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Cotton is an important natural fiber crop. The RF2 gene family is a member of the bZIP transcription factor superfamily, which plays an important role in plant resistance to environmental stresses. In this paper, the RF2 gene family of four cotton species was analyzed genome-wide, and the key gene RF2-32 was cloned for functional verification. A total of 113 RF2 genes were identified in the four cotton species, and the RF2 family was relatively conserved during the evolution of cotton. Chromosome mapping and collinear analysis indicated that fragment replication was the main expansion mode of RF2 gene family during evolution. Cis-element analysis showed that there were many elements related to light response, hormone response and abiotic stress response in the promoters of RF2 genes. The transcriptome and qRT-PCR analysis of RF2 family genes in upland cotton showed that RF2 family genes responded to salt stress and drought stress. GhRF2-32 protein was localized in the cell nucleus. Silencing the GhRF2-32 gene showed less leaf wilting and increased total antioxidant capacity under drought and salt stress, decreased malondialdehyde content and increased drought and salt tolerance. This study revealed the evolutionary and functional diversity of the RF2 gene family, which laid a foundation for the further study of stress-resistant genes in cotton. 

Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme in plants, which regulates carbon flow through the TCA cycle and controls protein and oil biosynthesis. Although it is important, there is little research on PEPC in cotton, the most important fiber crop in the world. In this study, a total of 125 PEPCs were identified in 15 Gossypium genomes. All PEPC genes in cotton are divided into six groups and each group generally contains one PEPC member in each diploid cotton and two in each tetraploid cotton. This suggests that PEPC genes already existed in cotton before their divergence. There are additional PEPC sub-groups in other plant species, suggesting the different evolution and natural selection during different plant evolution. PEPC genes were independently evolved in each cotton sub-genome. During cotton domestication and evolution, certain PEPC genes were lost and new ones were born to face the new environmental changes and human being needs. The comprehensive analysis of collinearity events and selection pressure shows that genome-wide duplication and fragment duplication are the main methods for the expansion of the PEPC family, and they continue to undergo purification selection during the evolutionary process. PEPC genes were widely expressed with temporal and spatial patterns. The expression patterns of PEPC genes were similar in G. hirsutum and G. barbadense with a slight difference. PEPC2A and 2D were highly expressed in cotton reproductive tissues, including ovule and fiber at all tested developmental stages in both cultivated cottons. However, PEPC1A and 1D were dominantly expressed in vegetative tissues. Abiotic stress also induced the aberrant expression of PEPC genes, in which PEPC1 was induced by both chilling and salinity stresses while PEPC5 was induced by chilling and drought stresses. Each pair (A and D) of PEPC genes showed the similar response to cotton development and different abiotic stress, suggesting the similar function of these PEPCs no matter their origination from A or D sub-genome. However, some divergence was also observed among their origination, such as PEPC5D was induced but PEPC5A was inhibited in G. barbadense during drought treatment, suggesting that a different organized PEPC gene may evolve different functions during cotton evolution. During cotton polyploidization, the homologues genes may refunction and play different roles in different situations.