1-Methylcyclopropene–Mediated Phosphorylation of Ethylene Receptor in Tomato Fruit: Implications for Inhibition and Recovery of Ripening Capacity, Poster Board #017

Application of 1-methylcyclopropene (1-MCP), an ethylene antagonist, to climacteric fruits is an effective technique for maintaining quality and extending shelf life. The effects of 1-MCP are most evident with fruits treated prior to ripening initiation. Fruits treated before ripening initiation exhibit significantly compromised ripening competence, resulting in excessive/abnormal texture, reduced pigment accumulation, and altered volatile evolution. Although delaying 1-MCP treatment until after ripening initiation can circumvent these problems, treatment with some fruits is less effective and faster ripening recovery is observed. Different degrees of ripening inhibition in response to 1-MCP have not been explained at the molecular level. One possible explanation is that 1-MCP, in binding irreversibly to the ethylene receptor (ETR), alters ETR conformation or other properties. Since previous reports have indicated that ETR exhibited autophosphorylation activity in vitro, we speculate that 1-MCP alters ETR phosphorylation state. Here, we propose a novel theory in which 1-MCP-mediated inhibition of ripening, and subsequent recovery, is due to ETR phosphorylation status. Phosphorylation state of ripening-related ETR in tomato, LeETR4, was analyzed using phosphate-affinity gel electrophoresis. LeETR4 was phosphorylated in untreated immature and mature-green fruit, and non-phosphorylated after ripening initiation. Interestingly, 1-MCP (2 µL·L^-1) treatment rapidly switched non-phosphorylated LeETR4 to the phosphorylated form whereas ethylene induced dephosphorylation. Exposure to 2,5-norbornadiene (NBD), a reversible cyclic-olefin ethylene antagonist, also induced LeETR4 phosphorylation, yet removal from NBD resulted in rapid dephosphorylation. The reversible phosphorylation state of LeETR4 in response to NBD application/removal is consistent with the reversible inhibitory effect of NBD unlike 1-MCP. Based on these observations, we assumed that the phosphorylation state of ETR is related to the progress of fruit ripening. We next examined the effect of 1-MCP on LeETR4 phosphorylation state in fruit subjected to extended storage. 1-MCP treatment at mature-green stage resulted in persistent phosphorylation state through nine days, which was consistent with strong inhibition of ripening. This observation indicates that 1-MCP–engaged LeETR4 was neither degraded nor dephosphorylated during storage. By contrast, in fruit treated with 1-MCP at turning stage, phosphorylated LeETR4 disappeared at four days and non-phosphorylated form appeared. This alternation of phosphorylation status is consistent with faster ripening recovery in 1-MCP–treated turning compared with mature-green fruit. Taken together, phosphorylation status of LeETR4 closely paralleled ripening progress. We conclude that the primary response to 1-MCP treatment is maintenance or induction of ETR phosphorylation, resulting in the suppression of ethylene signaling and inhibition of ripening.