The dehydration-responsive element binding protein 1 (DREB1)/C-repeat-binding factor (CBF) genes are key regulators of cold acclimation and freezing tolerance in the chilling tolerant Arabidopsis thaliana. Here, we investigated the function of three members of the 10 rice DREB1 genes, OsDREB1C, E, and G, in the chilling sensitive rice plants. Their loss of function (LOF) mutants were each more chilling susceptible compared to the wild type, and the LOF mutants of all three genes, dreb1ceg, were more chilling susceptible than any of the single mutants. Strikingly, these mutants were capable of cold acclimation, indicating that these rice DREB1 genes are important for basal chilling tolerance but not cold acclimation. Transcriptome and physiology analyses suggest that the OsDREB1C/E/G genes are involved in reactive oxygen species (ROS) scavenging and cell death regulation under chilling. Furthermore, these three rice DREB1 genes are found to promote tolerance to other abiotic stresses: the OsDREB1C/E/G genes are positive regulators of heat tolerance, OsDREB1C and OsDREB1G are positive regulators of salt tolerance, and OsDREB1G is a positive regulator of drought tolerance. These findings expand our knowledge of the roles of DREB1 proteins in plants, enhance our mechanistic understanding of abiotic stress tolerance and will facilitate the generation of stress-tolerant crop plants.

Arabidopsis cyclic nucleotide-gated channels (CNGC) 2 and 4 are shown to negatively regulate disease resistance and heat tolerance and to positively regulate plant growth. Whether or not their functions in these processes are interdependent is largely unknown. Here, using the mutation of phytoalexin deficient 4 (PAD4) to inhibit the enhanced defense response and programmed cell death (PCD), we assessed the contribution of the altered defense response to the heat tolerance and plant growth in the cngc2 and cngc4 single and double mutants. The pad4 mutation reverted the enhanced disease resistance of the cngc2 and cngc4 mutants at the normal temperature (22 °C) but not at the elevated temperature (28 °C). The pad4 mutation slightly alleviated the dwarfism of the cngc2 and cngc4 mutants at 22 °C but not at 28 °C, indicating a small contribution from the defense response to plant growth regulation. The pad4 mutation also reduced the enhanced heat tolerance in the cngc mutants, suggesting an involvement of PCD in heat tolerance. In addition, a higher heat tolerance was correlated with more opened stomata under heat treatment among the wild type and mutants of the PAD4, CNGC2 and CNGC4 genes. In sum, this study suggests that the regulation of heat tolerance and plant growth by CNGC2 and CNGC4 is almost independent of their regulation of disease resistance. It also reveals a PAD4-dependent role of CNGC2 and CNGC4 in stomatal aperture regulation and heat tolerance.

Chilling stress restricts the geographical distribution of rice and severely affects its growth and development, ultimately reducing both yield and quality. The plant hormone ethylene is involved in stress responses; however, its role in rice chilling tolerance has not been thoroughly explored. This study reveals that ethylene negatively regulates chilling tolerance in rice by antagonizing the tolerance-promoting effects of abscisic acid (ABA). Treatment with ethylene or its biosynthetic precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), resulted in a reduced survival rate after chilling and delayed stomatal closure in response to chilling. There are two Raf-like protein kinases related ethylene signalling, CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) and CTR2, that have overlapping functions in ethylene signalling, and their loss-of-function mutants exhibit constitutive ethylene responses. The ctr1 ctr2 double-mutant displayed lower survival rates and slower stomatal closure under chilling stress compared with the wild type. In contrast, ABA treatment significantly enhanced the survival rate of the wild type under chilling stress and promoted stomatal closure in response to chilling. Furthermore, ethylene inhibited the effects of ABA on chilling tolerance and stomatal closure. The ctr1 ctr2 double-mutant failed to respond to external ABA treatment in terms of stomatal closure and increased survival rate under chilling stress. Overall, our findings suggest that ethylene negatively regulates chilling tolerance in rice by inhibiting ABA-induced stomatal closure through the action of CTR1 and CTR2.