A SCARECROW-RETINOBLASTOMA protein network controls protective quiescence in the Arabidopsis root stem cell organizer

Abstract

Quiescent long-term somatic stem cells reside in plant and animal stem cell niches. Within the Arabidopsis root stem cell population, the Quiescent Centre (QC), which contains slowly dividing cells, maintains surrounding short-term stem cells and may act as a long-term reservoir for stem cells. The RETINOBLASTOMA-RELATED (RBR) protein cell-autonomously reinforces mitotic quiescence in the QC. RBR interacts with the stem cell transcription factor SCARECROW (SCR) through an LxCxE motif. Disruption of this interaction by point mutation in SCR or RBR promotes asymmetric divisions in the QC that renew short-term stem cells. Analysis of the in vivo role of quiescence in the root stem cell niche reveals that slow cycling within the QC is not needed for structural integrity of the niche but allows the growing root to cope with DNA damage.

Figure 1. WT QC cells divide infrequently and incorporate into the columella.

(A–D) 5 dpg pSCR::SCR:GFP plants showing S-phase label incorporation by F-ara-EdU for 1–4 dat. DAPI is shown in blue, F-ara-EdU staining is shown in red, and pSCR::SCR:GFP in green. (E–H) Pulse and chase experiment, plants were grown for 5 dpg in F-ara-EdU nucleoside analog, then transferred for 0–4 d into nonsupplemented medium. Arrowheads point to QC. (I) Quantification of entry in S-phase frequency for each cell type. QC, quiescent centre; CEI, cortex-endodermis initial; VSC, vascular stem cell; CSC, columella stem cell; SCD, stem cell daughter. (J–O) Confocal images of root meristems with a single WT BOB clone at 2 (J to L) and 7 (M to O) daHS. The 2 daHS single cell QC clone (arrow head in J) divided and its daughter cell incorporated to the columella region at 7 daHS (M). See also Figure S2.

Figure 2. The AmiGO concept for RBR silencing.

The AmiGO strategy. TS, Target sequence (A). Root apical meristem (RAM) of WT (B), 35S:amiGORBR at 8 (C) and 12 (D) dpg seedlings. Validation of the amiGORBR RNA interference by RT-PCR detection of endogenous RBR transcripts (E) and Western blot analyses for RBR protein levels (F). Mature amiGO-RBR synthesis detected by small-RNA Northern blot (G). The in planta action of amiGO-RBR was revealed by introducing the sensor construct 35S::vYFP:amiGORBR-TS in WT (H) and in the pRCH1::amiGORBR (J) backgrounds. Expression pattern of the pRCH1::GFP marker in the WT background (I) . AmiGORBR expression driven by different promoters generates distinct phenotypes in primary root meristems of 12 dpg seedlings. (K) p35S::amiGORBR causes overproliferation of QC, LRC (arrow heads), and CSC (asterisks) as well as cell death in vascular and columella cells. (L) pRCH1::amiGORBR shows overproliferation of the QC and LRC and cell death and (M) pSCR::amiGORBR shows extra periclinal divisions of the ground tissue (arrow heads) and extra division of the QC (asterisks), while (N) pWOX5::amiGORBR;pWOX5::GFP shows QC divisions that cause an increase in columella layers (asterisks). See also Figures S3, S4, S5.

Figure 3. RBR silencing induces asymmetric cell division of the QC.

Expression patterns of cell-fate markers in stem cell niche of WT (A, C, and E) and pWOX5:amiGORBR (B, D, and F) 5 dpg seedlings. Marker genotypes: cytoplasmic pWOX5:GFP and nuclear pSCR:SCR:YFP (A and B), pSMB::SMB:GFP (C and D), cytoplasmic pWOX5:GFP; nuclear pSCR:SCR:YFP and membrane pACR4:ACR4:GFP (E and F). Arrows in (D) indicate shift in SMB expression, and arrows in (E) and (F) point to plasma membrane-localized ACR4-GFP expression. Full rescue of the QC division in pWOX5:amiGORBR complemented with pWOX5:RBR:YFP and scr-4 complemented with pSCR::SCR:YFP (G, I) and no rescue by LxCxE mutant pWOX5:RBR^N849F-YFP or pSCR::SCR^AxCxA:YFP (H, J). Root phenotype of 12 dpg seedlings of Col-0 WT (K), pWOX5::amiGORBR (L), pWOX5::amiGORBR;pWOX5::RBR:vYFP (M), pWOX5::amiGORBR;pRBR::RBR^N849A:vYFP (N), and pSCR::SCR^AxCxA:YFP, scr-4 (O). Asterisks depict the columellla layers rootwards from the layer in contact with the QC and excluding the detaching distal layers. Production of extra columella stem cell in pSCR::SCR^AxCxA:YFP, scr-4 as shown with ACR4-GFP marker (P). Number of columella stem cell layers by lugol staining in pSCR::SCR^AxCxA:YFP, scr-4 (Q), Col-0 WT (R), scr-4 (S), and pSCR::SCR^AxCxA:YFP shr-2 scr-4 plants (T). See also Figure S6.

Figure 4. A modified RBR-SCR network in the QC.

(A) pCycD6::GUS:GFP expression in QC of pSCR::SCR^AxCxA:YFP, scr-4 background. (B) Down-regulation of RBR in the pSCR domain in pSCR::amiGORBR fails to induce pCycD6 transcription in the QC. (C) pSCR::SCR^AxCxA:YFP does not recover QC division in scr-4, shr-2 background. (D) pCycD6::GUS:GFP expression upon SHR induction in pSHR::SHR:GR, shr-2 background only in ground tissue and not in QC cells. (E) pCycD6::GUS:GFP expression in pSCR::IAAH line, treated with IAAM; note induction in endodermis but not in QC. (F) Auxin accumulation does not induce CycD6 transcription in the QC.

Figure 5. Dividing QC cells are more sensitive to DNA damage.

(A–C) Root stem cell niches at 25 dpg in WT Col 0 (A), pWOX5::amiGORBR (B), and pSCR::SCR^AxCxA:YFP, scr-4 (C) plants. (D) Quantification of QC division frequency upon hydroxyurea treatment. Roots were analyzed for QC divisions after treatment with replication stress inducer HU. Statistical significance (p<0.05) using student's t test for treatments (*) and genotypes (**). (E–G) Effect of zeocin treatment in root growth. Primary root growth after genotoxic stress is impaired in pWOX5::amiGORBR and pSCR::SCR^AxCxA:YFP, scr-4 (E–F) proportionally to the number of dead QC (G); blue column shows number of WT plants analyzed for cell death and red shows pWOX5::amiGORBR. Quantification of root growth after zeocin treatments (H) in WT (blue squares), pWOX5::amiGORBR (red triangles), and pSCR::SCR^AxCxA:YFP, scr-4 (green circles). Student's t tests indicate statistical significance (p<0.05) of all differences between genotypes at 48 and 72 h. See also Figure S7 and Tables 2 and 3.