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

Abstract

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 <i>mpk12</i>/<i>mpk4GC</i> 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, <i>osca1-2</i>/<i>1.3</i>/<i>2.2</i>/<i>2.3</i>/<i>3.1</i> Ca²⁺-permeable channel quintuple, <i>osca1.3</i>/<i>1.7</i>-channel double, <i>cngc5</i>/<i>6</i>-channel double, <i>cngc20</i>-channel single, <i>cngc19</i>/<i>20crispr</i>-channel double, <i>glr3.2</i>/<i>3.3</i>-channel double, <i>cpk-</i>kinase quintuple, <i>cbl1</i>/<i>4</i>/<i>5</i>/<i>8</i>/<i>9</i> quintuple, and <i>cbl2</i>/<i>3rf</i> double mutants showed wild-type-like stomatal VPD responses. A B3-family Raf-like mitogen-activated protein (MAP)-kinase kinase kinase, M3Kδ5/RAF6, activates the OST1/SnRK2.6 kinase in plant cells. Interestingly, B3 Raf-kinase <i>m3kδ5</i> and <i>m3kδ1</i>/<i>δ5</i>/<i>δ6</i>/<i>δ7</i> (<i>raf3</i>/<i>6</i>/<i>5</i>/<i>4</i>) quadruple mutants, but not a 14-gene <i>raf-kinase</i> 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 <i>ghr1</i> mutant alleles. These findings reveal genes and signaling mechanisms in the elusive high VPD-induced stomatal closing response pathway.