Abstract
Normal leaf margin development is important for leaf morphogenesis and contributes to diverse leaf shapes in higher plants. We here show the crucial roles of an atypical type II phosphatidylinositol 4-kinase, PI4Kγ5, in Arabidopsis leaf margin development. PI4Kγ5 presents a dynamics expression pattern along with leaf development and a T-DNA mutant lacking PI4Kγ5, pi4kγ5–1, presents serrated leaves, which is resulted from the accelerated cell division and increased auxin concentration at serration tips. Studies revealed that PI4Kγ5 interacts with and phosphorylates a membrane-bound NAC transcription factor, ANAC078. Previous studies demonstrated that membrane-bound transcription factors regulate gene transcription by undergoing proteolytic process to translocate into nucleus, and ANAC078 undergoes proteolysis by cleaving off the transmembrane region and carboxyl terminal. Western blot analysis indeed showed that ANAC078 deleting of carboxyl terminal is significantly reduced in pi4kγ5–1, indicating that PI4Kγ5 is important for the cleavage of ANAC078. This is consistent with the subcellular localization observation showing that fluorescence by GFP-ANAC078 is detected at plasma membrane but not nucleus in pi4kγ5–1 mutant and that expression of ANAC078 deleting of carboxyl terminal, driven by PI4Kγ5 promoter, could rescue the leaf serration defects of pi4kγ5–1. Further analysis showed that ANAC078 suppresses the auxin synthesis by directly binding and regulating the expression of auxin synthesis-related genes. These results indicate that PI4Kγ5 interacts with ANAC078 to negatively regulate auxin synthesis and hence influences cell proliferation and leaf development, providing informative clues for the regulation of in situ auxin synthesis and cell division, as well as the cleavage and functional mechanism of membrane-bound transcription factors.
Highlights
Phosphatidylinositol (PI) signaling pathway, as well as the relevant second messenger molecules inositol 1, 4, 5-trisphosphate and various phospholipid molecules, is important for multiple physiological processes in human, animals and plants
By studying a T-DNA mutant lacking an atypical type II phosphatidylinositol 4kinase (PI4Kγ5), pi4kγ5–1, which presents serrated leaves that resulted from the accelerated cell division and increased auxin concentration at serration tips, we here showed the crucial roles of PI4Kγ5 on the function of membrane-bound Transcription factors (TFs), possibly through a phosphorylation-dependent process
Biochemical analysis reveal that PI4Kγ5 phosphorylates ANAC078 and further genetic analysis confirmed that PI4Kγ5 is crucial for the cleavage and normal function of ANAC078, indicating that PI4Kγ5ANAC078 module downregulates auxin level during leaf development
Summary
Phosphatidylinositol (PI) signaling pathway, as well as the relevant second messenger molecules inositol 1, 4, 5-trisphosphate and various phospholipid molecules, is important for multiple physiological processes in human, animals and plants. PI 4-kinase (PI4K) catalyzes the synthesis of PI 4-phosphate (PI4P) by phosphorylating PI at the 4’ position of the inositol ring [1, 2], and play crucial roles in development and stress responses. Structural and biochemical analysis showed that PI4Kα1 and PI4Kβ1 contain the Pleckstrin Homology (PH) domain that binds to PI4P, and their enzymatic activities are negatively feedback regulated [4]. Physiological studies showed that PI4Kβ1 is involved in the polarized expansion of root hairs by interacting with RabA4b (a Rab GTPase) and pi4kβ pi4kβ double mutant presents shorter root hairs compared to wild type [5]. PI4Kβ1 is recruited to actin cytoskeleton following binding to PI phosphate kinase 1 [7] and both PI4Kβ1 and PI4Kβ2 act upstream of phospholipase C (PLC) to participate in the cold response [8]
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