integrated analysis of the effects of cold and dehydration ... · portant to improve tolerance to...

Integrated Analysis of the Effects of Cold andDehydration on Rice Metabolites Phytohormones andGene Transcripts1[W][OPEN]

Kyonoshin Maruyama Kaoru Urano Kyouko Yoshiwara Yoshihiko Morishita Nozomu SakuraiHideyuki Suzuki Mikiko Kojima Hitoshi Sakakibara Daisuke Shibata Kazuki Saito Kazuo Shinozakiand Kazuko Yamaguchi-Shinozaki

Biological Resources and Post-harvest Division Japan International Research Center for Agricultural SciencesTsukuba Ibaraki 305ndash8686 Japan (KM KY KY-S) Gene Discovery Research Group RIKEN Center forSustainable Resource Science Tsukuba Ibaraki 305ndash0074 Japan (KU KSa KSh) Department ofBiotechnology Research Kazusa DNA Research Institute Kisarazu Chiba 292ndash0818 Japan (YM NS HSuDS) Plant Productivity Systems Research Group RIKEN Center for Sustainable Resource Science YokohamaKanagawa 230ndash0045 Japan (MK HSa) Department of Molecular Biology and Biotechnology GraduateSchool of Pharmaceutical Sciences Chiba University Chiba 263ndash8522 Japan (KSa) and Laboratory of PlantMolecular Physiology Graduate School of Agricultural and Life Sciences University of Tokyo Bunkyo-kuTokyo 113ndash8657 Japan (KY-S)

Correlations between gene expression and metabolitephytohormone levels under abiotic stress conditions have been reported forArabidopsis (Arabidopsis thaliana) However little is known about these correlations in rice (Oryza sativa lsquoNipponbarersquo) despite itsimportance as a model monocot We performed an integrated analysis to clarify the relationships among cold- and dehydration-responsive metabolites phytohormones and gene transcription in rice An integrated analysis of metabolites and gene expressionindicated that several genes encoding enzymes involved in starch degradation sucrose metabolism and the glyoxylate cycle are up-regulated in rice plants exposed to cold or dehydration and that these changes are correlated with the accumulation of glucose (Glc)fructose and sucrose In particular high expression levels of genes encoding isocitrate lyase and malate synthase in the glyoxylatecycle correlate with increased Glc levels in rice but not in Arabidopsis under dehydration conditions indicating that the regulationof the glyoxylate cyclemay be involved inGlc accumulation under dehydration conditions in rice but not Arabidopsis An integratedanalysis of phytohormones and gene transcripts revealed an inverse relationship between abscisic acid (ABA) signaling andcytokinin (CK) signaling under cold and dehydration stresses these stresses increase ABA signaling and decrease CK signalingHigh levels of Oryza sativa 9-cis-epoxycarotenoid dioxygenase transcripts correlate with ABA accumulation and low levels ofCytochrome P450 (CYP) 735A transcripts correlate with decreased levels of a CK precursor in rice This reduced expression ofCYP735As occurs in rice but not Arabidopsis Therefore transcriptional regulation of CYP735As might be involved in regulatingCK levels under cold and dehydration conditions in rice but not Arabidopsis

Land plants must mount suitable responses toovercome the adverse effects of water stress caused byeither drought or low-temperature conditions (Levitt1980) Among the external stresses water stress is oneof the most important limitations of crop productivity(Bartels and Sunkar 2005) Discoveries of useful genesfor molecular breeding using metabolomics and tran-scriptomics promise to facilitate the improvement ofcrop yields under water stress conditions It is impor-tant to identify plant metabolites and transcripts thatrespond to water stress to determine the essential stepsin molecular processes related to the effective adapta-tion of plants to stress conditions Metabolomic andtranscriptomic data have provided much informationon the metabolite phytohormone and transcript net-works that control plant growth and developmentStudies using mass spectrometry (MS) -based metab-olomic techniques have identified and characterizedmany stress-responsive metabolites (Guy et al 2008Saito and Matsuda 2010 Urano et al 2010 Obata and

1 This work was supported by grants from the Ministry of Agri-culture Forestry and Fisheries of Japan (in part by Genomics forAgricultural Innovation Development of Abiotic Stress TolerantCrops by DREB Genes) and the Programme for Promotion of Basicand Applied Researches for Innovations in Bio-Oriented Industry(BRAIN) of Japan The Japan Advanced Plant Science Network pro-vided funding for the hormone analysis

Address correspondence to kyonosinaffrcgo jp andakysmaileccu-tokyoacjp

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (wwwplantphysiolorg) isKyonoshin Maruyama (kyonosinaffrcgojp)

KM designed and performed most of the experiments and ana-lyzed the data KU performed the metabolome experiments andKSh supervised her KY performed the microarray experimentYM NS and HS performed the metabolome experiments andDS and KSa supervised them MK performed the phytohormoneexperiments and HS supervised her KY-S designed the researchand KY-S and KM wrote the paper

[W] The online version of this article contains Web-only data[OPEN] Articles can be viewed online without a subscriptionwwwplantphysiolorgcgidoi101104pp113231720

Plant Physiology April 2014 Vol 164 pp 1759ndash1771 wwwplantphysiolorg 2014 American Society of Plant Biologists All Rights Reserved 1759

httpsplantphysiolorgDownloaded on December 11 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

Fernie 2012) Targeted analyses of phytohormoneshave been used to characterize signaling and metabolicnetworks (Muumlller et al 2002 Chiwocha et al 2003Kojima et al 2009) For example abscisic acid (ABA)plays an essential role in the response to water stress(Yamaguchi-Shinozaki and Shinozaki 2006 Hirayamaand Shinozaki 2007 Cutler et al 2010) and an increasein endogenous ABA is required for certain water stressresponses designated as ABA-dependent stress re-sponses (Raghavendra et al 2010 Weiner et al 2010Fujita et al 2011) Cytokinins (CKs) play major roles inseveral developmental and physiological processes inplants (for example in cell division regulation ofshoot growth and stress responses Mok et al 2000Sakakibara 2006 Ha et al 2012) Microarray-basedtranscriptomic analyses have identified many genesinvolved in the responses and tolerance to variousstresses in several plant species These genes encodemetabolic enzymes late embryogenesis-abundant(LEA) proteins detoxification enzymes chaperonesprotein kinases transcription factors and other geneproducts (Kilian et al 2007 Shinozaki and Yamaguchi-Shinozaki 2007 Cutler et al 2010 Maruyama et al2012)

Metabolite profiles have been reported for Arabidopsis(Arabidopsis thaliana) under dehydration conditionsThe levels of raffinose family oligosaccharides Probranched-chain amino acids (BCAAs) g-aminobutyratesaccharopine and agmatine were reported to be sig-nificantly higher in dehydration-treated plants thanin untreated plants (Urano et al 2009 Skirycz et al2010 Verslues and Juenger 2011) Increased levels ofseveral metabolites under dehydration conditions werecorrelated with the expression levels of their respectivebiosynthetic genes many of which are regulated byendogenous ABA (Urano et al 2009) Metabolite pro-files have also been determined for crops exposedto dehydration conditions The dehydration-inducedchanges in metabolites observed in Arabidopsis plantswere similar to those reported for wheat (Triticum aes-tivum Bowne et al 2012) legumes (Sanchez et al2012) and maize (Zea mays Sicher and Barnaby 2012)Several research groups have profiled changes in me-tabolite levels in Arabidopsis after exposure to lowtemperatures Metabolite analysis of natural accessionsof Arabidopsis after cold acclimation revealed a clearcorrelation between the accumulation of several carbo-hydrate metabolites (eg Glc Fru and Suc) and coldstress tolerance (Cook et al 2004 Hannah et al 2006)Gray and Heath (2005) used Fourier transform ioncyclotron MS and a nontargeted metabolic finger-printing approach to study the effects of cold accli-mation on the metabolome Gray and Heath (2005)found that a global reprogramming of metabolismoccurs as a result of cold acclimation An integratedanalysis using transgenic Arabidopsis plants over-expressing DRE-binding protein 1AC-Repeat bindingfactor3 which encodes a transcription factor thatfunctions in cold stress responses revealed that tran-scriptional regulation of the carbohydrate network is

necessary for the accumulation of specific carbohy-drates (eg Suc galactinol myoinositol and raffinose)The accumulation of these carbohydrates may be im-portant to improve tolerance to freezing stress in thesetransgenic plants (Cook et al 2004 Maruyama et al2009)

Rice (Oryza sativa lsquoNipponbarersquo) is important notonly as a major crop but also as a model monocot Inthis study we performed an integrated analysis of themetabolites phytohormones and gene transcripts inrice plants subjected to cold or dehydration treatmentsOur aim was to comprehensively survey the molecularresponses of rice to cold or dehydration stimuli Weused three types of MS systems gas chromatography(GC) coupled with time-of-flight MS (GC-TOF-MS)capillary electrophoresis coupled with MS (CE-MS)and liquid chromatography coupled with MS (LC-MS) We identified and characterized representativecold- and dehydration-responsive metabolites andphytohormones We also performed a transcriptomeanalysis using a rice oligonucleotide microarray Weanalyzed metabolite-gene and phytohormone-genecorrelations and identified several genes encodingmetabolic enzymes that might play key roles in theresponses of rice plants to cold or dehydration Wecompared the roles of the identified rice genes with theroles of their counterparts in Arabidopsis under coldor dehydration conditions

RESULTS

Metabolite Profiles of Rice Plants Exposed to Coldor Dehydration

We used GC-TOF-MS and CE-MS to determine theeffects of seven environmental conditions on whole riceseedlings grown in soil for 2 weeks The treatmentswere as follows 1- or 2-d exposure to cold at 10degC 2- or3-d exposure to dehydration without watering and nostress treatment (untreated for 1 2 or 3 d) The GC-TOF-MS and CE-MS analyses identified 152 differentmetabolites according to their retention time indicesand specific mass fragments (Supplemental Table S1)Relative to the levels in untreated plants the levels of38 52 24 and 91 metabolites were significantly in-creased in 1-d cold-treated plants 2-d cold-treated plants2-d dehydration-treated plants and 3-d dehydration-treated plants respectively (Benjamini and Hochbergfalse discovery rate [FDR] P 005) the levels of 26 1226 and eight metabolites respectively were signifi-cantly decreased in plants exposed to the same fourstress conditions (FDR P 005)

We used principal component analysis (PCA) tocompare the metabolite profiles of plants subjected toseven treatments (Fig 1A Supplemental Tables S1ndashS5) The cumulative contribution ratio of the PCA was744 up to the second principal component (PC2)The PCA analysis had two key features The firstprincipal component (PC1) reflected increases in the

1760 Plant Physiol Vol 164 2014

Maruyama et al


levels of metabolites in the rice plants subjected toseven treatments The plants subjected to the 3-d de-hydration treatment showed the highest PC1 valuePlants exposed to the 2-d cold treatment showed thesecond highest PC1 value The PC1 values for the threeclasses of untreated plants were similar and thosevalues were the lowest among seven treatments PC2reflected differences in the variety of metabolitesin plants among seven treatments The PC2 valueswere positive for cold-treated plants and negative fordehydration-treated plants The PC2 values for un-treated plants were nearly zero The results of the PCAindicated that the metabolite profiles of plants exposedto seven treatments were classified into three groupsaccording to plant growth conditions cold dehydra-tion and untreated

We selected representative metabolites showing thehighest and second highest eigenvector values Eigen-vector values correspond to the coefficients of eachPC The levels of these metabolites are displayed as barcharts (Fig 1B) The highest PC1 eigenvector valuewas for metabolite number 6 (unknown) The levels ofmetabolite number 6 were significantly higher in bothcold-treated and dehydration-treated plants than inuntreated plants Ile had the second highest PC1 eigen-vector value The Ile levels were also significantlyhigher in both cold-treated and dehydration-treatedplants than in untreated plants The plants subjectedto 3-d dehydration treatment showed the highest lev-els of metabolite number 6 and Ile Metabolite number14 (unknown) had the highest PC2 eigenvector valueThe levels of metabolite number 14 were significantlyhigher in both cold-treated and dehydration-treatedplants than in untreated plants Glc-6-P (G6P) had thesecond highest PC2 eigenvector value Cold-treatedplants contained significantly higher levels of G6P anddehydration-treated plants contained significantly lowerlevels of G6P compared with the levels in untreatedplants The plants subjected to 2-d cold treatmentshowed the highest levels of metabolite number 14and G6P

The levels of several sugars and amino acids werehigher in cold-treated and dehydration-treated plantsthan in untreated plants We analyzed the levels of alldetected sugars and amino acids and used heat mapsto illustrate the log ratio of each metabolite (Fig 1C and D) The levels of Glc Fru and raffinose were

Figure 1 Statistical analysis of 152 metabolites profiled in untreatedrice plants and rice plants subjected to cold or dehydration treatments

Levels of 152 metabolites were measured in rice plants subjected toseven treatments cold (10˚C) for 1 d (Cold 1d) or 2 d (Cold 2d) de-hydration (water withheld) for 2 d (Dehydration 2d) or 3 d (Dehydra-tion 3d) and untreated for 1 2 or 3 d (Untreated 1d 2d and 3drespectively) A PCA for metabolites Values for y and x axes arevalues for PC1 and PC2 respectively B Representative metabolitesshowing the highest and second highest eigenvector values In eachcase maximum level of metabolite was set to 100 Values are means(n = 3 experiments) error bars show SD C and D Relative levels ofrepresentative sugars (C) and amino acids after cold or dehydrationtreatments C1 Cold 1d C2 cold 2d D2 dehydration 2d D3 de-hydration 3d GABA g-aminobutyric acid G1P glucose 1-phosphate

Plant Physiol Vol 164 2014 1761

Integrated Molecular Analysis of Rice


significantly higher in cold-treated and dehydration-treated plants than in untreated plants Comparedwith their levels in untreated plants the levels of G6Ptrehalose and Suc were significantly higher in cold-treated plants and significantly lower in dehydration-treated plants The levels of BCAAs (Val Leu and Ile)and Pro were also significantly higher in cold-treatedand dehydration-treated plants than in untreatedplants The highest levels of these amino acids were inplants subjected to 3-d dehydration treatment

Phytohormone Profiles of Rice Plants Exposed to Coldor Dehydration

We used LC-MS to measure levels of phytohor-mones in plants subjected to seven different treatmentsand identified 14 phytohormones (Supplemental TableS6) PCA was used to compare the phytohormoneprofiles (Fig 2A Supplemental Tables S6ndashS10) Thecumulative contribution ratio of the PCA was 918up to PC2 Two key features emerged from the PCAFirst the PC1 value reflected decreases in the levels ofphytohormones in plants subjected to seven differenttreatments The lowest PC1 value was for plants sub-jected to 3-d dehydration treatment Plants subjectedto 2-d dehydration treatment showed the secondlowest PC1 value The PC1 values for the three classesof untreated plants were similar and those valueswere the highest among all treatments Second thePC2 value reflected differences in the variety of phy-tohormones in plants under seven different environ-mental conditions The PC2 values were positive forcold-treated plants and negative for dehydration-treated plants The results of the PCA indicated thatthe phytohormone profiles of plants subjected to sevendifferent treatments were classified into three groupsaccording to plant growth conditions cold dehydra-tion and untreated

Next we focused on representative phytohormoneswith the highest eigenvector values (Fig 2B) Thehighest PC1 eigenvector value was for trans-zeatinriboside-59-phosphatess (tZRPs) The level of tZRPs wassignificantly lower in both cold-treated and dehydration-treated plants than in untreated plants The lowestlevel of tZRPs was in plants subjected to 3-d dehy-dration treatment Isopentenyladenine (iP) had thehighest PC2 eigenvector value Compared with thelevel in untreated plants the level of iP was signifi-cantly higher in cold-treated plants but only slightlyhigher in dehydration-treated plants The highest levelof iP was in plants subjected to 2-d cold treatmentABA had the lowest PC2 eigenvector value the ABAlevel was significantly higher in cold-treated anddehydration-treated plants than in untreated plantsThe highest level of ABA was in plants exposed to the3-d dehydration treatment

Next we compared rice plants with Arabidopsis interms of the changes in phytohormone levels undercold or dehydration stress In Arabidopsis the ABA

Figure 2 Statistical analysis of changes in profiles of rice phytohor-mone levels after exposure to cold or dehydration A PCA for phyto-hormones Values for y and x axes are values for PC1 and PC2respectively B Relative levels of representative phytohormones aftercold or dehydration stress A small gray square indicates that thephytohormone could not be detected CKGs Cytokinin glucosides cZcis-zeatin cZR cis-zeatin riboside cZRPs cis-zeatin 59-Ps GA19gibberellin A19 GA44 gibberellin A44 GA53 gibberellin A53IAA indoleacetic acid iPR isopentenyladenosine iPRPs N6-(D2-isopentenyl) adenine ribotides JA jasmonic acid tZR trans-zeatinriboside


Maruyama et al


level in cold- and dehydration-treated plants was sig-nificantly higher than the level in untreated plantsConversely exposure of Arabidopsis plants to eithercold or dehydration significantly decreased the levelsof CKs including iP and trans-zeatin (tZ) In Arabi-dopsis the lowest levels of iP and tZ were in plantsexposed to the dehydration treatment (SupplementalFig S1 Supplemental Table S11)

Transcriptomic Profiles of Rice Plants Exposed to Coldor Dehydration

Previously we used oligonucleotide microarrays toidentify cold- and dehydration-responsive genes in riceplants exposed to 1-d cold and 3-d dehydration treat-ments (Maruyama et al 2012) The International RiceGenome Sequencing Project recently updated its ricegenome sequence information and the annotations formany rice genes have been revised in the rice annota-tion project database (httprapdbdnaaffrcgojpSupplemental Fig S2A) Given the update in the num-ber of rice genes we prepared a new to our knowledgeoligonucleotide microarray for transcriptomic analy-ses This array can detect 43175 genes (SupplementalFig S2B) We used themicroarray to identify additionalcold- and dehydration-responsive genes in rice plantsexposed to a 1- or 2-d cold treatment or a 2- or 3-d de-hydration treatment Overall 3576 (1-d cold treatment)4395 (2-d cold treatment) 2089 (2-d dehydration treat-ment) and 5927 (3-d dehydration treatment) genesweresignificantly up-regulated (FDR P 005 fold change2) and 3147 (1-d cold treatment) 4320 (2-d coldtreatment) 1678 (2-d dehydration treatment) and 6184(3-d dehydration treatment) genes were significantlydown-regulated (FDR P 005 fold change 05Supplemental Tables S12ndashS15)We used PCA to compare the transcript profiles

among plants subjected to seven different treatments(Fig 3A Supplemental Tables S16ndashS19) Two featuresemerged from the PCA First the PC1 values reflectedincreased levels of transcripts in plants subjected toseven different treatments The plants subjected to 3-ddehydration treatment showed the highest PC1 valueThe PC1 values for the four classes of untreated plantswere similar and represented the lowest values Sec-ond the PC2 value reflected differences in the typesof transcripts that accumulated under the differenttreatments The PC2 values were negative for cold-treated plants and positive for dehydration-treatedplants The PC2 values for untreated plants werenearly zero The results of the PCA indicated that thetranscript profiles of plants subjected to seven differenttreatments were classified into three groups accordingto plant growth conditions cold dehydration anduntreatedWe selected representative cold- or dehydration-

responsive genes with the first second and third high-est eigenvector values and displayed their transcriptlevels as heat maps (Fig 3B) In the transcriptome

profile the highest PC1 eigenvector value was forOs04g0583900 which encodes an Myb-type transcrip-tion factor Plants exposed to either cold or dehydrationshowed significantly increased Os04g0583900 tran-script levels compared with the levels in untreatedplants The highest transcript level of this gene was inplants subjected to the 3-d dehydration treatment(Fig 3B) TheOs04g0448401 andOs03g0184100 transcripts

Figure 3 Statistical analysis of changes in the profiles of rice transcriptlevels after exposure to cold or dehydration A PCA for transcript dataobtained from oligonucleotide microarrays Values for y and x axes arevalues for PC1 and PC2 respectively B Representative cold- anddehydration-responsive genes identified using oligonucleotide micro-arrays Genes shown are those genes with the first second and thirdhighest eigenvector values Heat maps illustrate transcript levels ofrepresentative cold- and dehydration-responsive genes MSU Michi-gan State University RAP The Rice Annotation Project




showed the second and third highest PC1 eigenvectorvalues respectively The levels of the Os04g0448401and Os03g0184100 transcripts were also significantlyhigher in both cold- and dehydration-treated plantsthan in untreated plants (Fig 3B) The Os10g0505900Os04g0610600 and OsLEA14water stress-induced(WSI) 18 (Os01g0705200) transcripts had the first sec-ond and third highest PC2 eigenvector values re-spectively Compared with their levels in untreatedplants the levels of these transcripts were significantlyhigher in dehydration-treated plants and the levels indehydration-treated plants were higher than thoselevels in cold-treated plants (Fig 3B) The OsMYB4(Os01g0695900) Os04g0465900 and Os02g0774100 tran-scripts had the first second and third lowest PC2eigenvector values respectively The levels of thesetranscripts were significantly higher in cold-treatedplants than in untreated plants and the levels in cold-treated plants were higher than those levels indehydration-treated plants (Fig 3B)

Molecular Functions of Cold-Responsiveand Dehydration-Responsive Genes in Rice

We used our in-house Gene Ontology database(Maruyama et al 2012) to annotate the molecu-lar functions of all identified cold- or dehydration-responsive genes (Supplemental Fig S3) We selectedthe helicase gene family to represent genes up-regulatedin plants subjected to cold treatments Helicases areinvolved in RNA metabolism and may be important forincreasing transcriptional and translational activitiesunder cold stress conditions The increase in the tran-script abundance of helicase genes up-regulated undercold stress was 306 (1-d cold treatment) and 421 (2-dcold treatment) We selected the dehydrinLEA genefamily to represent genes up-regulated in plants sub-jected to a dehydration treatment The increase in thetranscript abundance of genes encoding dehydrinLEA proteins up-regulated under dehydration stresswas 260 (2-d dehydration treatment) and 329 (3-ddehydration treatment) We selected genes involved inphotosynthesis to represent the genes down-regulatedin both cold- and dehydration-treated plants The de-crease in the transcript abundance of photosynthesis-related genes down-regulated under 1-d cold treatment2-d cold treatment and 3-d dehydration treatment was316 447 and 754 respectively

We focused on the cold- and dehydration-responsivegenes in the three selected representative molecularclasses (photosynthesis helicases and dehydrinLEAproteins) and used heat maps to illustrate the log ratioof these cold- or dehydration-responsive genes (Fig 4)Photosystem I subunit (psa) and photosystem II subunit(psb) encode subunits of PSI and PSII respectivelypsaE psaF psaG psaH psbP psbQ and psbY were sig-nificantly down-regulated in rice plants subjected to 1-dcold treatment 2-d cold treatment and 3-d dehydrationtreatment photosystem and electron transport system

E (plastocyanin) ferredoxin and cytochrome c6 werealso significantly down-regulated in rice plants sub-jected to these treatments Compared with the expres-sion levels in untreated plants those levels in plantssubjected to 2-d dehydration treatment were notsignificantly lower (Fig 4A) Helicases are classifiedinto three superfamilies superfamily (SF) 1 SF2 andSF3 (Gorbalenya and Koonin 1993) SF2 includes the

Figure 4 Molecular functions of cold-responsive and dehydration-responsive genes in rice plants Heat maps of up-regulated (red) ordown-regulated (blue) molecular functional classes indicate relativeabundance ( shading in boxes) in cold-treated (C1 and C2) anddehydration-treated (D2 and D3) plants Heat maps illustrate the logratios of cold- or dehydration-responsive genes in representative mo-lecular classes of photosynthesis (A) helicases (B) and dehydrinLEAproteins (C) psaE PSI subunit IV psaF PSI subunit III psaG PSIsubunit V psaH PSI subunit VI psbP PSII oxygen-evolving enhancerprotein 2 psbQ PSII oxygen-evolving enhancer protein 3 psbY PSIIPsbY protein


Maruyama et al


DEADDEAH box helicases which contain two con-served domains DEADc (PF00270) and Helicase con-served C-terminal domains (PF00271) There are twotypes of DEADDEAH box helicases only the type Iprotein has the helicase-associated domain 2 (PF04408)Representative members of DEADDEAH box heli-cases are shown in Figure 4B The transcript levels ofgenes encoding several members of DEADDEAH boxhelicases were increased under cold stress The levels oftheir corresponding transcripts in cold-treated plantswere higher than those levels in dehydration-treatedplants The transcript level of Os06g0697200 was sig-nificantly higher in both cold- and dehydration-treated plants than in untreated plants and higher indehydration- than cold-treated plants Representativemembers of the dehydrinLEA family are shown inFigure 4C LEA proteins are classified into six groupsin the Pfam database Many LEA proteins were in-duced by dehydration The transcript levels of genesencoding LEA proteins were higher in dehydration-than cold-treated plants For example whereas thetranscript level of Oryza sativa early methionine(Os05g0349800)was significantly higher in dehydration-treated plants than in untreated plants the level of thistranscript was lower in cold-treated plants than in un-treated plants The transcript level of Os01g0705200 wassignificantly higher in both cold- and dehydration-treated plants than in untreated plants and higher indehydration- than cold-treated plants

Expressions of Genes Involved in Carbohydrateand Amino Acid Metabolism

Our metabolome analyses revealed that the levelsof monosaccharides were significantly higher in bothcold- and dehydration-treated rice plants than in un-treated plants (Fig 1C) To predict candidate genesimportant for carbohydrate metabolism under coldor dehydration conditions we screened cold- anddehydration-responsive genes for those encoding en-zymes involved with starch degradation Suc metab-olism gluconeogenesis and the glyoxylate cycle (Fig 5AndashD Supplemental Figs S4ndashS6) The abundances ofseveral of the transcripts encoding starch degradationenzymes (eg a-amylase and b-amylase) were ele-vated in rice plants after exposure to cold or dehy-dration (Fig 5A Supplemental Fig S4) In particularthe levels of the b-amylase (Os03g0141200) tran-script were significantly up-regulated under both coldand dehydration conditions (Fig 5A SupplementalFig S4) The transcript level of the gene encodinga-glucanphosphorylase was increased under cold condi-tions but decreased under dehydration conditions com-pared with the transcript level in untreated plants (Fig 5ASupplemental Fig S4) Of the genes associated with Sucmetabolism several encoding alkalineneutral inver-tases showed increased transcript levels under bothcold and dehydration conditions (Fig 5B SupplementalFig S5) Among the genes encoding alkalineneutral

invertases the Os01g0332100 transcript was pre-sent at 10-fold higher levels in both cold-treated anddehydration-treated rice plants than in untreated plants(Fig 5B Supplemental Fig S5) Gluconeogenesis is animportant pathway for the synthesis of Glc from or-ganic acids and the glyoxylate cycle is an importantsource of the four-carbon compound succinate whichcan enter the gluconeogenesis pathway Phosphoenol-pyruvate carboxykinase and Fru-16-bisphosphataseplay key roles in gluconeogenesis (Rylott et al 2003)Expressions of the genes encoding phosphoenolpyruvatecarboxykinase and Fru-16-bisphosphatase did not in-crease markedly in either cold-treated or dehydration-treated rice plants (Supplemental Tables S12ndashS15)Conversely the levels of transcripts encoding isocitratelyase and malate synthase both key enzymes in theglyoxylate cycle increased significantly after dehydra-tion (Fig 5C Supplemental Fig S6) We confirmed thetranscript levels of isocitrate lyase and malate synthasegenes by quantitative real-time (qRT)-PCR The highestlevels of both transcripts were in plants subjected to the3-d dehydration treatment (Fig 5G) We also charac-terized the increases in raffinose Pro and BCAAs andscreened cold- andor dehydration-responsive genesfor those genes involved in the biosynthesis of galac-tinol raffinose and BCAAs and metabolism of Pro (Fig 5DndashF Supplemental Figs S7ndashS9) The levels of mostraffinose synthase-related transcripts increased underdehydration conditions but decreased under cold con-ditions (Fig 5D Supplemental Fig S7) The genesWSI76 (Os07g0687900) and Oryza sativa galactinolsynthase1 (OsGolS1 Os03g0316200) encode galactinolsynthases however probes corresponding to both ofthese genes were not included in our unique micro-array slide We analyzed the transcript levels of thesegenes using qRT-PCR The WSI76 transcript level in-creased significantly under dehydration conditions andincreased slightly under cold conditions The OsGolS1transcript level increased significantly under dehydrationconditions but did not increase under cold conditions(Fig 5H) The transcript level of OsP5CS (Os05g0455500)which encodes the Pro biosynthetic enzyme D1-pyrroline-5-carboxylate synthase increased under dehydrationconditions (Fig 5E Supplemental Fig S8) Branchedchain aminotransferase catalyzes the last step of BCAAbiosynthesis Expression of the Os03g0231600 a branchedchain aminotransferase family gene was induced by bothcold and dehydration (Fig 5F Supplemental Fig S9)

Expression of Genes Involved in ABA and CK Metabolism

Our phytohormone analysis indicated that exposure tocold or dehydration increased ABA levels in rice plantsCold treatment increased the level of iP and cold anddehydration treatments decreased the level of tZRPs(Fig 2B) We also investigated the expression of genesinvolved inABAandCKbiosyntheses under dehydrationand cold conditions (Fig 6 AndashC Supplemental Fig S10)Theenzyme9-cis-epoxycarotenoiddioxygenase (NCED)




which converts 9-cis-neoxanthin and 9-cis-violaxanthininto xanthoxin plays a key role in ABA biosynthesis(Schwartz et al 1997) Among the genes encoding NCED

familymembersOsNCED1 (Os03g0645900) andOsNCED3(Os07g0154100) showed significantly increased transcriptlevels in both cold- and dehydration-treated plants (Fig 6A

Figure 5 Carbohydrate and amino acid metabolic pathways A Starch degradation B Suc metabolism C Glyoxylate cycleD Raffinose biosynthesis E Pro metabolism F BCAA biosynthesis Heat maps illustrate transcript levels of representative cold-and dehydration-responsive genes A small gray square indicates the absence of a probe for that gene in the oligonucleotidemicroarray Heat maps illustrate accumulated levels of representative cold- and dehydration-responsive metabolites G Levelsof transcripts for genes encoding isocitrate lyase and malate synthase determined by qRT-PCR H Transcript levels of OsGolS1and WSI76 both encoding galactinol synthase Mean values are shown (n = 3 experiments) Error bars indicate SD AAMYa-Amylase ACO aconitase hydratase AHAS acetolactate synthase AN-Invs alkalineneutral invertase BAMY b-amylaseBCAT branched chain amino acid aminotransferase CS citrate synthase CWI apoplastic invertase F6P fructose 6-phosphateGP a-glucanphosphorylase GSA galactinol synthase A GWD glucan water dikinase HK hexokinase ICL isocitrate lyaseMDH malate dehydrogenase Mos malto-oligosaccharides MS malate synthase ProDH Pro dehydrogenase P5C D1-pyrroline-5-carboxylate P5CDH D1-pyrroline-5-carboxylate dehydrogenase P5CR D1-pyrroline-5-carboxylate reductase P5CS D1-pyrroline-5-carboxylate synthetase RS raffinose synthase SPS Suc-P synthase Sus Suc synthase S6P sucrose-6-phosphate TD Thrdehydratase UDPG UDP-galactose


Maruyama et al


SupplementalFigS10)ThemajorABAcatabolic pathwayis thought to involve hydroxylation of ABA to yield89-hydroxy-ABAby cytochromeP450 (CYP) 707A followedby the spontaneous isomerization of 89-hydroxy-ABAinto phaseic acid (Krochko et al 1998 Saito et al 2004)The transcript level of ABA8OX1 (Os02g0703600) aCYP707A family member was significantly higher inboth cold- and dehydration-treated rice plants than inuntreatedplants (Fig 6A)Comparedwith those levels inuntreated plants the levels ofABA8OX2 (Os08g0472800)and ABA8OX3 (Os09g0457100) transcripts were sig-nificantly higher indehydration- and cold-treatedplantsrespectively (Fig 6A Supplemental Fig S10) Thegenes CYP735A3 (Os08g0429800) and CYP735A4(Os09g0403300) encode CK transhydroxylases The tran-script level of CYP735A3was significantly lower in bothcold- and dehydration-treated plants than in untreatedplants (Fig 6B Supplemental Fig S10)However a probecorresponding to the CYP735A4 gene was not included inour unique microarray slide We analyzed the transcriptlevels of CYP735A4 and CYP735A3 by qRT-PCR andfound that both showed significant decreases under coldand dehydration conditions (Fig 6C)

To clarify the relationship between the accumulationof phytohormones and phytohormone-responsive geneexpression under cold or dehydration conditions weanalyzed the transcript levels of genes that are knownto be ABA and CK responsive (Fig 6D) Several genesfor LEA proteins are known to be involved in abioticstress tolerance in plants and OsLEA14WSI18 andOsLEA3 were reported to be ABA responsive (Moonset al 1997 Oh et al 2005) We found that OsLEA14WSI18 and OsLEA3 were induced by cold and dehy-dration The His-Asp phosphorelay system which con-sists of a sensor His protein kinase a His-containingphosphotransfer protein and a response regulatorplays important roles in CK signaling There are 13 typeA response regulators in rice all of which are up-regulated in response to CKs (Ito and Kurata 2006Jain et al 2006 Hirose et al 2007) However wefound that the CK-inducible marker genes Oryza sativaresponse regulator1 (OsRR1) and OsRR2 were down-regulated under cold conditions and dehydration con-ditions (Fig 6D)

Next we compared the transcriptional patterns ofgenes induced by ABA and CKs in rice plants withthose patterns of their counterparts in Arabidopsis Weanalyzed an Arabidopsis oligomicroarray and evalu-ated the transcript levels of the counterparts of thegenes identified in rice in Arabidopsis plants TheABA-inducible RD17 and RD29A genes were up-regulated in Arabidopsis after exposure to cold or de-hydration Conversely the CK-inducible ArabidopsisFigure 6 Pathways for ABA and CK biosyntheses A ABA biosynthesis

B CK biosynthesis C Levels of transcripts for genes encoding CYP735Asdetermined by qRT-PCR D Expression of genes encoding LEAs andOsRRs Heat maps illustrate transcript levels of representative cold- anddehydration-responsive genes A small gray square denotes the absenceof a probe for that gene in oligonucleotide microarray We referred tohttpricexprodnaaffrcgojp for expression of LEA andOsRR in ABA- orCK-treated rice plants Heat maps illustrate accumulated levels of rep-resentative cold-responsive and dehydration-responsive phytohormones

CKX Cytokinin oxidasedehydrogenase DMAPP dimethylallyl diphos-phate IPT adenosine P-isopentenyltransferase LOG lonely guy cyto-kinin NMP phosphoribohydrolase NSY neoxanthin synthase tRNA-IPTtRNA isopentenyltransferase




response regulator5 (ARR5) and ARR6 genes weredown-regulated by dehydration or ABA treatment(Supplemental Fig S11 Supplemental Tables S20and S21)

DISCUSSION

We performed an integrated analysis of the effects ofcold or dehydration stress on the levels of metabolitesphytohormones and gene transcripts in rice plants andcharacterized the events associated with changes intheir levels This analysis enabled us to identify im-portant genes encoding metabolic enzymes for carbo-hydrates amino acids and phytohormones that areinvolved in the responsesadaptation of rice plants tocold or dehydration In rice plants subjected to cold ordehydration treatments the expression of several genesencoding enzymes involved in starch degradation Sucmetabolism and the glyoxylate cycle increased dy-namically and these changes were correlated with theaccumulation of Glc Fru and Suc (Figs 1C and 5 AndashC)We predicted that Os03g0141200 which encodesb-amylase andOs01g0332100which encodes analkalineneutral invertase were important for starch degradationand Suc metabolism respectively in both cold- anddehydration-treated plants Their transcript levels weremore than 5-fold higher in both cold- and dehydration-treated plants than in untreated plants (Fig 5 A and BSupplemental Tables S11ndashS16) The high transcript levelsof both of these genes were correlated with Glc accu-mulation A similar relationship between high transcriptlevels of the counterparts of these genes and increasedlevels of Glc has been reported in cold- or dehydration-treated Arabidopsis plants (Kaplan and Guy 2004Kaplan et al 2007 Maruyama et al 2009)

Isocitrate lyase and malate synthase are key enzymesin the glyoxylate cycle and the expression levels of theirencoding genes play a role in activating gluconeogen-esis during postgerminative growth and plant patho-genesis (Eastmond et al 2000 Dunn et al 2009) Wepredicted that isocitrate lyase (Os07g0529000) andmalate synthase (Os04g0486950) would also play im-portant roles under dehydration conditions in rice Wedetected increased levels of isocitrate lyase and malatesynthase transcripts in rice plants subjected to dehy-dration treatments (20 times higher than those levelsin untreated plants Fig 5 C and G) The high expres-sion levels of these genes seemed to result in Glc accu-mulation (Figs 1C and 5 C and G) A query of the geneexpression database Genevestigator (Hruz et al 2008)confirmed that the isocitrate lyase and malate synthasegenes show increased expression under dehydrationconditions in several rice cultivars In contrast expres-sion of their counterparts in Arabidopsis plants doesnot increase after dehydration (Supplemental Fig S12Supplemental Table S21) These data imply that regu-lation of the glyoxylate cycle may be involved in Glcaccumulation in response to dehydration in rice but notArabidopsis

The Arabidopsis galactinol synthase family containseight genes Among them Arabidopsis thaliana galactinolsynthase3 (AtGolS3) and AtGolS2 are significantly up-regulated in cold- and dehydration-treated Arabidopsisplants respectively (Taji et al 2002 Maruyama et al2009) Raffinose probably acts as an osmoprotectant tostabilize cellular membranes and a scavenger of reactiveoxygen species to protect the photosynthetic complex inchloroplasts of Arabidopsis exposed to cold or dehy-dration (Nishizawa et al 2008) Transgenic Arabidopsisplants overexpressing a gene encoding galactinol syn-thase accumulated more endogenous raffinose andexhibited improved dehydration tolerance relative towild-type plants Hence genes for galactinol synthaselikely play a key role in increasing levels of endogenousraffinose under cold or dehydration conditions in Ara-bidopsis (Taji et al 2002) Our current analysis revealedcomparable increases in the levels of raffinose after ex-posure of rice plants to cold or dehydration (Fig 1C) Inrice galactinol synthase is encoded by only two genesWSI76 and OsGolS1 The WSI76 transcript level in-creased significantly in plants exposed to dehydrationbut increased only slightly after exposure to cold TheOsGolS1 transcript level increased significantly afterdehydration but not after cold treatment (Fig 5H)These data suggest that the roles of galactinol synthasegenes in raffinose accumulation under cold conditionsdiffer between rice and Arabidopsis

We identified tZRPs and iP as representative phy-tohormones involved in the responses of rice plants tocold or dehydration (Fig 2) Regarding changes in CKlevels the level of tZRPs decreased significantly in riceplants subjected to cold or dehydration treatmentsand tZ was not detected in any of the plants regard-less of the treatment Conversely the level of iP wassignificantly higher in cold-treated rice plants andslightly higher in dehydration-treated plants than incontrol plants (Figs 2B and 6B) In rice the transcriptlevels of CK-inducible genes encoding OsRR decreasedafter exposure to either cold or dehydration (Fig 6C)These results suggest that the CK-dependent tran-scription pathway is inactive in both cold-treated anddehydration-treated rice plants and that an increasedlevel of endogenous iP cannot activate the CK-dependenttranscription pathway under cold or dehydration stressWe predicted that CYP735A3 (Os08g0429800) andCYP735A4 (Os09g0403300) both of which encode CKtranshydroxylases play important roles in rice plantsunder cold or dehydration stress (Fig 6C) the low levelsof both transcripts were correlated with decreased levelsof tZRPs The decreased levels of tZRPs associated withthe down-regulation of CYP735A3 and CYP735A4 maybe related to growth retardation of rice plants undercold or dehydration conditions In Arabidopsis thetranscript levels of CYP735A1 (At5g38450) and CYP735A2(At1g67110) which encode CK transhydroxylases wereunchanged in plants subjected to cold or dehydrationtreatments However the CK levels decreased signifi-cantly in Arabidopsis plants subjected to cold or dehy-dration treatments (Supplemental Fig S13 Supplemental


Maruyama et al


Tables S20 and S21) These data imply that the roles ofthe CK transhydroxylase in the decreased level of CKsunder cold or dehydration conditions differ betweenArabidopsis and rice Transcriptional regulation ofCYP735A genes might be involved in regulating CKlevels under cold and dehydration conditions in ricebut not ArabidopsisIn Arabidopsis five NCEDs (NCED2 NCED3

NCED5 NCED6 and NCED9) are probably involvedin ABA biosynthesis (Iuchi et al 2001 Tan et al 2003)Among these proteins NCED3 is the one moststrongly induced by dehydration (Iuchi et al 2001)Transgenic Arabidopsis plants overexpressing NCED3show increased tolerance to dehydration whereas theNCED3 knockout mutant shows enhanced sensitivityto dehydration indicating that NCED3 plays a key rolein ABA biosynthesis in Arabidopsis exposed to dehy-dration (Iuchi et al 2001) Compared with the level incontrol plants the level of ABA in dehydrated riceplants was significantly higher whereas the level incold-treated plants was only slightly higher (Fig 2B)Rice harbors three OsNCED genes of these genesOsNCED1 and OsNCED3 showed expression levelsthat were correlated with the ABA level in both cold-and dehydration-treated plants (Figs 2B and 6A) Inaddition ABA-induced expression of LEA genes in-creased in rice plants exposed to cold or dehydration(Fig 6D) These results imply that OsNCED1 andOsNCED3 are key candidates for ABA biosynthesisand that the ABA-dependent transcription pathway isactive in rice plants exposed to cold or dehydrationCKs and ABA are involved in regulating several

processes in plant growth and development undervarious conditions including water stress AlthoughCKs are considered to antagonize ABA in dehydration-stressed Arabidopsis (Nishiyama et al 2011 Wanget al 2011) little is known about the cross talk betweenCK signaling and ABA signaling during water stress inrice Our integrated analysis of phytohormones andgene transcription indicated that there is an inverserelationship between CK signaling and ABA signalingin both rice and Arabidopsis plants exposed to dehy-dration stress (Figs 2B and 6D Supplemental Figs S1and S11) These results imply that the cross talkbetween CKs and ABA is fundamental to signal trans-duction in both rice and Arabidopsis under dehydra-tion conditions and that this cross talk evolved duringland colonization of plants before the divergence ofmonocots and dicots

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Wild-type rice (Oryza sativa lsquoNipponbarersquo) seedlings were grown in plasticpots filled with nutrient soil for 2 weeks under flooded lowland conditionsunder a 12-h-light (28degC)12-h-dark (25degC) photoperiod (approximately 1500mmol photons m22 s21) The plants were then subjected to (1) cold treatmentby transfer from 28degC to 10degC growing conditions for either 1 or 2 d or (2)dehydration treatment by growing them at 28degC without watering for either2 d until the soil moisture content was 187 (ww SD = 13 570 field

capacity) or 3 d until the soil moisture content was 156 (ww SD = 11476 field capacity) Wild-type Arabidopsis (Arabidopsis thaliana) plants weregrown in plastic pots filled with peat moss for 3 weeks (principal growthstage = 107ndash108) under a 16-h-light8-h-dark photoperiod (50 6 10 mmolphotons m22 s21) at 22degC The plants were then subjected to (1) a cold treat-ment by transfer from 28degC to 4degC with growth for 1 d or (2) a dehydrationtreatment by withholding water for 6 d at 28degC until the soil moisture contentdecreased to less than 10 (ww 472 field capacity)

GC-TOF-MS Analysis

Metabolites were extracted from the aerial parts of rice plants (100 mg persample) with methanol Extraction and derivatization were performed asdescribed previously (Kusano et al 2007) Metabolites were detected using aGC instrument (model 6890 Agilent Technologies) fitted with a 30-m DB-17ms column (025-mm id 025-mm film Agilent Technologies) coupled toa TOF mass spectrometer (Leco) Ribitol was used as the internal standard(Kusano et al 2007) The reproducibility of GC-TOF-MS analysis was assessedwith three biological replicates for each experiment

CE-MS Analysis

Metabolites were extracted from aerial parts of rice plants (100 mg) with achloroformmethanol solution (11) using a mixer and a 5-kD cutoff filter(Millipore) Extracted metabolites were detected using a CE-MS system con-sisting of an Agilent 1100 series MSD mass spectrometer an Agilent 1100series isocratic HPLC pump a G1603A Agilent CE-MS adapter kit and aG1607A Agilent CE-MS sprayer kit Met sulphone was used as an internalstandard The reproducibility of CE-MS analysis was determined using threebiological replicates in each experiment Unknown metabolites were quanti-fied using CE-MS

LC-Coupled MS

The levels of phytohormones in aerial parts of rice plants (100 mg) werequantified as described previously (Kojima et al 2009) using an LC-MS system(UPLCQuattro Premier XE Waters) fitted with an ODS column (17 mm21 3 100 mm AQUITY-UPLC BEH-C 18 Waters) Reproducibility wasassessed using three biological replicates in each experiment

Microarray Analysis

Total RNAs were isolated from aerial parts of rice or Arabidopsis plants andlabeled using Low RNA Input Linear AmplificationLabeling kit reagents(Agilent Technologies) Cy5-labeled complementary RNA experimental sam-ples and Cy3-labeled complementary RNA control samples were hybridized tothe microarray chips Biological and technical (dye-swap) replicates of thesample sets were analyzed After hybridization microarray slides werescanned (scanner model G2505C with scan control software version A851Agilent Technologies) and the data were analyzed using Feature Extractionsoftware version 101011 (Agilent Technologies) Raw data were analyzedusing GeneSpringGX software version 120 (Agilent Technologies) Expressionlog ratios and the Benjamini and Hochberg FDR P values were calculated usingGeneSpring GX (Maruyama et al 2014) The microarray design and datawere deposited at MIAMExpress (accession nos E-MEXP-3863 E-MEXP-3864E-MEXP-3865 E-MEXP-3862 E-MEXP-3999 and E-MEXP-4000)

Supplemental Data

The following materials are available in the online version of this article

Supplemental Figure S1 Levels of ABA and CKs after cold or dehydrationstress in Arabidopsis plants

Supplemental Figure S2 Rice genes in the rice annotation project databaseand our unique oligonucleotide microarray

Supplemental Figure S3 Molecular function of cold- and dehydration-responsive genes in rice plants

Supplemental Figure S4 Effects of cold or dehydration on transcript levelsof genes encoding starch degradation enzymes




Supplemental Figure S5 Effects of cold or dehydration on transcript levelsof genes encoding Suc metabolism enzymes

Supplemental Figure S6 Effects of cold or dehydration on transcript levelsof genes encoding glyoxylate cycle enzymes

Supplemental Figure S7 Effects of cold or dehydration on transcript levelsof genes encoding raffinose biosynthesis enzymes

Supplemental Figure S8 Effects of cold or dehydration on transcript levelsof genes encoding enzymes involved in Pro metabolism

Supplemental Figure S9 Effects of cold or dehydration on transcript levelsof genes encoding enzymes involved in BCAAs synthesis

Supplemental Figure S10 Effects of cold or dehydration on transcriptlevels of representative cold- and dehydration-responsive genes in-volved in ABA and CK biosynthesis

Supplemental Figure S11 Transcript levels of genes encoding rd17 rd29Aand ARRs

Supplemental Figure S12 Glyoxylate cycle in Arabidopsis

Supplemental Figure S13 CK biosynthesis in Arabidopsis

Supplemental Table S1 Mean and SD values for metabolites

Supplemental Table S2 Variance covariance matrix for metabolites

Supplemental Table S3 Eigenvalues for metabolites

Supplemental Table S4 Eigenvectors for metabolites

Supplemental Table S5 PC scores for metabolites

Supplemental Table S6 Mean and SD values for phytohormones in rice

Supplemental Table S7 Variance covariance matrix for phytohormones

Supplemental Table S8 Eigenvalues for phytohormones

Supplemental Table S9 Eigenvectors for phytohormones

Supplemental Table S10 Principal component scores for phytohormones

Supplemental Table S11 Mean and SD for phytohormones in Arabidopsis

Supplemental Table S12 Cold-treated rice plants (1 d)


Supplemental Table S14 Dehydration-treated rice plants (2 d)


Supplemental Table S16 Variance covariance matrix for transcripts

Supplemental Table S17 Eigenvalues for transcripts

Supplemental Table S18 Eigenvectors for transcripts

Supplemental Table S19 PC scores for transcripts

Supplemental Table S20 Cold-treated Arabidopsis plants (1 d)

Supplemental Table S21 Dehydration-treated Arabidopsis plants (6 d)

ACKNOWLEDGMENTS

We thank the Rice Genome Resource Center at The National Institute ofAgrobiological Sciences for use of the microarray analysis system and thetechnical support provided by Drs Yoshiaki Nagamura and Ritsuko MotoyamaWe thank Masami Toyoshima for skillful editorial assistance

Received November 1 2013 accepted February 6 2014 published February10 2014

LITERATURE CITED

Bartels D Sunkar R (2005) Drought and salt tolerance in plants Crit RevPlant Sci 24 23ndash58

Bowne JB Erwin TA Juttner J Schnurbusch T Langridge P Bacic ARoessner U (2012) Drought responses of leaf tissues from wheat

cultivars of differing drought tolerance at the metabolite level Mol Plant5 418ndash429

Chiwocha SD Abrams SR Ambrose SJ Cutler AJ Loewen M Ross ARKermode AR (2003) A method for profiling classes of plant hormonesand their metabolites using liquid chromatography-electrospray ioni-zation tandem mass spectrometry an analysis of hormone regulationof thermodormancy of lettuce (Lactuca sativa L) seeds Plant J 35 405ndash417

Cook D Fowler S Fiehn O Thomashow MF (2004) A prominent role forthe CBF cold response pathway in configuring the low-temperaturemetabolome of Arabidopsis Proc Natl Acad Sci USA 101 15243ndash15248

Cutler SR Rodriguez PL Finkelstein RR Abrams SR (2010) Abscisicacid emergence of a core signaling network Annu Rev Plant Biol 61 651ndash679

Dunn MF Ramiacuterez-Trujillo JA Hernaacutendez-Lucas I (2009) Major roles ofisocitrate lyase and malate synthase in bacterial and fungal pathogen-esis Microbiology 155 3166ndash3175

Eastmond PJ Germain V Lange PR Bryce JH Smith SM Graham IA(2000) Postgerminative growth and lipid catabolism in oilseeds lackingthe glyoxylate cycle Proc Natl Acad Sci USA 97 5669ndash5674

Fujita Y Fujita M Shinozaki K Yamaguchi-Shinozaki K (2011) ABA-mediated transcriptional regulation in response to osmotic stress inplants J Plant Res 124 509ndash525

Gorbalenya AE Koonin EV (1993) Helicases amino acid sequence comparisonsand structure-function relationships Curr Opin Struct Biol 3 419ndash429

Gray GR Heath D (2005) A global reorganization of the metabolome inArabidopsis during cold acclimation is revealed by metabolic finger-printing Physiol Plant 124 236ndash248

Guy C Kaplan F Kopka J Selbig J Hincha DK (2008) Metabolomics oftemperature stress Physiol Plant 132 220ndash235

Ha S Vankova R Yamaguchi-Shinozaki K Shinozaki K Tran LS (2012)Cytokinins metabolism and function in plant adaptation to environ-mental stresses Trends Plant Sci 17 172ndash179

Hannah MA Wiese D Freund S Fiehn O Heyer AG Hincha DK (2006)Natural genetic variation of freezing tolerance in Arabidopsis PlantPhysiol 142 98ndash112

Hirayama T Shinozaki K (2007) Perception and transduction of abscisicacid signals keys to the function of the versatile plant hormone ABATrends Plant Sci 12 343ndash351

Hirose N Makita N Kojima M Kamada-Nobusada T Sakakibara H(2007) Overexpression of a type-A response regulator alters rice mor-phology and cytokinin metabolism Plant Cell Physiol 48 523ndash539

Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L WidmayerP Gruissem W Zimmermann P (2008) Genevestigator v3 a referenceexpression database for the meta-analysis of transcriptomes Adv Bio-informa 2008 420747

Ito Y Kurata N (2006) Identification and characterization of cytokinin-signalling gene families in rice Gene 382 57ndash65

Iuchi S Kobayashi M Taji T Naramoto M Seki M Kato T Tabata SKakubari Y Yamaguchi-Shinozaki K Shinozaki K (2001) Regulationof drought tolerance by gene manipulation of 9-cis-epoxycarotenoiddioxygenase a key enzyme in abscisic acid biosynthesis in ArabidopsisPlant J 27 325ndash333

Jain M Tyagi AK Khurana JP (2006) Molecular characterization and dif-ferential expression of cytokinin-responsive type-A response regulatorsin rice (Oryza sativa) BMC Plant Biol 6 1

Kaplan F Guy CL (2004) b-Amylase induction and the protective role ofmaltose during temperature shock Plant Physiol 135 1674ndash1684

Kaplan F Kopka J Sung DY Zhao W Popp M Porat R Guy CL (2007)Transcript and metabolite profiling during cold acclimation of Arabi-dopsis reveals an intricate relationship of cold-regulated gene expres-sion with modifications in metabolite content Plant J 50 967ndash981

Kilian J Whitehead D Horak J WankeDWeinl S Batistic O DrsquoAngelo CBornberg-Bauer E Kudla J Harter K (2007) The AtGenExpress globalstress expression data set protocols evaluation and model data analysisof UV-B light drought and cold stress responses Plant J 50 347ndash363

Kojima M Kamada-Nobusada T Komatsu H Takei K Kuroha TMizutani M Ashikari M Ueguchi-Tanaka M Matsuoka M Suzuki Ket al (2009) Highly sensitive and high-throughput analysis of planthormones using MS-probe modification and liquid chromatography-tandem mass spectrometry an application for hormone profiling inOryza sativa Plant Cell Physiol 50 1201ndash1214


Maruyama et al


Krochko JE Abrams GD Loewen MK Abrams SR Cutler AJ (1998)(+)-Abscisic acid 89-hydroxylase is a cytochrome P450 monooxygenasePlant Physiol 118 849ndash860

Kusano M Fukushima A Arita M Jonsson P Moritz T Kobayashi MHayashi N Tohge T Saito K (2007) Unbiased characterization ofgenotype-dependent metabolic regulations by metabolomic approach inArabidopsis thaliana BMC Syst Biol 1 53

Levitt J (1980) Responses of Plants to Environmental Stress Ed 2 AcademicPress New York

Maruyama K Takeda M Kidokoro S Yamada K Sakuma Y Urano KFujita M Yoshiwara K Matsukura S Morishita Y et al (2009) Meta-bolic pathways involved in cold acclimation identified by integratedanalysis of metabolites and transcripts regulated by DREB1A andDREB2A Plant Physiol 150 1972ndash1980

Maruyama K Todaka D Mizoi J Yoshida T Kidokoro S Matsukura STakasakiH Sakurai T YamamotoYYYoshiwaraK et al (2012) Identificationof cis-acting promoter elements in cold- and dehydration-induced transcrip-tional pathways in Arabidopsis rice and soybean DNA Res 19 37ndash49

Maruyama K Yamaguchi-Shinozaki K Shinozaki K (2014) Gene ex-pression profiling using DNA microarrays In JJ Sanchez-SerranoJ Salinas eds Arabidopsis Protocols Ed 3 Vol 1062 Humana PressNew York pp 381ndash391

Mok MC Martin RC Mok DWS (2000) Cytokinins biosynthesis metab-olism and perception In Vitro Cell Dev Biol Plant 36 102ndash107

Moons A De Keyser A Van Montagu M (1997) A group 3 LEA cDNA ofrice responsive to abscisic acid but not to jasmonic acid shows variety-specific differences in salt stress response Gene 191 197ndash204

Muumlller A Duumlchting P Weiler EW (2002) A multiplex GC-MSMS tech-nique for the sensitive and quantitative single-run analysis of acidicphytohormones and related compounds and its application to Arabi-dopsis thaliana Planta 216 44ndash56

Nishiyama R Watanabe Y Fujita Y Le DT KojimaMWerner T Vankova RYamaguchi-Shinozaki K Shinozaki K Kakimoto T et al (2011) Analysis ofcytokinin mutants and regulation of cytokinin metabolic genes revealsimportant regulatory roles of cytokinins in drought salt and abscisic acidresponses and abscisic acid biosynthesis Plant Cell 23 2169ndash2183

Nishizawa A Yabuta Y Shigeoka S (2008) Galactinol and raffinose con-stitute a novel function to protect plants from oxidative damage PlantPhysiol 147 1251ndash1263

Obata T Fernie AR (2012) The use of metabolomics to dissect plant re-sponses to abiotic stresses Cell Mol Life Sci 69 3225ndash3243

Oh SJ Song SI Kim YS Jang HJ Kim SY Kim M Kim YK Nahm BHKim JK (2005) Arabidopsis CBF3DREB1A and ABF3 in transgenic riceincreased tolerance to abiotic stress without stunting growth PlantPhysiol 138 341ndash351

Raghavendra AS Gonugunta VK Christmann A Grill E (2010) ABAperception and signalling Trends Plant Sci 15 395ndash401

Rylott EL Gilday AD Graham IA (2003) The gluconeogenic enzymephosphoenolpyruvate carboxykinase in Arabidopsis is essential forseedling establishment Plant Physiol 131 1834ndash1842

Saito K Matsuda F (2010) Metabolomics for functional genomics systemsbiology and biotechnology Annu Rev Plant Biol 61 463ndash489

Saito S Hirai N Matsumoto C Ohigashi H Ohta D Sakata K MizutaniM (2004) Arabidopsis CYP707As encode (+)-abscisic acid 89-hydroxylase akey enzyme in the oxidative catabolism of abscisic acid Plant Physiol 1341439ndash1449

Sakakibara H (2006) Cytokinins activity biosynthesis and translocationAnnu Rev Plant Biol 57 431ndash449

Sanchez DH Schwabe F Erban A Udvardi MK Kopka J (2012) Com-parative metabolomics of drought acclimation in model and forage le-gumes Plant Cell Environ 35 136ndash149

Schwartz SH Tan BC Gage DA Zeevaart JA McCarty DR (1997) Specificoxidative cleavage of carotenoids by VP14 of maize Science 276 1872ndash1874

Shinozaki K Yamaguchi-Shinozaki K (2007) Gene networks involved indrought stress response and tolerance J Exp Bot 58 221ndash227

Sicher RC Barnaby JY (2012) Impact of carbon dioxide enrichment on theresponses of maize leaf transcripts and metabolites to water stressPhysiol Plant 144 238ndash253

Skirycz A De Bodt S Obata T De Clercq I Claeys H De Rycke RAndriankaja M Van Aken O Van Breusegem F Fernie AR et al(2010) Developmental stage specificity and the role of mitochondrialmetabolism in the response of Arabidopsis leaves to prolonged mildosmotic stress Plant Physiol 152 226ndash244

Taji T Ohsumi C Iuchi S Seki M Kasuga M Kobayashi M Yamaguchi-Shinozaki K Shinozaki K (2002) Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsisthaliana Plant J 29 417ndash426

Tan BC Joseph LM Deng WT Liu L Li QB Cline K McCarty DR (2003)Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid di-oxygenase gene family Plant J 35 44ndash56

Urano K Kurihara Y Seki M Shinozaki K (2010) lsquoOmicsrsquo analyses ofregulatory networks in plant abiotic stress responses Curr Opin PlantBiol 13 132ndash138

Urano K Maruyama K Ogata Y Morishita Y Takeda M Sakurai NSuzuki H Saito K Shibata D Kobayashi M et al (2009) Characteri-zation of the ABA-regulated global responses to dehydration in Arabi-dopsis by metabolomics Plant J 57 1065ndash1078

Verslues PE Juenger TE (2011) Drought metabolites and Arabidopsisnatural variation a promising combination for understanding adapta-tion to water-limited environments Curr Opin Plant Biol 14 240ndash245

Wang Y Li L Ye T Zhao S Liu Z Feng YQ Wu Y (2011) Cytokinin an-tagonizes ABA suppression to seed germination of Arabidopsis bydownregulating ABI5 expression Plant J 68 249ndash261

Weiner JJ Peterson FC Volkman BF Cutler SR (2010) Structural andfunctional insights into core ABA signaling Curr Opin Plant Biol 13495ndash502

Yamaguchi-Shinozaki K Shinozaki K (2006) Transcriptional regulatorynetworks in cellular responses and tolerance to dehydration and coldstresses Annu Rev Plant Biol 57 781ndash803




Fernie 2012) Targeted analyses of phytohormoneshave been used to characterize signaling and metabolicnetworks (Muumlller et al 2002 Chiwocha et al 2003Kojima et al 2009) For example abscisic acid (ABA)plays an essential role in the response to water stress(Yamaguchi-Shinozaki and Shinozaki 2006 Hirayamaand Shinozaki 2007 Cutler et al 2010) and an increasein endogenous ABA is required for certain water stressresponses designated as ABA-dependent stress re-sponses (Raghavendra et al 2010 Weiner et al 2010Fujita et al 2011) Cytokinins (CKs) play major roles inseveral developmental and physiological processes inplants (for example in cell division regulation ofshoot growth and stress responses Mok et al 2000Sakakibara 2006 Ha et al 2012) Microarray-basedtranscriptomic analyses have identified many genesinvolved in the responses and tolerance to variousstresses in several plant species These genes encodemetabolic enzymes late embryogenesis-abundant(LEA) proteins detoxification enzymes chaperonesprotein kinases transcription factors and other geneproducts (Kilian et al 2007 Shinozaki and Yamaguchi-Shinozaki 2007 Cutler et al 2010 Maruyama et al2012)

Metabolite profiles have been reported for Arabidopsis(Arabidopsis thaliana) under dehydration conditionsThe levels of raffinose family oligosaccharides Probranched-chain amino acids (BCAAs) g-aminobutyratesaccharopine and agmatine were reported to be sig-nificantly higher in dehydration-treated plants thanin untreated plants (Urano et al 2009 Skirycz et al2010 Verslues and Juenger 2011) Increased levels ofseveral metabolites under dehydration conditions werecorrelated with the expression levels of their respectivebiosynthetic genes many of which are regulated byendogenous ABA (Urano et al 2009) Metabolite pro-files have also been determined for crops exposedto dehydration conditions The dehydration-inducedchanges in metabolites observed in Arabidopsis plantswere similar to those reported for wheat (Triticum aes-tivum Bowne et al 2012) legumes (Sanchez et al2012) and maize (Zea mays Sicher and Barnaby 2012)Several research groups have profiled changes in me-tabolite levels in Arabidopsis after exposure to lowtemperatures Metabolite analysis of natural accessionsof Arabidopsis after cold acclimation revealed a clearcorrelation between the accumulation of several carbo-hydrate metabolites (eg Glc Fru and Suc) and coldstress tolerance (Cook et al 2004 Hannah et al 2006)Gray and Heath (2005) used Fourier transform ioncyclotron MS and a nontargeted metabolic finger-printing approach to study the effects of cold accli-mation on the metabolome Gray and Heath (2005)found that a global reprogramming of metabolismoccurs as a result of cold acclimation An integratedanalysis using transgenic Arabidopsis plants over-expressing DRE-binding protein 1AC-Repeat bindingfactor3 which encodes a transcription factor thatfunctions in cold stress responses revealed that tran-scriptional regulation of the carbohydrate network is

necessary for the accumulation of specific carbohy-drates (eg Suc galactinol myoinositol and raffinose)The accumulation of these carbohydrates may be im-portant to improve tolerance to freezing stress in thesetransgenic plants (Cook et al 2004 Maruyama et al2009)

Rice (Oryza sativa lsquoNipponbarersquo) is important notonly as a major crop but also as a model monocot Inthis study we performed an integrated analysis of themetabolites phytohormones and gene transcripts inrice plants subjected to cold or dehydration treatmentsOur aim was to comprehensively survey the molecularresponses of rice to cold or dehydration stimuli Weused three types of MS systems gas chromatography(GC) coupled with time-of-flight MS (GC-TOF-MS)capillary electrophoresis coupled with MS (CE-MS)and liquid chromatography coupled with MS (LC-MS) We identified and characterized representativecold- and dehydration-responsive metabolites andphytohormones We also performed a transcriptomeanalysis using a rice oligonucleotide microarray Weanalyzed metabolite-gene and phytohormone-genecorrelations and identified several genes encodingmetabolic enzymes that might play key roles in theresponses of rice plants to cold or dehydration Wecompared the roles of the identified rice genes with theroles of their counterparts in Arabidopsis under coldor dehydration conditions

RESULTS

Metabolite Profiles of Rice Plants Exposed to Coldor Dehydration

We used GC-TOF-MS and CE-MS to determine theeffects of seven environmental conditions on whole riceseedlings grown in soil for 2 weeks The treatmentswere as follows 1- or 2-d exposure to cold at 10degC 2- or3-d exposure to dehydration without watering and nostress treatment (untreated for 1 2 or 3 d) The GC-TOF-MS and CE-MS analyses identified 152 differentmetabolites according to their retention time indicesand specific mass fragments (Supplemental Table S1)Relative to the levels in untreated plants the levels of38 52 24 and 91 metabolites were significantly in-creased in 1-d cold-treated plants 2-d cold-treated plants2-d dehydration-treated plants and 3-d dehydration-treated plants respectively (Benjamini and Hochbergfalse discovery rate [FDR] P 005) the levels of 26 1226 and eight metabolites respectively were signifi-cantly decreased in plants exposed to the same fourstress conditions (FDR P 005)

We used principal component analysis (PCA) tocompare the metabolite profiles of plants subjected toseven treatments (Fig 1A Supplemental Tables S1ndashS5) The cumulative contribution ratio of the PCA was744 up to the second principal component (PC2)The PCA analysis had two key features The firstprincipal component (PC1) reflected increases in the


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DISCUSSION






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GC-TOF-MS Analysis


CE-MS Analysis


LC-Coupled MS


Microarray Analysis


Supplemental Data







































ACKNOWLEDGMENTS



LITERATURE CITED


























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DISCUSSION






Maruyama et al









GC-TOF-MS Analysis


CE-MS Analysis


LC-Coupled MS


Microarray Analysis


Supplemental Data







































ACKNOWLEDGMENTS



LITERATURE CITED


























Maruyama et al





















































Maruyama et al















Maruyama et al











DISCUSSION






Maruyama et al









GC-TOF-MS Analysis


CE-MS Analysis


LC-Coupled MS


Microarray Analysis


Supplemental Data







































ACKNOWLEDGMENTS



LITERATURE CITED


























Maruyama et al

















































Maruyama et al











DISCUSSION






Maruyama et al









GC-TOF-MS Analysis


CE-MS Analysis


LC-Coupled MS


Microarray Analysis


Supplemental Data







































ACKNOWLEDGMENTS



LITERATURE CITED


























Maruyama et al













































DISCUSSION






Maruyama et al









GC-TOF-MS Analysis


CE-MS Analysis


LC-Coupled MS


Microarray Analysis


Supplemental Data







































ACKNOWLEDGMENTS



LITERATURE CITED


























Maruyama et al











































GC-TOF-MS Analysis


CE-MS Analysis


LC-Coupled MS


Microarray Analysis


Supplemental Data







































ACKNOWLEDGMENTS



LITERATURE CITED


























Maruyama et al


































































ACKNOWLEDGMENTS



LITERATURE CITED


























Maruyama et al




































integrated analysis of the effects of cold and dehydration ... · portant to improve tolerance to...

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