Identification and expression analysis of OsHsfs in rice

Identification and expression analysis of OsHsfs in rice first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:huixia@zju.edu.cn †E-mail:huixia@zju.edu.cn huixia@zju.edu.cn State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China Heat stress transcription factors (Hsfs) are the central regulators of defense response to heat stress. We identified a total of 25 rice Hsf genes by genome-wide analysis of rice ( Oryza sativa L.) genome, including the subspecies of O . japonica and O . indica . Proteins encoded by OsHsfs were divided into three classes according to their structures. Digital Northern analysis showed that OsHsfs were expressed constitutively. The expressions of these OsHsfs in response to heat stress and oxidative stress differed among the members of the gene family. Promoter analysis identified a number of stress-related cis -elements in the promoter regions of these OsHsfs . No significant correlation, however, was found between the heat-shock responses of genes and their cis -elements. Overall, our results provide a foundation for future research of OsHsfs function. Heat shock Transcription factors Rice Protein structure Expression analysis INTRODUCTION 1 Heat stress transcription factors (Hsfs) are the central regulators of defense response. They control the expression of heat shock proteins (HSPs) in plants by specific binding to the highly conserved heat shock element (HSE) characterized by palindromic motifs of nGAAn (Miller and Mittler, 2006 2006 ). The basic structure and promoter recognition of Hsfs are highly conserved throughout the eukaryotic kingdom (von Koskull-Döring et al., 2007 2007 ). A typical Hsf protein contains a modular structure with an N-terminal DNA-binding domain (DBD), an adjacent bipartite oligomerization domain composed of heptads repeat of hydrophobic amino acid residues (HR-A/B), a nuclear localization signal (NLS) essential for nuclear uptake of the protein, a nuclear export signal (NES), and in many cases a less conserved C-terminal activation domain (CTAD) rich in aromatic, hydrophobic and acidic amino acids (AHA) that have been reported to be crucial for activation function (Nover et al., 2001 2001 ). Based on the conservative DBD and the HR-A/B regions, 21 putative Hsfs from the Arabidopsis , 23 from rice, and 18 from tomato have been identified through the genome-wide analysis (Baniwal et al., 2004 2004 ). Plant Hsf gene family is divided into three classes, HsfA , HsfB , and HsfC , according to their protein structures (Nover et al., 2001 2001 ). HsfA and HsfC have insertions of 21 and 7 amino acids, between the hydrophobic regions HR-A and HR-B, respectively. HsfB and HsfC are also characterized by lack of AHA motifs in their C-terminal regions (CTRs). Evidence from several important representatives in tomato and Arabidopsis displayed that plant Hsfs diversify in their biological functions (Kotak et al., 2007b 2007b ; von Koskull-Döring et al., 2007 2007 ). HsfA members are capable of transcriptional activation, while HsfB members act as repressors or as co-activators (e.g., HsfB1 ) of HsfA members (Bharti et al., 2004 2004 ; Czarnecka-Verner et al., 2004 2004 ). However, AtHsfA4 activity was reported to be repressed by AtHsf5 , which belongs to class A Hsfs (Baniwal et al., 2007 2007 ). Overexpression of Hsf genes in transgenic plants resulted in an up-regulation of heat stress-associated genes and an enhancement of thermotolerance, whereas the down-regulation of Hsf genes leads to a reduction in the thermotolerance (Charng et al., 2007 2007 ; Mishra et al., 2002 2002 ; Schramm et al., 2008 2008 ). In addition to control of heat stress response, Hsfs have also been reported to be involved in the defense response to pathogen attack, oxidative stress, heavy metals, dehydration and salinity, and in certain processes of development and differentiation (Larkindale et al., 2005 2005 ; von Koskull-Döring et al., 2007 2007 ). Rice is the most important cereal crop, which feeds more than a half of the world’s population (Jeon et al., 2008 2008 ). Molecular dissection of rice Hsf gene family would help to unravel the stress response mechanism in rice. Compared with the extensive studies done in Arabidopsis Hsf genes, only a few researches have involved monocots, such as rice and maize (Fu et al., 2006 2006 ; Yamanouchi et al., 2002 2002 ; Yokotani et al., 2008 2008 ). Although 23 Hsfs were identified in the Oryza japonica previously, the structure and expression profile of these OsHsfs have not been elucidated. In this study, we identified and classified 25 rice Hsf genes from both O . japonica and O . indica genomes. In addition, the expression of the individual genes was investigated through both digital expression analysis and semi-quantitative reverse-transcript polymerase chain reaction (RT-PCR). Our work will facilitate the function analysis of the OsHsfs genes. MATERIALS AND METHODS 1 Search for Hsf genes in rice genome and gene annotation 2 Consensus amino acid sequences of Arabidopsis heat shock factors, including the DNA-binding domain and HR-A/B region, were used to search the GenBank (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA; http://www.ncbi.nlm.nih.gov http://www.ncbi.nlm.nih.gov ), the International Rice Genome Sequencing Project (IRGSP; http://rgp.dna.affrc.go.jp http://rgp.dna.affrc.go.jp ), and Beijing Genomics Institute (BGI; http://btn.genomics.org.cn/rice http://btn.genomics.org.cn/rice ), using an E -value cutoff of 1.0. The full length of cDNA sequence, 1 kb DNA sequence upstream of the start-codon, and bacterial artificial chromosomes (BACs) or phage artificial chromosomes (PACs) containing OsHsf genes were obtained from the NCBI, IRGSP, or BGI. Expressed sequence tag (EST) sequences of all OsHsf genes collected from dbEST database ( http://www.ncbi.nlm.nih.gov/dbEST/ http://www.ncbi.nlm.nih.gov/dbEST/ ) were used for the identification of the tissue specific expression patterns of the OsHsfs (Audic and Claverie, 1997 1997 ). Finally, we compared all the OsHsf genes to identify redundant sequences. Promoters were analyzed by using PlantCARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). Sequence alignment and phylogenic analysis 2 Amino acid sequences of DBD and HR-A/B regions were used for multiple alignments by using ClustalX version 1.83 (Hicks et al., 1997 1997 ). To produce preferable alignments, the parameters were set as followings: for pairwise parameters, gap opening cost=30, gap extension cost=0.3; for multiple parameters, gap opening cost=20, gap extension cost=0.15; the Gonnet series were applied for protein weight matrices and defaulting parameters were used for other settings. Phylogenetic tree of OsHsf gene family was constructed using the N-J method. The ScHsf1 gene from Saccharomyces cerevisiae was selected as outgroup and bootstrap analysis was performed to measure the robustness of all nodes. Digital expression analysis 2 Digital expression of the OsHsfs was performed using the rice dbEST database. All ESTs were sorted by the library source, and normalized libraries were delimited for expression analysis. Frequencies of the ESTs in the corresponding library were calculated to represent the gene expression level. RT-PCR analysis 2 Sixty rice seedlings ( Oryza sativa L. cv. Nipponbare) were grown hydroponically with a 12-h photoperiod (250 µmol photons/(m 2 ·s)) and a day/night temperature of 28/22 °C. The 7-d-old seedlings were transferred to a growth chamber at 37 °C for heat shock treatment or immersed in 20 mmol/L H 2 O 2 solution for H 2 O 2 stress induction. The shoots of the seedlings were sampled at different time points (0, 1, 6, and 24 h after treatments) for isolating RNA. Total RNA was extracted with Trizol reagent (Gibco-BRL, Gaithersburg, MD, USA) according to the manufacturer’s instructions. cDNA was synthesized from 5 μg of the total RNA using moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA). Rice ACTIN gene OsACT1 (accession No. X63830) was served as a housekeeping gene control. Gene specific primers for corresponding OsHsf genes were designed for semi-quantitative RT-PCR analysis (Table A1 A1 ). RESULTS 1 Identification of OsHsf genes in rice genome 2 In order to identify rice Hsf genes, we used the conserved DBD and the HR-A/B regions to conduct basic local alignment search tool (BLAST) searches against the rice genome in NCBI, IRGSP, and GPI, and identified a total of 26 homologs in the O . japonica genome, which were highly similar to HSF proteins from other plants, and 27 in the O . indica . Of these Hsf genes, two gene loci Loc_Os06g66210 ( O . japonica ) and OsIBCD008764 ( O . indica ) were manually discarded because of the truncation in DBD region and the absence of corresponding ESTs in the dbEST, respectively. Another two Hsf genes from gene loci OsIBCD028792 and OsIBCD044667, which were previously annotated as two distinct genes, were proved to be identical in their cDNA sequences (Table 1 1 ). The remaining 50 sequences were analyzed for redundancy by performing pairwise comparisons of the genomic sequences in the coding regions ranging from start to the stop codon in each cultivar. Genes that share more than 95% similarity are considered as the same gene. It was found that all Hsf genes in O . indica have corresponding orthologs in O . japonica . BAC: bacterial artificial chromosome; PAC: phage artificial chromosome; EST: expressed sequence tag: ORF: open reading frame. Same genes with two cDNAs separated by intron; Two OsHsfB4d gene loci annotated in O. indica genome 2 Genes 2 Gene loci 2 BAC/PAC clone 2 Protein accession No. 2 Accession No. of cDNA 2 Total EST 2 ORF (aa) 2 Intron (bp) O. japonica O. indica   OsHsfA1 Os03g63750 OsIBCD012688 AC120506 NP_001051938 AK100430 50 506 1386   AP008209 AK106118   OsHsfA2a Os03g53340 OsIBCD011954 AC092558 AAP13005 AK069579 15 376 81   AP008209 AK109590   OsHsfA2b Os07g08140 OsIBCD022526 AP003826 NP_001059028 AK101824 10 372 1323   OsHsfA2c Os10g28340 OsIBCD030592 AC027658 NP_001064617 AK072391 10 358 1669   AP008216   OsHsfA2d Os03g06630 OsIBCD008955 AC105729 NP_001049047 AK066844 6 359 946   AP008209   OsHsfA2e Os03g58160 OsIBCD012247 AC092076 NP_001051552 AK068660 50 357 1199   AP008209   OsHsfA3 Os02g32590 OsIBCD006671 AP004777 NP_001047003 AK101934 7 498 1298   OsHsfA4a Os01g54550 OsIBCD003303 AP003076 NP_001044247 AK109856 21 440 582   OsHsfA4d Os05g45410 OsIBCD018681 AC111015 NP_001056127 AK100412 35 459 237   AP008211   OsHsfA5 Os02g29340 OsIBCD006509 AP004999 NP_001046889 AK072210 16 475 1475   AP008208 AK065643   OsHsfA6 Os06g36930 OsIBCD021096 AP005456 NP_001057889 − − 331 122   OsHsfA7 Os01g39020 OsIBCD002361 AP003308 NP_001043378 AK064271 7 402 1406   OsHsfA9 Os03g12370 OsIBCD009329 AC107226 NP_001049429 AK072571 24 406 1231   AP008209   OsHsfB1 Os09g28354 OsIBCD028792 a a AP006057 NP_001063364 AK101182 36 302 5880   OsIBCD044667 a a AP008215 AK061433   OsHsfB2a Os04g48030 OsIBCD015287 AL663003 NP_001053591 AY344483 4 305 102   OsHsfB2b Os08g43334 OsIBCD027305 AP004163 NP_001062423 AK101700 29 390 101   OsHsfB2c Os09g35790 OsIBCD029172 AP005681 NP_001063726 AK106545 19 454 121   AK106525   AK105409   OsHsfB4a Os08g36700 OsIBCD026821 AP004693 BAD09860 − − 380 93   OsHSFB4b Os07g44690 OsIBCD024341 AP005292 NP_001060424 AK063952 26 310 1665   AP008213 AK099354   OsHsfB4c Os09g28200 OsIBCD028788 AP005655 NP_001063356 AK241190 5 394 88   OsHsfB4d Os03g25120 OsIBCD010276 b b AC125784 ABF96133 − 1 305 1450   OsIBCD010275 b b AP008209   OsHsfC1a Os01g43590 − AP002744 AAQ23067 AK069479 1 348 109   OsHsfC1b Os01g53220 OsIBCD003218 AP003309 NP_001044160 AK066316 9 250 79   OsHsfC2a Os02g13800 OsIBCD005620 AP004070 NP_001046370 AK106488 9 298 86   OsHsfC2b Os06g35960 OsIBCD021036 AP003682 NP_001057843 − 1 278 1128 Protein structure and classification of OsHsfs 2 Protein sequence analysis detected a conserved DBD in the N-terminal region (Fig. 1 1 ) and an adjacent HR-A/B region in each of the OsHsf proteins (Fig. 2 2 ). The DBD domain contained three α-helix bundles and a small four-stranded antiparallel β-sheet as previously described in LpHsf24 (Fig. 1 1 ) (Schultheiss et al., 1996 1996 ). There was a conserved intron located near the 3′-end of the third helix (Fig. 1 1 ) with variant length (Table 1 1 ) in all the genes. All OsHsfs have only one intron between the invariant tyrosine residue and glycine residue, which is indicated by arrow. The secondary structure elements are showed below the sequence alignment: α 1 to α 3 , α-helix; β 1 to β 4 , β-sheet Based on the distance between HR-A and HR-B domains, OsHsfs are grouped into three classes: (a) OsHsfA, (b) OsHsfB, and (c) OsHsfC. The hydrophobic residues and unique amino acids (glycine and proline) are colored black and gray respectively. The positions a and d of heptapeptide repeats are marked below Further analyses of the HR-A/B domain revealed that there were, in the majority of cases, three or more repeated heptads in the HR-A domain and two incompletely repeated heptads in the HR-B domain (Fig. 2 2 ). HR-B in classes A and C had a very characteristic structure of overlapping sets of hydrophobic heptapeptide repeats (Fig. 2 2 ). According to the linker region between the HR-A and HR-B domains, OsHsfs family (Nover et al., 2001 2001 ) was divided into three classes, including 13 OsHsfAs, 8 OsHsfBs, and 4 OsHsfCs (Table 2 2 ). Numbers in brackets indicate positions of the putative function motifs; NLS: nuclear localization signal; NES: nuclear export signal; AHA: aromatic, large hydrophobic and acidic amino acids. Aromatic and large hydrophobic residues of AHA are set in boldface type Hsfs NLS NES AHA motifs  HsfA1 (231)RRR-5aa-KKRRLPK (493)LTEQMGLL AHA (449)DS FW EQ FL VA  HsfA2a (255)RNR-7aa-KKRRR (366)LVQSIYHL AHA (318)ES FW MQ LL SL  HsfA2b (239)RK-7aa-KKRRRR (356)LSEKMGYL AHA (328)DN FW EE LL NE  HsfA2c (227)KEKRK-7aa-KKRRR (345)LAQQLGYL AHA (315)DD FW AE LL VE  HsfA2d (223)KMK-7aa-KKRTR (346)LAQKLGYL AHA (315)DD FW EE LL NE  HsfA2e (224)RK-7aa-KKRRRR (340)LLSQKMGYL AHA (314)ED FW ED LL HE  HsfA3 (247)RQQK-7aa-KRKFLK (472)LDDGDLQL AHA (463)K FW E L D F QAL  HsfA4a (200)RKKRR (429)EKLGLL AHA1 (375)DG FW QQ FL TE   AHA2 (416)HL WW GKRNVE  HsfA4d (210)KKRRVPK (445)EQMGHL AHA (397)DV FW ER FL TE  HsfA5 (214)NKKRR (470)VEQLKL AHA (422)DK FW EQ FL TE  HsfA6 (231)RKKRR (319)LVQQIDCL AHA1 (271)M IWY E LL GEE   AHA2 (306)EP W EEMGEEE  HsfA7 (282)KNGLRGAAKRQR − AHA1 (349)DD VW EE L DAL   AHA2 (384)CG WV DDCPYL  HsfA9 (246)QRR-9aa-KKRR (371)QMGPPL AHA (380)DYD F PQ L EQD  HsfB1 (263)RKRAR − −  HsfB2a (265)REGKVRR (275)LSDLDVLAL −  HsfB2b (318)RKRMR − −  HsfB2c (327)LKRTR − −  HsfB4a (346)RKRS − −  HsfB4b (291)RKKRAHR − −  HsfB4c (269)RKKP (363)LVLECDDLSL −  HsfB4d (286)KKRRVQL − −  HsfC1a (229)RKRRR − −  HsfC1b (209)KRRR − −  HsfC2a (213)KRPRLLL − −  HsfC2b (219)KRARLLL − − Within the OsHsfs, the nuclear localization signals (NLS) composed of a cluster of arginine and lysine residues were detected adjacent to the HR-A/B domain (Table 2 2 ). There was a leucine-rich nuclear export signal (NES) at the C-terminus of all OsHsfAs except OsHsfA7, and of a small portion of OsHsfBs and OsHsfCs (Table 2 2 ). The C-terminal activation domain (CTAD) was only found in OsHsfA, suggesting that only class A Hsfs can activate autonomously. Phylogenetic analysis of OsHsfs 2 To determine the phylogenetic relationship among the OsHsfs , neighbor-joining phylogenetic trees were constructed using the amino acid sequences of DBD, the HR-A/B region, and the linker between them (Fig. 3 3 ). Arabidopsis Hsf genes were included in the phylogenetic tree as reference to classify rice Hsfs . As expected, the classes A, B and C Hsfs formed three individual clusters. Furthermore, the class A Hsfs were divided into two sub-clusters (Fig. 3 3 ). In a previous study, the N-terminal part and C-terminal part of DBD and HR-A/B regions were used separately to draw phylogenetic trees (Nover et al., 2001 2001 ). Although most proteins fixed their positions in the different phylogenetic trees, a few Hsfs changed their positions (Nover et al., 2001 2001 ). Similar phenomenon was also observed on the OsHsfs (data not shown). A more convinced relationship of the Hsfs was revealed by combining the DBD, HR-A/B, and the flexible linker between DBD and HR-A/B (Fig. 3 3 ). The tree was generated on the basis of the amino acid sequences of the N-terminal domains of Hsfs including the DBD region, the HR-A/B region and parts of the linker between both. ScHsf1 was set as the outgroup. Bar=substitutions/site Cis -acting elements in OsHsf promoters 2 One kilobase upstream regions of the OsHsf genes were analyzed for cis -acting elements using PlantCARE software (Higo et al., 1999 1999 ). A total of 9 different cis -acting elements commonly existed in the promoters of OsHsfs (Table 3 3 ). Seven of these cis -acting elements were related to stress responses. Two cis -acting elements with the highest frequency were G-box/Sp1 and abscisic acid (ABA) response element (ABRE). These two elements are involved in the light responsiveness and the ABA response, respectively (Table 3 3 ). CCGTCC/CAT-box, LTR, and ARE/GC-motif are cis -acting elements related to meristem expression, low temperature response, and anaerobic induction. Compared with classes B and C OsHsf genes, class A genes contained more these two cis -elements, suggesting that class A genes may play different roles from classes B and C genes (Table 3 3 ). In addition to these two elements, other cis -elements occurred at a similar frequency among the promoters of these three classes. T 1 : total number of each cis -element in HsfA ; T 2 : total number of each cis -element in HsfB and HsfC 2 Cis -elements 13 HsfA 2 T 1 8 HsfB 4 HsfC 2 T 2 2 Descriptions 1a 2a 2b 2c 2d 2e 3 4a 4d 5 6 7 9 1 2a 2b 2c 4a 4 4c 4d 1a 1b 2a 2b Skn-1 and GCN4-motif 2 2 0 1 0 0 1 1 1 1 1 1 4 15 1 1 3 0 0 3 0 2 1 4 2 0 17 Required for endosperm expression CCGTCC and CAT-box 2 1 2 1 1 0 0 1 1 1 1 3 0 14 1 0 0 0 0 0 2 0 0 2 0 1 6 Related to meristem expression G-box and Sp1 5 8 17 12 3 6 14 7 3 3 17 6 5 106 10 7 2 10 7 5 7 7 13 8 7 19 102 Involved in light response HSE 0 3 1 2 2 2 0 1 0 0 2 1 0 14 3 1 2 1 0 1 1 0 2 0 1 1 13 Involved in heat stress response LTR 0 2 1 1 1 0 1 0 1 0 0 0 0 7 0 0 0 0 1 1 0 0 0 0 0 0 2 Involved in low-temperature response ARE and GC-motif 2 5 4 3 2 1 4 1 1 2 5 3 0 33 4 1 1 2 1 0 3 0 1 2 0 3 18 Involved in the anaerobic induction MBS and CCAAT-box 1 1 0 1 2 1 0 2 1 1 3 2 1 16 1 1 1 2 3 2 1 3 1 1 0 1 17 MYB binding site, involved in drought-inducibility ABRE 0 1 3 4 2 3 8 0 1 0 14 0 1 37 0 2 0 5 11 3 0 5 5 2 1 3 37 Involved in the abscisic acid response CGTCA-motif 0 2 0 0 1 2 4 4 0 0 1 0 4 18 1 3 0 0 0 2 1 0 2 2 2 0 13 Involved in the MeJA-response Expression patterns of OsHsfs 2 The number of ESTs for a specific gene in a cDNA library is considered to be proportional to the transcript abundance of the mRNA. We used the EST sequences downloaded from the dbEST to analyze the expression pattern of OsHsfs in various plant tissues and different developmental stages. A total of 391 OsHsf ESTs corresponding to 23 of the 25 genes were identified except the OsHsfA6 and OsHsfB4a genes (Table 1 1 ). The majority of OsHsf genes had a low to moderate level of transcripts in most tissues, while some displayed a tissue-specific manner (Fig. 4 4 ). OsHsfA4a and OsHsfA4d were dominantly expressed in panicle and adult leaves, respectively. Overall, OsHsfs were expressed at a higher level in panicle and flower than in other tissues. ESTs of each Hsf are collected from dbEST and the transcript rations of all Hsfs in each library are calculated. Transcript levels of all OsHsfs in per million transcripts are counted according to the ration. Normalized libraries and libraries with less than 5000 ESTs are excluded. Detail data are shown in Table A2 A2 Under heat shock (HS) and H 2 O 2 treatment, the expression of class A Hsfs was higher than those of class B and class C Hsfs (Fig. 5 5 ). These results are consistent with those derived from the analyses of EST data. The lack of the detectable transcripts of several OsHsfs under our condition might have resulted from the tissue specific expression pattern ( OsHsfA4d ) or low expression levels of the genes (e.g., OsHsfB4a , OsHsfB4d , and so on) as suggested by the digital expression pattern. The transcription of OsHsfA1a , OsHsfA2b , OsHsfA3 , OsHsfA7 , OsHsfA9 , OsHsfB2c , and OsHsfC1b was up-regulated by both HS and H 2 O 2 treatments in a similar manner, while the up-regulation of OsHsfA2a , OsHsfA2c , OsHsfA2e , OsHsfA4a , OsHsfB2b , and OsSHFC1b was detected only under HS treatment, implying that there was a H 2 O 2 independent HS responsive pathway in rice. mRNA was extracted from shoots of 7-d-old O. japonica seedlings treated with heat (37 °C) and 20 mmol/L H 2 O 2 for different time points. The transcript level of OsACTIN1 was used as the housekeep gene control. Class A Hsfs and ACTIN genes were amplified for 28 cycles, while classes B and C Hsfs were amplified for 30 cycles DISCUSSION 1 Unlike yeast and Drosophila that only contain one Hsf gene, plant has a more complex family of Hsf genes (Baniwal et al., 2004 2004 ; Miller and Mittler, 2006 2006 ). In this study, we identified 25 OsHsfs based on the rice genome. Majority of the predicted OsHsfs were supported by the FL-cDNA and the EST sequences (Table 1 1 ). These 25 OsHsfs were divided into 3 classes (A, B and C) based on the protein structure and phylogenetic relationship (Figs. 2 2 ~ 3 3 ). The sequence analysis identified the conserved DBD, the HR-A/B domain, and the NLS that were the common motifs of OsHSFs. The NES and the AHA motifs were also the common motifs of class A genes, but they were only detected in a small portion of classes B and C genes (Table 2 2 ). Recently, great progress has been made in elucidating the functions of Hsfs (von Koskull-Döring et al., 2007 2007 ). AtHsfA9 is involved in seed development and controlled by seed-specific transcription factor abscisic acid-insensitive 3 (ABI3) (Kotak et al., 2007a 2007a ). The expression of AtHsfA3 is induced by drought and heat, and is dependent on the dehydration response element biding protein 2A (DREB2A) (Sakuma et al., 2006 2006 ; Schramm et al., 2008 2008 ). Promoter analysis showed that an array of OsHsf genes contains ABREs (Table 3 3 ). This indicates that an ABA pathway may be involved in Hsf induction and responsible for control of downstream processes, such as seed development or drought resistance (Kotak et al., 2007b 2007b ). The transcripts of AtHsfA2 increased under several stress conditions, especially in response to high light plus heat stress (Nishizawa et al., 2006 2006 ). During repeated cycles of heat stress and recovery, HsfA2 becomes a dominant Hsf in tomato and Arabidopsis (Mishra et al., 2002 2002 ; Schramm et al., 2006 2006 ). In contrast to tomato and Arabidopsis containing only one HsfA2 , rice has five HsfA2 genes (von Koskull-Döring et al., 2007 2007 ). Our results show that OsHsfA2a , OsHsfA2b , OsHsfA2c , and OsHsfA2e were induced by heat stress, while OsHsfA2b and OsHsfA2c were induced by H 2 O 2 also (Fig. 5 5 ). From the result of expression pattern, the OsHSfA2 subfamily was closely related with rice stress response. Overexpression of OsHsfA2e in Arabidopsis led to enhanced thermo and salt tolerance in transgenic plants (Yokotani et al., 2008 2008 ). Further studies to elucidate the functions of the other members in the subfamily may also have potential for the development of transgenic plants with improved stress tolerance. H 2 O 2 is reported to be an essential component in the heat stress signaling pathway (Volkov et al., 2006 2006 ), and the Hsf genes were considered as H 2 O 2 sensors in plant (Miller and Mittler, 2006 2006 ). Human and Drosophila Hsf1 was shown to sense hydrogen peroxide directly and assemble into a homotrimer in a reversible and redox-regulated manner (Storozhenko et al., 1998 1998 ; Zhong et al., 1998 1998 ). In Arabidopsis , the heat stress-induced H 2 O 2 is required for effective expression of heat shock genes (Volkov et al., 2006 2006 ), and the AtHsf4a may be an important sensors of H 2 O 2 (Davletova et al., 2005 2005 ). The intimate relationship between the HS and oxidative stress response suggests that some Hsfs might be responsive to both stresses. In this study, eight OsHsfs ( OsHsfA1a , OsHsfA2b , OsHsfC1b , OsHsfA3 , OsHsfA7 , OsHsfA9 , OsHsfB2c , and OsHsfC1b ) were found to be induced by both heat shock and H 2 O 2 (Fig. 5 5 ). Heat stress led to the accumulation of H 2 O 2 in tobacco and Arabidopsis culture cells (Volkov et al., 2006 2006 ) and mustard seedlings (Dat et al., 1998 1998 ). Hence, it is likely that Hsfs sense the H 2 O 2 level in cells and transfer the stress signaling. Mutation of OsHsfA4d led to a lesion mimic phenotype in mature leaves (Yamanouchi et al., 2002 2002 ). 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