ALA reverses ABA-induced stomatal closure by modulating PP2AC and SnRK2.6 activity in apple leaves

Abstract

5-Aminolevulinic acid (ALA), known as a new natural plant growth regulator, can reverse abscisic acid (ABA)-induced stomatal closure. The protein phosphatase 2A (PP2A) played an important role in regulation of stomatal movement by ALA and ABA; however, the underlying molecular mechanisms remain unclear. Here, we report that ALA promotes MdPP2A activity and gene expression in the leaf epidermis of apple (Malus × domestica Borkh.), and expression of the catalytic subunit MdPP2AC was most significantly correlated with stomatal aperture. Western blotting showed that ALA enhanced MdPP2AC protein abundance and phosphorylation. Y2H (yeast two hybrid), FLC (firefly luciferase complementation imaging) and BiFC (Bimolecular fluorescence complementation) assays showed that MdPP2AC interacted with several other MdPP2A subunits as well as MdSnRK2.6 (Sucrose non-fermenting 1-related protein kinase 2.6), and the latter interaction was further verified by pull-down and MST (microscale thermophoresis) assays. ALA downregulated ABA-induced MdSnRK2.6 gene expression, kinase activity, and protein phosphorylation. In transiently transgenic apple leaves, OE-MdPP2AC promoted stomatal aperture by reducing Ca2+ and H2O2 levels but increasing flavonol levels in guard cells. Conversely, OE-MdSnRK2.6 induced stomatal closure by increasing Ca2+ and H2O2 but reducing flavonols. Partial silencing of these genes had opposite effects on Ca2+, H2O2, flavonols, and stomatal movement. Application of exogenous ALA stimulated PP2A activity, which promoted SnRK2.6 dephosphorylation and lower kinase activity in wild-type and transgenic apple leaves. We therefore propose that PP2AC, which dephosphorylates SnRK2.6 and represses its enzyme activity, mediates ALA signaling to inhibit ABA-induced stomatal closure in apple leaves.

Transcriptional regulation of flavonol biosynthesis in plants

“…In tobacco, ABA treatment could inhibit expression of NtMYB184 (a flavonol-specific activator), which reduced production of flavonols and thus increased ROS levels to regulate stomatal closure [ 65 ]. ALA, known as a new natural plant growth regulator, can reverse ABA-induced stomatal closure [ 140 ]. In apple, ALA treatment enhanced protein abundance and phosphorylation of protein phosphatase 2AC (MdPP2AC), which promoted the interactions of different PP2A subunits and increased holoenzyme activity [ 140 ].…”

“…ALA, known as a new natural plant growth regulator, can reverse ABA-induced stomatal closure [ 140 ]. In apple, ALA treatment enhanced protein abundance and phosphorylation of protein phosphatase 2AC (MdPP2AC), which promoted the interactions of different PP2A subunits and increased holoenzyme activity [ 140 ]. Phosphorylated PP2A interacted with and dephosphorylated MdSnRK2.6 (sucrose non-fermenting 1-related protein kinase 2.6), which induced flavonol accumulation and thus reduced ROS levels in the guard cells to open stomata [ 140 ].…”

“…In apple, ALA treatment enhanced protein abundance and phosphorylation of protein phosphatase 2AC (MdPP2AC), which promoted the interactions of different PP2A subunits and increased holoenzyme activity [ 140 ]. Phosphorylated PP2A interacted with and dephosphorylated MdSnRK2.6 (sucrose non-fermenting 1-related protein kinase 2.6), which induced flavonol accumulation and thus reduced ROS levels in the guard cells to open stomata [ 140 ]. In addition, ALA treatment could inhibit expression of MdSCL8 (a flavonol repressor), which may promote expression of MdFLS1 and flavonol accumulation to participate in regulation of stomata opening [ 93 ].…”

ALA induces stomatal opening through regulation among PTPA, PP2AC, and SnRK2.6

“…Overexpression of AtPP2AC-5 promoted the PP2A activity and conferred better growth of root and shoot of Arabidopsis under salt condition ( Hu et al., 2017 ). Our previous pharmacological and genetic studies with detached apple leaves showed that MdPP2AC was involved in ALA-ABA modulating stomatal movement ( Chen et al., 2023 ). In the regulation, MdPP2AC is positive for ALA signaling but negative for ABA signaling.…”

“…SnRK2.6 phosphorylation and dephosphorylation controls stomatal closing and opening ( Mustilli et al., 2002 ; Yoshida et al., 2002 ). In the previous study, we demonstrated that SnRK2.6 functioned at the downstream of PP2A signaling, involved in ALA-ABA regulation of apple stomatal movement ( Chen et al., 2023 ). Based on these studies, we hypothesized that ALA positively regulated PTPA and PP2AC expression to enhance PP2A activity, and then negatively regulated SnRK2.6 activity, which finally promoted stomatal opening and improved leaf photosynthetic efficiency of plants.…”