Paracrine brassinosteroid signaling at the stem cell niche controls cellular regeneration

Abstract

Stem cell regeneration is crucial for both cell turnover and tissue healing in multicellular organisms. In Arabidopsis roots, a reduced group of cells known as the quiescent center (QC) act as a cell reservoir for surrounding stem cells during both normal growth and in response to external damage. Although cells of the QC have a very low mitotic activity, plant hormones such as brassinosteroids (BRs) can promote QC divisions. Here, we used a tissue-specific strategy to investigate the spatial signaling requirements of BR-mediated QC divisions. We generated stem cell niche-specific receptor knockout lines by placing an artificial microRNA against BRI1 (BRASSINOSTEROID INSENSITIVE 1) under the control of the QC-specific promoter WOX5. Additionally, QC-specific knock-in lines for BRI1 and its downstream transcription factor BES1 (BRI1-EMS-SUPPRESOR1) were also created using the WOX5 promoter. By analyzing the roots of these lines, we show that BES1-mediated signaling cell-autonomously promotes QC divisions, that BRI1 is essential for sensing nearby inputs and triggering QC divisions and that DNA damage promotes BR-dependent paracrine signaling in the stem cell niche as a prerequisite to stem cell replenishment.

Keywords: Brassinosteroid; Cell division; DNA damage; Paracrine; Quiescent center; Stem cell.

Fig. 1.

The stem cell niche of Arabidopsis roots and QC-specific expression of BR pathway components. (A) A stereotypical Arabidopsis WT primary root under confocal microscopy. The root stem cell niche is highlighted in color. (B) Detailed representation of the root stem cell niche. (C–H) Confocal images of 6-day-old WT and mutant Arabidopsis roots in control conditions. Green represents YFP-tagged pathway components. Red is PI counterstaining. Insets show the YFP channels at higher magnification. Scale bar: 50 µm.

Fig. 2.

The BR-regulated transcription factor BES1 promotes QC division in a cell-autonomous manner. (A–F) Confocal images of fixed 6-day-old WT and mutant Arabidopsis roots in control conditions. (G–L) Root anatomy of 6-day-old seedlings grown in medium supplemented with 4 nM BL. Arrows indicate the number of QC cell layers identified. (M) Quantification of QC division rate. ND, QC non-divided; PD, QC partially divided; D, QC totally divided. Asterisks indicate statistically significant differences due to genotype, comparing against WT either in control or 4 nM BL conditions. Frequencies in QC divisions were assessed with a two-sided Fisher's test. Values for all pairwise comparisons are provided in Table S1. Data are generated from three independent replicates (n>21). *P<0.05, **P<0.01, ***P<0.005. Scale bar: 50 µm.

Fig. 3.

The pWOX5:BRI1-amiR construct targets BRI1 and downregulates its transcription in the root stem cell microenvironment. Confocal images of 6-day-old Arabidopsis roots. (A,B) Genetic crosses between pWOX5:BRI1-amiR and pSCR:BRI1-GFP lines reveal that BRI1 is knocked down in the stem cell microenvironment. (C,D) Genetic crosses between pWOX5:BRI1-YFP and pWOX5:BRI1-amiR lines show that the amiRNA completely depletes BRI1 around the QC domain. (E–H) Genetic crosses of pWOX5:BRI1-amiR lines with pBRL1:BRL1-GFP and pBRL3:BRL3-GFP lines. Insets show the GFP channel separately. All crosses are F3 double homozygous plants. Scale bar: 50 µm.

Fig. 4.

BRI1 in the stem cells niche is required to promote QC divisions. (A,B) Confocal images of 6-day-old WT Arabidopsis roots grown in either control conditions or 4 nM BL show the change in QC division and organization. (C–F) pWOX5:BRI1-amiR transgenic lines grown in control conditions or in medium supplemented with 4 nM BL. Arrows indicate the number of QC cell layers identified. (G) Quantification of the QC divisions of WT and pWOX5:BRI1-amiR plants. ND, QC non-divided; PD, QC partially divided; D, QC totally divided. Asterisks indicate statistically significant differences due to genotype, comparing against WT either in control or 4 nM BL conditions (***P<0.005). Frequencies in division occurrence were assessed with a two-sided Fisher's test. Values for all pairwise comparisons are provided in Table 3. Data generated from three independent replicates (n>39). (H) Root growth dynamics of WT and pWOX5:BRI1-amiR lines. Asterisks denote significant differences with respect to the WT in a two-tailed t-test (*P<0.05). Data are generated from three independent replicates (n>46). Scale bar: 50 µm.

Fig. 5.

pWOX5:BRI1-amiR seedlings exhibit normal meristem divisions. Confocal images of fixed and EdU-stained 6-day-old Arabidopsis roots. (A–C) WT, pWOX5:BRI1-amiR#2 and pWOX5:BRI1-amiR#3 lines grown in control conditions. (D) bri1-116 line grown in control conditions as a negative control for QC division. (E–G) WT, pWOX5:BRI1-amiR#2, and pWOX5:BRI1-amiR#3 lines grown for 4 days in control conditions and 2 days in medium supplemented with 4 nM BL. (H) pWOX5:bes1-D-YFP line grown in control conditions as a positive control for QC division. Arrows indicate the number of QC cell layers identified. Scale bar: 50 µm.

Fig. 6.

BR receptors in the stem cell niche modulate QC divisions upon DNA damage. (A–D) Confocal images of 5-day-old seedlings treated with bleomycin for 24 h. (E–H) Confocal images of 5-day-old seedlings subjected to 24 h of bleomycin treatment and a subsequent 24 h of recovery. (I) The proportion of roots showing cell death in the root apex after 24 h of bleomycin treatment. HD, hard damage; MD, mild damage; ND, no damage. Asterisks indicate statistically significant differences respect to WT (***P<0.005). Differences in the proportion of damaged roots were assessed with a two-sided Fisher's test. Values for all pairwise comparisons are provided in Table S4. Data are generated from three independent replicates (n>25). (J) Quantification of QC divisions after 24 h of bleomycin treatment and 24 additional hours of recovery. ND, QC non-divided; PD, QC partially divided; D, QC totally divided. Asterisks indicate statistically significant differences with respect to WT (**P<0.01, ***P<0.005). Differences in division frequencies were assessed with a two-sided Fisher's test. Values for all pairwise comparisons are provided in Table S5. Data are generated from three independent replicates (n>24). Scale bar: 50 µm.

Fig. 7.

Working model: BR concentration as a limiting factor for QC divisions. In order to promote QC divisions when needed, a threshold concentration of BRs has to be reached in the root apical meristem. Upon reaching this threshold, the signal is transduced via BRI1 with enough strength to promote BES1 dephosphorylation. Dephosphorylated BES1, in turn, inhibits BRAVO and triggers QC division.