CaSnRK2.4-mediated phosphorylation of CaNAC035 regulates abscisic acid synthesis in pepper (Capsicum annuum L.) responding to cold stress.

Plant NAC transcription factors play a crucial role in enhancing cold stress tolerance, yet the precise molecular mechanisms underlying cold stress remain elusive. In this study, we identified and characterized CaNAC035, an NAC transcription factor isolated from pepper (Capsicum annuum) leaves. We observed that the expression of the CaNAC035 gene is induced by both cold and abscisic acid (ABA) treatments, and we elucidated its positive regulatory role in cold stress tolerance. Overexpression of CaNAC035 resulted in enhanced cold stress tolerance, while knockdown of CaNAC035 significantly reduced resistance to cold stress. Additionally, we discovered that CaSnRK2.4, a SnRK2 protein, plays an essential role in cold tolerance. In this study, we demonstrated that CaSnRK2.4 physically interacts with and phosphorylates CaNAC035 both in vitro and in vivo. Moreover, the expression of two ABA biosynthesis-related genes, CaAAO3 and CaNCED3, was significantly upregulated in the CaNAC035-overexpressing transgenic pepper lines. Yeast one-hybrid, Dual Luciferase, and electrophoretic mobility shift assays provided evidence that CaNAC035 binds to the promoter regions of both CaAAO3 and CaNCED3 in vivo and in vitro. Notably, treatment of transgenic pepper with 50 µm Fluridone (Flu) enhanced cold tolerance, while the exogenous application of ABA at a concentration of 10 µm noticeably reduced cold tolerance in the virus-induced gene silencing line. Overall, our findings highlight the involvement of CaNAC035 in the cold response of pepper and provide valuable insights into the molecular mechanisms underlying cold tolerance. These results offer promising prospects for molecular breeding strategies aimed at improving cold tolerance in pepper and other crops.

Salicylic acid-related ribosomal protein CaSLP improves drought and Pst.DC3000 tolerance in pepper.

The ribosomal protein contains complex structures that belong to polypeptide glycoprotein family, which are involved in plant growth and responses to various stresses. In this study, we found that capsicum annuum 40S ribosomal protein SA-like (CaSLP) was extensively accumulated in the cell nucleus and cell membrane, and the expression level of CaSLP was up-regulated by Salicylic acid (SA) and drought treatment. Significantly fewer peppers plants could withstand drought stress after CaSLP gene knockout. The transient expression of CaSLP leads to drought tolerance in pepper, and Arabidopsis's ability to withstand drought stress was greatly improved by overexpressing the CaSLP gene. Exogenous application of SA during spraying season enhanced drought tolerance. CaSLP-knockdown pepper plants demonstrated a decreased resistance of Pseudomonas syringae PV.tomato (Pst) DC3000 (Pst.DC3000), whereas ectopic expression of CaSLP increased the Pst.DC3000 stress resistance in Arabidopsis. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) results showed that CaNAC035 physically interacts with CaSLP in the cell nucleus. CaNAC035 was identified as an upstream partner of the CaPR1 promoter and activated transcription. Collectively the findings demonstrated that CaSLP plays an essential role in the regulation of drought and Pst.DC3000 stress resistance.