The cold-induced factor CBF3 mediates root stem cell activity, regeneration, and developmental responses to cold

Abstract

Plant growth and development involve the specification and regeneration of stem cell niches (SCNs). Although plants are exposed to disparate environmental conditions, how environmental cues affect developmental programs and stem cells is not well understood. Root stem cells are accommodated in meristems in SCNs around the quiescent center (QC), which maintains their activity. Using a combination of genetics and confocal microscopy to trace morphological defects and correlate them with changes in gene expression and protein levels, we show that the cold-induced transcription factor (TF) C-REPEAT BINDING FACTOR 3 (CBF3), which has previously been associated with cold acclimation, regulates root development, stem cell activity, and regeneration. CBF3 is integrated into the SHORT-ROOT (SHR) regulatory network, forming a feedback loop that maintains SHR expression. CBF3 is primarily expressed in the root endodermis, whereas the CBF3 protein is localized to other meristematic tissues, including root SCNs. Complementation of cbf3-1 using a wild-type CBF3 gene and a CBF3 fusion with reduced mobility show that CBF3 movement capacity is required for SCN patterning and regulates root growth. Notably, cold induces CBF3, affecting QC activity. Furthermore, exposure to moderate cold around 10°C-12°C promotes root regeneration and QC respecification in a CBF3-dependent manner during the recuperation period. By contrast, CBF3 does not appear to regulate stem cell survival, which has been associated with recuperation from more acute cold (∼4°C). We propose a role for CBF3 in mediating the molecular interrelationships among the cold response, stem cell activity, and development.

Keywords: cell fate; low temperature; organ patterning; protein movement; regeneration; stem cells.

Figure 1

The transcription factor C-REPEAT BINDING FACTOR 3 (CBF3) is regulated by SHORT-ROOT, shows enriched expression in the root endodermis, and regulates root system architecture. (A) Venn diagram comparing genes with enriched expression in the ground tissue (endodermis and cortex) with those in the SHR network. (B) Real-time PCR analysis of CBF3 expression in dissected root apical meristems (RAMs) of the WT and the shr mutant at 6 days post imbibition (dpi) under standard developmental conditions. Error bars denote SD of three biological replicates. ∗p < 0.05, one-way ANOVA. (C)CBF3 promoter drives expression to the endodermis (orange arrowheads) of the RAM and the lateral root meristem (LR-RAM), as well as to the QC of the LR-RAM (white arrowheads). Note that the fluorescent signal is scaled to depict comparable intensity values in the RAM and LR-RAM. E, epidermis; gt, ground tissue; v, vasculature. (D) Quantification of CBF3 expression (pCBF3::NLS:3X-mCherry) in RAMs and LR-RAMs. LR-RAMs correspond to the first and second emerged lateral roots. MIV/pixel, mean intensity value per pixel. (E) Seedlings of the WT and the loss-of-function mutant cbf3-1 at 6 dpi under standard developmental conditions. (F) Primary root length quantification at 6 dpi. (G) WT and cbf3-1 seedlings at 8 dpi showing lateral root outgrowth. (H) Lateral root length quantification of the first and second emerged lateral roots at 8 dpi. (I) Confocal images of WT and cbf3-1 RAMs at 6 dpi. Cell walls (in white) were stained with propidium iodide. (J and K) Quantification of RAM and LR-RAM (J) size and (K) cell number. RAMs and LR-RAMs were quantified at 6 and 8 dpi, respectively. LR-RAMs correspond to the first and second emerged lateral roots. In (D), (F), (H), (J), and (K), n ≥ 10. Error bars denote SD. In (D), (F), and (H), ∗p < 0.001, one-way ANOVA. In (J) and (K), ∗p < 0.001 by general linear model (GLM) and the least significant difference (LSD) post hoc test. Scale bars, 25 μm (C), 1 cm (E and G), and 100 μm (I).

Figure 2

CBF3 movement from the endodermis and QC regulates root development. (A and D) Confocal images showing CBF3-YFP protein localization under pCBF3 (in yellow) and the expression driven by pCBF3 (in red) in (A) the LR-RAM at 8 dpi or (D) the RAM at 6 dpi. Cell walls (in white) were stained with propidium iodide. Scale bars, 25 μm. (B and E) Confocal images showing the localization of the CBF3 protein fused to three fluorescent mCherry proteins (CBF3-3X-mCherry, in purple) and the expression driven by pCBF3 (in red) in (B) the LR-RAM at 8 dpi or (E) the RAM at 6 dpi. LR-RAMs correspond to the first emerged lateral roots. Note that CBF3-3X-mCherry is confined to the endodermis (orange arrowheads) and the QC (white arrowheads). The fluorescent signal is scaled to depict comparable intensity values. Scale bars, 25 μm. (C and F) Lateral (C) and primary (F) root length quantification of WT, cbf3-1, and cbf3-1 expressing CBF3-YFP or CBF3-3X-mCherry under pCBF3. First and second emerged lateral roots were quantified at 8 dpi. n ≥ 15. Error bars denote SD. Different letters denote p < 0.001 by GLM and LSD post hoc test.

Figure 3

CBF3 regulates SHR levels in roots. (A) Real-time PCR analysis (three biological replicates) of SHR expression in the RAM of the WT and cbf3-1 at 6 dpi. (B, D, F, H, and J) Graphs comparing (B) SHR expression, (D) SHR levels, (F) SCR levels, and (H) EN7 expression in the RAM of the WT and cbf3-1 at 6 dpi. (B, F, and H) n ≥ 10; (D) n ≥ 8. (J) SHR levels in the QC of the WT, cbf3-1, and cbf3-1 pCBF3::CBF3:3x-mCherry at 6 dpi. n ≥ 8. MIV/pixel, mean intensity value per pixel. In (A), (B), (D), (F), (H), and (J), error bars denote SD. ∗p < 0.001, one-way ANOVA (A, B, D, F, and H) or GLM and LSD post hoc test (H). (C, E, G, and I) Confocal images showing accumulation of (C) SHR (pSHR::SHR:GFP), (E) SCR (pSCR::SCR:YFP), and (G) EN7 (pEN7::NLS-3X-YFP) in the RAM of the WT and cbf3-1 at 6 dpi. (I) SHR (pSHR::SHR:GFP) in the stem cell niche (SCN) of the WT, cbf3-1, cbf3-1 pCBF3::CBF3:1x-mCherry, and cbf3-1 pCBF3::CBF3:3x-mCherry at 6 dpi. Cell walls (in white) were stained with propidium iodide. Scale bars, 25 μm.

Figure 4

CBF3 regulates QC organization non-cell autonomously. (A) Confocal images of the RAM (SCN area) of the WT, cbf3-1, cbf3-1 pCBF3::CBF3:YFP, and cbf3-1 pCBF3::CBF3:3X-mCherry at 6 dpi. The dashed yellow line delimits the QC. (B) Percentage of disorganized QC observed in WT, cbf3-1, cbf3-1 pCBF3::CBF3:1X-mCherry, and cbf3-1 pCBF3::NLS:3X-mCherry at 6 dpi. n ≥ 30. (C and D) Confocal images (C) and quantification (D) of WOX5 expression (pWOX5::ER:GFP, in yellow) in the RAM of the WT, cbf3-1, cbf3-1 pCBF3::CBF3:1X-mCherry, and cbf3-1 pCBF3::NLS:3X-mCherry at 6 dpi. n ≥ 17. White arrows: cells showing very low WOX5 expression. MIV/pixel, mean intensity value per pixel. In (A) and (C), cell walls (in white) were stained with propidium iodide. In (B) and (D), error bars denote SD. ∗p < 0.01 by GLM and LSD post hoc test. Scale bars, 25 μm.

Figure 5

CBF3 is involved in QC re-establishment after injury. (A) Time-course confocal imaging of WOX5 expression (pWOX5::ER:GFP, in yellow) upon laser ablation of cells above the QC at 5 dpi and the subsequent recovery period up to 72 h post ablation (hpa). (B–D) Confocal images showing WOX5 expression at (B) 24 hpa, (C) 48 hpa, and (D) 72 hpa in WT, cbf3-1, and cbf3-1 pCBF3::CBF3:3X-mCherry roots. (E) Graph showing the number of cells with relative maxima of WOX5 expression (pWOX5::ER:GFP) in WT, cbf3-1, and cbf3-1 pCBF3::CBF3:3X-mCherry roots at 0, 24, 48, and 72 hpa.In (A)–(D), cell walls (in white) were stained with propidium iodide. Two independent biological replicates, n ≥ 12. In (E), error bars denote SD. ∗p < 0.001 by GLM and LSD post hoc test. Scale bars, 25 μm.

Figure 6

SCN regulators are induced by moderate cold. (A and B) Confocal images (A) and quantification (B) of SHR-GFP accumulation (pSHR::SHR:GFP) at 6 dpi in the RAM of the WT and cbf3-1. (C and D) Confocal images (C) and quantification (D) of WOX5 expression (pWOX5::ER:GFP) at 6 dpi in the RAM of the WT and cbf3-1. In (A)–(D), plants were grown under standard conditions for 3 days and transferred to control (22°C) or cold conditions (10°C) for 72 h. n ≥ 10. In (B) and (D), error bars denote SD. ∗p < 0.01 by GLM and LSD post hoc test. MIV/pixel, mean intensity value per pixel.

Figure 7

Regeneration is promoted by moderate cold. (A) Time-course confocal imaging of SHR-GFP accumulation (pSHR::SHR:GFP) upon laser ablation of cells above the QC at 5 dpi and the subsequent recovery period up to 72 hpa. White boxes: 2× amplification of the distal domain of SHR-GFP. (B) Confocal images showing SHR-GFP accumulation upon laser ablation of cells above the QC in WT and cbf3-1 plants at 48 hpa following a 12-h pretreatment at 10°C. (C) Graph showing the percentage of WT and cbf3-1 plants at 0, 24, 48, and 72 hpa with distal nuclear localization of SHR-GFP upon laser ablation of cells above the QC following a 12-h pretreatment at 10°C. (D) Confocal images showing WOX5 expression (pWOX5::ER:GFP) upon laser ablation of cells above the QC in WT and cbf3-1 plants at 48 hpa following a 12-h pretreatment at 10°C. (E) Graph showing the number of cells with relative maxima of WOX5 expression in WT and cbf3-1 roots at 0, 24, 48, and 72 hpa upon laser ablation of cells above the QC following a 12-h pretreatment at 10°C. For (C) and (E), two independent biological replicates, n ≥ 10. Error bars denote SD. ∗p < 0.001 by GLM and LSD post hoc test. Scale bars, 25 μm.