INTRODUCTION
Rice (Oryza sativa L.) is a staple food for more than half of the world's population. Although total global rice production demonstrates annual increases, there are many biotic and abiotic factors affecting yield. The most influential abiotic stresses are drought, salinity, cold and heat stresses. In particular, drought stress resulting from the absence of rainfall for long periods or deficits in usable water resources can affect crop yield significantly (Hadiarto & Tran Reference Hadiarto and Tran2011; Joo et al. Reference Joo, Choi, Lee, Kim and Song2013). In plants, drought stress causes a decrease of water potential in tissue, which induces a series of physiological responses, such as growth and development inhibition, decrease in chlorophyll content, inhibition of photosynthesis and stomatal closure (Zhang et al. Reference Zhang, Zhang, Quan, Pan, Wan and Huang2013; Lim et al. Reference Lim, Lee and Jang2014). Meanwhile, the expression of unique genes or groups of genes and their expression patterns show different responses under drought stress (Do et al. Reference Do, Drechsel, Heyer, Hincha and Zuther2014; Oono et al. Reference Oono, Yazawa, Kawahara, Kanamori, Kobayashi, Sasaki, Mori, Wu, Handa, Itoh and Matsumoto2014).
In plants, APETALA2/ethylene-responsive factor (AP2/ERF) is a large family of transcription factors that include AP2, ERF, DREB and RAV sub-family members. The AP2 sub-family members possess two repeats of the AP2/ERF domain, ERF and DREB sub-family proteins contain a single AP2/ERF domain and RAV sub-family proteins have an additional B3 DNA-binding domain. The difference between ERF and DREB members is the binding sequence: the ERF proteins bind to AGCCGCC, while the DREB proteins recognize A/GCCGAC (Dey & Corina Vlot Reference Dey and Corina Vlot2015). Based on the conserved AP2/ERF DNA-binding domain, 170 AP2/ERF family genes have been identified by phylogenetic analysis of the rice genome (Rashid et al. Reference Rashid, Guangyuan, Guangxiao, Hussain and Xu2012). The AP2/ERF family proteins play a vital role in plant growth and enable plants to tolerate ambient changes (Licausi et al. Reference Licausi, Ohme-Takagi and Perata2013), and use different pathways in response to hormone changes and biotic and abiotic stresses (Mizoi et al. Reference Mizoi, Shinozaki and Yamaguchi-Shinozaki2012). It has previously been reported that an AP2/ERF factor, AtERF7, plays an important role in abscisic acid (ABA) responses and acts as a repressor of gene transcription (Song et al. Reference Song, Agarwal, Ohta, Guo, Halfter, Wang and Zhu2005). Two jasmonate-responsive AP2 factors, AaERF1 and AaERF2, positively regulate artemisinin biosynthesis in Artemisia annua L. (Yu et al. Reference Yu, Li, Yang, Hu, Wang and Chen2012). OsDERF1 modulates ethylene biosynthesis and drought tolerance through directly regulating two ERF repressors, OsERF3 and OsAP2-39, in rice and AtERF11 is a negative regulator of ABA-mediated ethylene synthesis via interaction with ACS2/5 promoters in Arabidopsis (Li et al. Reference Li, Zhang, Yu, Quan, Zhang, Zhang and Huang2011b ; Wan et al. Reference Wan, Zhang, Zhang, Zhang, Quan, Zhou and Huang2011). In addition, DREB1A and DREB2A have functions in low-temperature and drought stress responses in Arabidopsis, and SiDREB2 contributes to drought tolerance in foxtail millet (Liu et al. Reference Liu, Kasuga, Sakuma, Abe, Miura, Yamaguchi-Shinozaki and Shinozaki1998; Lata et al. Reference Lata, Bhutty, Bahadur, Majee and Prasad2011). DREB2A and DREB2B are induced by high-salt stress, while AtERF98 enhances salt tolerance through modulation of ascorbic acid synthesis (Nakashima et al. Reference Nakashima, Shinwari, Sakuma, Seki, Miura, Shinozaki and Yamaguchi-Shinozaki2000; Zhang et al. Reference Zhang, Wang, Zhang and Huang2012). RAV factors play roles in the disease defence pathway in tomato (Li et al. Reference Li, Su, Cheng, Sanjaya, You, Hsieh, Chao and Chan2011a ). Submergence tolerance regulator Sub1A is another ERF transcription factor, which also improves drought tolerance (Xu et al. Reference Xu, Xu, Fukao, Canlas, Maghirang-Rodriguez, Heuer, Ismail, Bailey-Serres, Ronald and Mackill2006; Fukao et al. Reference Fukao, Yeung and Bailey-Serres2011). The findings detailed above provide substantial evidence that each AP2/ERF family protein has a distinctive functional role in the regulation of diverse physiology processes, thus further dissection of the function of AP2/ERF proteins will deepen the understanding of plant responses to abiotic stresses.
Previously, 12 drought-responsive AP2/ERF genes (DERF) were identified using expression data for stress treatment in rice seedlings. Among these genes, OsDERF1 negatively modulates ethylene biosynthesis and drought tolerance through transcriptional regulation of OsERF3 and OsAP2-39 (Wan et al. Reference Wan, Zhang, Zhang, Zhang, Quan, Zhou and Huang2011). In the present study, the functional analysis of OsDERF2 (LOC_Os04g46440) using RNA interference (RNAi) knock-down transgenic plants is reported. The results provide evidence that OsDERF2 confers negative regulation in drought stress through transcriptional repression of ABA response-related genes in rice.
MATERIALS AND METHODS
Plant material and drought stress treatment
Rice (Oryza sativa) seeds of wild-type Nipponbare (WT) and transgenic lines were used in the drought stress treatment, as previously described (Wan et al. Reference Wan, Zhang, Zhang, Zhang, Quan, Zhou and Huang2011). After germination at 30 °C for 2 days, germinated seeds were transplanted on sandy soil in a greenhouse at 26 °C and 16 h light/8 h dark. The 2-week-old seedlings were exposed to successive drought by withholding water supply. For the control, all seedlings were maintained under normal growth conditions with water. When the seedlings began to wilt at the 6th day, drought phenotypes among different lines were observed. After all the seedlings showed varying degrees of stress symptoms up to the 9th day, plants were re-watered for 7 days to allow recovery, and then the growth status and rate of survival was recorded as images and analysed.
Phylogenetic analysis
Sequences of DREB members were searched using the basic local alignment search tool (BLAST) in the GenBank protein database (http://www.ncbi.nlm.nih.gov/BLAST/) and the results inspected manually. These sequences were aligned with ClustalW using default parameters. A phylogenetic tree was constructed using MEGA 5·0 with the neighbour-joining method. Bootstrap analysis was performed with 100 replicates, and bootstrap values on the tree are shown as percentages.
Sub-cellular localization analysis
The coding sequence of OsDERF2 was cloned into the pGDG vector to construct green fluorescent protein (GFP) fusion with OsDERF2. The fusion construct (35S::GFP-OsDERF2) and control (35S::GFP) were transformed into onion epidermal cells with an Agrobacterium-mediated system, incubated on 1/2 strength Murashige and Skoog (MS) medium for 24 h at 26 °C in darkness, and the fluorescence of GFP was observed using a Leica TCS-SP4 laser scanning confocal microscope.
Generation of transgenic plants
RNA interference transgenic plants were generated as described in Ding et al. (Reference Ding, Wang, Su, Zhai, Cao, Zhang, Liu, Bi, Qian, Cheng, Chu and Cao2007). The less conserved region at the C-terminus (located at amino acids 178-217 of OsDERF2) was used to interfere with gene expression. The resulting plasmid was transformed into Agrobacterium and WT Nipponbare calli were used as the recipients for Agrobacterium-mediated transformation. Transformed plants with reduced expression levels were detected using quantitative real-time polymerase chain reaction (qRT-PCR) as described by Wan et al. (Reference Wan, Zhang, Zhang, Zhang, Quan, Zhou and Huang2011). T3 transgenic lines were used in the present study and denoted as RIs, and the different lines are indicated by numbers.
Transcriptional activity detection in yeast
Different truncations (including the activating domain) were fused in-frame to the DNA-binding domain vector pGBKT7. The fusion plasmids were transformed into yeast AH109 as described by the manufacturer (Clontech, USA). The transformants were grown on selective medium plates at 30 °C for 3 days.
Abscisic acid and drought treatment for gene expression analysis
Wild-type seedlings cultured in water for 7 days were used for OsDERF2 gene expression analysis. For ABA treatment, plants were transferred into water with 50 µm ABA, while an equal volume of absolute ethanol was added to water for the controls. For the drought treatment, seedlings were taken out of the water and kept on filter paper. Samples were collected at 0, 0·5, 1, 2 and 3 h after drought treatment and 0, 0·5, 1, 2, 4 and 8 h after ABA treatment. Gene expression analysis was performed by qRT-PCR. The gene-specific primers are shown in Table 1.
Table 1. Oligo nucleotides used in the present study
Abscisic acid sensitivity test
For the ABA sensitivity test of transgenic rice seedlings, geminated plants of transgenic rice and the wild-type control at the same growth stage were transferred to MS medium containing different concentrations of ABA (0, 2 and 10 µm). The seedlings were grown for 3 days in a growth chamber and root lengths were measured.
Measurement of malondialdehyde and proline contents
The malondialdehyde (MDA) and proline contents of plants were detected following polyethylene glycol (PEG) treatment for 5 days as described in Madhava Rao & Sresty (Reference Madhava Rao and Sresty2000).
Detection of endogenous abscisic acid levels in plant
Leaves of 2-week-old seedlings (0·2 g) were frozen in liquid nitrogen and ground finely, followed by extraction with 1 ml extraction mixture (2-propanol:H2O:concentrated hydrochloric acid (HCl) = 2 : 1 : 0·002, vol/vol/vol). The extraction samples were shaken at 100 rpm for 30 min at 4 °C, followed with addition of 1 ml dichloromethane and shaken for 30 min at 4 °C. After centrifugation at 13 000 g for 5 min, 900 µl of the solvent was transferred from the lower phase and the solvent mixture concentrated. The samples were dissolved in 0·1 ml methanol and ABA was measured as described in Pan et al. (Reference Pan, Welti and Wang2010).
RESULTS
OsDERF2 belonging to the DREB family is inducible by drought stress
As described in the previous study, 12 drought-responsive ERF genes (DERF) were identified using expression data for stress treatment in rice seedlings (http://www.ricearray.org) (Wan et al. Reference Wan, Zhang, Zhang, Zhang, Quan, Zhou and Huang2011). Among these genes, OsDERF1 modulates drought response through negatively affecting ethylene production (Wan et al. Reference Wan, Zhang, Zhang, Zhang, Quan, Zhou and Huang2011). OsDERF2 (LOC_Os04g46440) encodes a 217 amino acid protein. Amino acids 46–109 contain a typical AP2 DNA-binding domain, and residues 34–40 contain a nuclear localization signal (Fig. 1(a)). NCBI (National Center for Biotechnology Information) BLASTp results and classification using MEGA 5·0 showed that OsDERF2 was similar to DREB members of the AP2/ERF family, such as LOC_Os02g43970, LOC_Os10g41130, OsDREB1A, OsDREB1B, OsDREB1C, AtTINY, AtABI4, AtDREB2A, AtDREB2B and ZmDBF2, which contain an AP2 domain (Fig. 1(b)).
Fig. 1. Sequence analysis of OsDERF2 protein. (a) Nucleotide and protein sequences of OsDERF2. Light grey box indicates nuclear localization signal, and dark grey box indicates APETALA2 domain; (b) phylogenetic relationships among plant DREB members from rice, maize and Arabidopsis. Bootstrap values from 100 replicated are shown at each node.
Sequence analysis of OsDERF2 showed that residues 34–40 PKKRPRN is a nuclear localization signal. To determine the sub-cellular localization of OsDERF2, the coding sequence of OsDERF2 was fused to GFP in the pGDG vector. The onion cells transformed with the control p35S::GFP displayed fluorescence throughout the cells, but fluorescence in the onion cells transformed with p35S::GFP-OsDERF2 was restricted exclusively to the nucleus (Fig. 2(a)), demonstrating that OsDERF2 is a nuclear-localized protein as predicted.
Fig. 2. Subcellular localization and transcriptional activity of OsDERF2 protein. (a) Nuclear localization of the OsDERF2 protein as revealed by GFP fusion protein. The constructs GFP and GFP-OsDERF2 were expressed in onion epidermal cells using an Agrobacterium-mediated system. GFP fluorescence was detected using confocal microscopy. Scale bars, 1 µm; (b) transcriptional activity of OsDERF2 in yeast. Left panel shows the schematic diagrams of various constructs used for transactivation. OsDERF1 was used as positive control. BD indicates GAL4 DNA-binding domain. Right panel shows the yeast growth on the SD/-Trp and SD/-His-Trp medium. Colour online.
The full-length, different deletions, including N-terminal of 190 amino acids, N-terminal of 149 amino acids and N-terminal of 110 amino acids of OsDERF2 were fused to the GAL4 DNA-binding domain resulting in the plasmids of pGBKT7-OsDERF2, pGBKT7-N190, pGBKT7-N149 and pGBKT7-N110, respectively. These plasmids were then transformed into yeast strain AH109, with plasmid pGBKT7 and the previously reported pGBKT7-OsDERF1 (Wan et al. Reference Wan, Zhang, Zhang, Zhang, Quan, Zhou and Huang2011) as negative and positive controls, respectively. The transformants of pGBKT7-OsDERF1, pGBKT7-OsDERF2 and pGBKT7-N190 could grow on the SD/-Trp and SD/-His-Trp medium, while the transformants of pGBKT7-N149, pGBKT7-N110 and the negative control pGBKT7 could not grow on the SD/-His-Trp medium (Fig. 2(b)). These results indicated that OsDERF2 contains an activation domain in amino acids 149–190, possibly functioning as a transcriptional activator.
The expression of OsDERF2 in different tissues of rice was determined with qRT-PCR and the results showed that transcripts of OsDERF2 were highly expressed in seedling leaves and sheaths, 25 and 10 times as much, respectively, as that in roots (Fig. 3(a)). Therefore, the tissues including leaves and sheaths were used to analyse the transcript level of OsDERF2 under different treatments. To investigate the function of OsDERF2, the promoter sequence (2000 base pairs upstream of the initiation codon) was analysed using the PLACE database (for motifs found in plant cis-acting regulatory DNA elements, now supplanted by TENOR – http://tenor.dna.affrc.go.jp/). The results showed that the promoter of OsDERF2 contains multiple putative stress-responsive cis-acting elements, including MYC, MYB and ABRE recognition sites. Further qRT-PCR analysis revealed that the gene expression of OsDERF2 was inhibited by ABA, and the solvent of ABA was used as a control. Although the expression of OsDERF2 obviously decreased at 0·5 h and then increased until 4 h under ABA treatment, it decreased seriously at 8 h (Fig. 3(b)). In drought-treated seedlings, OsDERF2 expression was strongly induced and peaked at 3 h (Fig. 3(c)). These data indicate that OsDERF2 might be involved in drought response through the ABA signalling pathway.
Fig. 3. Detection of OsDERF2 transcripts in different tissues of rice seedlings and in response to ABA and drought stress. (a) Expression of OsDERF2 in different tissues; (b) expression of OsDERF2 in response to 50 µm ABA for 8 h, ABA solvent is used as control; (c) expression of OsDERF2 in response to drought for 3 h. The bars represent (±) s.e.
OsDERF2 negatively modulates rice drought response
To determine the regulatory function of OsDERF2 in abiotic stress, OsDERF2 RNA interference transgenic rice (RI) were generated. The RI lines with expression levels of OsDERF2 decreased to 30–60% of WT were selected for the current research (Fig. 4). RI-2, RI-4, RI-5 and RI-6 showed 50, 60, 55 and 30% reductions in expression of OsDERF2, respectively. RI-2, RI-4 and RI-5 were used for the ABA sensitivity test. The lines, including RI-4, RI-5 and RI-6 were used in drought stress treatments and ABA-level detection. There are no obvious differences between these RI lines and WT in terms of plant development at the seedling and heading stages. However, in the drought stress tolerance treatment, when the WT seedling leaves withered, most of the RI lines still grew well. After the drought-treated seedlings were re-watered, the difference in growth status between WT and RI lines was increased (Fig. 5(a)). The survival rates of all RI lines (RI-4, RI-5 and RI-6) were more than 85%, whereas that of WT was only 55% (Fig. 5(b)). These results indicated that OsDERF2 regulated rice tolerance to drought negatively. Free radicals accumulating in plants under drought stress would lead to the damage of DNA, proteins and lipids, and MDA is an end product of membrane lipid peroxidation (Wan et al. Reference Wan, Zhang, Zhang, Zhang, Quan, Zhou and Huang2011). The MDA content of seedlings grown under normal and PEG treatment were investigated and the results showed that there were no obvious changes among WT and RI lines under normal growth conditions. After PEG treatment, although both WT and RI lines showed increased MDA content, the increase was higher in WT (up to two times) than in RI lines (1·5 times) (Fig. 5(c)), indicating that OsDERF2 enhances the production of oxidative stress. Meanwhile, proline is crucial for osmotic adjustment (Wei et al. Reference Wei, Hu, Deng, Zhang, Liu, Zhao, Luo, Jin, Li, Zhou, Sun, Wang, Yang and He2014). To determine whether OsDERF2 negatively modulates tolerance to drought through affecting osmolyte accumulation, the proline content was measured and found to increase up to 1·4 times in WT and 1·8 times in RI lines (Fig. 5(d)), suggesting that OsDERF2 reduces the accumulation of osmolytes to modulate drought tolerance.
Fig. 4. Relative expression levels of OsDERF2 in different RNAi transgenic lines. The gene expression in the WT was assigned a value of 1. The bars represent (±) s.e.
Fig. 5. Regulation of OsDERF2 in drought stress response. (a) WT and RI lines were subjected to drought stress for 9 days, followed by recovery for 7 days. Control: rice plants were grown under normal conditions. Drought: plants had the daily water supply withheld; (b) survival rate of all plants used in (a) after re-watering. The numbers in parentheses shows the survival seedlings/total tested plant; (c) MDA content of WT and RI plants under normal conditions or treatment with 15% PEG 6000 for 5 days; (d) proline content of WT and RI plants under normal condition or treatment with 15% PEG 6000 for 5 days. Data are the average of three replicates, and there were 10 plants per replicate. The bars represent (±) s.e. Colour online.
Enhanced response of OsDERF2 RNA interference lines to abscisic acid in root growth
Abscisic acid is a key regulator of plant adaptation to stress and different aspects of plant growth and development. Under stress conditions, plants synthesize ABA in various organs and initiate defence mechanisms, such as the regulation of stomatal aperture and expression of defence-related genes involved in resistance to environmental stresses. Since expression of OsDERF2 was inhibited by ABA, and RI lines of OsDERF2 were more tolerant to drought stress, further tests were conducted to investigate whether OsDERF2 is involved in ABA sensitivity, which is an important aspect of the ABA-mediated regulation pathway. Three RI lines (RI-2, RI-4 and RI-5) and WT were used to test the effect of ABA on seedling development. The germinated seedlings were treated with different concentrations of ABA (0, 2 and 10 µm), and the growth of RI lines was more inhibited than that of WT (Fig. 6(a)). The root length of RI lines was significantly shorter than WT plants under ABA treatment, and no apparent difference was observed under normal growth conditions. The root length decreased by 10 and 60% in WT at 2 and 10 µm ABA, and by 30 and 90% in RI lines (Fig. 6(b)). These results suggested that the RI lines of OsDERF2 were more sensitive to ABA than WT.
Fig. 6. ABA sensitivity of OsDERF2-RNAi transgenic rice. (a) Root growth of 3-day-old seedlings on MS medium with or without ABA (0, 2 and 10 µm); (b) root lengths of seedlings in (a). All experiments were repeated with three biological replicates. WT, wild type. RI-2, RI-4 and RI-5 are independent RNAi lines of OsDERF2. The bars represent (±) s.e. Colour online.
Increased abscisic acid levels in OsDERF2 RNA interference lines
Increase of ABA levels in plants could enhance drought stress tolerance, due to the closure of stomata and accumulation of numerous proteins, such as late embryogenesis abundant (LEA), for osmotic adjustment (Verslues & Bray Reference Verslues and Bray2006). Based on the enhanced ABA sensitivity of OsDERF2 RI lines, the ABA contents of two RI lines were measured, in order to confirm whether the drought tolerance of OsDERF2 RI lines is ABA mediated. The data showed that the endogenous ABA levels increased significantly in RI-5 and RI-6, compared with that in WT seedlings (Fig. 7(a)). Although the genes involved in ABA biosynthesis, including NCED3, NCED5, AAO2 and SDR1 in RI-6 had been slightly down-regulated, the expression of AAO3 was up-regulated 2·5-fold in RI-6 (Fig. 7(b)). These results proved that the transcriptional expression of the genes involved in ABA biosynthesis failed to contribute to ABA accumulation in OsDERF2 RNAi lines.
Fig. 7. ABA level of OsDERF2-RNAi transgenic rice. (a) ABA level of WT and OsDERF2-RNAi transgenic lines. FW indicates fresh weight. (b) Transcriptional expression levels of genes involved in ABA biosynthesis in rice detected by qRT-PCR. The expression of each gene in the WT was assigned a value of 1. ABA and RNA were extracted from the roots and leaves of 2-week-old seedlings. The bars represent (±) s.e.
Under abiotic stress, ABA concentration goes up and ABA receptors bind ABA, followed by release of SnRK2, which activates basic leucine zipper (bZIP) transcription factors (Kim et al. Reference Kim, Moon, Min, Choi, Kim, Koh, Yoon, Byun, Yoo and Kim2015). A total of 75 bZIPs have been identified and classified into 10 groups in Arabidopsis thaliana. Most of the ABRE binding bZIPs belongs to group A (Lu et al. Reference Lu, Gao, Zheng and Han2009); however, bZIPs in other groups also have functions in ABA response in rice (Liu et al. Reference Liu, Mao, Ou, Wang, Liu, Wu, Chu and Wang2014). To further confirm the difference of ABA levels between WT and RI lines, the expression levels of ABA-response genes, including bZIP transcription factor family genes were detected through qRT-PCR. It was found that the expression levels of OsbZIP15, OsbZIP33 and OsABA45 were up-regulated in the OsDERF2 RI lines (Fig. 8). OsbZIP15 and OsbZIP33 belong to group C. Gene expression levels under normal and drought treatment were also measured. The results showed that most OsbZIP genes and their downstream genes were up-regulated after drought treatment in both WT and RI-6 seedlings; however, the transcripts of OsbZIP20 and OsABA45 were up-regulated about five- and eightfold, respectively, in the WT seedlings, and 25- and 130-fold, respectively, in the RI-6 seedlings (Fig. 9). These results suggested that OsDERF2-modulated gene expressions involved in ABA response through regulated ABA accumulation, which may contribute to drought tolerance in rice.
Fig. 8. The up-regulated ABA-response genes in the OsDERF2-RNAi lines. The expression of each gene in the WT was assigned a value of 1. The bars represent (±) s.e.
Fig. 9. Transcriptional expression levels of ABA-response genes in WT and OsDERF2-RNAi line under normal and drought stress (indicated as D) by qRT-PCR. The expression of each gene under normal condition was assigned a value of 1. The bars represent (±) s.e.
DISCUSSION
In the current study, a rice AP2/ERF protein OsDERF2 was identified, which is located in the nucleus and has a transcriptional activity domain between amino acids 149–190. DREB sub-family members are involved in two separate signal transduction pathways under low temperature and drought. It has also been found that the expression of DREB genes is induced by abiotic stress at different time periods (Agarwal et al. Reference Agarwal, Agarwal, Reddy and Sopory2006). OsDERF2 is a novel transcription factor in the DREB sub-family. The expression of OsDERF2 is induced by drought, while its RNAi lines show more tolerance to drought stress, indicating that OsDERF2 acts as a negative regulator in drought stress. Wan et al. (Reference Wan, Zhang, Zhang, Zhang, Quan, Zhou and Huang2011) reported that OsDERF1 over-expression lines show more sensitivity to drought and RNAi lines show more tolerance to drought than the wild type. However, the expression of OsDERF1 is induced by drought stress (Wan et al. Reference Wan, Zhang, Zhang, Zhang, Quan, Zhou and Huang2011). Another example is OsbZIP71, which is repressed under saline conditions, while constitutive over-expression of OsbZIP71 improved plant tolerance to salt (Liu et al. Reference Liu, Mao, Ou, Wang, Liu, Wu, Chu and Wang2014). DREBs are generally positive regulators in abiotic stresses, but OsDERF2 negatively modulates drought tolerance in rice. Therefore, it is proposed that OsDERF2 may activate some repressors involved in drought response.
Many rice genes have been identified as drought responsive, which include the genes encoding for aquaporins, AP2/ERF-, bZIP-, NAC- and MYB-type transcription factors, LEA proteins, osmoprotectant-synthesizing enzymes, protein kinases, metallothionein and metallothionein-like proteins, and cytochrome P450 family proteins (Hadiarto & Tran Reference Hadiarto and Tran2011). The phytohormone ABA plays a crucial role in the adaptive response to abiotic stresses such as drought, cold and high salinity. It is also involved in various processes of plant growth, including seed maturation, dormancy, inhibition of cell division and germination (Zou et al. Reference Zou, Guan, Ren, Zhang and Chen2008). RNAi lines of OsDERF2 showed more sensitivity to ABA and accumulated more ABA than in the WT; meanwhile, these lines improved plant tolerance to drought stress. However, the expression of ABA biosynthesis genes is not related to the increased ABA level in OsDERF2 RNAi lines. It is known that ABA accumulation depends on production, degradation and transportation in roots (Shi et al. Reference Shi, Guo, Ye, Liu, Liu, Xia, Cui and Zhang2015); therefore, OsDERF2 may be involved in ABA catabolism or transportation. This should be the subject of further research. It implies that OsDERF2 has functions in ABA accumulation and ABA response, which is one reason for the higher tolerance of OsDERF2 RI lines under drought stress.
The biosynthesis of ABA is induced by drought and the resultant activation of two regulatory ABA-dependent gene expressions. One is the bZIP/ABRE system and the other is MYC/MYB. The bZIP family plays an important role in the ABA signalling pathway of abiotic stress. For example, OsbZIP23 (Xiang et al. Reference Xiang, Tang, Du, Ye and Xiong2008), OsbZIP46 (Tang et al. Reference Tang, Zhang, Li, Xiao and Xiong2012), OsbZIP72 (Lu et al. Reference Lu, Gao, Zheng and Han2009), OsbZIP12/OsABF1 (Amir Hossain et al. Reference Amir Hossain, Lee, Cho, Ahn, Lee, Jeon, Kang, Lee, An and Park2010), OsABI5 (Zou et al. Reference Zou, Guan, Ren, Zhang and Chen2008) and OsbZIP71 (Liu et al. Reference Liu, Mao, Ou, Wang, Liu, Wu, Chu and Wang2014) play important roles in ABA signal transduction and osmotic stress responses. In the current study, it was shown that the levels of OsbZIP15 and OsbZIP33 expression were up-regulated in OsDERF2-RNAi lines, which can form heterodimers with OsbZIP71 (Liu et al. Reference Liu, Mao, Ou, Wang, Liu, Wu, Chu and Wang2014). OsbZIP71 has no transcriptional activity and needs other bZIPs to activate downstream genes. Therefore, OsDERF2 may affect the activity of OsbZIP71 by modulating gene expressions of OsbZIP15 and OsbZIP33. Under drought stress, expression levels of OsbZIP20 and OsABA45 increased much more in the OsDERF2-RNAi line than that in WT plants. OsbZIP20 (RITA-1) displays broad binding specificity for palindromic ACGT elements, and plays a role in the regulation of rice genes expressed in developing rice seeds (Izawa et al. Reference Izawa, Foster, Nakajima, Shimamoto and Chua1994). The promoter sequence of OsbZIP20 (2000 base pairs upstream of the transcription start site) was analysed using the PLACE database. The results showed that the promoter of OsbZIP20 contains ABA-responsive cis-acting elements ABRE and AP2/ERF binding sites, including DRE, GCC and RAV1. Therefore, it is speculated that OsbZIP20 is not only involved in seed development but also in stress responses. OsABA45 is a GRAM domain containing an ABA-responsive protein, in which the promoter of this encoding gene contains two copies of the CGCG box (Wang et al. Reference Wang, Pan, Zhao, Zhu, Fu and Li2011). The CGCG box is regulated by calmodulin and involved in the transcription regulation of multiple abiotic stress responsiveness (Yang & Poovaiah Reference Yang and Poovaiah2002). In particular, many GC-rich motifs with a core motif of CGCG have been found to be over-represented in the promoter of the ABA- and stress-induced gene (Cuming et al. Reference Cuming, Cho, Kamisugi, Graham and Quatrano2007). These data further indicate that OsDERF2 has negative regulation in the response of the rice to drought environment through ABA-mediated pathway.
ACCESSION NUMBERS
The GenBank accession numbers are as follows: OsDERF2, LOC_Os04g46440; OsbZIP15, LOC_Os02g07840; OsbZIP20, LOC_Os02g16680; OsbZIP33, LOC_Os03g58250; OsbZIP52, LOC_Os06g45140; OsbZIP58, LOC_Os07g08420; OsbZIP71, LOC_Os09g13570; OsbZIP88, LOC_Os12g40920; OsABI5, LOC_Os01g64000; OsABA45, LOC_Os12g29400; OsDREB1A, LOC_Os09g35030; OsDREB1B, LOC_Os09g35010; OsDREB1C, LOC_Os06g03670; AtDREB2A, At5g05410; AtDREB2B, At3g11020; AtABI4, At2g40220; AtTINY, At2g44940.
The current work was supported by Grant Special Foundation of Transgenic Plants in China (grant numbers 2014ZX08009-15B and 2014ZX08001-003) and the National Science Foundation of China (grant number 31171465).
