SlECA4, an epsin-like clathrin adaptor protein, improves tomato heat tolerance via clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) is one of the main pathways for plant cells to internalize membrane proteins in response to changing environmental conditions. The Epsin-like Clathrin Adaptor (ECA) proteins play important roles in the assembly of the clathrin coat; however, their involvement in plant responses to heat stress remains unclear. Here we report that in tomato (Solanum lycopersicum), Epsin-like Clathrin Adaptor 4 (SlECA4) expression responded to heat stress. The silencing and knockout of SlECA4 increased tomato sensitivity to heat stress while the overexpression of SlECA4 enhanced tomato tolerance to heat stress. Treatment with a CME inhibitor, ES9-17, reduced tomato heat tolerance. SlECA4 localized to the plasma membrane, the trans-Golgi network/early endosomes, and the prevacuolar compartment/late endosomes. In a SlECA4 knockout line, both CME and recycling from the trans-Golgi network/early endosomes to the plasma membrane were inhibited. These data indicate that SlECA4 is involved in CME. After heat treatment, more punctate structures of SlECA4-green fluorescent protein accumulated in tobacco leaf epidermal cells by transient expression. Furthermore, compared with wild type, the rate of CME was inhibited under heat stress in the SlECA4 knockout line. Taken together, the ECA protein SlECA4 plays a positive role in tomato tolerance to heat stress via the CME pathway.