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Generation of chimeric repressors that confer salt tolerance in Arabidopsis and rice

Generation of chimeric repressors that confer salt tolerance in Arabidopsis and rice Introduction Plants exploit numerous strategies to adapt to changes in their environment. The development of plants with enhanced tolerance to abiotic environmental stress, such as drought, salinity and heat stress should enhance crop yield. Regulators of transcription are important in the acclimation of plants to environmental stresses, and several have been shown to confer tolerance to abiotic stress ( Hu , 2006 ; Divi and Krishna, 2009 ; Oh , 2009 ). Ethylene‐responsive element‐binding factors (ERFs) are involved in regulating responses to abiotic stress in Arabidopsis , in rice ( Oryza sativa ) and in cotton ( Gossypium hirsutum ) ( Karaba , 2007 ; Qiao , 2008 ; Wu , 2008 ). Ectopic expression of DREB1A, encodes an AP2/ERF, activates the expression of certain stress‐inducible genes in Arabidopsis , with resultant improved tolerance to drought, salt and freezing ( Kasuga , 1999 ). Basic HLH and NAC domain proteins, such as ICE1 and ANAC, respectively, act as regulatory factors that modulate the defence response ( Tran , 2004 ; Miura , 2007 ; Nakashima , 2007 ; Zheng , 2009 ). AtMYB102 is involved in integration of wounding and osmotic stress signalling ( Denekamp and Smeekens, 2003 ), and AtMYB41 , which is closely related to AtMYB102 , negatively regulates transcriptional responses to osmotic stress ( Lippold , 2009 ). The expression of AtMYB44 is induced not only by abscisic acid (ABA) but also by drought, salt and cold stress, and ectopic expression of AtMYB44 enhances drought and salt tolerance in Arabidopsis ( Jung , 2008 ). High levels of NaCl induce osmotic stress, oxidative stress and changes in nutrient uptake, and mechanisms of salt tolerance are closely related to those of tolerance to dehydration ( Seki , 2003 ). Factors that confer tolerance to NaCl, dehydration and other types of abiotic stress have been isolated ( Dubouzet , 2003 ; Verslues , 2006 ), but as signalling pathways of abiotic stress responses are complicated and overlap one another to some extent, it is difficult to isolate key regulators that confer tolerance to any given stress. We isolated novel transcription factors that are involved in various phenomena and developmental processes using our Chimeric REpressor gene Silencing Technology (CRES‐T; Hiratsu , 2003 ; Mitsuda , 2006 ). In CRES‐T, a transcription factor is converted to a strong repressor by fusion with the EAR‐motif repression domain (SRDX) and dominantly suppresses the expression of target genes, overwhelming the activation activity of corresponding endogenous and functionally redundant transcription factors to generate loss‐of‐function‐type mutant plants ( Hiratsu , 2003 ). Because some loss‐of‐function mutants have elevated tolerance to abiotic stresses ( Xin and Browse, 1998 ; Novillo , 2004 ; Wilson , 2009 ), we postulated that application of CRES‐T might allow the generation of factors that can improve tolerance to abiotic stress. The exploitation of natural genetic variations and the mapping of quantitative trait loci (QTL) have been widely used for trait modification of crops, in particular, in rice ( Leung, 2008 ; Swamy and Sarla, 2008 ). Transgenic approaches have been used in attempts to produce abiotic stress‐tolerant plants by the expression of various genes ( Gilmour , 2000 ; Garg , 2002 ; Zhang , 2005 ; Seong , 2007 ). However, it has proved difficult to identify and isolate useful factors that enhance abiotic stress tolerance without side effects, such as growth retardation and low yield. In this report, we show that five independent chimeric repressors derived from MYB, NAC, GARP, C2H2ZnF and ERF transcription factors confer tolerance to salt or osmotic stress in Arabidopsis . We also show that chimeric repressors derived from rice homologues of two of these factors confer salt tolerance on rice plants. Results Isolation of salt‐ and osmotic stress‐tolerant CRES‐T lines The coding region of the gene of each respective transcription factor was fused with cDNA for the SRDX repression domain that was short peptide modified from the EAR‐like motif repression domain of SUPERMAN ( Figure 1a , Hiratsu , 2003 ). The resultant chimeric repressor constructs ( Figure 1a ), driven by the CaMV 35S promoter, were introduced into Arabidopsis to yield CRES‐T lines. Then, T2 seeds of individual CRES‐T lines were used for the examination of tolerance to salinity and osmotic stress. 1 Tolerance to salt and osmotic stress of CRES‐T lines. (a) Schematic representation of transgene of chimeric repressor. 35S, TF, SRDX and NOS represent the CaMV 35S promoter, transcription factor, repression domain composed of 12 amino acids and NOS terminator, respectively. (b) Seedlings of wild‐type ( WT ) and of AtMYB102‐SRDX , ANAC047‐SRDX and HRS1‐SRDX plants, 2 weeks after seeding on MS plates prepared with 225 m m and 250 m m NaCl, as indicated. T2 seeds mixed from more than ten lines per each transgenic plant were used for the examination. (c) Seedlings of wild‐type ( WT ) and of ZAT6‐SRDX and AtERF5‐SRDX plants, 2 weeks after seeding on MS plates prepared with 600 m m and 650 m m mannitol, as indicated. T2 seeds mixed from more than ten lines per each transgenic plant were used for the examination. (d) Survival of seedlings, 2 weeks after seeding on MS plates prepared with 150 m m NaCl, as indicated. T3 seeds mixed with more than five lines of each T2 plant were used for the examination. (e) Survival of seedlings 2 weeks after seeding on MS plates prepared with 550 m m mannitol, as indicated. T3 seeds mixed with more than five lines of each T2 plant were used for the examination. (f) Columns show average germination rates for seeds of wild type ( WT ), AtMYB102‐SRDX , ANAC047‐SRDX and HRS1‐SRDX plants, respectively. Twenty T3 seeds in each group were subjected to each test, and examinations were replicated five times. Bars show standard deviations. (g) Columns show average survival rates for seedlings of wild‐type ( WT ), ZAT6‐SRDX and AtERF5‐SRDX plants, respectively. Twenty T3 seeds in each group were subjected to each test and examinations were replicated five times. Bars show standard deviations. Scales in (b) and (c) = 0.5 cm, and (d) and (e) = 1 cm. Significance level: ** P < 0.01 by t ‐test. Wild‐type seeds were barely able to germinate on MS plates supplemented with 225 m m NaCl, and seedlings were pale green and did not thrive. In the presence of 250 m m NaCl, almost no wild‐type seeds germinated. By contrast, T2 seeds from Arabidopsis CRES‐T lines for AtMYB102, ANAC047 and HRS1 ( AtMYB102‐SRDX , ANAC047‐SRDX and HRS1‐SRDX ) germinated, and green seedlings grew vigorously under high‐salt conditions ( Figure 1b ). These three CRES‐T lines grew well on soil, after transferring from MS plates, and developed normally without any morphological defects (data not shown). High concentrations of mannitol inhibit seed germination, and few wild‐type seeds germinated on MS plates prepared with 600 m m or higher concentrations of mannitol ( Figure 1c ). By contrast, T2 seeds from the CRES‐T ZAT6 and AtERF5 lines ( ZAT6‐SRDX and AtERF5‐SRDX ) germinated, and their cotyledons expanded in the presence of 650 m m mannitol ( Figure 1c ). The T3 generation of these CRES‐T lines tolerated salt or osmotic stresses similarly to the T2 generation, confirming that tolerance was heritable ( Figure 1d,e ). Germination or survival rates of the T3 generation of these CRES‐T lines were significantly higher than those of the wild type during treatment with salt or with mannitol, respectively ( Figure 1f,g ). The five CRES‐T lines ( AtMYB102‐SRDX , ANAC047‐SRDX , HRS1‐SRDX , ZAT6‐SRDX and AtERF5‐SRDX ) had no any growth retardation rather than developed vigorous larger rosette leaves than those of the corresponding wild‐type plants under normal condition (Figure S1). Then, we examined the salt tolerance of rosette plants of these CRES‐T lines. Four‐week‐old soil‐grown Arabidopsis CRES‐T lines were treated with 400 m m NaCl solution for 3 weeks. After 3 weeks, wild‐type plants treated with 400 m m NaCl were all dead, while all five CRES‐T lines remained viable, with the development of green rosette leaves ( Figure 2 ). Thus, transgenic plants that expressed a chimeric repressor retained stress tolerance from the seedling to the rosette stage. The CRES‐T lines for AtERF5 and ZAT6, which tolerated osmotic stress but not salt stress as seedlings, exhibited tolerance to salt stress as rosette plants. Our results suggest that mechanisms of acquisition of stress tolerance might differ depending on developmental stage of plants. Because osmotic stress is closely related to salt stress in terms of water‐stress response ( Quesada , 2000 ; Ma , 2006 ), our results suggest a link between responses to salt and osmotic stress. 2 Rosette plants of CRES‐T lines that exhibited salt tolerance. Panels show 7‐week‐old plants of wild‐type ( WT ), AtMYB102‐SRDX , ANAC047‐SRDX and HRS1‐SRDX , ZAT6‐SRDX and AtERF5‐SRDX . Photographs were taken at 3 weeks after the start of treatment with 400 m m NaCl. Untreated control and untreated‐five transgenic plants at 7 week old were presented in Figure S1. Scale = 2 cm. AtMYB102 , ANAC047 , HRS1 , ZAT6 and AtERF5 are stress‐response genes Quantitative RT‐PCR (qRT‐PCR) revealed that expression of AtMYB102 increased more than 100‐fold 6 h after the start of treatment of wild‐type plants with 200 m m NaCl and that of ANAC047 , ZAT6 and AtERF5 genes rose 2‐ to 15‐fold in response to salt stress ( Figure 3 ). By contrast, the level of expression of HRS1 fell to 10% of the pre‐treatment level at the 6‐h treatment ( Figure 3 ). 3 Relative levels of expression of AtMYB102 , ANAC047 , HRS1 , ZAT6 and AtERF5 genes in wild‐type plants in response to salt and osmotic stress. Black bars represent averages of relative levels of expression of the indicated genes, as determined by real‐time RT‐PCR, using rosette leaves of 3‐week‐old Arabidopsis plants. Expression level was determined at 2, 6, 12 and 24 h after the start of exposure to 200 m m NaCl (left part) or 300 m m mannitol (right part) on MS plates. Averages were calculated from three independent plants, and vertical lines indicate standard deviations. Values prior to treatment (0 h) were set at 1. Treatment with 300 m m mannitol induced the expression of all these genes, including HRS1 ( Figure 3 ). The level of expression of AtMYB102 rose more than 500‐fold in response to 300 m m mannitol. Levels of expression of ZAT6 and AtERF5 , whose corresponding CRES‐T lines tolerated osmotic stress, were increased by osmotic stress much more than by salt stress. Transcription factors of whose expression is induced by stress might generally increase tolerance to abiotic stress when they are converted to chimeric repressors. Expression of genes for DREB and ZAT was enhanced in AtMYB102‐SRDX plants We analysed gene expression in AtMYB102‐SRDX plants using a microarray. We compared expression profiles in three independent lines of 3‐week‐old AtMYB102‐SRDX seedlings with those in wild‐type seedlings under normal and salt‐stress conditions. Representative genes whose levels of expression were more than 25 times or <0.3 times that in the wild type (with P ‐values <0.01 by t ‐test) under stress are listed in Tables S1 and S2, respectively. The full data set discussed in this publication has been deposited in NCBI’s Gene Expression Omnibus ( Edgar , 2002 ) and is accessible through GEO Series accession number GSE19893 (). Levels of expression of DREB‐ related genes, namely, DREB1A , DREB2B , At1g22810 and At1g19210 , and of genes for zinc finger transcription factors, namely, ZAT11 , ZAT12 and ZAT7 , were elevated in AtMYB102‐SRDX plants (Table S1). These genes are involved in stress responses ( Nakashima , 2000 ; Ciftci‐Yilmaz and Mittler, 2008 ). We confirmed the enhanced expression of DREB1A , At1g22810 , ZAT11 and At1g19210 in AtMYB102‐SRDX plants by qRT‐PCR ( Figure 4a ). Expression of DREB1A , At1g22810 , and ZAT11 was enhanced not only by AtMYB102‐SRDX but also by salt stress. Synergistic induction was evident in the enhanced expression of the genes examined ( Figure 4a ). 4 Profiles of gene expression, as determined by microarray analysis of AtMYB102‐SRDX plants with and without exposure to salt stress. (a) Up‐regulated genes in AtMYB102‐SRDX plants. Grey and black columns indicate averages of levels of expression of each gene in 3‐week‐old wild‐type control ( WT ) and AtMYB102‐SRDX plants, respectively, as determined by qRT‐PCR. Levels are shown, on the left as indicated ‘untreated’ in each panel, for corresponding genes in plants incubated on regular MS plate for 24 h, Those in right part as indicated ‘salt’ represent expression levels from plants incubated on MS medium plus 200 m m NaCl for 24 h. Values for the WT without salt treatment were set at 1. (b) Down‐regulated genes in AtMYB102‐SRDX plants. See legend to Figure 4a for details. Values for the WT without salt treatment were set at 1. (c) Levels of expression of the AtMYB102 gene in wild‐type control ( WT ) and DREB1A –overexpressing transgenic plants ( DREB1A‐OX ) grown on regular MS plates. In addition, the expression of stress‐responsive genes for glutathione S‐transferase (At5g62480 and At1g78340), peroxidase (At5g06730), late embryogenesis abundant domain‐containing proteins (At2g18340 and At2g42560), dehydrin (RAB18: At5g66400), transporters and ABA‐related proteins was significantly elevated in AtMYB102‐SRDX plants, when compared with wild‐type plants under stress conditions (Table S1). The up‐regulated genes identified in this study might play critical roles in tolerance to salt stress in AtMYB102‐SRDX plants ( Dixon , 2002 ; Llorente , 2002 ; Puhakainen , 2004 ; Hong‐Bo , 2005 ). By contrast, expression of At4g10160 , BEE2 ( At4g36540 ) and At5g07690 , which encode a zinc finger protein and bHLH family proteins (the latter two genes), respectively, was suppressed in response to salt stress and was suppressed still further in AtMYB102‐SRDX plants under both normal and stress conditions ( Figure 4b , Table S2). Expression of BEE2 is suppressed in response to cold ( Friedrichsen , 2002 ), and our results suggest that At4g10160, BEE2 and At5g07690 might be involved in the negative regulation of the salt‐stress response. Both DREB1A and DREB2B play critical roles in abiotic stress responses and regulate the expression of stress‐ and defence‐responsive genes ( Knight and Knight, 2001 ; Agarwal , 2006 ). The high‐level expression of DREB1A , more than 600 times the wild‐type control level in response to salt stress, might contribute significantly to the enhanced stress tolerance of AtMYB102‐SRDX plants. To examine the relationship between AtMYB102 and DREB1A in detail, we made transgenic plants expressing 35S:DREB1A ( DREB1A ‐ OX ) and examined expression of AtMYB102 in DREB1A – OX plants. Our results showed that DREB1A slightly but clearly stimulated the expression of AtMYB102 under normal condition ( Figure 4c ). Repressive activity of HRS1 is involved in salt tolerance HRS1 includes an arrangement of amino acids similar to the EAR‐motif repression domain at its amino terminus (Figure S3). To determine whether HRS1 has repressive activity, we performed transient expression assays using a 35S‐GAL4:LUC reporter gene and an HRS1 effector fused to a GAL4 DNA‐binding domain ( 35S:GAL4DB‐HRS1 ) in Arabidopsis leaves. The HRS1 effector depressed the luciferase activity owing to the reporter gene to <25% of that of the GAL4DB control ( Figure 5a ), indicating that HRS1 had repressive activity. 5 Demonstration that HRS1 is a repressor of transcription. (a) The reporter construct and effectors used for transient expression assays are shown, with averages of relative luciferase activities after co‐bombardment of Arabidopsis leaves with each combination of reporter and effector genes. The activity of the reporter gene after bombardment with 35S:GAL4DB (GAL4‐DB) was taken as 100. Mean values were calculated from results of at least three replicates, and error bars indicate standard deviations. (b) Survival rates of the seedlings, 3 weeks after seeding on MS plates prepared with NaCl at 0, 150, 175 and 200 m m . Black, light grey, light green, orange and brown columns represent averages of survival rates for wild‐type ( WT ), DREB1A ‐ OX , HRS1‐SRDX , HRS1 ‐ OX and hrs1 plants, respectively. Twenty seeds from each group were subjected to each test, and examinations were replicated five times. Bars show standard deviations. Significance level: ** P < 0.01 by t‐test. Lower panels show seedlings of wild‐type ( WT ) and of DREB1A‐OX , HRS1‐SRDX , HRS1‐OX and hrs1 plants, 2 weeks after seeding on MS plates with 200 m m . Scale in (b) = 0.5 cm. Because HRS1 probably acts as a transcriptional repressor, plants in which it is overexpressed ( HRS1 – OX plants) and the corresponding chimeric repressor plants ( HRS1‐SRDX ) should have similar phenotypes, which should be the opposite to that of hrs1 loss‐of‐function mutants. To determine whether HRS1 acts as a transcriptional repressor in plants, we sowed seeds from HRS1 ‐ OX , HRS1‐SRDX and hrs1 mutant plants on MS plates supplemented with 150, 175 and 200 m m NaCl, using wild‐type and DREB1A ‐overexpressing plants ( DREB1A ‐ OX ) as controls for salt tolerance ( Kasuga , 1999 ). Survival rates under salt stress showed clearly that both HRS1‐SRDX and HRS1 ‐ OX plants tolerated salt stress, while hrs1 mutants had similar tolerance to salt stress to that of wild‐type plants ( Figure 5b ). Thus, HRS1 is probably a transcriptional repressor. Furthermore, HRS1‐SRDX plants had higher salt tolerance than HRS1 ‐ OX plants, suggesting that enhanced repressive activity of HRS1‐SRDX might confer higher tolerance to salt stress. DREB1A ‐ OX plants, which we used as a positive control, exhibited only modest tolerance to salt stress which was much lower than that of HRS1‐SRDX plants ( Figure 5b ). Thus, HRS1 appears to be a transcriptional repressor that might regulate responses to salt stress. The chimeric repressors derived from Os02g0325600 and Os03g0327800 improved salt tolerance in rice To determine whether chimeric repressors that conferred salt tolerance on Arabidopsis might have similar effects in rice, we isolated Os02g0325600 ( AK101809 ) and Os03g0327800 ( AK243514 ), which are rice paralogs of HRS1 and ANAC047 (Figure S2), respectively, and transformed rice with the corresponding chimeric gene constructs driven by the promoter of the rice actin1 ( ACT1 ) gene ( Os02g0325600‐SRDX and Os03g0327800‐SRDX ). We evaluated salt tolerance of transgenic rice plants by comparing growth ratios of regenerated roots and shoots with that of wild‐type rice in the presence of NaCl. We trimmed roots and shoots of 1‐week‐old rice seedlings grown on regular MS plates to exclude individual growth variability for comparative analyses. Thereafter, the largely trimmed seedlings were cultured on MS plates containing 200 m m NaCl for 4 weeks. The high concentration of NaCl inhibited regeneration and the development of roots and shoots of wild‐type seedlings ( Figure 6a ). By contrast, samples from Os02g0325600‐SRDX and Os03g0327800‐SRDX transgenic rice seedlings developed elongated roots and green leaves that were much longer and heavier than those of wild‐type plants, revealing tolerance to salt stress ( Figure 6a,b ). In addition, much lower levels of electrolyte leakage were examined in the shoot samples from Os02g0325600‐SRDX and Os03g0327800‐SRDX transgenic rice than those of wild‐type controls. ( Figure 6b ). The results suggest that remarkable reduction in membrane ion leakages may relate to enhancement of salt‐stress tolerance. 6 Salt tolerance of pAct:Os02g0325600‐SRDX and pAct:Os03g0325600‐SRDX transgenic rice. (a) Untransformed Oryza plants ( WT ) and chimeric repressor transgenic lines ( pACT:Os02g0325600‐SRDX and pAct:Os03g0325600‐SRDX ) after growth in MS plates prepared with 150 m m NaCl for 4 weeks after trimming. Refer to Experimental Procedure in details. (b) Shoot lengths, root lengths, fresh weights, dry weighs and ion leakage of wild‐type rice, and pAct:Os02g0325600‐SRDX and pAct:Os03g0325600‐SRDX transgenic rice plants 4 weeks after the removal of shoot and roots and replanting rice seedlings on MS plates prepared with NaCl of 0, 150 and 200 m m . Six regenerated seedlings in each treatment group were used for measurement. An average of ion‐leakage levels in each group was calculated from six samples and expressed as the percentage of the initial conductivity versus total conductivity. Bars show standard deviations. Scales in (a) = 5 cm. Significance level: * P < 0.05; and ** P < 0.01 by t ‐test. Wild‐type rice died within 1 month after the start of salt treatment, while Os02g0325600‐SRDX and Os03g0327800‐SRDX transgenic rice continued to grow and could bear seeds after they had been transferred to soil. Similar salt‐stress tolerance was confirmed in the transgenic rice that were grown on soil without any trimmings, treated with 150 m m NaCl solution for 10 days (Figure S4). Thus, the chimeric repressors derived from Os02g0325600 and Os03g0327800 conferred tolerance to salt stress on rice as did the chimeric HRS1 and ANAC047 repressors in Arabidopsis . Os02g0325600 has a region similar to the EAR‐like motif located near its amino terminus as does HRS1 (Figure S3). We confirmed the repression activity of Os02g0325600 in a transient expression assay ( Figure 5a ). Os02g0325600 is probably a transcriptional repressor that regulates the response to salt stress. Discussion We have engineered rice and Arabidopsis plants that tolerate abiotic stress using CRES‐T. The chimeric repressors derived from AtMYB102, ANAC047 and HRS1 induced tolerance to salt stress, and those derived from ZAT6 and AtERF5 conferred tolerance to osmotic stress in Arabidopsis seedlings ( Figure 1b–g ). Furthermore, rosette plants of these five CRES‐T lines were tolerant to treatment with 400 m m NaCl ( Figure 2 ). Our results indicate that mechanisms of tolerance to salt and osmotic stress overlap, perhaps at the level of water‐use efficiency. Tolerance of our CRES‐T lines was maintained from germination through the rosette stage and was heritable. The five transcription factors whose chimeric derivatives conferred tolerance to salt stress on Arabidopsis are encoded by salt‐ and osmotic stress‐responsive genes. Ectopic expression of our chimeric repressor possibly overcomes the induction of expression of the endogenous corresponding transcription factor in response to stresses. It could be interesting to address intrinsic functions of the transcription factor themselves and examine whether they act as negative regulators in stress responses by means of further studies. AtMYB102 , ZAT6 and AtERF5 are involved in stress responses ( Fujimoto , 2000 ; Denekamp and Smeekens, 2003 ; Devaiah , 2007 ). ANAC047 belongs to group III in the NAC family, which includes the stress‐related genes ATAF1 , NAP and RD26 ( Fujita , 2004 ; Lu , 2007 ). It is likely that ANAC047 is involved in stress responses as are other members of group III. The HRS1 gene whose expression was depressed by salt treatment is a transcriptional repressor ( Figure 5a,b ). Thus, HRS1 might act as a repressor of negative regulators that suppress stress responses. In addition, ZAT6 probably acts as a repressor because it includes an EAR‐motif repression domain in its carboxy‐terminal region. The repressive activity of native repressors is enhanced by fusion with SRDX ( Matsui , 2008 ), and the potentiated repressive activity might induce tolerance to distinct stresses in plants. Our results strongly suggest that negative regulators might participate in the acquisition of stress tolerance in Arabidopsis and rice. Expression of stress‐protective genes seemed to be induced upon suppression of a negative regulator by the activity of a chimeric repressor. It is reasonable to postulate that negative regulators act as fine tuners to suppress the bursts of expression of stress‐response genes because overexpression of such genes might have adverse effects on plant development ( Gilmour , 2000 ; Garg , 2002 ; Zhang , 2005 ; Xiong and Fei, 2006 ; Seong , 2007 ). Our microarray analysis revealed that the expression of DREB and related genes was dramatically enhanced in AtMYB102‐SRDX plants under salt‐stress condition, which might lead to the accumulation of stress tolerance‐related proteins. Looped regulation between AtMYB102 and DREB1A may contribute remarkable induction of the expression of DREB and related genes. The chimeric AtMYB102 repressor might suppress the expression of factors that negatively regulate the expression of DREB genes. Possible candidates for the negative regulatory genes are At4g10160, BEE2 and At5g07690 , whose expression was markedly depressed by salt stress in AtMYB102‐SRDX plants. In addition, the expression of ZAT11 , ZAT12 and ZAT7 was markedly elevated in AtMYB102‐SRDX plants under normal and salt‐stress conditions. Several zinc finger proteins, including ZAT11, ZAT12, ZAT7 and ZAT6, play key roles in regulating the defence responses of Arabidopsis ( Iuchi , 2007 ; Ciftci‐Yilmaz and Mittler, 2008 ). ZAT11, ZAT12, ZAT7 and ZAT6 include an EAR‐motif repression domain and repressive activity (in transient expression assays; data not shown), and the EAR‐motif repression domain of ZAT7 has been shown to confer stress tolerance ( Nakashima , 2000 ). It will be interesting to examine whether the EAR‐like motif of HRS1 and that of Os02g0325600 are essential for their effects on stress tolerance in Arabidopsis and rice, respectively. Salinity and osmotic stress cause drastic changes in ion and water homeostasis, and both types of stress are linked to defects in water utilization in plant cells. The results of ion‐leakage examination clearly showed that salt stress induced a vast leakage of electrolytes ( Figure 6b ). Many factors are involved in responses to abiotic stresses, such as chilling, freezing, drought and heat ( Xiong , 2002 ; Shinozaki , 2003 ; Kotak , 2007 ). Manipulation of these factors can successfully increase multistress tolerance in plants ( Karaba , 2007 ; Jung , 2008 ; Wu , 2008 ). We found a relationship between salt stress and osmotic stress in our analysis of ZAT6 and AtERF5 . Neither ZAT6‐SRDX nor AtERF5‐SRDX exhibited salt tolerance during germination, but rosette plants exhibited strong tolerance to salinity ( Figures 1 and 2 ). It is possible that these chimeric repressors might confer tolerance to other kinds of abiotic stress. Genetic manipulation has been widely utilized to modify the ability of crop plants to withstand salt stress ( Liu , 1998 ; Yamaguchi and Blumwald, 2005 ; James , 2008 ; Gao , 2009 ; Oh , 2009 ), and information obtained from Arabidopsis can be used to design functional crops because regulators of stress‐signal pathways are conserved to a significant extent in other plant species ( Mitsuda , 2006 ). Our results show that application of the Arabidopsis CRES‐T screening system to rice is quite effective, and rice is not only a model monocot but also an important crop. Our findings in Arabidopsis and rice may be applicable to other crops, such as maize, sugarcane and other gramineous plants. Manipulation of a transcription factor that is involved in adaptation to environmental conditions is an effective method for improving stress tolerance in plants ( Bhatnagar‐Mathur , 2008 ). Many efforts have been made to produce salt tolerant rice by overexpression of transcription factors ( Hu , 2006 ; Karaba , 2007 ; Oh , 2009 ). We have shown here that chimeric repressors derived from the GARP transcription factor Os02g0325600 and from the NAC family protein Os03g0327800 effectively induced salt tolerance in rice ( Figure 6a,b ). Our CRES‐T screening system in Arabidopsis appears to be an effective tool for the development of stress‐tolerant crops. Experimental procedures Preparation of constructs Protein‐coding regions of AtMYB102, ANAC047, At1G13300, ZAT6 and AtERF5 were amplified from an Arabidopsis cDNA library with appropriate primers and cloned into the binary vector pBCKH, as described previously ( Mitsuda , 2005 ). The cDNA clones AK101809 and AK243514 provided by the Rice Genome Resource Center (RGRC; Tsukuba, Japan), were used to prepare pAct:Os02g0325600‐SRDX and pAct:Os03g0327800‐SRDX . The coding regions of Os02g0325600 and Os03g0327800 was inserted into pAct:SRDXG that contained the promoter region of the rice ACT1 gene and transferred into the pBCKH vector ( Mitsuda , 2006 ). Reporter and effector constructs for 35S‐5xGAL4:LUC , 35S:GAL4DB and 35S:GAL4DB‐SRDX were described previously ( Hiratsu , 2002 ). The full‐length At1g13300 and Os02g0325600 constructs were prepared by PCR with appropriate primers (Table S3) and inserted into the effector plasmid ( Hiratsu , 2002 ). Primer pairs that used for the study were listed in Table S3. Plant growth and transformation Transformation of Arabidopsis ecotype Colombia (Col‐0) was performed as reported previously ( Matsui , 2004 ). Arabidopsis plants were grown on agar‐solidified Murashige–Skoog (MS) medium with 0.5% sucrose (MS plates), with or without NaCl or mannitol, and grown in soil under long‐day conditions (16 h of light and 8 h of darkness daily) at 22 °C. The pAct:Os02g0325600‐SRDX and pAct:Os03g0327800‐SRDX constructs were introduced separately into wild‐type rice cv. Nipponbare, as described previously ( Mitsuda , 2006 ). Rice was grown on MS plates and in soil with 14 h of light at 30 ± 2 °C and 10 h of darkness at 20 ± 2 °C daily. Stress tolerant assay Sterilized Arabidopsis seeds were plated on solid medium composed MS salts and different concentration of NaCl or mannitol. After 2 weeks of incubation, the percentage of seedlings that had germinated and developed green expanded cotyledons was determined. For assays in rosette plants, 4‐week‐old wild‐type and CRES‐T plants, which had been grown on soil, were treated with 400 m m NaCl solutions for 3 weeks. Phenotype of the 7‐week‐old plants was evaluated for comparison. For salt tolerance evaluation in rice, T2 plants of CRES‐T transgenic lines and wild‐type rice cv. Nipponbare were used. Roots and shoots of 1‐week‐old rice seedlings grown on regular MS plates were cut at 1 cm down and up, respectively, from the base position of the plants. The largely trimmed seedlings were cultured on MS plates containing 150 or 200 m m NaCl for 4 weeks. Thereafter, length of shoots and roots was measured, fresh weights of whole plants were taken, the samples were dried in an oven at 80 °C for 48 h, and dry weights were recorded. Ion‐leakage test was carried out according to the methods previously reported ( Yan , 2007 ), using 0.1 g shoot samples taken from wild‐type and transgenic rice seedling that had been trimmed and cultured with and without salt treatment as described previously. Analysis of RNA Preparation of total RNA and quantitative RT‐PCR (qRT‐PCR) were carried out as described previously ( Mitsuda , 2005 ), and the gene‐specific primers for qRT‐PCR are shown in Table S3. Relative amounts of transcripts were calculated by an absolute quantification method, with the UBQ1 gene as an internal control. At least three replicates from three independent lines were included in each experiment. Microarray analysis Three‐week‐old wild‐type and AtMYB102SRDX plants were transferred to regular MS medium and MS containing 200 m m NaCl, respectively, and incubated for 24 h. Total RNA was prepared from rosette leaves of three independent T2 lines of AtMYB102SRDX plants and wild‐type plants as described previously ( Mitsuda , 2005 ). For microarray analysis, preparation of Cy5‐ and Cy3‐labelled cDNA probes, hybridizations were performed as described previously ( Mitsuda , 2005 ). The data were analysed based on the criteria of a P ‐value cut‐off of 0.05, and a fold change cut‐off value was selected as more than 25 or <0.3. The full data set have been deposited in NCBI’s Gene Expression Omnibus ( Edgar , 2002 ) and are accessible through GEO Series accession number GSE19893 (). Transient expression assays Details of transient expression assays, after particle bombardment of Arabidopsis leaves, were described previously ( Ohta , 2001 ; Hiratsu , 2002 ). Acknowledgements The authors thank the Rice Genome Resource Center (RGRC) for providing clones, Ms. Junko Ishida for performing the microarray experiments, Ms Machiko Oonuki for skilled technical assistance and Mr Kazuhito Sato in GS and all members in Takagi‐Group in Advanced Institute Science and Technology (AIST) for helpful supports. This research was supported by the Research and Development Program for New Bio‐industry Initiatives at Bio‐oriented Technology Research Advancement Institutions. References Agarwal , P.K. , Agarwal , P. , Reddy , M.K. and Sopory , S.K. ( 2006 ) Role of DREB transcription factors in abiotic and biotic stress tolerance in plants . Plant Cell Rep. 25 , 1263 – 1274 . Bhatnagar‐Mathur , P. , Vadez , V. and Sharma , K.K. ( 2008 ) Transgenic approaches for abiotic stress tolerance in plants: retrospect and prospects . Plant Cell Rep. 27 , 411 – 424 . Ciftci‐Yilmaz , S. and Mittler , R. ( 2008 ) The zinc finger network of plants . Cell. Mol. 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Generation of chimeric repressors that confer salt tolerance in Arabidopsis and rice

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Wiley
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1467-7652
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10.1111/j.1467-7652.2010.00578.x
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21114612
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Abstract

Introduction Plants exploit numerous strategies to adapt to changes in their environment. The development of plants with enhanced tolerance to abiotic environmental stress, such as drought, salinity and heat stress should enhance crop yield. Regulators of transcription are important in the acclimation of plants to environmental stresses, and several have been shown to confer tolerance to abiotic stress ( Hu , 2006 ; Divi and Krishna, 2009 ; Oh , 2009 ). Ethylene‐responsive element‐binding factors (ERFs) are involved in regulating responses to abiotic stress in Arabidopsis , in rice ( Oryza sativa ) and in cotton ( Gossypium hirsutum ) ( Karaba , 2007 ; Qiao , 2008 ; Wu , 2008 ). Ectopic expression of DREB1A, encodes an AP2/ERF, activates the expression of certain stress‐inducible genes in Arabidopsis , with resultant improved tolerance to drought, salt and freezing ( Kasuga , 1999 ). Basic HLH and NAC domain proteins, such as ICE1 and ANAC, respectively, act as regulatory factors that modulate the defence response ( Tran , 2004 ; Miura , 2007 ; Nakashima , 2007 ; Zheng , 2009 ). AtMYB102 is involved in integration of wounding and osmotic stress signalling ( Denekamp and Smeekens, 2003 ), and AtMYB41 , which is closely related to AtMYB102 , negatively regulates transcriptional responses to osmotic stress ( Lippold , 2009 ). The expression of AtMYB44 is induced not only by abscisic acid (ABA) but also by drought, salt and cold stress, and ectopic expression of AtMYB44 enhances drought and salt tolerance in Arabidopsis ( Jung , 2008 ). High levels of NaCl induce osmotic stress, oxidative stress and changes in nutrient uptake, and mechanisms of salt tolerance are closely related to those of tolerance to dehydration ( Seki , 2003 ). Factors that confer tolerance to NaCl, dehydration and other types of abiotic stress have been isolated ( Dubouzet , 2003 ; Verslues , 2006 ), but as signalling pathways of abiotic stress responses are complicated and overlap one another to some extent, it is difficult to isolate key regulators that confer tolerance to any given stress. We isolated novel transcription factors that are involved in various phenomena and developmental processes using our Chimeric REpressor gene Silencing Technology (CRES‐T; Hiratsu , 2003 ; Mitsuda , 2006 ). In CRES‐T, a transcription factor is converted to a strong repressor by fusion with the EAR‐motif repression domain (SRDX) and dominantly suppresses the expression of target genes, overwhelming the activation activity of corresponding endogenous and functionally redundant transcription factors to generate loss‐of‐function‐type mutant plants ( Hiratsu , 2003 ). Because some loss‐of‐function mutants have elevated tolerance to abiotic stresses ( Xin and Browse, 1998 ; Novillo , 2004 ; Wilson , 2009 ), we postulated that application of CRES‐T might allow the generation of factors that can improve tolerance to abiotic stress. The exploitation of natural genetic variations and the mapping of quantitative trait loci (QTL) have been widely used for trait modification of crops, in particular, in rice ( Leung, 2008 ; Swamy and Sarla, 2008 ). Transgenic approaches have been used in attempts to produce abiotic stress‐tolerant plants by the expression of various genes ( Gilmour , 2000 ; Garg , 2002 ; Zhang , 2005 ; Seong , 2007 ). However, it has proved difficult to identify and isolate useful factors that enhance abiotic stress tolerance without side effects, such as growth retardation and low yield. In this report, we show that five independent chimeric repressors derived from MYB, NAC, GARP, C2H2ZnF and ERF transcription factors confer tolerance to salt or osmotic stress in Arabidopsis . We also show that chimeric repressors derived from rice homologues of two of these factors confer salt tolerance on rice plants. Results Isolation of salt‐ and osmotic stress‐tolerant CRES‐T lines The coding region of the gene of each respective transcription factor was fused with cDNA for the SRDX repression domain that was short peptide modified from the EAR‐like motif repression domain of SUPERMAN ( Figure 1a , Hiratsu , 2003 ). The resultant chimeric repressor constructs ( Figure 1a ), driven by the CaMV 35S promoter, were introduced into Arabidopsis to yield CRES‐T lines. Then, T2 seeds of individual CRES‐T lines were used for the examination of tolerance to salinity and osmotic stress. 1 Tolerance to salt and osmotic stress of CRES‐T lines. (a) Schematic representation of transgene of chimeric repressor. 35S, TF, SRDX and NOS represent the CaMV 35S promoter, transcription factor, repression domain composed of 12 amino acids and NOS terminator, respectively. (b) Seedlings of wild‐type ( WT ) and of AtMYB102‐SRDX , ANAC047‐SRDX and HRS1‐SRDX plants, 2 weeks after seeding on MS plates prepared with 225 m m and 250 m m NaCl, as indicated. T2 seeds mixed from more than ten lines per each transgenic plant were used for the examination. (c) Seedlings of wild‐type ( WT ) and of ZAT6‐SRDX and AtERF5‐SRDX plants, 2 weeks after seeding on MS plates prepared with 600 m m and 650 m m mannitol, as indicated. T2 seeds mixed from more than ten lines per each transgenic plant were used for the examination. (d) Survival of seedlings, 2 weeks after seeding on MS plates prepared with 150 m m NaCl, as indicated. T3 seeds mixed with more than five lines of each T2 plant were used for the examination. (e) Survival of seedlings 2 weeks after seeding on MS plates prepared with 550 m m mannitol, as indicated. T3 seeds mixed with more than five lines of each T2 plant were used for the examination. (f) Columns show average germination rates for seeds of wild type ( WT ), AtMYB102‐SRDX , ANAC047‐SRDX and HRS1‐SRDX plants, respectively. Twenty T3 seeds in each group were subjected to each test, and examinations were replicated five times. Bars show standard deviations. (g) Columns show average survival rates for seedlings of wild‐type ( WT ), ZAT6‐SRDX and AtERF5‐SRDX plants, respectively. Twenty T3 seeds in each group were subjected to each test and examinations were replicated five times. Bars show standard deviations. Scales in (b) and (c) = 0.5 cm, and (d) and (e) = 1 cm. Significance level: ** P < 0.01 by t ‐test. Wild‐type seeds were barely able to germinate on MS plates supplemented with 225 m m NaCl, and seedlings were pale green and did not thrive. In the presence of 250 m m NaCl, almost no wild‐type seeds germinated. By contrast, T2 seeds from Arabidopsis CRES‐T lines for AtMYB102, ANAC047 and HRS1 ( AtMYB102‐SRDX , ANAC047‐SRDX and HRS1‐SRDX ) germinated, and green seedlings grew vigorously under high‐salt conditions ( Figure 1b ). These three CRES‐T lines grew well on soil, after transferring from MS plates, and developed normally without any morphological defects (data not shown). High concentrations of mannitol inhibit seed germination, and few wild‐type seeds germinated on MS plates prepared with 600 m m or higher concentrations of mannitol ( Figure 1c ). By contrast, T2 seeds from the CRES‐T ZAT6 and AtERF5 lines ( ZAT6‐SRDX and AtERF5‐SRDX ) germinated, and their cotyledons expanded in the presence of 650 m m mannitol ( Figure 1c ). The T3 generation of these CRES‐T lines tolerated salt or osmotic stresses similarly to the T2 generation, confirming that tolerance was heritable ( Figure 1d,e ). Germination or survival rates of the T3 generation of these CRES‐T lines were significantly higher than those of the wild type during treatment with salt or with mannitol, respectively ( Figure 1f,g ). The five CRES‐T lines ( AtMYB102‐SRDX , ANAC047‐SRDX , HRS1‐SRDX , ZAT6‐SRDX and AtERF5‐SRDX ) had no any growth retardation rather than developed vigorous larger rosette leaves than those of the corresponding wild‐type plants under normal condition (Figure S1). Then, we examined the salt tolerance of rosette plants of these CRES‐T lines. Four‐week‐old soil‐grown Arabidopsis CRES‐T lines were treated with 400 m m NaCl solution for 3 weeks. After 3 weeks, wild‐type plants treated with 400 m m NaCl were all dead, while all five CRES‐T lines remained viable, with the development of green rosette leaves ( Figure 2 ). Thus, transgenic plants that expressed a chimeric repressor retained stress tolerance from the seedling to the rosette stage. The CRES‐T lines for AtERF5 and ZAT6, which tolerated osmotic stress but not salt stress as seedlings, exhibited tolerance to salt stress as rosette plants. Our results suggest that mechanisms of acquisition of stress tolerance might differ depending on developmental stage of plants. Because osmotic stress is closely related to salt stress in terms of water‐stress response ( Quesada , 2000 ; Ma , 2006 ), our results suggest a link between responses to salt and osmotic stress. 2 Rosette plants of CRES‐T lines that exhibited salt tolerance. Panels show 7‐week‐old plants of wild‐type ( WT ), AtMYB102‐SRDX , ANAC047‐SRDX and HRS1‐SRDX , ZAT6‐SRDX and AtERF5‐SRDX . Photographs were taken at 3 weeks after the start of treatment with 400 m m NaCl. Untreated control and untreated‐five transgenic plants at 7 week old were presented in Figure S1. Scale = 2 cm. AtMYB102 , ANAC047 , HRS1 , ZAT6 and AtERF5 are stress‐response genes Quantitative RT‐PCR (qRT‐PCR) revealed that expression of AtMYB102 increased more than 100‐fold 6 h after the start of treatment of wild‐type plants with 200 m m NaCl and that of ANAC047 , ZAT6 and AtERF5 genes rose 2‐ to 15‐fold in response to salt stress ( Figure 3 ). By contrast, the level of expression of HRS1 fell to 10% of the pre‐treatment level at the 6‐h treatment ( Figure 3 ). 3 Relative levels of expression of AtMYB102 , ANAC047 , HRS1 , ZAT6 and AtERF5 genes in wild‐type plants in response to salt and osmotic stress. Black bars represent averages of relative levels of expression of the indicated genes, as determined by real‐time RT‐PCR, using rosette leaves of 3‐week‐old Arabidopsis plants. Expression level was determined at 2, 6, 12 and 24 h after the start of exposure to 200 m m NaCl (left part) or 300 m m mannitol (right part) on MS plates. Averages were calculated from three independent plants, and vertical lines indicate standard deviations. Values prior to treatment (0 h) were set at 1. Treatment with 300 m m mannitol induced the expression of all these genes, including HRS1 ( Figure 3 ). The level of expression of AtMYB102 rose more than 500‐fold in response to 300 m m mannitol. Levels of expression of ZAT6 and AtERF5 , whose corresponding CRES‐T lines tolerated osmotic stress, were increased by osmotic stress much more than by salt stress. Transcription factors of whose expression is induced by stress might generally increase tolerance to abiotic stress when they are converted to chimeric repressors. Expression of genes for DREB and ZAT was enhanced in AtMYB102‐SRDX plants We analysed gene expression in AtMYB102‐SRDX plants using a microarray. We compared expression profiles in three independent lines of 3‐week‐old AtMYB102‐SRDX seedlings with those in wild‐type seedlings under normal and salt‐stress conditions. Representative genes whose levels of expression were more than 25 times or <0.3 times that in the wild type (with P ‐values <0.01 by t ‐test) under stress are listed in Tables S1 and S2, respectively. The full data set discussed in this publication has been deposited in NCBI’s Gene Expression Omnibus ( Edgar , 2002 ) and is accessible through GEO Series accession number GSE19893 (). Levels of expression of DREB‐ related genes, namely, DREB1A , DREB2B , At1g22810 and At1g19210 , and of genes for zinc finger transcription factors, namely, ZAT11 , ZAT12 and ZAT7 , were elevated in AtMYB102‐SRDX plants (Table S1). These genes are involved in stress responses ( Nakashima , 2000 ; Ciftci‐Yilmaz and Mittler, 2008 ). We confirmed the enhanced expression of DREB1A , At1g22810 , ZAT11 and At1g19210 in AtMYB102‐SRDX plants by qRT‐PCR ( Figure 4a ). Expression of DREB1A , At1g22810 , and ZAT11 was enhanced not only by AtMYB102‐SRDX but also by salt stress. Synergistic induction was evident in the enhanced expression of the genes examined ( Figure 4a ). 4 Profiles of gene expression, as determined by microarray analysis of AtMYB102‐SRDX plants with and without exposure to salt stress. (a) Up‐regulated genes in AtMYB102‐SRDX plants. Grey and black columns indicate averages of levels of expression of each gene in 3‐week‐old wild‐type control ( WT ) and AtMYB102‐SRDX plants, respectively, as determined by qRT‐PCR. Levels are shown, on the left as indicated ‘untreated’ in each panel, for corresponding genes in plants incubated on regular MS plate for 24 h, Those in right part as indicated ‘salt’ represent expression levels from plants incubated on MS medium plus 200 m m NaCl for 24 h. Values for the WT without salt treatment were set at 1. (b) Down‐regulated genes in AtMYB102‐SRDX plants. See legend to Figure 4a for details. Values for the WT without salt treatment were set at 1. (c) Levels of expression of the AtMYB102 gene in wild‐type control ( WT ) and DREB1A –overexpressing transgenic plants ( DREB1A‐OX ) grown on regular MS plates. In addition, the expression of stress‐responsive genes for glutathione S‐transferase (At5g62480 and At1g78340), peroxidase (At5g06730), late embryogenesis abundant domain‐containing proteins (At2g18340 and At2g42560), dehydrin (RAB18: At5g66400), transporters and ABA‐related proteins was significantly elevated in AtMYB102‐SRDX plants, when compared with wild‐type plants under stress conditions (Table S1). The up‐regulated genes identified in this study might play critical roles in tolerance to salt stress in AtMYB102‐SRDX plants ( Dixon , 2002 ; Llorente , 2002 ; Puhakainen , 2004 ; Hong‐Bo , 2005 ). By contrast, expression of At4g10160 , BEE2 ( At4g36540 ) and At5g07690 , which encode a zinc finger protein and bHLH family proteins (the latter two genes), respectively, was suppressed in response to salt stress and was suppressed still further in AtMYB102‐SRDX plants under both normal and stress conditions ( Figure 4b , Table S2). Expression of BEE2 is suppressed in response to cold ( Friedrichsen , 2002 ), and our results suggest that At4g10160, BEE2 and At5g07690 might be involved in the negative regulation of the salt‐stress response. Both DREB1A and DREB2B play critical roles in abiotic stress responses and regulate the expression of stress‐ and defence‐responsive genes ( Knight and Knight, 2001 ; Agarwal , 2006 ). The high‐level expression of DREB1A , more than 600 times the wild‐type control level in response to salt stress, might contribute significantly to the enhanced stress tolerance of AtMYB102‐SRDX plants. To examine the relationship between AtMYB102 and DREB1A in detail, we made transgenic plants expressing 35S:DREB1A ( DREB1A ‐ OX ) and examined expression of AtMYB102 in DREB1A – OX plants. Our results showed that DREB1A slightly but clearly stimulated the expression of AtMYB102 under normal condition ( Figure 4c ). Repressive activity of HRS1 is involved in salt tolerance HRS1 includes an arrangement of amino acids similar to the EAR‐motif repression domain at its amino terminus (Figure S3). To determine whether HRS1 has repressive activity, we performed transient expression assays using a 35S‐GAL4:LUC reporter gene and an HRS1 effector fused to a GAL4 DNA‐binding domain ( 35S:GAL4DB‐HRS1 ) in Arabidopsis leaves. The HRS1 effector depressed the luciferase activity owing to the reporter gene to <25% of that of the GAL4DB control ( Figure 5a ), indicating that HRS1 had repressive activity. 5 Demonstration that HRS1 is a repressor of transcription. (a) The reporter construct and effectors used for transient expression assays are shown, with averages of relative luciferase activities after co‐bombardment of Arabidopsis leaves with each combination of reporter and effector genes. The activity of the reporter gene after bombardment with 35S:GAL4DB (GAL4‐DB) was taken as 100. Mean values were calculated from results of at least three replicates, and error bars indicate standard deviations. (b) Survival rates of the seedlings, 3 weeks after seeding on MS plates prepared with NaCl at 0, 150, 175 and 200 m m . Black, light grey, light green, orange and brown columns represent averages of survival rates for wild‐type ( WT ), DREB1A ‐ OX , HRS1‐SRDX , HRS1 ‐ OX and hrs1 plants, respectively. Twenty seeds from each group were subjected to each test, and examinations were replicated five times. Bars show standard deviations. Significance level: ** P < 0.01 by t‐test. Lower panels show seedlings of wild‐type ( WT ) and of DREB1A‐OX , HRS1‐SRDX , HRS1‐OX and hrs1 plants, 2 weeks after seeding on MS plates with 200 m m . Scale in (b) = 0.5 cm. Because HRS1 probably acts as a transcriptional repressor, plants in which it is overexpressed ( HRS1 – OX plants) and the corresponding chimeric repressor plants ( HRS1‐SRDX ) should have similar phenotypes, which should be the opposite to that of hrs1 loss‐of‐function mutants. To determine whether HRS1 acts as a transcriptional repressor in plants, we sowed seeds from HRS1 ‐ OX , HRS1‐SRDX and hrs1 mutant plants on MS plates supplemented with 150, 175 and 200 m m NaCl, using wild‐type and DREB1A ‐overexpressing plants ( DREB1A ‐ OX ) as controls for salt tolerance ( Kasuga , 1999 ). Survival rates under salt stress showed clearly that both HRS1‐SRDX and HRS1 ‐ OX plants tolerated salt stress, while hrs1 mutants had similar tolerance to salt stress to that of wild‐type plants ( Figure 5b ). Thus, HRS1 is probably a transcriptional repressor. Furthermore, HRS1‐SRDX plants had higher salt tolerance than HRS1 ‐ OX plants, suggesting that enhanced repressive activity of HRS1‐SRDX might confer higher tolerance to salt stress. DREB1A ‐ OX plants, which we used as a positive control, exhibited only modest tolerance to salt stress which was much lower than that of HRS1‐SRDX plants ( Figure 5b ). Thus, HRS1 appears to be a transcriptional repressor that might regulate responses to salt stress. The chimeric repressors derived from Os02g0325600 and Os03g0327800 improved salt tolerance in rice To determine whether chimeric repressors that conferred salt tolerance on Arabidopsis might have similar effects in rice, we isolated Os02g0325600 ( AK101809 ) and Os03g0327800 ( AK243514 ), which are rice paralogs of HRS1 and ANAC047 (Figure S2), respectively, and transformed rice with the corresponding chimeric gene constructs driven by the promoter of the rice actin1 ( ACT1 ) gene ( Os02g0325600‐SRDX and Os03g0327800‐SRDX ). We evaluated salt tolerance of transgenic rice plants by comparing growth ratios of regenerated roots and shoots with that of wild‐type rice in the presence of NaCl. We trimmed roots and shoots of 1‐week‐old rice seedlings grown on regular MS plates to exclude individual growth variability for comparative analyses. Thereafter, the largely trimmed seedlings were cultured on MS plates containing 200 m m NaCl for 4 weeks. The high concentration of NaCl inhibited regeneration and the development of roots and shoots of wild‐type seedlings ( Figure 6a ). By contrast, samples from Os02g0325600‐SRDX and Os03g0327800‐SRDX transgenic rice seedlings developed elongated roots and green leaves that were much longer and heavier than those of wild‐type plants, revealing tolerance to salt stress ( Figure 6a,b ). In addition, much lower levels of electrolyte leakage were examined in the shoot samples from Os02g0325600‐SRDX and Os03g0327800‐SRDX transgenic rice than those of wild‐type controls. ( Figure 6b ). The results suggest that remarkable reduction in membrane ion leakages may relate to enhancement of salt‐stress tolerance. 6 Salt tolerance of pAct:Os02g0325600‐SRDX and pAct:Os03g0325600‐SRDX transgenic rice. (a) Untransformed Oryza plants ( WT ) and chimeric repressor transgenic lines ( pACT:Os02g0325600‐SRDX and pAct:Os03g0325600‐SRDX ) after growth in MS plates prepared with 150 m m NaCl for 4 weeks after trimming. Refer to Experimental Procedure in details. (b) Shoot lengths, root lengths, fresh weights, dry weighs and ion leakage of wild‐type rice, and pAct:Os02g0325600‐SRDX and pAct:Os03g0325600‐SRDX transgenic rice plants 4 weeks after the removal of shoot and roots and replanting rice seedlings on MS plates prepared with NaCl of 0, 150 and 200 m m . Six regenerated seedlings in each treatment group were used for measurement. An average of ion‐leakage levels in each group was calculated from six samples and expressed as the percentage of the initial conductivity versus total conductivity. Bars show standard deviations. Scales in (a) = 5 cm. Significance level: * P < 0.05; and ** P < 0.01 by t ‐test. Wild‐type rice died within 1 month after the start of salt treatment, while Os02g0325600‐SRDX and Os03g0327800‐SRDX transgenic rice continued to grow and could bear seeds after they had been transferred to soil. Similar salt‐stress tolerance was confirmed in the transgenic rice that were grown on soil without any trimmings, treated with 150 m m NaCl solution for 10 days (Figure S4). Thus, the chimeric repressors derived from Os02g0325600 and Os03g0327800 conferred tolerance to salt stress on rice as did the chimeric HRS1 and ANAC047 repressors in Arabidopsis . Os02g0325600 has a region similar to the EAR‐like motif located near its amino terminus as does HRS1 (Figure S3). We confirmed the repression activity of Os02g0325600 in a transient expression assay ( Figure 5a ). Os02g0325600 is probably a transcriptional repressor that regulates the response to salt stress. Discussion We have engineered rice and Arabidopsis plants that tolerate abiotic stress using CRES‐T. The chimeric repressors derived from AtMYB102, ANAC047 and HRS1 induced tolerance to salt stress, and those derived from ZAT6 and AtERF5 conferred tolerance to osmotic stress in Arabidopsis seedlings ( Figure 1b–g ). Furthermore, rosette plants of these five CRES‐T lines were tolerant to treatment with 400 m m NaCl ( Figure 2 ). Our results indicate that mechanisms of tolerance to salt and osmotic stress overlap, perhaps at the level of water‐use efficiency. Tolerance of our CRES‐T lines was maintained from germination through the rosette stage and was heritable. The five transcription factors whose chimeric derivatives conferred tolerance to salt stress on Arabidopsis are encoded by salt‐ and osmotic stress‐responsive genes. Ectopic expression of our chimeric repressor possibly overcomes the induction of expression of the endogenous corresponding transcription factor in response to stresses. It could be interesting to address intrinsic functions of the transcription factor themselves and examine whether they act as negative regulators in stress responses by means of further studies. AtMYB102 , ZAT6 and AtERF5 are involved in stress responses ( Fujimoto , 2000 ; Denekamp and Smeekens, 2003 ; Devaiah , 2007 ). ANAC047 belongs to group III in the NAC family, which includes the stress‐related genes ATAF1 , NAP and RD26 ( Fujita , 2004 ; Lu , 2007 ). It is likely that ANAC047 is involved in stress responses as are other members of group III. The HRS1 gene whose expression was depressed by salt treatment is a transcriptional repressor ( Figure 5a,b ). Thus, HRS1 might act as a repressor of negative regulators that suppress stress responses. In addition, ZAT6 probably acts as a repressor because it includes an EAR‐motif repression domain in its carboxy‐terminal region. The repressive activity of native repressors is enhanced by fusion with SRDX ( Matsui , 2008 ), and the potentiated repressive activity might induce tolerance to distinct stresses in plants. Our results strongly suggest that negative regulators might participate in the acquisition of stress tolerance in Arabidopsis and rice. Expression of stress‐protective genes seemed to be induced upon suppression of a negative regulator by the activity of a chimeric repressor. It is reasonable to postulate that negative regulators act as fine tuners to suppress the bursts of expression of stress‐response genes because overexpression of such genes might have adverse effects on plant development ( Gilmour , 2000 ; Garg , 2002 ; Zhang , 2005 ; Xiong and Fei, 2006 ; Seong , 2007 ). Our microarray analysis revealed that the expression of DREB and related genes was dramatically enhanced in AtMYB102‐SRDX plants under salt‐stress condition, which might lead to the accumulation of stress tolerance‐related proteins. Looped regulation between AtMYB102 and DREB1A may contribute remarkable induction of the expression of DREB and related genes. The chimeric AtMYB102 repressor might suppress the expression of factors that negatively regulate the expression of DREB genes. Possible candidates for the negative regulatory genes are At4g10160, BEE2 and At5g07690 , whose expression was markedly depressed by salt stress in AtMYB102‐SRDX plants. In addition, the expression of ZAT11 , ZAT12 and ZAT7 was markedly elevated in AtMYB102‐SRDX plants under normal and salt‐stress conditions. Several zinc finger proteins, including ZAT11, ZAT12, ZAT7 and ZAT6, play key roles in regulating the defence responses of Arabidopsis ( Iuchi , 2007 ; Ciftci‐Yilmaz and Mittler, 2008 ). ZAT11, ZAT12, ZAT7 and ZAT6 include an EAR‐motif repression domain and repressive activity (in transient expression assays; data not shown), and the EAR‐motif repression domain of ZAT7 has been shown to confer stress tolerance ( Nakashima , 2000 ). It will be interesting to examine whether the EAR‐like motif of HRS1 and that of Os02g0325600 are essential for their effects on stress tolerance in Arabidopsis and rice, respectively. Salinity and osmotic stress cause drastic changes in ion and water homeostasis, and both types of stress are linked to defects in water utilization in plant cells. The results of ion‐leakage examination clearly showed that salt stress induced a vast leakage of electrolytes ( Figure 6b ). Many factors are involved in responses to abiotic stresses, such as chilling, freezing, drought and heat ( Xiong , 2002 ; Shinozaki , 2003 ; Kotak , 2007 ). Manipulation of these factors can successfully increase multistress tolerance in plants ( Karaba , 2007 ; Jung , 2008 ; Wu , 2008 ). We found a relationship between salt stress and osmotic stress in our analysis of ZAT6 and AtERF5 . Neither ZAT6‐SRDX nor AtERF5‐SRDX exhibited salt tolerance during germination, but rosette plants exhibited strong tolerance to salinity ( Figures 1 and 2 ). It is possible that these chimeric repressors might confer tolerance to other kinds of abiotic stress. Genetic manipulation has been widely utilized to modify the ability of crop plants to withstand salt stress ( Liu , 1998 ; Yamaguchi and Blumwald, 2005 ; James , 2008 ; Gao , 2009 ; Oh , 2009 ), and information obtained from Arabidopsis can be used to design functional crops because regulators of stress‐signal pathways are conserved to a significant extent in other plant species ( Mitsuda , 2006 ). Our results show that application of the Arabidopsis CRES‐T screening system to rice is quite effective, and rice is not only a model monocot but also an important crop. Our findings in Arabidopsis and rice may be applicable to other crops, such as maize, sugarcane and other gramineous plants. Manipulation of a transcription factor that is involved in adaptation to environmental conditions is an effective method for improving stress tolerance in plants ( Bhatnagar‐Mathur , 2008 ). Many efforts have been made to produce salt tolerant rice by overexpression of transcription factors ( Hu , 2006 ; Karaba , 2007 ; Oh , 2009 ). We have shown here that chimeric repressors derived from the GARP transcription factor Os02g0325600 and from the NAC family protein Os03g0327800 effectively induced salt tolerance in rice ( Figure 6a,b ). Our CRES‐T screening system in Arabidopsis appears to be an effective tool for the development of stress‐tolerant crops. Experimental procedures Preparation of constructs Protein‐coding regions of AtMYB102, ANAC047, At1G13300, ZAT6 and AtERF5 were amplified from an Arabidopsis cDNA library with appropriate primers and cloned into the binary vector pBCKH, as described previously ( Mitsuda , 2005 ). The cDNA clones AK101809 and AK243514 provided by the Rice Genome Resource Center (RGRC; Tsukuba, Japan), were used to prepare pAct:Os02g0325600‐SRDX and pAct:Os03g0327800‐SRDX . The coding regions of Os02g0325600 and Os03g0327800 was inserted into pAct:SRDXG that contained the promoter region of the rice ACT1 gene and transferred into the pBCKH vector ( Mitsuda , 2006 ). Reporter and effector constructs for 35S‐5xGAL4:LUC , 35S:GAL4DB and 35S:GAL4DB‐SRDX were described previously ( Hiratsu , 2002 ). The full‐length At1g13300 and Os02g0325600 constructs were prepared by PCR with appropriate primers (Table S3) and inserted into the effector plasmid ( Hiratsu , 2002 ). Primer pairs that used for the study were listed in Table S3. Plant growth and transformation Transformation of Arabidopsis ecotype Colombia (Col‐0) was performed as reported previously ( Matsui , 2004 ). Arabidopsis plants were grown on agar‐solidified Murashige–Skoog (MS) medium with 0.5% sucrose (MS plates), with or without NaCl or mannitol, and grown in soil under long‐day conditions (16 h of light and 8 h of darkness daily) at 22 °C. The pAct:Os02g0325600‐SRDX and pAct:Os03g0327800‐SRDX constructs were introduced separately into wild‐type rice cv. Nipponbare, as described previously ( Mitsuda , 2006 ). Rice was grown on MS plates and in soil with 14 h of light at 30 ± 2 °C and 10 h of darkness at 20 ± 2 °C daily. Stress tolerant assay Sterilized Arabidopsis seeds were plated on solid medium composed MS salts and different concentration of NaCl or mannitol. After 2 weeks of incubation, the percentage of seedlings that had germinated and developed green expanded cotyledons was determined. For assays in rosette plants, 4‐week‐old wild‐type and CRES‐T plants, which had been grown on soil, were treated with 400 m m NaCl solutions for 3 weeks. Phenotype of the 7‐week‐old plants was evaluated for comparison. For salt tolerance evaluation in rice, T2 plants of CRES‐T transgenic lines and wild‐type rice cv. Nipponbare were used. Roots and shoots of 1‐week‐old rice seedlings grown on regular MS plates were cut at 1 cm down and up, respectively, from the base position of the plants. The largely trimmed seedlings were cultured on MS plates containing 150 or 200 m m NaCl for 4 weeks. Thereafter, length of shoots and roots was measured, fresh weights of whole plants were taken, the samples were dried in an oven at 80 °C for 48 h, and dry weights were recorded. Ion‐leakage test was carried out according to the methods previously reported ( Yan , 2007 ), using 0.1 g shoot samples taken from wild‐type and transgenic rice seedling that had been trimmed and cultured with and without salt treatment as described previously. Analysis of RNA Preparation of total RNA and quantitative RT‐PCR (qRT‐PCR) were carried out as described previously ( Mitsuda , 2005 ), and the gene‐specific primers for qRT‐PCR are shown in Table S3. Relative amounts of transcripts were calculated by an absolute quantification method, with the UBQ1 gene as an internal control. At least three replicates from three independent lines were included in each experiment. Microarray analysis Three‐week‐old wild‐type and AtMYB102SRDX plants were transferred to regular MS medium and MS containing 200 m m NaCl, respectively, and incubated for 24 h. Total RNA was prepared from rosette leaves of three independent T2 lines of AtMYB102SRDX plants and wild‐type plants as described previously ( Mitsuda , 2005 ). For microarray analysis, preparation of Cy5‐ and Cy3‐labelled cDNA probes, hybridizations were performed as described previously ( Mitsuda , 2005 ). The data were analysed based on the criteria of a P ‐value cut‐off of 0.05, and a fold change cut‐off value was selected as more than 25 or <0.3. The full data set have been deposited in NCBI’s Gene Expression Omnibus ( Edgar , 2002 ) and are accessible through GEO Series accession number GSE19893 (). Transient expression assays Details of transient expression assays, after particle bombardment of Arabidopsis leaves, were described previously ( Ohta , 2001 ; Hiratsu , 2002 ). 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Journal

Plant Biotechnology JournalWiley

Published: Sep 1, 2011

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