CsRCI2D enhances high-temperature stress tolerance in Camelina sativa L. through endo-membrane trafficking from the plasma membrane.

Rare Cold Inducible 2s (RCI2s) are hydrophobic proteins in cell membranes that participate in abiotic stress tolerance mechanisms. Additionally, they are used as traceable membrane trafficking markers in endocytosis studies. Plants regulate cell homeostasis through endocytosis by limiting the activity of plasma membrane transporter proteins to adapt to stressful conditions. In this study, we found high temperature (HT) stress-induced membrane trafficking of RCI2D in Camelina sativa L. The gene expression and protein synthesis were increased by HT stress at 37 °C. Moreover, rapid membrane trafficking of CsRCI2D was traced by multiple-phase membrane fractionation using sucrose density gradients and compared with CsRCI2E/F/G from the same protein family subgroup. The distribution of CsRCI2s was shown to be similar to that of the clathrin heavy chain, which is known as a major endocytosis protein. Subcellular localization of CsRCI2D was observed in the plasma membrane and endo-membranes and overlapped with membrane lipids. CsRCI2D co-localized with lipids, and its overexpression increased the intracellular lipid content compared to that of wild-type camelina. Moreover, transgenic camelina lines showed enhanced HT stress tolerance during germination and hypocotyl elongation when compared to the wild type. These results suggest that HT-induced CsRCI2D membrane trafficking enhances HT stress tolerance in camelina.