A membrane-associated NAC transcription factor OsNTL3 is involved in thermotolerance in rice

Xue-Huan Liu, Yu-Shu Lyu, Weiping Yang, Zheng-Ting Yang, Sun-Jie Lu, Jian-Xiang Liu

Plant Biotechnol. Journal; 2020 May;18(5):1317-1329. doi: 10.1111/pbi.13297.

Abstract

Heat stress induces misfolded protein accumulation in endoplasmic reticulum (ER), which initiates the unfolded protein response (UPR) in plants. Previous work has demonstrated the important role of a rice ER membrane-associated transcription factor OsbZIP74 (also known as OsbZIP50) in UPR. However, how OsbZIP74 and other membrane-associated transcription factors are involved in heat stress tolerance in rice is not reported. In the current study, we discovered that OsNTL3 is required for heat stress tolerance in rice. OsNTL3 is constitutively expressed and up-regulated by heat and ER stresses. OsNTL3 encodes a NAC transcription factor with a predicted C-terminal transmembrane domain. GFP-OsNTL3 relocates from plasma membrane to nucleus in response to heat stress and ER stress inducers. Loss-of-function mutation of OsNTL3 confers heat sensitivity while inducible expression of the truncated form of OsNTL3 without the transmembrane domain increases heat tolerance in rice seedlings. RNA-Seq analysis revealed that OsNTL3 regulates the expression of genes involved in ER protein folding and other processes. Interestingly, OsNTL3 directly binds to OsbZIP74 promoter and regulates its expression in response to heat stress. In turn, up-regulation of OsNTL3 by heat stress is dependent on OsbZIP74. Thus, our work reveals the important role of OsNTL3 in thermotolerance, and a regulatory circuit mediated by OsbZIP74 and OsNTL3 in communications among ER, plasma membrane and nucleus under heat stress conditions.

See: https://pubmed.ncbi.nlm.nih.gov/31733092/

Figure 1: Loss‐of‐function of OsNTL3 confers heat stress sensitivity in rice. (a–b) Up‐regulation of OsNTL3 by ER stress and abiotic stresses. Eight‐day‐old wild‐type rice Nipponbare seedlings were treated with various stresses (TM, 5 μg/ml; DTT, 2 mm; NaCl, 250 mm; ABA, 0.1 mm; PEG 4000,15%) for 4 hr and roots were harvested for OsNTL3 expression analysis (a). Time‐course experiments were performed under heat stress conditions (b). Relative gene expression is the expression level of OsNTL3 in stressed plants relative to that in non‐stressed plants, both of which were normalized to that of the internal control ACTIN. Error bars represent SE (n = 3). Asterisks indicate significance levels when comparing to the control in t‐test. (*, P < 0.05; **, P < 0.01; n.s., not significant at P < 0.05). (c‐e) Heat stress phenotypes of the OsNTL3 mutant plants. Eight‐day‐old wild‐type (WT) seedlings and two lines of targeted‐gene‐edited OsNTL3 (ntl3‐1 and ntl3‐2) mutant seedlings grown at 29 ºC were transferred to 45 ºC for 5 days and then photographed after recovering at 29 ºC for 7 days (d). Survival rate was count based on the appearance of newly developed green leaves (e). Totally, 141 rice plants under each temperature condition for each genotypes were examined for phenotype analysis. Protein domain structures of OsNTL3 in plants were depicted in c. (f–g) ROS accumulation and electrolytic leakage in WT and OsNTL3 mutant plants under different temperature conditions. Four‐week‐old wild‐type (WT) seedlings and OsNTL3 gene‐edited mutant seedlings grown at 29 ºC were transferred to 45 ºC for 16 hr, leaves were sampled for DAB staining (f) or transferred to 45 ºC for 5 hr, and leaves were sampled for electrolyte conductivity measurements (g). Error bars represent SE (n = 6 in e and n = 3 in g). Different letters indicate significant differences in comparisons between two samples as determined by LSD test following ANOVA analysis (P < 0.05). Bar = 1 cm.