The identification of gibberellin 2-oxidase gene family in cotton and functional analysis

Gibberellin (GA) homeostasis is critical for plant growth and development, with gibberellin 2-oxidases ( GA2oxs ) functioning as key regulators by deactivating bioactive GAs. Mepiquat chloride (MC), a widely used plant growth regulator in cotton production, effectively suppresses vegetative growth to improve yield and fiber quality, yet its molecular mechanisms, particularly concerning GA catabolism, remain incompletely understood. In this study, a genome-wide identification of GA2ox genes was performed in upland cotton ( Gossypium hirsutum ), followed by systematic analysis of their gene structures, conserved protein motifs, chromosomal locations, and expression patterns. Virus-induced gene silencing (VIGS) technology was used for perform functional validation of two candidate genes, GhGA2ox6 and GhGA2ox31 . RNA-sequencing was used to explore the differentially expressed genes and enriched pathways in GhGA2ox6 -silenced plants. Weighted gene co-expression network analysis (WGCNA) was further employed to explore the key gene modules and Hub genes associated with hormone pathways. A total of 39 GhGA2ox genes were identified and classified into three subfamilies: C20- GA2ox -I, C19- GA2ox -I, and C19- GA2ox -II. Expression profiling analysis revealed that tissue-specific patterns of GhGA2ox genes, and GhGA2ox6 and GhGA2ox31 were significantly up-regulated in response to MC treatment. VIGS-mediated silencing of GhGA2ox6 resulted in a marked increase in cotton plant height, while silencing GhGA2ox31 had no obvious phenotypic change. Further transcriptomic profiling analysis found that the height-promoting phenotype of GhGA2ox6 -silenced plants was driven by a coordinated multi-hormone regulatory network involving gibberllins, auxins, and cytokinins. WGCNA identified 3 gene modules were significantly associated with hormone contents, and 36 Hub genes involved in ethylene, auxin, jasmonate, and gibberenllin pathways. Collectively, our findings indicate that GhGA2ox6 acts as a negative regulator of cotton plant height and is involved in MC-mediated growth regulation. The WGCNA further complemented the multi-hormone coordinated regulatory mechanism underlying GhGA2ox6 function. This study provides novel insights into the molecular mechanisms underlying MC-regulated cotton growth and offers a potential target gene for modulating cotton plant growth via genetic engineering techniques.