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Link to original content: https://pubmed.ncbi.nlm.nih.gov/19717465
Multiple feedback loops through cytokinin signaling control stem cell number within the Arabidopsis shoot meristem - PubMed Skip to main page content
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. 2009 Sep 22;106(38):16529-34.
doi: 10.1073/pnas.0908122106. Epub 2009 Aug 26.

Multiple feedback loops through cytokinin signaling control stem cell number within the Arabidopsis shoot meristem

Sean P Gordon  1 , Vijay S Chickarmane, Carolyn Ohno, Elliot M Meyerowitz
Affiliations

Multiple feedback loops through cytokinin signaling control stem cell number within the Arabidopsis shoot meristem

Sean P Gordon et al. Proc Natl Acad Sci U S A. .

Abstract

A central unanswered question in stem cell biology, both in plants and in animals, is how the spatial organization of stem cell niches are maintained as cells move through them. We address this question for the shoot apical meristem (SAM) which harbors pluripotent stem cells responsible for growth of above-ground tissues in flowering plants. We find that localized perception of the plant hormone cytokinin establishes a spatial domain in which cell fate is respecified through induction of the master regulator WUSCHEL as cells are displaced during growth. Cytokinin-induced WUSCHEL expression occurs through both CLAVATA-dependent and CLAVATA-independent pathways. Computational analysis shows that feedback between cytokinin response and genetic regulators predicts their relative patterning, which we confirm experimentally. Our results also may explain how increasing cytokinin concentration leads to the first steps in reestablishing the shoot stem cell niche in vitro.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
CLV-independent regulation of WUS by cytokinin. (A) WUS/CLV interactions in a cross section of the SAM. WUS expression in RM cells promotes stem cell fate in overlying cells. Stem cells in turn secrete diffusible CLV3 ligand that binds to its receptor CLV1 in the RM leading to WUS downregulation. P labels organ primordia. (B) Hypothetical circuit in which cytokinin (CK) treatment leads to higher levels of WUS through suppression of CLV1. (C and D) CLV1 (C), or ARR5 (D) transcript after 24 h of mock or cytokinin treatment. (E and F) relative WUS transcript in wild-type, clv1–11, and clv3–2 seedlings after mock or cytokinin treatment for (E) 4 h, or (F) 24 h (P < 0.05). (G) Cytokinin induction of WUS for 4 h in absence (−) or presence (+) of 30 min cycloheximide (10 μM) pretreatment. (H) Enhancement of carpel number in cytokinin treated clv1 and clv3–2 mutants compared to wild type (two-way ANOVA, F = 81, P < 0.0001). qRT-PCR error bars indicate SEM from three biological replicates.
Fig. 2.
Fig. 2.
Feedback between cytokinin signaling and the WUS/CLV circuit influences patterning of gene expression. (A) Relative CLV1, ARR5, and WUS RNA transcript at varying cytokinin concentrations. (B–D) pTCS::GFP expression in the SAM (B), early flower bud (C), or cross section of SAM (D). (E) (1) Cytokinin activates WUS through suppression of CLV1 or (2) a CLV-independent pathway or (3) through both mechanisms. (F) Predicted steady state WUS levels at varying levels of cytokinin signaling for circuits (1, green line), (2, red line), and (3, blue line). (G) Steady state WUS levels for network (3) including CLV negative feedback compared to network 3 lacking the CLV pathway (3*). (H–J) spatial distribution of phosphorylated B-type ARR (H), WUS (I), or Type-A ARR (J) for network 2E (3). Axis of the plots correspond to a section of the meristem using arbitrary units in which 0,0 marks the center of the meristem. Error bars indicate SEM from two biological replicates. (Scale bars, 20 mm.)
Fig. 3.
Fig. 3.
AHK4 and WUS expression correlate in individual cells where ARR5 is suppressed. (A–C) ARR5 (green) reporter down regulation within the WUS domain (red) and organ primordia (AHP6 domain, Fig. S3). Inset in (C) plots ARR5 and WUS intensity (yellow line indicates profile). (D–F) Cytokinin receptor (AHK4, green) and WUS reporter (red) overlap within the SAM (center) or floral meristems (peripheral). Cross sections displayed below. (G) AHK4 and WUS overlap in single cells. (H) pixel intensity of AHK4 (x axis) and WUS (y axis) reporters in wild-type flowers (correlation coefficient R = 0.79, upward trend indicates positive correlation). (I) WUS (red) and AHK4 (green) in cytokinin treated clv3–2 SAM and floral meristems (arrows) compared to untreated clv3–2 mutants (J–L). [Scale bars, 20 μm except for 10 μm in (G) and 100 μm in (I).]
Fig. 4.
Fig. 4.
Cytokinin regulates domain of cytokinin signaling output, WUS and CLV3 expression. (A–D) live imaging of WUS reporter (green) before (A and B) and after 12 h of cytokinin treatment (1mM BAP) (C and D). Numbering in (A–D) registers cells in (A and B) to the same cells in (C and D) after 12 h. Arrow marks floral meristem. (E) CLV3 reporter (green) in plants after 24 h of mock treatment (n = 5) or (F) cytokinin treatment (n = 5). (G) pTCS::GFP reporter (green) after 24 h of mock treatment (n = 5) as compared to (H) 24 h of cytokinin treatment (n = 5). Membranes are marked with FM4–64 dye (A–D and G and H) or 29–1 membrane YFP marker (10) (E and F). (Scale bars, 50 μm.)
Fig. 5.
Fig. 5.
Cytokinin regulates WUS expression through an AHK2/AHK4 dependent mechanism while CLV1 suppression has no requirement for individual receptors. (A) Relative WUS or (B) CLV1 transcript levels in wild-type and individual cytokinin receptor mutants after 24 h of mock treatment or cytokinin treatment. (C) Cytokinin-induced clv mutant-like carpel number phenotypes in wild type (COL), ahk2–2, and ahk3–3 mutants. (D) Hypothetical positive feedback between apical stem cells and RM cells. Apical stem cells produce active cytokinins (CKs) perceived by RM cells expressing sufficient cytokinin receptor to activate WUS expression. WUS, in turn, promotes stem cell fate in apical cells. Negative feedback from the CLV pathway is also shown.
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