Heat stress-induced remodelling of lipid and microRNA networks in Brassica napus germinated pollen and pollen-derived small extracellular vesicles.

Heat stress (HS) is a primary factor limiting plant reproductive success, severely affecting pollen production and performance. In order to clarify the molecular alterations underlying HS-induced male sterility, lipidomic and small-RNA sequencing analyses were conducted on hydrated pollen, germinated pollen, pollensomes (PSs), and the vesicle-free medium of Brassica napus cv 'Phoenix CL' grown under both control and HS conditions. HS significantly reduced pollen germinability and increased PS externalization. Although particle size remained unchanged, a significant increase in PS abundance was observed in HS-derived samples. Lipid profiling revealed significant HS-induced remodelling across all samples, including an increase of saturated fatty acids in hydrated pollen and germinated pollen. Notably, triacontanoic acid, the dominant lipid in control PS, was lost under HS conditions and replaced by oleic acid. In parallel, small-RNA sequencing identified 70 miRNAs, 61 of which were differentially expressed. Hydrated pollen showed the strongest response to HS, while PS showed opposite trends, suggesting the selective retention or export of miRNAs. Among all, HS increased the levels of miR160, miR6030a, and miR319a in PS, while the release of miR399 shifted from being vesicular to non-vesicular. Target prediction revealed that these miRNAs regulate pathways associated with development, hormones, vesicles, and lipids. Overall, this study reveals that HS remodels lipid metabolism and miRNA-mediated regulation in pollen and PS, providing molecular signatures for improving crop heat tolerance.