Multi-genome comparative analysis and VIGS validation suggest that GhMAPKKK5 negatively regulates fruiting branch...

Plant architecture strongly influences planting density, canopy light interception, and mechanical harvesting efficiency in upland cotton (Gossypium hirsutum L.). Fruiting branch angle (FBA) is an important architectural trait, but its molecular regulation remains poorly understood. Here, we compared the MAPK/ERK kinase kinase (MEKK) subfamily across four upland cotton reference genomes and identified 44 MEKK members in each genome. Genomic positions and coding sequences were broadly conserved, whereas motif analysis distinguished 26 motif-conserved from 18 motif-variable homologous groups. Integrating this classification with phylogenetic relationships and expression differences between accessions with contrasting FBA prioritized GhMAPKKK5 (GhChrD12G0779) for functional analysis. GhMAPKKK5 expression was lower in large-angle accessions than in compact accessions. Virus-induced gene silencing of GhMAPKKK5 increased FBA at both the squaring-to-flowering and boll-setting stages, providing preliminary evidence that GhMAPKKK5 negatively influences branch orientation. Silencing was also associated with increased brassinosteroid content, reduced cytokinin-equivalent content, and altered expression of hormone- and branch angle-related genes, including GhLAZY1, GhTAC1, and GhSAUR. Together, these results link GhMAPKKK5 activity to FBA and to brassinosteroid- and cytokinin-related processes. GhMAPKKK5 is therefore a candidate for further mechanistic and agronomic evaluation of cotton branch architecture.