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

5-Aminolevulinic acid (ALA), as a new natural plant growth regulator, has been proved to regulate protein phosphatase 2A (PP2A) activity to promote stomatal opening in apple ( Malus domestica ) leaves. However, the molecular mechanisms underlying remain unclear. Here, we cloned and transformed MdPTPA , MdPP2AC , and MdSnRK2.6 of apple into tobaccos ( Nicotiana tabacum ) and found that over-expression (OE)- MdPTPA or OE- MdPP2AC promoted stomatal aperture while OE- MdSnRK2.6 induced stomatal closure under normal or drought condition. The Ca 2+ and H 2 O 2 levels in the guard cells of OE- MdPTPA and OE- MdPP2AC was decreased but flavonols increased, and the results in OE- SnRK2.6 was contrary. Exogenous ALA stimulated PP2A activity but depressed SnRK2.6 activity in transgenic tobaccos, leading to less Ca 2+ , H 2 O 2 and more flavonols in guard cells, and consequently stomatal opening. OE- MdPTPA improved stomatal opening and plant growth but impaired drought tolerance, while OE- MdSnRK2.6 improved drought tolerance but depressed the leaf P n . Only OE- MdPP2AC improved stomatal opening, leaf P n , plant growth, as well as drought tolerance. These suggest that the three genes involved in ALA-regulating stomatal movement have their respective unique biological functions. Yeast two-hybrid (Y2H) assays showed that MdPP2AC interacted with MdPTPA or MdSnRK2.6, respectively, but no interaction of MdPTPA with MdSnRK2.6 was found. Yeast three-hybrid (Y3H) assay showed that MdPTPA promoted the interactions between MdPP2AC and MdSnRK2.6. Therefore, we propose a regulatory module of PTPA-PP2AC-SnRK2.6 that may be involved in mediating the ALA-inducing stomatal aperture in green plants.