On-site single pollen metabolomics reveals varietal differences in phosphatidylinositol synthesis under heat stress conditions in rice

Although a loss of healthy pollen grains induced by metabolic heat responses has been indicated to be a major cause of heat-induced spikelet sterility under global climate change, to date detailed information at pollen level has been lacking due to the technical limitations. In this study, we used picolitre pressure-probe-electrospray-ionization mass spectrometry (picoPPESI-MS) to directly determine the metabolites in heat-treated single mature pollen grains in two cultivars, heat-tolerant cultivar, N22 and heat-sensitive cultivar, Koshihikari. Heat-induced spikelet fertility in N22 and Koshihikari was 90.0% and 46.8%, respectively. While no treatment difference in in vitro pollen viability was observed in each cultivar, contrasting varietal differences in phosphatidylinositol (PI)(34:3) have been detected in mature pollen, together with other 106 metabolites. Greater PI content was detected in N22 pollen regardless of the treatment, but not for Koshihikari pollen. In contrast, there was little detection for phosphoinositide in the single mature pollen grains in both cultivars. Our findings indicate that picoPPESI-MS analysis can efficiently identify the metabolites in intact single pollen. Since PI is a precursor of phosphoinositide that induces multiple signaling for pollen germination and tube growth, the active synthesis of PI(34:3) prior to germination may be closely associated with sustaining spikelet fertility even at high temperatures.