Maize MITOGEN-ACTIVATED PROTEIN KINASE 20 mediates high-temperature-regulated stomatal movement

High temperature induces stomatal opening; however, uncontrolled stomatal opening is dangerous for plants in response to high temperature. We identified a high-temperature sensitive (hts) mutant from the ethyl methane sulfonate (EMS)-induced maize (Zea mays) mutant library that is linked to a single base change in MITOGEN-ACTIVATED PROTEIN KINASE 20 (ZmMPK20). Our data demonstrated that hts mutants exhibit substantially increased stomatal opening and water loss rate, as well as decreased thermotolerance, compared to wild-type plants under high temperature. ZmMPK20-knockout mutants showed similar phenotypes as hts mutants. Overexpression of ZmMPK20 decreased stomatal apertures, water loss rate, and enhanced plant thermotolerance. Additional experiments showed that ZmMPK20 interacts with MAP KINASE KINASE 9 (ZmMKK9) and E3 ubiquitin ligase RPM1 INTERACTING PROTEIN 2 (ZmRIN2), a maize homolog of Arabidopsis (Arabidopsis thaliana) RIN2. ZmMPK20 prevented ZmRIN2 degradation by inhibiting ZmRIN2 self-ubiquitination. ZmMKK9 phosphorylated ZmMPK20 and enhanced the inhibitory effect of ZmMPK20 on ZmRIN2 degradation. Moreover, we employed virus-induced gene silencing (VIGS) to silence ZmMKK9 and ZmRIN2 in maize and heterologously overexpressed ZmMKK9 or ZmRIN2 in Arabidopsis. Our findings demonstrated that ZmMKK9 and ZmRIN2 play negative regulatory roles in high-temperature-induced stomatal opening. Accordingly, we propose that the ZmMKK9-ZmMPK20-ZmRIN2 cascade negatively regulates high-temperature-induced stomatal opening and balances water loss and leaf temperature, thus enhancing plant thermotolerance.