The Role of Gibberellin in the CBF1-Mediated Stress-Response Pathway

ORIGINAL PAPERThe Role of Gibberellin in the CBF1-Mediated Stress-Response PathwayShihui Niu &Qiong Gao &Zhexin Li &Xiaoyang Chen &Wei LiPublished online:11January 2014#Springer Science+Business Media New York 2014Abstract Constitutive expression of C-repeat/dehydration-responsive element binding factor genes confers drought,salt,and freezing tolerance in transgenic plants but causes dwarf-ism under normal growth conditions.Evidence has emerged that gibberellin (GA)plays a major role in these growth-regulatory mechanisms.However,the role and benefit of reducing the active GA level in abiotic stress responses are largely ing microarray analysis,we identified a subgroup of GA-regulated genes by comparing the tran-scriptome of GA over-accumulation and deficiency,CBF1constitutive expression,and wild-type tobacco plants (Nicotiana tabacum ).The results revealed that the geranylgeranyl diphosphate synthase is a regulatory site inte-grating environmental cues and is endogenous in the regula-tion of GA level and subsequent plant growth.Low levels of GA promoted the accumulation and gene activation of osmot-ic adjustment-related substances and enhanced the water-retention capability of leaves.We found that GA was associ-ated with SA/JA balance in the CBF1-mediated stress re-sponse,but inhibition of SA signaling is the principal regula-tor in this pathway.These results demonstrate that,in addition to regulating growth,GA has a much more complex role,andSA may be another important player in the CBF1-mediated stress-response pathway.Keywords Gibberellin .DREB1b/CBF1.Dwarfism .Abiotic stress .GGPPS.Salicylic acidIntroductionThroughout their life cycle,plants regularly face various en-vironmental conditions beyond their growth and productivity optimum range.To survive,plants must respond effectively to adverse conditions such as drought,cold,and salinity,referred to as abiotic stressors.Defense against these stressors is costly,often occurring at the expense of growth and development (Krasensky and Jonak 2012;Yang et al.2012a ,b ).However,studies have shown that the cost of growth inhibition is worthwhile to facilitate survival (Achard et al.2006;Achard et al.2008a ,b ).Additionally,overexpression of genes that significantly enhance abiotic stress tolerance is often accom-panied by severe growth inhibition under normal conditions (Achard et al.2008a ,b ;Gilmour et al.2004;Huang et al.2009;Jaglo-Ottosen et al.1998;Kasuga et al.1999;Mlynarova et al.2007).These studies indicate that growth retardation may result from a positive response mechanism and have adaptive significance (Munns 2002).Over the past decade,a group of AP2transcription factors known as the C-repeat/dehydration-responsive element-binding factor (DREB1/CBF)was characterized in the abiotic stress-response process (Stockinger et al.1997).Overexpression of the CBF genes results in enhanced drought,salt,and freezing tolerance of both nonacclimated and acclimated transgenic plants (Tillett et al.2012;Kasuga et al.1999;Novillo et al.2012;Oakenfull et al.2013).Additionally,constitutive expression of each CBF was found to have similar effects despite their being induced under specific stress conditions (Dubouzet et al.2003;Electronic supplementary material The online version of this article (doi:10.1007/s11105-013-0693-x)contains supplementary material,which is available to authorized users.S.Niu :Q.Gao :Z.Li :W.Li (*)National Engineering Laboratory for Forest Tree Breeding,Key Laboratory for Genetics and Breeding of Forest Trees andOrnamental Plants of Ministry of Education,College of Biological Sciences and Technology,Beijing Forestry University,Beijing 100083,People ’s Republic of China e-mail:bjfuliwei@X.Chen (*)Laboratory of Bio-Technology of Tropical and Subtropical Forestry,College of Forestry,South China Agriculture University,Guangzhou 510642,People ’s Republic of China e-mail:xychen@Plant Mol Biol Rep (2014)32:852–863DOI 10.1007/s11105-013-0693-xGilmour et al.2004;Maruyama et al.2004).Considerable overlap of plant responses to different stresses may be the cause of this redundant functional activity(Chen et al.2002; Krasensky and Jonak2012;Kreps et al.2002).Among the undesirable features of the ectopic expression of CBF genes are dwarfism and delayed flowering under normal growth condi-tions,and exogenous applications of gibberellin(GA)restored the wild-type phenotype in Arabidopsis and other species (Achard et al.2008a,b;Gilmour et al.2004;Huang et al. 2009;Jaglo-Ottosen et al.1998;Kasuga et al.1999;Magome et al.2004;Magome et al.2008;Suo et al.2012;Tillett et al. 2012).Several lines of evidence indicate that growth retardation of transgenic plants is mainly caused by reduced levels of bioactive GA(Achard et al.2008a,b;Magome et al.2004; Magome et al.2008),although the underlying molecular mech-anism remains only partially understood.Recent studies sug-gested that the expression of CBFs modulates GA catabolism by induction of GA2-oxidase,the key deactivation enzyme of GA, and the subsequent reduced growth phenotype(Achard et al. 2008a,b;Kendall et al.2011;Suo et al.2012).Nevertheless,no CBF binding CRT/DRE-like cis-elements were found in GA2ox gene-promoter sequences(Achard et al.2008a,b),and most members of the GA2ox gene family and GA synthase genes showed feedback regulation characteristics caused by GA defi-ciency(Achard et al.2008a,b;Huang et al.2009).Further study is needed to understand this discrepancy and the role of GA in CBF-mediated abiotic stress response.To this end,we compared the transcriptome of wild-type (WT)and transgenic tobacco plants overexpressing AtCBF1, PtGA20ox,and PtGA2ox1using12Agilent44K microarrays. We confirmed candidate downstream genes of GA using RT-PCR.These findings provide more comprehensive insight into the roles of GA in CBF1g-mediated abiotic stress response.Materials and MethodsPlant Materials and Growth ConditionsGenetic studies were carried out using clone W38from tobac-co(Nicotiana tabacum cultivar Wisconsin38).All plants were grown on hormone-free half-strength MS medium containing 0.7%carrageenan and2%sucrose and maintained in vitro at 25°C under a16:8-hour photoperiod.Two-month-old rooted plantlets were transferred to20-cm-diameter plastic pots and cultured in a mixture of peat moss,perlite,and vermiculite (2:1:1,v/v)at27±3°C with natural light.The microarray and GA analyses were done with in vitro cultured plants,whereas the height analysis and the water-deficit analysis such as superoxide dismutase(SOD),guaiacol peroxidase(GPX),soluble sugars,relative water content,and proline analysis were done with potted plants.In the water-deficit study,the soil water content was monitored by the time domain reflec-tometry system(Soilmoisture Equipment,USA),whereas the relative water content of leaves was determined by weighing leaves before and after drying in a drying oven.When the soil moisture was reached to10±3%(irrigation withholding after 10days),leaves sampled for analyzing.Vector Construction and TransformationThe open reading frames of PtGA20ox(Busov et al.2003)and PtGA2ox1(Israelsson et al.2005)were amplified from the cDNA of Populus tomentosa Carr.,and the AtCBF1were amplified from the cDNA of Arabidopsis thaliana using Pyrobest DNA polymerase.The AtCBF1,PtGA20ox,or PtGA2ox1cDNA fragments were cloned into pYL322-d1-CaMV35S and then recombined into pYLTAC380H,which contained the HPT II gene as a selectable marker,using the Cre/loxP system(Lin et al.2003).The constructs were trans-ferred into Agrobacterium strain LBA4404via the freeze/thaw method.The transformation of tobacco was performed as described previously(Dayan et al.2010).The leaves were transferred to fresh solidified MS medium containing 2mg L−16-BA,0.1mg L−1NAA,300mg L−1cefotaxime, and25mg L−1hygromycin B every2weeks until shoot regeneration.Regenerated shoots that rooted in the presence of20mg L−1hygromycin B were potted.The presence and expression of the inserted genes was confirmed by PCR and RT-PCR,respectively.At least three lines with the highest expression of the respective transgene were selected by qPCR. Once again,shoots were regenerated from leaves of these screened plants to produce aseptic transgenic plants. Regenerated plants were then transferred to antibiotic-free half-strength MS medium.Each line was represented by at least ten ramets in all experiments.Phytohormone AnalysisFor GA content analysis,3g of expanding leaves were col-lected from1-month-old plants,each line represented by three independent plants.The samples were immediately weighed (fresh),frozen,powdered in liquid nitrogen,and later analyzed with an LC-MS system.The approach was as previously described(Gou et al.2010).Biometric and Biochemical MeasurementsThe heights of greenhouse-grown plants were measured every 10days,with three lines and each line represented by six ramets.Activities of the antioxidant enzymes SOD and GPX were measured as described previously(Xie et al.2008).Soluble sugars and proline assays were analyzed as previously described(Zhou et al.2009).Microarray Assay AnalysisLeaves were harvested from both WT and transgenic plants grown under normal conditions,and each genotype was rep-resented by three independent transgenic lines.Total RNA was isolated and purified using the QIAGEN RNeasy Kit (Qiagen,USA).The total RNA was hybridized onto the4×44K Agilent tobacco(Nicotiana tabacum)microarrays and analyzed as described previously(Misra et al.2010).To identify statistically significant differentially expressed genes, cut-off criteria of P<0.05in the t tests and fold change(FC)≥1.5were adopted.The target sequences of each probe were obtained from the DFCI database(http://compbio.dfci. /cgi-bin/tgi/gireport.pl?gudb=tobacco),and the annotations and Arabidopsis homologs of target sequences were obtained as described previously(Misra et al.2010).For real-time RT-PCR analysis,total RNA was extracted using Trizol reagent and a SV total RNA Isolation Kit (Promega,USA).Two micrograms of total RNA were used to synthesize single-strand cDNA using the M-MLV reverse-transcriptase kit(Promega,USA).Real-time PCR assays were performed using the Quanti Tect SYBR Green PCR Kit (Qiagen,USA).We used the18s rRNA as the endogenous reference genes,previous studies has been shown that it was stable during the time period under both biotic and abiotic stress in tobacco(Schmidt and Delaney2010).PCR primers used can be found in Table S1in Supporting Information.The relative expression levels for microarrays data=value (transgenic)/value(WT);the relative expression levels for RT-PCR data=2(Ct(Wt)-Ct(18S rRNA))-(Ct(transgenic)-Ct(18S rRNA)). GenBank Accession NumbersSequence data from this article can be found in the GenBank data libraries under accession number(s):AJ001326 (PtGA20ox),JX102472(PtGA2ox1),AT4G25490 (AtCBF1),GQ911583.1(NtGGPPS1),GQ911584.1 (NtGGPPS2),AB032198.1(NtGA3ox1),EF471116.1 (NtGA3ox2),AB125232.1(NtGA2ox1),AB125233.1 (NtGA2ox2),EF471117.1(NtGA2ox3),AB016083.1 (NtGA20ox1),AB016084.1(NtGA20ox2),AM803721.1 (NtSDC1),AM791755.1(NtPHS1),AB625593.1(NtLTP1), FG642178.1(NtSNC-4),AF092915(encoding a cytochrome P450gene(CYP94B3)),X12737(NtPR-1a),FG135358 (NtLOX2),GQ859161.1(NtMYC2b),AF193770 (NtWRKY3),AF193771(NtWRKY4),and DQ460475.1 (NtWRKY12).ResultsCBF1Attenuated GA Signaling via Transcriptional Repression of GGPPSTransgenic tobacco plants were generated expressing AtCBF1, PtGA20ox,or PtGA2ox1under the control of the promoter of the cauliflower mosaic virus35S(Fig.1a,b)using the follow-ing constructs35S::CBF1,35S::PtGA20ox,and35S::PtGA2ox1 (hereafter referred to as35:CBF,35:G20,and35:G2,respec-tively).The level of major active GA in tobacco,GA1(Niu et al. 2013;Fukazawa et al.2000),was significantly altered in 35:G20and35:G2plants(Fig.1c),typically resulting in GA overproduction and GA deficiency syndrome,respectively (Fig.1a).Like35:G2,35:CBF plants showed a reduction in height and in GA1content albeit to a lesser extent(Fig.1a,c). The35:G20plants exhibited increases in internode length, whereas35:G2and35:CBF plants exhibited dwarfism (Fig.1d).To obtain more insight into the action of CBF1on the GA biosynthetic and catabolic pathways,we analyzed the expres-sions of GA pathway genes in WT and transgenic plants based on the microarray data and qPCR(Fig.1e).The results dem-onstrate that the GA synthase genes,GA20ox1(Tanaka-Ueguchi et al.1998)and GA3ox1-2(Itoh et al.1999),were upregulated,whereas the GA deactivating genes,GA2ox1-2 (Gallego-Giraldo et al.2008),were downregulated in both 35:G2and35:CBF plants,and the effects in35:G20were opposite(Fig.1e).This result is conferred by typical feedback and feedforward regulation in GA homeostasis.However,the expression pattern of GGPP synthase genes(GGPPS)in 35:CBF plants was similarly downregulated as in35:G20 plants(Fig.1e).GAs are synthesized from geranylgeranyl diphosphate(GGPP)via a multistep process,and the mRNA level of GGPPS1was more abundant than those of other genes in WT plants(Fig.1e),suggesting that the shortage of GGPP caused by CBF-mediated GGPPS1repression was responsible for the decrease in active GA and consequent growth retardation.The transcriptome remodeling of35:CBF,35:G20,and 35:G2plants and the overlap of GA and CBF1-regulated genesIn order to further understand the role of GA in the CBF1-mediated abiotic stress response,an exploratory microarray survey was carried out using12Agilent44K microarrays. When a1.5-FC with respect to WT plants in gene expression was used to filter our gene list,43genes were obtained. Fourteen genes were upregulated,and the other29genes were downregulated in35:CBF plants(Fig.2a).This result indi-cates that the low GA level in35:CBF plants primarily re-presses gene expression.All of these genes were annotated with gene ontology(GO)terms and are involved in severalbiological functions (Fig.2b ).The relative abundance distri-bution of genes of the specialized GO terms were mostly in the oxidoreductase activity,catalytic activity,peroxisome,plasma membrane,oxidation reduction process and metabolic process (Fig.2b ),indicating that reducing the active GA level is a positive response in adversity.The final list of 43candidate genes is shown in Table S2in Supporting Information.The Role of GA in Osmotic Adjustment Related to Substance Accumulation and Gene ActivationOverexpression of CBF1increased soluble sugar and proline content in transgenic plants,but GA had no effect on soluble sugar and proline content (Fig.3a,b ).This result suggests that CBF1activates the accumulation of soluble sugars andprolineFig.1Effects of endogenous GA level alterations and AtCBF1overex-pression on tobacco plants.WT wild-type plants,35:CBF 35S ::AtCBF1transgenic plants,35:G2035S ::PtGA20ox transgenic plants,35:G235S -::PtGA2ox1transgenic plants.a The height of WT and overexpressing AtCBF1,PtGA2ox1,and PtGA20ox transgenic plants.Bar =10cm.b Expression levels of PtGA20ox ,PtGA2ox1,and AtCBF1in transgenic tobacco.The expression levels are relative to the 18S rRNA and to the PtGA20ox of 35:G20value (set at 1).Values are means±SE of three biological replicates.The expressions of these genes were not detected in wild-type plants.c GA 1content in WT and transgenic plants.Values are means±SE of three independent biological replicates.*P <0.05;**P <0.01—significance as determined by Student's t test.d Heights ofWT and transgenic plants grown in a greenhouse.Bars show means and standard error over three events (at least six ramets per event).e The expression of GA pathway genes in WT and transgenic plants.The upper histogram shows expression levels of GA pathway genes in WT plants relative to 18S rRNA and to the GA2ox1value (set at 1);the relative expression levels=2(Ct(GA2ox1)-Ct(18S rRNA)]-[Ct(gene X)-Ct(18S rRNA)).The lower histogram shows expression levels of GA pathway genes in trans-genic plants normalized against WT (set at 1);the relative expression levels=2[Ct(Wt)-Ct(18S rRNA))-[Ct(transgenic)-Ct(18S rRNA)).Data represent the mean of three independent biological replicates per event.SE bars were not shown for being too small for identificationby a GA-independent mechanism.However,under low levels of GA,enhanced antioxidant enzyme activity occurred as a result of the constitutive expression of CBF1,whereas high levels of GA resulted in considerable inhibition (Fig.3c,d ).Glycine betaine (GB)is well known to mitigate oxidative damage and stabilize membranes of plant cells under stress,especially salt stress (Yang et al.2010).Choline is an impor-tant precursor substance for GB biosynthesis (Tabuchi et al.2006).Serine decarboxylase gene 1(SDC1)is a key gene in the choline synthesis pathway (Rontein et al.2001),and atsdc -1mutants showed necrotic lesions in leaves under normal conditions (Kwon et al.2012).We found that SDC1was activated in 35:CBF and 35:G2plants and repressed in 35:G20plants (Fig.3e ).A previous study indicated that the regulation of BvSDC1differed from that of other GB-related genes (Tabuchi et al.2006);therefore,it may play an impor-tant role in GB biosynthesis and stress response.The metabolism of starch is very sensitive to adverse environmental cues and can rapidly provide soluble sugars to protect cells against osmotic-stress damage (Krasensky and Jonak 2012).PHS1,which is involved in starch metabolism inleaves,is altered by CBF1and GA (Fig.3f ).PHS1encodes a glucanphosphorylase,and PHS1-deficient mutant plants dis-play water-stress sensitivity and enhanced formation of le-sions on their leaves (Zeeman et al.2004).The promotion of PHS1expression facilitates abiotic stress tolerance.GA Has a Negative Regulatory Role in Leaf Water Retention Both 35:CBF and 35:G2plants exhibited small,dark-green leaves,phenotypic hallmarks of GA-deficient mutants,where-as 35:G20typically had light-green leaves with long petioles (Fig.4a ).As a measure of drought resistance,the water-retention capability of both juvenile and expanded excised leaves of 35:G20plants were reduced relative to those of WT plants under normal conditions,whereas the effects of GA deactivation in leaves were opposite (Fig.4a ).The phe-notypes of 35:CBF,35:G2and 35:G20plants are similar,with relative water content of leaves indistinguishable from WT plants under control conditions.However,after a 10-d water withdrawal,35:G20had greatly reduced water contentinFig.2Overlap of genes between 35:CBF,35:G20,and 35:G2plant-array data.The total RNA of the three independenttransgenic lines per event were used for microarray analysis.a GA downregulated genes (left )and GA upregulated genes (right )overlapping with CBF-pathway-related genes were selected.b Gene Ontology (GO )distributions for candidate GA-regulated genes involved in the abiotic stress response.Therelative proportion of GO terms in the molecular function,cellular component,and biological process categories in the GO vocabulary.Results are from Blast2GO analysisleaves,whereas overexpression of CBF1and GA2ox1en-hanced the water-retention capability of leaves (Fig.4b ).We found that the expression of LTP1(lipid transfer protein 1)was altered in the 35:CBF,35:G2,and 35:G20plants (Fig.4c ).LTP1encodes an enzyme involved in lipid metab-olism and plays a role in the transport of extracellular lipo-philic material required for cutin deposition (Canevascini et al.1996).The lipophilic layer of cutin is intermeshed on the outer surface of the leaf,playing a critical role in leaf water-retention and dehydration-stress response (Kosma et al.2009).The changes in the cutin layers were measured by microscopy and seem to support the suggested changes in LPT 1expres-sion,however,quantitative data were difficult to produce because the layer thickness related to the developmental stage.LTP1was downregulated in 35:G20and upregulated in 35:CBF and 35:G2plants (Fig.4c ),further corroborating thatGA has a negative regulatory role in leaf water-retention and water-deficit response.GA Is Associated with SA/JA Signaling Balance in the CBF1-Mediated Stress-Response PathwaySesquiterpenoid phytoalexins are stress metabolites induced by biotic and abiotic elicitors in the Solanaceae plants (Mialoundama et al.2009).The capsidiol is the major sesquiterpenoid phytoalexin in tobacco and the induction of a sesquiterpene cyclase (5-epi-aristolochene synthase (EAS))is essential for capsidiol biosynthesis (Facchini and Chappell 1992;Mandujano-Chavez et al.2000).We found that seven EAS genes were downregulated by lack of GA (Fig.5a ).The EAS genes,with homologous genes designated TPS in Arabidopsis ,are involved in floral volatileterpeneFig.3The osmotic adjustment related to substance content and relative expression level of genes functioning against osmotic stress in WT and transgenic plants.a ,b Soluble sugars and proline content in WT and transgenic plants.c ,d SOD and GPX activities in WT and transgenic plants.Bars show means and standard error over three events (at least six ramets per event).e ,f Expression levels of NtSDC1and NtPHS1relative to 18S rRNA in WT and transgenic plants.Data represent the mean of threebiological replicates per genotypebiosynthesis (Hong et al.2012).Upregulation of TPS genes in Arabidopsis floral organs is certainly related to flower devel-opment (Tholl et al.2005).A recent study revealed that TPS genes are directly regulated by MYC2(Hong et al.2012),a basic helix –loop –helix domain-containing transcription factor that was reported to be a transcriptional activator of the JA signaling pathway (Abe et al.2003;Boter et al.2004).Moreover,suppressor of npr1-1,constitutive 4and CYP94B3,which are reported to be involved in SA/JA ho-meostasis (Bi et al.2010;Kitaoka et al.2011;Koo et al.2011;Yang et al.2012a ,b ),were downregulated by the absence of GA (Fig.5b ).To substantiate the regulation of SA/JA balance,we next analyzed the expressions of the SA-response marker gene PR -1a (van Verk et al.2008)and JA-response marker gene LOX2(Hong et al.2012;Yan et al.2009;Zhang et al.2012)(Fig.5c ).The result confirmed the attenuation of SA responses and the potentiation of JA responses in the CBF1-mediated abiotic stress response;very substantial decreases in transcript levels of the SA marker gene PR -1a indicated that inhibition of SA signaling is the principal regulator in this pathway.Our results show that the transcription factors WRKY3,WRKY4,and WRKY12were downregulated,and MYC2b was induced in 35:CBF and 35:G2plants (Fig.5d ).Previous studies have shown that WRKY3,WRKY4,and WRKY12acti-vate SA-responsive genes and repress JA-responsive genes(Li et al.2004;van Verk et al.2008;Yu et al.2001).By contrast,MYC2b is a transcriptional activator of the JA sig-naling pathway.These results indicate that the transcriptional regulation of these transcription factors may contribute to SA/JA signal transduction balance,and that this regulation is a component of the CBF1-mediated abiotic stress pathway.DiscussionGAs are well known phytohormones contributing to the reg-ulation of many growth and developmental aspects in plants,especially stem elongation and flowering (Plackett et al.2011;Sun 2010;Thomas and Sun 2004;Ubeda-Tomas and Bennett 2010;Yamaguchi 2008).It is therefore not surprising that GA is involved in growth retardation in the stress response and adaptation (Achard et al.2008a ,b ;Huerta et al.2008;Magome et al.2008).In fact,the association of compact plant growth patterns with abiotic stress has long been recognized in natural environments (Scott et al.2004).Recent studies dem-onstrated that this growth-regulatory mechanism,at least the CBF-mediated abiotic stress response,reduces bioactive GA,and the growth restraint is beneficial and facilitates survival (Achard et al.2006;Achard et al.2008a ,2008b ).It was suggested that GA 2-oxidases,the key deactivation enzyme of the GA biosynthesis pathway,contributes to thisGAFig.4The effect of GA and CBF1on leaf water-retention capability.a The water-retention capability of both juvenile (upper row )and expanded (lower row )excised leaves in WT and transgenic plants.The 35:G20plants had light-green leaves with long petioles,and both juvenile and expanded excised leaves from them withered faster under normal condi-tions than the wild type and 35:G2.The value on the left represents the time after the excised.b The relative water content of leaves of plants under control conditions and after a 10-day water withdrawal.*P <0.05,significance between values of WT and transgenes as determined by Student's t test.Values are means±SE of 18independent biological replicates.c The expression level of NtLTP1relative to 18S rRNA in WT and transgenic plants.***P <0.001,significance as determined by Student's t test.Values are means±SE of three independent biological replicatesreduction (Achard et al.2008a ,b ;Benedict et al.2006;Suo et al.2012).Nevertheless,in the present study,both microar-ray and RT-PCR analyses did not reveal any GA2ox genes upregulated in AtCBF1overexpressing tobacco plants.By contrast,all members of known and putative GA2ox genes were repressed,but GA20ox genes and GA3ox genes were upregulated (Fig.1e ).Previous studies confirm this observa-tion and suggest that this phenomenon might be a result of positive feedback and negative feedforward mechanisms in GA homeostasis regulation (Huang et al.2009).A plausible explanation for these results is that the lower active GA level may be a result of the limited supply of an upstream precursor substrate.To investigate this possibility,we analyzed the expressions of all diterpenoid biosynthesis genes based on the microarray data.The result showed that GGPPS are the likeliest candidate genes responsible for re-ducing bioactive GA levels.GGPPS are key enzymes cata-lyzing the synthesis of GGPP (geranylgeranyl diphosphate),a common precursor for both the carotenoid and GA pathways,which is produced mainly through the methylerythritol phos-phate pathway in plastids (Botella-Pavia et al.2004;Kasahara et al.2002;Yamaguchi 2008).The RT-PCR results further corroborate the hypothesis that GGPPS integrate environmen-tal cues and are endogenous in the regulation of GA level and subsequent plant growth.This regulatory mechanism seems to be a reasonable strategy to preserve resources by regulation of the more upstream genes.A prevailing theory posits that growth restraint improves survival due to the redirection of energy and resources under adverse conditions (Achard et al.2006;Harberd et al.2009).However,among the several fates for GGPP in plant metabolism,that of GA is the least likely in terms of allocated carbon flux,as the carotene content in fresh tissue is generally 1,000times higher than GA (Prisic and Peters 2007).Analysis of GGPPS mutants and the role of carotene in abiotic stress is necessary to understand the sig-nificance of this regulatory mechanism.It is clear that GA-signaling pathways regulate plant growth and stress tolerance,but the specific mechanism is complex.Our results and a previous study demonstrated that the absence of GA primarily represses,rather than promotes,gene expression (Bethke et al.2006).Therefore,although low levels of GA induced several genes that function against stress,this is only one part of the whole response mechanism.Inhibition of gene expression is also critical for plant survival under adversity,especially in the face of long-term adverse environments.Interactions between DELLAs and PIF3/PIF4transcription factors may be important components of this regulation (Harberd et al.2009;Zentella et al.2007).It is noteworthy that we found that GA exerts control over SA signaling under adverse environmental conditions.Previously,studies on SA as an important signaling molecule in plant defense mostly focused on responses against patho-gens.Recently,it was also found to be involved in the abiotic stress response (Rivas-San and Plasencia 2011;Scott et al.2004).Additionally,it was suggested that biosynthesis and signaling of SA were enhanced by GA and repressedbyFig.5Regulation of GA and CBF1involved in SA/JA signaling balance.a Theexpression of EAS genes in array data from WT and transgenic plants.Data represent the mean of three biological replicates.b –d Expression of SA and JAsignaling-pathway genes relative to 18S rRNA in WT andtransgenic plants.The expression levels of genes in transgenicplants are normalized against WT (set at 1).Data represent the mean of three biological replicates per genotype。