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.

Figure 1

ALA and ABA regulate stomatal aperture in apple leaves, possibly via PP2A activity. A: Photographs of stomata after different treatments. B: Stomatal aperture after ALA and/or ABA treatment. Data are means of 40 measurements ± standard error (SE). C: PP2A activity in apple epidermal strips after different treatments. Data are from three independent biological replicates. The same lowercase letters in B or C indicate no significant difference at p = 0.05.

Figure 2

Expression of genes that encode parts of PP2A subunits in apple leaves treated by ALA or/and ABA. Epidermis preparation and treatments were as described in Fig. 1. The data are the means of three independent biological replicates. The same lowercase letters indicate no significant difference at p = 0.05.

Figure 3

ALA significantly increases abundance and phosphorylation of the MdPP2AC protein. A: ALA promotes MdPP2AC abundance when α-tubulin (actin) was used as loading control. Molecular mass (kDa) markers are shown on the left. All experiments were repeated at least three times with similar results. B: Total plant proteins were extracted and immunoprecipitated with a phosphorylated MdPP2AC antibody. Image J software was used to estimate the relative abundance of total (C) and phosphorylated (D) MdPP2AC protein. Data are means of three independent biological replicates. The same lowercase letters in C or D indicate no significant difference at p = 0.05.

Figure 4

ALA promotes the interaction of MdPP2AC with other MdPP2A subunits. A: Quadruple dropout medium (SD/−Ade/–His/−Leu/−Trp supplemented with X-α-Gal and AbA) with 0.5 mg L⁻¹ ALA was used in Y2H assays to test potential interactions. BD-53 plus AD-T was applied for the positive control. The β-galactosidase activity was detected by ONPG (o-nitrophenyl β-D-galactopyranoside) assay. B: FLC assays. C: Intensity of the luciferase interaction signal was measured with ImageJ software. Values are means ± SE from three independent experiments (^*p < 0.05).

Figure 5

ALA inhibits the ABA-induced expression of MdSnRK2.6 and promotes MdSnRK2.6 dephosphorylation, and MdSnRK2.6 interacts with MdPP2AC. A: Expression of MdSnRK2.6 in apple leaves under ALA and/or ABA treatment. B: Western blotting assay of phosphorylated MdSnRK2.6, whose abundance was specifically induced by ABA but reduced by ALA. The total proteins of apple leaf epidermis under different treatments were extracted before immunoprecipitation with a specific phosphorylated SnRK2.6 antibody. C: Quantification of the phosphorylated SnRK2.6 levels in B. The same lowercase letters in A or C indicate no significant difference at p = 0.05. D: The subcellular localization of MdPP2AC and MdSnRK2.6 in tobacco leaves. The pCAMBIA1305-mCherry membrane marker and pCAMBIA1300-35S::GFP, 35S::MdPP2AC, and 35S:: MdSnRK2.6 recombinant plasmids were transiently co-expressed in 4-week-old N. benthamiana leaves. RFP: Red fluorescent protein. GFP: green fluorescent protein. Scale bar: 25 μm. E: A Y2H assay confirmed the interaction of MdPP2AC with MdSnRK2.6. The combination of BD-53 plus AD-T was used as a positive control, and BD-Lam plus AD-T was used as a negative control. F: The interaction of MdPP2AC and MdSnRK2.6 showed by an FLC assay. G: A BiFC assay showed the interaction of MdPP2AC and MdSnRK2.6. H: Pull-down assay. MdSnRK2.6-His and MdPP2AC-GST proteins were expressed in E. coli for immunoprecipitation. The anti-His and anti-GST antibody were used for detection of MdSnRK2.6-His and MdPP2AC-GST, respectively. I: Interaction between MdPP2AC and MdSnRK2.6 measured by MST. Despite small changes in the mass of MdPP2AC, a signal-to-noise ratio of 29.30 enabled accurate determination of the interaction strength, and the affinity between MdPP2AC and MdSnRK2.6 was determined to be 109.09 ± 0.53 nM. Three independent experiments were performed with similar results.

Figure 6

ALA treatment affects stomatal movement in transiently transgenic apple leaves. A: Stomatal aperture of apple leaves transiently overexpressing MdPP2AC or MdSnRK2.6 and treated with or without ALA. B: Stomatal aperture of apple leaves in which MdPP2AC or MdSnRK2.6 was transiently silenced, treated with or without ALA. Values are means ± SE of three biological replicates, and the same lowercase letters indicate no significant difference at p = 0.05.

Figure 7

Effects of ALA on MdPP2A (A, C) and MdSnRK2.6 (B, D) activities in the leaf epidermis of different transiently transgenic apple genotypes. Values are means ± SE of three biological replicates. The same lowercase letters indicate no significant difference at p = 0.05.

Figure 8

Western blotting assay of phosphorylated MdSnRK2.6 and MdPP2AC protein abundances in OE-MdPP2AC apple leaves treated with exogenous ALA and/or ABA. A: Western blotting assay. B and C: Quantification of grey levels of the phosphorylated SnRK2.6 and PP2AC protein bands. Proteins were extracted from the apple leaf epidermis under different treatments before immunoprecipitation. Molecular mass (kDa) markers are shown on the left. All experiments were repeated at least three times with similar results. The same lowercase letters indicate no significant difference at p = 0.05

Figure 9

Effects of ALA treatment on Ca²⁺, H₂O₂, and flavonol levels in stomatal guard cells of different genotypes of transiently transgenic apple leaves. A and C: Photographs of Ca²⁺, H₂O₂, and flavonol levels in stomatal guard cells. Scale bar: 10 μm. B and D: Relative fluorescence intensity of Fluo-3 a.m., H₂DCF-DA, and DPBA in guard cells. Each treatment was measured with ImageJ software. Values are means ± SE of 15 replicate measurements. Bars that share lowercase letters are not significantly different at p = 0.05.

Figure 10

A proposed model for the reversal of ABA-induced stomatal closure by ALA. → represents a positive effect, and —| represents a negative effect, − - - - represents speculation. Processes in red are studied in the present work, and those in black have been reported previously.