Fig 1.
UPB1 regulates MYB50 expression at the elongation zone.
(A) UPB1, MYB50, and CYCB1;1 expression in both of the meristematic and elongation zones in roots of 7-day old seedlings of Columbia (Col-0; gray) and upb1-1 (pink) as measured by RT-qPCR (n = 5). MZ and EZ indicate the meristematic and elongation zone, respectively. Bars show mean ± SD. Statistically significant differences were determined using Student’s t-test by comparing Columbia and upb1-1 in each zone (*** p < 0.001; ** p < 0.01). p value: comparison of UPB1 expression in MZ of Columbia and upb1-1, p = 0.002; comparison of UPB1 expression in EZ of Columbia and upb1-1, p < 0.001; comparison of MYB50 expression in EZ of Columbia and upb1-1, p = 0.0096. (B) UPB1 and MYB50 expression analysis of roots of 6-day old seedlings in pXVE::YFP-UPB1/Columbia with and without 5 μM estradiol (Est) treatment for 24 h measured by RT-qPCR (n = 5). Statistically significant differences were determined using Student’s t-test compared with and without estradiol treatment (*** p < 0.001; * p < 0.05). p value: comparison of UPB1 expression, p < 0.001; comparison of MYB50 expression, p = 0.016. (C) Root length in 4-day-old seedlings treated with 5 μM estradiol within the agar medium. Root length was measured every 30 min using time-lapse imaging (n = 5, means ± SD). (D) Root length in 4-day-old seedlings grown vertically on agar medium containing 5 μM estradiol. Root length was measured every 24 h for 72 h (Columbia, n = 38; pXVE::YFP-UPB1, n = 25, means ± SD). To assess statistical significance, we employed Student’s t-test for comparisons between Columbia and transgenic at each time (*** p < 0.001).
Fig 2.
MYB50 expression pattern in the primary root and the multi-color fluorescent protein expression of CFP-UPB1 and MYB50-GFP.
(A) Confocal microscopic images of primary root tip of 7-day old seedlings. White arrow heads indicate the nuclei with apparent GFP fluorescence. Scale bars, 100 μm. (B) Timepoint images every 60 min in the visualization of transcriptional repression of MYB50 by UPB1 in one nucleus. 6-day-old seedlings were stained by propidium iodide (PI) and CFP-UPB1 and each MYB50-GFP signal was independently detected. Apparent nuclei exhibiting detectable CFP or GFP fluorescence were visualized and enlarged. Left panels: merged images of CFP, GFP, and PI, Middle panels: CFP channel, Right panels: GFP channels. Scale bars, 5 μm. (C) Quantification of CFP-UPB1 and MYB50-GFP intensity after estradiol treatment (solid lines) and no-estradiol treatment (dashed lines) every 20 min. Each signal intensity was measured every 20 min using time-lapse imaging (n = 4: two nuclei from each of the two roots, means ± SD).
Fig 3.
Root phenotype of MYB50 induced-overexpressor (pXVE::YFP-MYB50).
(A) MYB50 expression after 24 h treatment of 6-day-old seedlings with 5 μM estradiol as measured by RT-qPCR (n = 3, mean ± SD). Significant differences were determined using Student’s t-test compared to Columbia (*** p < 0.001; ** p < 0.01). p-value: comparison of Columbia and #1, p < 0.001; comparison of Columbia and #2, p = 0.0052. (B) Root length increase in 4-day-old seedlings of Columbia (Col-0) and pXVE::YFP-MYB50/Columbia #1 treated with 5 μM estradiol within the agar medium. Root length was measured every 30 min using time-lapse imaging (n = 5, means ± SD). (C) Root length in 4-day-old seedlings grown vertically on agar medium containing 5 μM estradiol. Root length was measured every 24 h for 72 h (Columbia, n = 38; pXVE::YFP-MYB50 #1, n = 39; pXVE::YFP-MYB50 #2, n = 25; means ± SD). Statistically significant differences were determined using Student’s t-test by comparing Columbia and transgenic at each time (*** p < 0.001). (D) and (F) Confocal microscope images of 6-day-old roots treated with 5 μM estradiol (Est) for 24 h or untreated. Roots stained with propidium iodide. (D) The differentiation zone at a distance of 1 to 2 mm from each root tip. White asterisks indicate cortex cells with cell length measured. Scale bar, 100 μm. (E) Length of mature cortex cells. The black bar indicates the median. Mature cell length quantified for 10 to 15 roots, with approximately 3 to 10 cells per root. Measured cell numbers: Columbia—estradiol = 149, Columbia + estradiol = 141, pXVE::YFP-MYB50 #1—estradiol = 101, #1 + estradiol = 209, pXVE::YFP-MYB50 #2—estradiol = 130 and #2 + estradiol = 150. Statistically significant differences between samples determined using the Tukey’s honestly significant difference test (p < 0.001). (F) As (D) but showing the meristem. White arrowheads indicate the apparent end of the meristematic zone. Blue arrowheads indicate quiescent center cells. Scale bar, 100 μm. (G) Cortex cell number in the meristematic zone of 6-day-old roots treated with 5 μM estradiol (Est) or untreated. Counted root numbers: Columbia—estradiol = 41, Columbia + estradiol = 44, pXVE::YFP-MYB50 #1—estradiol = 42, #1 + estradiol = 53, pXVE::YFP-MYB50 #2—estradiol = 30, #2 + estradiol = 38. Statistically significant differences between samples determined using the Tukey’s honestly significant difference test (p < 0.001).
Fig 4.
(A) Biological processes in gene ontology (GO) enrichment analysis of upregulated genes in the MYB50 induced-overexpressor (FDR < 0.001). The color and size of each point represents the -log10 (FDR) value and the number of genes, respectively. (B) Common genes between upregulated genes in each timepoint of RNA-seq and MYB50-bound genes by ChIP-seq. (C) Common genes between downregulated genes in each timepoint of RNA-seq and MYB50-bound genes by ChIP-seq. (D) Common genes between MYB50- and UPB1-regulated genes. MYB50 RNA-seq (upregulation): Upregulated genes (368 genes) in at least a one-time point of estradiol induction of pXVE::YFP-MYB50 in RNA-seq. MYB50 ChIP-seq: Common genes (50 genes) between upregulated genes by MYB50 and MYB50-bound genes in pXVE::YFP-MYB50. UPB1 positive regulation: Positively regulated genes (1,208) by UPB1. UPB1 negative regulation: Negatively regulated genes (1,149) by UPB1. UPB1 data were retrieved from Tsukagoshi et al., 2010 [7].
Fig 5.
Root phenotype of PMEI8 induced-overexpressor (pXVE::YFP-PMEI8).
(A) MYB50 bound to the PMEI8 promoter region in ChIP-seq. “Blue boxes” indicate ORFs (thick box: exon; thin box: intron) and black arrows beneath each ORF represents the direction of transcription. (B) PMEI8 expression in 7-day-old roots after 24 h of 5 μM estradiol treatment as measured using RT-qPCR (n = 3, mean ± SD). Statistically significant differences were determined using Student’s t-test compared to Columbia (** p < 0.01). p value: comparison of Columbia and pXVE::YFP-MYB50 #1, p = 0.00131; comparison of Columbia and pXVE::YFP-MYB50 #2, p = 0.00702. (C) PMEI8 expression in 6-day-old roots after 24 h of 5 μM estradiol treatment as measured using RT-qPCR (n = 3, mean ± SD). Statistically significant difference was determined using Student’s t-test compared to Columbia (*** p < 0.001). (D) Root length in 4-day-old seedlings treated with 5 μM estradiol within the agar medium. Root length was measured every 30 min for 24 h using time-lapse imaging (n = 5, means ± SD). (E) Root length in 4-day-old seedlings grown vertically on agar medium containing 5 μM estradiol. Root length was measured every 24 h for 72 h (Columbia, n = 53; pXVE::YFP-PMEI8 #1, n = 33; pXVE::YFP-PMEI8 #2, n = 32; means ± SD). Statistically significant differences were determined using Student’s t-test by comparing Columbia and pXVE::YFP-PMEI8 at each time (*** p < 0.001). (F) and (H) Confocal microscope images of 6-day-old seedlings treated with 5 μM estradiol (Est) for 24 h or untreated. Roots were stained with propidium iodide (PI). (F) The differentiation zone is at a distance of 1 to 2 mm from each root tip. White asterisks indicate cortex cells with cell length measured. Scale bar, 100 μm. (G) Length of mature cortex cells in 7-day-old seedlings. Mature cell length was quantified for 10–15 roots, with approximately 3–10 cells per root. The black bar indicates the median. Measured cell numbers: Columbia -estradiol = 149; Columbia +estradiol = 141; pXVE::YFP-PMEI8 #1 -estradiol = 81; #1 +estradiol = 120; pXVE::YFP-PMEI8 #2 -estradiol = 131; #2 +estradiol = 119. Statistically significant differences between samples determined using the Tukey’s honestly significant difference test (*** p < 0.001). (H) As (F) but showing the meristem. White arrowheads indicate the apparent end of the meristematic zone. Blue arrowheads indicate the quiescent center cells (QC). Scale bar, 100 μm. (I) Cortex cell number in the meristematic zone of 6-day-old seedlings treated with 5 μM estradiol (Est) or untreated. Counted root numbers: Columbia -estradiol = 41; Columbia +estradiol = 44; pXVE::YFP-PMEI8 #1 -estradiol = 37; #1 +estradiol = 42; pXVE::YFP-PMEI8 #2 -estradiol = 39; #2 +estradiol = 42. Statistically significant differences between samples determined using the Tukey’s honestly significant difference test (*** p < 0.001).
Fig 6.
A schematic model of gene regulatory network under UPB1 and MYB50 control.
Black arrows and “T”s indicate transcriptionally direct target genes. The outline arrow and “T”s indicate an effect on the root phenotype. (MZ): Meristematic zone. (TZ): Transition zone. (EZ): Elongation zone. UPB1 directly represses PER57 in the TZ and negatively regulates the meristem size [7]. In the EZ, UPB1 directly represses MYB50 expression. The root phenotypic analysis using pXVE::YFP-MYB50 revealed that MYB50 negatively regulates the meristem size through a pathway separate from ROS regulation, whereas the cause remains unknown. However, MYB50 was expressed in the EZ close to the differentiation zone and directly activated PMEI8 expression. MYB50 regulatory network and PMEI8 inhibit cell elongation as pXVE::YFP-MYB50 and pXVE::YFP-PMEI8 shortened mature cell length. The MYB50-regulated transcriptional network includes several cell wall-associated genes, which may coregulate with PMEI8 to exert some coregulation.