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.

Acknowledgements

This work was supported by the Natural Science Foundation of China (32172512, 32272641), the Jiangsu Special Fund for Frontier Foundation Research of Carbon Peaking and Carbon Neutralization (BK20220005), the Jiangsu Agricultural Science and Technology Innovation Fund [CX(20)2023], and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. The authors greatly appreciate Miss Anqi Xing and her professional editorial team A&L Scientific Editing (www.alpublish.com) for helping us to polish the manuscript. Thanks to Prof. Yang Zhao and Dr. Qingzhong Li of the Shanghai Center for Plant Stress Biology and CAS Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, for kindly providing AtSnRK2.6 phosphorylated antibody.

Author contributions

LJW and YYA designed the experiments and revised the manuscript. ZC performed all the experiments and wrote the manuscript. All authors have read and approved the manuscript.

Data availability

Data supporting the conclusions of this work are available in the paper and its supplementary materials. Some datasets were derived from sources in the public domain: the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov) and the Arabidopsis information Resource (https://www.arabidopsis.org).

Conflict of interest

None declared.

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