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 Arabidopsis 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 Arabidopsis 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 transparent testa4 (tt4) mutant. In addition, exogenous flavonols recovered stomatal responses of tt4 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.

PP2A and microtubules function in 5‐aminolevulinic acid‐mediated H2O2 signaling in Arabidopsis guard cells

“…We found that flavonol accumulation decreased H 2 O 2 content in guard cells under ALA treatment (An et al 2016a), providing new information on how ALA controls H 2 O 2 content in guard cells. However, ALA-induced flavonol accumulation could not completely account for ALA-decreased H 2 O 2 content in guard cells (An et al 2016a). Here, we further showed that PP2A and MTs were also involved in ALA-regulated stomatal movement.…”

“…Flavonols are natural antioxidants in plants (Nakabayashi et al 2014). We found that flavonol accumulation decreased H 2 O 2 content in guard cells under ALA treatment (An et al 2016a), providing new information on how ALA controls H 2 O 2 content in guard cells. However, ALA-induced flavonol accumulation could not completely account for ALA-decreased H 2 O 2 content in guard cells (An et al 2016a).…”

“…ALA could induce stomatal opening and inhibit stomatal closure by down‐regulating H 2 O 2 in guard cells (An et al ). We further showed that flavonol played a role in mediating ALA‐decreased hydrogen peroxide (H 2 O 2 ) in guard cells (An et al ). These results provide valuable information on the mechanism by which ALA function.…”

“…Increasing attention has been paid to ALA because of its environmental-friendly Abbreviations -ABA, abscisic acid; ALA, 5-aminolevulinic acid; AsA, ascorbic acid; BR, brassinosteroid; CalA, calyculin A; CAT, catalase; DPI, diphenylene iodonium; GFP-TUA6, green fluorescent protein-tagged -tubulin 6 transgenic Arabidopsis; H 2 O 2 , hydrogen peroxide; MeJA, methyl jasmonate; MS, Murashige and Skoog; MTs, microtubules; OA, okadaic acid; OE, overexpression; PP2A, type 2A protein phosphatase; PP2A-C2, C2 subunit of type 2A protein phosphatase; PP2A-C2-OE, PP2A-C2 overexpression; PPFD, photosynthetic photo flux density; ROS, reactive oxygen species; VBT, vinblastine. characteristic and great application potential in agriculture (Wang et al 2003, Akram andAshraf 2013) and horticulture (Feng et al 2016, An et al 2016b, Zheng et al 2017, Zheng et al 2018. Our recent studies suggested a new role of ALA, regulating stomatal movement (An et al 2016a(An et al , 2016c. ALA could induce stomatal opening and inhibit stomatal closure by down-regulating H 2 O 2 in guard cells (An et al 2016c).…”

“…Increasing attention has been paid to ALA because of its environmental‐friendly characteristic and great application potential in agriculture (Wang et al , Akram and Ashraf ) and horticulture (Feng et al , An et al , Zheng et al , An et al , Zheng et al ). Our recent studies suggested a new role of ALA, regulating stomatal movement (An et al , ). ALA could induce stomatal opening and inhibit stomatal closure by down‐regulating H 2 O 2 in guard cells (An et al ).…”