Systematic analysis and comparison of CaLB genes reveal the functions of GhCaLB42 and GhCaLB123 in fiber development and abiotic stress in cotton

Ca2+-dependent lipid-binding (CaLB) family proteins, containing C2 domain, play an important role in plant growth, development, and abiotic stress response. There has been no systematic identification of CaLB family genes in cotton (Gossypium). In this study, 196 GhCaLBs were identified and divided into five groups by phylogenetic tree analysis in Gossypium hirsutum. Gene structure and conserved protein motif analysis supported the evolutionary conservation of GhCaLBs. Whole-genome or segmental duplications contributed to the expansion of GhCaLBs. 2144 orthologous and paralogous CaLB gene pairs among four cotton species were detected. And Ka/Ks values analysis showed that CaLBs experienced strong purification selection. Numerous hormone-regulated, stress-responsive, and growth and development cis-elements were detected in the 2 kb promoter regions of GhCaLBs. Besides, transcriptome data combined with qRT-PCR analysis showed some GhCaLBs were preferentially expressed in ovule or fiber. Subcellular localization experiment revealed that GhCaLB42-GFP was located in nucleus and cell membrane, while GhCalB123-GFP was in cell membrane. Overexpressing GhCaLB42 significantly promoted the elongation and density of leaf trichomes in transgenic Arabidopsis, while GhCaLB123 overexpression inhibited the elongation of leaf trichomes. Histochemical staining experiments showed that GhCaLB123 overexpression lines could significantly increase the thickness of xylem vessels and interfascicular fibers cell wall of stem compared with WT. GhCaLB42 may positively regulate fiber elongation, and GhCaLB123 inhibited elongation but promoted secondary cell wall (SCW) thickening in fiber. Yeast two-hybrid assays indicated that GhCaLB42 protein had multiple biological functions including fiber development and abiotic stress response, and GhCaLB123 might participate in substance transport for the cellulose synthesis. GhCaLB42 and its two interaction genes GhTHi1–1 and GhCIPK9 were induced to be highly expressed under ABA treatment. These findings provide an important foundation for understanding the functions of specific cotton CaLB genes and for the effective selection of fiber development related genes.