Gene Cloning And Characterization Of Chilling Sensitive Mutant Chs1in Arabidopsis

The plants have to deal with different stresses from environment during the whole lives, including biotic and abiotic stresses. Low temperature is an environmental factor that affects plant growth and development and plant-pathogen interactions. Plants have evolved a sophisticated system to cope with the environmental stresses. Although emerging evidence shows the tight link between cold and defense responses of plants, how temperature regulates plant defense responses is not well understood.In this study, we characterized chilling-sensitive mutant1(chs1), and functionally analyzed the role of the CHS1gene in plant responses to chilling stress. The chsl mutant didn’t show visible difference phenotype compared with Col when grown at normal temperature, however, when transferred to low temperature such as16℃, it displayed a chilling-sensitive phenotype, and also displayed defense-associated phenotypes, including extensive cell death, the accumulation of hydrogen peroxide and salicylic acid, and an increased expression of PR genes:these phenotypes indicated that the mutation in chs1activates the defense responses under chilling stress. A map-based cloning analysis revealed that CHS1encodes a TIR-NB-type protein, which lack LRR domain. CHS1mainly expressed in cauline leaves, flowers (mainly in sepals), old rosette leaves, and stems, but not in the siliques or roots. The chilling sensitivity of chs1was fully rescued by phytoalexin deficient4(pad4) and enhanced disease susceptibility1(eds1), but not by non-race-specific disease resistance1(ndr1), indicating that the chilling sensitive of chs1-2was fully depend on EDS1or PAD4, but not NDR1. The overexpression of the TIR and NB domains can suppress the chs1-conferred phenotypes, respectively. Interestingly, the stability of the transcriptional level of CHS1was not affected by temperature, but the accumulation of protein level of CHS1, chsl and CHS1TIR domain was positively regulated by low temperatures, and the accumulation of NB domain of CHS1was not affected by temperature. Notably, the TIR-GFP protein degraded much faster than the CHS1-GFP protein, suggesting that the NB domain of CHS1may stabilize the CHS1protein. The degradation of the TIR domain of CHS1was not dependent on the26S proteasome pathway. This study revealed the role of a TIR-NB-type gene in plant growth and cell death under chilling stress, and suggests that temperature modulates the stability of the TIR-NB protein in Arabidopsis.In order to identify new components involved in the chs1-mediated pathway, we screened mutants that resemble wild-type morphological phenotypes at16℃as chs1suppressors(soc, suppressor of chs1) in the M2population derived from EMS-mutagenized chsl-2seeds. One suppressor soc1suppressed all chs1-conferred phenotypes at chilling stress. Map-based cloning revealed SOC1encodes a TIR-NB-LRR protein. It is possible that SOC1was activated in chsl-2at low temperature by forming homo-dimmer or hete-dimmer. Another TIR-NB type gene AtTN2was also upregulated in chs1-2at16℃, implying that the induced-AtTN2may also contribute to the chilling sensitivity of chs1-2.