LRS1 phosphorylated in response to blue light regulates stomatal opening

Blue light receptor phototropin is a protein kinase that initiates various blue light signaling pathways, including the phosphorylation and activation of plasma membrane (PM) H⁺-ATPase for stomatal opening. Activation of PM H⁺-ATPase drives K⁺ uptake via K⁺_in channels. This influx of K⁺ is electrically balanced by intracellular anions such as Cl⁻ and malate^2-, increasing cellular osmotic potential and driving water influx and stomatal opening. Hypothesizing that unidentified phosphorylation events regulate this process, we performed phosphoproteomic analysis to elucidate the link between blue light signaling and stomatal opening. We found that blue light induces phosphorylation of Ser395 in Vicia faba LATERAL ROOT STIMULATOR 1 (VfLRS1) in guard cell protoplasts. VfLRS1 shows high similarity to Arabidopsis thaliana LATERAL ROOT STIMULATOR 1 (LRS1, AT3G05090), and the phosphorylation site is conserved as Ser393 in A. thaliana LRS1. Therefore, we investigated stomatal phenotype using a T-DNA-inserted knockout mutant of A. thaliana LRS1, lrs1. The lrs1 mutant exhibited normal blue light-induced stomatal opening and normal blue light-induced phosphorylation of Thr948 of PM H⁺-ATPase (numbering according to AHA1) in guard cells. The lrs1 mutant showed reduced starch accumulation during dark and red light illumination. Blue light-induced starch degradation was also severely suppressed in the lrs1 mutant. The reduced starch accumulation in lrs1 guard cells may have been associated with reduced levels of starch-derived metabolites, such as malate. We then examined blue light-induced stomatal opening in potassium gluconate (KGlu) buffer, in which most Cl^- was replaced by the poorly membrane-permeable gluconate anion. As expected, the lrs1 mutant showed significantly reduced blue light-induced stomatal opening under this condition. Taken together, our results indicate that LRS1 plays important roles in starch dynamics and contributes to stomatal opening.