Pan-genome analysis of the sunflower LOX gene family: evolution, expression, and salt stress-responsive candidate...

Sunflower (Helianthus annuus L.), the fourth largest edible oil-producing crop worldwide, has potential for application in the remediation of saline-alkali land. In this regard, lipoxygenase (LOX)-derived metabolites, particularly jasmonic acid, have been established to play positive roles in regulating the tolerance of plants to salt stress. However, using only a single reference genome provides limited genetic resolution, which is insufficient for a comprehensive elucidation of the evolutionary dynamics of the sunflower LOX family at the pan-genome level. In this study, we aimed to characterize the HaLOX gene family across multiple sunflower accessions and explore its evolutionary patterns in response to salt stress. Based on 10 sunflower genomes, we systematically identified the HaLOX family, revealing 220 genes clustered into 27 orthologous gene groups (OGGs) (12 core, 6 softcore, 7 shell, and 2 cloud OGGs). Together with the copy number variations analysis, these findings indicate that dispensable genes reflect the evolutionary diversity of the LOX family among sunflower accessions. Phylogenetic analysis further classified these OGGs into the 9-LOX and 13-LOX subfamilies (ratio, 1:2), a majority of which have undergone purifying selection. In contrast, panHaLOX10 and panHaLOX17 showed statistical support for episodic positive selection, suggesting possible functional divergence in specific sunflower lineages. Tandem duplication and whole-genome duplication (WGD)/segmental duplication were major drivers of HaLOX gene family expansion. Notably, only the 13-LOX subfamily underwent significant expansion via WGD/segmental duplication, whereas the 9-LOX subfamily did not experience such expansion events. In addition, transposon analysis suggested that DNA/hAT, LTR/Copia, and LTR/Gypsy may have contributed to HaLOX evolution, with proportions of 37.6%, 16.5%, and 16.9%, respectively. We also identified a high proportion of putative stress-responsive cis-regulatory elements (CREs) in HaLOX gene promoters (43.9%), among which MYB, MYC, and ABRE were the most abundant CREs. Moreover, RT-qPCR validated the expression patterns of nine 13-LOX genes exposed to salt stress, particularly panHaLOX10/17. Overall, our findings provide preliminary insights into the potential evolutionary implications of dispensable genes, thereby contributing to a better understanding of the evolutionary characteristics and functional differentiation of the HaLOX family, and potentially facilitating the identification of candidate genes for salt tolerance molecular breeding.