Genome-wide study uncovering the role of GPAT gene family in watermelon and functional validation of ClGPAT8 against cold stress.

Glycerol-3-phosphoacyltransferase (GPAT) plays role in abiotic stress responses. An inclusive bioinformatics methodology identified 11 GPAT genes in watermelon (Citrullus lanatus L.), scattered across seven chromosomes. Gene structure analysis revealed that the documented genes contained 1-13 introns, with most genes containing 1-2 introns. Phylogenetic investigation clustered the GPATs into three clades (GPAT9-like, ATS1-like, and others), displaying evolutionary resemblance to Arabidopsis homologs. Promoter regions were supplemented with abiotic-stress-responsive cis-elements and hormones, indicating regulation by phytohormonal and environmental stressors. Collinearity investigation identified one localized duplication episode among Cla97Chr09 and Cla97Chr10, while synteny investigation exposed nine orthologous sets with Arabidopsis, representative of few duplications but strong but stable conservation. Moreover, 30 miRNAs were predicted to target 10 ClPAT genes via translational inhibition or cleavage, suggesting potential post-transcriptional regulation that will require further experimental validation. qRT-PCR examination showed that 10 ClPATCLGPAT genes responded to cold stress, with ClPATCLGPAT8 particularly showed strongest induction. To confirm its function, virus-induced gene silencing (VIGS) was employed to suppress ClGPAT8 expression in watermelon. In cold stress (4°C), ClGPAT8-silenced plants exhibited greater membrane injury, enhanced leaf wilting, and increased production of reactive oxygen species (ROS) compared with control plants. Physiological assays revealed markedly elevated relative electrical conductivity (REC) and malondialdehyde (MDA) content. Histochemical staining established excessive accumulation of O₂·⁻ and H₂O₂ in silenced plants. These conclusions demonstrate that ClGPAT8 positively regulates cold tolerance by reducing oxidative damage, maintaining membrane integrity, and providing a theoretical foundation for developing cold-resilient watermelon genotypes.