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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.