic outline of peroxisome biogenetic mutant selection is presenting in Figure 2.3. 58 Wild-type strain Peroxisome biogenesis I mutant selection EMS mutagenesis double plating 1 A Positive selection SUGY SGY I Positive strains SUGY-/SGY+ double plating * ole-/uri- SOY ^ SGY 6 strains I SOY-/SGY+ Genetic analysis I i 34 Strains Biochemical analysis Figure 2.3. The outline of yeast S. cerevisiae mutagenesis and peroxisome biogenesis mutant isolation and identification. 59 2.4.1 The peroxisome biogenesis mutant failed to grow on the uric acid as sole nitrogen source and oleic acid as carbon source Cells recovered from treatment with EMS were plated on the positive selection medium and incubated for 4-5 days at 28° C. The colonies that failed to grow on the oleic acid containing medium but grew on the glycerol medium were putative pas mutants or ^-oxidation catabolic mutants. From the colonies that appeared on the plates, 935 colonies were tested for their inability to grow on the plates containing minimal oleic acid medium (SOY) and on the plates containing mineral glycerol medium (SGY). Among them, a total of 34 oleate-negative and glycerol-positive mutants (oleic acid non-utilizer, ole-) were obtained and subsequently tested for their abili^ to utilize uric acid as the sole nitrogen source. To avoid any mutation in the P-oxidation pathway or fatty acid metabolic enzymes, uric acid was used as sole nitrogen source to determine the ole- mutants survival ability on uric acid. Uricase is known to be a typical peroxisomal matrix protein (Nguyen, et al., 1995; Voet and Voet, 1990). Urate oxidation is carried out inside peroxisomes where uric acid is converted into allantoin which can be utilized as the nitrogen source for cell growth. Peroxisome deficiency will abolish the urate oxidation due to the possible reasons of the ill-assembled peroxisomes, enzyme mislocalization or non-peroxisome proliferation. Using uric acid as a sole nitrogen source as selective medium and ammonium sulfate as the positive control medium, the 34 oleic acid non utilizing strains were double plated onto uric acid-containg selective plates and SGY plates. After 4-5 days growth, most of them showed equal growth well on uric acid as the wild-type strain, only six strains showed different levels of deficiency of utilizing uric acid. The selection scheme is summerized in Table 2.1. 60 Selection steps after EMS Mutagenesis Number of mutants recovered Positive selection on lauric acid, 935 maltose, and 3-AT Selection for oleic acid~and 34 (3.6%) glycerol+ phenotype Selection for uric acid" and NH4+ (ole*/uri") 6 (0.64%) Table 2.1 Yeast Mutant isolation scheme. 61 f wt ole- \ \Spb1 Spb2 7 Figure 2.4. The growth ability on uric acid-containing medium of the peroxisome biogenesis mutants generated in this study. A. Dextrose/Ammonium sulfate minimal medium used as control. B. Dextrose/Uric acid minimal medium. The enenly distributed white spots were the crystals of uric acid in the medium, w t wild-type strain, which is grown normally on uric acid; ole-, oleic acid deficient mutant strain which grows normally as the wild-type; Spbl, this strain is completely defective for utilization of uric acid; Spb2, this strain show poor growth on uric aicd with tinny colonies. 62 2.4.2 Spbl contains a lecesive mutation at a single allele In order to test the dominance and loci number of the mutated genes, yeast genetic analysis was performed. The ole-Zuri- mutants were crossed with a wild-type strain and resulting diploids were able to grow on oleic acid and uric acid, respectively, indicating that the mutations studied are recessive. The diploids were spomlated and 10-12 randomly selected tetrads were dissected. To determine the number of allele of the mutation, tetrad analysis of these diploids were carried out. Four of them showed a normal 2:2 segregation for the oleic acid-minus phenotype showing that these properties were caused by monogenetic defects in the nuclear genomes. Two of them had abnormal segregation, which may have more than one mutation occurring in different genes. The four mutants were further crossed with each other in all combinations. They fall into 3 different complementation groups. Strain Spbl and Spb2 showed no complementation with each other and appeared different growth ability on uric acid. Spbl stain shows no growth on uric acid, however, Spb2 shows poor growth on it and forms tiny colonies (Figure 2.4). Other two strains SpbB and Spb4, show partial growth on uric acid and no growth, respectively (data not shown here). Due to the phenotype of Spbl and its low reverse mutation efficiency, Spbl, therefore, was chosen to isolate plant genes via a functional complementation. 63 Spbl Spb2 Spb3 Spb4 Strains Figure 2.5. Catalase activities of yeast strains in cell-free extracts. The catalase activities of peroxisomal fractions and cytosol fractions of Spb mutants, ole- non-utilizers and a wild-type strain. The Black bars represent catalse activities of the peroxisomal fractions, and the bars with shade represent catalase activities in the cytosol fraction. * The enzyme activity is represented by the changes in absorbance ( A240) of H2O2 degradation per mg protein per minute. 64 % Figure 2.6. Enzyme activities of Spbl mutant cells under different growth conditions. Cell-free extracts were prepared from S. cerevisiae wild-type and Spbl mutant cells after 16-18 hours induction in oleate as the sole carbon source, urate as the sole nitrogen source, and galactose/ammonium as control, respectively. The enzyme activity comparisons are represented in; (A) Catalase activity; (B) Uricase activity; (C) Peroxidase activity. 65 2.4.3 Biochemical analysis of mutants by fractionation of peroxisomal marker enzymes Oleic acid is able to induce peroxisome proliferation as well as catalase activity. Previous studies have shown that only peroxisomal catalase A is oleic acid inducible, whereas catalase T is not (Van Der Leij et al., 1990). Peroxisome deficiency could cause catalase activity reduction in peroxisome or catalase mislocalization in cytosol. To determine the ability of induction by oleic acid, the catalase activity was determined. Catalase activity measurements in cell-free extract of four independently isolated Spb strains revealed a 1.5- to three- fold reduction in peroxisomal fraction in comparison to that of wild type cells (Figure 2.5). Cytosolic catalase activity level of all these mutants were near to that of the wild type. Thus, the four mutants that have an abnormal fraction pattern for peroxisomal enzymes, display different biochemical phenotypes. Mutants contain low catalase activities in peroxisomes, are the cellular distribution of this enzyme probably do not reflect an import deficiency but more likely is caused by lowered amount of peroxisomal catalase (catalase A), and unaltered amount of cytosolic catalase (catalase T). A question is raised based on the catalase enzyme assay, that whether the mutation with such phenotype is due to the catalase deficiency. To avoid catalase deficiency mutation a cell free enzyme assay was carried out in Spbl. Oleic acid is known to be used as a peroxisome proliferator of S. cerevisiae . In this study, it was used to determine the deficiency of the mutant Spbl on the induction of peroxisomal enzyme activity, as well as to determine the induction ability of the peroxisome proliferation by uric acid. The ability of induction by oleic acid and uric acid were tested in both wild-type and Spbl strains. The medium contained dextrose and ammonium sulfate (GS) was used as control. An early log phase (about 12 hr) of YPD cultured cells were prepared before being transferred to mineral media containing either dextrose/, oleic acid/ or galactose/uric acid. After 16-18h induction, these cell were spun down and cell-firee extract were made. Catalase activities were measured and revealed 66 down and cell-firee extract were made. Catalase activities were measured and revealed that the catalase activity of wild-type cell was significantly induced by oleic acid, but showed very small induction by uric acid (Figure 2.6). In addition, peroxidase was induced by oleic acid but in a much amount. Uric acid could significantly induce uricase activity in both wild type and mutant cells. However, in mutant cells, the induction level of Spbl by oleate and urate were dramatically low (Figure 2.6). The mutant Spbl, therefore, has less catalase, uricase, and peroxidase activity than those of the wild type cell. However, these enzymes activities were shown to be induced by comparing with the control. Therefore, it is suggested that peroxisomal protein expression might be impaired in the mutant cells due to proliferation deficiency. Comparison among wild type, Spbl and Spb2 are shown in Table 2.2. Yeast strain wt Spbl Spb2 Cytosolic catalase T + + + Peroxisome catalase A + +/- +/- Growth on glycerol + + + Growth on oleic acid + - - Growth on uric acid + - tinny colonies Table 2.2. The features of the peroxisome biogenesis mutants Spbl and Spb2. 67 2.4.5 Plant cDNA isolation by functional complementation The S. cerevisiae Spbl mutant was used for functional complementation with a soybean nodule cDNA library constructed in a yeast expression system. The transformation efficiency of this yeast strain by using was 10* -10^ cu/^g of DNA. The first selection was carried out onto the uracil drop-out SD medium amended with IM sorbital for selection of positive transformants, and then the transformants were transferred onto uric acid-containing medium or oleic acid-containing medium by replica plating technique. The colonies were able to grow on these medium were selected and further analyzed. 2.4.5.1 Selection of transformants on the uric acid-containing medium There were two truncated cDNA firagments isolated firom colonies grown on uric acid-containing selective medium. These two cDNA showed poorly recomplementation with Spbl mutant on uric acid-containing medium. Both cDNA fragment were sequenced. One is about SOObp long, and its deduced amino acid sequence showed to be homologous with cytochrome P450s. The other is about 1.2kb long, and its deduced amino acid sequence showed a homology with a copper-containing amine oxidase (a diamine oxidase). Both polypeptides contain a SKL peroxisome targeting signal near or at their C-tenninal. Therefore, they were considered as peroxisomal proteins. According to the amino acid sequence, both polypeptides contains the essential active domains of each protein. These two cDNAs could not rescue the mutant growing on the oleic acid- containing mediiun. 2.4.4.2 Selection of transformants on the oleic acid-containing mediiun One cDNA was isolated firom cells grown on oleic acid-containing selective medium. It contains a basic Helix-Loop-Helix (bHLH) motif and appears to be a 68 transcriptional activation factor which has a homology with a common bean phaseolin G- box binding protein, maize myc-Iike regulatory R and rice transcriptional activator R. The bHLH motif is responsible for the DNA binding activity of several transcriptional activators (see chapter 3 for more details). 2.5 Discussion This study described a procedure for generating and isolating peroxisomal mutants following a positive selection scheme. Using this procedure we have isolated 34 ole- mutants showing disturbed in peroxisome functions or oleate utilization pathway, of which 6 show double mutation phenotypes, i.e. the deficiency in utilization of oleic acid as well as uric acid. To avoid the double mutation, the genetics of this mutation was studied. Four of these six mutants demonstrated single loci mutation and the allele segregation demonstrated a ratio of 2:2. Within these 4 strains, Spbl and Spb2 fell into the same complementation group. However, they showed different abilities of utilizing uric acid (Figure 2.4), Spbl shows no growth on uric acid, and Spb2 shows poor utilization of uric acid and form tinny colonies. However, one ole- strain was able to grow on uric acid equal to wild-type strain. Spbl is the only strain that showed the most restriction in utilizing oleic acid as the sole carbon source and uric acid as the sole nitrogen source, and has very low firequency of reverse mutation. This mutant, therefore, became the most desirable strain for further studies. 69 Figure 2.7. The growth ability of peroxisome biogenesis mutants pebl, peb2, peb3 and peb4 on the uric acid as the sole nitrogen source. 1, pebl strain shows con^letely defective of utilization of uric acid; 2, peb 2 strains show relatively poor growth; 3, peb3 strain shows shows even poor gowth that peb2; 4, peb4 strain shows no growth on uric acid; 5, pebS growth normally on uric acid and behaves as a wild-type. Their ability of utilize uric aicd as the sole nitrogen source are ranked like: peb5 > peb3 > peb2>peb4 > pebl. 70 Any defect in peroxisome assembly, biogenesis and proliferation would possibly cause a phenotype of defective utilization of oleic acid and uric acid. For example, the peroxisome cluster mutants (pebs) (Zhang et al., 1993) generated by Lazarow’s group were determined in this study to test the ability of utilizing uric acid as the sole nitrogen source, their phenotypes on uric acid utilization were observed (Figure 2.6). These peb mutants (pebl, peb2, peb3 and peb4) demonstrated various levels of utilization of uric acid as the sole nitrogen source. Pebl, which showed no growth on the uric acid- containing medium (Figure 2.7), contains normal catalase activity, but peroxisomes are clustered (Zhang et al., 1993). Gene cloning study has shown that pebl cDNA encodes a protein that has homology with Pas7p which functions as a putative PTS2 receptor for importing peroxisomal protein with PTS2 (Zhang and Lazarow, 1995). Peb2 and Peb4 mutants showed poor growth on uric acid (Figure 2.7), which lack peroxisomes and hence peroxisomal enzymes may be mislocalized in cytosol (Zhang et al., 1993). Peb3, demonstrated fair growth on uric acid (Figure 2.7), this mutant contains less number of peroxisomes under oleic acid induction (Zhang et al., 1993), which rendered slower rate of growth than the wild-type. However, PebS showed normal growth under uric acid (Figure 2.7), in which peroxisomes are normal but clustered (Zhang et al., 1993). This mutation may not effect the ability of cells to utilize uric acid. Thus, peroxisome deficiency mutant do effect the utilization of uric acid as the sole nitrogen source. Uric acid-containing medium can be used as a selective medium to identify peroxisome biogenesis mutation which also could related nitrogen metabolism. Due to inability to observe the morphology of peroxisomes of Spb mutant cells, biochemical method was used to characterize mutants. Data reveal that catalase and uricase activities were highly induced in wildtype cells tmder oleic acid induction (Figure 2.6). However, Spbl shows no significantly induction on catalase and uricase activities when cells were under oleic acid induction. In the peroxisomal firaction, that catalase 71 activities are low in both Spbl and Spb2 (Figure 2.5) indicates a mutation may occur in a gene involved in peroxisome biogenesis that causes an effection on the peroxisome proliferation. Three soybean cDNA fragments were isolated via functional complementation with a putative peroxisome biogenesis mutant of S. cerevisiae (Spbl). A cDNA encoding a putative G-box binding protein was isolated, which can rescue the mutant growth on oleic acid containing medium and as well as uric acid containing medium. This complementation is likely happened on the gene level. Two other cDNAs encoding a P450 and a diamine oxidase were isolated which involve in the alternate urate- degrading enzyme system and amine oxidation. Their complementation are likely happened on the enzyme functional by-pass'. The mutant Spbl generated from this study likely contains a mutation in a transcriptional activator which regulate peroxisome biogenesis and peroxisomal enzyme expressions. In this mutant, the genes encode proteins in peroxisome biogenesis, proliferation and peroxisomal enzymes became non-inducible by oleic acid or uric acid. As a result, peroxisomes are not be able to proliferated or assembled. The substrates required functional peroxisome become non-utilizable. Based on these results, a similar model of peroxisome proliferation and regulation in plants was realized as established in rodents and yeast peroxisome studies (Figure 2.8). Therefore, the deficiency of peroxisome proliferation activated receptor (maybe a transcriptional activator in this yeast mutant) cause Spbl to fail to utilize oleic acid as the sole carbon source and uric acid as the nitrogen source, respectively. 72 Peroxisome Periiiferator Fatty acid j j Purines I Xanoblotic compounds | Hypoxanthine (Oleic acid) I I Xanthine Uric acid Peroxisome proliferator activated receptor (PAR*) I i I / X i Peroxisome biogenesis genes and enzyme gene expression I I jp^tty acid oxidation genes ! ]' ' I Urate oxidation genes iPAS I Igenes i 1 ) Uricase I _____ 1 2) Peroxidase (P450) 3) Amine oxidase 2.8. The hypothetical model of peroxisomal protein gene regulations. The oleic acid and uric acid non-utilizer could be due to the mutation in the peroxisome proliferator activated receptor. A mutation in a transcription factor which involves in regulation of peroxisome biogenesis and peroxisome enzyme expression will cause a nonfunctional peroxisome as well as deficiency in utilizing its substrates. 73 CHAPTERS A PUTATIVE G-BOX BINDING PROTEIN WITH BASIC HELIX-LOOP-HELIX APPEARS TO BE INVOLVED IN REGULATION OF PEROXISOME PROLIFERATION IN SOYBEAN 3.1 Abstract Expression of peroxisomal proteins involved in peroxisome biogenesis are under developmental and environmental controls primarily at the level of transcription. To understand plant peroxisome biogenesis and proliferation, a yeast peroxisome biogenesis mutant was used to isolate a soybean cDNA by functional complementation. DNA sequence analysis showed that a cDNA (spbl) encodes a DNA binding protein which contains a basic region/helix-loop-helix (bHLH) domain. The deduced amino acid of SPB 1 shows some homologies with other plant bHLH containing transcription factors, e.g. phaseolin G-box binding protein, myc-like-R factor and rice transcription activator Ra. This bHLH containing putative G-box (CACGTG) binding protein contains 150 amino acid residues and has a molecular weight of 16.4 kDa. Sequence comparison with other known plant bHLH proteins, SPPB1 represents a new member of bHLH protein family in plants, which may involve in regulation of peroxisome proliferation in plants. 74 3.2 Introduction Peroxisome proliferation and peroxisomal protein expressions are known to be mainly controlled at the levels of transcription (Einerhand et al., 1995; Igual et al., 1992; Simon et al., 1991; Wang, 1994). In S.cerevisiae, peroxisomal protein gene expressions are found to be repressed by glucose, derepressed by glycerol and induced by oleic acid. Several transcriptional factors have been suggested to be involved in tuning the expression of entire spectrum of peroxisomal proteins (Einerhand et al., 1993; Wang, 1994). A well-studied example is the peroxisomal acyl-CoA oxidase encoded by POXl gene which is controlled by multiple cis-acting elements in the promoter region, including two repression sequences and an activating sequence (Wang, et al., 1994). In S. cerevisiae , two transcription factors (Adrl and Rtgl) have so far been characterized to positively control peroxisomal protein expression (Simon, et al, 1995; Kos, et al., 1995) and other two yeast transcription factors (ABFl and RP-A) are involved in the repression of FOX gene expression by glucose (Einerhand et al., 1995). ADRl was initially found to control alcohol dehydrogenase 2 (ADH2) gene expression, is shown to be involved in the regulation of peroxisome proliferation and the utilization of oleic acid (Simon et al., 1995). Many genes encoding peroxisomal enzymes are under the control of ADRl, including catalase A, thiolase, and PASl (Simon, et al., 1991; 1995). In addition, a oleate-response element (ORE) commonly found in the 5'-flanking regions of a number of genes encoding peroxisomal enzymes and proteins involved in peroxisome biogenesis, but the factor(s) binding to ORE is not yet known (Kos, et al., 1995). In mammals, the peroxisomal proliferator activated receptor (PPAR) has been shown to be important in the induction of peroxisome proliferation, which is a member of the TR/RAR/RXR subclass of ligand-dependent transcription factors (Isseman and 75 Green, 1990; Dreyer et al., 1992; Tugwood et ai., 1992). Peroxisome proliferators cause a rapid peroxisome proliferation and coordinated transcriptional activation of genes encoding the enzymes of the peroxisomal ^-oxidation pathway in rat and mice. A DNA binding site, the cis-acting peroxisome proliferator response element (PPREs) which consists of AGGTCACTGGTCA, have been identified in the 5’-flanking region of H202"producing rat acyl-CoA oxidase (ACOX) gene and in other genes inducible by peroxisome proliferators (Osumi et al., 1991; Tugwood et ai., 1992). Binding PPARs to its PPREs is via cooper-acting with retinoid X receptor which enhances the gene expression involved in lipid metabolism (Kliewer et al., 1992; Gearing et al., 1993). PPARs bind to cognate response elements through heterodimerization with retinoid X receptors (RXRs) (Bardot et al., 1993). Heterogeneous expression of human RXR alpha and mouse PPAR in yeast S. cerevisiae are able to synergistically activate transcription via cognate elements. This demonstrates that at least part of the mammalian peroxisome proliferator-signaling pathway can be faithfully reconstructed in yeast and suggests that the transcriptional regulation of fatty acid metabolism are conserved in yeast and mammals (Marcus et al., 1995). Proteins that possesse a basic Helix-Loop-Helix domain generally is defined as a DNA binding protein in eukaryotic cells, and acts as a transcription activator. Its regulation of transcription occurs by binding to the specific DNA cis-acting element sequences in the gene's promoter regions. A series of studies have shown that major proteins of the transcription apparatus are conserved among eukaryotes, and that proteins involved in transcription, as well as promoter elements with which such protein interact, are evolutionary related between yeast and mammals (Harsman et al., 1988; Marcus et al., 1995). PPAR is a bHLH containing protein which interacts with DNA by combining with RXRs factors. Rtgl, a transcriptional factor regulates a peroxisomal enzyme citrate 76 synthase gene expression, is a bHLH-containing DNA binding protein (Kos, et al., 1995). Adrl contains a bZIP DNA binding domain (Simon et al., 1995). Spbl fails to grow on the medium containing oleic acid as sole carbon source or uric acid as sole nitrogen source. A mutation likely occurs to the genes directly or indirectly involved in peroxisome assembly, proliferation and protein import. In this study, a soybean cDNA was able to restore the ability of this mutant to grow on oleic acid or uric acid-containing media. This demonstrated that it would be possible to isolate plant pas genes by complementation approach. Sequence analysis of pSPBl indicates that it contains a basic helix-loop-helix motif near the N-terminus and shares sequence homology with phaseolin G-box binding protein and rice transcriptional factor R as well as myc-like regulatory proteins. Thus, this gene may regulate the transcription of genes involved in peroxisome biogenesis. This study may open up the avenue to study plant peroxisome biogenesis. 77 3.3 Results Biochemical analysis of Spbl based on subfiraction of peroxisomal marker enzymes catalase and uricase revealed that these two enzymes are not defective since they are able to be induced at a low level. The mutation may effect the proliferation of peroxisomes. 3.3.1 SPB1 cDNA isolation by functional complementation The mutant was selected on the oleic acid containing medium (SOY) as described in the previous chapter. Oleic acid is known to induce peroxisome proliferation. Yeast and mammals utilize fatty acid and are similar in regulation of peroxisomal enzyme expression (Marcus, 1995). Therefore, plant must also share a similar regulation in terms of utilizing fatty acid. Functional complementation with a soybean cDNA library with Spbl was performed. The transformants were first selected on ura drop-out minimal medium. The plasmid-containing positives then were transferred onto oleic acid- containing medium. Four positive colonies were isolated from approximate 200,000 plasmid-containing positive transformants and were further streaked onto a SOY plate to confirm the growth. Only one positive colony showed constant growth on SOY. The plasmid (pSPBl) was isolated and retransformation was performed. Complementation with pSPBl, showed partial growth on SOY (data no shown) or uric acid-containing selective medium (Figure 4.1). A Spbl complemented with pYEUra3 plasmid showed no growth in both media. pSPBl contains about 750 base pairs long cDNA fragment and its nucleotides sequence was determined. In order to obtain a larger cDNA, pSPBl Xbal and Xhol fragment was used as probe to screen a soybean nodule cDNA library constructed in AZapn. Among the eight positive colonies, the plasmids contained the largest cDNA 78 insert still was about 750 base pairs long. The nucleotide sequence of the newly isolated cDNA showed identical sequence to the previous isolated SPB 1 cDNA. A primary northern data showed the mRNA was in a low molecular weight and with a very low expression rate (data is not shown). 3.3.2 SPB 1 is a DNA binding protein containing a bHLH motif As shown in Figure 5.1, SPBl has 759 nucleotides. One open reading frame was identified, which consists o f450 bp, and encodes a polypeptide of 150 amino acids with deduced molecular weights of 16,525 Daltons. The ATG codon at nucleotides 61- 63 most probably represent SPB 1 translation initiation site. The sequence context of the first ATG (AGAAGCATGGAT> matches well to the consensus translation start site (TAAACAAIQGCT) defined by Joshi (1987) for plant genes. Leucine is the most frequently used amino acid residue in the peptide, which occupies 10% of the total amino acids. Alanine, glutamic acid, leucine, glutamine, serine and valine are the second most frequently used amino acids. There is no histidine in this protein. Only one tyrosine, two cysteine and two proline are existing in this peptide. A highly hydrophilic domain is residing near the N-terminus and a hydrophobic domain near the C-terminus (Figure 3.2A). A basic amino acids containing-domain is found at N-terminus and form a hydrophilic domain (Figure 3.2 B). 79 GCiU^i:AACCATCGAGAAiUX:AAAAACCAAAATCAATTTAGaGGAAAACGTTGGAAGAAGC3ffî;GATTCTrGGCX3G 75 H D S W R 5 CGTGGXAAAAACGCATCI?UrcC7^GCXX3U^GTTGCAACAACTTCGCTCGGTTACAAATTCCAGTGCTGTGAACAAA 150 RGKNASMQRKr.QQI.RSVTNSSAVNK 30 GCCnxrAATTATTGTGGATGACACCAGAXACATAGAGGAGCTGAASCAAAAAGTGGATGGCTTGAACTCAGAGCTA 225 ASIIVDDTRYIEELKQKVDGLNSEL 55 GGAACCX3CGGAATCATCAATCTCCCAAGACGAACTACCXATGGTCACTGTAGAAACCCTAGAAAGGGGATTCCTC 300 GTAESSISQDELPMVTVETLERGFL 80 ATTAAKàTGTTTTCAGAAAGGAAITGTCCœGiaTGCTrrGGGGCAAIACTasaTCCAarrTGflAGAACrGGGACrT 375 INVFSERNCPGMLGAILDAFEELGL 105 GA!IX3TGCTTGAIGCTAGGGTTTCrTGCGAAGACACTTTCCAGCTTGAAGCAGTCGGAGGftGAAAGTCAAGAGAAC 450 DVLDARVSCEDTFQLEAVGGESQEN 130 GAAAGCATTGCXXSCGCAAGTGGTAAAGCAAGCAGTGCTTCAAGCAAiœAAAACATGGArTAATCGAAGAACACA 525 ESIAAQVVKQAVLQAIQNMD* 150 TGTGCTAA3AA3AATGTACCTCAGACTCTCTrAATTTCTTAAAATCTTTGTATTAAArAEAAAAGTTACGTACCC 600 AAGCTTAASTGAIGAGAGAATTGAAGTGTGAIACTAIAITGTGCCCAAATGTAAGTACCACAATGGCCCTTAAAT 675 GTGATGTGGCTTCTTCGTTTGTAGGAAAGAATGAATGGACAGGGATCGTCCAGGTTAArTGGEAAAAAAAAAAAA 7 5 0 AAAAAAAAA Figure 3.1. Nucleotide and deduced amino acid sequence of soybean putative G-box binding protein (SPBl). Nucleotide is numbered in 5’ to 3' direction from the first identified base. The first start codon Met is underlined. A stop codon is indicated by a *. 80 A 1 0 0 2 1 O -1 -2 -2 -3 -3 1 0 0 B 1 o o : I ' ' I ' _ ' I I I. I _ _ I—I A A B B T 1 T 1 O O Figurse 3.2. The profiles of hydrophobicity, and acidic and basic amino acid usage of SPB I. A. The SPB I hydrophobicity profile. SPB I contains a hydrophilic N-terminus and a hydrophobic domain near C-terminus. B. The acidic and basic amino acid usage profile. Except the second amino acid D is an acidic amino acid, there is no acidic amino acid residue found within the 35 amino acid sequence at N-terminus. (A) represents acidic amino acid residues; (B) represents basic amino acid residues. 81 S P B l 1 K D SRRRGKNASMQR _____ P61 441 EPEKRPRKRGRKPGKGRBEPLNHVB A ^ g Q l R i c e R 3 98 K------NHVMS ^ H r I M a iz e LC 4 1 5 K------NHVHS ^ H k I MÏC 1 K ------K H 'V H S R a 1 M S ^ B r I basfc region S P B l 20 | r | v t n M s a - h n V D A T R K Q K # D P G l 4 80 Ir A V v B m V s K M o 6 D AK S K L S E K G - Rice R 418 Ik H v H k P o E K r H e M aize LC 465 k p L h r Q R r H q MYC 2 1 Ik I l h K H D E Q r H b R a 2 1 I k I v v ^ B i k k H d E K r H b Loop Helix I S P B l 55 P G l 520 LEKQL~“ ™““ *~ |LVKKELELAT{ SP SP PP G P Rice R 452 S P C P L TRSR ------R K C R ITGRKVSAGA RKAPAPBV M aize LC 475 A S R P S TTTRLITRPSRGNN SVRKEVCAGsl R K --S P E L MYC 61 S R - P S T R G R ------RR-H lAG — — — ISG a I RKASSEPG R a 57 S P C P L T R - R ------RKSR i t g k r v s a v a I I I I RKASTPEV S P B l 55 GTABSSISQOELPMVTVETLBRGFLIN-- P — — — PG l 546 PPSNKEARETTSKLIOL-ELEVKIIG-HDAMIRZ Rice R 492 ASDDDTO GERRHCVSNVHVTZMDHKE Q W K M aize LC 513 GRO-DVERPPVLTMDAGTSNVTVTVSDKD Myc 90 6RDVBRERLWALSMDGPSNVNVTVM0-KE G W K R a 90 ASDDDTO GVH-HCVSKVMVTIMDN-E S P B l 92 GMLGAZLDAFVELGLDVLOARVSCEDTFQLEAVGGESQEM P G l 584 HPAARLMAALKELDLDVKHASVSVVNDLMXQQATVNHGNR Rice R 529 LLMTRVFDAIKGVSLDVLSVQASTSDGLLGLKIQAKFASS M a iz e LC 552 LLMTRVFDAZRSLHLDVLSVQASAPD6FMGLRIRAQFAGS MYC 129 M L M T R V R a 125 L L S P B l 13 1 ESIAAQVVKQAVLQAIQNMD 150 PGl 184 FYTQEQLRSARSSKIGNAL 642 R ic e R 569 AAVEPGMITEALRKAIAS 586 M a iz e LC 592 GAVVPWMXSEALRKAIGKR 610 MYC 134 Ra 127 Figure 3.3 Comparison of SPB 1 with other bHLH containing transcription regulatory proteins in plants. The most conserved amino acids are blocked. The Basic region helix-loop-helix is underlined. SPB 1, this study; PGl, phaseolin G-box binding protein 1 (UI8348); Rice ^ rice transcriptional activator (U39680); Maize LC, anthocynanin regulatory LC protein 0P13526); MYC, Phyllostachys acuta myc-like re^atory R gene product (U11448); ROj Oryza anstraliensis transcriptional activator Ra ^39863). 82 SPB 1 amino acid sequence shows a homology with common bean phaseolin G- box binding protein and other plant transcriptional activators, which contains a basic helix-loop-helix motifs. The amino acid sequence of SPB 1 and other partial amino acid sequence of known proteins were compared and aligned (Figure 3.3). Data reveals that SPB 1 contains a bHLH domain at its N-terminus which is conserved with the bHLH domain near the C-terminus of phaseolin G-box binding protein l(PGl), rice transcriptional activator R and maize transcriptional factor LC. The similarity of amino acid sequence of SPBl with PGl C-terminal peptide (441-642) is 31%, with rice R factor (398 to 642) is 28%, with maize LC (415 to 586) is 29%, with Phyllostachys acuta myc-like regulatory R is 31% and rice activator Ra is 25%. SPB 1 shows the most close relationship to the phaseolin G-box binding protein 1 (441 to 642) and maize LC protein (415 to 610) C-terminal bHLH containing regions. A Cys^^ (SPBl numbering) residue near C-terminus is conserved in all sequences. Based on the data of amino acid sequence comparison, SPB 1 cDNA likely encodes a new member of the bHLH protein family. This peptide only contains a DNA binding domain, and contains a active DNA binding motif. Possibly, the N-terminal region of SPBl is missing, and likely SPBl is an incomplete protein. 3.4 Discussion: Sequence comparison reveals that SPBl contains a bHLH DNA binding domain and shows homology with the bHLH-containing domain of the common bean phaseolin G-box binding protein. This may suggest that SPBl possibly acts as a DNA binding protein through bHLH motif, and the DNA binding target possibly is a G-box (CACGTG) like DNA segment. The overall homology with these known plant bHLH- containing proteins is less than 40%, therefore, SPB 1 represents a new member of 83 bHLH protein family, and is suggested to be a putative G-box-binding protein involved in the regulation of peroxisome biogenesis related proteins and peroxisomal enzymes in plants. A further study needs to be carried on the protein activation and its target sequence. S. cerevisiae PAS 19 has been isolated and encodes a positive transcriptional regulator ADRl, which has been found to regulate of several protein expressions involved in peroxisome biogenesis and peroxisomal proteins (Kos, et al., 1995). In S. cerevisiae, more than one positive regulators have been found to regulate peroxisomal gene expressions (Einerhand, et al., 1993; Wang, et al., 1994). Thus, a mutation occurs on one of these transcriptional factors would result in peroxisome defect. A transcriptional factor mutation on a DNA binding domain can cause a defective binding to the promoter regions of the genes which are under the control of this factor. Expression of SPB 1, therefore, allows the transcription apparatus activated at a low level. And thus, the genes are expressed and allow cells to grow on oleic acid or uric acid, respectively. bHLH protein family is a class of transcription factors ubiquitously distributed in eukaryote from yeast, plants to mammals (Murre, et al., 1989; Kawagoe and Murai, 1996). These proteins form either homo- or hetero-dimers and play major roles in cell- type specifically determination, cell growth and development. Therefore, a plant bHLH DNA binding protein replace a yeast transcriptional factor is feasible. Whether SPB 1 involves in the regulation of expressions of the genes encoding the proteins related to peroxisome biogenesis or peroxisomal enzymes remain to be elucidated. Peroxisome proliferation is a complex procedure, and requires a multiple gene regulation activators (Einerhand et al., 1993; Wang, et al., 1994). A yeast model has been proposed via studies of regulation of ADH2 (Yu et al., 1989, Donoviel, 1995). Two cff-acting upstream activation sequences (UAS) act synergistically in the derepression of ADH2 gene expression in S. cerevisiae (Simon, 1995). UAS 1 is the 84 binding site for the transcriptional regulator ADRl which interacts with the protein bound to UAS2, and together enhance ADH2 expression. ADRl is suggested not the only positive regulator of many genes and may have an even more general function as a transcriptional activator under suboptimal growth conditions. An alternate yeast model has also been raised via study of 3-oxoacyl-CoA thiolase gene FOX3 (Einerhand, et al., 1993). This model is similar to the PPAR/retinoid X receptor-a induction of gene expression in higher eukaryotic cells (Kliewer at al., 1992). A m-acting element, oleate response element (ORE) commonly exists in yeast genes induced by oleate. The factors binding to the ORE element could therefore be involved in the coordination of expression of oleate-inducible genes and in the proliferation of peroxisomes. The models of peroxisomal gene expression and regulation in mammalian (Kliewer at al., 1992) and yeast (Simon et al., 1995; Donoviel et al., 1995), are presented in Figure 3.4. The common regulation is performed by the bHLH containing DNA binding protein, PPAR, in the mammalian, and ADRl (bZIP protein) in yeast. This common regulator is required for the genes involved in fatty acid metabolism and peroxisome biogenesis. Other regulators must be required to determine the specificity expression of a particular gene under diverse substrates. In plants, proteins that specifically interact with a G-box are defined as G-box binding proteins, which have been isolated in many plants. A c/s-acting promoter element, G-box (CACGTG), is an essential functional component of many stimulus- responsive promoters existing in diverse plant genes, which are switched on in response to the environmental stimuU, such as light, anaerobiosis, p-coumaric acid, and hormones such as abscisic acid, auxin, ethylene and methyl jasmonate (Hong et al., 1995; Meier et al., 1995). The mechanisms of how the G-box element functions in these differently regulated genes is unclear. One mechanism of G-box participating in specific gene expression has been proposed to act as a common regulatory element, that is, G-box via 85 GBP binding to this element simply acts as a general regulation of transcription, functioning in response to signals that are perceived by proteins bound to surrounding sites (Hong et al., 1995; Meier et al., 1995). Thus, GBP is suggested to act in general regulation of transcriptional factor and regulate gene expression synergistically with other regulators. Obviously, plants must also share a common regulatory mechanism of peroxisome biogenesis and peroxisomal protein expression with their yeast and animal counterparts. Based on the feature of the G-box and GBP, a G-box binding protein acts as a common regulatory activator, is feasible in plants. Unlike the most other GBPs which contain a bZIP motif, SPB 1 is a putative GBP containing a bHLH motif, which may represent a different gene regulation. Even though, this study did not provide further information on the DNA binding site and regulatory mechanism on this soybean putative GBP. A potential hypothetical model is worthy to be raised and discussed. This will help the future studies on the plant peroxisomal gene regulations. SPB 1, therefore, is proposed to function as a common transcriptional regulator which may interact with a cis-acting G-box elements of the genes coding peroxisomal enzymes or proteins involved in peroxisome biogenesis. Gene specific expression will be determined by other factors which may perceive the signal generated by developmental and environmental stimuli (Figure 3.4). Overall, the proliferation of the peroxisomal compartment is genetically programmed and controlled. To get the insight of the controlling of peroxisome proliferation and biogenesis in plant cells, future studies have to be carried out on the promoters of genes coding for typical plant peroxisomal matrix proteins and the corresponding transcription factors required for their controlled expression. 86 Promoter regions Peroxisomal proteins PPAR I ^ R A. Rodent a s. cerevisiae — I u^i uaS2 ^ SSSSJâ^SSSSSS C. Soybean Figure 3.4. Hypothetical models of regulation of genes coding for peroxisomal proteins. A. Fatty acids and peroxisome proliferators activate peroxisome proliferator activated receptor (PPAR). PPAR binds to cognate response element (PPRE) through heterodimerization with retinoid X receptor (RxR) which enhances the gene expression. B. Two cis-acting element UASs (upsteam activation sites) is residing in the promoter region of ADH2 gene. UASl is the binding site for ADRl which combined with the tmknown factor X bound to UAS2. Thus, UAS 1 and UAS2 synergistically regulate ADH2 gene expression. C. In plants, G-box binding protein binding to the G-box element may act as a common transcription factor which interacts wi6 other unknown factors (XBP)bound to surrounding sites (X-box), and synergistically regulates gene expression. 87 CHAPTER4 ISOLATION AND CHARACTERIZATION OF A NOVEL CYTOCHROME P450 cDNA ENCODING A PEROXISOMAL H2O2-DEPENDENT URATE-DEGRADING PEROXIDASE IN SOYBEAN 4.1 Abstract Tropical legumes convert fixed nitrogen into purines. The purines (xanthine or hypoxanthine) are then exported to the uninfected cells where they are oxidized into allantoin and allantoic acid (ureides) which are then transported to the shoot. We propose that purines, thus produced, induce peroxisome proliferation in the uninfected cells and the synthesis of enzymes necessary for the oxidation of purines. Our study suggested that xanthine can induce peroxisome proliferation in Saccharomyces cerevisiae, but uric acid could only induce uricase activiQr. Complementation of a yeast mutant defective in peroxisome proliferation and utilization of uric acid as sole nitrogen source resulted in isolation of a cytochrome P450 cDNA from a soybean nodule cDNA library. A full length cDNA of 1.9-kilobase contains 1533 base-pair long open reading frame encoding a protein with a molecular weight of 67 kDa. This protein was identified as a H 2O2- dependent urate-degrading peroxidase (P450W). This finding confirms the existence of an alternative urate-degrading diamine oxidase and peroxidase enzyme activity in soybean. A highly hydrophobic domain at the N-terminus and two peroxisomal targeting signals residing near the C-terminus indicate that P450W is a putative peroxisomal 88 membrane-bound protein. Amino acid sequence comparison reveals that P450W shares less than 40% homology with other known P450s and forms a new gene family of the P450 superfamilies. The feature of P450W implies that the mode of action as a peroxidase or a monooxygenase may be regulated by the ratio of O 2/H2O2 in the compartment of peroxisomes. 89 4.2 Introduction Cytochrome P450, generally, functions as a monooxygenase. It is widely distributed from primitive bacteria and yeast to highly developed plants and animals. It participates in a diverse biosynthetic and metabolic reactions, catalyzing a variety of compounds from endogenous or exogenous sources like fatty acids, phenolics, purine and xenobiotics (West, 1980; Blee, et al., 1984; Ortiz de Montellano, 1986). That cytochrome P450 can mediate hydroperoxide-dependent oxidation as a peroxidase was initially described by O'Brien and collaborators in 1970's ( Hrycay et al., 1972; Kadlubar et al., 1973; Rahimtula, et al., 1974). Thereafter, a wide variety of organic hydroperoxides including fatty acid peroxides have been shown to be catalyzed by P450's (Weiss et al. 1987; Ullrich, et al, 1982; Hecker and Ullrich, 1989). A peroxidase is defined as a hemoperotein with the activity of the oxidation of inorganic and organic substrata at the expense of a biogenic hydroperoxide (including hydroperoxide- H2O2, allqfl hydroperoxide or acyl hydroperoxide) (Saunders et al., 1964). Therefore, the discovery and association of the peroxidase function of P450s have opened up a new perspective for its mechanisms. In terms of prosthetic group and catalytic mechanism, peroxidases and cytochrome P450's share many similarities (see review, Mamett and Kennedy, 1995). In the reaction of hydrogen peroxide-dependent hydroxylation and epoxidation by P450, this enzyme is associated with ferric state (Fe^+) and does not require electron transfer firom NADPH (Smith, 1991), and peroxide oxygen is transferred to the substrate (Nordblom, 1976, Rahimtula, 1978) as equation (1): ROH + XH2 ------► RO +X/ XH2O (1) 90 However, as a monooxygenase, P450 utilizes NAD(P)H as the source of reducing equivalent for the oxygenation, involving adding a oxygen atom to the substrates, an electron transfer pathway is involved during the reaction. The oxidizing agent generated in both reactions is the same based on the similarity in the regiochemistry and stereochemistry. Noteworthy, P450 catalyzed hydroperoxide-dependent oxidation, mechanistically, is less complex than the NADPH-dependent oxidations due to the absence of electron transfers from P450 reductase. According to Blee et al.'s studies (1984) on soybean cotyledon enzyme activities, the plant peroxidase could be a cytochrome P450. The function of cytochrome P450s as NADPH-dependent monooxygenases has been well-established. However, the function of P450s as a peroxidase has just begun to be explored (Capdevila et al, 1995). In plants, the first well-defined P450 peroxidase is allene oxide synthase (AOS) from flax seeds, which derives allene oxide from the 13- hydroperoxide of linoleic acid (Song, 1991; Song, 1993). Allene oxide is the key precursor in the biogenesis of jasmonic acid. The first plant P450 cDNA encoding a H202-dependent peroxidase was isolated from flaxbean seed (Song, 1993), one of the examples that completely specialized for the metabolism of endogenous peroxide substrate. Most plant cytochrome P450s were discovered in association with studies on the biosynthesis or metabolism of cellular constituents like lignin, phenolics, membrane sterols, terpenoids and phytoalexin (West, 1980). About 100 sequences within 30 gene families of plant P450s have been registered in the Genbank, these P450s are found to catalyze a wide range of substrates. Many leguminous plants are known to produce phytoalexin and glyceollin as plant defense molecules. Previous study of isolation of P450s showed that the synthesis of the phytoalexin, glyceollin, in fungal infected soybean is a process depending on several cyt P450 enzymes (Kochs, 1989). Within the 91 last five years, many cyt P450 cDNAs have been cloned and are suggested to be involved in pbenopropanoid/fiavonoid metabolism and related to the plant defense mechanism. These enzymes are, licodione synthase (LS) (Octani, 1994), and isofiavone synthase (IPS) (Kochs, 1986; Hashim, 1990) participating in divergent pathways to retrochalcone and isofiavone biosynthesis; a jasmonic acid-induced soybean cytP450 93Al (Suzuki, 1996); trans-cinnamic acid 4-hydroxylases (cytP450 71C) from difference species (Teutsch, et al., 1993; Mizutani, 1993; Fahrendorf, 1993; Hotze, 1995); a femlic acid 5-hydroxylase from Arabidopsis thaliana (Mayer, 1996), and AOS from flaxbean seed producing the jasmonic precursor (Song, 1993). Also, the last step of salicylic acid biosynthesis pathway is catalyzed by a P450, benzoic acid 2-hydroxylase (Le6n et al., 1995). The P450s are commonly found to be in the microsome fraction preparations (Donaldson, 1993). They are membrane bound proteins mostly existing in ER, a few found in mitochondria and in animal peroxisomes (Gutierrez, et al., 1988). In plant, P450 localized in glyoxysome membrane has been suggested by Donaldson’s group on the castor bean endosperm glyoxysome membrane components and electron transport system studies (Dick, 1987). Recently, non-microsomal P450 is found to be a major rubber particle protein (Pang et al., 1995). A horse-radish peroxidase has been reported to be able to catalyze the oxidation of uric acid. A P450 peroxidase could also catalyze uric acid. Generally, urate oxidation into allantoin is catalyzed by a high Km peroxisomal matrix protein, uricase, which is highly induced during nitrogen fixation and nodule formation in soybean. Symbiotically-reduced nitrogen in tropical legumes is assimilated via ureides. In soybean nodules, there is a proliferation of peroxisome in the uninfected cells where the final steps of ureide biogenesis take place. The expression of glutamine synthetase, uricase and PRPP-AT is highly induced after nitrogen fixation 92 (Kim & Venna, 1995). In the young roots, the high Km uncase is lacking, a urate- degradation enzyme system has been reported to exist in soybean radicals, which is defined as a diamine oxidase-peroxidase urate-degrading enzyme system ( Tajima et al., 1985). The alternative urate oxidation is catalyzed by a low Km peroxidase coupling with a diamine oxidation. This enzyme system is enhanced by addition of a diamine, cadaverine, which is required as a cofactor for this reaction (Tajima, 1985; 1978). In this enzyme system, it has been suggested that the hydrogen peroxide is generated as a by-product firom cadaverine oxidation by a diamine oxidase, and its formation is coupled with uric acid degradation by a peroxidase (Tajima, 1985). Wounding has shown to enhance this enzyme system through the increasing in the level of cadaverine (Tajima, 1978). The significance of ureide generation by this system is not known. In this smdy, we have cloned a putative peroxisomal cytochrome P450 firom soybean {Glycine max) nodule cDNA library by functional complementation of a yeast mutant. According to this study, the cytochrome P450 we cloned may function as a peroxidase involved in ureide metabolism. This study confirms the existence of a urate- degrading diamine oxidase and peroxidase enzyme system which may be involved in nitrogen assimilation during plant under stressed, cell defense and early seedling growth. The significant of this alternative urate-degrading enzyme system to the plant development may eventually be elucidated. 4.3 Materials and Methods 4.3.1 Strain, plasmids, media and culture conditions Strains used in this study were S. cerevisiae DBY1034 ( MAT a, lvs2-801. his4- 539, am3-52 ) (Seger and Botsein, 1987), and an ethylmethane sulfonate (EMS) mutated 93 strain of DBY1034 named Spbl which can not utilize oleic acid as sole carbon source or uric acid as sole nitrogen source. A soybean nodule cDNA library was constructed in LAMDA MAXI which contains a pYEUra3 (Clontech) yeast expression shuttle vector. Host strain was E. coli XL 1-Blue (Hanahan, 1983). Minimal media contains 0.67g yeast nitrogen base without amino acids (YNB) (Difco laboratories, Detroit, MI) and amino acid lys (40pg/ml) his (40|i,g/ml), ura (20pg/ml) as needed, supplemented with 2% glucose (SG), 2% dextrose (SD) or 2% (wt/vol) glycerol plus 0.05% yeast extract (SGY) or (0.1) oleic acid, 0.5% Tween plus 0.05% yeast extract (YNO) as described by Van Der Leij et al. (1992). Rich media are YPD and YEPD as described by Guthrie and Fink (1991). The inducive medium contains 0.17% nitrogen base without amino acid and (NH4)S0 4 (Difco, Detroit), 0.05% yeast extract, ImM xanthine (the N source) and 2% of dextrose (NDYX) or 2.5 mM uric acid (UGY), 2% (wt/vol) glycerol, 0.05% yeast extract, and amino acid lys (40pg/ml) his (40p,g/ml), ura (20pg/ml) as needed. The uric acid containing selective medium (NUGY) included 0.17% nitrogen base without amino acid and (NH 4 )S0 4 (Difco, Detroit), 2% galactose 2.5 mM uric acid (added after autoclaving), 0.05% yeast extract, 2% agar, and amino acids including lysine 50|ig/ml, 40|ig /ml of histidine, arginine 40p,g/ml , leucine 50pg/ml , phenylalanine 50pg/ml, threonine 50pg /m l, 40pg/ml and tyrosine 50|Xg/ml. 4.3.2 Functional complementation The 5. cerevisiae mutant Spbl was used for functional complementation with a soybean nodule cDNA library. A standard electroporation procedure was employed for the plasmid transfer into S. cerevisiae (Becher and Guarente, 1991 ). Transformation efficiency of yeast ranged between 10^ -10^ cfu/p.g of DNA. The first round selection was carried out onto the uracil drop-out mineral medium (SD with IM sorbital, and 94 40mg/l histidine and 40mg/l lysine) for selection of uracil prototropy. Plates were incubated at 30° C for three days, and then the colonies were transferred onto uric acid- containing selective medium (NUGY) by replica plating technique for selecting positive clones. Plasmids were isolated from yeast positive clones according to the method for the bacterial plasmid minipreparation as described (Sambrook, et al., 1989) with the following modification. Yeast cells were incubated in SD-uracil drop out medium in 2ml for 24-36 hr and collected by centrifugation. Cells were resuspended in 200ml buffer I (50mM Tris-Cl, pHS.O, lOmM EDTA, 0.6 Sorbitol), and then added Img/ml lyticase (Sigma) incubating on ice for about 4 hr. The procedure was the same as plasmid mini preparation from bacteria. Then the plasmid recovered from yeast were used to transform into E. coli and to received the larger amount and higher quality plasmids. Retransformation of Spbl mutant was carried out to test the ability of recomplementation. 4.3.3 Full length cDNA isolation and DNA sequencing A cDNA isolated by the functional complementation was sequenced by progressive sequencing on both strands using Sequenase 2.0 (United State Biotechemical). The cDNA insert received was used as a probe to screen a soybean nodule cDNA library to isolate its full length cDNA. The positive clones were excised into plasmid in vivo following the standard procedure (Stratagene). One longest clone (pYP450) was completely sequenced. 4.3.4 Sequence alignment, comparison and phylogenetic trees The multiple amino acid sequences were aligned by CLUSTAL method of DNA- STAR computer program. The multiple aligrunent used to construct phylogenetic tree 95 and distance comparison were made with CLUSTAL method. Hydrophobicity was made by DNA-strider program. 4.3.5 RNA isolation and Northern hybridization Total RNAs from soybean tissues were prepared by hot phenol treatment (Verwoerd et al., 1989). Soybean tissues were one, two and four days germinated seed cotyledons, ten days stems and leaves, root nodules at different stages, one-day's germinated root radicles, two day’s germinated cotyledon-free seedling, four, five and seven-day old roots. Northern blotting were performed at 68° C using DNA probes (pBl-P450W Xbal-Xhol fragment) labeled with a^^P-dATP following the method described by Mahmoudi and Lin (1989). 4.3.6 Expression of P450W cDNA in E .co li, protein extraction and SDS-PAGE Soybean P450 was inserted in pRSET expression system. Two oligonucleotides (CGGGATCCATGTTGCTGGAACTTGCA and T7 primer ) were used as primer to synthesis P450W cDNA. BamH I and Xho 1 restriction enzymes were used to cut the PCR product. The BamHl and Xhol P450W fragment was cloned into the BamHl/Xhol site of bacterial expression vector pRSETA (Invitrogen) to give plasmid pEP450W. The transformed cells were grown in LB medium and the proteins were induced by adding O.SmM IPTG. One ml of overnight cell cultures without IPTG induction was inoculated into 500ml flask with 200ml LB media. After 3-5 hour growth, when cell culture reached ar ODgoo of about 0.5, IPTG (0.5mM/ml) was added into one flask, the one without IPTG was used as control. After 5 hour induction, cell cultures were pelleted at 10,000 for 10 minutes and washed twice with cold distilled water. Cell pellet was dissolved in phosphate buffer, pH 7.0. Cells were broken by sonication. Sonicated cell solutions 96 were spun down at 5,000 rpm for 5 minutes, and the supernatant were centrifuged at 13,000 rpm for 10 minutes. After separation of pellet and supernatant, pellets were dissolved in 500 |xl phosphate buffer (pH 7.0). Both supernatant and pellet solution were stored on ice for enzyme assay. SDS-PAGE was performed following the standard method. 4.3.7 Cell-ftee fractionation and induction Wild-type S. cerevisiae was used to determine the induction ability of catalase and uricase activities by xanthine used as sole N-source. Cell grew in YPD overnight and the cell cultures were harvested and washed twice in cold sterilized distilled water. Then same aliquot was transferred into 500ml flask with 200 ml XGY-induction medium or SG-non-induction medium and incubated at 30°C by shaking at 225 rpm for 36 hr. After induction, cell cultures were collected by centrifugation at 5,000 rpm for 10 min., cells resuspended in 50 ml water and washed twice. Cell culture was then finally resuspended in =lml 5mM 2[N-morpholino] ethane sulfonic acid (MES), pH 5.5 and ImM EDTA buffer with 0.6M sorbitol buffer pH 8.0 (Lui, et al., 1993) to digest spheroplast. 4.3.8 Enzyme assay Xanthine-induced cells were broken by glassbeads as described by Lui, et al (1993). The Buffer was used with 5mM 2[N-morpholino] ethane sulfonic acid (MES), pH 5.5 and ImM EDTA with 0.6M sorbitol (Lui, 1993). Enzyme activities were measured with a spectrophotometer at room temperature. Catalase activity was determined using H 2O2 as a substrate according to the method described by Luck et al, (1963). Uricase activity was determined using uric acid as substrate, and measuring the decrease in absorption at 293 nm according to the method described previously (Suzuki 97 and Verma, 1991). Peroxidase activities were determined at 450nm using o- phenylenediamine as the substrate (Tajima et al., 1985). Protein concentrations were determined using Bio-Rad reagent (Bradford, 1976). 4.3.9 Determination of H2O2 affection on cell growth Yeast strains including wildtype, mutant, mutant with pYP450W or wildtype with plasmid pYEUraB. H2O2 was added at ImM, 2mM, 3mM, 4mM and 5mM concentrations to the uric acid-containing liquid medium with galactose as C-source. The same aliquot of cell solution was transferred into each treatment with 2ml in total volume, and each treatment had three duplicates. Test mbes were the incubated at 30°C with 225 rpm shaking for 36 hr. The ability of cells growth was detected by photospectrometer at 600nm. 98 a S - - o - - XGB M E4 - YNB e S 3 - I S 2 - T« > a i a V 0 10 20 30 40 a . . . XGB i M B YNB S 10- 3 5 - > i 5 0 10 20 30 40 Time (hr) Figure 4.1. Induction of yeast peroxisomal enzyme activities by xanthine. Wild-type strain S. cerevisiae cells were grown on xanthine or ammonia (control) as the sole nitrogen source. The pellet faction of cell-free extract after centrifugation at 20,000 rpm for 30 min were used to determine the peroxisomal marker enzyme activities. (A) Catalase activity; (B) Uricase activity. The enzyme activities induced by xanthine or ammonia are represented by dashed or solid lines, respectively. 99 Figure 4.2. Functional complementation of Spbl mutant with pP4S0W grown on uric Acid medium (NUGY agar). The ability of utilizing uric acid as the sole nitrogen source were compared. A. Control medium (SG, galactose/ammonia); B. selective medium (NUGY, galactose/uric acid). Wt, wild-Qrpe strain grew normal on SG and NUGY; mt, the Spbl mutant was not able to grow on a NUGY agar plate; mut/P4S0, Spbl expression of pYP450W results in a functional "by-pass' at enzyme level which can rescue cells growth on the uric acid; mt/GXl, Spbl expression of pYSPBl (a putative G-box binding protein) results a functional complementation; mut/V, Spbl expression of plasmid pYEUra3 does not rescue the mutant. 100 4.4 Results 4.4.1 Xanthine can act as a peroxisome proliferator in 5. cerevisiae Xanthine is an intermediate in the purine metabolism pathway, and is oxidized into uric acid by a cytosolic enzyme —xanthine dehydrogenase (Datta et al., 1991). Uric acid is transferred into peroxisomes to be further oxidized into allantoin and allantoic acid in soybean, fish and other mammals (Tajima, 1975; Tanaka et al., 1977; Voet and Voet, 1990). In this study, the wild-type strain of S. cerevisiae was cultured in the induction medium which contained xanthine as the sole nitrogen source and dextrose (YNDX) as the carbon source. The non-induction medium (control medium) contained ammonium sulfate and dextrose (SD). The induction of catalase activities in yeast by xanthine and NH4+ are shown in Figure 4.1 A. Catalase activities were measured in peroxisomal fractions of control and induced yeast cells. The catalase activity was increased about 3.5 times in the peroxisomal firaction of xanthine-induced cell fraction comparing with the non-induced one (Figure 4.1 A). The peroxisomal fraction of control in which NH4+ was used as the nitrogen source, showed no significant changes in catalase activity with culturing time. However, the activity of catalase began to show induction after 10-hour incubation, the enzyme activity was increased dramatically during 16-24-hour incubation period and reached at the highest and most stable level at 32 hours of incubation. The induction of uricase activities are represented in Figure 4. IB. Uricase activities were measured by using the classic method as described in the Method section. Uricase activity was increased 4 times in the xanthine-induced peroxisomal fraction as compared with the control one (Figure 4. IB). The uricase had no significant change in the NH4-incubated yeast cells. Similar to the catalase activity induction, in the xanthine- induced culture, uricase activity was induced after a 10-hour incubation period. 101 Thereafter, the enzyme activities were induced dramatically and reached about 4-fold after 32 hours of induction. Two peroxisome marker enzymes showed the same induction trends which implied that both enzyme activities are induced as a result of peroxisome proliferation (Amaiz, 1995). 4.4.2 Isolation of a cytochrome P450 cDNA by functional complementation The Spbl yeast mutant which carries the autotrophic marker ura3 was transformed with soybean nodule cDNA library maintained in the E. co//-yeast shuttle vector pYEUraS. Uracil prototrophy colonies were firstly selected on the URA drop-out mineral plates and subsequently scored for their ability to grow on uric acid selective medium (NUGY). Among 70,000 colonies, two clones were restored the ability of growth on uric acid. Colonies that could grow on uric acid as the sole nitrogen source were selected. The plasmids isolated firom these two strains and retransformed into the Spbl which regained the ability of growth on NUGY. The plasmid contained the insert of 0.8 and 1.2 kb, respectively. The retransformed colonies showed partial complementation. After retransformation was demonstrated (Figure 4.2), the DNAs were sequenced and found to contain incomplete ORFs and included the poly T, the 3 end of cDNAs. Deduced amino acid sequence firom the cDNA firagment of 800 bp long showed homology to cytochrome P450s. The other cDNA which had 1.2kb long insert encodes a copper-containing amine oxidase (see next chapter). 102 ACCACATTCAACCACMTCTCAtTTCTTATCTATACGCAAACCACTTCCTTCACCATGCCTTMCCAACCTTGTCTTaUlCamCTTCCCTCCrCCCT rT 100 CGACaACAGCTTCAACAACTCAGCCCrCCACCCAACGACCCrTGCCATCCAGCCGAACGKCAGAGAGAATAACACGCCAACATCTTCCATACTAArrAA 2 0 0 MLLBLALCLCVLAH rT T Cr CCACTTCCCTCCCACACCAACTCCAAAATCAAAACCACTTCCCCACCTCCCAAACCCTCCAACCCCAAACCCrCCrCCTCCCTTCATTCCCCACC 4 00 rLHLR?TPSAKStCALRHLPHPPSP!CPRPPPICH LHLLKOICLLHrALIDLSKKRCPLPSLSrCTMATV CCGCCCCTCCACCCCTCT&TTCTTCAACCTCTTCCTCCAAACCCACGACtgAACTTCCrtCAACACAACCTTCCMACCTCTCCCATAAGACCCCTCACT 600 CCSrPBLrKLrLOTRBCrSFHTRFQTSAIRRLT TACCACAACTCTGTCCCCATCCTTCCATTCCGACCrTACTCCAACrTCCTCACCAACCTCATCATGAACCACCTTCTCAACGCCACCACCCACAACAACC 7 00 YDNSVAMVPrCPTWKFVRKLIHIVDLLirATTOKK TCACCCCrTTCACCACCCAACAGArCXCCAACTTCCTTAGCCrTATCCCCCAAACCCCACACCCCCACMGCCCCTTCACCTCACCGACGACCTTCTCAA 8 0 0 LRPLRTQOIRKFLRVMAQSAeAQKPLOVrBBLLK ATGGACCAACAGCACCATCrCCATGATGATCCrCCGCCAGGCrGAGATGATCAGAGACATCGCTCGCGACGTTCTTAAGATCTTCCGCGAATACACCCTC 900 irTNSTtSMMHLGEABHt RD lAREVLKIFCBYSL ACTCACTTCATCTCCœ TTTGAACrATCTCAACCTTCCAAACTATCAGAACAGCATTGATGACATCTTGRACAACTTCCACCCTGTCCTTGAAACCCTCA 1000 TDFIWPLKYLKVCKYEKRIODILHKrDPVVBRV CCACCACa^CCATCCACaiTCAAAATTACCAACGAGCAAATCAACGCCCTTCTTGTCGACTTTTTCTCTCCACCCACAGAYTCCACACCCCTCCCAACAGAC 1200 OETMEtK ITKEOI KCLVVDFFSACTDSTAVATE TCCCCATTCCCAGACCTCCTCACCACGTCTACACCrcrTGTCCCCAAACATAGACTCCTTCaCGAACrTCRCRCTCAAAACCTTCCTTACATTAGCCCCA 1300 WALAELVRRSTAVVCKDRLVDEVDTONLPYIRA TTGTGAAGCACACATTCCCAATGCACCCACCACTCCCACTGCrCAAAACAAACrcCACACAAGACTCTCACATTAATCCGTATCTGATCCCAGAGCCACC 1 400 I VKETFRMHPP LPVVKRKCTEECEINCYVIPEGA ATTCCTTCTTTTCAAtGTTtCCCAACTACCAACGGACCCCAAATACTGCGACACACCATCAGAATTCCCTCCCCACAGCTTCTTACAAACTCCTCCTGAA 1 5 0 0 LVLFNVWQVGRDPKYWDRPSEFRPERFLETCAE GCCGAAGCAGGCCCTCTTGATCTTACGGGCCAGCATTTCCAACTCCTCCCATTTCCCTCTGCCAGCAGAATCrCCCCTGGCGTCAATTTGCCTACrTCAC 1 6 0 0 GEACP LDLRGQKFQ LLPFG5GRRM©PGVHLATS 1 7 0 0 CAATCGCAACACTTCTTGCATCTCTTATCCAATGCTTTGACCTCCAAGrGCTGCCCCCTCAACCACAAATATTGAAACCTCATGATCCCAAAGTTAGCAT CHATLLA5LICCF0LQVLGPQGQILKCD0PICVSM BERACLTVPRAHSLVCVPL ART G V A S K L L S • TAATCATCATATACAATACTACTCrCTTCCCATCCCAGTTGCrTTTTATCrATTCATAATCATCATTTCAATAAGCTGTGACTGGTACTTAATCAACTAA 1900 TTAAGCTTACATAAAAAAAAAAAAAAAAA 1 9 2 9 Figure 4.3. Nucleotide sequence of cDNA and deduced amino acid sequence of P450W. The translation start site ATG is indicated by bold letters, and the termination codon is marked with an asterisk. The circled residues indicates the conserved cysteine, the heme- binding site. The peroxisome targeting tripeptides ARI and SKL are underlined. 103 P450WU AWFLH ] S o y b e a n [j—ICLV------SnVc*AYXLWRKQSKK— ---- ] Arabidopsis SSISQfTLSKLSDPTTSLVIWSLFIFISFITRR-RRPl A v o c a d o —A T T .U ^Ir.P — — rATRT/^^^VT.r.tn'.MkkKR- T o b a c c o LTTYAAVF------LGTLFLLFIiSKL- P450WU S o y b e a n Arabidopsis A v o c a d o QV 126 T o b a c c o P450Wn iIHKFLRVMAQSAEAQKPLDVTEELIiWrNSTISMMMLGE------195 S o y b e a n kklcmsellsgrmmdqfl : tETKEFISRyFRKGVAGBAVDFGDELMTLSNNXVSRHrLSQKTSENDNQ 198 A r a b i d o p s i s RRVCVHKVFS -CNVGKPINVGBQXFALTRNXTYRAAFGSACEKGQD- 203 Avocado RKICVLELLSXKRVNSYRSII c^ P450W11 ------AFMI kn~ S o y b e a n ------AEEMKKU Arabidopsis ------EFIRILQEF! A v o c a d o K NKEADLATEL' T o b a c c o SFVNPEEFKKMLDl P450WU RKNGEWEGEAS— I ETM------EEKXTKEQI S o y b e a n RKNKETGTAKQF— ] ENA------EIKLDKKNI A r a b i d o p s i s KUMuHAVDl SEBAKLVSETADLCtlSXKLTRlXlI A v o c a d o RKANGS DGWEQKDD T o b a c c o R— NGV——! Ir-ADDPKL------EVKLERH P450WU WK- S o y b e a n Arabidopsis XL-HETAEDTSIC A v o c a d o T o b a c c o 4-KATECRENi P450WU XPBGAL' TS 447 S o y b e a n IPAKTRLFVNVWAXj A rabidopsis IPKKSRVMINAFAI Y 466 A v o c a d o IPAKTRVFINAHAZ IS 451 T o b a c c o VQKGTRVLVSVWTl] SLGLK 452 P450WU S o y b e a n Arabidopsis —MKPSE— A v o c a d o -LTKED— T o b a c c o WTPED— Figure 4.4. Comparison of the amino acid sequence of P450W with other plant cytochrome P450s. Amino acid sequences of P450W and of plant P450s from soybean (CYP93A1, GenBank Acc# D83968), Arabidopsis (Ferulate-5-hydroxylase, U38416), avocado (CYP71A1, A35867), and tabacco (HSR515, X95342) were aligned by Clustal method. Residues conserved in all sequences are blocked. The Thr-containing region is indicated by doubled horizontal lines. The conserved threonine residue is embrassed. The most conserved Cys-containing region is under lined and the Cys is indicated by an asterisk. 104 To get the full length cDNA, the nodule cDNA library (LAMDA MAXI) was screened with plaque hybridization method using the truncated SOObp long P450 cDNA as the probe. A full length cDNA (one out of 24 positives) was isolated from 25,000 plaques and its sequence was determined for both stands using a progressive primer design strategy. This full length P450 cDNA contains 1929 base pairs and has a 1533 base pairs long open reading frame, which encodes a protein with 511 amino acid residues with approximate 57 kDa in molecular weight. This P450 protein is named as P450W. 4.4.3 P450W is a novel member of the P450 superfamily The nucleotide sequence and deduced amino acid sequence are presented in Figure 4.3. The ATG codon at nucleotides 258-260 most probably represent P450W translation initiation site. It should be noted a second in-frame ATG (position at 490- 492) occurs 77 codons into the coding region and could also represent the translation start site. The sequence context of the second ATG (GGTACCATGGCA) matches better than the first ATG (TTCACGAT GTTGl to the consensus translation start site (TAAACAATGGCT) defined by Joshi (1987) for plant genes. However, in eukaryotes the first ATG encontered by the translation machinery is usually selected as the initiation site. After the first Met, there is a hydrophobic domain and then followed by a sequence PPG/SPK/PPRPP, which matches the consensus for the proline-rich sequence found in many P450s (Chen and Kemper, 1996). There are 77 mostly conserved residues mainly within two regions: the Thr- containing helix I region near center and the cysteine-containing region near the C- terminal end. Sequence alignment in the primary structure reveals that three Cys- containing regions are highly conserved among mammalian microsomal cytochromes. Among the three Cys-containing region, the one near the C-terminus is the most highly 105 conserved region in all currently known-primary structures of prokaryotic and eukaryotic cytochromes (review, see Makino, 1994). This Cys and the corresponding Cys residues in other cytochromes are suggested to be the proximal thiolate ligand to the heme. From this primary sequence comparison (Figure 4.4), we observed that plant cytochromes contain only this most conserved Cys residue near the C-terminus, which is the presumed heme-binding cysteine. The deduced amino acid sequence of P450W was compared with other P450s (Figure 4.4) showing highly conserved in this heme binding region. This heme-binding domain resides between residues Pro 431 and Gly 441 in P450W, which is identified as cysteine-containing domain including Cys 439 (P450W numbering). The other highly conserved homologous sequence was found in the region of long distal helix I which contains a threonine-containing region (Poulos, 1985; Nelson, 1988). The corresponding sequence is found among residues 297 and 313 (P450W numbering), which is shown in Figure 4.5. The region is believed to be situated in the vicinity of the heme and constmcts the oxygen binding pocket residue 248-249 of P450cam which are always a Gly-Gly or Ala-Gly sequence (Makino, 1994). It has been found that Thr 252, which forms an unique hydrogen bond with Gly 248, this Gly 248- Thr 252 hydrogen bond disrupts the normal helix hydrogen bond pattern and forms the local deformation of helix I which serves as the oxygen pocket (Makino, 1994). The conserved threonine is functionally important in the hydroxylation by P450s. P450W lacks conserved Thr308 in the helix I, instead of Thr308, it is Ser308 (Figure 4.5). This could have a function different from the one which has Thr at this conserved position. From previous study on the mutation of oxygen binding pocket, rat liver P450©-i was mutated from thr252 to His resulting in a complete loss of enzyme activity. When it was replaced with Ser or Asn, the enzyme activity was either partially restored or higher than the original native protein (frnai, 1989). P450cam» changing Thr 252 to Ser 252, showed that the rate of oxygen consumption was reduced about 62% from the native type, that 106 H%02 generation was 15% for Ser in compared with 3% in the native type and that the ROH generation was 85% compared with 97% in the native type (Makino, 1994). Its reaction suggests that there is not complete dependence on O2 Also, more than one Thr in this helix I region imphed a broad substrate of P450W. * ** * * P450 Wu V D F F SA GT D g3oa T A V A T E W Tobacco HSR515 Q DM LAGGT E S 3 0 9 S A V TV EW Soybean CYP93A1 MD I F V A GT D S A V S I E w Arabidopsis FAHl M D V M F GGT E T 3 2 4 V A S A I E w F3.5H LN LF T A G T D T*®* S SS A I E w P450cam G LL L V G G L D 7 2 5 2 V V N F L S p Flaxseed AOS CF N S W G GF K L F P S LM K Guayule RPP CF NT F GGV K X 2 8 3 L FP N T MK Figure 4.5. Amino acid sequences of P450s in the distal helix region. The primary sequence comparison of threonine-containing region in the distal helix I. Tobacco HSR515, hypersensitive response protein; Soybean CYP 93A1, jasmonic acid induced P450; Arabidopsis FAHl, ferulic acid 5-hydroxylase {Arabidopsis thaliana)-, F3.5H, flavonoid-3', 5'-hydroxylase {Petunia hybrida) ; P450cam, the first isolated P450 from Pseudomonas putida; Flax seed AOS, flax seed anllene oxide synthase; and Guayule RPP, rubber particle protein. The position for Thr is indicated by the numbered residues at this site in its protein sequence. The conserved region are indicated by asterisks. 107 4.4.4 P4S0W contains a peroxisomal targeting motif and is a membrane bond protein Among these known plant P450s, P450W shows the most sequence homology with the jasmonic acid-induced soybean CYP93A1. However, the percent of similarity between these two proteins is 35% which is less than the threshold value of 40% that delineates P450 families (Nelson, 1993). We analyzed the phylogenetic relatedness of P450W with other closely related P450s available in Swiss-Prot databank. A subset of 18 sequences were aligned by the cluster method. The data demonstrates that P450W is a novel P450 among other known P450s (Figure 4.6A). Another interesting feature is that the C-terminus of P450W contains a peroxisomal targeting signal SKL (Ser-Lys-Leu) within the last five amino acids. According to the degenerate forms of peroxisomal targeting sequence S/A/C/- K/H/R/-I/L (Gould, 1990), another peroxisomal targeting analog ARI is found residing within the last 11 amino acid residues of the C-terminus (Figure 4.2). Therefore, these sequences likely serve as the topogenic information for P450W targeting to peroxisomes. P450W N-terminus contains an acidic residue (E) at 4th position which is subsequently followed a cluster of a hydrophobic domain (15 residues) and then a basic amino acid rich domain. This N-terminus feature of P450W seems to fit well with the common feature of the microsomal P450s which contains 25-35 amino acid residues in the amino-terminal region: an acidic residue closely neighboring the amino-terminus is followed by a cluster of hydrophobic residues and a basic residue is not found until the 20-35th residue (Imai, 1993). The hydrophobic N-terminus may serve as a transmembrane anchor according to a previous smdy (Nelson and Strobe, 1990). The P450W's hydrophobicity profile is similar to the other membrane-bound P450s. Figure 4.6B represents the hydrophobicity of P450W, a highly hydrophobic domain resides at the N-terminus. 108 Avocado CYF71A1 ^ g p lan t CVP71A1 ZM CYP71C1 ZM CYP71C4 Potato CYP Arabidopsis FAHl Tobacco H9^515 Tobacco MO CM-HYCRO Human CYP1A PIG CYP1A2 Human CYP450c ftbbit CYP1 CcCVPIA Scup CYP1A Soybean CYP93A1 Soybean CYP93A2 P450W 64.0. —r —T" T" —r —r —r 60 50 40 30 20 10 Figure 4.6. The phylogenetic trees of P450W with other P450s and the hydrophobicity profile. A. The phylogenetic trees of 17 P450 proteins calculated by the CLASTAL method. These protein sequences are released from GenBank or Swiss-Prot, their access number is indicated. Avocado CYP71A1, (GenBank Acc#) A35867; eggplant CYP71A1, X70981; ZMCYP71C1, Zea mays, X81827; ZMCYP71C4, Zea mays, X81831; potato CTP, U48434; Arabidopsis FAifi, U38416; tabacco ftSF5i5, X95342; tobacco MO, P450-dependent monooxygenase, X96784; CM-HYDRO, flavonoid 3', 5'-hydroxylase D 14590; Human CYPIA, X02612; pig CYP1A2, D50457; rabbit CYPl X05685 CcCYPlA, teleost, U19855; scup CYPIA, U14162; soybean CYP93A1, D83968 soybean CYP93A2, D86351; P450W, this study. B . The hydrophobicity profile of P450W. N-terminus contains a highly hydrophobic domain which may serve as a membrane anchor. 109 Figure 4.7. RNA blot analysis of P450W gene expression in soybean tissues. The RNAs were isolated from soybean different tissues are presented from left to right. Lane 1, 2 and 3 are RNAs from cotyledons of 1,2 and 4 days old seedlings grown in the dark; Lane 4, 10-day old seedling stems. Lane 5, 10-days old leaves; Lane 6, 7, and 8 are the RNAs from nodules of 10, 20 and 35 days old plants; Lane 9 is 1-day old root radicles; Lane 10 is two-day old cotyledon-free seedlings; Lane 11, 12,13 are the roots of four, five and seven days old plants. 1 1 0 4.4.5 Expression of P450W correlates with purine metabolism in plants From Northern blot data (Figure 4.7), P450W is highly expressed in the germinated seed cotyledon, radicals, root, leaf and mature nodules where HR and peroxisomes are dominated. There was no expression of P450W in soybean stem tissues. The soybean radicles showed the highest expression rate within the root tissues. The expression of P450W varied in soybean nodules. In the early nodule formation stage, P450W was expressed at a very low level and was induced during nodule maturation and nitrogen fixation, and declining in the late nodule stage. Generally, the cotyledon and root tissues are rich with glyoxysomes, the leaf is rich with leaf peroxisomes and nodules are full of nodule peroxisomes. Within these tissues cells are undergoing at a high development rate which must require a high rate of purine metabolism. It is true that purine metabolism is carried out at a high rate during cell division and growth, as well as in the nitrogen fixation. This data showed that the tissues are dominant with P450W generally in the high all growth rate or contains high population of peroxisomes. P450W, therefore is corresponding to the purine and nitrogen metabolisms. In the purine metabolism pathway, xanthine is produced and released into cytosol, oxidized into uric acid which is further oxidized into ureides by urate oxidation enzymes. I l l É H Peroxisomal Fraction Q Soluble Fraction 2P 7.5 Figure 4-8. Peroxidase and uricase activities of P450W expressed in yeast cells. Cell-free extracts were prepared from the yeast wild-type, Spbl, Spbl with pYEUraS and Spbl with pYP450W after induction by uric acid as the sole nitrogen source, respectively. Peroxisomal fraction and soluble fraction were separated by centrifugation at 20,000g for 30min. The enzyme activities of (A) peroxidase, and (B) uricase activity were determined for the peroxisomal fractions (dark bars) and the soluble fractions (light bars), respectively. 112 10 Q Peroxidase $ ■ Uricase 5 - B e t 2 .5 - Î 0 J ± L _ ^ control P450 * Peroxidase activity = nmoles products formed/mg x min. Uricase activity= xlO nmoles of uric acid degraded/mg x min. B M A B 9 6 66 . 5 7 . 4 6 3 2 1 Figure 4.9. SDS-PAGE analysis of P450W expression in E. coli and the enzyme assays. A. Cell-free extract was prepared from the cell cultures with or without IPTG induction. The enzyme activities were determined with 5mM H2O2. Peroxidase (white bars) and uricase activities (black bars). B. 10% SDS-PAGE gel of electrophoretic analysis of P450W expression. M. represent the standard molecular weight marker; A. with IPTG induction; B. without IPTG induction. The arrow is indicating the band of P450W which has a proximate MW of 57 kDa. 113 4.4.6 P450W has peroxidase activity The preliminary enzyme assay was determined by measuring the catalase activity as P450W complimented the yeast cells. Cell cultures were induced by uric acid- containing NUGY liquid medium and induced for 16 hr. The Spbl with P450W expression exhibited significantly high level of H2O2 decomposition measured spectrophotometrically. This observation implied that P450W could decompose H2O2 as a peroxidase. The peroxidase and uricase activities were determined and the data are shown in Figure 4.8. Spbl complemented with P450W cDNA demonstrated significantly higher peroxidase activity than that of wild-type and mutants in the peroxisomal fractions (Figure 4.8A). Apparently, soluble fraction of wild-type cells contained higher peroxidase activity than that of the mutant with pYP450W. Over expression of P450W in Spbl mutant, the peroxisomal fraction demonstrated higher peroxidase activity than that of soluble fractions of both wild-type and mutant. This implied that P450W protein mostly impacted on the membranes. Thus, increased enzyme activity of membrane fraction truly presented the feature of this protein. Obviously, the uricase activity was also increased in the microsomal fraction of the mutant with pYP450W expression (Figure 4.8B). The uricase activity of peroxisomal firaction of mutant is lower than that of wild- type cells. However, the soluble fractions which represent the cytosol fraction show higher enzyme activity of uricase, indicates uricase is mislocalized in the cytosol in the mutant cells and the uricase expression is not defective. This result also agrees the previous findings about Spbl which is defective in the peroxisome proliferation. Another study was performed to determine the peroxidase activity in E. coli of P450W expression. P450W expressed by the pRSETA Express system™ (Invitrogen) showed high uricase activity and as well as peroxidase activity when H2O2 was provided in Figure 4.9A. The specific activity of uricase is higher than that of peroxidase. The 114 activity of P450W expressed in E. coli was detected by SDS-PAGE gel. The protein has 57 kD molecular weight according to the SDS-PAGE (Figure 4.9B). The optimum pH is 7.0 based on the peroxidase enzyme assay. 4.4.7 Expression of P450W confers H2O2 tolerance in yeast With the observation of P450W acting as a peroxidase in S. cerevisiae, a further experiment was carried out to establish the role of P450W in vivo. In this experiment, the effect of growth of yeast by different H2O2 concentrations (0-5 mM) was determined. The ability of cell growth was measured by spectrophotometer at an absorption of 600nm. Results obtained from this experiment clearly showed differences within the wild-type, mutant, the mutant with expressing P450W (Figure 4.10). All strains, except mutant, grew equally well in the media contaiiting H2O2 up to 2mM. The growth ability of wild-type and wild-type with pYEUraS stains declined significantly when H2O2 concentrations exceeded 2mM. In contrast, the mutant with P450W expression could grow just as well in an H2O2 concentration of 0-4 mM, but grew poorly when H2O2 was at 5mM which seemed to be the threshold level of H2O2 for cell growth with pP450W expression. The mutant showed no obvious growth in any of these media. 115 8 < 0.5 - G 1 2 3 4 5 6 The concentration of H2O2 (mM) Figure 4.10. Spbl expression of P450W growth in H202-containing medium. The growth abilities of yeast cells were determined by the absorbance of OD at 600 nm. The liquid medium contained uric acid as the sole nitrogen source amended with H2O2 of 1,2, 3,4 and 5mM, respectively. The tested cells were wild-type (-0 —), wild-type with pYEUraS (-» -), mutant Spbl(-A—), and Spbl with pYP450W (-Ar-). 116 4.5 Discussion In eukaryotes, the cytP450 hemoproteins are membrane bound and often inactivated by proteolysis and oxygen (Donaldson and Luster, 1991). For all these reasons, cytP450s have been difGcult to isolated and characterize. Using yeast mutant to clone plant gene is potentially a way to smdy gene-encoding membrane proteins which are present in very low abundance and often difGcult to purify. It is noteworthy that gene function can be received through funcGonal complementation approach, while genes identiGed by using PCR-based approaches usually represent an unknown function. 4.5.1 A putative peroxisomal cytochrome P450 isolated from soybean A soybean cytP450 cDNA was isolated by functional complementation with a yeast S. cerevisiae mutant. P450W shows homology with cytochrome P450s and contains the heme-binding domain which is highly conserved among known P450s. A peroxisomal targeting signal residing near C-terminus and a highly hydrophobic domain of N-terminus suggests that P450W is a membrane-bound protein with the C-terminus flowing inside peroxisomes. Since the amino acid sequence of P450W shows less than 40% similarity with corresponding sequences of other known P450s, its cDNA may represents a new member of the P450 gene family and encodes an H202-dependent peroxidase involved in urate oxidation. Northern blot data suggested that P450W is a constitutive protein existing in cotyledons, roots, leaves and nodules where peroxisomes are the most abundant among the plant tissues. P450W could be a membrane protein in cotyledon glyoxysomes, leaf peroxisomes and nodule peroxisomes. P450W has a molecular weight of 57 kDa. Previous studies have shown that a 56-57 kDa protein has been found to exist in all kinds of peroxisome membranes in several plant peroxisome and glyoxysome membranes 117 (Liang et ai., 1993; del Rio et al., 1992 ). Further, from the previous study on castor bean glyoxysomes, it was discovered that cytochrome bs and a P420 (the denatured form of P450) are in the glyoxysome membrane and as well as NADP oxidation enzymes, it is very likely that P450W is a peroxisomal protein. P450W contains a putative peroxisome targeting signal residing near the C- terminus of the protein. A C-terminus tripeptide, acting as peroxisome targeting signal, has been defined by Gould et. al (1989).. The tripetide signal Ser-Lys-Leu (SKL) is degenerate and conforms to the consensus sequence S/A/C/-K/H/R-L/I which naturally occurs in a variety of peroxisomal proteins at or near the C-terminus (Gould, 1989; Hayashi, 1997). SKL which acts as a peroxisomal targeting signal in soybean has been found in the soybean uricase C-terminus (Nguyen, et al., 1985). Many peroxisomal proteins containing this targeting signal at an internal position also act as topogenic information. The targeting signal of firefly luciferase resides at the 15th amino acid from the C-terminus (Gould et al., 1987). Yeast Candida boidinii PMP 20 contains a peroxisomal targeting signal within its C-terminus 12 amino acids (Gould, 1989). The carboxyl terminal 27 amino acids of human catalase that are able to direct a heterologous protein into the microbody contain an internal Ser-His-Leu (Subramani, 1992). In this case, it has been suggested that the internal tripeptide, acting as peroxisomal targeting signal, may be dependent on the context of the adjacent amino acid sequences. This P450W was found to contain a SKL sequence located 2 and a degenerate form of ARI sequence located 8 amino acids from the carboxyl terminus, respectively (Figure 4.2). Within the last twenty amino acids, fifteen residues are conserved for the targeting signals according to S/A/C/-K/H/R/-L/I peroxisomal targeting analogs. The C- terminal region of P450W acting as peroxisomal topogenic information is strongly suggested. However, a definite conclusion must await a direct evidence by in vivo import system or immunolocalization methods. The features of P450W suggest that its 118 C-terminus guide protein into peroxisomes facing to the peroxisome matrix, and its hydrophobic N-terminus serves as a membrane anchor which hooks onto the peroxisome membranes. 4.5.2 P450 H202-dependent hydroxylation activity is possibly regulated by the O2/H2O2 ratio Both serine and throenine residues contain a hydroxyl group suggested to be important in hydroxylation activity (Mokino and Shimada, 1993). The existence of a Thr-rich region is commonly found in many known P450s (Figure 4.5). Like other known P450s, P450W contains the most conserved Cys-containing heme-binding domain. However, P450W contains a Ser instead of TTir residue in the conserved Thr- containing helix I region which is known to form the oxygen pocket. An interesting connection has been made is that the number of Thr-residues in the helix I region may reflect the catalytic specificity of P450 enzyme activity. P450cam has only the conserved Thr in this region and exhibits strict substrate specificity and regioselection (Mokino and Shimada, 1993). It can be found firom the sequence alignment that most plant P450s contain more than one Thr-residues surrounded by Ser-residues. The implication is that plant P450s may have a broad range of substrate specificity. Plant P450s cloned thus far are involved mostly in phenopropanoid pathway. The specificity of some P450s to their substrates are not quite clear (Donaldson and Luster, 1991; Akashi et al., 1997). Changes of amino acid at Thr282 of P450cam have been found to greatly influence enzyme activity. Site-directed mutagenesis on the oxygen pocket of P450cam demonstrated that replacement of a hydrophobic amino acid with Thr can abolish the enzyme activity (Mokino and Shimada, 1993). Replacement of Ser with Thr in the helix- I region dramatically reduces the oxygen consumption by about 40%. Oxygen consumption can be further reduced to 72% by replacing Val to Thr. 119 Recently, a study by Pang et ai. (1995) showed that rubber particle protein, a homology with P450, lacks threonine in the helix-I region, which shows high homology with AOS. AOS and RPP both lack the conserved Thr, but instead contain De in the helix-I region. Their enzyme activities do not require molecular oxygen nor NADPH- dependent cytochrome reductase. However, the activi^ of AOS (allene oxide synthase) has been shown to be H202*dependent and does not require consumption of oxygen (Song, 1991; Song, 1993). This study demonstrated that expression of P450W could endorse cells to growth under high H2O2 concentration. Based on the feature of helix-I region of P450W, a possible hypothesis about its function is postulated. P450, behaves as a monooxygenase or a H202-dependent peroxidase, likely is regulated by the intracellular molecular ratios of oxygen and H2O2 Possibly the enzyme activity does not completely require molecular oxygen or a NADPH-dependent cytochrome P450 reductase. When oxygen is avaüable, P450 may behave as a monooxygenase as an electron acceptor, however, when H2O 2 is high, P450 is likely to behave as a peroxidase. Therefore, the mode of reaction of P450 may be regulated by the ratio of O2/H2O2 inside the peroxisomes. It is known that the ratio of P450 and NADPH cytochrome P450 reductase is 20:1 in the micorsomal membrane (Takemori et al., 1993). Hence, these two kinds of activities could exist in the peroxisomal P450s. 4.5.3 Catabolism of uric acid through the alternate pathway Early study has detected two types of uricase activities during development of soybean roots: one in young seedlings and ± e other in nodules (Tajima, 1975). They show, however, no common physical or chemical properties (Bergmann, 1983). 120 Urate oxidase +Catalase : Urate + 2H%0 + 0% ^ Allantoin +H2O2 +CO2 H2O2 -----► H2O + I/2O2. (I) Diamine Oxidase +Peroxidase: RH2NH2 + H2O ^ RCHO +NH3 +H2O2; Urate+ H2O2 ' ^ Allantoin+H2O. (2) In root nodules, urate oxidation is generally catalyzed by a uricase using the uricase and catalase enzyme system in peroxisomes (Equation 1) which are widely distributed in bacteria, yeast, plants and animals (Tajima, 1975; Tanaka et al., 1977; Voet and Voet, 1990; ). Whereas, a urate degrading enzyme system has been found in soybean radicals (Equation 2), and contains a diamine oxidase and a peroxidase (Tajima, 1975). The urate degrading activity was found to be localized in roots throughout their growth period and regulated by a cofactor (cadaverine). The diamine oxidase catalyzes the conversion of polyamine and amines into aldehyde, ammonia and hydrogen peroxide (Mclntire, 1993). The peroxidase degrades uric acid by using the hydrogen peroxide as an oxidant. Therefore, expression of H202-dependent peroxidase may confer the ability of the cells to grow on urate, even in the absence of functional peroxisomes. This would allow rescue of a non-uric acid-utilizing mutant. Mutant Spbl has lower catalase, uricase and peroxidase than that of wild-type strains. Spbl has been identified as a mutant, defective in peroxisome proliferation due to a transcriptional factor mutation (chapter 2 and 3) which likely causes the deficiency of proliferation of peroxisome as well as induction of some peroxisomal proteins (Kos et al., 1995; Karpichev et al., 1997). This functional complementation did not occur at the gene level, but likely happened at the enzyme level through a functional typass' made possible by the low catalase and uricase activities natural to this mutant phenotype. 121 Results of this study show that uric acid can not induce peroxisome proliferation and catalase activity, but does induce uricase activity. Induction of peroxisomal enzyme by a substrate or a proliferator is accomplished via different mechanisms (Kos et al., 1995; Karpichev et al., 1997). Without the catalase activity, uricase activity could be inactivated by the H2O2 or cells may die due to the lethal level of H2O2 generated by urate oxidation (equation 1). Unlike uricase, P450W catalyzes uric acid by consumption of the same amount of H2O2 (equation 2). 4.5.4 Xanthine is a peroxisome proliferator Peroxisome proliferators are a group of structurally diverse compounds that cause a dramatic increase in both number and size of the peroxisome as well as the induction of peroxisomal enzymes (Hess et al., 1985). For example, marker enzymes such as catalase and uricase can be induced by a peroxisome proliferator (Amaiz, 1995, ). When 5. cerevisiae is grown on oleic acid as the sole C-source, a remarkable peroxisome proliferation occurs. As a result, there is a considerable induction of peroxisomal enzymes in yeast. Oleic acid is thus far considered the only peroxisome proliferator in 5. cerevisiae (Gould, 1992). Enzymatic analysis of S. cerevisiae after induction by xanthine as the sole nitrogen source, demonstrated 3.5- to 4.0- fold increase in catalase and uricase activities. These enzyme activities were induced at about the same time and at about the same level. This suggested that the increased enzyme activities may be accounted for by the increase in peroxisome number and size of individual peroxisomes (Amaiz, 1995; Tamura, 1990). Our results demonstrated that xanthine can be an inducer of peroxisome proliferation in S. cerevisiea . We further suggest that xanthine may function as a peroxisome proliferator in soybean nodules during nodule formation and nitrogen fixation. 122 Uric acid has been identified as a good antioxidant which accumulates inside peroxisomes when uricase is absent (Ames et al., 1981). Tan et al.(l993) found that uric acid can significantly decrease the oxidation of xanthine to uric acid and O2" formation. Uric acid is an efiective inhibitor of the formation of superoxide and hydrogen peroxide by xanthine oxidase as found in human plasma. In tropical legumes, purine biosynthesis is highly induced during nodule formation and nitrogen assimilation, as well as purine oxidation (Kim, 1996). As a result of purine biosynthesis, hypothanthine and xanthine production are much enhanced. According to a resent study, xanthine oxidase is identified as a peroxisome matrix protein (del Rio et al., 1992) which converts hypoxanthine into xanthine and xanthine into uric acid. Thus, hypoxanthine and xanthine likely serve as peroxisome proliferators since they both are substrates of xanthine oxidase. Nodule-specific enzyme, nodulin-35, a subunit of uricase, is highly induced during nodule formation, and such induction is coupled with purine biosynthesis ( Kim, 1996). H2O2 concentration can become dramatically high inside peroxisome due to purine oxidation as shown in the purine metabolism pathway in Figure 1.10. Therefore, H2O2 and O2" together may cause highly oxidative stress inside peroxisomes. This oxidative stress, in turn, may cause peroxisome proliferation (Palma et al., 1991). The excess H2O2 and O2" permeate through peroxisome membranes into cytosol (Boveris, et al., 1972; Heupel, et al., 1991). 4.5.5 Ureides formation by diamine oxidase-peroxidase enzyme system may be involved in nitrogen supplement during seedling growth or under stress conditions. Allantoin formation occurs in the root portion of many plants (Mothes, 1961, Tajima, 1977), though its mechanism is unclear. We suggeste that nitrogen supplement is provided by the diamine oxidase and peroxidase enzyme system in plants under developmental and environmental stress conditions. Allantoin generation in soybean root 123 radicles is catalyzed through aa alternative pathway which is regulated by a polyamine, cadaverine. Whereas, this enzyme system is only activated when the catalase activity is low (Tajima, 1985). Catalase is the key enzyme to scavenge the H2O2 both in peroxisomes and in cytosol. A common feature has been shown that catalase activity is inhibited in plants under stress conditions such as heavy metals, salinity, and pathogen attack (review, see del Rio, 1992). Interestingly, that catalase activity remains low during soybean seedling growth (Tajima et al., 1985). Polyamine levels are known to regulate cell growth and development. In growing seedlings where cells are actively dividing, purine biosynthesis and metabolism are also important. A high content of cadaverine enhances the urate degrading enzyme system. The diamine oxidase-peroxidase urate-degrading system may carry out other important cellular functions during soybean seedling growth apart from nitrogen supplies since it is regulated by polyamines. When a mature soybean plant (6-weeks-old) was cut off at the uppermost portion of the hypocotyl, just below the cotyledons, urate degrading activity was elicited in the root (Tajima, 1977). The increase in the degrading activity paralleled the increase in the cofactor. It is wound inducible. Polyamine is associated with plant defense and wound resistance. An early study with pea seedling extract, showed that peroxidase with the presence of cadaverine can inhibit the amine oxidase activity. The mechanism of this enzyme inhibition was thought to be due to peroxidase degraded products of the intermediates catalyzed by amine oxidase, but other amines are not afrected by this peroxidase. However, Tajima's study (1987) in the soybean radicles, found that cadaverine regulates the peroxidase and diamine oxidase urate-degrading enzyme system. That soybean radicles contain such unique urate-degrading enzyme system must have its 124 unique cellular function during plant development A human diamine oxidase-peroxidase enzyme system reported is found involved in anti-virus processes (Klebanoff and Kazazi, 1995). In conclusion, this study suggested that polyamine, amine oxidation and urate oxidation are catalyzed under a diamine oxidase-peroxidase enzyme system within plant peroxisomes which may be involved in regulation of cellular proliferation, cellular toxicity, oxidative stress, and plant cell defense during seedling growth. 125 CHARPTER5 ISOLATION AND CHARACTERIZATION OF A SOYBEAN PEROXISOMAL AMINE OXIDASE cDNA 5.1 Introduction Polyamines are found to play an important role in plant growth and development, from organogenesis to protection of plant against stresses (Bagni et al., 1982; Smith, 1985). Increase in polyamine biosynthesis activities have been observed in a variety of developmental and stress situations, such as cell division, fruit ripening, senescence, chilling injury, UV-B radiation, salt, osmotic and ozone stresses (Evans and Malmberg, 1989; Maccarron et al., 1997; Tiburcio et al., 1994; Kramer et al., 1992; McDonald and Kushad, 1986; Galiba et al., 1993). Corresponding changes of polyamine levels and spectra during a response or a developmental event are generally observed . For example, high levels of free polyamines appears in the cells undergoing division, whereas, low level appears when cells undergoing expansion (Galston, 1995). Apparently, polyamine are present in all plant cells, in free, soluble conjugated or bound conjugated forms. The soluble conjugated polyamines can represent up to 90% of the total polyamines content, and generally are in high content in young tissues and decreasing with tissue development and maturiQr (Slucum and Galston, 1985; Rastogi and Davies, 1991). Thus, it is believed that the alternation cellular level of polyamines 126 may correspond to the changes in the concentration of the enzymes responsible for biosynthesis and catabolism of these amines (Seiafini-hracassini, 1991). Polyamine biosynthesis has been relatively well understood, whereas, the control of turnover of polyamines by polyamine and diamine oxidase is less understood. A resent study has shown that ozone induced polyamine accumulation can be due to the down regulation of amine oxidase (Maccarrone et al., 1997). Copper/topa quinone amine oxidases (EC 1.4.3.6) are widely distributed in all kind of living organisms from prokaryotic bacteria to eukaryotic cells, fungi, plants and animals (Mclntire and Hartmemn, 1993). It catalyzes oxidative deamination of a wide variety of biogenic mono-, di- and polyamines to corresponding aldehydes, ammonia and hydroperoxide as for the following Equation 1 (Mclntire, 1992). RCH2NH2 + O2 ► RCHO + NH3 + H2O2 (1) Copper-containing amine oxidases are homodimers of 70-95 kDa subunits, each subunit contains a tightly bound Cu(II) and a quinone cofactor, 6-hydroxydopa (TOPA) formed tyrosine(Mu et al, 1992; Tanizawa et al, 1994; Matsuzaki, 1994). This active- site cofactor is formed posttranslatonally with the modification of tyrosine to topa quinone through a common pathway existing in all organisms (Cai and Klinman, 1994; Choi, 1996). In mammalian cells, peroxisomes contain polyamine oxidases which convert spermine and N-acetylspermine to spermidine, and spermidine and N-acetylspermidine to putrescine (Beard etal., 1985). Whether the enzyme plays an active role in the regulation of cellular polyamine levels is unclear (hayashi et al., 1989). Also, copper- containing amine oxidases are peroxisomal enzymes in some fungi and yeast (Bruinenberg, et al., 1989; Haywood and Large, 1981). In microbes as well as yeast. 127 amine oxidases could allow cells growth on amines as a sole nitrogen source. In plants, whereas, peroxisomal or interacellular amine oxidases have not yet been reported. Plant copper-containing amine oxidases (diamine oxidase, DAO) have been studied are mostly found in the apoplast and loosely associated with the cell membranes and walls (Federico et al., 1985; Smith, 1985). In many tissues, both DAO and polyamines are dominant in the apoplast (Slocum, 1985), therefore, the physical functions of DAO is believed to provide H 2O2 in the cell wall for a peroxidase-dependent cell wall lignification and stiffening (Angelini, et al., 1990; 1993). Recently, a lentil seedling amine oxidase (LSAO) is found to be responsible for the catabolism of intracellular poly amines (Lahiri, 1992). It is suggested that the functions of in cell wall stiffening (Robert, 1984), plant growth regulation (Saftner 1989) and response to stress factor (Saftener, 1990) are through the control of intracellular level of polyamines. In soybean radicles, a urate-degrading diamine oxidase-peroxidase enzyme system is found to catalyze the conversion of urate into allantoin, the final step of purine metabolism which is generally mediated by a uricase in peroxisomes (Tajima, et al., 1985). The content of cadaverine, a diamine, provides an alternate route catalyzed by diamine oxidase (Tajima, et al., 1983). The hydrogen peroxide derived from cadaverine oxidation accelerate the peroxidase activity of urate-degradation (Tajima, et al., 1985; Tajima and Yamamoto, 1977). Our study has shown a cytochrome P450 is the corresponding peroxidase that catalyze the H 2Ü2-dependent urate degradation in soybean peroxisomes. In the present study we have isolated and identified a copper-containing amine oxidase from soybean cDNA library through functional complementation with S. cerevisiae . Based on the deduced amino acid sequence, this protein contains a typical peroxisomal targeting signal (PTSl) at its C-terminus which likely serves as the topogenic signal. This finding further confirmed our study that the existence of urate 128 degrading system is present in soybean peroxisomes. Therefore, plant peroxisomes containing amine oxidation like its animal and yeast counter part is found by this study. Thus, our results identified and characterize the first plant peroxisomal amine oxidase. It is likely that the intracellular levels of polyamines are regulated by the peroxisomal amine oxidase which may play a key role in generation of intracellular signals to regulate plant growth and development. Polyamines are known to be the major nitrogen source stored in plant seeds. Ureide generation correlated with polyamine oxidation through urate-degrading diamine oxidase-peroxidase enzyme system may provide a nitrogen source during cell growth and development. A peroxisomal copper amine oxidase isolated in this study could shed the light of getting insight of the mechanisms of polyamine in the regulation of plant growth and development. 129 5.2 Materials and Methods 5.2.1 Functional Complementation The S. cerevisiae mutant Spbl was used for functional complementation with a soybean nodule cDNA library (in LAMDA MAXI) as described in chapter 4. Uric acid selective medium (NUGY) contained 0.17 nitrogen base without amino acid and ammonium sulfate, 2% galactose, 2.5mM uric acid (added after autoclaving), 0.05% yeast extract, 2% agar, and amino acids 50pg/ml of lysine, 40pg /ml of histidine, 40pg /ml arginine, 50|ig/ml leucine, 50pg/ml phenylalanine, 50p.g /ml threonine, 40p,g/ml and 50|ig/ml tyrosine. 5.2.2 Soybean nodule cDNA library screening and DNA sequencing A cDNA fragment isolated by the functional complementation was sequenced by progressive sequencing on the both strands using Sequenase 2.0 (United State Biotechemical). The cDNA insert (Xbal and Xhol fragment) was used as a probe to isolate its full length copy. A unidirectional cDNA library constructed from RNA of 3- week-old soybean nodules in A, Zap II (Delauney and Verma, 1990) was screened using the 32p-iabeled cDNA. Hybridization was carried out followed by the standard method (Sambrook, 1989). Positive clones were converted into recombinant pBluescrip phagemids by in vivo excision (Short, et al., 1988), and the full length cDNA was isolated. 5.2.3 Sequence alignment, comparison and phylogenetic trees Multiple amino acid sequences were aligned by using CLUSTAL method of DNA-STAR computer program. The multiple alignment used to construct 130 phylogenetic trees were made with CLUSTAL method. Hydrophobicity was calculated by DNA-strider program. 5.2.4 RNA isolation and Northern hybridization Total RNAs from soybean tissues were prepared by hot phenol treatment (Verwoerd et al., 1989). Soybean seeds germinated for one, two, four, five seven and ten days. Tissues from one-, two- and four-day old cotyledons, ten-day old stems and leaves, root nodules at different stages, one-day's germinated radicles, two day’s germinated cotyledon free seedling, four-, five- and seven-day old roots were used. Northern blotting was performed at 68 ^ C using DNA probes (pBl-SPAO Xbal-Xhol fragment) labeled with a32p_jATP following the method described by Mahmoudi and Lin (1989). For comparison of RNA expression levels, a pea small rDNA was used as internal control. 5.2.5 Expression of soybean peroxisomal amine oxidase (SPAO) cDNA in E. coli SPAO cDNA was constructed in pRSET expressing system. The BamHl and Xhol of SPAO cDNA fragment was cloned into the BamHl/Xhol site of bacterial expression vector pRSET C (Invitrogen) to give plasmid pESPAO. pESPAO was transformed in to a E. coli strain lys B21(ChlO- The transformed cells were grown in LB medium containing ampicillin 75-100 p.g/ml and chloramphenicol 30iig/ml. Proteins were induced by adding 0.5mM IPTG and 0.5mM CUSO 4 and incubated in 25°C for 5 hrs at 250 rpm. Cells were then collected by centrifugation and washed with ice cold water. Finally, cells were suspended in 0.1 M potassium phosphate buffer containing 0.5mM CUSO 4, pH 7.2 (2 v buffer /v cell). Cells were broken by sonication. The unbroken cells and cell debris were discarded after centrifugation at 5,000 rpm for 131 10 min. The supernatant was collected and stored on ice for enzyme assay. The level of protein expression and molecular weight of fusion protein were determined by SDS- PAGE following a standard procedure using 15% gel. 5.2.6 Enzyme assay Amine oxidase activity was assayed at 37° C using benzylamine as the substrate. lOp.1 of cell extract containing about 20^g protein was added to 5mM benzylamine hydrochloride (Sigma) in O.IM potassium phosphate, pH 7.2, and product benzyldehyde formation was measured spectrophotometrically by monitoring increase in absorbance at 250nm (E=12.8mM*lcm’^) (Cai and Klinman, 1994). Protein concentrations were determined using the Bradford protein assay (Bio-Rad) with bovine serum albumin as a standard. The ability to catalyze different substrates by SPAO was measured by a peroxidase coupling methods described by Trivedi (1978) and Tissier (1994) with some modifications. The reaction mixture consisting of 100 mM K 2HPO4, pH 7.2, 8 U/ml horse-radish peroxidase, 25mM p-hydroxybenzoate, l.OmM 4-amino-antipyrine, ImM cadaverine, or putrecine, spermidine, benzylamine, lysine and uric acid, respectively. The reaction was carried out at 37°C. The reaction mix was prewarmed in 37°C for Imin, and then added lOfxl cell extract in the reaction mixture. The absorbance 500nm was measured as the starting point, then keet the reaction at 37°C water-bath for 4min and measured the absorbance as the stopping point. The enzyme activities were determined by the difference of the absorbance. 132 5.3 Results 5.3.1 Isolation of a soybean peroxisomal amine oxidase (SPAO) via functional complementation Early study has shown that a copper amine oxidase gene from a methylotrophic yeast Hansenular polymorpha has been successfully expressed in S. cerevisiae under the control of the AD HI promoter (Cai and Klinman, 1994). Some yeast, like H. polymorpha, is capable of utilizing amine as the sole nitrogen source, multiple amine oxidase can be induced by a single type of amine (Green, et al., 1982; Zwart, 1983). Unlike H. polymorpha, S. cerevisiae cannot utilize monoamines or diamines as a nitrogen source to support cell growth (Large , et al., 1986; Cai and Klinman., 1994) because it lacks any endogenous amine oxidases and any quinoproteins (Cai and Klinman, 1994). S. cerevisiae therefore provides an excellent background for the complementation of heterologous amine oxidase gene. This study was initially designed to select the genes involved urate degradation or a gene involved in peroxisome biogenesis. A peroxisomal biogenesis mutant Spb 1 isolated from previous study can not utilize oleic acid as sole carbon source or uric acid as sole nitrogen source due to a defect in a putative transcription factor invovled in the synthesis of peroxisomal proteins. As result it causes a defect in peroxisome proliferation as well as some peroxisomal enzyme deficiency. The mutant strain carrying the auxotrophic markers ura3, lys2 and his3 was transformed with a soybean cDNA library and Ura+ colonies were selected on SG minimal medium amended with lysine and histidine. Those colonies that contained plasmid were then subsequently transferred onto uric acid selective medium (2.5 mM uric acid) which contained lysine, histidine and other amino acids (totally up to 2.5mM). 133 mut-i-SPAOi 'V ' I . w t| m ut ? mut+pYEUra3 Figxare 5.1. Functional complementation of Spb 1 mutant with pYSPAO; growth on uric acid-amino acid amended medium. In contrast to the wild-type strain (wt), the Spbl mutant (mut) show no growth. Mutant with pYSPAOi (mut+pYSPAOi) is able to grow and form small size colonies. Expression of vector pYEUraS (mut+pYEura3) did not rescue the mutant. 134 Two positive clones showed growth on the uric acid selective medium but not on oleic acid-containing medium were isolated. One of these plasmid was shown to contain a 1.2 kb long cDNA insert. The retransformation by this plasmid was confirmed by the complementation (Figure 5.1). The DNA was sequenced from both ends. The deduced amino acid sequence of the cDNA fragment encoding a polypeptide showed similarity to a truncated copper-containing amine oxidase with C-terminal region which contains a typical peroxisomal targeting signal SKL-COOH at C- terminus. Thus this incompleted peptide appears to be a peroxisomal protein which is named soybean peroxisomal amine oxidase (SPAOi). The fact that S. cerevisiae is lacking any endogenous amine oxidase, and no amine is presenting in the medium except amino acids, and also uric acid is not the substrate of amine oxidase, apparently, ammonia ion releasing from the deamination and transamination of amino acids catalyzed by amine oxidase was belived to served as the nitrogen source to rescue the cells on this selective medium. 5.3.2 Isolation and characterization of SPAO full length cDNA Using the incomplete amine oxidase cDNA fragment from pSPAOi (1.2 kb long) as probe, a soybean nodule cDNA library constructed in X ZapII (Delauney and Verma, 1990) was screened. Of the 5x10^ plaques of the cDNA library screened, 26 gave positive hybridization signals after the primary screening. Several clones were taken through two rounds of screening and pBluscrip phagemids were excised from XL 1-Blue in vivo. Three clones finally obtained all contained a 2.6 kilobases long inserts which represent the full length cDNAs of the amine oxidase gene. These three clones showed the same restriction enzyme site patterns. They were then assumed the same cDNAs. 135 c#agtmgt9qtggtggcggcgqcqt#c#gcqqtggt9qcggcqt##q#*cgg###camc##ctggc#gcattatt 7 5 gttgctgtacgccagtgg#acmccmctgeagtggg#cttactacgctg#gcttgct#accetctccaatggccac 150 #gctcaggma#a##cg#cgecatgtcgegccmcccamamt#acaacmmggtcgc#ctegcagcacctccaacttc 2 2 5 tccttettcegcgccacaacaacaetcecaatcacaaceacggccctctgttgccaccttcatttccgccatcga 3 0 0 tteeecteceeaaaacegettcctgeeaaaggtaceaetgtcaftfgtgagageteaaaecageeaeecCCtggac 3 7 5 KVRAQTSKPLD 11 eeaecaaetgctgetgaaataccagcagctgtagegaccgttegagcageCggggeaacacetgaggCgagggat 4 5 0 PLTAAStSVAVAT VRAACATPeVRD 3 5 ggeatgegctttatcgaagtagaectggtagaaccagaaaagcaagttgtcgcatcageagacgcatatttctte 5 2 5 CMRFICVOLVEPEKQVVALAORrrP 51 ceteetttecaaecateactgcteectaggacaaaaggtggaeetg^gatteeaaetaaaetteetccaaggaaa • 5 0 0 PPFOPSLLPRTKGGPVIPTKLPPRK • 5 gcaagactagttgtttacaacaaaaageegaafcgagacaageaeatggattgttgaacttagagaagttcatqca 5 7 5 ARLVVrVKKSKBTSTV tVELRCVHA 111 acaactcgaggtggmcaccataggggcmaagtgattccatctacagttgttcctgatgttcagcctccaatggac 7 2 5 TTRGCRBRGKVISSrVVPDVQPPMD 1 2 5 getgtggagCatgctgagtgCgaageCgttgtaaaggaetttectccctctegtgaagegaatgaaggaagagga 8 0 0 AVETAECBAVVKDPPPrREAirEGRC 1 5 1 gggattgaggaaatggatetegtgatggtagatccctggtgtgctggaCateaeagtgaageCgacgetectagc 8 7 5 GIEEMOLVMVDPWCAGYHSBADAPS 1 8 5 cggagacttgctaaaeeactaatcttttgtaggaetgaaagtgactgcccCAtggaaaacggctatgcecgtcca 9 2 5 RRLAKPLIPCRTESDCPMEMGYARP 211 gttgagggaatccacgtacctgtcgacacgcaaaacacggtcgttcttgaatctgaagategcaaactcgtcccc 1000 VBGIHVLVDMQMMVVLEPBORRLVP 2 3 5 cttccaeetgcCgacccactaagaaattatacttctggtgaaaeccaaggaggagttgaeegaagtgatgtgaaa 1 0 7 5 LPPAOPLRKYTSGBTQGGVDRSDVK 2 5 1 eeeetgeagattattcagcctgaaqgtccaagttttegtgttaaCgggeaetteattgaaeggeagaaqtggaac 1 1 5 0 P L Q I lOPEGPSPRVRGHPZeWOKVR 2 8 5 ettegtattggattcaeteetaqqqagqgtccgqttatteattcaqtaqcetatattgatggaaqteqqgqacqg 1 2 2 5 PRIGPTPREGLVI RSVATIDGSRGR 3 1 1 agaccagtgqcceatagaCtqaqctttqttqaqaCgqtqgtcccACatqqaqatcctaatgatceccaetacagg 1 3 0 0 RPVAHRLSPVBMVVPTGDPVOPRYR 3 3 5 aaaaacgettCtgatgctggggaagatggcCtgqgtaaaaactctcattctctgaagaagggctgtgactqttta 1 3 7 5 KRAPDAGBDCZ.GRHSB5LKKCC0CL 3 5 1 ggctacateaaatactttgatgcgcatttcacaaacttctatggagqtgttgaaacaattgaaaactgtgtttgc 1 4 5 0 CriKTFDAHPTNPTCCVBrtBBCVC 3 8 5 ctgcacgaagaagaeeatggcattttatggaageateaagatcggaqaaeaggttcggctgaagttegaaqgccc 1 5 2 5 I.KeeOHCILIflC0QO«RTCLKBVilRS 4 1 1 agaacgccgaeagtctetttcataegcaetgtggetaactatgaqcatggacttttetggcaettctateaggac 1 5 0 0 RTtTVSFICTVRII©E*CPPI»HPÏQD 4 3 5 ggaaaaatagaagcagaggteaagetcaeaggaattcteagcttaqgageactgeaaccaggtgaaactcgaaaa 1 5 7 5 CKt EXBVKLTGZ LSLCAL0P6BTRR 4 5 1 catggeaeaaccattgeacccggactacacgcgcctgtccaceaacattttttcgttgctegtatggacacggca 1 7 2 5 Figure 5.2. cDNA and deduced amino acid sequence of soybean peroxisomal copper-containing amine oxidase. The translation start site ATG is in bold, and the termination codon is marked with an asterisk. Potential polyadenylation signals are underlined. Circled residues indicate the tyrosine (Y) that is modified to TPQ, and the four conserved histidines. The peroxisome targeting tripeptide SKL is in bold. 136 SPAO W- -or- -SHFIDPLXAAEISVAVAIVRAAGAIFCVROaffinEV- -DLVEPEXOWA- 5 5 AGPAO ! rp- -ST lor ASPnaASACEISEW QGILBTAGUCP EKRIAStlr- -GVUPARGA------5 2 M A O X l iIL- -NAESEALVGV SHPUPLSfVEIARAVAIlKEGPAAAE SFRFTSV- -ELREPSKDDL— 5 8 ECHO teSPSI^YSARKTHJUAVALSFANQAPVPAHGCCAHMVEMDKTLKEPCADVaNDOyAQUTLIiaxaiXVICVKPCAarrAIVNCQPL 85 HFAO ERLRQIASOATAASAAPAHP AHPIDPLSTAEIKAAUnVKSYFACKKIS FHTV ------TIREPAHK ------S 3 PSAO AS ------TTWRIA—— ------— ' ------t P S V I X L L S F ------K A W S V T P L 2 9 SPAO ------U DA X FPPPFQ PSU f- -RTKGGPVIPTKLPPRICARLWYNXKSNEISTWI E I R 1 0 7 A C P A O ------G S E A E D ------RRFRVTIHOVSGARPQEVl i | S V - 8 0 A M A O X I ------R A G V A V A ------READAVLVDRAOARSFEAIlV , DI I L - 8 7 ECM O ALOVPW M KDNK AH V SD TFIIO VFQ SCU X nTQ VEK RPH PIM A LTA D BIKaA VEIV K ASA DFK FN T-RFTEIStJfPD II K E A . M A F 1 6 9 H P A O ------AY ------IQ ------WKEQGCP ------LPPRIAY YV ILEACKPCV XEClU D I I A 9 9 P S A O H V Q ------H PIÛPLTKEÊFIA VQ nVQ NK Y PISM naA FH nG LD OPEK Dli I L R Y 7 9 SPAO EVHAITRGGHHRGKVISSTW PDVQ ------PPHM VEYAECEAW KDFPPFREANEGRG-GIEEM DLV 1 6 9 A C P A O ------T N C rV IS A V E IO T A A T G — E ------LPV 1 EEEFEW EQLIAXDEPM LKALAA»N— L-DVSKV 1 3 2 AMAOXI EAGTVDSMCLLAENI Q ------PPnODEFAECEOACRKDPEVIAALAKRG— LINLOLV 1 3 8 ECHO ALENKPVDQPRKADVIM IiXaaaiEAW DLQNM KU.SNQPIKDAHGM VLL— DOFASVQNinOiSEEFAAAVKKRGI— TDAKlW 2 5 0 H P A O S L ------5 VIETRALETVQ ------PILTVED ICSTEEVIIV OPA VIEQ CVESCIPAN EM HK V 1 5 1 PSAO ETHPTLVSIPRKIFW AIIN SQ rH EILIN LRIRSIV S-O NIHN GY GFPII^EQ SIAIKLPLKY PPFIDSV KK SGL N LSEI 1 6 0 S P A O H V DDPHCA: I YHSI EA Q A PSR r- I lA K PLIFC R TESD CPM EN G Y A IfiV EG IH V U .V : M O W tW LEFEDRKLV PIPPAO PLBN YTSG ETQ 2 5 2 A G P A O r v a p LSA: : VFEYAEEI; b g r —I ILRGLAFVQDFPE OSAIiN A l iV D G L^.VAYVqvVSKEVTRW IDTGVFPVPAEHG— N YTDPEI.T 2 1 0 AM AOXI CFEPW 3 ^[^(FG-ED NEG RT-M lM RA D.VFVROEAD OSPYAIC lENFIVFYnuaGIKW RIEDOQAIPVPSARC— NYL-PKYV 2 1 4 ECMO ITTPL' SYUVGDC NYNAIf lENLVMLV\AlLEQ KK IV KIEEG PW PVPM r-A RP-FD G RO RV 3 2 9 HPAO YCOPW dt^Y-OEBHGIGK— H UQ AU .VYYRSOED DSQY!: s i i U } - i f c p :IV RIEEK K VIFIDIPN RRRK VSK HK HA NFY PKm i 2 2 9 PSAO VCSSFngM F GEEKinBCVRUXFMKESTV NIYVI# r r c i 'TIVAQUllU M aVEY HD RDIEA VPTA -EM rEY aVSK QS 2 3 6 SPAO GGVD— RSDVKPLQIII :c i E : PISFE M : -H FIE I E 2K N M F R KG PT P I E ; L.V I IH-SVA YIOG SRO W PVA H EILSFV EM V/PI SDPN DP 3 3 3 A G P A O G P L ------KRQKPISI';tc i E : P!SFT V If SNH H I SmSU NG FOV f e : w l hl-M h IA FIO X E D EtlA -PIIN R A SIA E m /PI G O PSPI 2 9 0 A M A O X I G E A ------RIDLKPINI':T ( ! E : A S F T V If -N H in I M MK i SFE W G FTtf E : L ,V1 IH-OLK FKD QG VD RrPVIHRASLSEM V /PI GDTAPV 2 9 3 E C M O A P ------A V K P M Q I I lh e : K N Y T Ia ' -CMa I EWHDID FH L S tM S I V : P M I::S-TV TYN -O NG TKE S P A O H Y R K N A f : A : E O G If RNSKSinCGCfiCLGYIICYFDAHFTNnpsVETIENCVCLipEDHGIIMIf|QDH-l'II RIGLAE— VRRSRTLT 4 1 5 A G P A O E tSH Q N Y f ! I! E Y U IITYLSPVIJI 5 D A E : N PiR l E IE IN S Il:CM I EON Gi:L A K SDL-W SGIHY— TRRNREW V 3 7 2 A M A O X I QAKKM AE : S ! E B nH m A H SlJL G C flC LG E II:ICYFDGHSVDSI H O E PffriE M A II:CM E EO D Si:I N K I FDF-REG tA E— TRRSRKLV 3 7 5 ECHO WYFKAYIISID'XatiTLTSPIMUatteSNAVLUIETIAO'iY i;V PM EIPP R A I AkV ! F! RXA GPI EYlÿ QBCQPNVSIERR ELV 4 8 6 HPAO HQEUODUf i : e y :GA( : noNPLSLGc : ckgvxhyumhfsdi D E » : 01PIT V K N A V C II:t I SD O G LLFK i ISD F-RDN FATSLVTRAIKLV 3 9 5 PSAO FYFKTFEtJstiEIPGEULSTVSLIPNEOCPIPHA Q FIDTY VH SArOIPILLK NA ICVEBQX G NIIW RIlTENG IPN ESIEESRIEV NLI 4 0 2 SPAO V SFI^A ^ E Y G Ff I H FY O E: : E I EA EV K LT : IL S L G A L Q - -PG ETR ECY G in-l v :: k p g 492 AGPAO ISFTII I($/ipYCFYHYLYU#MEFEAKAlHvVFriS A F P - -EG GSD NISQ L— APGLGAPE AMAOXI ISFIA M V A M; y EX j A nM H Ifl## E F L V K A l : ILSTAGOL- -PGEKNPYGQSIMOGLYAP: ECMO VRUfl;:î i V(C^YIETÿIFH E rU n GIDAGA 3 : lEAVKGVKAEOM HDETAKDOTRYGTLIDI HPAO VSQIlEplA A m EY C Ü . 4 V EM OC : A I R L D IR I 3 : ILNTYILG ------DDEEACPWGTRVYPI -GOG 472 PSAO VRTIVnVCMPNVIIllEFKAaaKPSIAL« ILEIKGINIK—HKDEIKEOLH-GKLVSANSIG: SPAO EAFNQW EVNVKVEXPGDNNVHNNAFYAEEK-LLKSEM EAM ITCOPLSARPHIVF I rimnni-TGHL'.TGIKLI ■VP-G SNCLPLAG S 5 7 4 AGPAO GFTNRVEEEO W RQIM GPG NERGN AFSRXRI-VLTRESEA VREAD ARIG RIW XIS I PIESKNR-I -IM Ë PV C I K IH A.-H - NQ PTLLA DP 5 2 6 AM AOXI GVKNAVYEVDHBYPEHNPT— G IAFM AV DR-LLCTEO KA IRKINEAK H RnaaA I HESIi k n l - v n e p v M r l i i P-TN G IO LA /IR D 5 2 8 ECMO CENNSLVAM D-PW KPNIAGCPRTS— TI 4 I]VNQYHIGMEQDAAQKF 0 P G T I R L L S I PNK ENEt-M G NPV ^: IIIPYA G GTHPV AK G 6 4 8 HPAO N SA A ACD/UCSSPYPLGSPEW EYG N AFY SEICI-TFKIV K DSLIN YESAIGRSN DIE I PtO O /N PY SG K PPS I K LI .V S-TQCPPLLA KE 5 5 5 PSAO G TH NSFEK rSLKIV EUK DG SSKRK SY fRTEIQIAK IESDA K m G LA PAEIr-V VnW N IICIA-V G NEVEV tLII P-A IPA H PLLTE 5 6 2 S P A O E A K - • F U i A A F I K K N U I ' E l A R DEM E H Pq^PNQ N PRV G-EG LAIM VK QN RS-LEEA : rV UH Y VE'VT: 6 5 2 A G P A O G S S - lA F l A A FA IX D I* I EWDOl IFVNQHSGGA— G LPSY IA QD RD -IOG C : I W W H T l t 6 0 3 AMAOXI DAY VIS I I A Q FA EM iU i I rA |$)RIRII IER FA A M EY PN 0 A TG AD -EX aJEIW IQK DRN-IV D 1 : . W W Y TEIMEI: l 6 0 6 ECMO AQFAPDIlEM IYl I L SnO K Q U I T E t^E R F P dlK Y IPNRSTHDT— G LG OY SKD N ES-Um : A W VK TlH lT 7 3 0 H P A O G S L ------V A II AEW ASHSVII V W D N RLY PS|$)IiHV PQ H SG D GV RCM REHIGD GSBNU Xn : ILFFHTE; 6 3 5 PSAO DDYPQI BGAFTNYNVli m ^lRIEK NA ^iLYV DH SRG DD — TLAVW IKQNRE-IVNit i v i w h v m ; 6 3 9 SPAO VERIIIGEEIH H C n H C S i A V D V P P N - —OSDLDOKENGLP/UCPIQNGLIAKL 7 0 1 AGPAO VDIVGII F t l E ! E (fE ORE I V IO V PA N - P S ------QSGSHCHG 6 3 8 AM AOXI R O N IG fl##|H (ÿl I riM L P T S - —TST-----TOTGEADICCHNGK 6 4 8 ECHO l E l l I L ------G A L K K ------OK 7 5 7 HPAO A EPITI!$|I#U M IEIM G U ):IQ PSYA M ITSE/U CRA VH KETKD iCTSRLA FEG SCCG K 6 9 2 PSAO LLSTSFEUHeneEEVOVL- E C T L S P R 0 VAWPG ------CSN 6 7 4 Figure 5.3. Comparison of the amino acid sequences of SPAO with other amine oxidases. SPAO, soybean peroxisomal amine oxidase, (this study); AGPAO, phenylethyiamine oxidase of A. glob^ormis (U035I7); AMAOXI, amine oxidase (MA05Q) of Arthrobacter sp. (L12983); ECAO, E. coli tyramine oxidase (EC 1.4.3.6) (P46883); HPAO, Hansenulapolymorpha peroxisomal copper amine oxidase (ECl.4.3.6.) (X15111); PSAO, pea seedling amine oxidase Ô-39931). Sequence alignment by Clustal method. Residues conserved in all sequence are boxed. The conserved histidine residues are indicated by asterisks . The conserved TPQ-containing NYD/E region is underlined. 137 -AGPAO -AGAO -AMAOXI - m -HHAO -HFDAO -ECMO .PSAO -HPAO 60.6 T T 50 30 20 10 B Percent Similarity 1 2 3 4 5 6 7 8 9 1 33.2 33.8 31.4 15.4 19.3 2 4 .9 21.1 13.8 1 SPAO 2 6 2 .6 37.3 59.6 15.5 23.4 27.1 20.7 14.4 2 AGPAO 3 5 9 .2 57.1 42.7 15.3 2 5 2 2 7 .9 23.6 13.1 3 AMAOXI 4 63.1 38.0 52.0 m 1 5 2 2 4 .9 2 6 .6 2 2 4 1 5 2 4 AGAO 5 7 9 .8 7 9 .2 7 8 .4 79.8 162 14.5 15.1 36.1 5 HRAO 6 7 5 .9 73.1 71.5 73.6 78.2 202 23.6 14.4 6 ECMO 7 6 9 .2 6 8 .2 6 4 .7 68.2 82.8 77.1 19.6 13.6 7 HPAO I 8 7 4 .0 7 6 .6 73.4 73.8 79.4 69.1 77.0 B |13.9 8 PSAO 9 8 1 .8 80.3 79.7 80.6 57.0 78.4 83.5 77.1 9 HPDAO 1 2 3 4 5 6 7 8 9 Figure 5.4. The genetic distance of SPAO with other amine oxidases. A. Phylogenetic tree of 9 copper-containing amine oxidase proteins calculated by the CLASTAL method; B. The distance of these proteins with each other. SPAO, soybean peroxisomal amine oxidase, (this study); AGPAO, phenylethyiamine oxidase of A. globtformis (U03517); AMAOXI, amine oxidase (MA05Q) of Arthrobacter sp. (L12983); ECAO, E. coli tyramine oxidase (EC 1.4.3.6) (P46883); HPAO, Hansenula polymorpha peroxisomal copper amine oxidase (EC 1.4.3.6.) (XI5I11); PSAO, pea seedling amine oxidase ^39931); HRAO, human retina- specific amine oxidase (D88213); AGAO, copper amine oxidase, monoamine oxidase and histamine oxidase o f Arthrobactr sp. (D38508). HSDAO, H. sapiens diamine oxidase (X78212). 138 The DNA sequence of one full length cDNA insert in pSPAO is shown in Figure 5.2, which was determined for both strands using a progressive primer design strategy. The total length of SPAO cDNA is 2665 base pairs. A long open reading &ame starts from nucleotide 343 to 2441 which encodes a 701 amino acid long peptide. The ATG codon at nucleotides 343-345 ( ACTGTCATGGGTl matches well the consensus translation start site (TAAACAATGGCD defined by Joshi (1987) for plant genes with approximately molecular weight of 78,643 Daltons. It should be noted that a second in frame ATG (position at 454-456) occurs 37 codons into the coding region and could represent the translation start site. However, based on the sequence comparison of N-terminal amino acids with other known amine oxidases, the first ATG most probably represent the amine oxidase translation initiation site. There is one polyadenylation signal at 3'-untranslated region (2503-2508) which is exactly matches the AATAAA consensus. 5.3.3 SPAO contains the conserved region for topa quinone cofactor formation There are 63 most conserved residues mainly within two regions: the central region and the C-terminal end. 12 of the conserved residues are glycine, and seven residues are proline. There are four histidine residues, one is near the center and the two histidine form a HXH motif in the center region and near the C-terminus, the fourth histidine is residing in a high conserved C-terminal region of the protein. The internal sequence from Asn^^4 iq oiu426 agrees with the conserved Asn-Tyr-Asp/Glu sequence, in which the middle Tyr is the precursor to the topa quinone cofactor covalently bound to copper amine oxidases from various sources (Mu et al, 1992, Copper et al, 1992, Mu et al, 1994, Cai and Klinman, 1994, Choi, 1995). On the basis of numerous evidences, the enzyme-bound Cu(II) has three axial histidine ligands (Mu, et al., 1994). Histidine residues aligned with these at position His^'^^, His^^"^, and His^^of the SPAO 139 numbering are conserved in all 9 sequences (Figure 5.3). Thus, the C-terminal region of the protein contains active site of the enzyme (Mu et al, 1992; Harman and Mclntire, 1997). The deduced amino acid sequence of SPAO shows overall homology ranging from 33.8% to 13.8% with other eight known copper-containing amine oxidases firom different sources (Figure 5.4B). SPAO shows closer relation to Arthrobacter methylamine oxidase than that to the pea seedling amine oxidase. The identity of the translated soybean amine oxidase was confirmed by comparison with the two most conserved regions within copper-containing amine oxidases, including the TPQ- containing N-Y-D/E region, and the H-X-H motif. SPAO, thus, likely indicates a new subfamily within copper-containing amine oxidases. 5.3.4 A conserved sequence exists at N-terminus of some amine oxidases Both lentil seedling amine oxidase and pea seedling amine oxidase have been defined as extracellular enzymes (Tipping and McPhenersonm, 1995). Their N- terminal shows a conserved region which is X-T-P-L-H-V-Q-H-P-L-D. The amino acid sequence preceding this region has been defined as a signal peptide which contains 25 amino acid residues (Von heijine, 1986). Most amine oxidase identified so far are extracellular, except a peroxisomal amine oxidase from yeast H. polymorpha. Proteins secreting outside the plasma membrane requires a signal peptide as topogenic information for their transportation. If SPAO is an extracellular polypeptide, it must contain a secret leading sequence at its N- terminus. To investigate the feature of N-terminal region amino acid sequence of SPAO, several other amine oxidase N-terminal amino acid sequences were further analyzed. A sequence was found to be very conserved among these amine oxidases by this study, see Figure 5.5. This conserved amino acid sequence is defined as XgH-P-L- 140 D-P-L-T/S-X-X-E-I (X represents any amino acids). The amino acid sequence preceding this region has been identified as targeting sequence in yeast amine oxidase (Faber, 1995) and pea seedling amine oxidase (von Heijine, 1986; Choi, 1996). The conserved N-terminal region of pea and lentil seedling amino oxidase was determined to be XTPLHVQHPLD (Choi, 1996), preceding this region of amino acid sequence by a putative signal sequence of 25 residues with characteristics expected of a secretion signal (von Heijine, 1986). Coincidentally, a recent study has identified the topogenic information is residing at the first 16 amino acids residues at N-terminus of yeast H. polymorpha peroxisomal amine oxidase. These 16 amino acids are able to direct one cytosolic protein bacterial ^-lactamase, into H. polymorpha peroxisomes (Faber, 1995). Therefore, the sequences preceding of -X6-H-P-L-D-P-L-T/S-X-X-E-I likely act as secretion/targeting signal in most of the amine oxidases, if not all. Interestingly, SPAO lacks of any preceding sequence in between the initial codon Met and this conserved sequence (Figure 5.5). Obviously, the topogenic information is not residing at N- terminus, this suggests it must be residing at internal sequence or at the carboxyl- terminus. 5.3.5 A peroxisomal targeting signal SKL/AKL resides at the C-terminus of SPAO A tripeptide Ser-Lys-Leu sequence is defined as peroxisomal targeting signal PTSl which is conserved in all eukaryotic cells (Gould, 1989). More significantly, a typical peroxisome targeting tripeptide SKL sequence is found at SPAOi C-terminus, and an AKL, a degenerate form of PTS1 is found at SPAO C-terminus (Figure 5.6A). Both SKL and AKL serve as peroxisomal targeting signals in plants (Hayashi, 1997). This observation strongly suggestes SPAO is most likely a peroxisomal protein. 141 Amine oxidase Sequence GenBank Acc# SPAO 0 -M7RAQTSHPLDPLTAAEISVAVA-22 T h is s tu d y AMAOXI 9 -EALVGVSHPLDPLSRVEIARAVA-31 L12983 AGAO 21 -LVHAAAQHPLEQLSAEEIHEARR-44 D38508 HPAO 16 -AAPARPAHPLDPLSTAEIKAATN-3 8 X15111 PSAO 2 6 -VT PLHVQHPIiDPLTKEKFIiAVQT-48 L39931 ECMO 117 -FQVEKRPHPLNALTADEIKQAVE -1 3 9 P46883 HSDAO 7 5 -MLLPKKYHVLRFLDKGERHPVRE-9 7 X78212 Figure 5.5 The conserved region of N-terminual region of several amine oxidases. An alignment of amino acid sequences at N-terminal region of several amine oxidases which are mentioned in Figure 5.4. The conserved residues are in bold. The amino acid sequence preceding this region is suggested to be the signal leading sequence. 142 SPAO 632 VLWYVFGVTHIPRLEDWPVM SPAOi 632 SLWYVIGVTHIPRLEDWPVI SPAO 642 PVERIGFMLMPHGFFNCSPA SPAOi 642 PVERIGFILIPHGFFNCSPA SPAO 662 VDVPPNQSDLDDKENGLPAK SPAOi 662 VDVPPNPSDLDDQENGLPTK SPAO 682 P I Q N G L I A K L 701 SPAOi 682 P N Q N G L I S K L 701 Figure 5.6. Comparison of C-terminal region amino acid sequence soybean peroxisomal amine oxidase isomers. SPAOi, the C-terminal region of the incomplete amine oxidase isolated by complementation; SPAO, the C-terminal region of the soybean Peroxisomal amine oxidase isolated by using SPAOi cDNA as probe. The difference of the amino acids are underlined. The peroxisome targeting signal tripeptides are in bold. 143 The different peroxisomal targeting analogs at the C-termini of these two amine oxidase isomers imply that they may represent tissue specificity or targeting efficiency. A previous smdy has suggested that the peroxisome-targeting signal is slightly different within different specialized peroxisomes. There are four different types of peroxisome in plant cells and it is possible that seed glyoxysomes, leaf peroxisomes, nodule peroxisomes and non-specialized peroxisomes may have their different peroxisomal targeting signal analogs to guide proteins into their destiny. Glyceraldehyde phosphate dehydrogenase firom Trypanosoma brucei glycosome, contains a carboxyl-terminal AKL sequence (Michels, 1986). AKL act as peroxisome-targeting signal but has 78% efficiency of SKL showed in the firefly luciferase (Gould et al., 1990). Therefore, SPAO contains its topogenic information at C-terminus and lacks of any targeting information at its N-terminus. There is no typical transmembrane domain residing in this amino acid sequence based on the hydrophobicity of SAG (Figure 5.6B). Together, these data suggest that this soybean amine oxidase is a typical peroxisomal matrix protein. 5.3.6 SPAO is possible a inducible enzyme in soybean tissues Total RNAs were prepared from the tissues described in materials and methods (Figure 5,7). Generally, SPAO exists in all young tissues at very low levels, and it shows relatively high level in leaves, mature nodules, four-, five-and seven-day old roots. The level of SPAO is very low in one-day old radicles and two-day old cotyledon-free tissues. Which agreeed the result made by Tajima et al. (1985) that cadaverine is dramatically increased in the roots after after 3-day planting. In comparison with the distribution of P450W, SPAO exists in plant tisssues at very low level in normal conditions. This data suggestes that SPAO may be inducible by the intracellular level of polyamines. 144 1 2 3 4 5 6 7 ,8 9 10 11 12 13 ^ _____ Figure 5.7. RNA blot analysis of SPAO gene expression in soybean tissues. The RNAs were isolated from soybean different tissues are presented from left to right. Lane 1,2 and 3 are RNAs from cotyledons of 1, 2 and 4 days old seedlings grown in the dark; Lane 4 ,10-day old seedling stems. Lane 5, 10-days old leaves; Lane 6,7, and 8 are the RNAs from nodules of 10,20 and 35 days old plants; Lane 9 is 1-day old root radicles; Lane 10, is two-day old cotyledon-free seedlings; Lane 11, 12, 13 are the roots of four, five and seven days old plants. MB 96 78 66 46 — Figure 5.8. The SDS-PAGE of SPAO expression in E.coli. The SPAO expression in E. coli lysB2l cell was determined by a SDS-PAGE. Lane M, represents standard molecular weight (kDa); Lane A, represents the pellet fraction with IPTG induction; lane B, represents the pellet fraction without IPTG induction. 145 5.3.7 Diamines are highly efficient substrates of SPAO The production of an active quinone-containing form of the recombinant diamine oxidase greatly dependent on the presence of Cu^+ ions in the medium for the transformed E. coli cells (Choi et al., 1995). SPAO cDNA was subcloned infirame in a E. coli expression system. Amine oxidase activity of SPAO expressed in E. coli was determined by using benzylamine as a substrate. The product benzyldehye generated was detected by absorbance at 250nm. SPAO showed amine oxidation specific activity of 2.95fiM/mg. min. In addition, the catalyzing abilities of SPAO towards cadaverine, putresine, spermidine, benzylamine, lysine and uric acid were determined by using peroxidase-coupled enzyme assay. In this assay, H 2O2 generated from amine oxidation is decomposed by a coupled horse-radish peroxidase. The product was measured spectrophotometrically as increase in absorbance at 500nm. The efficiency of amines as substrates of SPAO is presented in Table 5.1. In legumes, amine oxidase is most active towards putrecine and cadaverine (Floris, et al., 1983; Angelin, 1985; Suzuki, 1973). This study also demonstrated that SPAO catalyzed very efficiently the oxidative deamination of diamines such as putrescine and cadaverine, and with low efficiency to polyamine like spermidine. L-lysine oxidation was not detectable by this method. Previous study has shown that L-lysine is able to be catalyzed by soybean, pea and lentil copper amine oxidases (Suzuki, 1973; Medda et al., 1996). However, soybean copper amine oxidase catalyzed the oxidation of L-lysine without oxygen consumption, indicates no H 2O2 production in L-lysine oxidation. Uricase activity was not detected in SPAO cmde extract, thus, uric acid is likely not the substrate of SPAO. Based on the SDS-PAGE gel, the molecular weight of this protein is about 78 kD (Figure 5.8 ). 146 Substrates Percentage of degradation (%) Putrecine 100 Cadaverine 71 Spermiding 19 Benzylamine 9.5 Lysine ND Uric acid ND Table 5.1 Comparison of the relative catalytic ability of SPAO to the different substrates 147 5.4 Discussion The peroxisome biogenesis mutant Spbl we isolated showed the deficiency of peroxisome proliferation as well as a series of peroxisomal enzymes, e. g. uricase and catalase. Apparently, peroxisomal transaminase and amino acid oxidase are also not induced. Thus, the amino acids amended in the uric acid containing selective medium possibly became the substrates of amine oxidase which catalyzes deamination of amino acids resulting of ammonium ion releasing, which then was used as nitrogen source to support Spbl growth. Unlike using alkylated amine as substrate, amino acid oxidative product is ketoacid and (o-amino group which can serve as C and N source for cell growth. However, in the heterologous expression of H. polymorpha amino oxidase in S. cerevisiae, even though, a native enzyme can be received, S. cerevisiae is still unable to form visible colonies using alkylated amine as sole nitrogen, its product aldehyde accumulation might become toxic to the cells under amine-containing plates (Cai and Klinman, 1994). S. cerevisiae is unable to utilize amines as the sole N-source due to its lack of any endogenous amine oxidases (Cai and Klinman, 1994). High concentration of lentil copper amine oxidase is able to catalyze the deamination of amino acids (Mendda et al., 1996). When medium is lacking a ready to use nitrogen source, a non-specific deamination or transamination catalyzed by highly expressed soybean copper amine oxidase in S. cerevisiae , may occur. The cDNA (pY-SPAOi)recovered from the positive transformant of yeast is about 1.2 kb in length with poly-A terminus. It encodes the C-terminal region of this amine oxidase from residue 387 to 701 which contains the active site of the enzyme (Hartmann and Mclntire, 1997; Mu et al., 1992 ). 148 The most conserved region TPQ-containing and H-X-H motif are residing in SPAOi- Thus, the amine oxidase active catalytic site is in the truncated protein which resulted in a functional complementation. A full length soybean copper-amine oxidase was received by using SPAOi cDNA as a probe. Comparison of deduced am ino acid sequence with other copper- containing amine oxidases revealed that SPAO contains the conserved topa quinone precursor tyrosine. The overall homology with eight known copper-containing amine oxidases were less than 40% the threshold value of a new subfamily. Another striking feature within this protein is its containing a conserved peroxisomal targeting signal at its C-terminus . Hence, we believe SPAO is a peroxisomal protein which may involve in the regulation of intracellular polyamine oxidation. In pairwise comparison with pea seedling copper-containing amine oxidase, the overall homology is only 21.1%, which has been defined as an extracellular am ine oxidase. Both pea and soybean are legume plants, thus, these two amine oxidases must have distinct cellular functions. Current data have shown that the amine oxidase is an extracellular enzyme in Leguminoseae (Federico and Agelini, 1986; Federico et al., 1988; Soldum and furey, 1991; Tipping, McPherson, 1995). Whereas, our data strongly indicates a peroxisomal am ine oxidase existing in soybean and other legumes. This finding opens up a new prospective of amine oxidase in cellular functions in plants. Plant copper amine oxidases have traditionally be considered essentially as diamine oxidase, LSAO shows able to catalyze oxidative deamination of aliphatic monoamines, substituted alkylamine and some amino acids (Medda, 1996). Pervious study (Suzuki, 1973) has shown in soybean seedling, diamines, e.g., cadaverine, putrecine, spermidine and agmatine are the most ready oxidized compounds for soybean amine oxidases, polyamine and monoamines are oxidized slowly. L-lysine can be definitely oxidized by pea amine oxidase (Mann, 1955) and soybean amine oxidase 149 (Suzuki, 1973). Lentil amine oxidase is able to catalyze the deamination of some amino acids (Medda et al., 1996). Using horse-radish peroxidase coupled enzyme assay, data reveals by this study that putrecine showed higher catalytic efficiency than cadaverine to SPAO. During soybean seed germination, the major polyamine present in soybean radicles is always cadaverine (Tajima, 1985). Therefore, in soybean radicles the contribution of a diamine, cadaverine, is believed to be coupling with peroxidase for urate degradation (Tajima, 1978). SPAO has no activity toward uric acid, whether SPAO is able to utilize amino acids as substrates is not determined. A further study on the enzyme kinetics and substrate specificity may be performed in the future. Based on our Northern data, this peroxisomal amine oxidase was expressed at a extremely low level in all the tissues, and its RNA was very unstable. Secretary proteins generally contain a proteolytically cleavable leading peptide at their N-termini. Unlike these extracellular amine oxidases, SPAO does not contain such a cleavable secretary leading peptide at its N-terminus. Amino acid sequence comparison of this study reveals that a conserved region is residing at the N-terminus of many amine oxidases. It is defined as -X6-H-P-L-D-P-L-T/S-X-X-E-I, the sequences preceding this conserved region is suggested to be served as a signal peptide (Tipping and McPherson, 1995; Faber et al., 1995). The C-terminal conserved peroxisomal targeting signals have been well defined which compose of three amino acids Ser-Lys-Leu (SKL) and its degenerate form S/A/C/-K/H/R-L (Gould, 1989; Gould, 1990). A recent smdy on the carboxyl terminus of microbodies function as PTSl in higher plants further reveals that the tripeptide sequence of the form [C/A/S/P]-[K/R]-[I/L/M] function as a microbody-targeting signal ( Hayashi, 1997 ). Thus, that both SKL and AKL were found in SPAOi (incomplete 150 sequence) and SPAO isomers in this study (Figure 5.6) strongly suggests amino oxidation occurring in plant peroxisomes which may involved in regulation of intracellular polyamine levels. This study shows a new perspective of amine oxidase due to its peroxisomal localization. Further more, this enzyme may give a new function to the plant peroxisomes in which amine oxidation has not been determined. So far, plant amine oxidases are generally found to be extracellular enzymes loosely associated with membrane and cell walls in the Leguminoseae, and also polyamines are majorly stored in apoplasts (Federico, 1986; Federico; 1988; Slocum, 1991). The major role of the extracellular amine oxidases has been defined to regulate the level of di- and polyamine in the apolplast to form a H202-producing system. Peroxidases within the apoplast are correlated with cell wall compounds oxidation with decomposing of H 2O2 The evidence from the Leguminoseae, has shown a role for copper amine oxidase in conjunction with peroxidases in lignification and cross-linking of cell wall components during normal growth, in wounding and resistance response (Angelini et al., 1990, 1993). We suggests that the alternate urate-degrading diamine oxidase-peroxidase enzyme system is operated when plants are under stress conditions and regulated by the intracellular polyamine levels. 151 When plant is under developmental and enviromental stimulations Purine Catabolism Increase in polyamine Hypoxanthine Xanthine oxidase Polyamines Xanthine diamine Xanthine oxidase oxidase Uric Acid HoOo+NH-i+R.CO CytoP450 peroxidase catalase X uricase X Aiiantoin~| H2Q2 accumulation ^ peroxisome N-source Induce plant response systems Figure 5.9. The hypothetical model of the alternate urate-degrading enzyme system in soybean peroxisomes. The urate-degrading enzyme system is regulated by the intracellular polyamine concentration which is increased when plants are under developm ent and environmental induction. The mechanism of polyamines in the regulation of ceU growth and development may regulate their levels via a peroxisoitl diamine oxidase- peroxidase urate-degrading enzyme system in plants. 152 5.5 Perspectives Isolation of soybean peroxisomal P450 peroxidase and amine oxidase reveals that the subcellular localization of this alternative urate-degrading enzyme system is in peroxisomes which agrees with the fact that uric acid is generally produced and oxidized inside in peroxisomes (Figure 5.9). Polyamines are known to be a major form of nitrogen source stored in plant seeds (Rostogi and Davies, 1990). One possible function of the urate-degrading diamine oxidase peroxidase enzyme system may involve in provide nitrogen supplement during plant cell growth and development under the regulation by polyamines. Increasing in polyamine biosynthesis plays an important role in response to developmental and environmental stimuli. The intracellular levels of polyamines regulated by polyamine oxidation are suggested to be essential in the polyamine regulatory functions. One of the mechanism regulated by polyamines may be due to the production of molecules such as H 2O2 and amine oxidative intermediates generated by amine oxidation. H 2O2 has been found to be as secondary messengers in cellular signal transduction pathways (Saran, 1989, Schreck, 1991) and intermediates of polyamine oxidation can act as negative regulator in human (Cona et al., 1991; Gramzinski et al., 1990; Parchement et al., 1990), plant (Hill, 1967) and yeast (Zwart et al., 1980). In conclusion, we propose that H 2O2 generated from peroxisomal polyamine oxidation may play a central signaling role in regulating cell growth, development and plant defense response. 153 CHAPTER 6 GENERAL DISCUSSION AND SUMMARY Intensive studies on yeast and m a m m a l i a n cell peroxisomes have made great progresses on determining function and biogenesis of their organelles. Peroxisomes have been marked as an essential organelle for cell growth, development and cellular signaling. Due to the lack of studies on plant peroxisomes, many important functions of peroxisomes still remain undiscovered, especially the roles are played under the stress conditions. Fatty acid ^-oxidation carried out inside peroxisome has been long well-known mechanism and its mechanism of induction of peroxisome proliferation has been also well studied. Purine metabolism is also known to be carried out inside peroxisomes, but the mechanism of induction by purines is unknown. 6.1 Xanthine is a peroxisome proliferator The priority goal of my research was to find out and explain the phenomenon of peroxisome proliferation in uninfected cells of nodules during the assimilation of symbiotically reduced nitrogen, whether purines, xanthine or uric acid, are able to induce the peroxisome proliferation was tested in yeast cells of S. cerevisiae, which was used in this research as eukaryotic model to study plant peroxisome biogenesis and functions. The enzymes catalyzing the oxidation of purines are located in the 154 uninfected cells of nodules. Recently, xanthine dehydrogenase has been changed into xanthine oxidase and is suggested to be present in peroxisomes and catalyze the reaction generating oxygen free radical O 2" (del Rio, 1992). This study showed that xanthine was able to induce the proliferation of peroxisomes in S. cerevisiae, as well as peroxisomal enzymes activity involved in purine metabolism, e.g., uricase activity and catalase activity. Uric acid, on the other hand, was able to induce uricase activity, but not the proliferation of peroxisome. Uric acid induces uricase expression which may increase the volume of the peroxisomes but not the number of peroxisomes. Xanthine oxidation acts upstream of urate oxidation which results of production of uric acid and superoxide inside peroxisome. Uric acid accumulation induces uricase activity which converts uric acid into allantoin and generates H 2O2 as a by-product. Therefore, oxidation of xanthine and uric acid results in H2O2 and 0 2 " level increasing, and the oxidative stress inside peroxisomes then causes the proliferation of peroxisomes. Therefore, uninfected cells consist high population of peroxisomes, and that uricase is highly induced concomitantly with nitrogen assimilation (Kim, 1996). A evidence has been provided for the present study of pea plant metabolism of activated oxygen species under effect of senescence, that ± e O2" generating enzyme xanthine oxidase and H 202-generating enzyme uricase were induced in pea leaf peroxisomes. The level of H 2O2, therefore, was increased dramatically inside peroxisomes, and peroxisome proliferation was observed (Pastoris and del Rio, 1994). Xanthine causes oxidative stress which in turn causes peroxisome proliferation and uric acid production, and finally results of induction of uricase activity inside peroxisomes. A situation likely occurs in soybean nodules (Figure 6.1). 155 Ureide Synthesis & Transport Î Fix N Peroxisome Proliferation Purine Purines Oxidative Stress Synthesis oxidation (H2Q2 , O2 ) Low 0 2 High Salinity Figure. 6.1. Xanthine is a peroxisome proliferator. In nodule, nitrogen assimilation enhances piuine biosynthesis which in turn cause purine metabolism accelerated inside peroxisome of non-infected cells. Xanthine/xanthine oxidase is a source of producing superoxide and generate uric acid, which increases O2 and H2O2 level and results of oxidative stress inside peroxisomes. Peroxisome proliferation is induced, as well as uricase and catalase activity. Ureide biosynthesis is, therefore, induced as uricase activity is increased. In general, purine metabolism is induced when cells undergoing cell division and in high growth rate. 156 Uricase Uric acid Ureide + HgO^ Growth (1) induced catalase HgO+Pg Induced No growth (2) H2Q2+' Ureide Growth (3) Yeast Mutant Uric acid P450 Peroxidase Spbl •PPAR Uric acid Ureide + 2 2 Peroxisomal H O Growth - (4) enzymelnduced Peroxisome His Bigenesis Mutant Lys Diamine oxidase*’ "eto acld + NHg Growth (5) Figure 6.2. Functional complementation to peroxisomal biogenesis mutant. 6.2 The mutant and its functional complementation A mutant with a single allele mutation causes cell defective in using oleic acid or uric acid as carbon or nitrogen source, two unrelated metabolism pathway involved in the same compartment organelle. Such defective must be due to the deficiency of this organelle. Three genes were isolated via functional complementation, they encode a G-box binding protein, a positive regulatory protein may involved in peroxisome biogenesis, and two enzymes involved in the alternate urate-degrading pathway, a P450 peroxidase and a diamine oxidase. Figure 6.2 summarized the complementation occurs in this yeast peroxisome biogenesis mutant 6.3 Urate degrading alternative pathway in peroxisomes One of the objectives was to isolate genes involved in metabolism of purine. One striking finding of my dissertation was the isolation of the genes involved in urate- degrading enzyme system via the functional complementation of yeast. This finding generated two very important peroxisomal enzymes, and confirmed the existence of a new urate oxidation system inside peroxisomes, and suggested possibly new function of peroxisomes. Urate-degrading alternative pathway: Diamine Oxidase RH2NH2 + H2O — ► RCHO +NH3 +H2O2; P450peroxidase Urate+ H 2O2 ~~ Allantoin+H 2O. In this study, a cytochrome P450 cDNA has been cloned and its protein demonstrated a peroxidase and uricase activity with present of H 2O2. Unlike uricase, it catalyses uric acid by expending of H 2O2 instead of generating H 2O2 Cytochrome P450s catalyze H 202-dependent oxidation have been reported in many studies. 158 However, P450 involves in purine metabolism has not been studied yet. P450 has been found in the glyoxysomal membrane by Donaldson group's studies (Hicks and Donaldson, 1982). And a 57 kD membrane protein has been found in all kinds of peroxisomes in cotton plant studied by Jiang (1994). Based on the deduced amino acid sequences, both P450W and SPAO contain the PTSI near or at C-termini of the proteins which indicate that this diamine oxidase-peroxidase urate-degrading enzyme system existing in peroxisomes. Northern blot data suggested, that P450W exists in cotyledon, root leaves and root nodules more abundant than that of SPAO in the above tissues. Polyamine, cadaverine, acts as a regulator of diamine oxidase as the core reaction to regulate the cellular level of poly amines. P450 is a membrane bound enzyme which may decompose H 2O2 during H 2O2 passing through the peroxisomal membrane. It may serve as a protection barrier to prevent H 2O2 releasing into cytosol. 6.4 The hydroxylation activity of peroxisomal P450W may be regulated by the ratio of O 2 /H 2 O 2 Based on P450W's primary structure feature threonine-containing region and its ability of utilize H 2O2 under low aeration, it is suggested that P450W may functionally regulated by the ratio of O 2/H2O2 inside peroxisomes, in terms of as a monooxygenase or a H 202-dependent peroxidase. Monooxygenase; RH + 02 ------► ROH4- H2O (3) ; Peroxidase: RH+H 2O2 ------► ROH+ H2O (4) A hypothetical regulation of P450 and redox potential of peroxisomes is proposed to be controlled by the existence of O 2/H2O2 in peroxisomes. When peroxisome proliferator 159 present or under stress conditions, H 2O2 generating oxidase is induced and H 2O2 level is increased, therefore, P450s are altered to catalyze H 202-dependent hydroxylation. As normal condition cellular respiration ir> under going in peroxisome, peroxisomal P450 possibly utilize O 2 to catalyze substrate as a monooxygenase. Such features may give P450 a sensor function in peroxisomal membrane which is regulated by the ratio of O2/H2O2. Therefore, I propose that peroxisomal membrane P450s are possibly regulated by the availability of O 2 and H2O2 in peroxisomes and may act as a sensible regulator to alter the reaction in peroxisomes and generates secondary signal molecules 6.5 A possible linkage of salicylic acid biosynthesis and polyamine oxidation Salicylic acid biosynthesis requires H 2O2, which is catalyzed by a P450 in tobacco plants (Le 6n et al., 1995). In terms of biochemical reaction, benzoic acid 2- hydroxylase (BA2H) catalyzed reaction is similar to the P450W's, H 202-dependent hydroxylation on a cyclic carbon bound. Many P450s that mediate a H 202-dependent peroxidation under stress conditions may be peroxisomal, however, none has been proven. It is known that P450s can be induced by peroxisome proliferators and have a broad range substrates. We propose that salicylic acid is synthesized inside plant peroxisomes. Our preliminary data showed that the production of SA in vitro was catalyzed by P450W-SPAO-cadaverine enzyme system. Thus, P450 catalyzed oxidative reaction may generate some cellular regulatory signal molecules, e.g., salicylic acid, which acts as a second messenger to bind to catalase and in turn to elevate H2O2 level inside cells. H2O2. then acts as a secondary messenger to induce corresponding defense gene expressions. That catalase is inhibited in plants under stresses (del Rio, 1992) can be explained by the production of SA inside peroxisomes, see Figure 6.3. 160 Overall, this study suggested that polyamine, amine oxidation and urate oxidation are catalyzed by a diamine oxidase-peroxidase enzyme system within plant peroxisomes which may be involved in regulation of cellular proliferation, cellular toxicity, oxidative stress, and plant cell defense during seedling growth. Thus, the present results reveal a new cellular function of peroxisomes, and this new function will be elucidated by further smdy of plant peroxisome biogenesis. 161 Purine Developmental response Salicylic acid catabolism & environmental stimuli biosynthesis Phenylalanine f a l I Hypoxanthine Cinnamic acid Xanthine oxidase Increase in polyamine 02- Benzy lamine AOJ4 diamine Xanthine oxidase oxidase Benzylaldehye 02- Perkin 1 5 reaction w Uric Acid H202*+NH3+ RCO Citmamic acid tease X m02* CA4H (P450) Allantoin NH3 Benzoic acid H Z 0 2 * BA2H (P450) Nitrogen source Transammation Salicylic acid SOD X — — >. catalase X NADF peroxisome 02- 0 2 -, H2O2 Redox Induce plant defence response systems Systemic Acquired Resistance Figure 6.3 The hypothetical model of peroxisomes in plant defense through a diamine oxidase-peroxidase urate-degrading enzyme system. 162 Figure 6.3 (continued), 1) Xanthine oxidation inside peroxisomes (del Rio et al., 1992); 2) P450-peroxidase urate oxidation (this study); 3) Urate oxidation catalyzed by uricase (Nguyen et al., 1985); 4) Amine oxidation by a peroxisomal amine oxidase (this study); 5) Perkin reaction, benzylaldehyde is converted into cinnamic acid; 6) Cinnamate hydroxylation catalyzed by CYP73, trans-cinnamic acid 4-hydroxylase (CA4H) (Teutsch et al., 1993; Mizutani, et al., 1993). 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