Fig. 1. Phenotype, physiological and transcription analysis of wild type (WT) and OsCYL4 transgenic plants. A, Identification of genes OsCYL4a and OsCYL4b on agarose gel. B, Gene structures of OsCYL4a and OsCYL4b. The boxes and lines represent exons and introns, respectively. C, Relative expression levels of OsCYL4b in response to abiotic stress and abscisic acid (ABA) treatment at the seedling stage in WT type rice variety Zhonghua 11 (ZH11). D, Subcellular localization of OsCYL4b-YFP fusion protein in rice protoplast, analyzed together with the cytoplasm (CP) marker OsRac1-C212S-CFP and the plasma membrane (PM) marker CBL1-mCherry. Scale bars, 10 μm. E, Drought sensitivity test of OsCYL4b-overexpression (4bOE) and RNAi (4bRNAi) plants at the four-leaf stage. F, Transcript levels of OsCYL4b in OsCYL4b transgenic plants. G, Survival rates of WT and OsCYL4b transgenic plants after drought stress. H, Drought sensitivity test of OsCYL4b- and OsCYL4a-overexpression plants (4bOE and 4aOE, respectively) at the four-leaf stage. I, Survival rates of WT, OsCYL4b-, and OsCYL4a- overexpression plants after drought stress. J, Water loss rate from detached leaves of WT, OsCYL4b-overexpression, and RNAi plants at indicated time points. K, Rates of three stomatal opening levels in ZH11, 4bOE, and 4bRNAi plants under normal and drought stress conditions. L, Stomatal conductance of 4bOE and 4bRNAi lines. M, Detection of ABA content in 4bOE plants. N, Expression of ABA biosynthesis-related genes in WT, 4bOE, and 4bRNAi plants. O, Detection of H2O2 accumulation with nitro blue tetrazolium (NBT) staining in leaves of ZH11 and OsCYL4b transgenic plants under drought treatment. The upper panel is the normal control and the lower panel is the NBT staining. P, H2O2 contents in WT and OsCYL4b transgenic plants under normal and drought stress. Q, Superoxide dismutase (SOD) activity in WT, 4bOE, and 4bRNAi plants. R, Heat map showed expression levels of reactive oxygen species-scavenging-related and stress responsive genes in WT, 4bOE, and 4bRNAi plants under normal and drought stress conditions. Relative expression levels of genes in F, N, and R were normalized by a log2 calculation method. Total RNA was extracted from four-week-old leaves. The rice UBQ gene (LOC_Os03g13170) was used as the internal control. Data are Mean ± SE (n = 3). Asterisks in C, F, G, I, J, L, and M indicate significant differences using the two-tailed Student’s t-test (*, P < 0.05; **, P < 0.01). Significant differences indicated by different lowercase letters in K, N, P, and Q were calculated using the Duncan’s new multiple range test.
Fig. 1. Phenotype, physiological and transcription analysis of wild type (WT) and OsCYL4 transgenic plants. A, Identification of genes OsCYL4a and OsCYL4b on agarose gel. B, Gene structures of OsCYL4a and OsCYL4b. The boxes and lines represent exons and introns, respectively. C, Relative expression levels of OsCYL4b in response to abiotic stress and abscisic acid (ABA) treatment at the seedling stage in WT type rice variety Zhonghua 11 (ZH11). D, Subcellular localization of OsCYL4b-YFP fusion protein in rice protoplast, analyzed together with the cytoplasm (CP) marker OsRac1-C212S-CFP and the plasma membrane (PM) marker CBL1-mCherry. Scale bars, 10 μm. E, Drought sensitivity test of OsCYL4b-overexpression (4bOE) and RNAi (4bRNAi) plants at the four-leaf stage. F, Transcript levels of OsCYL4b in OsCYL4b transgenic plants. G, Survival rates of WT and OsCYL4b transgenic plants after drought stress. H, Drought sensitivity test of OsCYL4b- and OsCYL4a-overexpression plants (4bOE and 4aOE, respectively) at the four-leaf stage. I, Survival rates of WT, OsCYL4b-, and OsCYL4a- overexpression plants after drought stress. J, Water loss rate from detached leaves of WT, OsCYL4b-overexpression, and RNAi plants at indicated time points. K, Rates of three stomatal opening levels in ZH11, 4bOE, and 4bRNAi plants under normal and drought stress conditions. L, Stomatal conductance of 4bOE and 4bRNAi lines. M, Detection of ABA content in 4bOE plants. N, Expression of ABA biosynthesis-related genes in WT, 4bOE, and 4bRNAi plants. O, Detection of H2O2 accumulation with nitro blue tetrazolium (NBT) staining in leaves of ZH11 and OsCYL4b transgenic plants under drought treatment. The upper panel is the normal control and the lower panel is the NBT staining. P, H2O2 contents in WT and OsCYL4b transgenic plants under normal and drought stress. Q, Superoxide dismutase (SOD) activity in WT, 4bOE, and 4bRNAi plants. R, Heat map showed expression levels of reactive oxygen species-scavenging-related and stress responsive genes in WT, 4bOE, and 4bRNAi plants under normal and drought stress conditions. Relative expression levels of genes in F, N, and R were normalized by a log2 calculation method. Total RNA was extracted from four-week-old leaves. The rice UBQ gene (LOC_Os03g13170) was used as the internal control. Data are Mean ± SE (n = 3). Asterisks in C, F, G, I, J, L, and M indicate significant differences using the two-tailed Student’s t-test (*, P < 0.05; **, P < 0.01). Significant differences indicated by different lowercase letters in K, N, P, and Q were calculated using the Duncan’s new multiple range test.
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