Late embryogenesis abundant gene GhLEA-5 of semi-wild cotton positively regulates salinity tolerance in upland cotton

The productivity and quality of cotton are significantly compromised by salt stress. In this study, the full length of encoding region and genomic DNA sequences of GhLEA_5A/D (Gh_A10G166600 and Gh_D10G188300), which belong to the late embryogenesis abundant gene family in allotetraploid upland cotton (Gossypium hirsutum L.) and semi-wild cotton (Gossypium purpurascens), were isolated and their salt tolerance was experimentally confirmed. Analysis of sequence alignments and phylogenetic trees indicated a significant level of homology between GhLEA-5A and GhLEA-5D. Additionally, a conserved protein motif was consistently identified across these sequences. The transcriptome data analysis showed that the expression level of GhLEA-5A/D was substantially enhanced in the leaves of salt-tolerant G. purpurascens accessions compared to salt-sensitive materials. In the real-time quantitative reverse transcription PCR (qRT-PCR) assays, notable expression levels of the GhLEA-5D gene were detected in salt-tolerant upland cotton materials following exposure to salt stress at 3 and 12-hour time points. The suppression of GhLEA-5A/D transcription via Virus-induced Gene Silencing (VIGS) technology significantly exacerbates salt sensitivity in cotton. This is evidenced by the nearly 50 % increase in malondialdehyde (MDA) content alongside a 60 % reduction in peroxidase (POD) levels in salt-treated plants when compared to the control group. The overexpression of the GhLEA-5A/D gene conferred enhanced salt tolerance in Arabidopsis, resulting in a 25 % increase in root length, a 30 % improvement in survival rate, a 15 % increase in water retention, and a 15 % boost in photosynthetic efficiency. The chlorophyll fluorescence parameters, enzyme activities, diaminobenzine, and nitroblue tetrazolium staining suggested that GhLEA-5A/D likely exhibited a positive regulatory role for cotton responding to salt stress. Furthermore, we identified 76 candidate proteins that potentially interact with GhLEA-5 in the yeast two-hybrid screening library. These results provide a theoretical basis for studying the mechanism of cotton salt tolerance and offer new resources for improving cotton salt tolerance genes.