Fig 1.
STTM165/166 plants exhibit drought stress resistance phenotype.
(A) Drought resistance phenotype. 3-week-old plants (upper panel) were grown under the same conditions but without irrigation for 14 days (middle panel), and then rewatered for 2 days (lower panel). (B) Quantification of the survival rate. Forty plants of wild type and STTM165/166 were used in each experiment, and the survival rate was calculated from the results of four independent experiments. (C) Water loss assay. Aerial parts of 3-week-old plants were detached and weighed at the indicated time points. Water content at any time point was calculated as percentage of the fresh weight at time zero. Data were derived from four independent experiments (±SD).
Fig 2.
STTM165/166 plants display cold tolerance phenotype.
(A) Comparison of the survival rates of freezing-treated wild type and STTM165/166 seedlings after 5 days of recovery at 23°C. (B) Leakage of electrolytes from excised leaves of wild type and STTM165/166 plants exposed to indicated freezing temperatures. Wild type and STTM165/166 plants were grown under normal conditions for three weeks and then subjected to cold acclimation (4°C for 7 days) before freezing treatment. (C) Comparison of the expression of CBF genes in wild type and STTM165/166 under cold treatment. (D) Comparison of the expression of cold responsive genes in wild type and STTM165/166 under cold treatment.
Fig 3.
STTM165/166 plants are hypersensitive to ABA during seed germination and early seedling development.
(A) The phenotype of wild type and STTM165/166 plants grown on MS medium supplemented with 1.0 μM ABA. Seedlings were photographed 9 days after stratification. (B, C) Germination and cotyledon greening analysis of the STTM165/166 and wild type plants in response to different concentrations of ABA (0, 1.0, 1.5 and 2.0 μM). Germination was scored at 4 days and greening was scored at 9 days after stratification. Three independent experiments were performed, and >100 seeds for each treatment were used for each experiment. Values are means ± standard deviation. (D, E) Quantitative RT-PCR analysis of the expression of both mature miR165/166 and its targets at seedling stage. Three independent experiments were performed, and values are means ± standard deviation.
Fig 4.
Quantitative RT-PCR analysis of ABA-responsive genes in wild type and STTM165/166 seedlings.
(A) The expression of ABA-responsive genes was analyzed in wild type and STTM165/166 2-day-old seedlings grown on MS medium. Three independent experiments were performed and values are means ± standard deviation. (B) The expression of ABA-responsive genes was analyzed in wild type and STTM165/166 2-day-old seedlings grown on MS medium with 10 μM ABA. Three independent experiments were performed and values are means ± standard deviation.
Fig 5.
PHB promotes ABI4 expression by directly binding to its promoter.
(A) ABI4 expression was analyzed in wild type and STTM165/166 2-day-old seedlings using qRT-PCR. (B) ABI4 expression was analyzed in PHB:PHB G202G-YFP lines using qRT-PCR. (C) Analysis of ABI4 promoter. A 3.0kb fragment upstream of ATG was chosen for the promoter analysis. (D) EMSA assay showed that PHB binds to an ABI4 promoter region. Labeled probe of the ABI4 promoter region was incubated with GST-PHB fusion protein. For the competition test, a non-labeled probe was added at 10-fold and 100-fold concentrations.
Fig 6.
ABA content is altered in STTM165/166 plants.
(A) Quantitative RT-PCR analysis of the expression of genes involved in ABA conjugation and de-conjugation. (B) Quantitative RT-PCR analysis of BG1 expression in various tissues of wild type and STTM165/166 plants. (C) Comparison of the ABA content between wild type and STTM165/166 plants.
Fig 7.
PHB directly upregulates the expression of BG1.
(A) BG1 expression was analyzed using qRT-PCR in PHB:PHB G202G-YFP lines. (B) Analysis of BG1 promoter. The diagram shows a 4.0kb fragment upstream of the first ATG codon. (C) ChIP-qPCR was performed using specific primers corresponding to different promoter regions. (D) EMSA assay showed that PHB binds to the BG1 promoter region. Labeled probes of the BG1 promoter region were incubated with GST-PHB fusion protein. For the competition test, a non-labeled probe was added at 10-fold and 100-fold concentrations.