Arabidopsis Type II Phosphatidylinositol 4-Kinase PI4Kγ5 Regulates Auxin Biosynthesis and Leaf Margin Development through Interacting with Membrane-Bound Transcription Factor ANAC078
Fig 6
ANAC078 negatively regulates auxin synthesis by directly binding to the promoters of YUC2, YUC4 and GH3.5.
A. qPCR analysis confirmed the expression of ANAC078(-C) in WT [ANAC078(-C)ox] (left), which results in the decreased IAA amount (right). ACTIN7 gene was used as an internal reference and ANAC078 transcription level in WT was set as 1.0. IAA content was measured by LC/MS using the 7th and 8th rosette leaves (~1 cm in length) of 20-day-old plants. The experiments were repeated three times and statistically analyzed (**, p<0.01). Data are presented as means ± SD (n = 3). B. qPCR analysis revealed the opposite expressions of auxin-synthesis related genes (YUC2, YUC4, and GH3.5) in ANAC078(-C)ox and pi4kγ5–1 plants, indicating the negative effects of ANAC078(-C) in auxin synthesis. ACTIN7 gene was used as an internal reference and transcription level of tested gene in WT was set as 1.0. The experiments were repeated three times and statistically analyzed (**, p<0.01). Data are presented as means ± SE (n = 3). C. ANAC078 presents transcriptional repression activity. Transient transcription dual luciferase (Dual-LUC) assay was performed by using YUC2 promoter in the absence or presence of GFP-ANAC078. Relative LUC activities normalized to the REN activity (LUC/REN) was calculated. The experiments were repeated three times and data are presented as means ± SD (n = 3). D. EMSA assays showed that ANAC078 directly binds to the promoters of YUC2, YUC4, and GH3.5. Arrows indicated the shifted bands of DNA fragments. E. ChIP assay confirmed that ANAC078 directly binds to the promoters of YUC2, YUC4, and GH3.5. qPCR was performed to detect the DNA abundance. Input was added as positive control (PC) and IP samples without antibody were used as negative control (NC). R1, IP samples by using GFP antibody. The amplified DNA abundance using PC as template was set as 1.0. The experiments were repeated three times and data are presented as means ± SE (n = 3). F. A hypothetic model how PI4Kγ5-ANAC078 module regulates auxin synthesis and leaf development. ANAC078 is an important negative regulator of auxin synthesis by down-regulating auxin-synthesis genes YUC2 and YUC4, or up-regulating auxin-metabolism gene, GH3.5. Proteolysis of transmembrane region and C-terminal of ANAC078 results in the translocation of ANAC078(-C) into nucleus, while interaction with PI4Kγ5 is crucial for ANAC078 proteolysis, possibly through phosphorylation, to maintain the normal ANAC078 cleavage and proper auxin levels in rosette leaves, leading to the fine-controlled leaf margin development and morphogenesis (upper panel). In pi4kγ5–1 mutant, defective interaction with PI4Kγ5 results in the suppressed ANAC078 cleavage, possible due to the reduced phosphorylation, and hence enhanced auxin synthesis, which leads to the promoted cell proliferation at leaf teeth and highly serrated rosette leaves (bottom panel). This provides a complex regulation of in situ auxin synthesis and leaf development.