FUNCTIONAL IDENTIFICATION AND CHARACTERIZATION OF AN ARABIDOPSIS THALIANA GENE INVOLVED IN VITAMIN B 6 BIOSYNTHESIS Except where reference is made to the work of others, the work described in this thesis is my own or was done in collaboration with my advisory committee. This thesis does not include proprietary or classified information. ______________________________________ Charmaine L. Porter Certificate of Approval: _____________________ _____________________ Joe H. Cherry Robert D. Locy, Chair Professor Professor Biological Science Biological Science _____________________ ______________________ Joanna Diller Narendra K. Singh Assistant Professor Professor Biological Science Biological Science ____________________________ Stephen McFarland Dean Graduate School FUNCTIONAL IDENTIFICATION AND CHARACTERIZATION OF AN ARABIDOPSIS THALIANA GENE INVOLVED IN VITAMIN B 6 BIOSYNTHESIS Charmaine L. Porter A Thesis Submitted to the Graduate Faculty of Auburn University in Partial Fulfillment of the Requirements for the Degree of Master of Science Auburn, Alabama December 16, 2005 iii FUNCTIONAL IDENTIFICATION AND CHARACTERIZATION OF AN ARABIDOPSIS THALIANA GENE INVOLVED IN VITAMIN B 6 BIOSYNTHESIS Charmaine L. Porter Permission is granted to Auburn University to make copies of this thesis at its discretion, upon request of individuals or institutions and at their expense. The author reserves all publication rights _______________________________ Signature of Author _______________________________ Date of Graduation iv VITA Charmaine LaShawn Porter, daughter of Claude Porter and Charles and Shari Brown, was born July 30, 1981 in Baton Rouge, Louisiana. She graduated with honors from Baton Rouge Magnet High in 1999. From there she attended Tuskegee University in Tuskegee, Alabama for 1 1/2 years and then transferred to Auburn University in January, 2001. She graduated cum laude from Auburn University with a Bachelor of Science degree in Microbiology, May 2003. Immediately after graduation, she entered graduate school at Auburn University in the Department of Biological Science, in May 2003, to pursue her Master of Science degree in Molecular Biology. v THESIS ABSTRACT FUNCTIONAL IDENTIFICATION AND CHARACTERIZATION OF AN ARABIDOPSIS THALIANA GENE INVOLVED IN VITAMIN B 6 BIOSYNTHESIS Charmaine L. Porter Master of Science, December 16, 2005 (B.S., Auburn University, 2003) 59 Typed Pages Directed by Robert D. Locy Vitamin B 6 (pyridoxine) is a biological coenzyme seemingly involved in environmental stress tolerance and an important vitamin in human nutrition produced in plants as well as fungi and bacteria. A series of studies from these different organisms revealed that SNZ1/SNO1 homologues are the genes involved in the production of pyridoxine. We decided to functionally define SNO1 and SNZ1 orthologs and/or paralogs in the Arabidopsis thaliana genome by using transgenic yeast expressing putative SNZ1 or SNO1 homologs to suppress the growth defects of yeast ?snz1 and ?sno1. Using a blast search we were able to find Arabidopsis homologs of yeast SNZ1/SNO1. Moreover, we have obtained knockout deletion mutants of SNZ1 and SNO1 from S. cerevisiae and were able to confirm that the SNZ1 and SNO1 deletions did not grow as efficiently as wild type in media lacking pyridoxine. In addition, we were able to clone the putative A. thaliana SNZ1 into a high expression yeast shuttle vector (p426 GPD). Our results indicate that the AtSNZ1 does complement phenotypic expression of the SNZ1 knockout in yeast. vi ACKNOWLEGEMENTS The author would like to thank her advisory committee, Dr. Locy, Dr. Cherry, Dr. Singh and Dr. Diller for their time and support in writing this thesis. She would also like to express appreciation for Dr. Jose Barbosa for the his advice and help throughout this investigation. This thesis is dedicated to Shayne O?Reilly for his consistent encouragement and support. vii Style manual or journal used: Plant Physiology Computer Software used: Microsoft Word, Excel, Power Point, Adobe Photoshop 2 and Sigma Plot 2000 viii TABLE OF CONTENTS LIST OF TABLES???????????????????????????..ix LIST OF FIGURES???????????????????????????.x I. LITERATURE REVIEW??????????????????????..?...1 II. FUNCTIONAL CHARACTERIZATION OF ARABIDOPSIS SNZ GENES Introduction?????????????????????????........15 Materials and Methods....???????????????????...?....17 Results?????????????????????????.??.......22 Discussion????????????????????????.??.....28 References????????????????????????..??....32 APPENDIX?????????????????????????????....44 ix LIST OF TABLES 1. Description of various types of vitamins??????????????????1 2. List of SNZ1 and SNO1 verification primers????????????????.19 3. Expected PCR product sizes for verification???????????????.... 23 4. BLAST searches of the putative Arabidopsis SNZ1p and SNO1p???????...26 x LIST OF FIGURES 1. Proposed pyridoxine synthesis pathway in E.coli???????????????3 2. Direction of the Open Reading Frame for SNZ1 and SNO1 in yeast?????.??..9 3. Proposed pyridoxine synthesis pathway in S.cerevisiae????????????11 4. Verified PCR products A. WT (wild type)????????????????????????36 B. ?SNZ1???????????????????????????...37 C. ?SNO1?.??????????????...????????...??...38 5. WT, ?SNZ1 and ?SNO1 growth on minimal media A. Serial dilutions on minimal media????????????????....39 B. Control: growth on synthetic complete media???????????.?.39 6. 48 hr growth curve analysis..?????????????????????.?40 7. Effects of methylene blue on the growth of Wt, ?SNZ1 and ?SNO1 A. WT, ?SNZ1 and ?SNO1 streaked on methylene blue plates?????........41 B. Control- WT, ?SNZ1 and ?SNO1 streaked on methylene blue plates and placed in the dark???????????????????????...... 41 8. Sequence alignments of Yeast and Arabidopsis SNZ1p and SNO1p????..??..42 9. Phenotypic complementation of the AtSNZ1 gene on minimal media??.??.??43 Appendix I. Deletion Strategy. ???????????????...????....?.45 Appendix II. Flowchart of the Six B 6 Vitamers?????????????....?.....46 Appendix III. Summary of Eukaryotic Pyridoxine Studies?????????.??..47 Appendix IV. AtSNZ1 Complementation????????????????.??.48 Appendix V. Pyridoxal Metabolism in Plants????????????.??...?..49 LITERATURE REVIEW Vitamins are indispensable dietary compounds that are required by many organisms. Most unicellular organisms and plants are autotrophic for the production of vitamins. However, as heterotrophs, humans are not able to synthesize vitamins and therefore we must obtain vitamins from other sources, such as fruits and vegetables. Vitamins are essential, in that, they are the substances that we need in order to grow and develop properly. Each group of vitamins has a specific role to play in our development. For instance, vitamin D in milk helps our bones become stronger, vitamin A in carrots aids night vision, vitamin C in oranges assists the body in healing and the B vitamers, which are found in green leafy vegetables, help the body make protein and energy. Table 1. Description of the various types of vitamins Vitamin Function A For vision, eyes, clear skin, healthy bones, hair, teeth A (Beta carotene) Antioxidant which turns into vitamin A when the body needs it D Calcium and phosphorous metabolism needed for strong bones and teeth E Prevents oxidation of proteins, fats, and vitamin a, protects red blood cells K Helps the blood clot properly C An antioxidant that helps create the immune system B-1 (Thiamine) Functioning of nervous system, appetite and energy B-2 (Riboflavin) For the skin, eyes, and energy B-3 (Niacin) For the skin, the nervous system and mental performance B-5 (Panthothenic Acid) develops acetylcholine B-6 Helps metabolize protein and fat, needed for the hemoglobin formation Biotin Metabolize amino acids and stimulate hair growth Folic Acid Red blood cell formation, metabolizes fat, amino acids, and protein Choline Prevents fat accumulation in the liver Inositol Involved in calcium mobilization 1 2 One member of the eight soluble B vitamers, vitamin B 6 , has become of great interest to the scientific community in recent years. This attention is due to the fact that vitamin B 6 has been implicated in a variety of biological processes throughout nature. Vitamin B 6 is a combined term for, pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL) and their phosphorylated derivatives pyridoxine-5?-phosphate (PNP), pyridoxamine-5?- phosphate (PMP) and pyridoxal-5?-phosphate (PLP) (Dakshinamurti, 1990). These six vitamers are all B 6 -vitamers but pyridoxine is the vitamer that is commonly referred interchangeably as vitamin B 6 . Vitamin B 6 , principally in the form of the coenzyme pyridoxal 5'-phosphate (PLP), is involved in a wide range of biochemical reactions, including the metabolism of amino acids and glycogen; the synthesis of nucleic acids, hemoglobin, sphingomyelin and other sphingolipids; and the synthesis of the neurotransmitters serotonin, dopamine, norepinephrine and gamma-aminobutyric acid (GABA) ( Dakshinamurti, 1990). Current research has suggested that vitamin B 6 is involved in, but not limited, to the growth of cells, maintenance in the immune systems, and the balancing of hormonal changes in women (Wyatt et al., 1999). Relative to other vitamins, only a minuscule amount of information is known about the biosynthesis of vitamin B 6 . The first biochemical pathway for pyridoxine biosynthesis was elucidated in E. coli (Laber et al., 1999). This pathway was based on biochemical evidence that indicated that in E. coli, the active form of vitamin B 6 , pyridoxal 5? phosphate (PLP) was synthesized de novo by a pathway involving the condensation of the amino acid 4- (phosphohydroxy)-L- threonine and the sugar 1-deoxy-D-xylulose-5-phosphate (Laber et al., 1999). The genes pdxA and pdxJ code for enzymes that have been implicated in the intramolecular condensation reaction between the two substrates to yield PNP. PNP is then oxidized by the gene product of PdxH to form the active coenzyme PLP (Zhao et al., 1995). It has also been shown that Rhizobium meliloti uses the same precursors, 1-deoxy- d-xylulose and 4-hydroxy-L-threonine, as E. coli, in order to synthesize pyridoxine (Tazoe et al., 2000). The original work with E. coli involving pyridoxine auxotrophic mutants (Osami et al., 1999) has been followed by extensive bioinformatics analyses that have shown that most bacteria and archea contain this pathway. However, this predominantly bacterial pathway has never been demonstrated in Eurkaya and some Archea. 4-phospho-hydroxy-L threonine + 1-Deoxy-D-xyulose-5-P PdxA/ PdxJ Pyridoxine-5-P Pyridoxal-5-P PdxH Pyridoxamine-5-P A summary of the E.coli pyridoxine pathway (conserved E.coli genes are colored blue) E.coli transaminases Figure 1. The proposed pyridoxine synthesis pathway in E.coli Precursors to the de novo pathway of pyridoxine (vitamin B 6 ) involves the condensation of the amino acid, 4-phospho-hydroxy-L threonine and sugar, 1-Deoxy-D-xyulose-5-P which are then catalyzed by the enzymes PdxA and PdxJ to form pyridoxine-5-P (PNP). 3 4 The biosynthesis of pyridoxine has evolved via two different pathways during the evolution of eubacteria (Ehrenshaft et al., 1999). Some bacteria utilize the previously defined E. coli pathway and other organisms (primarily Archea and Eukarya) use an, at the present, a poorly defined Eukarya pathway. Radiolabling experiments support the notion of two mutually conserved pathways for pyridoxine synthesis. However, the point at which the divergence occurred between prokaryotes and eukaryotes is not well understood. We do know from feeding studies using 15 N-labeled substrates that glutamic acid is donor of the amide group in bacterial pyridoxine biosynthesis (Tanaka et al., 2000). Contrarily, in the yeast Saccharomyces cerevisiae as well in three additional eukaryotes, 15 N ?labeling revealed that the nitrogen atom of pyridoxine arises from the amide group of glutamine and not glutamic acid (Tanaka et al., 2000). Additional evidence indicated a divergence of pathways when 13 C labeling provided evidence that the C 5 carbon skeleton of vitamin B 6 in yeast originates from a glucose-derived pentulose or pentose intermediate (Gupta et al., 2001). On the other hand, in E. coli, the C 5 chain of pyridoxine originates by the condensation of pyruvic acid with glyceraldehyde 3- phosphate which leads to the production of one of the major building blocks of pyridoxine in E. coli, 1-deoxy-D-xylulose 5- phosphate (Gupta et al., 2001). Although the E. coli pathway of pyridoxine formation has been extensively studied and understood, this isotopic labeling has given us a start point in understanding the mechanism of eukaryotic pyridoxine biosynthesis. More information on the genetics of pyridoxine synthesis became available when bioinformatics analysis using the BLAST search utility at NCBI revealed that 5 microorganisms either contain homologues to E. coli pdxA/pdxJ or homologues to a gene called SOR1; but does not contain copies to both types of genes (Ehrenshaft et al., 1999). SOR1 is the important gene that became the catalyst in understanding the genetic and phenotypic relationship of pyridoxine in eukaryotes. SOR1 (singlet oxygen resistance) is a gene first identified in the filamentous fungal pathogen of plants, Cercospora nicotianae, with linkage to resistance against compounds that produced singlet oxygen (Ehrenshaft et al., 1999). Cercospora produces a potent toxin called cercosporin as part of its mechanism of plant pathogenesis. Cercosporin is a photosensitizer that is activated by light and produces singlet oxygen (O 2 singlet ). This singlet oxygen can cause damage to critical cellular components, such as membranes, proteins and DNA which often leads to cellular death. Organisms have developed methods to protect themselves against reactive oxygen species (ROS) that are reduced in form, such as superoxide (O 2 - ), hydrogen peroxide (H 2 O 2 ) and the hydroxyl radical (OH?). Naturally, because these ROS are by products of cellular metabolism, they have received a tremendous amount of attention in the literature. Conversely, most organisms are unable to tolerate the non-radical singlet oxygen (O 2 singlet ) and there are only a few biological defenses that have been identified for scavenging (O 2 singlet ) (Daub 1998). Because Cercospora is unique in using a (O 2 singlet ) ?generating toxin as part of its mechanism of pathogenesis, it must have mechanisms to protect itself from toxicity of the (O 2 singlet ). The mechanism that Cercospora has evolved utilizes the gene product of SOR1 gene to detoxify (O 2 singlet ). Furthermore, SOR1 has another role other than the conferring resistance to ROS in C. nicotianae (Ehrenshaft et al., 1999). Studies have revealed that 6 SOR1 is involved in a de novo pathway of vitamin B 6 synthesis. Experiments involving C. nicotianae sor1 mutants demonstrated that such mutants were unable to grow in the absence of pyridoxine but pyridoxine prototrophy was restored by a SOR1 transformation (Ehrenshaft et al., 1999). In addition, because of the studies with the Cercospora SOR1gene, pyridoxine was implicated in the quenching (O 2 singlet ) through a mechanism that consumed pyridoxine inside the cells (Ehrenshaft et al., 1999). Actually, the authors were able to show that pyridoxine quenches (O 2 singlet ) at a rate comparable to two of the most highly efficient antioxidants, vitamins C and E. This was the first demonstrations that showed pyridoxine had antioxidant properties and was capable of quenching or protecting against (O 2 singlet ) . Thus, the reports with Cercospora nicotianae on the SOR1 gene was the beginning of characterizing a novel biochemical function for pyridoxine in eukaryotes. The fact that studies with the SOR1 gene have indicated that pyridoxine aids Cercospora in resisting (O 2 singlet ) production is consistent with results that have been published in other systems. The antioxidant properties of vitamin B 6 (pyridoxine) were examined in Schizosaccharomyces pombe by treating cells with menadione, which is a generator of superoxide (Chumnantana et al., 2004). It was shown that pyridoxal-5?- phosphate, pyridoxamine-5?-phosphate and pyridoxamine demonstrated higher antioxidant activity than vitamin C. Moreover, numerous studies in humans have indicated that a pyridoxine deficiency occurs in both type 1 and type 2 diabetic patients (Ellis et al., 1991) and a study using red blood cells (RBC) showed that pyridoxine could inhibit (O 2 singlet ) and radical production, which helps prevent lipid peroxidation, protein glycosylation and lowers hyperglycemia in diabetic patients (Jain and Lim, 2000). Thus, 7 it seems that pyridoxine has the dual function of supplying the coenzyme PLP to many enzymatic reactions along with providing resistance against active oxygen species. As a result of widespread genomic sequencing, it is now understood that SOR1 gene of unknown biochemical function, is a highly conserved gene throughout the three domains of life (Stolz and Vielreicher, 2003). Sequence alignments revealed that this gene exhibits approximately 60% homology throughout the archea, eubacteria and eukaryotes kingdoms. All of the organisms in these kingdoms have evolved a different name for the gene involved in vitamin B 6 production. A study in Neurospora crassa was able to effectively indicate that PDX1 is the SORI homologue through genomic analysis of the fungi?s genome; they were also able to demonstrate that mutations in the PDX1 gene conserved regions caused the pyridoxine mutant phenotype that has been documented in the literature (Bean et al., 2001). In Aspergillus nidulans, pyro A, is the name used for the SOR1 homologue. Interestingly, mutations of the pyroA, cause increased sensitivity to the (O 2 singlet ) generated by the photosensitizer, methylene blue (Osmani et al., 1999). However, the pyroA auxotrophic mutants were able to grow in the methylene blue at media concentrations of pyridoxine of 0.5 ug/ml in the media. Thus, comparable to the studies cited above in other fungi, this data indicates that in A. nidulans, pyroA is required for pyridoxine biosynthesis and for resistance to (O 2 singlet ). Since several independent experiments provided data to show that SOR1 and its homologues are involved in pyridoxine biosynthesis, SOR1 and its homologues are now commonly referred to as PDX (pyridoxine requiring) gene, as a measure to give a consensus name in the literature among the homologues. Some organisms have more than one copy of PDX1 gene. In fungus, Neurospora crassa contains two copies of the PDX1 8 homologue, i.e. pdx1/pdx2, which are in close physical proximity to each other (Bean et al., 2001). Additionally, other organisms, such as Bacillus subtilis and Haemophilus influenzae, also encode this second gene that is in close proximity to its PDX1 homologue (Ehrenshaft and Daub, 2001). Moreover, in the yeast Saccharomyces cerevisiae, the analysis of the PDX genes becomes more complicated, in that yeast have three unlinked PDX1 homologues called SNZ1 (Snooze), SNZ2 and SNZ3 respectively. In addition there are three PDX2 homologues that are adjacent to the PDX1 homologues and have been named SNO (Snz proximal ORF). Paralogous gene families, such as the SNZ/SNO family, comprise approximately 40% of the yeast genome (Blandin et al., 2000). Sometimes these gene redundancies involve genes with exact functional redundancy although there are many cases in which the paralogous genes have undergone evolutionary divergence where the genes are only partially functionally redundant or not functionally redundant at all (Rodriguez-Navarro et al., 2002). This divergence is what is believed to have happened to the SNZ gene family in yeast. SNZ1 ?snooze? (YMR096c) was originally identified when it was shown that the SNZ1p protein was expressed during the stationary phase of the yeast life cycle (Braun et al., 1996). Additional homologues, SNZ2 (YNL333w) and SNZ3 (YFL059w) were confirmed after publication of the entire yeast genome. SNZ2 and SNZ3 encode proteins that exhibit approximately 99% homology with each other and approximately 80% identity to the SNZ1 amino acid sequence (Padilla et al., 1998). Furthermore, analysis of yeast genomic sequences has led to the discovery of another conserved gene family located on the same chromosome immediately adjacent to each SNZ gene that is called SNO (SNZ-proximal reading frame) (Figure 2). Comparably, SNO2 (YNL334c) and SNO3 (YFL069c) encode for proteins that are nearly 100% identical to each other and approximately 72% identical to the SNO1 (YMR095c) protein (Braun et al., 1996). SNZ1 SNO1 Chromosome 13 Figure 2. Direction of the Open Reading Frames for SNZ1 and SNO1 On Chromosome 13 in Saccharomyces cerevisiae. In yeast, the paralogous members of the SNZ and SNO family, i.e. SNZ1 and SNO1, are located adjacently on the same chromosome and are transcribed in divergent directions. Now, a conundrum exists in the literature about SNZ homologues. Bioinformatics studies have shown SNZ and its homologues to be one of the most highly conserved proteins present within bacteria, fungi and higher plants but these proteins have not had a previously defined function. Usually a high degree of conservation throughout evolutionary time signifies the importance of a protein in nature. This is not the case for the SNZ/SNO family in yeast. In fact, single null deletions of each gene results in viable cells. Ironically, the expression of these genes increases during stationary phase which is a time when most genes decrease in expression (Braun et al., 1996). This observation 9 10 along with evolutionary conservation has led to the hypothesis that the SNZ/SNO gene families are involved in the universal nature of the growth response. As mention previously, the three different studies from Aspergillus nidulans, Neurospora crassa, and Cercospora nicotiniae have revealed that the SNZ/SNO homologous genes (PDX1/2, PyroA, and SOR1) are related to the biosynthesis of pyridoxal (vitamin B 6 ). To support the hypothesis that the yeast SNZ/SNO genes have an analogous phenotypic expression to their homologues involving pyridoxine biosynthesis, it was demonstrated that SNZ1 and SNO1 mutants were sensitive to methylene blue, a producer of singlet oxygen (Padilla et al., 1998). Surprisingly, the other mutant alleles snz1, 2, 3?3, sno1, 2, 2?3, snz2, 3?3, sno2, 3?3, were not sensitive to growth on methylene blue (Padilla et al., 1998). Furthermore, only SNZ1 and SNO1 are required for growth in media lacking pyridoxine, but SNZ2, SNZ3, SNO2 or SNO3 are not required for growth. Copies 2 and 3 of SNZ/SNO are activated by vitamin B 1 (thiamin) biosynthesis, and thus may play a role in the synthesis of that vitamin (Rodriguez-Navarro et al., 2002). In addition, yeast 2-hybird analysis indicated that there was a strong interaction between Snz1p and Sno1p (Padilla et al., 1998). Thus, it can be concluded that only the SNZ1 and SNO1 copies are responsible for the metabolism of pyridoxine in yeast, while the other copies are involved in some other growth response. The conserved aspects of the SNZ/SNO homologues in nature has led to the quest of understanding the manner in which SNZ1/SNO1 participates in the pyridoxine biosynthetic pathway. Moreover, yeast has become the model system in which the eukaryotic pyridoxine pathway is being studied and understood. O OP OH OH OH CH 2 0-P H-C-OH H-C-OH C=O CH 2 OH Putative Pyridoxine Synthetic Pathway in Yeast Taken from H.Kondo et. al N OH OH OH HOH 2 C Gln NH4 Sno1p Snz1p D-ribose 5- phosphate D-ribulose 5- phosphate 2?hydroxypyridoxine CH 2 0H CH 2 0H N H 3 C OH Pyridoxine rki1 Ribose 5-Phosphate ketol- isomerase snz1/sno1 Rki1p Figure 3. Proposed pyridoxine pathway in Saccaharomyces cerevisiae The pyridoxine pathway in yeast begins with D-ribose 5?-phosphate by which ribose 5?-phosphate ketol-isomerase (Rki1p) catalyzes the compound into D- ribulose 5?-phosphate. The Snz1p and the Sno1p complex release ammonia from glutamine, where the nitrogen is incorporated into the ring, leading to the formation of 2?-hydroxypyridoxine. Currently, data strongly suggest that the Snz1p and Sno1p complex functions as a glutaminase to supply ammonia for the ring nitrogen of pyridoxine in yeast (Dong et al., 2004). The Snz1p and Sno1p complex is believed to participate in a glutamine amidotransferase reaction with Sno1p serving as a glutaminase (Dong et al., 2004). Recently, it has been shown through a cross complementation experiment using the E. coli pdxJ with Cercospora SOR1 (Pdx1/SNZ1) gene, that PDX1 is the responsible for pyridoxine ring closure reaction (Wetzel et al., 2004). Since this finding, a putative pathway of the synthetic pathway of pyridoxine in yeast has been proposed in the literature (Kondo et al, 2004). The synthesis starts with D-ribose 5-phosphate being 11 12 converted to D- ribulose 5-phosphate by a ribose 5-phosphate ketol-isomerase, ultimately providing the C 5 carbon skeleton of the vitamin (Kondo et al., 2004). From this point, the Sno1p and Snz1p releases the ammonia from glutamine and the nitrogen atom is incorporated in the ring that formed by the condensation of D-ribulose 5-phosphate with an unidentified substrate forming, 2-hydroxypyridoxine, the starting biosynthetic precursor of vitamin B 6 (Zeidler et al., 2001). There is a commonality among both the prokaryotic and eukaryotic pathway in which these organisms have a resourceful salvage pathway that converts the three natural free forms of vitamin B 6 , pyridoxine (PN), pyridoxal (PL), and pyridoxamine (PM), to the major operating coenzyme, pyridoxal-5?- phosphate (PLP). This salvage pathway involves the use of two enzymes, pyridoxal kinase and pyridoxine-phosphate oxidase. Pyridoxal kinase catalyzes the phosphorylation of the 5-hydroxymethyl group of all three vitamins while pyridoxal ?phosphate oxidase catalyzes the oxidation of the alcohol of pyridoxine (PNP) and the amine of pyridoxamine (PMP) to an aldehyde liberating ammonia in the latter case (McCormick and Chen, 1998). In addition, there are phosphatases that are able to convert the phosphates of the vitamins to back to the free form of the vitamin. Pyridoxine involving genes, such as SNZ and SNO, are not found in the genome of higher eukaryotes (animals), with the exception of the marine sponge Suberites domuncula (Seack et al., 2001). Sponges (Urmetazoa) were one of the first organisms to diverge from the common ancestor of all Metazoa (Seack et al., 2001). Since animal lineages have obviously lost the SNZ and SNO genes, it is possible loss of SNZ and SNO in animals occurred at the Poriferal (sponges) lineage (Tanaka et al., 2004). Interestingly, 13 it has been reported that there is mammalian pyridoxal kinase and oxidase activity. A study indicated that the mammalian pyridoxal oxidase is developmentally regulated in the liver and brain. Moreover, it was shown that there is an absence of pyridoxal oxidase in certain tumors (Ngo et al., 1998). It was hypothesized that the mammalian pyridoxal kinases and oxidases help to facilitate entry of vitamin B 6 into the cell (McCormick and Chen, 1998). This observation is appealing in that humans can not synthesize vitamin B 6 and must uptake the vitamin nutritionally from other sources, such as plants, fungi or bacteria. From these experiments, it is apparent that vitamin B 6 plays major roles in mammals that are not yet fully identified. As we are constantly looking for means to improve the longevity of humans, logically, it would be appropriate to study how humans obtain and metabolize their vitamin B 6 source, which is plants, and to examine how the source of vitamin B 6 could be manipulated in plants. Pyridoxine 5??-D-glucoside (PNG), is a natural occurring glycosylated derivative of vitamin B 6 , which is found in most fruits, vegetables, and cereal grains comprising approximately 75% of the total amount of vitamin B 6 found in plants (Gregory,1998). Unfortunately, there is little information known about pyridoxine biosynthesis in plants. Bioinformatic searching of the genomes of different plants species has shown that there are several genes that have been identified which show a high degree of homology to PDX1. However, none of these genes have a function identified, and none of the genes have been extensively characterized. One study, attempted to characterize the PDX1 orthologue in the plant Phaseolus vulgaris. The results indicated that the pvPDX1 (putative PDX I) gene of P. vulgaris, was able to partially reverse pyridoxine auxotrophy in null mutants of C. nicotianae (Graham et al., 2004). This is the first data to suggest 14 that plants also use the eukaryotic divergent pathway of pyridoxine biosynthesis. However, further work is needed in order to elucidate the pyridoxine biosynthetic pathway of plants. Since the genome has already been sequenced, Arabidopsis thaliana is a useful model system in order to learn more information about the intrinsic factors of vitamin B 6 . Also, Arabidopsis plants are easily grown in the laboratory, making the plant an appealing system for studying the SNZ1/ SNO1 (Pdx1/Pdx2) genes functional characteristics in plants. In, Arabidopsis mutants, it was shown that the SOS4 (salt overly sensitive) gene encodes a pyridoxine, pyridoxal or pyridoxamine (PN/PL/PM) kinase homolog that functions in the biosynthesis of PLP (Shi et al., 2001). In addition, a blast search revealed that Arabidopsis genome contains at least three homologs to SORI/ PDXI/ SNZ1 that may putatively function as the protein involved in pyridoxine biosynthesis with the gene accession numbers as follows: At2g38240, At3g16050, and At5g01410. In addition, At5g60540 and At4g26900 represent putative SNO1 (Pdx2) homologs. Detailed knowledge of vitamin synthesis in plants might lead to better nutritional quality in foods which would indirectly enhance the life of humans. With this perspective in mind, current studies were initiated in an attempt to elucidate the pyridoxine biosynthesis pathway in plants. 15 INTRODUCTION Eukaryotic and certain prokaryotic organisms have a gene family homologous to the SOR1 gene of Cercospora nicotianae (yeast SNZ) that expresses a highly conserved protein which exhibits at least 60% homology in Archea, bacteria, and eukaryotes. Despite this conservation, only a minuscule amount of information is known about its function. However, it has been shown in Saccharomyces cerevisiae (Baker?s yeast) that SNZ synthesis increases dramatically during stationary phase (Braun et. al., 1996). SNZ is a member of a paralogous gene family consisting of three members, SNZ1, SNZ2 and SNZ3. In addition, each of these genes is found adjacent to members of another conserved family named SNO (SNZ-proximal reading frame). Studies show that both SNZ1 and SNO1 are involved in pyridoxine (pyridoxal) biosynthesis, which is the first step of vitamin B 6 production. Vitamin B 6 is an essential nutrient that provides the coenzyme pyridoxal-5-phosphate (PLP) to most biological systems. Furthermore, it has been documented that PLP plays a role in protective responses to stress in certain organisms. Using bioinformatics analysis, we have found that the Arabidopsis thaliana genome contains homologues to the SNZ1 and SNO1 genes found in yeast and other fungi. We have used PCR to obtain cDNA clones of the coding sequence of these Arabidopsis genes, and we have obtained single gene deletions of the Saccharomyces cerevisiae SNZ1 and SNO1 genes. These mutants are unable to grow as efficient, in comparison to the WT strain, when placed in media lacking pyridoxine/pyridoxal phosphate. We have expressed an A.thaliana homologue of yeast SNZ1 in these yeast deletion mutants and 16 demonstrated functional complementation of the phenotype of the SNZ1 growth and biochemical phenotypes. 17 MATERIALS AND METHODS Strains and Media The following strains S. cerevisiae were used in this study: BY4742 (Mat? his?1leu2?2lys?2ura3?3), BY4742::?SNZ1 (Mat? his?1leu2?2lys?2ura3?3) and BY4742::?SNO1 (Mat?his?1leu2?2lys?2ura3?3). The following media was used for the cultivation of yeast in this study: YPD (1% yeast extract, 2% peptone, 2% glucose), SC (Synthetic Complete) (0.67% yeast nitrogen base without amino acids, 2% glucose, and supplemented with auxotrophic requirements which included histidine, leucine, lysine, and uracil), SC-Ura ( same as SC except lacking uracil supplementation); Minimal Medium (vitamin B 6 deficient media) composed of the same components as SC media except that vitamin B 6 was specifically excluded from the yeast nitrogen base mixture used; and methylene blue medium (where YPD was supplemented with 37 ?g of methylene blue per ml and subjected to a light source. All solid media used for Petri Plates contained 2% agar. Strain Cultivation All strains grown on plates were incubated at 30?C for 2-3 days unless otherwise indicated. In experiments, where liquid culture was conducted, the yeast cells were shaken at 250 rpm at 30?C and samples taken from the culture were assayed for growth at time points shown in the various figures and the text. 18 Yeast Knock Out Strains Both the ?SNZ1 and ?SNO1 single deletion knockouts were purchased from the American Type Culture Collection (ATCC). The knock out strains were constructed based on a PCR-based gene deletion strategy which was use to generate a start-to-stop deletion of the open reading frame of SNZ1 and SNO1. The deletion module consisted of a KanMX4 cassette that was constructed with the addition of the 45 base pairs immediately upstream of the ATG start codon and the 45 base pairs of sequence immediately downstream of the stop codon. During the deletion process, both the SNZ1 and SNO1 genes were replaced with a KanMX4 module via homologous recombination. The KanMX4 module was uniquely tagged with two 20mer sequences which were detected via hybridization to a high density oligonucleotide array. Yeast Knock Out Strain Verification For verification of specific deletions, specific primers used for polymerase chain reaction verification were designed between lengths of 17 and 28 bases and a melting temperature (T M ) of around 65?C. Primers A and D were designed to be homologous to the region which is about 200 bases upstream or downstream of the open reading frame. Primers B and C were designed to bind to regions within the open reading frame and yield a PCR product of 300-1000 bases when used with the A or D primers. The KanB and KanC primers were picked from regions within the KanMX4 cassette. Specific primer combinations for both SNO1, SNZ1, and KanB and KanC are shown in Table 2. 19 Table 2. List of SNZ1 and SNZ1 specific verification primer combinations Primer Name Primer Sequence SNO1-A GCGTCCTCCTATTTATATCGAAAAT SNO1-B AAACTACCTTTTCCGGATTATGAAC SNO1-C AATTAGAAAACGAAAGTGCCCTAGT SNO1-D CACACAAGAATAAAAGAGCTTGACA SNZ1-A TTTATGGCACTAGTTGGAATAGCTC SNZ1-B GCCTTTATCTCCTCTGTAATTCTCC SNZ1-C GGAGAATTACAGAGGAGATAAAGGC SNZ1-D ATATTTGACCCTGAGGTACTGTTGA KanB TAATGCCGAGGAGGGACGTC KanC TGATTTTGATGACGACGAGCGTAAT All strains were verified using a standard 25ul PCR reaction which included template DNA obtained from the appropriate strain to be tested, 2.5mM dNTPs, 50mM MgCl 2 , 10X PCR buffer, Taq polymerase (Qiagen) and primers consisting of the combinations A and B, C and D, A and D, A and KanB, KanC and D. KanB and KanC primers were used in order to verify that the SNZ1 and SNO1 genes were correctly knocked out and replaced by a kanamycin resistance cassette. A successful deletion yielded PCR products using primers A and KanB, and KanC and D, but did not yield PCR products using primers A and B or C and D. The A-D primers yield a product regardless if the strain was WT or a deletion, but the size of this product could vary between wild type and mutant strains depending on the length of the gene in question. Arabidopsis cDNA Arabidopsis SNZ1 and SNO1 homologs were prepared by PCR using an Arabidopsis cDNA, purchased from the Ohio State stock center, as template. Primers for the 5? and 3? ends of the putative AtSNZ1 and AtSNO1 were designed that contain restriction sites 20 on their 5? ends (AtSNZ1-Xho1 (5?-primer) and EcoRI (3?-primer); AtSNO1-XhoI (5?- primer) and SpeI (3?-primer)). The amplified PCR product were cloned into pGEM T- Easy and sequenced to verify the PCR product. The cloned pGEM T-Easy vector was then cut with appropriate restriction enzymes, and inserts were then cut out, separated from plasmid DNA using agarose gel electrophoresis, cleaned from the gel, and directionally cloned into the p426 GPD shuttle vector. This construct was grown in E. coli, and subsequently transformed into appropriate yeast strains for phenotypic evaluation. E. coli Transformation E. coli transformations involving the pGEM T-Easy plasmid and yeast shuttle vector (p426 GPD) were performed by using standard heat shock protocol. First, 50ng of plasmid DNA was mixed in a 1.5ml microfuge tube with 50ul of E. coli DH5? competent cells and incubated on ice for 30 minutes. Then the tubes containing the mixture of plasmid and competent cells was placed into a water bath at 42 o C for 45 seconds. The tubes were then put back on ice for 2 minutes to reduce the damage to the E. coli cells. Following the previous step, 500ul of LB (minus antibiotic) was added to the tubes which were then incubated for 1hr at 37 o C. After the 1 hr incubation period, 100ul of the culture was spread on LB plates containing ampicillin and incubated overnight. Yeast Transformation Transformation of yeast deletions with the putative Arabidopsis SNZ1 gene was accomplished by the lithium acetate method (Gietz, 1994). 21 Sequencing Sequencing was done by Auburn University Genomic and Sequencing Lab using appropriate primers for each sequenced construct. Serial Dilutions The yeast strains were grown in the appropriate media for about 1-3 days until the cells reached a density of 2.0 at OD 600 . These cells were then diluted to approximately a 1.0 OD 600 and then aliquoted to five 1:10 serial dilutions and plated on 100mm 2 plates. Growth Curve WT and mutants were inoculated into test tubes containing minimal media at a starting OD of 0.02. To minimize error, three replicates of each growth curve were made and the standard error of each time point was calculated using the statistical package in Microsoft XP. The OD was taken every two hours for 48 hrs. The control followed the procedure above but used synthetic complete media instead of minimal media. 22 RESULTS Verification of the Saccharomyces cerevisiae snz1 and sno1 Deletion Mutants Strain deletion was performed by using a PCR deletion strategy that allowed the SNZ1 and SNO1 genes to be replaced by a KanMX4 module which breaks down the antibiotic, kanamycin and geneticin (a kanamycin analog more toxic to yeast). The yeast knockouts were initially screened based on the fact that the deletion containing the KanMX4 module would confer resisitance to geneticin. A successful deletion results in the replacement of the sites where the B and C primers land in the wild type SNO or SNZ genes with sites where the KanB and KanC primers would land in the KanMX4 module. The A and D primers which are outside the reading frame of SNO and SNZ will remain. The wild type and knockout strains were verified by using the A/D, A/KanB, A/B, KanC/D, and C/D primers in a PCR reaction. For the knockout strains only the A/D, A/KanB and KanC/D PCR products were obtain which indicated that the gene was knocked out (Figure 4b and 4c ). WT did not produce any PCR product with the KanB or KanC primers but did with the B and C primers (Figure 4a). Table 3 shows the expected size of the PCR products for each gene and primer combination. The appropriate size of the PCR products obtained confirmed that we had appropriate yeast deletion strains containing the sno1 deletion and the snz1 deletion. 23 Table 3. Expected Sizes of PCR product for verification by using the designated deletion primers Strain A-D A-KanB KanC-D A-B_WT C-D_WT WT 1450 bp ________ ________ 805 bp 666 bp ?SNZ1 2140 bp 592 bp 890 bp ________ _______ ?SNO1 2182 bp 655 bp 869 bp ________ _______ The Phenotype of Yeast sno1 and snz1 Deletion Mutants Yeast cells are potentially a model system for understanding the eukaryotic pathway for pyridoxine biosynthesis. It has been described that the yeast SNZ1 and SNO1 are required for growth in media lacking vitamin B 6 (Rodriguez- Navarro et al., 2002). However this phenotype is difficult to demonstrate. Thus, in order to use yeast as a model system to functionally characterize the Arabidopsis SNZ1 and SNO1 homologues, it was necessary to verify that yeast sno1 and snz1 mutants cannot grow on media lacking pyridoxine. Yeast deletion mutants and wild type cells were cultivated overnight in liquid SC media without pyridoxine and subsequently plated on Petri plates on the same SC minimal media minus pyridoxine. The cells were washed 2X to remove traces of pyridoxine pre-cultured media and diluted to an OD 600 of 0.2. The cells were then plated in a set of serial dilutions of this titer, and the results of this experiment are shown in Figure 4a. 24 From this experiment, it was apparent that both sno1 and snz1 deletion mutants were not able to grow as efficiently as wild type on minimal media lacking supplied pyridoxine. The wild type had growth even at the last 10 -4 dilution. There was no growth of the ?snz1 after the 10 -2 dilution while ?sno1 growth was lost at the 10 -3 dilution. These results confirm that the ?snz1 and ?sno1 knockout are sensitive to the lack of pyridoxine and more interestingly, that ?snz1 has a slightly more drastic phenotype than the ?sno1 on minimal media (Fig. 5a). When WT and the 2 deletion mutant strains were plated on SC medium at the same serial dilutions as described, both of the mutants as well as the WT grew comparably on SC medium containing pyridoxine (Fig. 5b). This demonstrates that the growth defect in the mutants observed on minimal medium lacking pyridoxine is only related to the absence of pyridoxine in the medium. To further substantiate the phenotype of ?snz1 and ?sno1, quantitative measurements of the growth of ?snz1 and ?sno1 in liquid media with and without pyridoxine were made. The growth curve was carried out for 48 hrs in minimal media without pyridoxine. Our results show that after 48 hrs there was a significant difference of growth between WT and the ?snz1 and ?sno1 mutant (Fig. 6). There was a slight difference of growth between WT and the SNO1 mutant. This growth curve analysis is consistent with the observation that mutants are not able to grow as efficiently as WT on media lacking pyridoxine. This phenotype further substantiates that these genes do play a role in the synthesis of pyridoxine. The original discovery of the SOR1 gene has led to the subsequent finding that the pyridoxine and its metabolites have antioxidant properties. Methylene blue, under visible 25 light, produces toxic singlet oxygen which can be damaging to the cellular components, leading to apoptosis. Padilla et al. reported that out of all the three SNZ/SNO homologues of yeast, only SNZ1 and SNO1 were sensitive to methylene blue in the light (Padilla et al., 1998). To further analyze the defects of the yeast ?snz1 and ?sno1 deletions, we subjected the cells to methylene blue-induced singlet oxygen stress. Figure 6 indicates, that the ?snz1 and ?sno1 deletion mutants are more sensitive to methylene blue than wild type. Again, we were able to show ?snz1 mutant was more sensitive than ?sno1 (Figure 7). Heterologous Expression of the Arabidopsis putative SNZ1 gene Now that the phenotypic characteristics of yeast SNZ1 and SNO1 genes and their deletion mutants have been determined, it is possible to use these mutant strains to examine the phenotype of the strains expressing the homologs of SNO and SNZ from the plant Arabidopsis thaliana. This will permit the identification of functional homologs of these genes from this plant. To begin these experiments a bioinformatic analysis was done using the WU-BLAST2 search utility at The Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org/). The results of these BLAST searches are given in Table 4. Note that there are 3 genes in the Arabidopsis genome that code for proteins that show greater than 60% similarity to the yeast SNZ1p amino acid sequence. The alignment of these sequences with the sequence of yeast SNZ1p is shown in Figure 8. Note that while there is significant divergence of the Arabidopsis and yeast sequences at both the amino- terminal and carboxy-terminal regions, there is substantial identity of the sequences in the 26 middle regions of the protein. There is only one gene that codes for a protein with greater than 50% similarity to the yeast SNO1p amino acid sequence. Table 4. Results of BLAST searches using the yeast SNO1p and SNZ1p amino acid sequences to detect homologs of those genes in the genome of Arabidopsis thaliana. Gene (Bit score) E-value Length Identity Similarity Arabidopsis homologs to Yeast SNZ1 (NP_013814) 68418.m00054 stress-responsive protein, putative similar to ethylene- inducible protein HEVER [Hevea brasiliensis] SWISS-PROT:Q39963 At5g01410 (851) 3.9e-86 310 aa 57% (165/284) 74% (212/285) 68415.m04695 stress-responsive protein, putative similar to ethylene- inducible protein HEVER [Hevea brasiliensis] SWISS-PROT:Q39963; contains Pfam domain, PF01680: SOR/SNZ family At2g38230 (NP_181358.1) (830) 6.5e-84 310 aa 58% (162/276) 75% (209/276) 68416.m02029 stress-responsive protein, putative similar to ethylene- inducible protein HEVER [Hevea brasiliensis] SWISS-PROT:Q39963; contains Pfam domain, PF01680: SOR/SNZ family At3g16050 (NP_188226.1) (632) 6.2e-63 315 aa 43% (121/276) 64% (183/276) Arabidopsis homologs to Yeast SNO1 (NP_013813) (238) 3.5e-21 256 aa 38% (66/172) 54% (93/172) Having found sequences for genes from Arabidopsis that are putatively functional homologs of both yeast SNZ1 and SNO1, clones of these genes were obtained. One of the putative Arabidopsis SNZ1 genes was functionally characterized and used for the functional characterization in the yeast system. It was demonstrated that the Arabidopsis gene functionally complements the yeast ?sno1 deletion mutant. Our focus was on the SNZ1 because our previous research has provided strong evidence that SNZ1 has the stronger phenotype and Arabidopsis contains multiple genes that are putatively SNZ- homologs. We were able to engineer the At5g01410, putative Arabidopsis SNZ1 gene, into a p426 GPD, which is a multicopy expression vector with a strong yeast promoter. 27 This plasmid was then transformed into its perspective knockout mutant. The presence of the glyceraldehyde 3 ?phosphate dehydrogenase (GPD) promoter in the vector, allowed for the plant gene to be constitutively expressed in yeast. Our results indicate that the AtSNZ1 gene functionally complements the yeast SNZ1 knockout phenotype in minimal media. When we plated WT, ?SNZ1 and the ?SNZ1: AtSNZ1 on minimal media, it was clear that cells complemented with the AtSNZ1 grew comparably to WT (Fig 9), while the mutant showed inhibition of growth at an earlier dilution. This is the first direct experimental evidence that indicates that the putative AtSNZ1 gene is actually involved in pyridoxine biosynthesis. 28 DISCUSSION Vitamin B 6 and its intermediates are important coenzymes in many enzymatic reactions. The implication of vitamin B 6 importance in many aspects of nature has led scientists to research the synthesis of the vitamin. Bioinformatics studies have revealed that most organisms use one of the following two pyridoxine biosynthetic pathways that have been discovered to date, i.e. the E. coli pathway or eukaryotic pathway. In addition, 15 N ?labeling has shown that glutamine was incorporated into pyridoxine ring of yeast unlike E. coli which uses glutamate, confirming that two different pathways exist (Tazuya et al., 1995). The E. coli pathway has been well studied, and how pyridoxine is formed in this bacterium is well understood. However, more research needs to be conducted in order to grasp the full picture of pyridoxine biosynthesis in eukaryotes. We do know that the eukaryotic pathway contains a conserved PDX1 (SORI/SNZ1) gene family that has been proven to be important for vitamin B 6 production. SOR 1, originally known for protecting the fungus Cercospora nicotianae from singlet oxygen production, mutants were unable to grow unless they were supplied with pyridoxine. It was the study of SOR 1 gene in Cercospora that has uncovered a novel role of dual function in which pyridoxine was involved in antioxidant properties as well as pyridoxine biosynthesis. Genomic sequencing has exposed the fact that SOR 1is a gene that has homologues in many organisms and is distributed widely in nature, but the function of this gene has not been extensively characterized. We now know that SORI/PDX1 genes are orthologues to a larger family of genes that are commonly referred to as SNZ genes. 29 SNZ (Snooze) gene family was found in the yeast, Saccharomyces cerevisiae, and its members consist of three unlinked PDX1 homologues, SNZ1, SNZ2 and SNZ3. In addition, there is an SNO (Snz- proximal reading frame) family with each member adjacent to the open reading frame of one SNZ gene and transcribes in opposite directions (Fig.2). This family consists of three homologues to the PDX2 gene, SNO1, SNO2, and SNO3. As mentioned previously, little is known about the function of SNZ/SNO, but a few reports have given some insight to the function of the gene families. It has been shown that both SNZ1/SNO1 increases in synthesis during stationary phase (Braun et al., 1996). Furthermore, it was shown that only the SNZ1/SNO1 homologues were sensitive to methylene blue (Padilla et al., 1998). Lastly, functional analysis showed that both SNZ1 and SNO1 were required for growth in media lacking vitamin B 6 , and SNZ2/SNO2, SNZ3/SNO3 genes pairs seemed to be involved in vitamin B 1 (thiamin) biosynthesis (Rodriguez-Navarro et al., 2002). Our results have confirmed that both the SNZ1 and SNO1 in yeast are involved in the vitamin B 6 biosynthesis. We were able to go back and repeat some of the experiments in the literature that were done on SNZ1 and SNO1 mutants. Our experiment with vitamin B 6 less media (minimal media) clearly indicated that both SNZ1 and SNO1 were needed for growth in media lacking pyridoxine. We became more convinced of this phenotype when we quantitatively assessed the growth curve between WT and the mutants in minimal media; where SNZ1 and SNO1 grew less than the WT strain. Additionally, we were able to show that SNZ1 and SNO1 are sensitive to growth on methylene blue plates. Thus, it is very likely that pyridoxine functions as an antioxidant against singlet oxygen production in yeast. In all of our experiments, it was clear that 30 SNZ1 mutant had a more pronounced phenotype that SNO1. There is strong evidence that suggest that SNO1 is a glutaminase possibly supplying the nitrogen atom for the pyridoxine ring when it is in a complex with SNZ1 (Dong et al., 2004). However, it is not clearly defined as to what role SNZ1 plays in the formation of pyridoxine but we believe that this stronger phenotype may be due to the fact that SNZ1 may function in one of the early steps in the formation of the pyridoxine ring. More research needs to be done in order to characterize the function of SNZ1. Our goal in this investigation was to characterize the function of the putative Arabidopsis thaliana SNZ1 and SNO1 genes. Since SNZ1 is the gene that is more important in pyridoxine biosynthesis we decided to focus on the just the AtSNZ1 gene. Our complementation of the AtSNZ1 gene on minimal media strongly suggests that the AtSNZ1 may play a role in the formation of pyridoxine as its homologous counterparts. We attempted to demonstrate that the AtSNZ1 construct complemented the methylene blue sensitive phenotype of the yeast ?snz1 mutant, but were unable to demonstrate this (data not shown). This may be the result of the fact that the AtSNZ1 gene does not fully complement the yeast mutant phenotype. This could be related to expression levels in yeast, or possible targeting sequences that wrongly target some or all of the AtSNZ protein. Additionally, in our hands the methylene blue phenotype is not very strong, and thus it was not clear that there is actually a strong methylene blue phenotype in the mutants. In literature, there have not been many reports involving the production of vitamin B 6 in plants. This is surprising since humans obtain most of our dietary vitamin B 6 source from plants. 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(2002) 2?- Hydroxypyridoxol, a Biosynthetic Precursor of Vitamins B 6 and B 1 in Yeast. J. Am. Chem. Soc. 124, 4542-4543. Zhao, G., Pease, A., Bharani, N. and Winkler, M. (1995) Biochemical Characterization of gapB-Encoded Erythrose 4-Phosphate Dehydrogenase of Escherichia Coli K-12 and Its Possible Role in Pyridoxal 5?-Phosphate Biosynthesis. J. Bacteriol. 177, 2804-2812. MW A/D A/KB C/D KC/D A/B Figure 4a. WT verification PCR products obtained with Saccharomyces cerevisiae strain BY4742 (WT) the molecular weight markers (MW) are shown along with the products of reactions using the primer pairs A/B, A/KB, AB, KC/D and CD. Note that only products were obtained with the WT primers. 36 MW A/B A/KB C/D A/D KC/D Figure 4b. SNZ1 mutant verification PCR products obtained with Saccharomyces cerevisiae strain BY4742 (?snz1) the molecular weight markers (MW) are shown along with the products of reactions using the primer pairs A/B, A/KB, AB, KC/D and CD. Note that besides the A/D primer pair, no products were obtained with the WT primers. 37 MW A/B A/KB C/D A/D KC/D Figure 4c. SNO1 mutant verification PCR products obtained with Saccharomyces cerevisiae strain BY4742 (?sno1) the molecular weight markers (MW) are shown along with the products of reactions using the primer pairs A/B, A/KB, AB, KC/D and CD. Note that besides the A/D primer pair, no products were obtained with the WT primers. 38 ________Fig 5a________ ________Fig 5b________ Dilution WT ?SNZ1 ?SNO1 WT ?SNZ1 ?SNO1 1.0 0.1 0.01 0.001 0.0001 Figure 5a & b. Minimal Media Phenotypic Experiments The growth of wild type (BY4742), BY4742:: ?SNZ1, and BY4742:: ?SNO1 was determined by plating a culture of the appropriate yeast strain on Petri plates in either minimal medium (Figure 4a, see methods) or SC medium (Figure 4b, see methods) at varying dilutions (as indicated). The reduced ability of the 2 deletion mutant to grow on media lacking pyridoxine is shown in Figure 4A, while the lack of a growth defect on media containing pyridoxine is shown in Figure 4b. 39 Time (hours) 0 1020304050 OD 60 0nm 0 1 2 3 4 5 WT min SNZ min SNO min WT comp SNZ comp SNO comp Figure 6. 48hr Growth Curve Analysis Forty eight hour growth curves of WT (BY4742) (diamond), BY4742 ?SNZ1 (square), and BY4742 ?SNO1 (triangle) on minimal (closed symbols) and synthetic complete media (open symbols). Growth of 3 replicate cultures of each treatment were estimated by determining the optical density of the culture at 600 nm at the time points indicated in the figure. Error bars shown represent the standard error of 3 replicate measurements at each time point. 40 WT ?SNO1 ?SNZ1 WT ?SNO1 ?SNZ1 Figure 7. Methylene Blue Experiments WT, ?SNZ1, and ?SNO1 streaked on methylene blue plates. Incubation of the plate in the light leads (left plate) to the production of singlet oxygen which reduces the growth of the snz1 deletion mutant, but not the sno1 deletion mutant compared to wild type. The plate on the right was incubated in the dark, where no singlet oxygen is produced. 41 CLUSTAL W (1.82) multiple sequence alignment At5g01410 -----MEG--TGVVAVYGNGAITEAK-KSPFSVKVGLAQMLRGGVIMDVVNAEQARIAEE 52 At2g38230 -----MAG--TGVVAVYGEGAMTETKQKSPFSVKVGLAQMLRGGVIMDVVNAEQARIAEE 53 At3g16050 MADQAMTDQDQGAVTLYSGTAITDAKKNHPFSVKVGLAQVLRGGAIVEVSSVNQAKLAES 60 SNZ1p -----MTG--------------------EDFKIKSGLAQMLKGGVIMDVVTPEQAKIAEK 35 * . *.:* ****:*:**.*::* . :**::**. At5g01410 AGACAVMALERVPADIRAQGGVARMSDPQMIKEIKQAVTIPVMAKARIGHFVEAQILEAI 112 At2g38230 AGACAVMALERVPADIRAQGGVARMSDPEMIKEIKNAVTIPVMAKARIGHFVEAQILEAI 113 At3g16050 AGACSVIVSD----PVRSRGGVRRMPDPVLIKEVKRAVSVPVMARARVGHFVEAQILESL 116 SNZ1p SGACAVMALESIPADMRKSGKVCRMSDPKMIKDIMNSVSIPVMAKVRIGHFVEAQIIEAL 95 :***:*:. : :* * * **.** :**:: .:*::****:.*:********:*:: At5g01410 GIDYIDESEVLTLADEDHHINKHNFRIPFVCGCRNLGEALRRIREGAAMIRTKG-EAGTG 171 At2g38230 GVDYVDESEVLTLADEDNHINKHNFKIPFVCGCRNLGEALRRIREGAAMIRTKG-EAGTG 172 At3g16050 AVDYIDESEIISVADDDHFINKHNFRSPFICGCRDTGEALRRIREGAAMIRIQGDLTATG 176 SNZ1p EVDYIDESEVLTPADWTHHIEKDKFKVPFVCGAKDLGEALRRINEGAAMIRTKG-EAGTG 154 :**:****::: ** :.*:*.:*: **:**.:: *******.******* :* :.** At5g01410 NIIEAVRHVRSVNGDIRVLRNMD-DDEVFTFAKKLAAPYDLVMQTKQLGRLPVVQFAAGG 230 At2g38230 NVVEAVRHVRSVNGAIRLLRSMD-DDEVFTYAKKIAAPYDLVVQTKELGRLPVVQFAAGG 231 At3g16050 NIAETVKNVRSLMGEVRVLNNMD-DDEVFTFAKKISAPYDLVAQTKQMGRVPVVQFASGG 235 SNZ1p DVSEAVKHIRRITEEIKACQQLKSEDDIAKVAEEMRVPVSLLKDVLEKGKLPVVNFAAGG 214 :: *:*:::* : :: ..:. :*:: . *::: .* .*: :. : *::***:**:** At5g01410 VATPADAALMMQLGCDGVFVGSGIFKSGDPARRARAIVQAVTHYSDPEMLVEVSCGLGEA 290 At2g38230 VATPADAALMMQLGCDGVFVGSGVFKSGDPVKRAKAIVQAVTNYRDAAVLAEVSCGLGEA 291 At3g16050 ITTPADAALMMQLGCDGVFVGSEVFDGPDPFKKLRSIVQAVQHYNDPHVLAEMSSGLENA 295 SNZ1p VATPADAALLMQLGCDGVFVGSGIFKSSNPVRLATAVVEATTHFDNPSKLLEVSSDLGEL 274 ::*******:************ :*.. :* : ::*:*. :: :. * *:*..* : At5g01410 MVGINLNDEKVERFANRSE---- 309 At2g38230 MVGLNLDD-KVERFASRSE---- 309 At3g16050 MESLNVRGDRIQDFGQGSV---- 314 SNZ1p MGGVSIESISHASNGVRLSEIGW 297 * .:.: . . Figure 8. Sequence Alignments The amino acid sequences for yeast SNZ1p and the proteins coded for by Arabidopsis genes At5g01410, At2g38230, and At3g60650 were obtained from the GenBank database at NCBI (http://www.ncbi.nlm.nih.gov/) and used to conduct an alignment using the CLUSTAL-W program online at EMBL (http://www.ebi.ac.uk/clustalw/) the alignment is shown above. An (*) under a column indicates an exact match of sequence, while (:) or (.) show stronger and weaker similarity of amino acids. 42 WT ?snz1 ?snz1:AtSNZ1 1.0 43 0.1 0.01 0.001 0.0001 Figure 9. Arabidopsis SNZ1 complementation Growth of Wild type (WT), the snz1 deletion mutant (?snz1), and the snz1 deletion mutant containing the putative Arabidopsis SNZ1 homolog transformed into the mutant on p426 shuttle vector driven by the yeast GPD promoter (?snz1: AtSNZ1). APPENDIX www.yeastgenome.orgww.ye Appendix I. Deletion Strategy 45 Vitamin B 6 Naturally Occurring Compounds Phosphorylated Derivatives Pyridoxine (PN) Pyridoxal (PL) Pyridoxamine (PM) Pyridoxine 5?-Phosphate (PNP) Pyridoxal 5?-Phosphate (PLP) Pyridoxamine 5?-Phosphate (PMP) Appendix II. Flowchart of the six B 6 vitamers 46 Yes (copy 1) Auxotrophic mutants; Methylene blue SNZ 1, 2, 1,2, 3 3 SNO Saccharomyc es cerevisiae ?N/APutative SNZ1; SNO1 Arabidopsis thaliana YesGenomic analysis; Auxotrophic mutants Pdx1/ Pdx2Neurospora crassa YesAuxotrophic mutants; methylene blue Pyro AAspergillus nidulans YesAuxotrophic mutants; antioxidant properties Sor 1Cercospora nicotianae Involved in Pyridoxine Biosynthesi s Experiments Pdx (Pyridoxine Requiring) Name Studies Appendix III. Summary of Eukaryotic Pyridoxine Studies 47 ?SNO1 ?SNZ1 WT ?SNO1: ?SNZ1: AtSNO1 AtSNZ1 Appendix 1V. AtSNZ1 complementation 48 Pyridoxal Metabolism in Plants ATP ADP ATP ADP O 2 H 2 O 2 O 2 H 2 O + NH 4 + PDX3 Pyridoxamine 5?-phosphate Oxi dase PDX3 Pyridoxamine 5?-phosphate Oxi dase SOS4 Pyridoxal Kinase SOS4 Pyridoxal Kinase ?-ketoacids ?-amino acids Aminotrnasferases P i Pyridoxine Phosphatase Pyridoxal Phosphatase P i SNZ ?? SN0 ?? Pyridoxamine Phosphatase N H 3 C HO CH 2 -O- CH 2 OH 5'-O-(?-D-glucopyranosyl)pyridoxine O HO CH 2 OH OH OH N H 3 C HO CH 2 OPO 3 2- CH 2 OH Pyridoxine Phosphate N H 3 C HO CH 2 OH CH 2 OH Pyridoxine CH 2 OH CHO Pyridoxal N H 3 C HO CH 2 OPO 3 2- CH 2 NH 3 + Pyridoxamine Phosphate N H 3 C HO CH 2 OPO 3 2- CHO Pyridoxal Phosphate N H 3 C HO CH 2 NH 3 + Pyridoxamine CH 2 OH N H 3 C HO UDP-glucose UDP Gl ucose Pyridoxine glucosyl transferase ATP ADP SOS4 Pyridoxal Kinase P i NADP + NADPH + H + + H + NADPHNADP + Pyridoxal Reductase Pyridoxine ?-glucosidase 49 Appendix V. Proposed pathway for the biosynthesis of pyridoxine in plants