近畿大学学術情報リポジトリ

全文

(1)Doctoral Dissertation. Functional analysis of Nudix hydrolase family in Arabidopsis thaliana. Takahisa Ogawa. Graduate School, Kinki University Division of Agricultural Science (Major: Applied Bioscience).

(2) 9.@4. Functional analysis of Nudix hydrolase family in Arabidopsis thaliana Takahisa Ogawa March, 2007 Graduate School, Kinki University Division of Agricultural Science Major: Applied Bioscience (Advisor: Prof: Shigeru Shigeoka). .@4.

(3) 08:/ . =3. ". "? <"57 6. (21"#, %' ;! *$A>& ) -+ Submitted to the Graduate School, Kinki University, to fulfill the requirement for the Doctorate Degree..

(4) Acknowledgements I wish to express my science graduate to Dr. Shigeru Shigeoka, Professor of Faculty of Agriculture, Kinki University, for his kind guidance, valuable advice, stimulating discussion and critical review throughout the work including the manuscript of this thesis. I am grateful to Dr. Tamo Fukamizo and Dr. Ryutaro Utsumi, Professors of Faculty of Agriculture, Kinki University, for reading the entire text in its original form. I am thankful to Dr. Kazuya Yoshimura, College of Bioscience and Biotechnology, Chubu University, for his valuable help,. kind. suggestion, and stimulating discussion throughout the work. I wish to thank Dr. Toru Takeda, Dr. Masahiro Tamoi, and Dr. Yabuta, Faculty of Agriculture, Kinki University, for their valuable discussions and suggestions throughout the work. Finally, special thanks are due to Kazuya Ishikawa, Hiroe Miyake, and Yayoi Ueda, for their many helpful collaborations.. Thanks are. also due to all the past and present members of our laboratory of Plant Molecular Physiology in the Faculty of Agriculture, Kinki University, for their kind cooperations. This research was supported by a Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists..

(5) ABBREVATIONS. ADP-glucose. Adenosine 5’-diphosphoglucose. ADP-ribose. Adenosine 5’-diphosphoribose. Ap3A. P1, P3-Di(adenosine-5’)triphosphate. Ap4A. P1, P4-Di(adenosine-5’)tetraphosphate. Ap5A. P1, P5-Di(adenosine-5’)pentaphosphate. ATP. Adenosine 5’-triphosphate. CoA. Coenzyme A. dNTP. 2’-Deoxynucleoside 5’-triphosphate. FAD. Flavin adenine dinucleotide. H2O2. hydrogen peroxide. IPTG. isopropyl--D-thiogalactopyranoside. LB. Luria-Bertani broth. NAD+. Nicotinamide adenine dinucleotide. NADH. Nicotinamide adenine dinucleotide, reduced form. Nudix. Nucleoside diphosphates linked to some moiety X. 1. singlet oxigen. O2. O2. -. superoxide. 8-oxo-dGTP. 8-oxo-2’-deoxyguanosine 5’-triphospate. PAGE. polyacrylamide gel electrophoresis. PAR. Poly(ADP-ribosyl)ation. PARP. Poly(ADP-ribose)polymerase. PARG. Poly(ADP-ribose)glycohydrolase. PVDF. polyvinylidene difluoride. RT-PCR. reverse transcription-PCR. UDP-glucose. Uridine 5’-diphosphoglucose. UDP-galactose Uridine 5’-Diphosphogalactose.

(6) CONTENTS. CHAPTER I. Introduction. 1. CHAPTER II. Identification of genes involved in tolerance to oxidative stress by activation tagging. CHAPTER III. Molecular characterization of Nudix hydrolase family in A. thaliana. CHAPTER IV. Prevention. of. 16. replicational. and. transcriptional. errors by Arabidopsis 8-oxo-dGTPase, AtNUDX1. CHAPTER V. 4. 35. Physiological role of Arabidopsis ADP-ribose/NADH pyrophosphatases, oxidative stress. AtNUDX2. and. AtNUDX7,. under 53. REFERENCES. 72. PUBLICATIONS. 87.

(7) CHAPTER I INTRODUCTION. Plants are aerobic organisms that require molecular oxygen for survival. However, oxygen is inherently dangerous to plants because it can be readily reduced to active oxygen species (AOS) (Figure I-1). The term AOS is generic, embracing not only free radicals but also hydrogen peroxide (H2O2) and singlet oxygen (1O2). AOS lead to damage of important cellular components such as protein, DNA, and lipid. They are involved in virtually all major areas of aerobic biochemistry (e.g. respiratory and photosynthetic electron transport; oxidation of glycolate, xanthine, and glucose) and are produced in copious quantities by several enzyme systems. [e.g.. plasmalemma-bound. NADPH-dependent. superoxide. (O2-). synthase and superoxide dismutase (SOD; 1.15.11) even under optimal conditions (Alvarez and Lamb 1997, Bowler et al., 1992).. The chief. toxicity of O2- and H2O2 is thought to reside in their ability to initiate cascade reactions that result in the production of hydroxyl radical (OH) and other destructive species such as lipid peroxides. Furthermore, a wide range of environmental stresses including light, temperature, drought, and salinity lead to enhanced production of AOS. Much of the injury to plants associated with stress exposure is caused by AOS at the cellular level (Alscher et al., 1997; Bowler et al., 1992; Foyer et al., 1994; Shigeoka et al., 2002).

(8). . .

(9) . . .

(10).

(11) .

(12)

(13) . . . .

(14). Figure I-1. Generation of active oxygen species.. . .

(15) This dangerous cascade is prevented by efficient operation of several defense systems in the cells. Higher plants contain a number of enzymes involved in the defense system. A lot of genes of these defense systems have been isolated from various photosynthetic organisms including higher plants and were demonstrated their abilities to scavenge AOS.. The. transgenic tobacco plants overexpressing thylakoid membrane-bound ascorbate peroxidase (tAPX) or catalase from Escherichia coli in chloroplasts showed increased tolerance to oxidative stress caused by paraquat treatment (Yabuta et al., 2002 and Miyagawa et al., 2000). The recent Arabidopsis genome sequence (The Arabidopsis Genome Initiative, 2000) has brought out a lot of information on the molecular mechanisms of the defense systems involved in the oxidative stress. DNA microarray technique is being used to identify complete genes that are induced or suppressed during oxidative stress. However, it is likely that there are many genes that are constitutively expressed at low levels but have key physiological roles are unclear, because it is difficult to identify a novel gene and/or enzyme that using the conventional methods. One of the most direct ways of dissecting complex physiological processes in plants is generation and analysis of genetic mutants. Mutations, resulting from T-DNA or transposon tagging or chemical mutagenesis, recessive.. usually. cause. loss-of-function. and. are,. therefore,. Consequently, the mutant phenotype can only be observed. following selfing of the mutated plants.. This demands a substantial. amount of effort and is impossible for all plant species.. Another. drawback of loss-of-function mutagenesis is that mutation of functionally redundant genes does not lead to phenotypically altered plants. Many of these disadvantages are circumvented by an alternative approach to generate mutants, called T-DNA activation tagging. T-DNA activation tagging is a method to generate dominant mutations in plants. . .

(16) or plant cells by random insertion of a T-DNA carrying strong constitutive enhancer elements, which can cause transcriptional activation of flanking genes. Therefore, in contrast to the conventional mutagenesis, mutants generated by T-DNA activation tagging allow direct selection for the desired phenotype in the primary transformants.. This means that the. mutant phenotype can appear in the T1 generation because gain-of-function mutants. behave. dominantly.. The. advantages. of. characterizing. gain-of-function mutants are as follows: (1) the candidate gene can be isolated easily by plasmid rescue or by TAIL-PCR; (2) the mutant phenotype can easily be reproduced by introduction of the candidate genes into the wild type under the control of a strong promoter; (3) inactivation of a certain pathway in loss-of-function mutants might kill a plant, but activation of the same pathway in gain-of-function mutants might still be compatible with survival of the plants. Therefore, it is likely that T-DNA activation tagging method can be very useful in studying the antioxidative systems in higher plants. In this work, to identify the novel genes involved in the defense system against oxidative stress, I studied the following: (1) identification of genes involved in tolerance to the oxidative stress by activation tagging, (2) molecular characterization of Nudix hydrolase family in A. thaliana, (3) prevention of replicational and transcriptional errors by Arabidospsis 8-oxo-dGTPase, AtNUDX1, and (4) physiological role of Arabidopsis ADP-ribose/NADH pyrophosphatases, AtNUDX2 and AtNUDX7, under oxidative stress.. . .

(17) CHAPTER II Identification of genes involved in tolerance to the oxidative stress by activation tagging Introduction. For activation-tagging technology, a T-DNA vector that possesses the enhancers of cauliflower mosaic virus (CaMV) 35s gene was first developed by Walden and colleagues (Hayashi et al., 1992). Their T-DNA contained four copies of tandem arranged CaMV 35S enhancers in the proximal part of the right border. This T-DNA vector was used to introduce into cultured cells.. Following the development of in planta transformation of. Arabidopsis using vacuum infiltration and floral dipping, this T-DNA is being introduced into plants to produce mutant lines or activation-tagged lines. The activation of genes by the CaMV 35S enhancers causes dominant phenotypes. This method has been used successfully for the isolation of genes that are involved in hormone signaling (Kakimoto, 1996), phenylpropanoid biosynthesis (Borevitz et al., 2000), and morphogenesis (Nakazawa et al., 2003) in higher plants. Therefore, it is likely that T-DNA activation tagging method could be very useful for studying the antioxidative systems in higher plants. In this study, in order to identify the genes involved in tolerance to oxidative stress, I generated Arabidopsis activation-tagged lines and screened for paraquat-resistant mutants.. . .

(18) Materials and Methods Materials The Agrobacterium tumefaciens GV3101 strains and activation-tagging T-DNA vector, pPCVICEn4HPT, were provided by Dr. Kakimoto (Osaka university). The pDH123 vector was received from Dr. Shikanai (Kyusyu university). Restriction enzymes and modifying enzymes were purchased from TaKaRa (Kyoto, Japan). All other chemicals were of analytical grade and were used without further purification.. Plant growth conditions and generation of activation-tagging lines Arabidopsis thaliana (Columbia ecotype) plants were grown at 25˚C in long-day conditions (16 h light / 8 h dark period) under white light (100 E/m2/s).. The activation-tagging T-DNA vector, pPCVICEn4HPT, was. introduced into Agrobacterium tumefaciens GV3101 (pMP90) and was transformed to Arabidopsis plants using vacuum infiltration method (Bechtold and Pelletier. 1998).. T1 seedlings were selected on basic. Murashige and Skoog (MS) medium in Petri dishes containing 3% sucrose and 20 mg l-1 hygromycin for 3 weeks and were transferred into soil. T2 seeds were harvested from 12,500 individual lines.. Screening of paraquat-resistant mutants T2 seeds from 50 individual activation-tagged lines were mixed as a screening pool and were selected on MS medium with 3% sucrose and 20 mg l-1 hygromycin for 7 days. The selected T2 seedlings were transferred to another medium containing 3 M paraquat (PQ), which generates AOS under light, and were grown for 7 days under the long-day conditions. The survived plants were transferred to a fresh medium without PQ and grown for 2weeks and were then transferred to soil to obtain T3 seeds.. . .

(19) Identification of T-DNA insertion sites The T-DNA insertion sites in the paraquat-resistant mutant (pqr mutant) were identified by the plasmid rescue technique. Genomic DNA was extracted from leaves of pqr mutant using Dneasy plant Mini Kit (QIAGEN). Four hundred nanograms of genomic DNA was digested with Bam HI in a final volume of 400 l at 37˚C overnight and precipitated by ethanol, and was dissolved in 50 l of distilled water. The digested DNA was self-ligated using T4 DNA ligase (700 units, TaKaRa) in final volume of 400 l at 16˚C overnight. The ligated DNA was ethanol-precipitated and was dissolved in 10 l of distilled water. Fourty microlitres of E.coli DH10B electro-competent cells (Invitrogen) were transformed with 5 l of the ligated DNAs solution and was spread on LB agar medium containing 50 mg l-1 ampicillin. Colonies appeared after incubation at 37˚C overnight were picked up and cultured in 4 ml LB medium containing ampicillin. Plasmid DNA was prepared from these liquid cultures. Genomic fragments in the plasmids were sequenced using a primer at Left-border in the T-DNA, 5’-ATAACGCTGCGGACATCTAC-3’. The T-DNA insertion site in the Arabidopsis thaliana. genome. was. identified. using. BLASTN. program. (http://www.ddbj.nig.ac.jp).. Cloning of genes and cDNAs in the vicinity of T-DNA insertion site The genes encoding AtNUDX2 proteins that located in the vicinity of T-DNA insertion site in pqr-216 were isolated from the obtained genomic DNA of wild type plants by PCR using specific primers designed from the respective. 5’-. and. 3’-untranslational. (5’-TCTAGATGATGGGTTCTCTG-3’),. regions:. AtNUDX2-XbaI-F AtNUDX2-SacI-R. (5’-TGTGAGGAGAGCTCTGTTTG-3’). PCR amplification was performed for 30 cycles of denaturation at 95˚C for 60 s, annealing at 55˚C for 60 s, and elongation at 72˚C for 60 s, followed by 72˚C for 10 min.. . .

(20) Total RNA was isolated from the leaves of 4-week-old wild-type plants (1.0 g fresh weight) as previously described (Yoshimura et al., 1999). First. strand. cDNA. was. synthesized. using. ReverTra. transcriptase; Toyobo) with an oligo dT primer.. Ace. (reverse. The reaction was. performed in 20 l mixture containing the buffer supplied by the manufacture (Toyobo), 5 g total RNA, 0.25 M oligo (dT) primer, 1 mM dNTPs, and 100 units of the reverse transcriptase. The reaction mixture was incubated at 42˚C for 60 min and then 99˚C for 5min. The open reading frame of AtNUDX2 (At5g47650) was amplified from the first strand cDNA using the primers mentioned above. PCR amplification was performed for 30 cycles of denaturation at 95˚C for 60 s, annealing at 55˚C for 60 s, and elongation at 72˚C for 60 s, followed by 72˚C for 10 min. The amplified cDNA encoding AtNUDX2 protein was cloned into pSTBlue T-vector (Novagen). DNA sequencing was performed by the dideoxy chain terminator method using an automatic DNA sequencer (ABI PRISMTM 310, Applied Biosystems).. Northern blot analysis Total RNA (30 g each) from the leaves of 4-week-old T3 generation of pqr-216 and transformants overexpressing AtNUDX2 was subjected to electrophoresis on 1.2% (w/v) agarose gel containing 2.2 M formaldehyde and transferred to Hybond N+ membrane (Amersham Pharmacia Biotech). The RNA-blot membrane was hybridized with. 32. P-labeled cDNA encoding AtNUDX2. in a buffer containing 6 X SSC, 5 X Denhardt's solution, 1% (w/v) SDS, and 100 g ml-1 denatured salmon sperm DNA at 55˚C for 12 h. The membrane was then washed twice with 2 X SSC, 0.1% SDS at room temperature for 10 min, and in 0.1 X SSC, 0.1% SDS at 60˚C for 30 min. Autoradiography was carried out using a phosphor imager (Mac BAS 1000; Fuji Photofilm). The transcript levels were estimated from the densitometric readings of three independent experiments and expressed as relative expression ratios.. . .

(21) Generation of transgenic plants The cDNA encoding AtNUDX2 was cloned into a binary vector, pDH123, under CaMV 35S promoter in Xba I / Sac I to get pDH123/AtNUDX2. The vector for AtNUDX2-suppressed plants using the RNAi method was constructed using GATEWAY cloning technology (Invitrogen, Paisley, U.K.). The cDNA encoding the 5’-end of AtNUDX2 cDNA was cloned into the donor vector, pDONR201, and then re-cloned into the destination vector, pGWB80. The specific primers with attB1 and attB2 sequences were as follows: attB1-AtNUDX2-5’ (5’-AAAAAGCAGGCTTCACTCATCATACGTTGT-3’), attB2-AtNUDX2-5’ (5’-AGAAAGCTGGGTCCTTAACAGCAGTTTCAG-3’). PCR and in vitro BP and LR recombination reactions were carried out according to the manufacturer’s instructions (Invitrogen, Paisley, U.K.). The constructs were introduced into A. tumefaciens by electroporation, which were then used to transform A. thaliana by the vacuum infiltration method as described above. T1 seedlings were selected on basic MS medium in Petri dishes containing 3 % sucrose and 20 mg l-1 hygromycin for 3 weeks and transferred into soil.. T2 seeds were harvested and used for the. paraquat tolerant experiments.. Western blot analysis Polyclonal mouse antibodies against AtNUDX2 was prepared using His-tagged recombinant AtNUDX2 protein, synthesized as described previously (Ogawa et al., 2005). Western blot analysis was carried out as described previously (Yoshimura et al., 2004). The AtNUDX2 protein was detected by using specific polyclonal antibody as the primary antibody, and anti-mouse IgG-HRP conjugate (Bio-Rad, CA, USA) as the secondary antibody. Protein bands were detected using the ECLTM detection system (GE Healthcare, Bucks, U.K.). The protein concentration was determined by the method of Bradford (1976).. . .

(22) Measurement of chlorophyll content Chlorophyll was extracted with acetone at 4 ˚C from 0.2 g of seedling and measured by the method of Arnon (1949).. Results Isolation of paraquat resistant mutants I generated approx. 12,500 activation-tagged lines (T2) of Arabidopsis (ecotype Columbia) using the pPCVICEn4HPT, which contains four copies of 35S enhancer at the right border of the T-DNA. These activation-tagged lines were screened by 3.0 M paraquat treatment under long-day conditions (16 h light; 100 E m-2 s-1/8 h dark period) for oxidative stress-resistant mutants. Out of 10,000 activation-tagged lines, I isolated two mutants (pqr-216 and 236) showing tolerance to the paraquat treatment (Figure II-1A and B). Approximately, three-fourth of the T3 seeds from the pqr-216 and 236 showed resistance to hygromycin and increased tolerance to the paraquat treatment (data not shown).. These results suggest that the. phenotype was due to an increased gene expression caused by the 35S enhancers in the T-DNA.. . .

(23) Figure II-1. Paraquat-resistant mutants (pqr-216 and 236) obtained from activation-tag lines. (A) Phenotype of the wild-type plant (WT) and pqr-216 and 236 mutants under the oxidative stress conditions caused by the PQ treatment.. T2 seeds of activation-tag lines were. selected on MS medium containing 20 mg l-1 hygromycin for 7 days, transferred to MS medium containing 3 M PQ, and were grown for 7 days under long-day conditions. The wild-type plant was grown under the same conditions, except for the selection by hygromycin. (B) Chlorophyll content of the wild-type plant and pqr-216 mutant under normal and stressful conditions. Different letters indicate significant differences (P < 0.05). (C) T-DNA insertion site of pqr-216 and 236. The line and arrow represent genomic DNA and the gene, respectively. Gray boxes show the four copies of tandem arranged 35S enhancers. RB, right border; LB, left border. (D) Northern blot analysis of the AtNUDX2 transcript in 4-week-old wild-type and pqr-216 plants. Detailed procedures are described in the Materials and Methods section.. . .

(24) Identification of the ge ne responsible for paraquat resistant phenotype in pqr-216 and 236 To identify the gene responsible for the phenotype of pqr-216 and 236 mutants, the sites of T-DNA insertion were analyzed. I cloned the flanking genomic sequence of the T-DNA insertion site in pqr-216 and 236 by the plasmid rescue technique. Sequence analysis of the genomic fragment revealed that the T-DNAs were inserted at 2.1 and 3.0 kbp-upstream from the predicted translation start point of the gene At5g47650 encoding Nudix hydrolase (AtNUDX2) and At3g54900 encoding calcium exchanger interacting protein 1(CXIP1) in pqr-216 and 236, respectively (Figure II-1C). Recently, it has been reported that CXIP1 is reclassified as a member of the monothiol glutaredoxins, AtGRXcp, and loss of AtGRXcp in Arabidopsis leads to protein oxidation in chloroplasts and seedlings sensitivity to external oxidant, such as H2O2 (Cheng et al., 2006). Therfore, I concluded that PQ-resistant phenotype of pqr-236 is caused by the overexpression of CXIP1. I analyzed the expression levels of AtNUDX2 in the leaves of 4-weeks-old pqr-216 and wild type plants by Northern hybridization. The expression level of AtNUDX2 in pqr-216 was 10.62.7-fold higher than those in the wild-type plants (Figure II-1D). To determine whether oxerexpression of AtNUDX2 gene causes the paraquat-resistant phenotype in Arabidopsis plants, I generated Arabidopsis plants overexpressing AtNUDX2 (Pro35S:AtNUDX2) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. In the T3 generation of Pro35S:AtNUDX2-20-1, -20-2, and -21-1, the level of the AtNUDX2 transcript was approx. 26.62.9-, 14.62.5-, and 8.71.3-fold higher, respectively, than that in the control plants (Figure II-2A). Western blot analysis showed the presence of a polypeptide of 32 kDa, which corresponded to the deduced molecular mass of the AtNUDX2 protein (31.6 kDa), in the extracts prepared from. . .

(25) the leaves of Pro35S:AtNUDX2-20-1 and -20-2 plants (Figure II-2A). Unfortunately, the band corresponding to the AtNUDX2 protein was not detected in the extracts from the control and Pro35S:AtNUDX2-21 plants, because the expression level of AtNUDX2 was very low. No difference was observed in growth or morphology between the control and these transgenic plants. As evaluated by the survival rate and the chlorophyll content, like the pqr-216 mutant, the transgenic plants clearly showed enhanced tolerance to oxidative stress compared with the control plants (Figure II-2B-D). These results lead to the conclusion that the PQ-resistant phenotype of pqr-216 mutant is caused by the overexpression of AtNUDX2. Unfortunately, an Arabidopsis mutant containing a T-DNA insert within AtNUDX2 was not found. A strain of Arabidopsis containing a Ds transposon insert about 100 bp upstream from the initiating ATG codon of AtNUDX2 has been registered in the RIKEN Arabidopsis Transposon mutant lines (http://rarge.gsc.riken.go.jp/db_home.pl); however, the levels of AtNUDX2 mRNA were not decreased in the mutant (data not shown).. Therefore,. I generated Arabidopsis plants (RNAi- AtNUDX2) transformed with an RNAi construct using the 5’-end of AtNUDX2. The expression of AtNUDX2 in the transgenic plants was reduced by approx. 80% compared to that in the control plants (transformed with the empty vector); however, the degree of tolerance to oxidative stress was not changed in the transgenic plants (data not shown).. . .

(26) Figure II-2. Effect of overexpression of AtNUDX2 on oxidative stress tolerance. (A) Northern blot (upper) of AtNUDX2 transcript and western blot (lower) of AtNUDX2 protein in the Pro35S:AtNUDX2 plants. The independent transformed lines (T3 generation) were used for the analysis. C, control plants transformed with the empty vector (pDH123). (B) Phenotypes of the control (left) and Pro35S:AtNUDX2 (right) plants during the PQ treatment. Seven-day-old seedlings were grown on MS medium containing 3 M PQ for 7 days under long-day conditions. The plants were transferred to MS medium without PQ and were then grown for an additional 7 days. (C) Survival rates of Pro35S:AtNUDX2 plants under the PQ treatment. Data are the mean values ± SD of three individual experiments (n = 3). Different letters indicate significant differences (P < 0.05). Detailed procedures are described in the Materials and Methods section. (D) Chlorophyll content of the control and Pro35S:AtNUDX2 plants under normal and stressful conditions. Different letters indicate significant differences (P < 0.05).. Discussion. To identify the genes involved in the tolerance to oxidative stress, I screened activation-tagged lines for paraquat-resistant mutants. Out. . .

(27) of 10,000 lines of Arabidopsis mutants, I found two mutants (pqr-216 and 236) showing the phenotype of increased tolerance to oxidative stress caused by paraquat treatment (Figure II-1A). The T-DNA was inserted at 2.1 and 3.0 kbp-upstream from the predicted translation start point of the gene At5g47650 encoding a Nudix hydrolase (AtNUDX2) and At3g54900 encoding calcium exchanger interacting protein 1(CXIP1) in pqr-216 and 236, respectively (Figure II-1C). Recently, it has been reported that CXIP1 is reclassified as a member of the monothiol glutaredoxins, AtGRXcp, and loss of AtGRXcp in Arabidopsis leads to protein oxidation in chloroplasts and seedlings sensitivity to external oxidant, such as H2O2 (Cheng et al., 2006). This report lead to conclusion that PQ-resistant phenotype of pqr-236 is caused by the overexpression of CXIP1. Actually, the expression level of CXIP1 increased in pqr-236 mutant compared with wild-type plants (data not shown). On the other hand, Nudix hydrolases use a wide spectrum of substrates, which are mostly nucleoside diphosphate derivatives that include dinucleoside polyphosphates, ADP-ribose, NADH, nucleotide sugars, or ribo- and deoxynucleoside triphosphates (Dunn et al., 1999). As all substrates are either potentially toxic, cell signaling molecules, or metabolic intermediates whose concentrations require modulation during the cell cycle, it has been postulated that the role of Nudix hydrolases is to sanitize or regulate the accumulation of these metabolites. The expression level of AtNUDX2 in pqr-216 was 10.62.7-fold higher than those in the wild-type plants (Figure II-2A). Furthermore, the Pro35S:AtNUDX2 plants clearly showed enhanced tolerance to oxidative stress compared with the control plants (Figure II-2B-D). These results lead to the conclusion that the PQ-resistant phenotype of pqr-216 is caused by the overexpression of AtNUDX2. However, in higher plants, little is known about the physiological roles of Nudix hydrolases. Therefore, I. . .

(28) characterized the molecular properties of Arabidopsis Nudix hydrolases.. Summary I have isolated two mutants (pqr-216 and 236) from an Arabidopsis activation-tagged line showed a phenotype of increased tolerance to oxidative stress after treatment with 3 M PQ. The T-DNAs were inserted at 2.1 and 3.0 kbp-upstream from the predicted translation start point of the gene At5g47650 encoding Nudix hydrolase (AtNUDX2) and At3g54900 encoding calcium exchanger interacting protein 1(CXIP1) in pqr-216 and 236, respectively. It has been reported that CXIP1 is a member of the monothiol glutaredoxins, AtGRXcp, and loss of AtGRXcp in Arabidopsis leads to protein oxidation in chloroplasts and seedlings sensitivity to external oxidant, therefore, I concluded that PQ-resistant phenotype of pqr-236 is caused by the overexpression of CXIP1. In pqr-216, judging from the phenotype of transgenic plants overexpressing the AtNUDX2 flanking the T-DNA insert, it was clear that the enhanced expression of AtNUDX2, was responsible for the oxidative stress tolerance.. . .

(29) CHAPTER III Molecular characterization of Nudix hydrolase family in A. thaliana Introduction. Nudix (nucleoside diphosphates linked to some moiety X) hydrolases that are characterized by a conserved Nudix motif: GX5EX7REVXEEXGU, where U is usually Ile, Leu or Val (Bessman et al., 1996), are widely distributed in over 120 species, ranging from viruses to humans (Xu et al., 2001). It has been proposed that Nudix hydrolases may be divided into subfamilies based on their major substrates: dinucleoside polyphosphates, ADP-ribose, NADH, nucleotide sugars, or ribo- and deoxy-nucleoside triphophates (Dunn et al., 1999). Since the accumulations of their substrates are often toxic to the cell, their intracellular levels need to be precisely regulated by these enzymes (Bessman et al., 1996). Among the Nudix hydrolases, E. coli MutT protein hydrolyzes all canonical nucleoside triphosphates with a preference for 8-oxo-dGTP (8-oxo-2’-deoxyguanosine-5’-triphosphate). and. 8-oxo-GTP. (8-oxo-2’-guanosine-5’-triphosphate), the oxidized form of the free guanine nucleotide, by attacking an active oxygen species, such as a superoxide radical (O2-), H2O2 or a hydroxyl radical (•OH), to the monophosphate form. Since 8-oxo-G (8-oxo-7, 8-dihydroguanine) can be incorporated into the nascent strand opposite the adenine and cytosine in the template with almost equal efficiency, resulting in an A:T to C:G transversion mutation (Tajiri et al., 1995; Maki and Sekiguchi, 1992), MutT functions in the prevention of the misincorporation of such mutagenic nucleotides into DNA or mRNA during DNA replication or transcription (Maki. . .

(30) and Sekiguchi, 1992; Taddei et al., 1997).. In human cells, the. accumulation of 8-oxo-G may be responsible for a significant portion of spontaneous mutations that lead to the induction of cancer, as well as age-related disorders (Ames et al., 1993). Homologues of E. coli MutT in human (hMTH1) and rodent cells were identified, and they complement the function of MutT in E. coli (Sakumi et al., 1993; Furuichi et al., 1994; Cai et al., 1995; Kakuma et al., 1995). In contrast to human and E. coli, little is known about the functions of Nudix hydrolases and defense systems toward oxidative damage of nucleotides by MutT proteins in higher plants (Hays, 2002). There are important differences between the life strategies of plants and most eukaryotes.. Especially,. even. under. optimal. photosynthetic. electron. conditions,. transport. in. chloroplasts. is. the. inevitably. accompanied by the reduction of O2 to O2-, which is followed by the production of other active oxygen species such as H2O2 and •OH, on the reducing side of Photosystem I, although light, as the energy source for photosynthesis, is essential for plant life (Asada, 1997; Foyer et al., 1994). Furthermore, a wide range of environmental stresses, such as drought, high salinity, and low temperature result in the enhanced production of active oxygen species (Foyer et al., 1994; Alscher et al., 1997; Asada, 1999; Bowler et al., 1992; Shigeoka et al., 2002). Based on a BLAST search of the DNA database, it is likely that 27 open reading frames encoding potential homologues of Nudix hydrolases exist in Arabidopsis thaliana. Among them, AtNUDX1 (At1g68760) was initially characterized as a NADH pyrophosphatase (Dobrzanska et al., 2002). In addition, Klaus et al. (2005) recently reported that AtNUDX1 was the closest homologue of Lactococcus lactis YlgG and showed activities toward both dihydroneopterin triphosphate, a precursor of folate, and (deoxy) nucleoside triphosphates including 8-oxo-dGTP (Klaus et al., 2005).. . .

(31) However, little attention has been given to the effects of MutT-type proteins, including AtNUDX1, on the sanitization system of oxidized nucleotides. Furthermore, several members of the Nudix hydrolase family have recently been described as having either a high specificity for ADP-ribose or including ADP-ribose within their substrate specificity range (Dunn et al., 1999). Although it is yet to be proven, it is assumed that these enzymes play an important role in maintaining the concentration of intracellular ADP-ribose at a sub-toxic level, since the accumulation of ADP-ribose can be potentially cytotoxic due to its ability to modify protein and to bind to ATP-activated K+ channels (Kwak et al., 1996). It has been demonstrated that the gene products from Methanococcus jannaschii MJ1149, E. coli orf186 and Saccharomyces cerevisiae YSA1, and the human and mouse Ysa1p homologues, NUDT5 and Nudt5, respectively, have activities towards ADP-ribose (Dunn et al., 1999; Sheikh et al., 1998; 0’Handley et al., 1998; Yang et al., 2000). In this chapter, I characterized the molecular properties of cytosolic Nudix hydrolases in Araibdopsis, including AtNUDX2 identified from activation-tagged paraquat resistant mutant, pqr-216.. Materials and Methods. Materials and plant growth conditions The E. coli strain, CC101, and the mutT deficient strain, CC101T, were kind gifts from Prof. Maki (NARA INSTITUTE of SCIENCE and TECHNOLOGY). The pTrc100 plasmid for the complementary assay was obtained from Prof. Nakabeppu (Kyushu University).. The 8-oxo-dGTP was purchased from. TriLink Biotechnologies (San Diego, USA).. Restriction enzymes and. modifying enzymes were purchased from TaKaRa (Kyoto, Japan). All other. . .

(32) materials and enzymes were of analytical grade and were obtained from commercial sources.. Arabidopsis thaliana (Columbia ecotype) plants. were grown at 25˚C in long-day conditions (16 h light / 8 h dark period) under white light (100 E/m2/s).. Construction of expression plasmids of recombinant AtNUDX Total RNA was isolated from the leaves of 4-week-old wild-type plants (1.0 g fresh weight), as previously described (26). The first strand cDNA was synthesized using ReverTra Ace (reverse transcriptase; Toyobo) with an oligo dT primer according to the manufacturer’s instructions. The open reading frames of AtNUDX111 were amplified from the first strand cDNAs, using the primer sets shown as follows; AtNUDX1-Nde I-F (5’-CATATGTCGACAGGAGAAGC-3’),. AtNUDX1-Nde. I-R. (5’-CATATG. TTAACTCTTACATC-3’), AtNUDX2-Nde I-F (5’- CATATG TCTGCTTCTAGTTC-3’), AtNUDX2-Nde. I-R. (5’-CATATGGAAAGCTCTGTTTG-3’),. AtNUDX4-Nde. I-F. (5’-CATATGACAGGGTTCTCTGT-3’),. AtNUDX4-BamH. I-R. (5’-GGATCCAGTGCTACTAATTC-3’),. AtNUDX5-Nde. I-F. (5’-CATATGGACGGTGAAGCTTT-3’),. AtNUDX5-Nde. I-R. (5’-CATATGAGCAGAAACACTTT-3’),. AtNUDX6-Nde. I-F. (5’-CATATGGACAATGAAGATCA-3’),. AtNUDX6-Nde. I-R. (5’-CATATGCACGTTCTGAAGAA-3’),. AtNUDX7-Nde. I-F. (5’-CATATGGGTACTAGAGCTCA-3’),. AtNUDX7-Nde. I-R. (5’-CATATGTCGGAATAATATAG-3’),. AtNUDX8-Nde. I-F. (5’-CATATGGATTCTGTTTCTCT-3’),. AtNUDX8-Nde. I-R. (5’-CATATGGAAGAGAGAGTATC-3’),. AtNUDX9-Nde. I-F. (5’-CATATGGCGAAACTAGCAGAAGAGAT-3’),. AtNUDX9-BamH. I-R. (5’-GGATCCCAATAATTCACAACTAGGTG-3’),. AtNUDX10-Nde. I-F. (5’-CATATGTCAGACCAAGAGGCTCCTCT-3’),. AtNUDX10-BamH. I-R. (5’-GGATCCAATGACGATGTTCTTGGAAG-3’),. AtNUDX11-Xho. I-F. . .

(33) (5’-CTCGAGATGTCTTCAACAACAACAGA-3’),. AtNUDX11-Xho. I-R. (5’-CTCGAGCTCAATCAGATCAAGTCAAG-3’). The sequences were homologous to the cDNAs of AtNUDX1~11 except for the replacement of the original nucleotides that introduced the desired restriction sites (bold sequences).. The amplified DNA fragments were ligated into pSTBlue. T-vectors (Novagen). DNA sequencing was performed using the dideoxy chain terminator method with an automatic DNA sequencer (ABI PRISMTM 310, Applied Biosystems). The resulting constructs were digested with Nde I/ Nde I for AtNUDX 1, 2, 4, 5, 6, 7 and 8, Nde I/BamH I for AtNUDX9 and 10, and Xho I/Xho I for AtNUDX11, and were ligated into the expression vectors,. pET16b. (Novegen). or. pCold. II. (TaKaRa),. to. produce. Histidine-tagged proteins. The resulting plasmids, pET16b/AtNUDX 1, 2, 5, 6, 7 and 8, and pCold II/AtNUDX 4, 8, 9, 10 and 11 were introduced into E. coli strain BL21 (DE3) pLysS cells.. Expression and purification of recombinant AtNUDX proteins E. coli strain BL21 (DE3) pLysS transformed with pET16b/AtNUDX1, 2, 5, 6, 7 or 8, or pCold II/AtNUDX 4, 8, 9, 10 or 11 was grown in 50 ml of LB medium containing 50 g ml-1 of ampicillin and 34 g ml-1 of chloramphenicol. After an overnight culture at 37˚C, the cultures were transferred to 500 ml of LB medium (with the antibiotics) and grown to an A600 of 0.6. Isopropyl-1-thio--D-galactopyranoside was added to a concentration of 0.4 mM and the cells were incubated for 16 h at 16˚C. The harvested cells were resuspended in Tris-HCl (pH 8.0) containing 0.5 M NaCl, 5 mM imidazole and 1 mM 2-mercaptoethanol, and were sonicated (10 kHz) using 20-s strokes with 30-s intervals and were centrifuged at 15,000 x g for 15 min.. The hexahistidine-tagged recombinant AtNUDX. proteins were purified from the soluble fraction using a HiTrap chelating HP column (Amersham Biosciences) according to the manufacturer’s. . .

(34) instructions. Protein contents were determined following the method of Bradford (Bradford, 1976).. Enzyme assay and HPLC analysis The hydrolytic activities of AtNUDX proteins towards to 8-oxo-dGTP, ADP-ribose and NADH were assayed according to the method described in Tassotto and Mathews (2002) with some modifications. Sixty microliters of the reaction mixture, containing 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 25 M substrate and 0.2~1.0 g of recombinant proteins was incubated at 37˚C for 10 min. The reaction was terminated by adding 10 l of 100 mM EDTA. The mixture was then analyzed with HPLC using a COSMOSIL C18 column (4.6 X 250 mm, Nacalai tesque) at a flow rate of 0.6 ml min-1 for the mobile phase buffer, which contained 73 mM KH2PO4, 5 mM tetrabutylammonium dihydrogenphosphate, and 20 % methanol.. The substrates and their. reaction products were detected according to their UV absorbance, as follows; 8-oxo-dGTP: 293 nm, dGTP: 252 nm, dCTP: 271 nm, dTTP: 264 nm, ADP-ribose, NADH, FAD, CoA, ApnA, UDP-Glucose, and UDP-galactose: 260 nm.. Complementation assay of the E. coli mutT mutation AtNUDX cDNAs were amplified with the RT-PCR using the specific primer sets. as. shown;. AtNUDX1-KpnI-R. AtNUDX1-NcoI-F. (5’-GGATCCTTAACTCTTACATC-3’),. (5’-GAGCTCTCTGCTTCTAGTTC-3’), AAGCTCTGTTTG-3’), AtNUDX7-BamHI-R. (5’-CCATGGCGACAGGAGAAGCG-3’), AtNUDX2-SacI-F. AtNUDX2-XbaI-R. AtNUDX7-EcoRI-F. (5’-TCTAGAGA. (5’-GAATTCGGTACTAGAGCTCA-3’),. (5’-GGATCCTCGGAATAATATAG-3’).. The. amplified. DNA. fragments were digested with Nco I/Kpn I (AtNUDX1), Sac I/Xba I (AtNUDX2) and Eco RI/Bam HI (AtNUDX7), respectively, and were ligated into pTrc100 vectors.. . .

(35) The complementation assay was carried out according to the method described in Sakumi et al. (1993). E. coli strains CC101 (wild type) or CC101T (mutT-) were transformed with empty plasmids, or the plasmids containing AtNUDX cDNAs. A single transformant was grown in 5 ml of LB medium containing 100 g ml-1 ampicillin at 37˚C overnight. The culture solutions were diluted 1 x 106 fold and 100 l of each culture was grown in 6 ml of LB medium containing 1 mM IPTG for 1416 hours. Mutation frequencies toward streptomycin resistance were measured by plating aliquots of these cultures on LB medium with or without the antibiotic (100 g ml-1).. Analysis of AtNUDX expression Total RNAs were isolated from several tissues (roots, stems and leaves) of 2-week-old wild-type A. thaliana plants (1.0 g fresh weight). The cDNAs encoding AtNUDX proteins were amplified with the RT-PCR (20~28 cycles). using. the. following. primer. sets;. AtNUDX1-F. (5’-ATAATGTCGACAGGAGAAGC-3’), AtNUDX1-R (5’-CATCTA TTAACTCTTACATC-3’), AtNUDX2-F. (5’-TTCACAGTTGCTCCACCACG-3’),. AtNUDX2-R. (5’-AGAATCTGTGAGGAAAGCTC-3’), AtNUDX3-F (5’-CATATGGCGGAGGAGCACTT-3’), AtNUDX3-R. (5’-GGATCCGCTGAAACTGAAAC-3’),. AtNUDX4-F. (5’-CATATGACAGGGTTCTCTGT-3’), AtNUDX4-R (5’-GGATCCAGTGCTACTAATTC-3’), AtNUDX5-F. (5’-TGTAGCATGGACGGTGAAGC-3’),. AtNUDX5-R. (5’-CTAGTCAGAGAGAGAGGTGG-3’), AtNUDX6-F (5’-TGGACAATGAAGATCAGGAG-3’), AtNUDX6-R. (5’-CAACCAGAGGTGGAGGCTAG-3’),. AtNUDX7-F. (5’-TGAGATGGGTACTAGAGCTC-3’), AtNUDX7-R (5’-CAGAGAGAAGCAGAGGCTTG-3’), AtNUDX8-F. (5’-CCACATAGATCTTCTTGATC-3’),. AtNUDX8-R. (5’-GAGGATGAAGAGAGAGTATC-3’),. AtNUDX9-F. (5’-CATATGGCGAAACTAGCAGAAGAGAT-3’),. AtNUDX9-R. (5’-GGATCCCAATAATTCACAACTAGGTG-3’),. AtNUDX10-F. . .

(36) (5’-CATATGTCAGACCAAGAGGCTCCTCT-3’),. AtNUDX10-R. (5’-GGATCCAATGACGATGTTCTTGGAAG-3’),. AtNUDX11-F. (5’-CTCGAGATGTCTTCAACAACAACAGA-3’),. AtNUDX11-R. (5’-CTCGAGCTCAATCAGATCAAGTCAAG-3’),. Actin2-F. (5’-GAGATCCACATCTGCTGG-3’),. Actin2-R. (5’-GCTGAGAGATTCAGGTGCCC-3’).. The actin transcript was used as a constitutive control.. PCR. amplification was performed with 20~26 cycles of 95˚C for 60s, 55˚C for 60s, and 72˚C for 60s, followed by 72˚C for 10 min. The PCR products were analyzed on 1 % agarose gels. Equal loading of each amplified gene sequence was determined with the control Actin2 PCR product.. RESULTS. Nudix hydlolase genes in A. thaliana A search for the Nudix hydrolase genes in the National Center for Biotechnology Information Data Base (http://www.ncbi.nlm.nih.gov/) showed that 27 genes (AtNUDX1~27) encoding the Nudix domain existed in A. thaliana. The portions of the open reading frames of 27 genes are characterized by a signature sequence of highly conserved amino acids spanning a region of 23 amino acids called the Nudix box, which acts as the catalytic and nucleotide binding sites (Figure III-1). Among them, only AtNUDX1 has been characterized as an NADH, dihydroneopterin triphosphate, and (deoxy) nucleoside triphosphate pyrophosphatases (Dobrzanska et al., 2002; Klaus et al., 2005). The deduced amino acid sequences of AtNUDXs showed low homology (11.6~25.9%) with those of E. coli MutT and human MTH1, and their sizes ranged from 16.4 KDa (147 amino acids) to 86.9 kDa (772 amino acids). Furthermore, it was predicted that these AtNUDXs were classified into 3 types by their subcellular localization in the TargetP (http://www.cbs.dtu.dk/services/TargetP/). . .

(37) as follows; cytosol (AtNUDX1~11, 25), mitochondrion (AtNUDX12~18) and chloroplast (AtNUDX19~24, 26, 27). Among them, the product of AtNUDX3 is not likely a typical Nudix hydrolase enzyme, since its predicted sequence shows an extremely low homology to those of other AtNUDXs, and its molecular weight (86.9 kDa) is much higher than those of the enzymes in other organisms, such as E. coli and human. In eukaryotic cells, pools of their substrates are present mainly in the cytosol (Tassotto and Mathews, 2002).. Therefore, I focused on cytosolic types of AtNUDXs. (AtNUDX1, 2 and 4~11) and studied their molecular properties.. Nudix motif. Figure III-1. Partial sequence alignment of AtNUDX proteins with ADP-ribose and 8-oxo-dGTP pyrophosphatases of the Nudix family.. Polypeptides identified in a Blast search against the Nudix signature sequences are shown. Amino acids that are fully conserved or substitutive are shown by grey box. The Nudix motif is shown below the sequence. The sequences used here are as follows AtNUDX1, At1g68760; AtNUDX2, At5g47650; AtNUDX3, At1g79690; AtNUDX4, At1g18300; AtNUDX5, At2g04430; AtNUDX6, At2g04450; AtNUDX7, At4g12720; AtNUDX8, At5g47240; AtNUDX9, At3g46200; AtNUDX10, At4g25434; AtNUDX11, At5g45940; AtNUDX12, At1g12880; AtNUDX13, At3g26690; AtNUDX14, At4g11980; AtNUDX15, At1g28960; AtNUDX16, At3g12600; AtNUDX17, At2g01670; AtNUDX18, At1g14860; AtNUDX19, At5g20070; AtNUDX20, At5g19460; AtNUDX21, At1g73540; AtNUDX22, At2g33980; AtNUDX23, At2g42070; AtNUDX24, At5g19470; AtNUDX25, At1g30110; AtNUDX26, At3g10620; AtNUDX27, At5g06340; E.coli MutT, P08337; Homo sapiens MTH1, P36639; Homo sapiens NUDT5, AF218818; Mus musculus Nudt7, AF338424; Synechococcus sp. PCC7002 nuhA, AB105878; Methanococcus jannnaschii MJ1149, D64443; S. cerevisiae YLR151c, NP_013252.. . .

(38) Expression and purification of the His-tagged recombinant AtNUDX proteins The cDNAs encoding cytosolic AtNUDXs were obtained from total RNA prepared from rosetta leaves of A. thaliana. The entire ORF regions of AtNUDX1, 2 and 4~11 were cloned into pET16b or pCold II for production of their recombinant proteins fused with a hexahistidine-tag in E. coli. The recombinant proteins of AtNUDX1, 2, 4, 5, 6, 7, 9, 10 and 11 were produced with high efficiency in the soluble fraction. The molecular masses of each recombinant AtNUDX proteins agreed with their predicted molecular masses. Therefore, these recombinant proteins were purified using a HiTrap chelating HP column to apparent homogeneity, as judged with SDS-PAGE (Figure III-2). The production of recombinant proteins of AtNUDX8 was detected only in the insoluble fraction because of the formation of inclusion bodies.. Figure III-2. Purification of recombinant AtNUDX proteins. Recombinant AtNUDX proteins were overexpressed in E.coli, purified with Ni2+ affinity chromatography, and verified using SDS-PAGE with Coomassie blue staining. The experimental conditions are described in Materials and Methods section. Left lanes and right lanes of all AtNUDXs contain 15 g crude extract and 2 g of purified recombinant proteins, respectively. M: molecular mass standards (Amersham Bioscience) as indicated on the left.. Substrate specificities of recombinant AtNUDX proteins To analyze the properties of cytosolic AtNUDXs as Nudix hydrolases,. . .

(39) I measured the hydrolytic activities of these enzymes in the presence of 5 mM Mg2+ for deoxyribonucleoside triphosphates and various types of nucleoside diphosphate derivatives with HPLC.. The results are. summarized in Table III-1. The recombinant proteins of AtNUDX2, 6, 7 and 10 hydrolyzed both ADP-ribose and NADH to AMP. These proteins showed a high affinity for ADP-ribose compared with the other ADP-ribose pyrophosphatases, hNUDT5 (Yang et al., 2000), Methanococcus jannaschii ADP-ribose. pyrophosphatase,. MJ1149. (Sheikh. et. al.,. 1998). and. cyanobacterium Synechococcus sp. PCC 7002 ADP-ribose pyrophosphatase, NuhA (Okuda et al., 2004) (Table III-2). The AtNUDX2, 6 and 7 proteins also showed a high affinity for NADH.. TableIII-1 Substrate specificities of AtNUDX proteins Substrate. AtNUDX2. AtNUDX4. AtNUDX5. AtNUDX6. AtNUDX7. AtNUDX9. 8-oxo-dGTP. 0.94 ± 0.04. AtNUDX1. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. dGTP. 1.48 ± 0.07. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. dATP. 0.65 ± 0.04. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. dTTP. 0.90 ± 0.03. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. < 0.01. n.d.. dCTP. n.d.. NADH. n.d.. 0.10 ± 0.01. n.d.. n.d.. 0.28 ± 0.03 0.07 ± 0.01 0.19 ± 0.01 0.11 ± 0.01. ADP-ribose. n.d.. 0.19 ± 0.02. n.d.. n.d.. ADP-glucose. n.d.. < 0.01. n.d.. n.d.. Ap3A. n.d.. 0.02 ± 0.01. n.d.. n.d.. < 0.01. Ap4A. n.d.. 0.02 ± 0.01. n.d.. n.d.. < 0.01. Ap5A. n.d.. < 0.01. n.d.. n.d.. n.d.. < 0.01. UDP-glucose. n.d.. n.d.. n.d.. n.d.. n.d.. UDP-galactose. n.d.. n.d.. n.d.. n.d.. CoA. n.d.. n.d.. n.d.. n.d.. FAD. n.d.. n.d.. n.d.. < 0.01. 0.06 ± 0.01. n.d.. 0.02 ± 0.01. n.d.. AtNUDX10. 0.08 ± 0.02. AtNUDX11 n.d.. n.d.. n.d.. < 0.01. n.d.. n.d.. < 0.01. n.d.. n.d.. < 0.01. n.d.. n.d.. < 0.01. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. n.d.. < 0.01 0.02 ± 0.01. 0.02 ± 0.01. n.d.. < 0.01. n.d. 0.42 ± 0.01 n.d.. All substrates were at a concentration of 25 M and 0.5~1 g of the recombinant proteins were used. The activities of the recombinant AtNUDT proteins were measured at 37 ˚C with 5 mM Mg2+, as described in “EXPERIMENTAL PROCEDURES”. Data are the means of 3 independent determinations S.D. Ap3A, adenosine (5’) triphospho (5’) adenosine. Other dinucleoside polyphosphates are abbreviated in an analogous manner.. All specific activities were. mol/min/mg. n.d.; not detected.. AtNUDX1 was previously reported to have activity toward NADH in the presence of 5 mM Mn2+ (Dobrzanska et al., 2002). However, this activity was not detected in the presence of 5 mM Mg2+ (Table III-1). In addition, AtNUDX1 showed hydrolysis activity with 5 mM Mg2+ toward 8-oxo-(d)GTP to. . .

(40) 8-oxo-(d)GMP, with a high affinity (Table III-3). Furthermore, the Km value for 8-oxo-dGTP was lower than that of human MTH1 (Fujikawa et al., 1999) and S. cerevisiae YLR151c (Nunoshiba et al., 2004). Interestingly, the AtNUDX1 protein also showed a high affinity for dGTP, dATP and dTTP compared with other MutT-type enzymes (Table III-3 ).. The AtNUDX11. protein showed activity toward CoA-like mouse Nudt7 (Nudt7-M), human Nudt7 (Nudt7-H), and D. radiodurans DR-CoAase (Xu et al., 2001; Gasmi and Mclennanm, 2001). No activity to any of the substrates tested was detected in the AtNUDX4, 5 and 9 proteins.. Table III-2. The kinetic parameters of ADP-ribose and NADH pyrophosphatases in. thaliana,human, M. jannaschii, Synechococcus PCC7002, and yeast Protein Km Vmax kcat M mol/min/mg s-1 ADP-ribose AtNUDX2 16.9±2.3 0.20±0.01 0.12 AtNUDX6 23.0±0.9 0.18±0.01 0.11 AtNUDX7 23.2±6.3 0.24±0.02 0.12 AtNUDX10 27.4±2.1 0.10±0.01 0.06 hNUDT5 a 31.6±5.1 9.50±2.11 MJ1149 b 340 6.2±0.3 1.98 NuhA c 94 23.6 1.8 NADH AtNUDX2 22.3±1.1 0.16±0.01 0.01 AtNUDX6 13.7±0.4 0.32±0.01 0.18 AtNUDX7 37.2±5.3 0.09±0.01 0.06 NPY1 d 170 2.0 1.5 hNUDX12 e 11 12.0 11.0. A.. kcat/Km s-1 M-1 7.0103 4.6103 5.3103 6.3102 5.8103 1.9104 4.3103 1.4104 1.5103 8.5103 1.0106. The standard assay was used with concentrations of 5-300 M for ADP-ribose and NADH at 37 ˚C with 5 mM Mg2+ as described in Materials and Methods section. Data are the means of 3 independent determinations S.D. et al., 2004;. . d, e. a,. Yang et al., 2000; b, Sheikh et al., 1998; c, Okuda. Abdelraheim et al., 2001 and 2003.. .

(41) Table III-3 Comparison of the kinetic parameters of AtNUDX1 in A. thaliana with those of MutT-like proteins in E. coli, human, and yeast Protein AtNUDX1 8-oxo-dGTP dGTP dATP dTTP E. coli MutT a 8-oxo-dGTP dGTP dATP dTTP hMTH1 b 8-oxo-dGTP dGTP YLR151c c 8-oxo-dGTP. Km M. Vmax mol/min/mg. kcat s-1. kcat/Km s-1 M-1. 0.8±0.1 2.7±0.2 5.8±0.1 0.8±0.1. 0.25 0.83 0.17 0.25. 3.7104 1.4104 1.1104 1.5104. 0.48 1100 1800 1700. 4.2 4.8 1.3 0.28. -. -. 15.2 870. -. 12.3 -. 8.0105 -. 23.8. -. 0.13. 5.6103. 6.8±0.9 58.3±2.5 16.1±1.3 15.6±3.0. The standard assay was used with concentrations of 5-300 M for all substrates at 37 ˚C with 5 mM Mg2+ as described in Materials and Methods section. Data are the means of 3 independent determinations S.D.. a,. Maki et al., 1992; b, Fujikawa et al., 1999; c, Nunoshiba. et al., 2004.. Most of the characterized Nudix hydrolases require the presence of various divalent ions to become fully active. Therefore, I analyzed the requirement for divalent ions of AtNUDX1. Mg2+ was the most effective divalent ion. The presence of Mn2+ resulted in 79.5 %~90.2 % of the activity compared with the presence of Mg2+ (data not shown). Recently, Klaus et al. (2005) reported that the typical cytosolic levels of Mg2+ and Mn2+ were ~1 mM and ~1 M, respectively, in plants. The AtNUDX1 activities toward both 8-oxo-dGTP and other dNTPs in the presence of 1 mM Mg2+ were shown to be approx. 80 % of those in 5 mM Mg2+ (data not shown). The activities in the presence of 1 M Mn2+ were approx. 10 % of those in 5 mM Mg2+. Similar results were observed in all of the other AtNUDX proteins. These results indicated that the divalent ion essential for AtNUDX1 activity is Mg2+.. . .

(42) Complementation of the E. coli mutT mutation by AtNUDX1 Next, I examined the effects of the expression of AtNUDX1 on the ratio of spontaneous mutation frequency toward streptomycin-resistance in the E. coli mutT- strain, CC101T, which was devoid of its own 8-oxo-dGTPase (MutT) activity (Furuichi et al., 1994). As shown in Table III-4, the mutation frequency of CC101T carrying pTrc100/AtNUDX1 was completely suppressed to the same extent as that of the wild-type E. coli strain, CC101.. However, the mutation frequency of CC101T cells carrying. pTrc100/AtNUDX2 and 7 were almost the same as those of the cells with an empty pTrc100.. These results clearly indicated that the AtNUDX1. protein has the ability to prevent mutation via the sanitization of 8-oxo-dGTP, and thus acts as a functional homologue of E. coli MutT in A. thaliana.. Table III-4 Suppression of E. coli mutT mutator activity by the expression of AtNUDX1 E.coli strain Mutation frequency Relative ratio CC101 (wild-type) 2.24 ± 0.5510-10 1.0 CC101T (mutT-) with pTrc100 (vector) 5.46 ± 0.9910-7 2438 CC101T (mutT-) with pTrc100/AtNUDX1 1.31 ± 0.9010-9 5.8 CC101T (mutT ) with pTrc100/AtNUDX2 5.90 ± 1.3110-7 2634 CC101T (mutT-) with pTrc100/AtNUDX7 4.38 ± 0.3910-7 1955 The mutation frequency was calculated according to the number of colonies on the streptomycin plates, as described in Materials and Methods section. Wild-type cells and mutT- cells are the E. coli strains CC101 and CC101T, respectively, and carry either pTrc100 (vector), pTrc100::AtNUDX1, pTrc100::AtNUDX2, or pTrc100::AtNUDX7.. Data are the means of 3. independent determinations ± S.D. Tissue-specific expression of AtNUDX transcripts To confirm the tissue-specific expression of cytosolic AtNUDXs, I analyzed the transcript levels of cytosolic AtNUDXs in various tissues with the semi-quantitative RT-PCR.. As shown in Figure III-3, the. transcripts of all cytosolic AtNUDXs were detected in leaves, stems and roots. The transcript levels of AtNUDX6 in the roots were lower than those in leaves and stems. On the other hand, the transcript levels of. . .

(43) AtNUDX3, 8 and 10 in leaves were lower than those in roots and stems. These results suggest that the expression of each AtNUDX is individually regulated in different tissues. s es s ot em eav Ro St L. s es s ot em eav L Ro St. AtNUDX1. AtNUDX7. AtNUDX2. AtNUDX8. AtNUDX3. AtNUDX9. AtNUDX4. AtNUDX10. AtNUDX5. AtNUDX11. AtNUDX6. Actin2. Figure III-3 Expression of the cytosolic AtNUDX genes in different plant tissues. Semi-quantitative RT-PCR was performed using specific primers for AtNUDX genes and Actin2 on total RNA from roots, stems and leaves. PCR amplification was performed with 20~26 cycles of 95˚C for 60s, 55˚C for 60s, and 72˚C for 60s, followed by 72˚C for 10 min. Aliquots of the products were analysed on 1 % agarose gel.. Discussion. The Nudix hydrolases are widely distributed in over 200 species ranging from viruses to humans (Xu et al., 2006). In A. thaliana, there were 3 types of Nudix hydrolases that were potentially localized in the cytosol, chloroplasts and mitochondria (Figure III-1).. In eukaryotic cells,. pools of their substrates, especially dNTPs, are present mainly in the cytosol (Bestwick et al., 1982) and human MTH1 is mostly localized in the cytosolic fraction (Kang et al., 1995). Therefore, I analyzed here the molecular and enzymatic properties of cytosolic Nudix hydrolases. AtNUDX1, whose molecular weight (16.4 kDa) was very similar to that of E. coli MutT (14.9 kDa) and hMTH1 (17.9 kDa) had activities toward 8-oxo-dGTP with a higher affinity than those for other substrates (Table III-3). These results suggest that AtNUDX1 is involved in the prevention of spontaneous mutation. In a mutT-deficient E. coli strain, the rate of spontaneous occurrence of A:T to C:G transversions increases to 1,000-fold that of the wild type level and also further increases. . .

(44) transcriptional errors (Taddei et al., 1997).. Furthermore, hMTH1,. homologues of E. coli MutT in human and rodent cells, can complement the function of MutT in E. coli (Sakumi et al., 1993; Furuichi et al., 1994; Cai et al., 1995; Kakuma et al., 1995). In addition, it has been reported that hNUDT5, which has activities toward not only 8-oxo-dGDP, but also ADP-ribose, suppresses the mutations of the E. coli mutT- strain (Ishibashi et al., 2003).. Interestingly, AtNUDX1 suppressed the. mutation frequency of the E. coli mutT- strain to the same extent as that in the wild-type E. coli strain (Table III-4). Klaus et al. have recently suggested that AtNUDX1 is likely to be a bifunctional enzyme that may act as a MutT-type Nudix hydrolase and a dihydroneopterin triphosphatase in the folate synthesis pathway (Klaus et al., 2005). My result clearly suggests that AtNUDX1 functions as an 8-oxo-dGTPase in A. thaliana hydrolyzing 8-oxo-dGTP that accumulates in the nucleotide pool due to oxidative conditions. AtNUDX1 was constitutively expressed in various tissues, suggesting that sanitization of the oxidized nucleotide pool in the cytosol may be necessary not only in photosynthetic tissues but also in non-photosynthetic ones. However, the kcat/Km of AtNUDX1 is approx. 20-fold lower than that of hMTH1; although, that is approx. 7-fold higher than that of YLR151c (Table III-3 ).. Interestingly, A. thaliana contained two different. 8-oxo-G glycosylases (AtMMH, AtOgg1) encoded by the homologous genes of E.coli MutM, and yeast and human Ogg1, for the repair of 8-oxo-G from damaged DNA (Ohtsubo et al., 1998; Garcia-Ortiz et al., 2001). Therefore, the low ability of AtNUDX1 may be substantially complemented by the functions of AtMMH and AtOgg1. Furthermore, it is likely that some Nudix hydrolases localized in the chloroplasts and mitochondria may also function as a MutT-type enzyme to sanitize the nucleotide pools in each organelle.. . .

(45) On the other hand, AtNUDX2, 6, 7 and 10 had ADP-ribose pyrophosphatase activities with high affinity (Tables III-1 and 2). Furthermore, the kcat/Km of these proteins were almost equal to that of MJ1149. Free ADP-ribose is produced during the reverse processes of degrading protein bound mono- or poly-(ADP-ribose) or cyclic ADP-ribose and is also produced by the turnover of -NAD+ (Olivera et al., 1989). ADP-ribosylation is a regulatory modification of protein in which an ADP-ribose moiety in -NAD+ is transferred to a specific amino acid residue of the acceptor protein by poly (ADP-ribose) polymerase, in response to DNA strand break and resealing (Amé et al., 1999). The free ADP-ribose is a highly reactive molecule that causes non-enzymatic mono-ADP-ribosylation of proteins (Jacobson et al., 1994).. Furthermore, mono-ADP-ribosylation of the. protein by bacterial toxins leads to an immediate cytotoxic effect (MacDonald and Moss, 1994). Despite lacking knowledge about the possible specific roles of free ADP-ribose in cellular processes, it seems reasonable to conclude that the level of ADP-ribose in the cell should be carefully maintained to minimize the potential of the detrimental effect of free ADP-ribose (Bessman et al., 1996; Sheikh et al., 1998). Therefore, it is likely that the AtNUDXs (AtNUDX2, 6, 7 and 10) that have ADP-ribose pyrophosphatase activity can cooperatively play roles in removing the free ADP-ribose. In addition, AtNUDX2, 6, 7 and 10 also had NADH pyrophosphatase activity (Tables III-1 and 2). It has been reported that NADH pyrophosphatase is involved in the regulation of the cellular NADH/NAD+ ratio, an important factor in maintaining the balance between the anabolic and catabolic pathways in the cell (Frick and Bessman, 1995), although the contribution of these AtNUDX proteins to the regulation is still unknown. AtNUDX6 was expressed at a low level in the roots, whereas AtNUDX2 and 7 were constitutively expressed in various tissues, suggesting that the expression of these enzymes with overlapping. . .

(46) substrate spectra were individually regulated under different mechanisms (Figure III-3). Interestingly, only AtNUDX11 had activity toward CoA, although the physiological function of the enzyme is not yet understood. Recently, it was reported that CoA and its derivatives are the substrates for Nudix hydrolase in D. radiodurans (Xu et al., 2001), mice and humans (Gasmi and Mclennanm, 2001).. All of. them. have the conserved motif,. (LLTXR(SA)X3RX3GX3FPGG), which was found N-terminally adjacent to the Nudix motif. This motif was also conserved in AtNUDX11 (Figure III-1). There was no correlation between the conserved amino acid sequences and the enzymatic properties of the AtNUDX proteins, although the enzymes contained the Nudix motif, which was common among Nudix hydrolases in living organisms (Figure III-1 and Table III-1). In addition, AtNUDX 2, 6, 7 and 10 showed an activity as the ADP-ribose pyrophosphatase, whereas these proteins had no proline residues at the 15 or 16th amino acid from the C-terminus glycine residue of the Nudix motif, which is characteristic of the subfamily of ADP-ribose pyrophosphatases (Lin et al., 2002). The results obtained here suggest that the plant Nudix family has evolved in a specific manner, different from that of yeast and human.. Summary. Nudix hydrolases are a family of proteins that catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Twenty-seven genes of the Nudix hydrolase homologues (AtNUDXs) with predicted localizations in the cytosol, chloroplasts and mitochondria exist in Arabidopsis thaliana. Here, we demonstrated the comprehensive analysis of 9 types of cytosolic AtNUDX proteins (AtNUDX1, 2, 4, 5, 6, 7, 9, 10 and 11). The recombinant proteins of AtNUDX2, 6, 7 and 10 showed both ADP-ribose and. . .

(47) NADH pyrophosphatase activities with significantly high affinities compared with those of animal and yeast enzymes. The expression of each AtNUDX is individually regulated in different tissues. These findings suggest that most cytosolic AtNUDXs may substantially function in the sanitization of potentially hazardous ADP-ribose and the regulation of the cellular NADH/NAD+ ratio in plant cells. On the other hand, the AtNUDX1 protein had the ability to hydrolyze 8-oxo-dGTP with a Km value of 6.8 M and completely suppress the increased frequency of spontaneous mutations in the E. coli mutT- strain, indicating that AtNUDX1 is a functional homologue of E. coli MutT in A. thaliana and is involved in the prevention of spontaneous mutation.. . .

(48) CHAPTER IV Prevention of replicational and transcriptional errors by Arabidopsis 8-oxo-dGTPase, AtNUDX1 Introduction. Even under optimal conditions, many metabolic processes including chloroplastic, mitochondrial, and plasma membrane-linked electron transport systems in higher plants produce active oxygen species (AOS), such as superoxide radical (O2-), H2O2, and hydroxyl radical (•OH) in higher plants (Asada, 1997; Foyer et al., 1994). In addition, the imposition of biotic and abiotic stress conditions can give rise to excess concentrations of AOS and, consequently, leads to imbalance between AOS production and AOS-scavenging mechanisms, that is, oxidative damage at the cellular level. Therefore, plants have developed efficient AOS-scavenging mechanisms involving a large number of antioxidants and antioxidative enzymes. Cellular DNA, RNA, and their precursor nucleotides are exposed to a high risk of being oxidized by AOS. Among the various types of oxidized damage in nucleic acids, 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxo-G) is one of the dominant forms of oxidatively generated base modifications produced by reaction of a •OH on the C8 position of 2’-deoxyguanosine (dG) in DNA or on the free nucleotide form of guanine in the nucleotide pools (Hayakawa et al., 1995; Haghdoost et al., 2006). 8-oxo-G has a potential to alter genetic information, since it can pair with adenine and cytosine at almost equal efficiencies (Wood et al., 1990; Moriya et al., 1991; Shibutani et al., 1991). 8-Oxo-G-containing nucleotides (8-oxo-dGTP and 8-oxo-GTP) can be incorporated into DNA as well as RNA. . .

(49) and would cause both replicational and transcriptional errors (Ames et al., 1991; Sekiguchi and Tsuzuki, 2002). In addition, 2-OH-dATP can be incorporated into the nascent strand of DNA opposite guanine as well as tymine, resulting in a transversion mutation (Kamiya and Kasai, 2000). To prevent the mutagenic consequences of oxidized nucleotide, living organisms have developed cellular defense systems. In Escherichia coli, these are comprised of MutT, MutM, and MutY proteins (Michaels and Miller., 1992). The MutT protein hydrolyzes all canonical nucleoside triphosphates with a preference for 8-oxo-dGTP and 8-oxo-GTP to the monophosphate form in the nucleotide pool, and functions in the prevention of the misincorporation of such mutagenic nucleotides into DNA or mRNA during DNA replication or transcription (Maki and Sekiguchi., 1992; Taddei et al., 1997). In a mutT-deficient strain, the mutation rate increases 100 to 1,000-fold compared with wild-type cells (Tajiri et al., 1995). In addition to the sanitation of nucleotide pools by the MutT protein, there is the base excision repair pathway. The MutM protein, which is originally identified as a formamidopyrimidine DNA glycosylase/lyase, acts upon several modified purines, including 8-oxo-G (Boiteux et al., 1992; Chung et al., 1991).. The MutY protein is also. a DNA glycosylase, but it removes adenine from A:G, A:(8-oxo-G), and A:C mispairings (Michaels et al., 1992; Au et al., 1989). As a result of actions of MutT, MutM, and MutY proteins, E.coli efficiently protects the occurrence of spontaneous mutations, which are caused by 8-oxo-G. Mammalian cells have developed defense systems similar to those of E. coli. The cDNAs encoding the MutT protein homologue (MTH1) with the 8-oxo-dGTPase activity have been cloned from human, mouse, and rat (Sakumi et al., 1993; Kakuma et al., 1993; Cai et al., 1995). The human MTH1 (hMTH1) protein, but not E.coli MutT protein, has an ability to efficiently hydrolyze oxidized (d)ATP, such as 2-OH-(d)ATP, as well as 8-oxo-(d)GTP. . .

(50) (Fujikawa et al., 2001). A human gene encoding the MutY protein homologue (MYH) has been identified (Boiteux and Radicella, 1999). However, there is no MutM protein homologue in animals and yeast cells. Instead of the MutM protein, these cells possess MutM analogues, 8-oxo-G DNA glycosylases/lyase (OGG), which do not share significant sequence identity with bacterial MutM proteins (van der Kemp et al., 1996). In mammalian cells, more than one genome in a single cell has to be maintained throughout the entire life of the cell, one in the nucleus and the other in mitochondria. The genome in the mitochondria is likely to be more susceptible to AOS-induced oxidative damage, since oxygen metabolism is high. Therefore, all three gene products, MTH1, OGG1, and MYH proteins, which are considered to minimize oxidative DNA damage in human cells located both in nuclei and mitochondria of human cells (Kang et al., 1995; Nishioka et al., 1999; Ohtsubo et al., 2000). In contrast to E. coli and human, little is known about the defense systems toward oxidative DNA damage in higher plants (Hays, 2002). Interestingly, in chapter III, I showed that one of Arabidopsis Nudix hydrolases, AtNUDX1 protein, has an activity toward 8-oxo-(d)GTP with high affinity and completely suppresses frequency of spontaneous mutations in the E. coli mutT- strain. In addition, it has been reported that various plants including Arabidopsis have the genes for both MutM and OGG proteins, designated as AtMMH and AtOGG1, respectively (Ohtsubo et al., 1998; Dany and Tissier, 2001; Garcia-Ortiz et al., 2001; Murphy and Gao, 2001). The recombinant AtMutM and AtOGG1 proteins specifically cleaves duplex DNA containing an 8-oxo-G:C mispair with similar characteristic as other bifunctional DNA glycosylases/lyases (Murphy and George, 2005). Furthermore, AtOGG1 suppresses the mutator phenotype of an E. coli strain deficient in 8-oxo-G repair enzymes (Garcia-Ortiz et al., 2001). A gene encoding adenine DNA glycosylase-like protein. . .

(51) (At4g12740, designated AtMYH) is also registered in the Arabidopsis genome database. Therefore, in this chapter, I analyzed the ability of the AtNUDX1 protein to prevent misincorporation of oxidized ribonucleotide to mRNA leading to transcriptional errors. Furthermore, I demonstrated the physiological function of the AtNUDX1 protein using knockout AtNUDX1 mutants (KO-nudx1). In addition, I studied the sub-cellular localization of AtNUDX1 protein and the proteins involved in the base excision repair pathway (AtOGG1, AtMMH, and AtMYH) and the expression of these proteins under oxidative stressful conditions. The results obtained here indicate that the AtNUDX1 protein is a central component for cellular defense systems and the defense systems against oxidative DNA damages are distributed in several organelles in Arabidopsis cells.. Materials and Methods Complementation assay of transcriptional errors in E.coli mutT- strain The E. coli CC101 (wild-type) and CC101T (mutT-deficient) strains carry an amber mutation in codon 461 of the lacZ gene, where the A:T to C:G transversion mutation is reversed phenotypically to produce the -galactosidase (Taddei et al., 1997). The E.coli CC101 cells yield white colonies, since they are unable to produce an active -galactosidase when cells are cultured in the presence of 5-Bromo-4-chloro-3-indolyl- -D-galactopyranoside (X-gal). On the other hand, the CC101T cells, which carry a mutT mutation in addition to the lacZ amber mutation, produce blue colonies, probably due to the partial phenotypic suppression of the lacZ mutation caused by the misincorporation of 8-oxo-G into mRNA. The pTrc empty vector and the pTrc100/AtNUDX1 plasmid, which was constructed as described in chapter III, were transformed into the E. coli CC101 and. . .

(52) CC101T. The obtained transformants were grown on LB medium containing 20 mg /ml Ampicillin and 0.5 mg/ml X-gal. Quantitative assays for -galactosidase activity were performed by measuring the UV absorbance at 420 nm, which represents the hydrolysis of o-nitrophenyl- -D-galactoside to o-nitrophenol as a substrate. Three independent clones from each transformant were used, and at least four experiments were performed with each clone.. Sub-cellular localization of GFP fusion protein The vectors for the generation of the GFP fusions were constructed using GATEWAY cloning technology (Invitrogen, Paisley, U.K.). The cDNA encoding the open reading frame of AtNUDX1, AtOGG1, and AtMMH were cloned into the donor vector, pDONR201, and then re-cloned into the destination vector, pGWB5. The specific primers with attB1 and attB2 sequences were as follows; attB1-AtNUDX1cGFP (5’-AAAAAGCAGGCTTAATGTCGACAGGAGAAG-3’), attB2-AtNUDX1cGFP (5’-AGAAAGCTGGGTAGTCTCCACCACCATGAG-3’), attB1-AtOGG1cGFP (5’-AAAAAGCAGGCTGATGAAGAGACCTCGACC-3’), attB2-AtOGG1cGFP (5’-AGAAAGCTGGGTATGGCTTCAACGTATCAC-3’), attB1-AtMMH-1cGFP (5’-AAAAAGCAGGCTATGCCGGAGCTTCCAGAG-3’), attB2-AtMMH-1cGFP (5’-AGAAAGCTGGGTAACTTTTTCTTCCTTTTG-3’). PCR and in vitro BP and LR recombination reactions were carried out according to the manufacturer’s instructions (Invitrogen, Paisley, U.K.). A. tumefaciens, which was transformed with the obtained constructs by electroporation, was used to infect Arabidopsis via the vacuum infiltration method as described above. T1 seedlings were selected on basic MS medium in Petri dishes containing 3 % sucrose and 20 mg l-1 hygromycin for 2 weeks and transferred to soil. T2 seeds were harvested and used for the experiments. Leaf protoplasts were prepared from 2-week-old transgenic plants by the method of Abel and Theologis (1994).. . .

(53) The fluorescence was observed under Radiance 2100 confocal fluorescence microscope (Bio-Rad, CA, USA). Pictures were processed using LaserSharp2000 software (Carl Zeiss, Inc., Tokyo, Japan).. Isolation of the T-DNA knockout line for AtNUDX1 The knockout Arabidopsis line (SALK_025320: KO-nudx1) containing a T-DNA insert in the AtNUDX1 gene was obtained through the SIGnAL project (http://signal.salk.edu/tabout.html). The KO-nudx1 plants were outcrossed and self-fertilized to check for segregation and to obtain a purely homozygous line, respectively. Semi-quantitative RT-PCR analysis of AtNUDX1 transcripts was performed using specific primers for AtNUDX1 in a reaction by 30 cycles of 95˚C for 30s, 55˚C for 30s, and 72˚C for 60s, followed by 72˚C for 10 min. Aliquots of the products were analyzed on 1 % agarose gel. Actin2 was used to normalize the transcript levels in each sample. The specific primers with AtNUDX1 and Actin2 sequences were as follows: AtNUDX1-F (5’-ATAATGTCGACAGGAGAAGC -3’), AtNUDX1-R (5’-CATCTATTAGTCTCCACCAC-3’), Actin2-F (5’-GAGATCCACATCTGCTGG -3’), Actin2-R (5’-GCTGAGAGATTCAGGTGCCC-3’).. Stress treatments Two-week-old Arabidopsis plants were subjected to various types of stress treatment with PQ and high light irradiation. PQ treatment was imposed by transferring the plants to MS medium containing the agent at 3 M and growing them under normal conditions or under exposure to illumination at 1,600 E m-2 s-1. High light stress was accomplished by exposure to illumination at 1,600 E m-2 s-1.. Analysis of 8-oxo-G in DNA with an HPLC-Electrochemical detector Measurement of 8-oxo-G in genomic DNA was carried out according to. . .

(54) Minowa et al. (2000) with some modifications. Genomic DNA was extracted from ~1.0 g of plant tissues. Two hundreds fifty g of DNA suspended in 165 l of 10 mM sodium acetate solution and digested with 4 units of nuclease P1 (Sigma) and 2 units of Alkaline phosphatase at 37˚C for 2 hours. The resulting deoxynucleoside mixture was filtered with Ultrafree-MC centrifugal filter units (MILLIPORE).. The. mixture was lyophilized, resuspended in 500 l of water, and analyzed with an HPLC-ECD system: column, COSMOSIL C18 column (4.6 X 250 mm, Nacalai tesque); eluent, 50 mM NaH2PO4 buffer, containing 5 mM tetrabutylammonium dihydrogenphosphate, 10 M EDTA, and 4 % methanol; flow rate, 1 ml/min. The amount of deoxyguanosine (dG) and 8-oxo-deoxyguanosine (8-oxo-dG) in the DNA samples were measured with UV at 290 nm and ECD.. Semi-quantitative RT-PCR analysis Total RNA (50 g) extracted from Arabidopsis leaves, as described above, was purified with an RNeasy Plant Mini Kit (Qiagen, MD, USA), and then treated with DNase I to eliminate any DNA contamination (Takara, Kyoto, Japan), and was converted into first strand cDNA using ReverTra Ace (Toyobo, Osaka, Japan) with the oligo (dT)20 primer. Semi-quantitative RT-PCR analysis of AtNUDX1, AtOGG1, AtMMH, and AtMYH transcripts were performed using specific primers in a reaction by ~27 cycles of 95˚C for 30s, 55˚C for 30s, and 72˚C for 60s, followed by 72˚C for 10 min. Aliquots of the products were analyzed on 1 % agarose gel. Actin2 was used to normalize the transcript levels in each sample. The specific primers with AtNUDX1, AtOGG1, AtMMH, AtMYH, and Actin2 sequences were as follows; AtNUDX1-F (5’-ATAATGTCGACAGGAGAAGC -3’), AtNUDX1-R (5’-CATCTATTAGTCTCCACCAC-3’), AtOGG1-F (5’-ATGAAGAGACCTCGACCTAC-3’), AtOGG1-R (5’-TCATGGCTTCAACGTAGCAC-3’), AtMMH-F (5’-GATTAAGCGAGTCATTATCG-3’), AtMMH-R (5’-GCTAGTAGCTTTCTCTTCAG-3’), AtMYH-F. . .

(55) (5’-TATCGGAGATTATGCTTCAG-3’), AtMYH-R (5’-TATACAACTAATAACTCCAC-3’), Actin2-F (5’-GAGATCCACATCTGCTGG -3’), Actin2-R (5’-GCTGAGAGATTCAGGTGCCC-3’).. Results Prevention of the transcriptional errors by the AtNUDX1 protein 8-Oxo-GTP can be incorporated into RNA by the normal action of RNA polymerase which leads to transcriptional errors and the E.coli MutT protein has the ability to prevent the misincorporation depending on its activity toward 8-oxo-GTP (Taddei et al., 1997). Therefore, I examined the ability of AtNUDX1 protein to eliminate the mismatch-evoking oxidized nucleotides from the RNA precursor pool using E.coli lacZ- strain (Ishibashi et al., 2005). CC101T cells, which carried a mutT mutation, produced blue colonies on the plates containing X-gal by the partial phenotypic suppression of the lacZ amber mutation caused by the misincorporation of 8-oxo-G into mRNA (Figure IV-1A). On the other hand, when the AtNUDX1 cDNA was introduced into the CC101T cells, the formation of blue colonies was mostly suppressed to the same extent as that of the CC101 cells (Figure IV-1A). More quantitative data were obtained by measuring the actual -galactosidase activities in the cultures of CC101T cells harboring plasmids bearing the AtNUDX1 cDNA. The -galactosidase activity in the CC101T cells increased approx. 40,000-fold compared with the CC101 cells. The increase in the enzyme activity in the CC101T cells was completely suppressed by the expression of the AtNUDX1 cDNA (Figure IV-1B).. . .