Integrative Regulation of Drought Escape through ABA-Dependent and -Independent Pathways in Rice  Hao Du, Fei Huang, Nai Wu, Xianghua Li, Honghong Hu, Lizhong Xiong  Molecular Plant  Volume 11, Issue 4, Pages 584-597 (April 2018) DOI: 10.1016/j.molp.2018.01.004 Copyright © 2018 The Author Terms and Conditions

Figure 1 Phenotypical Features of Drought Escape for Different Rice Varieties upon Low Water Treatments. (A) Schematic design of the experimental timetable for low water-deficit treatment (LWT) and sampling period (described in Methods). (B) Performance of three rice varieties under different soil water content (SWC) conditions. (C) Statistical data of the days to flowering, tiller number, plant height, and seed-setting percentage of three rice varieties under different SWC. Values are mean ± SD (n = 6). **P < 0.01 (t-test). Molecular Plant 2018 11, 584-597DOI: (10.1016/j.molp.2018.01.004) Copyright © 2018 The Author Terms and Conditions

Figure 2 ABA Biosynthesis and Signaling Are Essential for DE under LWT. (A) Quantification of endogenous ABA contents in ZH11 leaves under normal and LWT conditions. Leaves were harvested every 4 h for 2 days at the 45th day after germination (DAG) and at different developmental stages. (B) Expression patterns of OsNCED4 and OsNCED5 under normal and LWT conditions at different developmental stages by qRT-PCR analysis. (C) Statistical analysis of the relative ABA level in the ABA-deficient lines. (D) Performance and days to flowering of phs3-1, PDS-RNAi, and WT controls under normal and LWT conditions. (E) Expression patterns of OsbZIP23 under normal and LWT conditions at different developmental stages by qRT-PCR analysis. (F) Performance and days to flowering of osbzip23, bZIP23-OE, and WT under normal and LWT conditions. (G) Expression patterns of OsTOC1, Ghd7, Ehd1, RID1, HD3a, and RFT1 in the OsbZIP23 overexpression line and ZH11 under normal and LWT conditions at different developmental stages by qRT-PCR analysis. (H) Expression patterns of OsTOC1, Ghd7, Ehd1, RID1, HD3a, and RFT1 in osbzip23 mutant and ZH11 under normal and LWT conditions at different developmental stages by qRT-PCR analysis. Values are mean ± SD (n = 3). *P < 0.05, **P < 0.01 (t-test). Molecular Plant 2018 11, 584-597DOI: (10.1016/j.molp.2018.01.004) Copyright © 2018 The Author Terms and Conditions

Figure 3 The Circadian Clock Components OsTOC1, OsPRR37, OsGI, and OsELF3 Are Required for DE. (A) Expression patterns of OsTOC1, OsPRR37, OsGI, and OsELF3 in ZH11 under normal, LWT, and ABAT conditions at different developmental stages by qRT-PCR analysis. (B) Performance and days to flowering of the OsTOC1-OE lines and WT′ (wild-type genotype segregated from the OsTOC1-OE lines) under normal growth conditions, LWT, and ABA treatment (ABAT). (C) Performance and related days to flowering of OsPRR37 NIL (ZS97 background) and ZS97 under normal and LWT conditions. (D) Performance and days to flowering of the OsGI-RNAi lines and WT′ (wild-type genotype segregated from OsGI-RNAi lines) under normal and LWT conditions. (E) Performance and days to flowering of elf3 and WT′ (wild-type genotype segregated from the heterozygous oself3 mutant) under normal and LWT conditions. (F) Expression patterns of RFT1 and HD3a in the OsTOC1-OE line and WT′ under normal growth conditions, LWT, and ABAT by qRT-PCR analysis at different developmental stages. (G) Expression patterns of OsTOC1 in phs3-1, PDS-RNAi, and its controls under normal and LWT conditions. (H) Expression patterns of HD1, Ehd1, and HD3a in the OsPRR37 NIL (ZS97 background) and ZS97 under normal and LWT conditions at different developmental stages by qRT-PCR analysis. (I) Expression patterns of HD1, Ehd1, and HD3a in the OsGI-RNAi lines and WT′ under normal and LWT conditions at different developmental stages by qRT-PCR analysis. (J) Expression patterns of OsPRR37, Ghd7, Ehd1, HD3a, and RFT1 in elf3 and its WT′ control at different developmental stages under normal and LWT conditions by qRT-PCR analysis. Values are mean ± SD (n = 3). *P < 0.05, **P < 0.01 (t-test). Molecular Plant 2018 11, 584-597DOI: (10.1016/j.molp.2018.01.004) Copyright © 2018 The Author Terms and Conditions

Figure 4 MADS-Box Genes Are Involved in DE Signaling. (A) Expression patterns of OsMADS50 in ZH11 under normal, LWT, and ABAT conditions at different developmental stages by qRT-PCR analysis. (B) Performance and relative days to flowering of osmads50 and WT′ under normal and LWT conditions. Values are mean ± SD (n = 3). **P < 0.01 (t-test). (C) Expression patterns of OsMADS14, OsMADS15, OsMADS18, Ehd1, HD3a, and HD1 in osmads50 and WT′ under normal and LWT conditions at different developmental stages by qRT-PCR analysis. Molecular Plant 2018 11, 584-597DOI: (10.1016/j.molp.2018.01.004) Copyright © 2018 The Author Terms and Conditions

Figure 5 PhyB and Ghd7 Play a Negative Role in DE, which Is Partially Dependent on ABA. (A) Expression patterns of PhyB in ZH11 under normal growth, LWT, and ABAT at early developmental stages by qRT-PCR analysis. (B) Performance of the phyb mutant and WT under normal and LWT conditions. (C) Expression patterns of Ghd7, Ehd1, and HD3a in the phyb mutant and ZH11 under normal and LWT conditions at different developmental stages by qRT-PCR analysis. (D) Expression patterns of Ghd7 in ZH11 under normal growth, LWT, and ABAT at early developmental stages by qRT-PCR analysis. (E) Performance and relative days to flowering of Ghd7-OE and Ghd7 NIL (ZS97 background) and its controls under normal and LWT conditions. (F) Expression patterns of Ehd1, RFT1, and HD3a in Ghd7-OE and WT under normal and LWT conditions by qRT-PCR analysis. (G) Performance of Ghd7-OE and Ghd7 NIL (ZS97 background) and its controls under normal and ABAT conditions. (H) Performance of the phyB mutant and its control under normal and ABAT conditions. (I) Expression patterns of Ehd1, RFT1, and HD3a in Ghd7-OE and WT under normal and ABAT conditions by qRT-PCR analysis. (J) Expression patterns of Ghd7, Ehd1, and HD3a in the phyb mutant and ZH11 under normal and ABAT conditions by qRT-PCR analysis. (K) Expression patterns of Ghd7 and PhyB in phs3-1, PDS-RNAi, and its controls at different developmental stages under normal and LWT conditions by qRT-PCR analysis. Values are mean ± SD (n = 3). *P < 0.05, **P < 0.01 (t-test). Molecular Plant 2018 11, 584-597DOI: (10.1016/j.molp.2018.01.004) Copyright © 2018 The Author Terms and Conditions

Figure 6 LWT Inhibits the Generation of Tillers in Rice. (A) Expression patterns of D10, D14, D17, and D27 under normal and LWT conditions at early developmental stages (samples from stem base) detected by qRT-PCR. (B) Tiller numbers and days to flowering of the d10 mutant and D3-RNAi line under normal and LWT conditions. (C) Expression patterns of OsTB1 in the stem base at early developmental stages detected by qRT-PCR. (D) Expression patterns of OsTB1 in axillary buds detected by qRT-PCR. (E) Performance and tiller numbers of OsTB1-RNAi and WT′ (wild-type rice segregated from the OsTB1-RNAi lines) under normal and LWT conditions. (F) Expression patterns of OsMADS57 and D14 in leaf blades and axillary buds of OsTB1-RNAi and WT′ under normal and LWT conditions. Values are mean ± SD (n = 3). **P < 0.01 (t-test). Molecular Plant 2018 11, 584-597DOI: (10.1016/j.molp.2018.01.004) Copyright © 2018 The Author Terms and Conditions

Figure 7 Schematic Model of Regulatory Interactions in the DE Pathway in Rice. (A) OsNCED4/5 are induced by LWT, which leads to an increase of endogenous ABA biosynthesis. ABA can activate the transcript levels of OsbZIP23 and downstream gene OsTOC1. OsTOC1 promotes flowering under LWT by increasing the expression levels of HD3a and RFT1. OsbZIP23 functions to upregulate HD3a and RFT1 by repressing the transcript level of Ghd7, and inducing the flowering switch gene RID1. Moreover, ABA also can repress the transcript levels of Ghd7 and PhyB directly, which may release the suppression of Ehd1, HD3a, and RFT1, and PhyB can also regulate Ghd7 (Osugi et al., 2011). (B) LWT can induce the transcript levels of OsMADS50, OsGI, and OsELF3, and downregulates OsPRR37. OsMADS50 promotes DE by repressing HD1 but inducing the transcript levels of Ehd1 and OsMADS14/15/18/22, which finally contributed to the induction of HD3a and RFT1. OsGI can also activate Ehd1 but suppress HD1, thus helping in the upregulation of HD3a and RFT1. OsELF3 represses the negative regulator Ghd7, OsPRR37, and HD1 (Yang et al., 2013). OsPRR37, repressed by LWT, suppresses the expression of Ehd1 but induces HD1. (C) SL biosynthesis genes D10/D17/D27 (Lopez-Obando et al., 2015) are induced by LWT, which can increase the transcript levels of D3 and D14, and thus repress tillering. OsTB1 is induced by LWT, which regulates the negative regulators of tillering (OsMADS57 and D14) in rice. Molecular Plant 2018 11, 584-597DOI: (10.1016/j.molp.2018.01.004) Copyright © 2018 The Author Terms and Conditions