Introduction Drought and highly saline soils are among the most serious challenges to crop production in the world today. This is particularly the case in developing countries, where these abiotic stresses severely limit crop growth and productivity. Both traditional breeding and genetic engineering of crop plants have been utilized to improve drought and high-salinity tolerance or resistance with the goal of increasing agricultural productivity in affected regions. Understanding plant responses to abiotic stresses at the genomic level provides an essential foundation for future breeding and genetic engineering efforts. Arabidopsis 2000 2003 2002 2001 2002) 2002 2002 1999 2003 1996 2004 cis 2003 2004) Arabidopsis 2001 Arabidopsis 2002a 2002b Arabidopsis 2002 2002 Oryza sativa 2002 2002 2003 2002 2002 2001 2003 2004 2003 2005 2005 2005b 2005 Materials and methods Plant material and stress treatments O. Sativa   indica  1962 RNA isolation, probe labeling, and hybridization 2005b 2005 Microarray and initial data analysis 2005b 2005 2005 t 2004 P 2000 2003 P RT-PCR analysis of genes Actin1 Functional classification 2004 http://www.arabidopsis.org/tools/aracyc Arabidopsis 2003 Arabidopsis Arabidopsis 2005 2005b S3 cis cis  indica  cis 1999 http://www.dna.affrc.go.jp/htdocs/PLACE/signalscan.html cis cis http://www.cse.ucsc.edu/∼kent/improbizer/ t 2004 Gel shift assays 1998 Clustering and chromosomal location analysis of genes involved in stress responses 1998 indica 9 Results Identification of drought and high-salinity stress responsive genes using a rice whole genome oligomer microarray 2005b 2005 S1 1962 S1 t- P 1 2 Fig. 1 A B C  Fig. 2 A y B  y C P D  Most of the previously known genes responsive to both drought and high-salinity stress have been recovered from our microarray analysis. Those include the LEA protein (OsJRFA063984), aquaporin (OsIRUA001311), OsNAC1 (OsJRFA108080), dehydrin rab 16b (OsIFCC035025) and DREB1 (OsJRFA067313). Many other responsive genes have been identified for the first time in this study (Table S1C). Among those newly revealed genes, some are expected to be involved in general cell function or known to be involved in other stress responses, while others have putative or unknown function. The annotation for some of those genes suggested that they include transcription factors from multiple families, heat shock proteins, various stress (drought, high-salinity, disease, cold, and ABA) responsive genes, protein kinases, transporters, photosynthesis enzymes, and other metabolic pathways (Table S1C). The diversity of affected processes suggests a high level of complexity in regulation. There is a significant overlap between genome expression profiles in response to drought and high-salinity stress 2 2 S2 S3 S3 S3 Arabidopsis S3 Arabidopsis  E  S3 2 Distinct groups of genes are induced during rehydration after drought stress 2 3 3 1 Fig. 3 A B C P A B C D y  Table 1 Genes induced by 48-h rehydration and inhibited by drought stress in shoot and panicle Gene name Putative functions D1 D2 D3 D3R Shoot OsIFCC006273 Unknown 0.596 −1.611 −3.497 1.920 OsIFCC021002 Glycosyl hydrolases family 17 0.440 −1.105 −1.775 2.470 OsJRFA107373 Unknown −1.521 −0.487 −4.348 2.470 OsJRFA101949 AP2 domain 0.216 −0.166 −2.456 2.026 OsJRFA106016 Kelch motif −0.315 −0.582 −2.604 1.865 OsJRFA070715 Peroxidase −0.189 −0.499 −2.456 2.215 OsJRFA065471 Folate/biopterin transporter 0.331 −0.533 −2.595 3.095 OsIFCC041459 Chitinase −0.460 −0.136 −2.125 1.972 Panicle OsIFCC023423 Photosystem I reaction center subunit IV −0.067 −0.293 −2.610 2.206 OsIFCC032344 Photosystem I reaction center subunit n −0.141 −0.626 −1.700 2.390 OsIFCC001915 Photosystem II polypeptide −0.119 −0.934 −1.774 1.787 OsJRFA061606 Pathogenesis–related protein PR-10a −1.811 −0.007 −1.841 4.117 OsJRFA107373 Unknown −3.693 0.395 −1.978 5.605 OsJRFA109398 a b −1.867 −1.348 −1.899 1.905 OsIFCC025509 Photosystem II 10 kDa polypeptide PsbR −1.077 −0.966 −1.735 1.831 OsIFCC038501 Chloroplast precursor −1.087 −0.966 −1.805 1.837 OsIFCC022062 Photosystem I reaction center subunit VI −0.540 −0.206 −1.996 1.811 OsIFCC024081 a b −0.595 −0.685 −1.814 2.298 OsIFCC014461 Unknown −0.794 −0.660 −1.722 1.790 OsIFCC017469 Unknown −0.879 −1.619 −1.764 2.417 OsIFCC012276 Catalase −1.567 −0.594 −1.893 2.592 OsJRFA060135 Unknown −0.922 −0.491 −2.540 1.793 OsIFCC033400 Pathogenesis-related protein PR-10a −0.115 −0.032 −2.550 2.008 OsJRFA059435 Magnesium-protoporphyrin IX monomethyl esteraerobic oxidative cyclase −0.344 −0.348 −1.837 2.851 OsIFCC022709 Protease inhibitor/seed storage/LTP family −0.091 −0.300 −1.684 1.842 OsJRFA061968 Geranylreductase −0.334 −0.244 −1.804 2.001 OsJRFA071762 Aluminium-induced protein 0.217 −0.594 −1.849 1.752 OsIFCC029079 Blight-associated protein p12 precursor −1.110 0.379 −1.848 2.980 OsJRFA106991 Unknown −0.899 −0.142 −1.874 1.715 Log2 transformed ratios of all genes at three stages of drought stress and rehydration stage were listed a b Limited overlap of stress responsive genes among rice organs 4 4 4 4 Fig. 4 A B A B C D C D  a b 2 Table 2 Genes up- or down-regulated by both drought and high-salinity stresses in all three organs Gene name Putative functions Flag leaf Shoot Panicle D(h) S(h) D(h) S(h) D(h) S(h) Up-regulated OsJRFA058518 Protein kinase domain 2.591 1.939 3.939 3.443 3.134 3.553 OsJRFA058851 Unknown 5.430 3.900 5.894 4.600 2.042 3.878 OsJRFA062356 Unknown 4.959 2.477 4.699 4.660 3.314 5.727 OsJRFA062972 a b 4.758 2.542 1.895 2.931 2.291 2.204 OsJRFA063156 Unknown 1.837 1.743 4.405 3.921 2.606 2.866 OsJRFA063334 Unknown 3.208 2.140 4.521 3.188 1.809 2.167 OsJRFA063578 ABA-responsive protein 2.743 3.352 4.169 3.733 1.859 2.748 OsJRFA063889 Unknown 3.340 3.263 2.186 3.344 2.061 3.712 OsJRFA063984 LEA protein 5.513 4.730 3.763 4.629 3.971 6.773 OsJRFA068381 Unknown 2.648 1.830 3.630 2.833 1.820 1.786 OsJRFA070577 Unknown 3.036 2.877 3.065 2.899 1.883 2.372 OsJRFA070872 Unknown 8.485 7.733 3.446 6.346 3.963 4.107 OsJRFA071812 Unknown 2.748 2.268 3.015 2.244 1.767 2.559 OsJRFA072568 Unknown 3.001 3.121 2.411 3.326 1.714 2.005 OsJRFA106307 Unknown 4.284 3.493 4.399 2.511 2.316 2.960 OsJRFA106562 Unknown 3.797 4.009 2.544 3.467 2.010 3.978 OsJRFA107065 Unknown 2.770 1.751 4.859 3.648 2.984 2.292 OsJRFA108083 Unknown 2.274 2.597 4.853 4.241 3.077 4.631 OsIFCC031279 Alpha-galactosidase 3.407 3.087 3.819 3.538 2.389 2.206 OsIFCC036408 Unknown 3.137 3.987 3.038 3.111 4.742 4.192 OsIFCC035028 Dehydrin 7.974 7.689 5.148 6.767 8.045 6.382 OsIFCC018156 Abscisic acid-induced protein 5.167 7.224 3.243 4.911 2.329 2.327 OsIFCC003263 CHY zinc finger 1.709 2.147 1.994 2.282 3.791 1.956 OsIFCC018343 Homeobox domain 4.290 3.955 3.432 3.959 2.683 3.017 Down-regulated OsIFCC033098 Arabidopsis −2.676 −4.719 −2.542 −2.346 −1.701 −2.519 OsIFCC015113 Phosphoribulokinase /Uridine kinase family −2.456 −2.689 −1.863 −1.717 −1.853 −1.899 The highest ratio of each gene at three stages under drought (D(h)) or high-salinity (S(h)) were log2 transformed and listed 4 2 The shoot samples we used consisted largely of young leaves and were thus physiologically closer to flag leaf than panicle. Indeed, shoot and flag leaf generally exhibited similar patterns of gene up-regulation in response to drought and high-salinity stress. However, this does not appear to be the case for repressed genes, where overlap between shoot and flag leaf gene expression is minimal. 5 5 Fig. 5 RT-PCR analysis of the representative drought-induced genes among the three rice organs. Total RNA samples were prepared from the shoot (S1, S2, and S3), flag leaf (F1, F2, and F3), and panicle (P1, P2, and P3) and taken from plants at three stages of drought treatment and the untreated control plants (S0, F0, and P0). The corresponding log2 transformed median ratio of microarray data is shown at the bottom of each mRNA blot line. N/A: no expression. The * corresponds to cases where the microarray data was not confirmed by the mRNA blot results  cis cis 2001 2002a 2003 2003 2003 2005 2004 2005 1995 2005 cis 6 6 6 Fig. 6 x y A B C D E F  6 6 6 6 cis cis 1 7 7 Fig. 7 A B C  8 Fig. 8 Three motifs associated with genes repressed during drought and induced by rehydration in panicle. The core sequence logos and the position of the motifs in each of the 11 promoters are listed  Transcription factor genes under drought and high salinity stress are expressed in a largely organ specific manner 3 Table 3 Transcription factor genes induced in each organ by drought or high-salinity stress Gene name  Putative function   Panicle: induced by high-salinity only S(h) D(h) OsIFCC018668 bHLH transcription factor 1.673 −0.850 OsIFCC029156 Helix-loop-helix DNA-binding domain 1.720 −0.599 OsJRFA110611 No apical meristem (NAM) protein 2.234 −1.500 OsJRFA105079 CCAAT-box binding factor HAP5 homolog 3.521 −0.850 OsJRFA070817 RING zinc finger protein 1.708 −0.685 OsIFCC039583 Zinc finger, C3HC4 type (RING finger) 2.278 0.530 OsJRFA108605 Helix-loop-helix DNA-binding domain 4.713 0.240 OsJRFA108208 AP2 domain 2.429 N/A OsJRFA101136 C3HC4-type zinc finger 5.559 −0.753 OsIFCC008718 No apical meristem (NAM) protein 2.542 N/A OsJRFA066984 Dof domain, zinc finger 1.846 0.168 OsJRFA110661 Zinc finger C-x8-C-x5-C-x3-H type 1.732 N/A OsJRFA106969 Myb-like DNA-binding domain 2.010 N/A OsIFCC016263 Zinc finger, C2H2 type 2.636 N/A Panicle: induced by drought only S(h) D(h) OsIFCC042866 AP2 domain N/A 3.050 Panicle: induced by both drought and high-salinity S(h) D(h) OsJRFA107283 NAM-like protein 7.773 2.080 Shoot: induced by drought only S(h) D(h) OsJRFA105599 DRE-binding protein 1A N/A 1.784 OsIFCC031932 WRKY DNA -binding domain −2.746 2.186 OsIFCC031182 Myb factor N/A 2.565 OsIFCC042758 Helix-loop-helix DNA-binding domain −0.024 2.367 OsJRFA100208 Helix-loop-helix DNA-binding domain −0.836 3.132 OsJRFA107524 Dof domain, zinc finger N/A 2.294 OsJRFA106333 Helix-loop-helix DNA-binding domain 0.430 1.762 OsJRFA106282 WRKY DNA -binding domain N/A 2.026 OsIFCC043271 Helix-loop-helix DNA-binding domain 0.245 2.259 OsIFCC000715 Myb-like DNA-binding domain 0.949 2.617 OsJRFA110587 Similar to DNA-binding protein WRKY3 0.905 4.075 OsJRFA107146 AP2 domain 0.625 2.746 OsIFCC038336 Zinc finger transcription factor ZF1 0.529 2.407 Shoot: induced by both drought and high-salinity S(h) D(h) OsJRFA072192 Zinc-finger protein 2.074 2.059 OsIFCC000984 WRKY DNA -binding domain 1.744 2.153 Shoot: induced by high-salinity only S(h) D(h) OsIFCC017057 B3 DNA binding domain 2.026 N/A OsJRFA067496 TRAF-type zinc finger 2.199 0.801 Flag leaf: induced by drought only S(h) D(h) OsIFCC029554 Zinc finger, C2H2 type −0.553 2.032 Flag leaf: induced by high-salinity only S(h) D(h) OsIFCC001054 Zinc finger C-x8-C-x5-C-x3-H type 1.705 0.582 The highest ratio of each gene among three stages of drought (D(h))or high-salinity (S(h)) were log2 transformed and listed Minimal co-regulation of neighboring genes in drought or high-salinity stress responses 2004 2001 2002 Drosophila 2002  Arabidopsis 2003 2005a 2000 2005b 2005 indica 9 2005 9 Fig. 9 A B  Discussion This study provides new insight into the rice response to drought and high salinity stresses at the whole genome level. Using a whole genome microarray, we monitored the expression of 36,926 unique or known rice genes or gene models in three different organs under drought and high salinity stress. Our work thus offers the first comprehensive picture of genome expression modulation in response to drought and high salinity stress in three distinct rice organs. Genome expression reprograming showed significant overlap between drought and high salinity responsive genes  Arabidopsis  2002a b 2003 2003 S1 Reprograming of genome expression in response to drought and high-salinity stresses is largely organ specific In this study, we examined whole genome expression profiles under drought and high-salinity conditions in three organs: four-tiller stage shoot, filling stage flag leaf and panicle. Rice plants at these two growth stages, particularly the late one, are sensitive to drought and high-salinity stress. It is well known that drought or high-salinity stress at the heading and early panicle stages can severely compromise rice growth and development and reduce crop yield even with late rehydration. It is evident that the rice genome is subject to significant reprograming with regard to which portion of genome is expressed under drought or high salinity stress. 4 2002 S2 4 3 6 S2 Rice may possess specific mechanisms to facilitate plant recovery during rehydration after drought 3 Arabidopsis 2003 2005 2005b cis Arabidopsis 2002 2003 3 cis cis 8 cis 2002 2004 2003 Electronic supplementary material Below are the electronic supplementary materials.
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