Phosphorylation of Serine 186 of bHLH Transcription Factor SPEECHLESS Promotes Stomatal Development in Arabidopsis

The initiation of stomatal lineage and subsequent asymmetric divisions in Arabidopsis require the activity of the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). It has been shown that SPCH controls entry into the stomatal lineage as a substrate either of the MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) cascade or GSK3-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2). Here we show that three serine residues of SPCH appear to be the primary phosphorylation targets of Cyclin-Dependent Kinases A; 1 (CDKA; 1) in vitro, and among them Serine 186 plays a crucial role in stomatal formation. Expression of an SPCH construct harboring a mutation that results in phosphorylation deficiencies on Serine 186 residue failed to rescue stomatal defects in spch null mutants. Expression of a phosphorylation-mimic mutant SPCHS186D complemented stomatal production defects in the transgenic lines harboring the targeted expression of dominant-negative CDKA; 1.N146. Therefore, in addition to MAPK-and BIN2-mediated phosphorylation on SPCH, phosphorylation at Serine 186 is positively required for SPCH function in regulating stomatal development.