Table_2_Functional Characterization of Cotton C-Repeat Binding Factor Genes Reveal Their Potential Role in Cold Stress Tolerance.DOCX

Low temperature is a common biological abiotic stress in major cotton-growing areas. Cold stress significantly affects the growth, yield, and yield quality of cotton. Therefore, it is important to develop more robust and cold stress-resilient cotton germplasms. In response to climate change and erratic weather conditions, plants have evolved various survival mechanisms, one of which involves the induction of various stress responsive transcript factors, of which the C-repeat-binding factors (CBFs) have a positive effect in enhancing plants response to cold stress. In this study, genomewide identification and functional characterization of the cotton CBFs were carried out. A total of 29, 28, 25, 21, 30, 26, and 15 proteins encoded by the CBF genes were identified in seven Gossypium species. A phylogenetic evaluation revealed seven clades, with Clades 1 and 6 being the largest. Moreover, the majority of the proteins encoded by the genes were predicted to be located within the nucleus, while some were distributed in other parts of the cell. Based on the transcriptome and RT-qPCR analysis, Gthu17439 (GthCBF4) was highly upregulated and was further validated through forward genetics. The Gthu17439 (GthCBF4) overexpressed plants exhibited significantly higher tolerance to cold stress, as evidenced by the higher germination rate, increased root growth, and high-induction levels of stress-responsive genes. Furthermore, the overexpressed plants under cold stress had significantly reduced oxidative damage due to a reduction in hydrogen peroxide (H2O2) production. Moreover, the overexpressed plants under cold stress had minimal cell damage compared to the wild types, as evidenced by the Trypan and 3,3′-Diaminobenzidine (DAB) staining effect. The results showed that the Gthu17439 (GthCBF4) could be playing a significant role in enhancing cold stress tolerance in cotton and can be further exploited in developing cotton germplasm with improved cold-stress tolerance.

低温是全球主产棉区常见的非生物胁迫。低温胁迫会显著影响棉花的生长、产量及纤维品质，因此培育抗逆性更强、耐低温胁迫的棉花种质资源具有重要意义。为应对气候变化与极端天气频发的现状，植物演化出多种生存策略，其中一类涉及多种胁迫响应转录因子的诱导表达，而C-重复结合因子（C-repeat-binding factors, CBFs）在增强植物低温胁迫响应过程中发挥积极作用。本研究针对棉花CBF家族基因开展了全基因组鉴定与功能表征分析：在7个棉属物种中，共鉴定到29、28、25、21、30、26和15个CBF基因编码的蛋白。系统发育分析显示该家族可分为7个进化支，其中进化支1和6所含成员最多。此外，多数CBF编码蛋白被预测定位于细胞核中，少数蛋白分布于细胞其他区域。通过转录组与实时荧光定量PCR（RT-qPCR）分析，发现Gthu17439（GthCBF4）的表达量显著上调，并通过正向遗传学实验得到验证。过表达Gthu17439（GthCBF4）的转基因棉花植株对低温胁迫的耐受性显著提升，具体表现为更高的种子萌发率、更发达的根系，以及胁迫响应基因的高诱导表达水平。此外，低温胁迫下的过表达植株体内过氧化氢（hydrogen peroxide, H2O2）生成量减少，氧化损伤显著缓解。进一步通过台盼蓝染色与3,3′-二氨基联苯胺（3,3′-Diaminobenzidine, DAB）染色实验证实，与野生型植株相比，低温胁迫下的过表达植株细胞损伤程度显著更低。本研究结果表明，Gthu17439（GthCBF4）在提升棉花低温胁迫耐受性方面发挥关键作用，可进一步用于培育耐低温胁迫能力更优的棉花种质资源。