Genome-wide identification of the <i>Gossypium hirsutum</i> CAD gene family and functional study of <i>GhiCAD23</i> under drought stress

Cinnamyl alcohol dehydrogenase (CAD) is a crucial enzyme in the final stage of lignin monomer biosynthesis. This study focuses on the CAD gene family within <i>Gossypium hirsutum</i>. Through comprehensive genomic analysis, we identified 29 <i>GhiCAD</i> genes within the <i>Gossypium hirsutum</i> genome using a bioinformatics approach. Phylogenetic analysis revealed that the GhiCAD family can be categorized into four subgroups, which are closest to the evolutionary relationship with <i>Arabidopsis thaliana</i>. There are multiple <i>cis</i>-acting elements on the promoters of <i>GhiCAD</i> genes associated with abiotic stress responses. Some <i>GhiCAD</i> genes demonstrated high expression in various tissues like root, leaf, and sepal, as well as in fiber and ovule at different developmental stages (10 days post anthesis (DPA), 15 DPA, 20 DPA, 25 DPA). The transcript levels of <i>GhiCAD23</i> were notably elevated when exposed to PEG treatment and drought stress (DS). <i>GhiCAD23</i> is also co-expressed with many known drought response genes, suggesting its involvement in the plant's reaction to DS. Employing virus-induced gene silencing (VIGS) technology to silence the <i>GhiCAD23</i> gene, it was found that silencing <i>GhiCAD23</i> reduced the tolerance of cotton to DS. Under DS, the relative leaf water content, superoxide dismutase (SOD), and catalase (CAT) enzyme activities of the <i>GhiCAD23</i>-silenced cotton plants were decreased by 31.84%, 30.22% and 14.19%, respectively, while malondialdehyde (MDA) was increased by 72.16% compared with the control cohort. Drought promotes the accumulation of lignin, and it was found that silencing the <i>GhiCAD23</i> reduces lignin accumulation in cotton under DS. The analysis of phenotypic and physiological indicators indicates that <i>GhiCAD23</i> is vital in cotton's resistance to DS. This investigation provides an important reference for future comprehensive exploration of the <i>GhiCAD23</i> gene's function in cotton's DS response mechanism.