OsMADS57 together with OsTB1 coordinates transcription of its target OsWRKY94 and D14 to switch its organogenesis to defense for cold adaptation in rice

Abstract

Plants modify their development to adapt to their environment, protecting themselves from detrimental conditions such as chilling stress by triggering a variety of signaling pathways; however, little is known about how plants coordinate developmental patterns and stress responses at the molecular level. Here, we demonstrate that interacting transcription factors OsMADS57 and OsTB1 directly target the defense gene OsWRKY94 and the organogenesis gene D14 to trade off the functions controlling/moderating rice tolerance to cold. Overexpression of OsMADS57 maintains rice tiller growth under chilling stress. OsMADS57 binds directly to the promoter of OsWRKY94, activating its transcription for the cold stress response, while suppressing its activity under normal temperatures. In addition, OsWRKY94 was directly targeted and suppressed by OsTB1 under both normal and chilling temperatures. However, D14 transcription was directly promoted by OsMADS57 for suppressing tillering under the chilling treatment, whereas D14 was repressed for enhancing tillering under normal condition.We demonstrated that OsMADS57 and OsTB1 conversely affect rice chilling tolerance via targeting OsWRKY94. Our findings highlight a molecular genetic mechanism coordinating organogenesis and chilling tolerance in rice, which supports and extends recent work suggesting that chilling stress environments influence organ differentiation.

Keywords: D14; OsMADS57; WRKY94; cold tolerance; gene network; organogenesis; rice (Oryza sativa); trade-off.

Figure 1

OsMADS57 is required for chilling tolerance. (a) Diagram of OsMADS57 gene structure. The triangles represent the T‐DNA insertion sites in osmads57‐1 (m57‐1) and osmads57‐2 (m57‐2) mutants. (b) Chilling tolerance phenotype of the gain‐of‐function mutant osmads57‐1 (m57‐1), the loss‐of‐function mutant osmads57‐2 (m57‐2) and the wild‐type (WT) Dongjin (DJ, Oryza sativa ssp. japonica cv Dongjin). Seedlings were incubated at 4°C for 84 h, then transferred back to the normal condition for recovery. Bars, 2.5 cm. (c) Chilling tolerance phenotype of the loss‐of‐function mutant d14, the double mutant d14 osmads57‐1 (d14 m57‐1) and the WT Shiokari (Shi, Oryza sativa ssp. japonica cv Shiokari). Seedlings were incubated at 4°C for 96 h, then transferred back to the normal condition for recovery. Bars, 2.5 cm. (d) Expression analysis of OsMADS57 in osmads57‐2 (m57‐2) using quantitative reverse transcription polymerase chain reaction. Values are expressed as the mean ± SD, n = 3 (three technical replicates per biological repeat). **, P < 0.01 (Student's t‐test). (e) Survival rate (percentage of seedlings surviving) of seedlings from (a) following the chilling treatment. Values are expressed as mean ± SD, n = 3, *, P < 0.05 (Student's t‐test). (f) Survival rate of seedlings from (b) following the chilling treatment. Values are expressed as mean ± SD, n = 3, **, P < 0.01 (Student's t‐test).

Figure 2

OsMADS57 directly upregulates OsWRKY94 expression during chilling treatment. (a) Schematic of the OsWRKY94 promoter showing the CArGboxes in red (S1 and S2). The S1 sequence is CTTTTTATAG and the S2 sequence is CTTTAAAAAG. Black lines S1–S4 indicate the sequences tested in the chromatin immunoprecipitation (ChIP) assays. (b) In vitro electrophoretic mobility shift assay (EMSA) showing that the GST‐OsMADS57 fusion protein binds to the OsWRKY94 promoter (WRKY94S2). The oligos were synthesized by fusing three copies of the S2 motif and their flanking sequence. The mutant probe (AGGGCCCCCT) served as the negative control. (c) ChIP to measure OsMADS57 occupancy at the OsWRKY94 promoter in vivo. The DNA isolated using ChIP was analyzed using quantitative PCR. Values are expressed as mean ± SD, n = 3. The UBIQUITIN (UBQ) promoter was used as a negative control.*, P < 0.05; **, P < 0.01 (Student's t‐test). (d) Yeast one‐hybrid analysis. D14p represents the positive control. WRKY94p _▵ is the truncated promoter of OsWRKY94 in which the CTTTAAAAAG sequence is absent. CYCp is the negative control of the blank vector. (e) OsWRKY94 expression in response to the chilling treatment in lines expressing various OsMADS57 constructs. Values are expressed as mean ± SD, n = 3. *, P < 0.05;**, P < 0.01 (Student's t‐test). (f) Transient transcriptional assay of OsWRKY94 driven by OsMADS57 in Arabidopsis protoplasts.*, P < 0.05; **, P < 0.01 (Student's t‐test). (g) Diagram of OsWRKY94 gene structure. The triangles represent the T‐DNA insertion sites in oswrky94‐1 (w94‐1) mutant. (h) Phenotype and (i) survival rate of oswrky94‐1 (w94‐1) and the wild‐type, Dongjin (DJ, Oryza sativa ssp.japonica cv Dongjin) following 84 h of the chilling treatment. Values are expressed as mean ± SD, n = 3, **, P < 0.01 (Student's t‐test). Bars, 2.5 cm.

Figure 3

The oswrky94‐1 osmads57‐2 (w94‐1 m57‐2) double mutant is sensitive to chilling stress. (a) Phenotype of oswrky94‐1 osmads57‐2 (w94‐1 m57‐2) double mutant following 72 h of chilling treatment. Bars, 2.5 cm. (b) Survival rate of the oswrky94‐1 osmads57‐2 (w94‐1 m57‐2) double mutant following the chilling treatment. Values are expressed as mean ± SD, n = 3, *, P < 0.05; ***, P < 0.001. Student's t‐test. (c) OsWRKY94 expression level in the oswrky94‐1 osmads57‐2 (w94‐1 m57‐2) double mutant. Values are expressed as mean ± SD, n = 3. *, P < 0.05; **, P < 0.01 (Student's t‐test).

Figure 4

OsTB1 directly targets OsWRKY94. (a) Schematic of theOsWRKY94 promoter showing the OsTB1 binding motif. The P1 sequence is TGGTCC and the P2 sequence is TGGGCC. (b) Electrophoretic mobility shift assay (EMSA). The GST protein and the mutant probe (Pm) served as the negative control. In Pm, TGGGCC was mutated to AAAAAA. Excessive amounts (×50) of unlabeled oligos were added as the competitors. (c) Yeast one‐hybrid analysis. The effectors contained the GAL4 activation domain. (d) Expression activity assay of OsWRKY94 driven by OsTB1 in Arabidopsis protoplasts. **, P < 0.01. Student's t‐test. (e) Quantitative reverse transcription polymerase chain reaction analyses of OsWRKY94 transcript levels in seedlings of ostb1 and the wild‐type, Taichung 65 (TC65, Oryza sativa L. ssp. japonica cv Taichung 65) following 24 h of chilling treatment. The expression levels in TC65 both before and after chilling treatment were defined as 1. Values are expressed as mean ± SD, n = 3. **, P < 0.01 (Student's t‐test). (f) Phenotype and (g) survival rate of ostb1 following 84 h of chilling treatment. Values are expressed as mean ± SD, n = 3, **, P < 0.01 (Student's t‐test). Bars, 2.5 cm. (h) Chilling treatment increases tiller growth in ostb1. The arrows indicate the tillers that developed after the chilling treatment.

Figure 5

OsMADS57 interacts with OsTB1 to coordinate rice growth and chilling tolerance. (a) Electrophoretic mobility shift assay (EMSA) of the effect of OsTB1 on OsMADS57 binding to the OsWRKY94 promoter. Excess amounts (×50) of unlabeled oligos were added as the competitors. GST protein served as the negative control. (b) Effect of OsTB1 and OsMADS57 on the transcriptional regulation of OsWRKY94 in Arabidopsis protoplasts. OsTB1m was used as the negative control. **, P < 0.01 (Student's t‐test). (c, d) Kinetic binding of OsMADS57 to the OsWRKY94 promoter subfragment WRKY94S2 under (c) 25°C and (d) 4°C. The raw data curves are shown in red and the fitted curves in dotted black lines. (e) Kinetic and association constants of OsMADS57 binding to the promoter of OsWRKY94; k_a, association rate constant; k_d, dissociation rate constant; K_D, dissociation constant; M, mol l⁻¹; s, second. (f) OsTB1 effect on OsMADS57 binding to WRKY94S2 at 25°C and 4°C. A mixture of 1 μM OsMADS57 and 5 μM OsTB1 was placed in an ice bath for 30 min then passed over the biosensor chip. (g) Assay of D14 as a competitor for OsMADS57 binding to WRKY94S2 at 25°C and 4°C. The D14 probe contained the CATTAAAAAG CArG‐box. (h) Temperature‐dependent regulation of OsWRKY94 and D14 by OsMADS57 interacting with OsTB1 to balance rice growth and chilling tolerance. OsMADS57 activates OsWRKY94 under chilling stress, and represses D14 under normal conditions.