2010.10.30 E3链接酶OsPUB15参与过氧化应激 [Plant J]

This is an Accepted Article that has been peer-reviewed and approved for publication in the The Plant Journal, but has yet to undergo copy-editing and proof correction. Please cite this article as an “Accepted Article”; doi: 10.1111/j.1365-313X.2010.04416.x Received Date : 02-Sep-2010 Revised Date : 30-Sep-2010 Accepted Date : 18-Oct-2010 Article type : Full Paper OsPUB15, an E3 ubiquitin ligase, functions to reduce cellular oxidative stress during seedling establishment Jong-Jin Park1,2, Jakyung Yi1,2, Jinmi Yoon1,2, Lae-Hyeon Cho1,2, Jin Ping1,2, Hee Joong Jeong1,2, Seok Keun Cho3, Woo Taek Kim3 and Gynheung An1, 2,4 * Running title: Abnormal germination in rice seed 1 Department of Life Science, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea 2Crop Biotech Institute, Kyung Hee University, Youngin 446-701, Republic of Korea 3Department of Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea 4Department of Plant Molecular Systems Biotechnology, Kyung Hee University, Youngin 446- 701, Republic of Korea * For correspondence (fax +82-31-204-3178; e-mail genean@khu.ac.kr). The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors is Gynheung An (genean@khu.ac.kr). Subject areas: development. SUMMARY The plant U-box protein functions as an E3 ligase to poly-ubiquitinate a target protein for its degradation or post-translational modification. Here, we report functional roles for OsPUB15, which encodes a cytosolic U-box protein in the Class II PUB family. Self-ubiquitination assays showed that bacterially expressed MBP-OsPUB15 protein has E3 ubiquitin ligase activity. A T-DNA insertional mutation in OsPUB15 caused severe growth retardation and a seedling lethal phenotype. Mutant seeds did not produce primary roots, and their shoot development was significantly delayed. Transgenic plants expressing the OsPUB15 antisense transcript phenocopied these mutant characters. The abnormal phenotypes were partially rescued by two antioxidants, catechin and ascorbic acid. Germinating seeds in the dark also recovered the rootless defect. Levels of H2O2 and oxidized proteins were higher in the knockout mutant compared with the wild type. OsPUB15 transcript levels were increased upon H2O2, salt, and drought stresses; plants over-expressing the gene grew better than the wild type under high salinity. These results indicate that PUB15 is a regulator that reduces ROS stress and cell death. Keywords: E3 ligase, rice, ROS, seedling lethal, U-box INTRODUCTION Reactive oxygen species (ROS) are radical derivatives of molecular oxygen; they include hydrogen peroxide (H2O2), superoxide radical (O˙2¯ ), hydroperoxyl radicals (HO˙2), and hydroxyl radical (·OH), which are produced either by ionizing radiation of water or through inefficient reduction of oxygen during energy generation (Puntarulo and Cederbaum, 1988). ROS function as signaling molecules that control various cellular processes, including pathogen defense, programmed cell death, and stomatal behavior (McAinsh et al., 1996; Maxwell et al., 2002; Tiwari et al., 2002). ROS levels are heightened by various stresses, e.g., increased light and salt, drought, cold, heat, pathogen infection, and mechanical damage (Apel and Hirt, 2004). High quantities cause oxidative damage to proteins, DNA, and lipids, which eventually leads to cell death (Mittler, 2002). Thus, excess ROS must be rapidly removed by various enzymatic and nonenzymatic systems so that an equilibrium is tightly controlled between their production and scavenging. In addition, the ubiquitin (Ub) proteasome system (UPS) functions during the stress response (Cho et al., 2008). Ubiquitination is accomplished by three enzymes: E1, Ub- activating enzyme; E2, Ub-conjugating enzyme; and E3, Ub-ligase enzyme. Ubiquitin is activated by E1 in an ATP-dependent manner, and this activated Ub is conjugated to a target protein by E2. E3 then mediates covalent conjugation of the target protein to Ub for degradation (Moon et al., 2004; Smalle and Vierstra, 2004; Dreher and Callis, 2007). The ubiquitination system is present in all eukaryotes and is implicated in many cellular processes, such as differentiation, cell division, and hormone responses (Zeng et al., 2008; Yee and Goring, 2009). Plant U-box (PUB) E3 proteins contain a conserved region that resembles the RING finger domain, except that the zinc-chelating cystein and histidine residues are absent (Aravind and Koonin, 2000; Ohi et al., 2003). There are 77 members in rice and 63 in Arabidopsis (Zeng et al., 2008). The SPL11 (SPotted Leaf 11) is a rice PUB that represses HR-associated cell death and the pathogenic defense (Zeng et al., 2004). BnARC1, a PUB member in Brassica, interacts with S-locus kinase, which regulates self-incompatibility (Gu et al., 1998; Stone et al., 1999, 2003). AtPUB17 is an Arabidopsis homolog of BnARC1. Atpub17 knockout plants display decreased resistance to avirulent Pseudomonas syringae pv. tomato (Yang et al., 2006). PHOR1 functions as a positive regulator during GA signaling; its antisense suppression produces semi-dwarf plants with higher endogenous GA levels and decreased sensitivity to exogenous GA whereas PHOR1-overexpressing lines show greater GA sensitivity (Amador et al., 2001). Under low light, SAUL1 suppresses premature senescence of young seedlings and enhances ABA biosynthesis (Raab et al., 2009). AtPUB22 and AtPUB23 have been proposed as negative regulators of abiotic stresses; loss of their expression confers drought tolerance whereas their overexpression results in hypersensitivity to salt and drought (Cho et al., 2008). Finally, the pub22 pub23 pub24 triple knockout mutants accumulate higher levels of ROS, causing cell death (Trujillo et al., 2008). Here, we report that ospub15 knockout plants are defective in seedling growth while OsPUB15 over-expressers are tolerant to high salt. We propose that OsPUB15 is a negative regulator of cell death and plant responses to abiotic stresses. RESULTS Isolation of a rootless mutant We identified a rootless mutant line, 4A-02107, from the T-DNA insertional population in japonica rice cv. Dongjin (Jeon et al., 2000; Jeong et al., 2002; An et al., 2003; Lee et al., 2003; Ryu et al. 2004). Mutant seeds developed normal shoots, but the seedlings did not produce roots (Figure 1a). Longitudinal sections from 3-day-old seedlings showed that radicle growth was hindered and those tissues eventually turned brown (Figure 1b, c). Although a coleoptile and three leaves appeared, their growth was significantly retarded compared with their segregating wild-type (WT) siblings (Figure 1d). Mutant shoots carried hairs, lamina joints, and the 4th leaf primordium, which turned yellow within two weeks after germination (Figure 1e; Figure S1). To observe radical growth in detail, imbibed seeds were cross-sectioned. In the WT, scutellum cells swelled after imbibition, creating an empty space between radicle and scutellum (Figure 1g). That space continuously enlarged during the first 18 h (Figure 1g, h, o). Radicles were approximately 0.5 mm long at 6 h after imbibition (Figure 1n). They continued to elongate to 1.0 mm, eventually filling the empty space at 24 h (Figure 1i, o). During the first 18 h, radicles of the mutant elongated normally, but scutellum had less swelling, resulting in a smaller space between radicle and scutellum (Figure 1j-l, n, o). At 24 h, the radicles stopped growing due to the lack of any more space (Figure 1m, o). Magnified images of the seeds at 24 h showed that root-cap cells from the WT were arranged radially, whereas those from the mutant did not elongate and were aligned in straight lines (Figure S2). T-DNA was inserted into OsPUB15 Sequence analysis of the T-DNA flanking region in the mutant revealed that T-DNA was inserted into LOC_Os08g01900 (http://tigrblast.tigr.org/euk-blast/) on Chromosome 8 (An et al., 2003, Jeong et al., 2002). The gene comprises five exons and four introns (Figure 2a). Its full-length cDNA was identified as AK106557 and AK102080 in the Knowledge-based Oryza Molecular Biological Encyclopedia (KOME) (http://cdna01.dna.affrc.go.jp/cDNA). T- DNA was inserted 2419 bp downstream from the ATG start codon, in the fifth exon of the gene. The predicted protein encoded by the gene is OsPUB15, a member of the Class II subfamily of U-box proteins (Zeng et al., 2008). Among the members of that subfamily in rice, OsPUB15 is most closely related to OsPUB16 (Figure S3). Functional-domain analysis with Pfam 7.0 (http://www.sanger.ac.uk/Software/Pfam) showed that the region between the 232nd and 295th amino acid residues shares high similarity to the consensus U-box domain sequence, and the region between the 559th and 812th residues is highly homologous to the armadillo repeat motif (ARM) found in β-catenin of Drosophila (Riggleman et al., 1989). OsPUB15 transcripts were ubiquitous from the young seedling stage through maturity, although levels were higher in shoots than roots during early development (Figure 2b). During seed imbibition, transcripts were rapidly increased, reaching the maximum at 2 h before declining to a steady state at 12 h (Figure 2c). OsPUB15 complementation rescued the mutant phenotypes To examine whether the abnormal seedling phenotypes observed from the T-DNA insertional line were due to a mutation in OsPUB15, we made the antisense construct using the 381-bp region of the 3’ UTR that started at 17 bp upstream of the stop codon (Figure 3a). The fragment was placed between the maize ubiquitin (ubi) promoter and nopaline synthase (nos) terminator, and the molecule was transferred to embryonic calli via Agrobacterium-mediated transformation (An et al., 1985; Kim et al., 2009). Thirteen independently transformed plants were regenerated and expression was measured for introduced antisense transcripts and those of the endogenous OsPUB15 (Figure 3b). We selected transgenic plants #4 and #12, in which levels for OsPUB15 were severely reduced due to strong expression of the antisense transcript (Figure 3c). We also chose transgenic plant #5, in which expression was not significantly reduced. Whereas plant #5 grew almost normally, #4 and #12 were semi-dwarf (Figure 3d). Seedlings of the latter two showed retarded development of roots and shoots (Figure 3e). These results support the theory that the retardation phenotypes observed from the T-DNA insertion line were due to the mutation in OsPUB15. To further confirm that the phenotypic changes observed from knockout plants were due to this defect, we complemented the mutant by expressing OsPUB15. Full-length OsPUB15 cDNA was placed under the 35S promoter (Figure S4a) and the molecule was transferred to embryonic calli derived from seeds of ospub15 heterozygote plants. Seeds of the heterozygous plants were used because homozygous plants were lethal. Among 36 T2 transformants, 8 were in ospub15 homozygous plants, 10 were in heterozygotes, and the remaining 18 were in the WT (Figure S4b). Transgenic plants expressing OsPUB15 in the ospub15 homozygous background grew normally (Figure S4c, plants #6 and #27). This demonstrated complementation of the mutant phenotypes with OsPUB15. Among the transformants in the WT background, those (#11 and #16) from one group weakly expressed the introduced OsPUB15 while the others (#2, #6, #27, and #35) had high expression (Figure S4d). However, both groups had normal and similar patterns of growth (Figure S4c). Therefore, it appears that overexpression of OsPUB15 does not affect plant development under standard growing conditions. OsPUB15 is a member of the U-box E3 ligases Plant U-box proteins are E3 ligases that poly-ubiquitinate target proteins for degradation (Yee and Goring, 2009). To check whether OsPUB15 has E3 ligase activity, we performed in-vitro self-ubiquitination assays. Full-length OsPUB15 cDNA was fused to the coding region of maltose binding protein (MBP), and this construct was expressed in E. coli. The OsPUB15- MBP protein was purified on an MBP affinity column. After the fusion protein was incubated with ubiquitin, E1 (Arabidopsis UBA1), and E2 (human UBC5c), the reaction mixture was separated on a polyacrylamide gel and incubated with antibodies against MBP. Human E2 was used because Arabidopsis E2 (UBC5, UBC8, and UBC10) did not ubiquitinate our protein. A polyubiquitination tail was detected when E1, E2, and ubiquitin were present (Figure 4a). However, when E1 or E2 was absent from the reaction mixture, that tail was not observed. Analyses with ubiquitin antibodies also showed the tail only when E1, E2, and ubiquitin were included in the reaction (Figure 4b). To confirm further that the tail was due to ubiquitination, we substituted amino acid residue methionine at position 242 to valine within the conserved U-box domain (Figure S5). Ubiquitination experiments with this mutated protein showed that the protein had little E3 ligase activity (Figure 4c). These results indicated that OsPUB15 is an E3 ubiquitin ligase. To learn the cellular location, we constructed two fusion vectors, YFP–OsPUB15 and OsPUB15–GFP. As a positive control, we used the previously characterized mRFP that is localized to the nucleus and cytoplasm (Park et al., 2004).When these chimeric molecules were co-introduced into rice mesophyll protoplasts prepared from 10-day-old seedlings, the GFP signal from OsPUB15–GFP coincided with the RFP signal driven by the mRFP protein in the cytoplasm (Figure S6a-c). Similar results were obtained with OsPUB15–YFP (Figure S6d-f). These experimental results suggested that OsPUB15 is a cytosolic protein. Hydrogen peroxide and oxidized protein levels were elevated in ospub15 The ospub15 mutants were lethal due to severe growth retardation and a failure of root development. Because radicles did not emerge from the embryos, that phenotype perhaps resulted from an inability to penetrate a physical barrier. To examine this possibility, we removed the coleorhizae region that covered the radicle, but this did not facilitate radicle growth (data not shown). Because the radicles and shoots turned brown, we considered another possibility that toxic compounds, such as ROS, were accumulating, which would cause cell death when over-produced. ROS accumulation is prompted by various stresses, e.g., salt and drought (Spickett et al., 1993; Hernandez et al., 2001; Cruz de Carvalho, 2008). Similar observations were made when plants were treated with 250 nM paraquat or 250 mM NaCl. Mutant plants also accumulated high levels of H2O2 (Figure 5a). The levels of oxidized proteins were also increased in mutants or as a result of paraquat treatment (Figure 5b). Therefore, we tested whether OsPUB15 expression is regulated by those stresses. Seven- day-old seedlings were exposed to 0.1 mM H2O2, 250 mM NaCl, or drought, and were sampled at 0, 30, 60, and 120 min post-treatment. Transcript levels were increased approximately 2- to 3-fold by both salt and H2O2 (Figure 5c). Drought stress also induced this expression, peaking at 60 min. These observations implied that OsPUB15 functions during stress responses and that a lack of expression causes ROS to accumulate to levels that are toxic to cell growth. If these mutant phenotypes were a consequence of high amounts of ROS, they could possibly be rescued by catechin and ascorbic acid. The former, a flavonoid compound, is known as an antioxidant reagent while the latter offsets ROS reactivity by accepting surplus electrons. When ospub15 mutant seeds were grown on a 1/2 MS medium, 3% of the seedlings produced abnormal primary roots (Figure 5d). By contrast, 32% of such seeds germinated abnormal roots on media containing 1 μM catechin. Similarly, 41% of seeds developed abnormal roots on media containing 10 μM ascorbic acid. Our results supported the theory that these growth- retardation phenotypes were due to ROS accumulation but that its removal could partially rescue the defect. Because light generates ROS (Shohael et al., 2006), we held our seeds in the dark. As expected, a large number of the mutant seeds produced roots, further supporting the hypothesis that ROS is a factor in growth retardation (Figure 5d, e). Over-expressers were tolerant to salt and paraquat stresses We examined whether the OsPUB15 over-expressers are tolerant to salt and paraquat. The latter generates O2˙ in the chloroplasts, which is dismutated to H2O2 (Asada, 2006), while the former causes an ion imbalance and hyperosmotic stress (Zhu, 2001). Over-expressers grew 1.4- and 1.9-fold faster than the WT under NaCl and paraquat treatment, respectively (Figure 6a, b). The amount of H2O2 was about 30% lower in over-expressers grown with paraquat, and 25% lower in salt-stressed plants (Figure 6c). Levels of oxidized proteins also were lower in the over-expressers after paraquat treatment (Figure 6d). Under paraquat exposure, over- expresser shoots were about 1.8-fold taller than those from the WT (Figure 6e). Whereas WT plants were severely wilted and shrunken by salt and drought stresses, over-expresser plants were much less susceptible (Figure 6f, g). ROS-related genes were induced in the ospub15 mutant Because H2O2 production was induced in the mutants, we examined transcript levels for stress-related genes that encode superoxide dismutase 2 (SOD2), glyoxalase, oxidoreductase, BAX inhibitor, EL5, ALDH7, and alanine transferase. The SOD2 enzyme converts superoxide oxygen (O2-) to H2O2 (Lee et al., 2008). Glyoxalase detoxifies methylglyoxal with glutathione (Ranganathan et al., 1995), and oxidoreductase is an enzyme that accepts surplus electrons (Baker and Lawen, 2000). Although its role in plants is unknown, the BAX inhibitor suppresses Bax-induced cell death in yeast (Xu and Reed, 1998). Gene expression is triggered by both biotic and abiotic stresses in Arabidopsis (Watanabe and Lam, 2006). The EL5 gene is induced by treatment with N-acetylchitoheptaose, which causes various cellular responses, including ROS generation (Takai et al., 2001). ALDH7 removes toxic compounds generated by oxidative stress during late-stage seed development. Its expression is dramatically increased under cold, drought, heat, methyl viologen, and high salt (Shin et al., 2009). We also monitored the genes encoding catalase, cytochrome C oxidase, glutathione peroxidase, and glutathione reductase, because they are involved in the removal of ROS (Apel and Hirt, 2004). Quantitative real-time PCR analyses showed that transcript levels of these genes were increased in ospub15 mutant plants (Figure 7). We also observed that Pi-d2, OsSERK1, 07g35580, and 08g03020 were higher in the mutant. Proteins encoded by these receptor-like kinase genes interact with OsPUB15 or OsPUB16 (Ding et al., 2009), and Pi-d2 and OsSERK1 are involved in antifungal immunity (Chen et al., 2006; Hu et al., 2005). Our analyses showed that these genes were also strongly induced in the mutant (Figure 7). DISCUSSION A defect in OsPUB15 causes seedling lethal We observed that mutations in OsPUB15 caused severe defects. Mutant seedlings stopped growing after three leaves were formed, and radicle growth was retarded, failing penetratio