Identification of drought and high-salinity stress responsive genes using a rice whole genome oligomer microarray
A previously reported rice genome microarray containing 70mer oligomer probes for 36,926 unique genes or gene models (Ma et al., 2005b; Jiao et al., 2005) was used for genome-wide profiling of gene expression responses to drought and high-salinity stresses. Total RNAs were isolated from four-tiller stage shoots and from flag leaves and panicles (as described in Experimental procedures) at three different time points (or sampling stages) after initiation of drought or high-salinity stress treatments (Fig. S1). The sampling stages for both drought and high-salinity treatments were based on morphological phenotypes (leaf rolling state) and physiological states (relative water content, RWC, see Bare and Weatherley, 1962). Stage one is defined as exhibiting slightly rolled leaves with 90–95% RWC. Stage two is defined as exhibiting halfway rolled leaves with 80–85% RWC. Stage three is defined as exhibiting completely rolled leaves and with 70–75% RWC. For drought treatment, the three stages were designated as D1, D2, and D3, while the three stages for the high-salinity treatment were designated as S1, S2, and S3 (see Fig. S1). The cDNA probes produced from the stress-treated and corresponding untreated control sample pairs were hybridized to microarray slides. For each sample point, three replicates were performed with dye-swap to correct for uneven dye effects.
The microarray data processing includes LOESS normalization within slide and quantile normalization between slides to remove the systematic errors (see Experimental procedures for details). To identify the differentially expressed genes, Limma R implementation was used to perform pair-wise comparison, and a moderated t-statistics was computed for each gene of treated sample versus control sample. Genes with FDR-adjusted P value less than 0.05 were considered to indicate significant differences (see Experimental procedures for details). Numbers of genes up- or down-regulated by drought or by high-salinity stress at each stage in each organ were shown in Fig. 1. In total, 582, 1,257 and 614 drought up-regulated genes and 795, 646 and 1,305 drought down-regulated genes were identified in flag leaf, shoot and panicle, respectively; and 1,676, 817 and 1,310 high-salinity up-regulated genes and 1,270, 1,323 and 2,284 high-salinity down-regulated genes were identified in flag leaf, shoot and panicle, respectively (Fig. 2A, B). The complete lists of these genes are available in the supplemental data (Tables S2–S13).
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
To compare the gene expression profiles of the drought and high-salinity stress responses, genes commonly regulated by both stresses at each organ type were examined. A total of 322, 415, and 174 genes were up-regulated and 215, 173, and 372 genes were down-regulated by both stresses in flag leaves, shoots and panicles, respectively (Fig. 2A, B). The common genes represent 55, 33 and 28% (induced) and 27, 27 and 29% (repressed) of all drought-responsive genes in flag leaf, shoot and panicle, respectively. These results indicate that about one-third, even half in flag leaf, of the drought responsive genes in each organ are also regulated by high salinity stress.
To further compare gene expression under the two abiotic stresses, all drought regulated genes were selected for cluster analysis (see Experimental procedures). As shown in Fig. 2C, up to half of the genes up- or down-regulated by drought stress also exhibit a similar expression pattern under high-salinity stress. The remainder of the drought responsive genes exhibits minimal expression changes or distinct expression patterns in response to high-salinity stress.
Among genes specifically induced by drought stress, some have known functions while others were previously predicted to have functions related to drought stress or the ABA response. For example, our experiments showed that genes with putative functions including NAM, HLH, G-box binding, Zn-finger, AP2 transcription factors, and protein kinases (including MAPK family genes) were all affected by drought stress. Quite a few known drought-responsive genes such as DREB1A, LEA protein, WSI76 protein, MAP65 (microtubule associated protein), and ubiquitin were also included. These genes were specifically induced by drought and may function exclusively in the response to drought stress in rice. To gain an overall view of the effects of drought and high-salinity stress on various functional gene groups, gene ontology (GO) categories for drought and high-salinity stress induced genes in three organs were examined (Fig. S2). For the majority of these functional categories, the relative numbers of genes responsive to the two stresses were similar.
We also examined the effects of drought and high-salinity on rice genes coding enzymes in known metabolic pathways using shoot as a model (Fig. S3). Some pathways are similarly regulated by the two stresses, including the Calvin cycle, TCA cycle variation I, brassinosteroid biosynthesis II, gibberellin biosynthesis, and IAA biosynthesis I, and the de novo biosynthesis of pyrimidine ribnucleotides/pyrimidine deoxyribonucleotides/purine nucleotides (Fig. S3 and data not shown). While other pathways respond differentially to drought or high-salinity stresses, the representative pathways include sterol biosynthesis, sugars and polysaccharides, ABA biosynthesis, lignin biosynthesis, octane oxidation, cutin biosynthesis, and starch degradation (Fig. S3). For example, some steps in these pathways were repressed or invariable under high-salinity stress, but were induced by drought stress. It is noticeable that one step in the ABA-biosynthesis pathway (corresponding to Arabidopsis NCED3) showed slightly inhibited in gene expression under high-salinity stress (only at stage 3), but was induced under drought treatment (Fig. S3c). Our informatic analysis suggested that OsJRFA107649 is the closest Arabidopsis NCED3 homolog, with 66% identity (E = 1.8E–195). From our analysis, OsJRFA107649 expression ratios (log2 transformed) were 0.278 (D1), 3.461 (D2) and 2.53(D3) under drought treatment and 3.626 (S1), 2.283 (S2) and –1.958 (S3) under high salinity treatment. The expression ratios of rice gene at D3 and S3 stage were used for the diagram in Fig. S3. It is interesting to note that under high salinity this gene was initially induced and then inhibited at stage 3 (S3 ratio: –1.958), while under drought treatment this gene was induced starting at stage 2 and maintained after stage 3 (D3 ratio: 2.53). This example illustrated a distinct response kinetics for different responsive genes in response to drought and high-salinity stresses.
By comparing transcriptomes under drought and high-salinity stresses in three distinct organs, we found that drought responsive genome expression in flag leaf is closer to that for high salinity stress than to the drought expression response observed in any other organ (Fig. 2C, D). In general, there seems to be a closer relationship between the transcriptome-level responses to the two abiotic stresses in the same organ than between or among transcriptomes in distinct organs in response to the same stress. However, the extent of overlap in the responses to drought or high-salinity stresses varies for the different rice organs. For example, it is more divergent in the responses to the two abiotic stresses in panicle, while flag leaves show the greatest overlap.
Distinct groups of genes are induced during rehydration after drought stress
In its natural environment, a plant’s ability to respond to rehydration after drought stress is important for its survival, although little is known about the gene expression changes that occur during rehydration. Our experiments showed that after a period of drought (third stage), all the rice leaves rolled and turned yellow, and the RWC dropped to 70–75%. When we applied water to those severely stressed plants, rice leaves started to unroll after 5 h, and exhibited normal flatness within 24-48 hours of rehydration. We collected tissue samples 48 h after rehydration and extracted total RNA for gene expression analysis (Fig. 2C).
Comparing to samples from normal grown rice plants, in flag leaf, shoot and panicle, 807, 281 and 224 genes were up-regulated after 48 h of rehydration following drought stress, respectively (Fig. 3, Table S14–S16). We further compared gene expression profiles during drought treatment with those after rehydration (Fig. 3A–C). The expression patterns of those genes seem to belong to three groups, with flag leaf exhibiting the greatest number of gene expression changes following rehydration. Group I, which included 71, 98 and 68 genes from flag leaf, shoot and panicle, respectively, was induced during both drought treatment and rehydration. Group II, which included 71, 8 and 21 genes in flag leaf, shoot and panicle, respectively, was repressed during drought stress but induced by rehydration (for shoot and panicle, see Table 1). Group III, which included all remaining 665 genes in flag leaf, 175 genes in shoot, and 135 genes in panicle, showed no significant expression changes under drought stress but were up-regulated during rehydration. We suspected that group I genes must be induced by drought stress, but have yet to return to normal levels after 48-h rehydration. Those genes in groups II and III were specifically induced by rehydration.
Table 1 Genes induced by 48-h rehydration and inhibited by drought stress in shoot and panicle
The down-regulation of group II genes in response to drought led us to speculate that they may play an important role in conferring drought stress tolerance, whereas the up-regulation of group II and group III genes during rehydration could be important for recovery. In both group II and III genes, we found that two classes of genes were over-represented among the rehydration-inducible genes in shoot and panicle: transporter genes and photosynthesis-related genes. These transporter genes included: OsJRFA065471 (folate/biopterin transporter), OsJRFA066919 (putative potassium transporter), OsJRFA067899 and OsIFCC019970 (ABC transporter, putative), OsJRFA068003 and OsJRFA106202 (Transmembrane amino acid transporter protein), OsJRFA068765 (H-ATPase), OsIFCC040482 (phosphate:H+ symporter), OsJRFA072183 (Sodium:sulfate symporter transmembrane region), OsJRFA102086 (putative lipid transfer protein) and OsJRFA103807 (aquaporin). The photosynthesis related genes cover most of the gene components of the two photosystems, such as genes for putative chlorophyll a/b-binding protein, Photosystem I or II reaction center subunits, and plastocyanin. These photosystem genes and transporter genes represent a large portion of all genes induced by rehydration, and their physiological roles in plants fit well with a potential contribution toward plant recovery from drought stress.
Limited overlap of stress responsive genes among rice organs
We also examined the degree of overlap in expression of stress responsive genes in two or more rice organs under drought or high-salinity stress. Venn diagrams revealed that only a small portion of genes was shared between each pair of organs (Fig. 4). The greatest overlap occurred between shoot and flag leaf, which shared more inducible genes than shoot and panicle or flag leaf and panicle under both drought and high-salinity conditions. Under drought stress, one-third of the genes up-regulated in flag leaf (197/582) were also induced in shoot (Fig. 4A), while under high salinity stress, over half of the induced genes (494/817) in shoot (mostly young leaves) were also up-regulated in flag leaf (Fig. 4B). Only 21 and 23% of genes up-regulated in panicle were also induced in flag leaf under drought and high salinity stresses respectively. The percentage of genes shared between shoot and panicle is similar to that between flag leaf and panicle (Fig. 4A, B).
Interestingly, only a small fraction of stress responsive genes were expressed in all three organs examined. For example, 42 and 151 genes were induced in all three organs under drought and high-salinity stress respectively. In general, most of those genes exhibited high levels of expression as well as strong inducibility. Among them, 27 genes were induced by both drought and high-salinity stress in all three organs. This group of 27 genes includes a protein kinase (OsJRFA058518), chlorophyll a/b binding protein (OsJRFA062972), ABA-responsive protein (OsJRFA063578, OsIFCC018156), LEA protein (OsJRFA063984), dehydrin (OsIFCC035028), CHY zinc finger protein (OsIFCC003263), and homeobox protein (OsIFCC018343) (Table 2).
Table 2 Genes up- or down-regulated by both drought and high-salinity stresses in all three organs
Analysis of stress down-regulated genes (Fig. 4C, D) revealed a similar small overlap among the three organs. Only 68 and 129 out of 795 total genes down-regulated in flag leaf under drought stress were also inhibited in shoot and panicle, respectively. For high-salinity stress, 135 and 214 out of 1,270 down-regulated genes in flag leaf were also repressed in shoot and panicle respectively. Only 16 and 38 genes were down-regulated in all three organs under drought and high salinity stress respectively, while only two genes were inhibited in all three organs under both drought and high salinity stress (Table 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.
To validate the microarray data, RT-PCR analysis was used to verify the transcription response of representative genes from microarray results. For this purpose, we picked a group of eight representative genes and designed primers for RT-PCR analysis (Table S18). The cDNA templates were synthesized from total RNA samples prepared from shoot, flag leaf and panicle under drought and unstressed controls. The semi-quantitative RT-PCR results for the eight representative genes were shown in Fig. 5. The expression patterns of all eight genes reflected changes observed by microarray analysis fairly and accurately for all three organs examined, except in three cases where RT-PCR failed to confirm the microarray results (Fig. 5), indicating that our microarray data is reliable.
Enrichment of ABRE and DRE cis-regulatory elements in abiotic stress-induced gene promoters
Previous reports have suggested that several well-characterized drought-, high-salinity and cold inducible gene promoters contain two common cis-regulatory elements, the ABA-responsive element (ABRE) and the dehydration-responsive element (DRE) (Seki et al., 2001, 2002a, b; Yazaki et al., 2003; Rabbani et al., 2003; Dubouzet et al., 2003; Nakabayashi et al., 2005; Maruyama et al., 2004; Yamaguchi-Shinozaki and Shinozaki, 2005). ABRE and DRE confer ABA-dependent and ABA-independent gene expression in response to water stress. It has been reported that a single copy of ABRE in the promoter region only induces relatively minor elevation of expression level, while multiple copies of ABRE strongly activate expression (Shen and Ho, 1995; Nakabayashi et al., 2005).
To elucidate the relationship between copy numbers of DRE and ABRE cis-regulatory elements in gene promoter regions and abiotic stress-induced gene transcription, we divided the genes into groups based on their expression patterns. Genes induced specifically by drought or high-salinity stress in each of the three organs defined six groups, while genes induced by both drought and high-salinity stress in each organ defined another three groups. Common inducible genes shared by at least two organs under drought or high-salinity stress defined another nine groups. We only selected those genes among all those groups with full-length cDNA sequences available for further promoter analysis. In brief, the 2 kb upstream regions (–1 to –2,000 bp) preceding the ATG start codon of genes were selected for promoter motif analysis (see Experimental procedures). A similar number of rice genes with full length cDNA available that lack stress responsive expression were used as control. Copy number of ABRE and DRE core motifs on each promoter region were counted. The percentages of genes in each group with 1 to 6 copies of ABRE core or DRE core elements were then calculated.
Compared to control genes, copy numbers of the ABRE and DRE core motifs in the promoter regions of genes with organ-specific expression in response to drought or high-salinity stress were not markedly different (Fig. 6A, D). The promoter regions of genes responsive to both drought and high-salinity stress in each organ were enriched for ABRE and DRE core motifs compared to the promoters of control genes (Fig. 6B, E). For example, only 25% of control genes have over four copies of ABRE core motif in their promoter regions, but 48, 44 and 48% of genes induced by both drought and high-salinity stresses in flag leaf, panicle and shoot respectively have over four copies of the ABRE core motif in their promoter regions (Fig. 6B).
When genes expressed in at least two organs under drought or high-salinity stress were compared to control genes, the ABRE and DRE core motif copy numbers also showed significant differences (Fig. 6C, F). For example, 50% of these genes contain over four copies of the ABRE core motif in their promoter regions, compared with 25% for the control genes (Fig. 6C). A similar pattern was found for the DRE core element (Fig. 6F).
Genes induced in an organ-specific manner by drought or high-salinity stresses did not contain a significantly different number of copies of ABRE or DRE core motifs compared to control genes (Fig. 6A, D). Thus it is plausible that the stress-induced expression in this fraction of genes may rely less on transcriptional activation mediated by ABRE and DRE motifs. Unidentified organ-specific cis-regulatory elements may exist in the promoter regions of these genes and play a more important role in transcription activation under drought or high-salinity stress.
Identification of two novel cis-regulatory elements that respond to rehydration after drought stress
As shown in Table 1, there are 8 and 21 genes with full-length cDNA sequences repressed by drought stress but induced by rehydration in shoot and panicle, respectively. An analysis of the upstream region of these eight shoot genes identified a novel motif (motif-SP, GGCAGCCG) located near to the translation start codon (–73 to –250) in five of them (Fig. 7A). To investigate the potential functional role of this novel motif, we performed a gel shift mobility assay which involved incubating a 24 bp probe containing motif-SP from one of those five genes (OsJRFA070715) and a control containing a single base mutation at an invariable position with nuclear extracts (see Experimental procedures). Only nuclear extracts from drought-treated shoot (at the third stage), but not from unstressed shoot or shoot under rehydration, showed a sequence specific binding activity with the probe (Fig. 7C). A single base mutation at an invariable position in the motif-SP core of the probe completely abolished protein binding activity, suggesting high specificity of interaction with the core motif included in this 24 bp sequence. This specific binding activity was only observed in drought-treated plants, suggesting that it is involved in transcriptional repression under drought conditions.
We also searched promoter regions of 21 genes inhibited by drought stress but induced by rehydration in panicle. Three conserved motifs were found through this blind search in 11 of the 21 genes: ABRE motif, the above-mentioned motif-SP, and another novel element, motif-P (Fig. 8). The presence of the ABRE motif suggests that a gene may respond to drought stress by employing an ABA-dependent pathway, while motif-SP may play an important role in drought-stress repression and subsequent activation during rehydration. We failed to detect specific binding activity using similar nuclear extracts (data not shown) and thus the role of motif-P remains to be defined.
Transcription factor genes under drought and high salinity stress are expressed in a largely organ specific manner
We further examined transcription factor genes whose expression are regulated by drought and high-salinity stresses. Among all genes induced by drought or high-salinity treatment in the three organs, a total of 186 genes (Table S17) were predicted to be transcription factors with a DNA-binding domain. These transcription factors belong to various families, including AP2, bHLH, MYB, HB, NAC, zinc finger, MADS, bZIP, WRKY and HSF families. These transcription factors could also be divided into several groups depending on their expression patterns. Among the 186 transcription factor genes, 12 genes were induced in all three organs in at least one stage following either drought or high-salinity stress, whereas the remaining 174 genes were mainly up regulated in one or two organs. Transcription factors induced only in one individual organ were listed in Table 3. It is interesting to note that over half of the transcription factors were expressed in at least two organs, while other transcription factors were activated in an organ-specific manner. This suggests that the expression of different transcription factors may play a key role in common or organ-specific gene expression in response to drought or high-salinity conditions.
Table 3 Transcription factor genes induced in each organ by drought or high-salinity stress
Minimal co-regulation of neighboring genes in drought or high-salinity stress responses
Co-regulation of adjacent genes has been observed in several organisms (Hurst et al., 2004), including human (Caron et al., 2001; Lercher et al., 2002), Drosophila (Spellman and Rubin, 2002), Arabidopsis (Birnbaum et al., 2003, Ma et al., 2005a), and yeast (Cohen et al., 2000). All these studies identified co-regulation of neighboring gene clusters on chromosomes (chromatin domains). In rice, analysis of organ-specific gene expression revealed that about 10% of the genome belongs to chromatin domains that exhibit this co-expression pattern (Ma et al., 2005b). However, similar analysis of light-regulated genes failed to reveal significant co-regulation (Jiao et al., 2005).
To investigate possible co-regulation of neighboring genes in the drought and high-salinity responses, we mapped rehydration induced genes in three organs and drought and high-salinity induced genes in shoot to all 12 indica chromosomes (Figs. 9, S4). This analysis indicated that significant gene clustering among rehydration, drought, and high-salinity induced genes did not occur at the chromosomal level, similar to the pattern characteristic of light-regulated genes (Jiao et al., 2005). Among the 12 rice chromosomes, only three regions showed significant enrichment of 10 or more co-regulated genes within any given 2 Mb domain. For example, the 33–35 Mb region on chromosome 2, the 11–13 Mb region in chromosome 3, and the 31–33 Mb region in chromosome 6 (Fig. 9B) contain significant enrichment of rehydration regulated genes in all three organs, although even in those cases genes are not directly adjacent.