Genome-wide identification and functional characterization of the CP12 gene family in cotton reveals its critical role in heat stress response

Chao LiYanhai Zhao^*Yanqin Wang^*Shuguang LiJuan XuZiling HanWenlong Li

• Tarim University, Alar, Xinjiang, China

Calvin Cycle Protein 12 (CP12) plays a key role in the dark/light regulation of the Calvin-Benson-Bassham (CBB) cycle responsible for CO2 assimilation. Beyond its canonical role, emerging evidence suggests CP12 may function as a molecular chaperone and participate in stress responses. We performed genome-wide identification and characterization of the CP12 gene family in Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, and Gossypium raimondii. A total of 11, 10, 5, and 4 of the CP12 genes were identified in the four types of cotton. Phylogenetic analysis classified them into three distinct clades (I-III), which was further supported by analyses of conserved motifs and gene structures. Synteny analysis revealed that whole-genome duplication (WGD) and segmental duplication were the primary drivers for the expansion of this gene family. Promoter cis-element analysis uncovered an abundance of stress-responsive elements. Utilizing RNA-seq data from flowers, leaves, and buds, we discovered a striking clade-specific expression pattern: Clade I genes were significantly upregulated under heat stress, with Ghir_CP12_10 exhibiting a dramatic ~10-fold induction in flowers. In contrast, Clade II genes were generally down-regulated. This molecular pattern was closely associated with changes in physiological indicators, including a decrease in net photosynthetic rate and increases in malondialdehyde, catalase, and peroxidase. Our study provides the first evidence that the CP12 gene family in cotton has undergone functional divergence. Clade I members act as positive regulators of thermotolerance, potentially by stabilizing photosynthetic complexes and protecting key enzymes from oxidative damage under heat stress.