Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces a morphological response to drought stress

Abstract

The mechanisms for plant growth restriction during stress conditions remains unclear. Here, we demonstrate that a phytochrome-interacting factor-like protein, OsPIL1/OsPIL13, acts as a key regulator of reduced internode elongation in rice under drought conditions. The level of OsPIL1 mRNA in rice seedlings grown under nonstressed conditions with light/dark cycles oscillated in a circadian manner with peaks in the middle of the light period. Under drought stress conditions, OsPIL1 expression was inhibited during the light period. We found that OsPIL1 was highly expressed in the node portions of the stem using promoter-glucuronidase analysis. Overexpression of OsPIL1 in transgenic rice plants promoted internode elongation. In contrast, transgenic rice plants with a chimeric repressor resulted in short internode sections. Alteration of internode cell size was observed in OsPIL1 transgenic plants, indicating that differences in cell size cause the change in internode length. Oligoarray analysis revealed OsPIL1 downstream genes, which were enriched for cell wall-related genes responsible for cell elongation. These data suggest that OsPIL1 functions as a key regulatory factor of reduced plant height via cell wall-related genes in response to drought stress. This regulatory system may be important for morphological stress adaptation in rice under drought conditions.

Drought is the biggest constraint of growth and development in plants, often resulting in reduced crop yields (1, 2). To produce crops with high yields even under drought conditions, it is essential to unravel the molecular mechanisms of growth reduction in response to drought stress. Growth reduction under stress conditions has been considered a secondary effect of reduced photosynthetic activity and stomatal closure (3). However, it is now accepted that plants actively reduce their growth to save photosynthetic resources and decrease transpiration area as a stress adaptation response (4).

Under stress conditions, plants induce expression of a wide variety of genes. Among these genes, key factors that function in stress responses have been identified (5–8). These factors include: DRE binding 1 (DREB1)/C-repeat binding factor-type transcription factors that are involved in response to low-temperature stress; DREB2-type transcription factors that play important roles in drought and high-temperature stress-responsive gene expression; ABA responsive element binding /ABA binding factor-type transcription factors that modulate the ABA-dependent transcriptional network; and NAC-type transcription factors that are involved in response to drought and high-salinity stresses. These factors are known to function as transcriptional activators to enhance abiotic stress tolerance. Constitutive expression of these transcription factor genes in transgenic Arabidopsis or rice confers tolerance to abiotic stresses (9–11). Moreover, overexpression of these genes causes growth reduction phenotypes in transgenic plants, but the molecular mechanisms of these phenotypes remain unclear.

Knowledge of physiological and molecular mechanisms for growth reduction in response to abiotic stresses is still fragmented. Drought decreases leaf area by reducing both cell number and size (3, 4, 12). This reduced cell number is attributed to the inhibition of the G1-to-S transition, mediated by a specific cyclin-dependent kinase (4, 13). Cell size modulation under stress conditions is accompanied by a change in cell wall rheology via cell wall-related genes, including expansins (3, 14, 15). Key factors that function in the transcriptional network of growth reduction in response to abiotic stresses are still unclear.

To dissect transcriptional networks in abiotic stress responses, it is important to study transcription factors that are differentially expressed between stressed and nonstressed plants. Although most transcription factors that function in stress tolerance are up-regulated by abiotic stresses, we detected candidate transcription factors down-regulated by these stresses. Here, we report a gene for a phytochrome (phy)-interacting basic helix-loop-helix transcription factor (PIF)-like protein down-regulated under drought stress conditions. One of the PIF family transcription factors was initially isolated as a protein that interacted with phytochrome through a yeast two-hybrid screen (16, 17). PIFs have been shown to be components of various developmental responses, including seed germination, seedling growth, and cell fate (18). We propose that the rice PIF-like protein can act as an internodal growth regulator and plays an important role in a drought-associated growth-restriction mechanism that functions as a morphological adaptation to drought stress.

Results

Expression Patterns of OsPIL1.

A rice PIF-like gene (OsPIL1/OsPIL13, LOC_Os03g56950) was identified by microarray analyses (19) as one of the stress-responsive transcription factor genes that were down-regulated by drought stress. First, we confirmed the stress-responsive expression pattern of the OsPIL1 gene. The level of OsPIL1 mRNA in rice seedlings grown under nonstressed conditions with 12-h light/12-h dark cycles oscillated in a circadian manner, with peaks in the middle of the light period (Fig. 1A). Intriguingly, when drought stress started in the middle of the dark period, OsPIL1 expression was not elevated during the light period. When drought stress started early in the light period, OsPIL1 expression was drastically decreased to a level similar to that observed in the dark period. Cold stress also inhibited expression during the light period (Fig. 1B). High-salinity (NaCl), high-temperature, ABA and gibberellic acid (GA) did not obviously change the OsPIL1 expression pattern. When the rice seedlings were treated with ethephon, OsPIL1 expression reached a peak earlier than in the control plants.

Fig. 1.

Expression analysis of OsPIL1 by quantitative RT-PCR. Bars indicate the SD of three to four replicates. Arrows indicate the start point of the stress treatment. (A) Expression patterns of OsPIL1 under nonstress (control) or drought stress (drought) conditions. (B) Expression patterns of OsPIL1 when rice seedlings were kept at 4 °C (cold) or 37 °C (hot), or the roots were immersed in 250 mM NaCl, 25 μM ABA, 25 μM GA, or 100 μM ethephon.

Histochemical Analysis and Subcellular Localization of OsPIL1.

Promoter-glucuronidase (GUS) analyses were performed to reveal histological localization of OsPIL1 expression. GUS signals in seedlings were detected in the leaves and the basal part of shoots (Fig. 2A). At the early heading stage, GUS signals were observed in the nodal regions, especially in the nodal septum (Fig. 2 B and C). To confirm these GUS signals at the heading stage, the mRNA levels of OsPIL1 were investigated using quantitative RT-PCR. In agreement with the promoter-GUS analyses, much higher OsPIL1 mRNA levels were detected in the node region compared with the internode region (Fig. 2D).

Fig. 2.

Promoter-GUS staining analysis of OsPIL1. Similar expression patterns were observed in multiple independent transgenic lines. (A) Expression of the GUS gene in 2-wk-old seedlings. (Upper) The leaf region. (Lower) A longitudinal section at the basal part of the seedling. (Scale bar, 2 mm.) (B) Expression of GUS in the node and internode portions of the stem at heading stage. Photos show longitudinal sections. (Scale bars, 20 mm.) (C) Expression of GUS in the node portion of the stem at heading stage. (Scale bars, 5 mm.) ls, leaf sheath; lsp, leaf-sheath pulvinus; mc, medullary cavity; ns, nodal septum. (D) Quantitative RT-PCR analysis of OsPIL1 expression in panicles, first internodes, first nodes, and second internodes at heading stage. Bars indicate the SD of three to four replicates.

To examine the subcellular localization of OsPIL1 protein, we performed a transient expression assay using rice protoplast cells prepared from shoots. Clear GFP-tagged OsPIL1 protein signals were detected in the nucleus (Fig. S1A), suggesting that the OsPIL1 protein was localized to the nucleus. We also examined transgenic rice seedlings expressing GFP-tagged OsPIL1. GFP signals were detected in the nucleus of cells in the basal part of shoots (Fig. S1C).

Morphological Phenotypes of Transgenic Plants Overexpressing OsPIL1 or Expressing an OsPIL1 Chimera Repressor.

To further elucidate the function of the OsPIL1 protein, we generated transgenic rice plants overexpressing OsPIL1 (OsPIL1-OXs) or expressing OsPIL1 fused to a repression domain (RD) that consists of only 12 amino acids to convert a transcriptional activator into a transcriptional repressor and to overcome potential functional redundancy conferred by homologs (OsPIL1-RDs) (20). We obtained seven independent OsPIL1-OX candidates and six independent OsPIL1-RD candidates, and selected transformants with high transgene expression levels for further analyses (OsPIL1-OX#2, OsPIL1-OX#17, OsPIL1-RD#3, and OsPIL1-RD#5) (Fig. S2A). Although we also tried to generate RNAi transgenic rice, we could not obtain transformants in which the expression level of OsPIL1 was reduced. OsPIL1-OX and OsPIL1-RD rice seedlings showed a slight increase and decrease in plant height, respectively (Fig. S2 B and C). At 30 d after imbibition, OsPIL1-OX and OsPIL1-RD rice plants showed a significant increase and decrease, respectively, in stem length between the shoot base and the uppermost lamina joint (Fig. S2 D and E). At the adult stage, OsPIL1-OXs displayed enhanced stem elongation, leading to a strikingly tall plant (Fig. 3 A and C and Fig. S2I). This result was mainly because of increased elongation in each internode (Fig. S2J). In contrast, adult OsPIL1-RD plants were shorter than vector control plants. The sheath lengths of flag leaves were not different between vector control plants and OsPIL1-OXs or OsPIL1-RDs (Fig. S2G). Consequently, in OsPIL1-OXs the sheath length was shorter than the internode length (Fig. S2 F, H, and K). In OsPIL1-RDs the sheath length was greater than the internode length, resulting in imperfect emergence of panicles (Fig. S2 F, H, and K).

Fig. 3.

Phenotypes of OsPIL1 transgenic rice plants. (A) Morphology of mature vector control plants (Con), transgenic rice plants overexpressing OsPIL1 (OX), and plants expressing an OsPIL1 chimeric repressor that contains an additional repression domain (RD). Some leaves and leaf sheaths were removed to check the position of each node. PN, panicle node; 1N, first node; 2N, second node; 3N, third node. (Scale bars, 30 cm.) (B) Longitudinal sections at the first internodes in mature Con, OX, and RD plants. The left side of each photo indicates silicified epidermis (se) and sclerenchymatous fiber tissue (sft), and the right edge shows a central lacuna (cl). (Scale bars, 0.1 μm.) (C) Heights of mature Con, OX, and RD plants. **P < 0.001 compared with the control value in Scheffé’s test. Bars indicate the SD of 7–17 replicates. (D) Vertical lengths of parenchyma cells in Con, OX, and RD plants. **P < 0.001 compared with the control value in Scheffé’s test. Bars indicate the SD of 30 replicates.

To determine whether the abnormal internode elongation was caused by alterations in cell number or cell size, longitudinal sections of internodes were examined. The first internode cells of OsPIL1-OXs were larger than those of nontransgenic control plants (Fig. 3 B and D). Smaller internode cells were found in OsPIL1-RDs. These results suggest that the differences in internode lengths between control plants and OsPIL1-OXs or OsPIL1-RDs primarily resulted from alterations in cell size. The vertical seed lengths were longer in OsPIL1-OXs and slightly shorter in OsPIL1-RDs compared with those of nontransgenic control plants (Fig. S2 L and M). The yield was not significantly different between the transgenic plants and the vector controls (Fig. S2N).

Transgenic Arabidopsis plants overexpressing OsPIL1 (35S:OsPIL1) showed longer hypocotyls than vector control plants (Fig. S2 O and P). In contrast, transgenic Arabidopsis plants expressing OsPIL1 fused to the RD (35S:OsPIL1:RD) had short hypocotyls. At the adult stage, the 35S:OsPIL1 and 35S:OsPIL1:RD plants showed no increase or decrease in plant height (Fig. S2Q).

Downstream Genes of OsPIL1.

To identify downstream genes regulated by the OsPIL1 transcription factor, we performed a microarray analysis using the OsPIL1 transgenic rice plants. Because OsPIL1-OXs showed increased internode elongation (Fig. 3) and the OsPIL1 gene was highly expressed in the first node portion (Fig. 2), only the first node portions of OsPIL1-OXs and vector control plants were used for the microarray analysis. After RNA extraction from the first node portion, large-scale expression profiles were compared between OsPIL1-OXs and vector control plants. The transcriptome analysis identified 1,396 genes up-regulated [formal concept analysis (FCA) > 2.0] and 1,358 genes down-regulated (FCA < 2.0) in the first node portion of OsPIL1-OXs (Dataset S1). Expression of more than half of the up-regulated genes was decreased under drought stress (790 of 1,396 genes), and expression of large numbers of the down-regulated genes was increased by drought stress (480 of 1,358 genes) in our microarray data (Fig. 4A). A gene-expression search engine, GENEVESTIGATOR, also confirmed the decreased expression of the OsPIL1-OX up-regulated genes under drought conditions (Fig. 4B).

Fig. 4.

Downstream genes of OsPIL1. (A) Expression profiles of the OsPIL1 downstream genes by GENEVESTIGATOR (https://www.genevestigator.com/gv/user/gvLogin.jsp). The responses of the up-regulated (FCA > 2.0) genes in the node portion of OsPIL1 OX plants to external stimuli or perturbations were analyzed. (B) Expression changes under drought conditions of up- (FCA > 2.0) or down- (FCA < 2.0) regulated genes in the node portion of OsPIL1 OX plants, from our earlier microarray data (19). (C) Expression levels of OsPIL1, the representative OsPIL1 downstream genes (Expansin S1, Expansin S2, OsEXPA2, OsEXPA4, Extensin, and 1-ACC oxidase) and drought-inducible marker genes (OsNAC6 and OsDREB2B2) in the node portions of wild-type rice plants at heading stage under nonstressed (Con) or drought-stressed (DS) conditions by quantitative RT-PCR. Wild-type rice plants at heading stage were subjected to drought stress for 40 h after uprooting. Bars indicate the SD of three to five replicates. (D) Design of constructs for transient expression assay. (E) Transient expression assay using rice protoplast cells. Intrinsic long promoters (−1,000 to −1 bp, designated Long Exp), intrinsic short promoters (G-box location to −1 bp, designated Short Exp), intrinsic G-box-deleted promoters (fragments without a G-box, designated Del G Exp), and artificial promoters that include three G-box elements (designated G3 Exp) were used. Constructs containing the LUC gene driven by the ubiquitin promoter were used to normalize transfection efficiency. Bars indicate the SD of five to seven replicates.

To obtain an ontological profile of the up- or down-regulated genes in OsPIL1-OXs, ontological terms were assigned (Fig. S3A), and the enrichment significance was analyzed by AgriGO. Sixteen Gene Ontology (GO) terms were significantly enriched in the up-regulated gene set (Table S1). Of the enriched GO terms, we noted ones related to cell wall development because activated cell elongation was observed in OsPIL1-OXs (Fig. 3 B and D). Four GO terms were related to cell wall development: cell wall organization or biogenesis (GO:0071554), cell wall organization (GO:0071555), cellulose metabolic process (GO:0030243), and cellulose biosynthetic process (GO:0030244). These GOs included 39 genes, such as expansins and cellulose synthases (Table S2). Most of these genes were down-regulated under drought conditions (Table S2). Furthermore, we investigated gene-expression levels in nonstressed and drought-stressed node portions. In drought-stressed node portions, the expression levels of Expansin S1 (Os03g0336400), Expansin S2 (Os04g0228400), OsEXPA2 (Os01g0823100), OsEXPA4 (Os05g0477600), and Extensin protein-like (Os03g0637600), as well as OsPIL1, were lower than in the nonstressed node portions (Fig. 4C). Higher expression levels of OsNAC6 (21) and OsDREB2B2 (22) in drought-stressed node portions compared with nonstressed ones supported the effectiveness of this stress manipulation. These data suggest that the cell wall-related genes identified here are ideal candidates for OsPIL1 downstream genes.

We searched for cis-element candidates in the upstream regions of the OsPIL1 downstream gene ORFs using RiCES. CACGTG (G-box) sequences, which have been shown to be recognition sites for Arabidopsis PIF proteins (23), were found in the upstream regions of OsPIL1 downstream genes (Table S3). Other sequences were also detected, including CAAT[GC]ATTG and CCA[ACTG]TG, which are binding sites for HD-ZIP and LEAFY, suggesting that these transcription factors might be also involved in the OsPIL1 downstream cascade.

Transactivation Activity of OsPIL1.

To verify whether OsPIL1 functions as a transcriptional regulator, we investigated the transactivation activity of OsPIL1 using the transient assay system (22, 24). Protoplasts were cotransfected with GUS reporter constructs containing the promoter region from OsEXPA4 (Os05g0477600), which was identified above as one of the OsPIL1 downstream genes, and effector plasmids containing OsPIL1 cDNA fused to the ubiquitin promoter (Fig. 4D). In the presence of the effector, the −1 kb OsEXPA4 promoter (Long Exp) increased GUS activity (Fig. 4E). This promoter region contained a copy of the G-box element. A short OsEXPA4 promoter (Short Exp) containing the G-box element similarly increased GUS activity. A promoter lacking the G-box element (Del G Exp) showed decreased GUS activity compared with Long Exp or Short Exp. When a construct containing additional copies of the G-box element (G3 Exp) was used, GUS activity was higher than in Long Exp or Short Exp. Furthermore, we analyzed the promoter of another gene, 1-aminocyclopropane-1-carboxylate (1-ACC) oxidase (Os09g0451400), because we predicted that it was one of the downstream candidate genes of OsPIL1. This gene was up-regulated in the OsPIL1-OXs (FCA = 3.5) (Dataset S1) and down-regulated in the drought-stressed node (Fig. 4C). We obtained similar data to the OsEXPA4 promoter using the GUS gene fused to the −1 kb 1-ACC oxidase promoter (Fig. S3C). These results suggested that OsPIL1 could activate expression of the OsEXPA4 and 1-ACC oxidase genes via the G-box element. To check whether the OsPIL1-GFP protein was functional, we performed a transactivation assay using the G3 Exp promoter-GUS construct. The reporter GUS activity was enhanced by the addition of OsPIL1-GFP proteins, suggesting that the fusion protein was functional (Fig. S3E).

We also examined the effect of OsPhyB on the transcriptional activity of OsPIL1 using the transient reporter assay. Addition of OsPhyB did not change the GUS activity under either dark or light conditions, whereas PhyB decreased the transcriptional activity of PIF4 (Fig. S3F). This finding suggested that OsPhyB does not affect the transcriptional activity of OsPIL1. This hypothesis was supported by the observation that OsPIL1 did not interact with OsPhyB in the yeast two-hybrid assay (Fig. S3G) or the bimolecular fluorescence complementation assay (Fig. S3H), and by the fact that one of the four residues important for PhyB binding in Arabidopsis PIFs (25) was not conserved in OsPIL1 (Fig. S3I).

Hormone Content.

Growth modulation by PIF4 was reported to be mediated by indole-3-acetic acid (26) or GA (27) signaling. To check the involvement of plant hormones in OsPIL1-regulated stem elongation, we measured hormone content in the OsPIL1 transgenic rice plants (Table S4). We used growing stem regions from the shoot base to the uppermost lamina joint at 30 d after imbibition as materials (Fig. S2 D and E). The levels of GA1, GA19, GA20, GA44, and GA53 were slightly decreased in OsPIL1-OXs but not significantly changed in OsPIL1-RDs. The levels of other hormones were not significantly different between the OsPIL1 transgenic rice plants, suggesting that the hormones measured here were not involved in the regulation of stem elongation by OsPIL1. Thus, OsPIL1 internode elongation may be modulated by a different pathway from Arabidopsis PIF4.

Discussion

The molecular mechanisms for the regulation of plant growth and development during stress conditions remains unclear. Our data provide evidence that OsPIL1 functions as a key regulator of reduced plant height in rice during stress conditions. Several results support this conclusion: (i) OsPIL1-OXs showed activated internode elongation via increased cell size, whereas OsPIL1-RDs had short internode sections resulting from decreased cell size (Fig. 3); (ii) OsPIL1 downstream genes included a number of the cell wall-related genes (Table S2), which have been reported to be involved in plant growth regulation by cell elongation (28, 29); and (iii) light-dependent expression of OsPIL1 was clearly inhibited during stress conditions (Fig. 1).

The activated internode elongation observed in OsPIL1-OXs is the most intriguing phenotype (Fig. 3 A and C). Internode elongation is considered to be caused by two kinds of regulatory mechanisms (15). One mechanism is an enhanced cell-division rate in the meristematic region, initiated by GA-induced expression of cyclin genes and a p34cdc2-like histone H1 kinase (15, 30). The other mechanism is cell elongation achieved by regulation of microtubule orientation, creep of cell-wall polymers, and biosynthesis, transport, and incorporation of new cell-wall components (15, 31). We concluded that cell elongation caused the activated internode elongation in OsPIL1-OXs, because of alterations in internode cell size (Fig. 3 B and D). This conclusion was further supported by the finding that the set of up-regulated genes in OsPIL1-OXs included a number of genes associated with cell wall biosynthesis and development (Table S2). Expansins have been shown to regulate cell elongation by cell wall expansion, which is caused by acid-induced cell wall relaxation (15, 32–34). α-Expansin OsEXPA4 overexpressors show an elongated shoot, coleoptile, and mesocotyl, resulting from changes in cell size (29). A network of extensins is thought to be involved in the control of cell elongation and cell-wall architecture (35, 36). Cellulose synthase is also involved in cell wall formation and plant growth. A rice cellulose synthase mutant showed significant reduction in plant growth (37). These factors reinforce the possibility that the cell wall-related genes identified here as OsPIL1 downstream genes caused the abnormal internode elongation in OsPIL1 transgenic rice plants. Most of the up-regulated genes in OsPIL1-OXs were significantly expressed in aerobic and anaerobic seed germination (Fig. 4B), which suggests that they are involved in germination processes. Germination processes cause cell proliferation and enlargement, and are adversely affected by drought conditions.

The set of down-regulated genes in OsPIL1-OXs included many drought-inducible genes in rice plants (Fig. 4A). We compared these down-regulated genes with genes downstream of the transcription factors OsDREB1A, OsNAC6, and OsbZIP23, which have been reported to positively regulate expression of stress-responsive genes in rice plants. The number of genes downstream of OsDREB1A, OsNAC6, and OsbZIP23 was 81, 158, and 743, respectively (38–40). Among these genes, only 13, 37, and 61 genes overlapped with the down-regulated genes in OsPIL1-OXs, respectively. These results suggest that the pathways regulated by OsDREB1A, OsNAC6 and OsbZIP23 act in parallel to the pathway regulated by OsPIL1.

The OsPIL1 mRNA level in rice seedlings grown under nonstressed conditions with light/dark cycles oscillated in a circadian manner with peaks in the middle of the light period (Fig. 1). This finding is similar to Arabidopsis PIF4 and PIF5 expression patterns (41). Phylogenetically, Arabidopsis PIF4 and PIF5 are the closest orthologs of OsPIL1 (38). Transgenic Arabidopsis plants overexpressing PIF4 or PIF5 showed increased hypocotyl elongation (42, 43). We observed a similar morphological phenotype in transgenic Arabidopsis seedlings overexpressing OsPIL1 (Fig. S2 O and P). These results imply that rice OsPIL1 may have a similar role to Arabidopsis PIF4 and PIF5 in plant developmental processes. PIF4 was shown to control photoperiodic growth (41, 44), stomatal development under high light intensity (45), and thermal acceleration of flowering (46) and petiole elongation (47). These studies suggest that PIF4 functions as a node that connects development with external stimuli such as light and temperature.

Arabidopsis PIF4 and PIF5 have also been shown to function in shade avoidance (43). These transcription factors accumulate in the dark and act as constitutive repressors of photomorphogenesis in seedlings. In response to red light these proteins are selectively degraded, which requires their interaction with light-activated PhyB. The proteins are stable in the shade and required for shade avoidance that promotes shade-induced acceleration of hypocotyl elongation (44). In contrast to Arabidopsis PIFs, OsPIL1 was shown not to interact with OsPhyB (Fig. S3), suggesting that OsPIL1 is more stable and active upon exposure to light than Arabidopsis PIFs and may affect plant growth more strongly. However, as the OsPIL1 mRNA level was decreased by drought stress, internode elongation does not occur under the stressed conditions in rice. This strategy gives plants an advantage in that chances of shading by neighbor plants are significantly increased. Shading provides plants with lower temperature conditions, reducing transpiration rate and increasing survivability when plants are rewatered. A similar strategy has been reported in the response to UV-B exposure. UV-B decreases stem elongation, increasing opportunities to use shade provided by neighbor plants (48, 49). Because the exogenous OsPIL1 expression in OsPIL1-OXs is expected to be constitutive even under abiotic stress conditions, morphological studies of OsPIL1-OXs under the stress conditions may provide a better understanding of the physiological function of OsPIL1 in rice.

In conclusion, our data provide a model for OsPIL1 regulation of plant height via cell wall-related genes under drought stress conditions (Fig. S4). Under nonstress conditions, light exposure increases the expression of OsPIL1. This expression leads to increased expression of cell wall-related genes, and consequently cell elongation, resulting in internode elongation. Conversely, drought stress decreases the expression of OsPIL1 under light illumination, leading to decreased expression of cell wall-related genes. This process inhibits cell elongation, resulting in dwarf height. Down-regulation of OsPIL1 expression may function as a reliable morphological adaptation system for reducing plant height under drought stress conditions.

Materials and Methods

Plant Materials and Stress Treatments.

Rice (Oryza sativa cv. Nipponbare) plants were grown as described previously (21, 39). Drought, high-salinity and low-temperature stress experiments were conducted as described in ref. 21. A high-temperature stress experiment was performed as described in ref. 22). Ethephon was used as an alternative to ethylene.

Generation of Transgenic Plants.

To develop OsPIL1-OXs, we inserted the OsPIL1 cDNA ORF into a plant expression vector (50) containing a ubiquitin promoter region (51). This construct was introduced into wild-type rice cv. Nipponbare by Agrobacterium-mediated transformation (52). OsPIL1-RDs were generated using the CRES-T system (20, 53). To develop transgenic rice plants expressing a GUS gene driven by the OsPIL1 promoter, we inserted the OsPIL1 promoter region into a GUS expression vector and introduced the construct into rice plants (21)

Transient Expression in Rice Mesophyll Protoplasts.

Protoplasts from rice shoots were prepared as described in ref. 54. PEG-calcium transfection was performed for transient expression assay (54). After incubation, reporter activity was measured as described previously (22, 24).

Microarray Analysis.

Total RNA for microarray analysis was isolated from node portions of OsPIL1-overexpressing and vector control plants at the heading stage. The microarray analysis was performed as described in ref. 19. Microarray data are available at the European Bioinformatics Institute (http://www.ebi.ac.uk/microarray-as/ae), with the accession number E-MEXP-3605.

Quantitative RT-PCR Analysis.

Total RNA was prepared using RNAiso reagent (Takara Bio). PCR product levels were normalized by the expression value of 18S rRNA as an internal standard. The OsPIL1 primers were 5′-GCAAACAGTGCCACCACAGG-3′ (1,092–1,111 nt from ATG) and 5′-CTAAATTCCATCAGAGGTTGG-3′ (1,213–1,233 nt from ATG). The 18S rRNA primers were 5′-ATGGTGGTGACGGGTGAC-3′ and 5′-CAGACACTAAAGCGCCCGGTA-3′.

Measurement of Plant Hormone Levels.

The plant hormone levels in 100 mg of tissue from each plant was quantified as described in ref. 55 using a liquid chromatography-MS system (UPLC/Quattro Premier XE; Waters) with an ODS column (AQUITY-UPLC BEH-C18, 1.7 μm, 2.1 × 50 mm). Three biological replicates were used in each experiment.