Table_3_Characterization of Phytochrome-Interacting Factor Genes in Pepper and Functional Analysis of CaPIF8 in Cold and Salt Stress.DOC

As a subfamily of basic helix-loop-helix (bHLH) transcription factors, phytochrome-interacting factors (PIFs) participate in regulating light-dependent growth and development of plants. However, limited information is available about PIFs in pepper. In the present study, we identified six pepper PIF genes using bioinformatics-based methods. Phylogenetic analysis revealed that the PIFs from pepper and some other plants could be divided into three distinct groups. Motif analysis revealed the presence of many conserved motifs, which is consistent with the classification of PIF proteins. Gene structure analysis suggested that the CaPIF genes have five to seven introns, exhibiting a relatively more stable intron number than other plants such as rice, maize, and tomato. Expression analysis showed that CaPIF8 was up-regulated by cold and salt treatments. CaPIF8-silenced pepper plants obtained by virus-induced gene silencing (VIGS) exhibited higher sensitivity to cold and salt stress, with an obvious increase in relative electrolyte leakage (REL) and variations in the expression of stress-related genes. Further stress tolerance assays revealed that CaPIF8 plays different regulatory roles in cold and salt stress response by promoting the expression of the CBF1 gene and ABA biosynthesis genes, respectively. Our results reveal the key roles of CaPIF8 in cold and salt tolerance of pepper, and lay a solid foundation for clarifying the biological roles of PIFs in pepper and other plants.

作为碱性螺旋-环-螺旋（basic helix-loop-helix, bHLH）转录因子的一个亚家族，光敏色素互作因子（phytochrome-interacting factors, PIFs）参与调控植物的光依赖型生长与发育过程。然而，目前关于辣椒中PIFs的研究信息仍较为匮乏。本研究通过基于生物信息学的方法，鉴定得到6个辣椒PIF基因。系统发育分析结果显示，辣椒与其他部分植物的PIFs可划分为3个清晰的类群。基序分析表明，这些PIF蛋白含有大量保守基序，这与PIF蛋白的分类结果相一致。基因结构分析提示，CaPIF基因包含5至7个内含子，其内含子数量相较于水稻、玉米、番茄等其他植物更为稳定。表达分析结果显示，CaPIF8的表达可被低温与盐胁迫诱导上调。通过病毒诱导基因沉默（virus-induced gene silencing, VIGS）技术获得的CaPIF8沉默辣椒植株，对低温和盐胁迫的敏感性显著升高，其相对电解质渗漏率（relative electrolyte leakage, REL）明显增加，且胁迫相关基因的表达模式发生改变。进一步的胁迫耐受性实验表明，CaPIF8分别通过促进C-repeat结合因子1（CBF1）基因与脱落酸（abscisic acid, ABA）生物合成基因的表达，在低温与盐胁迫响应中发挥不同的调控作用。本研究结果揭示了CaPIF8在辣椒低温与盐胁迫耐受性中的关键作用，为阐明辣椒及其他植物中PIFs的生物学功能奠定了坚实基础。