E3泛素连接酶

Plant Science 180(2011)775–782Contents lists available at ScienceDirectPlantSciencej o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /p l a n t s ciOverexpression of OsRDCP1,a rice RING domain-containing E3ubiquitin ligase,increased tolerance to drought stress in rice (Oryza sativa L.)Hansol Bae a ,1,Sung Keun Kim a ,1,Seok Keun Cho a ,Bin Goo Kang b ,Woo Taek Kim a ,∗a Department of Systems Biology,College of Life Science and Biotechnology,Yonsei University,Seoul 120-749,Republic of Korea bReSEAT Program,Korea Institute of Science and Technology Information,Seoul 130-741,Republic of Koreaa r t i c l e i n f o Article history:Received 5February 2011Received in revised form 21February 2011Accepted 21February 2011Available online 4March 2011Keywords:Drought stress E3ubiquitin ligase Oryza sativaOsRDCP1-overexpressing lines Rice osrdcp1knock-out mutant RING domaina b s t r a c tCaRma1H1was previously identified as a hot pepper drought-induced RING E3Ub ligase.We have identi-fied five putative proteins that display a significant sequence identity with CaRma1H1in the rice genome database (/cgi-bin/RiceGE ).These five rice paralogs possess a single RING motif in their N-terminal regions,consistent with the notion that RING proteins are encoded by a multi-gene family.Therefore,these proteins were named OsRDCPs (Oryza sativa RING domain-containing proteins).Among these paralogs,OsRDCP1was induced by drought stress,whereas the other OsRDCP members were constitutively expressed,with OsRDCP4transcripts expressed at the highest level in rice seedlings.osrdcp1loss-of-function knockout mutant and OsRDCP1-overexpressing transgenic rice plants were developed.Phenotypic analysis showed that wild-type plants and the homozygous osrdcp1G2mutant line displayed similar phenotypes under normal growth conditions and in response to drought stress.This may be due to complementation by other OsRDCP paralogs.In contrast,35S:OsRDCP1T2transgenic rice plants exhib-ited improved tolerance to severe water deficits.Although the physiological function of OsRDCP1remains unclear,there are several possible mechanisms for its involvement in a subset of physiological responses to counteract dehydration stress in rice plants.©2011Elsevier Ireland Ltd.All rights reserved.1.IntroductionTurnover of a wide range of eukaryotic proteins is regulated by the ubiquitin (Ub)-26S proteasome pathway.This pathway plays a critical role in the control of multiple cellular processes as diverse as cell cycle,differentiation,hormone responses,protein trafficking,and responses to environmental stresses [1–3].Multiple Ub chains are attached to the target proteins by E1,E2,and E3enzymes and serve as degradation tags,leading to the proteolysis of targeted proteins by the 26S proteasome complex [4,5].Based on subunit composition,E3s can be classified into two groups.The HECT and RING/U-box E3classes are comprised of single subunits,whereas the SCF and anaphase-promoting-complex (APC)E3ligases consist of multiple polypeptides [1–5].In most cases,E3Ub ligases are responsible for identifying specific target proteins.Abbreviations:ABA,abscisic acid;MeJA,methyl jasmonate;RT-PCR,reverse transcriptase-polymerase chain reaction;SA,salicylic acid;Ub,ubiquitin.∗Corresponding author.Tel.:+82221232661;fax:+8223125657.E-mail address:wtkim@yonsei.ac.kr (W.T.Kim).1These two authors equally contributed to this work and are listed in alphabetical order.A number of RING-containing proteins function as E3Ub ligases in responses to abiotic environmental stresses.Arabidopsis HOS1works as a negative regulator for cold-stress signal transduction [6,7],while XERICO participates positively in salt-and osmotic-stress responses through the overproduction of abscisic acid (ABA)[8].Arabidopsis SDIR1and DRIP RING E3Ub ligases are involved in the regulation of positive and negative drought-stress responses,respectively [9,10].Ryu et al.[11]recently reported that AtAIRP1RING E3is a positive regulator of an ABA-dependent defense mech-anism against drought stress.Compared to the studies of Arabidopsis RING E3ligases,research elucidating the roles of these Ub ligases in crop plants has been scarce.The hot pepper CaRma1H1RING E3ligase confers strong tolerance to dehydration stress when it is ectopically expressed in Arabidopsis [12].More recently,the rice OsDSG1RING protein was reported to regulate high-salt and drought stress responses [13].Collectively,these results are indicative of cellular functions of RING E3Ub ligases in defense against abiotic environmental stresses in higher plants.We aimed to unravel the physiological roles of RING E3Ub lig-ases with respect to abiotic stresses,such as water deficit.In this report,we used rice as a molecular genetic model system and identified five homologous OsRDCP (Oryza sativa RING domain-containing protein)family members,all of which contain a single0168-9452/$–see front matter ©2011Elsevier Ireland Ltd.All rights reserved.doi:10.1016/j.plantsci.2011.02.008776H.Bae et al./Plant Science180(2011)775–782RING motif in their N-terminal regions.Among these rice par-alogs,OsRDCP1was induced by drought stress.Loss-of-function rice mutant plants(osrdcp1)containing a T-DNA copy integrated into the OsRDCP1gene and OsRDCP1-overexpressing transgenic rice plants(35S:OsRDCP1)were developed.Phenotypic analysis showed that wild-type plants and the homozygous osrdcp1G2 mutant line displayed similar phenotypes under normal growth conditions and in response to drought stress.In contrast,OsRDCP1-overexpressing T2transgenic rice plants exhibited markedly improved tolerance to severe water deficits.Although the physi-ological function of OsRDCP1has not yet been determined,there are several possibilities for the involvement of OsRDCP1in a subset of physiological responses to counteract dehydration stress in rice plants.2.Materials and methods2.1.Plant materialsRice(O.sativa var.Japonica cv.Dongjin)seeds were germi-nated on Murashige and Skoog medium containing MS basal salts (Wako Pure Chemical,Osaka,Japan),3%sucrose,0.2%phytogel,and 0.55mM myo-inositol as described previously[14].Seedlings were grown for1–2weeks at27◦C under continuous light,transplanted to soil in a greenhouse,and raised to maturity.The rice mutant line osrdcp1that contained a T-DNA insertion within OsDRCP1was developed from a population of T-DNA-tagging lines produced from O.sativa var.Japonica cv.Dongjin[15,16]using a gene-specific primer M1(5 -TTGACTTTGCACACATAGGACA-3 )and a T-DNA right border primer LBa-1(5 -ACAAGCCGTAAGTGCAAGTG-3 ).Plants homozygous for the T-DNA insertion were identified through mul-tiple PCRs with primers M2(5 -CTGGTGCCATTGTATGGTCGT-3 ) and LBa-1.The35S:OsRDCP1transgenic rice plants overexpress-ing OsRDCP1under the control of the35S CaMV promoter were generated by Agrobacterium-mediated co-cultivation methods as previously earlier[17].Regenerated plants were grown in a green-house at approximately30◦C during the day and20◦C at night.The light/dark cycle in the greenhouse was14/10h[18].2.2.Reverse transcriptase-polymerase chain reaction(RT-PCR)RT-PCR was performed in25␮l reaction buffer(10mM Tris, pH8.0,50mM KCl,1.5mM MgCl2,0.01%gelatin,and200␮M deoxynucleotides)containing1␮l of thefirst-strand cDNA reaction products,1␮M gene-specific primer sets,and2.5units Taq poly-merase(Promega,Madison,WI,USA).Gene-specific primers were designed based on the nucleotide sequences of OsRDCP1,OsRDCP2, OsRDCP3,OsRDCP4,and OsRDCP5(Table1).PCR conditions con-sisted of25cycles of45s at95◦C,1min at60◦C,and90s at72◦C in an automatic thermal cycler(Perkin-Elmer/Cetus,Norwalk,CT, USA).PCR products were separated on1.2%agarose gels and visu-alized under UV light.2.3.Application of abiotic stresses and plant hormonesRice plants were subjected to various abiotic stresses based on the method of Cho et al.[19]with significant modifications as described previously[20].For drought stress treatments,rice seedlings were grown for2weeks on agar plates under light con-ditions,harvested,and dehydrated on Whatman3MMfilter paper at room temperature.The degree of water stress was determined as the decrease in the fresh weight(5–30%)of the seedlings.For high-salinity treatments,2-week-old whole seedlings were soaked in solution containing150mM NaCl for0,2,6,or24h with gentle shaking.For low-temperature treatments,2-week-old plants were incubated at4◦C for0,2,6,or24h.For hormone treatments,leaf Table1Primer sequences for RT-PCR and genotyping PCR.Gene Primer sequenceRT-PCROsRDCP1(Os04g44820)Forward5 -GGTTCTTTGTTCTTGTCAGTGCTG-3Reverse5 -GGAATGGGCACACCATTCAG-3 OsRDCP2(Os02g42690)Forward5 -TGAGACGGCAGCACATGGAG-3Reverse5 -TCACTACTTAACAGCGTCCGCTC-3 OsRDCP3(Os01g56070)Forward5 -CAGCATGTTGATGTGTTCTTGAAG-3Reverse5 -CTACAAACAAGACTTGCAACTGCAC-3 OsRDCP4(Os03g47500)Forward5 -GTTTCACTATGGGTATGGCCATG-3Reverse5 -CATGCAAATATTCTATGAACACTTCATG-3 OsRDCP5(Os12g43930)Forward5 -ATATGGACACGGCCACGGT-3Reverse5 -CTCTAGTTCATTCAGACAGAGCTTCC-3 OsRab16b(Os11g26780)Forward5 -ACAAGGGCAACAACCACCAG-3Reverse5 -GCTTGCAATGGCATCACAAG-3 OsActin(Os03g50885)Forward5 -GTCAGCAACTGGGATGATATGG-3Reverse5 -TCTCCTTGCTCATCCTGTCAG-3 Genotyping PCROsRDCP1M15 -TTGACTTTGCACACATAGGACA-3M25 -CTGGTGCCATTGTATGGTCGT-3 LBa-15 -ACAAGCCGTAAGTGCAAGTG-3tissues of4-week-old plants were sprayed with100␮M salicylic acid(SA),100␮M methyl jasmonate(MeJA),or100␮M ABA and harvested at different time points.For ethylene treatments,light-grown2-week-old intact plants were enclosed in6-l jars containing air or air plus50␮l/l ethylene for various time periods.Proper ethylene concentrations were confirmed by gas chromatography [21].2.4.RNA gel blot analysisTotal RNA was obtained from rice plants as described previ-ously[22].RNA(10␮g per lane)was separated by electrophoresis on1.0%-agarose gels,blotted onto nylon membranefilters(Amer-sham,Arlington Heights,IL,USA),and hybridized with32P-labeled cDNA probes for OsRDCP1,OsEXP,OsLEA3,OsPHGPX,or OsActin under high-stringency hybridization and washing conditions.Blots were visualized by autoradiography at−80◦C using Kodak XAR-5film and an intensifying screen.Hybridization signals were quanti-fied with a PhosphorImager(Fuji,Tokyo,Japan).2.5.In vitro ubiquitination assaysIn vitro ubiquitination assays were conducted as described pre-viously[23]with minor modifications.Briefly,500ng bacterially expressed MBP-OsRDCP1fusion protein was incubated with150␮l ubiquitination reaction buffer(50mM Tris–HCl,pH7.5,2.5mM MgCl2,and0.5mM DTT)containing4mM ATP and20ng each of human E1and human E2UbcH5B.Arabidopsis E1(UBA1)and Arabidopsis E2(UBC8)were previously used for our in vitro ubiqui-tination assays.However,in this study,we obtained better results of rice E3Ub ligase assay with human E1and human E2UbcH5B as described by Park et al.[24].Reaction mixtures were incubated at 30◦C for0–120min,separated by8%SDS–PAGE,and subjected to immuno-blotting using an anti-MBP antibody(New England Bio-Labs,Ipswich,MA,USA)as described previously[25].2.6.Phenotypic analysis of wild-type,osrtbp1mutant,and35S:OsRDCP1transgenic rice plantsWild-type,osrtbp1mutant,and various independent 35S:OsRTBP1transgenic rice plants were subjected to drought stress as described previously[12,23]with modifications.Six-week-old plants were grown in pots under greenhouse conditions. The soil was then allowed to dry by withholding water for15 days until plants displayed wilting.Dehydration sensitivity wasH.Bae et al./Plant Science180(2011)775–782777Fig.1.Identification offive homologous rice RING E3OsRDCP family members.(A)Schematic representation of predictedfive rice OsRDCP paralogs.The GenBank accession numbers of OsRDCP1,OsRDCP2,OsRDCP3,OsRDCP4,and OsRDCP5are Os04g44820,Os02g42690,Os01g56070,Os03g47500,and Os12g43930,respectively.N-terminal RING domain and C-terminal membrane-anchoring domain are indicated by green bars and red bars,respectively.(B)Sequence alignment of the RING domain of the OsRDCP paralogs.Amino acids identical in allfive proteins are shown in black.Amino-acid residues that are conserved in at least three of thefive sequences are shaded.Putative Zn2+-interacting Cys and His residues are indicated.The numbers on the right indicate the amino-acid residues.(C)Comparison of the predicted amino-acid sequences offive full-length rice OsRDCP paralogs.The green box and red box indicate N-terminal RING domain and C-terminal membrane-anchoring domain,respectively.(For interpretation of the references to color in thisfigure legend,the reader is referred to the web version of the article.)scored as the capacity of plants to resume growth when returned to normal water conditions with re-watering.3.Results and discussion3.1.Identification and expression offive homologous rice OsRDCP gene family membersCaRma1H1was previously identified as a hot pepper drought-induced RING E3Ub ligase[12].Transgenic Arabidopsis plants that ectopically expressed CaRma1H1were highly resistant to severe water deficits.Searches of the rice genome database (/cgi-bin/RiceGE)revealed that there are at leastfive putative proteins that display a significant sequence identity with CaRma1H1and Arabidopsis AtRma1[26]homologs (Fig.1A).Thesefive rice paralogs possess a single RING motif in their N-terminal regions(Fig.1B),consistent with the notion that RING proteins are encoded by a large multi-gene family[2,4,5].In addition,they contain a putative membrane-anchoring domain in their C-terminal regions.Thus,these paralogs were named OsRDCPs (O.sativa RING domain-containing proteins).The predicted OsRDCP paralogs were25–79%identical to each other at the amino acid level (Fig.1C).To examine whether these rice RING E3members were induced by water deficits,2-week-old rice seedlings were subjected to drought stress and expression of each member was monitored by RT-PCR using gene-specific primer sets(Table1).The degree of water stress was monitored as the loss of fresh weight(0–30%)of the seedlings.The results showed that,among thefive paralogs,778H.Bae et al./Plant Science180(2011)775–782Fig.2.RT-PCR analysis offive OsRDCP family members in rice seedlings.Light-grown2-week-old seedlings were subjected to drought stress.Expression of each gene member in shoot and root tissues was analyzed by RT-PCR using gene-specific primer sets(Table1).The degree of water stress was monitored as loss of seedling fresh weight(0or30%).Rice Rab16b was used as a positive control for a drought-induced gene and actin was used as a loading control.OsRDCP1was significantly induced by drought stress in both shoot and root tissues,while the other OsRDCP members were constitu-tively expressed(Fig.2).The level of the OsRDCP4transcript was the highest in rice seedlings.3.2.OsRDCP1is a drought-inducible RING E3Ub ligase in riceThe OsRDCP1gene(GenBank accession number AK070732)is located on chromosome4and contains one intron(2093bp)in the5 -untranslated region(Fig.3A).The coding region of OsRDCP1 is762bp and encodes253amino acids with a molecular mass of 28.1kDa and a calculated p I of7.2.Full-length OsRDCP1is37%, 29%,and31%identical to hot pepper CaRma1H1[12],Arabidopsis AtRma1[26],and human RING protein HsRma1[27],respectively (Fig.3B).In addition,the RING domain of OsRDCP1is48–78%iden-tical to those of hot pepper,Arabidopsis,and human RING proteins. Thesefindings suggest that the domain is critical for Ub ligase activ-ity in rice plants.Like CaRma1H1,AtRma1,and HsRma1,OsRDCP1 has a single putative membrane-anchoring domain in its extreme C-terminus,suggesting that it is a membrane-associated protein.These structural conservations suggest that OsRDCP1is a RING domain-containing E3Ub ligase.Therefore,we characterized the OsRDCP1gene in more detail at the molecular level.Since OsRDCP1 was identified initially as a homolog of water stress-induced hot pepper CaRma1H1,we hypothesized that the expression of OsRDCP1is modulated by abiotic stresses in rice plants.To test this hypothesis,the OsRDCP1mRNA accumulation profile was monitored by RNA gel blot analyses under various abiotic stress conditions.Two-week-old light-grown rice seedlings were sub-jected to dehydration stress.OsRDCP1transcript(approximately 1.2kb)levels were significantly augmented in response to5–30% water loss(Fig.3C).This increase was seen in both shoots and roots with similar degrees of induction.The OsEXP gene,a homolog of the Arabidopsis EXPANSIN gene[28],was included as a positive control for drought stress.OsEXP was induced following20–30%water loss (Fig.3C).Thus,the induction kinetics of OsEXP was distinct from that of OsRDCP1.Significant induction of OsRDCP1transcripts was also detected following cold stress(2,6,and24h at4◦C)(Fig.3D). In contrast,expression of OsRDCP1was unaffected by high salin-ity(150mM NaCl)for at least24h,even though the expression of OsLEA3,a positive control for salt stress responses[29],was up-regulated after a2-h treatment(Fig.3E).We next examined the changes in OsRDCP1mRNA levels in response to various plant hormones.As shown in Fig.3F,OsRDCP1 transcription was activated by100␮M MeJA after a2–6-h incu-bation,with induction kinetics different than that of OsPHGPX,a positive control for MeJA induction[30].On the other hand,expres-sion of the OsRDCP1gene was not affected by100␮M SA(Fig.3F), 100␮M ABA,or20␮l/l ethylene.Thus,OsRDCP1is inducible by a subset of abiotic stresses,including drought,cold,and MeJA.3.3.In vitro self-ubiquitination assaysTo test whether OsRDCP1contained E3Ub ligase activity,full-length OsRDCP1was expressed in Escherichia coli as a fusion protein with maltose binding protein(MBP).Purified MBP-OsRDCP1was incubated at30◦C in the presence or absence of Ub,ATP,human E1,and the human E2protein UbcH5B for various time periods, and subjected to immuno-blot analysis with an anti-MBP antibody. MBP-OsRDCP1produced high-molecular-mass smear ladders in a time-dependent manner(Fig.4A).In contrast,no ubiquitina-tion was detected when the E1,E2,ATP,or Ub was excluded from the reaction mixture(Fig.4B).The MBP-OsRDCP1C54S and MBP-OsRDCP1C86S mutant proteins,in which the Cys54and Cys86 residues,respectively,were replaced with Ser residues,displayed a negligible level of Ub ligase activity(Fig.4C).The Cys54and Cys86 residues are highly conserved among different RING proteins[4,5]. In contrast,mutation of K23to R23did not exert any inhibitory effects on the Ub ligase activity.Collectively,the results depicted in Figs.3and4strongly suggest that rice OsRDCP1is a drought-inducible RING E3Ub ligase.3.4.Isolation of a T-DNA insertion mutant of OsRDCP1and construction of35S:OsRDCP1transgenic rice plantsTo elucidate the possible functions of OsRDCP1,a single loss-of-function rice mutant line containing a T-DNA insertion in the OsRDCP1gene was isolated and referred to as osrdcp1.The inser-tion was annotated within the intron located in the5 -untranslated region of OsRDCP1on chromosome4(line1A-175-10)(Fig.5A).The homozygous mutant line was subsequently identified by multiplex PCR(Fig.5B).RNA gel blot analysis showed that the G2rice mutant seedlings contained an undetectable amount of OsRDCP1mRNA. The osrdcp1mutant plants did not exhibit any detectable mor-phological differences as compared to the wild-type plants under normal growth conditions.This may be due to complementation by other OsRDCP paralogs.Transgenic rice plants that overexpressed OsRDCP1under the control of the CaMV35S promoter were also constructed. Markedly heightened mRNA levels of OsRDCP1were observed inH.Bae et al./Plant Science180(2011)775–782779Fig.3.Structure and expression OsRDCP1.(A)Restriction enzyme map analysis of the rice OsRDCP1gene.Solid bars indicate coding regions and the solid line indicates the intron.The gene-specific probe used for RNA gel blot analysis is indicated.The green bar and red bar indicate N-terminal RING domain and C-terminal membrane-anchoring domain,respectively.(B)Phylogenetic relationship of RING Ub-ligase homologs from rice(OsRDCP1,OsRDCP2,OsRDCP3,OsRDCP4,and OsRDCP5),Arabidopsis(AtRma1, At4g28270,and At4g27470),hot pepper(CaRma1H1),and humans(HsRma1).(C–F)RNA gel blot analyses of OsRDCP1mRNA.Light-grown2-or4-week-old rice plants were subjected to(C)drought(0–30%loss of fresh weight),(D)cold temperatures(4◦C for0–24h),(E)high salinity(150mM NaCl for0–24h),(F)SA(100␮M for0–6h),or MeJA (100␮M for0–6h).Total RNA was isolated,separated by electrophoresis,and blotted.Filters were hybridized with32P-labeled OsRDCP1,OsEXP,OsLEA3,OsPHGPX,or OsActin under high-stringency conditions.(For interpretation of the references to color in thisfigure legend,the reader is referred to the web version of the article.)different independent35S:OsRDCP1T2transgenic lines (Fig.5C).These osrdcp1loss-of-function mutant and OsRDCP1-overexpressing transgenic rice plants were used for drought tolerance analysis.3.5.Over-expression,but not knock-out,of OsRDCP1alteredplant responses to water stressSix-week-old wild-type,G2osrdcp1mutant,and T2 35S:OsRDCP1(lines#8,#9,and#10)plants were grown in pots where water covered up to approximately40%of each plant. These plants were then subjected to drought stress by withholding water until the soil in the pots was completely dry.To maintain constant experimental conditions,the drought stress experiments were carried out in a growth chamber at26◦C and70%humidity, which resulted in the gradual dehydration of the soil.These growth conditions are similar to those of domesticfields where rice plants are cultivated in May and June in South Korea.After12–15days of dehydration,both wild-type and osrtbp1mutant plants displayed wilting(Fig.6).Thus,no significant phenotypic differences were detected between the wild-type and osrtbp1plants.In contrast,the 35S:OsRDCP1transgenic lines exhibited considerably less visual symptoms of drought stress.After15days of drought stress,these transgenic lines appeared healthy and showed reduced desiccation damage relative to the wild-type and osrtbp1plants(Fig.6).After re-watering for5–15days,the wild-type and osrtbp1plants were unable to recover and died(Fig.6).Under these experimental conditions,however,OsRDCP1-overexpressing lines successively survived and continued to grow.These results indicate that the OsRDCP1-overexpressing plants were more tolerant to water deficit as compared to the wild-type and osrdcp1plants.Overall, these results suggest that OsRDCP1is involved in plant responses to unfavorable water-stress conditions.Previous studies indicated that transgenic rice plants over-expressing stress-related proteins,including Ca2+-dependent protein kinase and trehalose-metabolizing enzymes,became more tolerant to abiotic stresses[31,32].In addition,constitutive expres-sion of stress-inducible transcription factors conferred improved resistance to water stress in rice.For example,enhanced toler-ance to drought and high salinity was observed in transgenic rice plants that over-expressed CBF3/DREB1A and ABF3,Arabidop-sis transcription factors in ABA-independent and ABA-dependent stress-response pathways,respectively[33].Transgenic rice plants were developed that constitutively expressed plant-specific NAC transcription factors,resulting in increased resistance to drought and salt stresses with the concomitant induction of a large num-ber of stress-related genes[34,35].Based on these previous results, along with our current data,we considered the possibility that780H.Bae et al./Plant Science180(2011)775–782Fig.4.In vitro self-ubiquitination analysis of OsRDCP1.(A)Bacterially expressed MBP-OsRDCP1was incubated at30◦C for0–120min in the presence of E1,E2,ATP,and Ub. Reaction mixtures were subjected to immuno-blot analysis with the anti-MBP antibody.(B)MBP-OsRDCP1was incubated for60min in the presence or absence of E1,E2, ATP,and/or Ub.(C)Wild-type MBP-OsRDCP1and its various mutants were analyzed in Ub ligase assays.K23R,C54S,and C86S are mutant proteins in which the Lys23,Cys54, and Val86residues were changed to Arg,Ser,and Ser,respectively.Fig.5.Molecular characterization of the loss-of-function osrdcp1T-DNA mutant line and OsRDCP1-overexpressing transgenic rice plants.(A)Schematic diagram of OsRDCP1 with the T-DNA insertion.Solid bars and the solid line indicate exons and the intron,respectively.Integration of T-DNA in the intron is shown as a triangle.Gene-specific (M1and M2)and T-DNA-specific(LBa-1)primers used in genotyping PCRs are indicated with arrows.(B)Genotyping of the osrdcp1mutant plants.Gene-specific and T-DNA-specific primer sets used for genomic PCR are shown on the left.W,wild-type;T,T-DNA insertion.(C)RNA gel blot analysis of OsRDCP1mRNA in wild-type,the homozygous G1osrdcp1mutant line,and several independent35S:OsRDCP1T1transgenic rice plants.rRNAs are shown as loading controls.H.Bae et al./Plant Science180(2011)775–782781Fig.6.Overexpression of OsRDCP1increased tolerance to drought stress in transgenic rice plants.Six-week-old wild-type,G2osrdcp1mutant,and T235S:OsRDCP1(lines#8, #9,and#10)plants were grown in pots where water covered approximately40%of each plant.These plants were then subjected to drought stress by withholding water for 15days to dry soil completely.Thereafter,plants were re-watered and their growth and morphologies were monitored for another15days.OsRDCP1,a stress-inducible RING E3Ub ligase,may be involved in turn-over or degradation of a negative transcription factor(s)that inhibits the expression of water stress-induced genes.Constitutive expression of OsRDCP1in rice cells may quicken the ubiquitin-dependent degradation of a certain transcriptional repressor(s), thereby enhancing the cellular level of stress-responsive proteins. The elevated levels of stress-responsive proteins would enhance tolerance against water stress.Alternatively,over-expression of OsRDCP1may induce the ubiquitination and subsequent degrada-tion pathway of a protein(s)that is responsible for the inactivation or degradation of water stress-related proteins,which would also result in increased levels of defense proteins in rice cells.These hypotheses are partially supported by recent results that rice RING E3OsDSG1and U-box E3OsPUB15are involved in the regulation of salt,drought,and oxidative stress responses[13,24].Though the actual mechanism remains to be defined,our results suggest that OsRDCP1plays a critical role in rice cellular responses to water deficits.In addition to OsRDCP1,there are four homologous OsRDCP paralogs(Fig.1).We speculate that these paralogs may also play a role in cellular processes in rice.This assumption is consistent with the notion that osrdcp1knock-out mutant plants display nor-mal phenotypes possibly due to complementation by other OsRDCP members.The basal levels of mRNAs for these paralogs are quite low except for OsRDCP4transcript(Fig.2).Thus,we are making an effort to obtain knock-out mutant and overexpressing transgenic rice plants to analyze rice RING-domain containing OsRDCP E3Ub ligase members.4.Concluding remarksHigher plants are continuously faced with diverse environmen-tal stresses during their life cycle.Dehydration stress can seriously impair the growth and development of many soil plants and is often responsible for large reductions in crop yields globally[36–40]. Thus,it is of immense importance to unravel the roles of stress-inducible genes and develop stress tolerant crop plants.Although the physiological function of OsRDCP1remains to be elucidated, we propose that OsRDCP1is involved in a subset of physiological responses that counteract dehydration stress in rice plants.Thus, OsRDCP1may be a useful target protein for the development of drought-tolerant rice plants.Future experiments will focus on pre-cisely defining the cellular and physiological roles of OsRDCP1with respect to drought tolerance.AcknowledgementsThis work was supported by grants from the National Research Foundation(Project No.2009-0078317funded by the Min-istry of Education,Science,and Technology,Republic of Korea), the BioGreen21Program(funded by the Rural Development。