Raf-like kinases and receptor-like (pseudo)kinase GHR1 are required for stomatal vapor pressure difference response

Stomatal pores close rapidly in response to low-air-humidity-induced leaf-to-air vapor pressure difference (VPD) increases, thereby reducing excessive water loss. The hydroactive signal-transduction mechanisms mediating high VPD-induced stomatal closure remain largely unknown. The kinetics of stomatal high-VPD responses were investigated by using time-resolved gas-exchange analyses of higher-order mutants in guard-cell signal-transduction branches. We show that the slow-type anion channel SLAC1 plays a relatively more substantial role than the rapid-type anion channel ALMT12/QUAC1 in stomatal VPD signaling. VPD-induced stomatal closure is not affected in mpk12/mpk4GC double mutants that completely disrupt stomatal CO2 signaling, indicating that VPD signaling is independent of the early CO2 signal-transduction pathway. Calcium imaging shows that osmotic stress causes cytoplasmic Ca2+ transients in guard cells. Nevertheless, osca1-2/1.3/2.2/2.3/3.1 Ca2+-permeable channel quintuple, osca1.3/1.7-channel double, cngc5/6-channel double, cngc20-channel single, cngc19/20crispr-channel double, glr3.2/3.3-channel double, cpk-kinase quintuple, cbl1/4/5/8/9 quintuple, and cbl2/3rf double mutants showed wild-type-like stomatal VPD responses. A B3-family Raf-like mitogen-activated protein (MAP)-kinase kinase kinase, M3K delta 5/RAF6, activates the OST1/SnRK2.6 kinase in plant cells. Interestingly, B3 Raf-kinase m3k delta 5 and m3k delta 1/delta 5/delta 6/delta 7 (raf3/6/5/4) quadruple mutants, but not a 14-gene raf-kinase mutant including osmotic stress-linked B4-family Raf-kinases, exhibited slowed high-VPD responses, suggesting that B3-family Raf-kinases play an important role in stomatal VPD signaling. Moreover, high VPD-induced stomatal closure was impaired in receptor-like pseudokinase GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1) mutant alleles. Notably, the classical transient wrong-way VPD response was absent in ghr1 mutant alleles. These findings reveal genes and signaling mechanisms in the elusive high VPD-induced stomatal closing response pathway.

Automated summary

The study investigated the kinetics of high VPD-induced stomatal responses, revealing that SLAC1 plays a more significant role in stomatal VPD signaling compared to ALMT12/QUAC1. The research also showed that VPD-induced stomatal closure is independent of the CO2 signal-transduction pathway. Calcium imaging demonstrated transient changes in cytoplasmic Ca2+ in guard cells under osmotic stress.