ALA-Induced Flavonols Accumulation in Guard Cells Is Involved in Scavenging H2O2 and Inhibiting Stomatal Closure in Arabidopsis Cotyledons

Abstract

5-aminolevulinic acid (ALA), a new plant growth regulator, can inhibit stomatal closure by reducing H₂O₂ accumulation in guard cells. Flavonols are a main kind of flavonoids and have been proposed as H₂O₂ scavengers in guard cells. 5-aminolevulinic acid can significantly improve flavonoids accumulation in plants. However, whether ALA increases flavonols content in guard cells and the role of flavonols in ALA-regulated stomatal movement remains unclear. In this study, we first demonstrated that ALA pretreatment inhibited ABA-induced stomatal closure by reducing H₂O₂ accumulation in guard cells of <i>Arabidopsis</i> seedlings. This result confirms the inhibitory effect of ALA on stomatal closure and the important role of decreased H₂O₂ accumulation in this process. We also found that ALA significantly improved flavonols accumulation in guard cells using a flavonol-specific dye. Furthermore, using exogenous quercetin and kaempferol, two major components of flavonols in <i>Arabidopsis</i> leaves, we showed that flavonols accumulation inhibited ABA-induced stomatal movement by suppressing H₂O₂ in guard cells. Finally, we showed that the inhibitory effect of ALA on ABA-induced stomatal closure was largely impaired in flavonoid-deficient <i>transparent testa4</i> (<i>tt4</i>) mutant. In addition, exogenous flavonols recovered stomatal responses of <i>tt4</i> to the wild-type levels. Taken together, we conclude that ALA-induced flavonol accumulation in guard cells is partially involved in the inhibitory effect of ALA on ABA-induced H₂O₂ accumulation and stomatal closure. Our data provide direct evidence that ALA can regulate stomatal movement by improving flavonols accumulation, revealing new insights into guard cell signaling.