Heat shock transcription factor A1b regulates heat tolerance in wheat and Arabidopsis through OPR3 and jasmonate signalling pathway

Competing interests

The authors declare no competing interests.

Author contributions

M.X. and H.P. designed the experiments. X.T., F.W., Y.Z., T.L., K.Y., L.Z. and Z.Q. performed the research. Z.H., Y.Y. and Z.N. analysed the data. Q.S. V.R. H.P. and M.X. wrote the paper.

Dear Editor,

High temperature adversely affects plant growth and severely causes crop yield loss worldwide, especially for chimonophilous wheat (Triticum aestivum L.; Akter and Islam, 2017). Heat shock transcription factors (HSFs) and plant hormones play regulatory roles in plant responses to heat stress (Baniwal et al., 2004). In this study, we found that TaOPR3 contributes to heat tolerance in wheat probably via regulating JA level.

To investigate the biological function of TaOPR3 in heat responses, we generated TaOPR3 RNAi (Ri1‐3) and overexpression (OE1‐3) lines (Figure 1a). Under normal conditions, no obvious phenotypic variation was detected between TaOPR3 transgenic lines and ‘KN199’ (CK) (Figure 1b). However, under heat stress conditions, Ri and OE lines were more sensitive and resistant to heat stress than CK, respectively (Figure 1b). Specifically, Ri and OE lines exhibited a reduction (0.047 g vs. 0.068 g, on average) and increase (0.133 g vs. 0.075 g, on average) of fresh weight, respectively (Figure 1c). In addition, electrolyte leakage level was higher (67%) in Ri lines and lower (43%) in OE lines compared with CK (53%) (Figure 1d). Furthermore, the heat‐sensitive phenotype, in terms of electrolyte leakage level, of Ri lines was reduced by exogenous application of MeJA (from an average of 33% to 61%; Figure 1e).

Figure 1.

OPR3 contributes to heat tolerance in wheat and Arabidopsis (a) Expression analysis of TaOPR3 in KN199 (CK), TaOPR3 RNAi and overexpression transgenic lines. (b) Phenotypic analysis of KN199, TaOPR3 RNAi and overexpression transgenic lines in response to heat stress. Statistical analysis of fresh weight (c) and electronic leakage (d) for CK, Ri and OE seedlings after heat stress. (e) Statistical analysis of electronic leakage of CK, Ri and OE seedlings with or without MeJA treatment after heat stress. (f) Schematic diagram of T‐DNA insertion and HSE elements in AtOPR3 gene in opr3‐3 mutant as well as GUS reporter construction. (g) Expression analysis of AtOPR3 in response to heat stress in WT and opr3‐3 by qRT‐PCR. (h) Endogenous JA levels in WT and opr3‐3 seedlings before (NC) and after (HS) heat treatment. (i) Statistic analysis of survival rate for WT and opr3‐3 seedlings with or without MeJA treatment after heat treatment. (j) Expression analysis of TaOPR3 in WT, opr3‐3 and 35S:TaOPR3/opr3‐3 by qRT‐PCR. (k) Heat tolerance analysis of WT, opr3‐3 and 35S:TaOPR3/opr3‐3 seedlings. (l) GUS staining analysis of pOPR3:GUS /WT and pdHSE2:GUS /WT seedlings before (NC) and after (HS) heat treatment. (m) Expression analysis of AtOPR3 in hsfa1a, hsfa1b, hsfa2 and hsfa3 mutants before (NC) and after (HS) heat stress by qRT‐PCR. (n) GUS staining analysis of pOPR3:GUS /WT and pOPR3:GUS /hsfa1b before (NC) and after (HS) heat treatment. (o) Yeast one‐hybrid detects HSFA1b and HSE‐2 interaction. (p) Expression analysis of DREB2A in response to heat stress in WT and opr3‐3 by qRT‐PCR. (q) Statistic analysis of survival rate for WT, opr3‐3 and 35S:DREB2A/opr3‐3 seedlings after heat treatment. Each experiment was performed in triplicate. (r) Expression analysis of TaDREB2A in KN199, TaOPR3 RNAi and overexpression transgenic lines in response to heat stress. Data represent mean ± SD. n.s. indicates P > 0.05, * indicates P < 0.05 and ** indicates P < 0.01 (student's t‐test). Letters indicate significant differences (P < 0.05, LSD test). Scale bars = 1 mm.

To better understand how plants respond to elevated temperatures via JA pathway, we identified a new T‐DNA insertion mutant allele of AtOPR3 in Arabidopsis (SALK_053805; hereafter named as opr3‐3) (Figure 1f). Unlike previously reported opr3 mutant alleles (Chehab et al., 2011), AtOPR3 expression is slightly increased under normal conditions but no obvious phenotypic variation is observed in opr3‐3. However, the transcript level of AtOPR3 is decreased in opr3‐3 after heat treatment (Figure 1g). Accordingly, a reduction in JA level to heat stress was detected in opr3‐3 compared to WT (Figure 1h). The opr3‐3 plants were more sensitive to heat stress than WT (Figure 1i). Nevertheless, the heat‐sensitive phenotype of opr3‐3 was rescued by exogenous application of MeJA (Figure 1i). In addition, overexpression of wheat TaOPR3 re‐establishes heat tolerance in Arabidopsis opr3‐3 mutant, suggesting that OPR3‐mediated thermotolerance may be conserved between Arabidopsis and wheat (Figure 1j and k).

To elucidate the underlying mechanism responsible for the transcriptional regulation of AtOPR3 under heat stress conditions, the putative AtOPR3 promoter sequence (1500 bp) was analysed using Plant CARE interface programme. Two potential heat shock elements (HSE‐1 and HSE‐2) at the position of ‐175 bp and ‐903 bp were identified (Figure 1f). Interestingly, the opr3‐3 T‐DNA insertion is located between the two HSEs. Next, GUS reporter was fused with integral 1500‐bp promoter sequence (pOPR3:GUS; including both HSE‐1 and HSE‐2) and with a 620 bp sequence only including HSE‐1 but not HSE‐2 (pdHSE‐2:GUS) (Figure 1f). Results showed that deletion of HSE‐2 affects GUS expression in response to heat stress (Figure 1l).

HSEs are usually bound by HSFs to regulate gene expression (Wu, 1995). Among four hsf mutants (hsfa1a, hsfa1b, hsfa2 and hsfa3), we found that AtOPR3 transcript abundance is reduced only in hsfa1b mutant after heat treatment (Figure 1m). Next, we introduced pOPR3:GUS construct into both WT and hsfa1b mutant and found that pOPR3:GUS/WT lines exhibited up‐regulated GUS expression level after heat treatment, but not for pOPR3:GUS/hsfa1b lines (Figure 1n). Yeast one‐hybrid assay indicated that yeast strain co‐transformed with vector expressing HSFA1b plus vector containing canonic HSE‐2 sequence grow on selective media (media lacking leucine and containing 200 ng/ml AbA), while strain co‐transformed with mHSE‐2 (a substitute of HSE‐2) is unable to grow (Figure 1o).

To further shed light on the mechanisms linking JA signalling pathway with heat stress tolerance, we investigated the expression pattern of JA inducible gene DREB2A and found that DREB2A mRNA level is lower in opr3‐3 than in WT after 1 h and 2 h heat treatment, (Figure 1p). Constitutively expressing DREB2A in opr3‐3 mutants (35S:DREB2A/opr3‐3) exhibited enhanced heat tolerance with higher survival rate (84%‐90%) than that of opr3‐3 (31%) and WT (62%) after heat treatment (Figure 1q). These results suggest that JA affects heat tolerance by regulating DREB2A. We also found that the expression level of TaDREB2A is impaired in TaOPR3 RNAi lines compared with WT at 3 h, 6 h and 9 h after heat stress, whereas it is enhanced in TaOPR3 overexpression lines (Figure 1r), further indicating a potentially similar mechanism of heat stress tolerance in wheat and Arabidopsis.

Limited information is available about molecular mechanisms of the JA‐mediated thermotolerance. In present study, we provide information which improves knowledge regarding the mechanistic link between heat stress/HSFs and JA signalling pathway. When plants perceive heat stress, HsfA1b might convert into a functional homo‐trimer (Peteranderl et al., 1999; Wu, 1995) and activates AtOPR3 expression. This leads to increased JA biosynthesis and accumulation. Subsequently, JA‐mediated signalling pathway activates a cascade resulting in increased DREB2A expression and enhanced heat tolerance in plants. Our study provides a potential approach to improve crop heat stress tolerance by increasing OPR3 expression level appropriately under heat stress conditions.

Tian, X. , Wang, F. , Zhao, Y. , Lan, T. , Yu, K. , Zhang, L. , Qin, Z. , Hu, Z. , Yao, Y. , Ni, Z. , Sun, Q. , Rossi, V. , Peng, H. and Xin, M. (2020) Heat shock transcription factor A1b regulates heat tolerance in wheat and Arabidopsis through OPR3 and jasmonate signalling pathway. Plant Biotechnol. J., 10.1111/pbi.13268