Fig. 4: MYB3R4 activates CSLD5 expression in dividing cells.

a A schematic diagram of the CSLD5 genomic region, with colored circles indicating the positions of the MSA elements. “I” and “II” are the regions analysed by ChIPqPCR. b Genome browser tracks of MYB3R1 and MYB3R4 ChIP-seq coverage at CSLD5 genomic region. c ChIP-qPCR analysis to confirm MYB3R1 and MYB3R4 binding to the promoter regions of CSLD5. Data were presented as mean values  ±  SD of three independent replicates. d, e Electrophoretic mobility shift assay (EMSA) showing that MBP-MYB3R1 and MBP-MYB3R4 could bind to the Cy5-labeled DNA probes. f Dual-luciferase assay demonstrating transcriptional activation of CSLD5 by MYB3R1 and MYB3R4. n = 6 biological replicates, with error bars representing mean ± s.e.m. g Comparison of CSLD5 transcript levels in WT and myb3r1 myb3r4 SAMs by qRT-PCR. Data represent means ± SD, n = 3 biological replicates. h RNA FISH to compare CSLD5 mRNA expression in WT and myb3r1myb3r4 SAMs. i Expression patterns of pCSLD5::GFP-CSLD5 at the SAM. Scale bars, 20 µm (left two panels), 5 µm (right panel). j Time-lapse imaging of GFP-CSLD5 during cell division. Scale bar, 5 µm. k Partial co-localization of GFP-CSLD5 with VHA-RFP. Pearson correlation coefficient (PCC) = 0.895 ± 0.006 (n = 60 ROIs from three individual SAMs). Scale bar, 5 µm. l, m RNA FISH to compare HIS4 (l, top panel) and CYCB1;2 (l, bottom panel) mRNA expression in wild-type and csld5 SAMs. Quantification of RNA FISH signals is shown in (m). Scale bar, 50 μm. n A schematic illustration of molecular events during cell division. o A proposed model of feedback between cell wall synthesis and cell cycle progression. Mutation of CSLD5 resulted in delayed cell plate formation and softer cell walls, which feed back to the cell cycle machinery, leading to cell cycle arrest at early mitosis. Boxplots (h, m) indicate median (box center line), 25, 75% (box), and the whiskers extend to the minima and maxima. Unpaired two-tailed Student’s t-test (c, f, g, m), ^****P < 0.0001. Source data are provided as a Source Data file.