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Irreversible fate commitment in the Arabidopsis stomatal lineage requires a FAMA and RETINOBLASTOMA-RELATED module.

Matos JL, Lau OS, Hachez C, Cruz-Ramírez A, Scheres B, Bergmann DC - Elife (2014)

Bottom Line: In the Arabidopsis stomatal lineage, a transient self-renewing phase creates precursors that differentiate into one of two epidermal cell types, guard cells or pavement cells.We found that irreversible differentiation of guard cells involves RETINOBLASTOMA-RELATED (RBR) recruitment to regulatory regions of master regulators of stomatal initiation, facilitated through interaction with a terminal stomatal lineage transcription factor, FAMA.Disrupting physical interactions between FAMA and RBR preferentially reveals the role of RBR in enforcing fate commitment over its role in cell-cycle control in this developmental context.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Stanford University, Stanford, United States.

ABSTRACT
The presumed totipotency of plant cells leads to questions about how specific stem cell lineages and terminal fates could be established. In the Arabidopsis stomatal lineage, a transient self-renewing phase creates precursors that differentiate into one of two epidermal cell types, guard cells or pavement cells. We found that irreversible differentiation of guard cells involves RETINOBLASTOMA-RELATED (RBR) recruitment to regulatory regions of master regulators of stomatal initiation, facilitated through interaction with a terminal stomatal lineage transcription factor, FAMA. Disrupting physical interactions between FAMA and RBR preferentially reveals the role of RBR in enforcing fate commitment over its role in cell-cycle control in this developmental context. Analysis of the phenotypes linked to the modulation of FAMA and RBR sheds new light on the way iterative divisions and terminal differentiation are coordinately regulated in a plant stem-cell lineage.

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Generation of transgenic lines expressing Myc-tagged RBR driven by FAMA promoter, in vivo immunoprecipitation of the RBR-Myc protein and phenotypic analysis of the transgenic lines.(A) Western detection of RBR-Myc in transgenic plants harboring the FAMAp:RBR-MYC construct in either Col (lines 1–4) or FAMALGK (lines 1–3) backgrounds. Total protein was extracted from 5-dpg seedlings of the indicated genotypes and probed with α-Myc antibody. Recombinant RBR-Myc has a calculated M.W. of 128.5 kDa. (B) In vivo pull-down assay of stomatal lineage expressed RBR-MYC from transgenic plants. Total soluble protein from Col and FAMAp:RBR-MYC (in Col) was incubated with an anti-Myc antibody. Precipitated samples were probed with the same antibody in Western analysis. IB: Immunoblot, IP: Immunoprecipitation. (C–F) Confirmation that expression of FAMAp:RBR-MYC does not alter guard cell development. (C–F) Confocal images of 6-dpg cotyledons and DIC images of 12-dpg cotyledons of wild type and FAMAp:RBR-MYC plants. Transgenic plants harboring the FAMAp:RBR-MYC construct (E–F) do not exhibit changes in GC divisions (neither fewer, nor more divisions are found) and are indistinguishable from wild type (C–D) at 6 and 12-dpg. Cell outlines (purple) in confocal images were visualized with propidium iodide. All images are at the same magnification. Scale bar, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.03271.014
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fig5s2: Generation of transgenic lines expressing Myc-tagged RBR driven by FAMA promoter, in vivo immunoprecipitation of the RBR-Myc protein and phenotypic analysis of the transgenic lines.(A) Western detection of RBR-Myc in transgenic plants harboring the FAMAp:RBR-MYC construct in either Col (lines 1–4) or FAMALGK (lines 1–3) backgrounds. Total protein was extracted from 5-dpg seedlings of the indicated genotypes and probed with α-Myc antibody. Recombinant RBR-Myc has a calculated M.W. of 128.5 kDa. (B) In vivo pull-down assay of stomatal lineage expressed RBR-MYC from transgenic plants. Total soluble protein from Col and FAMAp:RBR-MYC (in Col) was incubated with an anti-Myc antibody. Precipitated samples were probed with the same antibody in Western analysis. IB: Immunoblot, IP: Immunoprecipitation. (C–F) Confirmation that expression of FAMAp:RBR-MYC does not alter guard cell development. (C–F) Confocal images of 6-dpg cotyledons and DIC images of 12-dpg cotyledons of wild type and FAMAp:RBR-MYC plants. Transgenic plants harboring the FAMAp:RBR-MYC construct (E–F) do not exhibit changes in GC divisions (neither fewer, nor more divisions are found) and are indistinguishable from wild type (C–D) at 6 and 12-dpg. Cell outlines (purple) in confocal images were visualized with propidium iodide. All images are at the same magnification. Scale bar, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.03271.014

Mentions: By what molecular mechanism might this specific loss of commitment take place? Analysis of chromatin states in maturing leaves revealed H3K27me3 (a chromatin mark associated with transcriptional repression) in the genomic regions of SPCH, MUTE, FAMA, EPF1 and other stomatal genes (Lafos et al., 2011), and a recent report showed that manipulation of a member of the POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) can alter developmental regulation of H3K27me3 deposition at SPCH and MUTE loci (Lee et al., 2014b). Animal and plant Rb/RBR proteins can serve as interaction bridges between chromatin modifying enzymes and specific genomic contexts (Burkhart and Sage, 2008; Gutzat et al., 2012) and RBR was previously found to be associated with SPCH regulatory regions via ChIP in whole seedlings (Weimer et al., 2012). Therefore it is plausible that, as the final master regulator bHLH in the stomatal pathway, FAMA (with RBR) ensures terminal differentiation of GCs by facilitating stable repression of early stomatal lineage genes. To test this model, we assayed the co-association of FAMA and RBR with regulatory regions of three key stomatal lineage genes that have significant H3K27me3 coverage (SPCH, FAMA and EPF1) and, as specificity controls, two cell cycle genes known to be RBR targets (Nowack et al., 2012). Because RBR is essential and expressed in nearly all cells, to accurately assay its role as a potential partner of FAMA in the stomatal lineage, we generated a Myc-tagged version of RBR expressed under the FAMA promoter (FAMAp:RBR-MYC). We confirmed that expression of this transgene did not alter stomatal development and that we could effectively immunoprecipitate it from plants (Figure 5—figure supplement 2). In ChIP assays, SPCH, FAMA and EPF1 were all targets of FAMA and of stomatal lineage-expressed RBR (Figure 5B–C and Figure 5—figure supplement 3). Further dissection of the binding regions at SPCH and EPF1 indicates that FAMA and RBR are enriched in the same pattern, suggesting that they bind as part of the same complex (Figure 5—figure supplement 4). We then tested the key prediction of our model–that association of RBR with a stomatal target gene is dependent on its interaction with FAMA. ChIPs of FAMAp:RBR-MYC in a FAMALGK background showed that RBR enrichment at the promoters of SPCH and FAMA, but not of the general RBR target gene PCNA, was significantly reduced (Figure 5D and Figure 5—figure supplement 4), consistent with our recruitment model. RBR enrichment at the promoter of a negative regulator of stomatal development, EPF1, was more variable in our assays, sometimes showing little change (Figure 5D), but failing to associate with RBR in other replicates (Figure 5—figure supplement 3C).


Irreversible fate commitment in the Arabidopsis stomatal lineage requires a FAMA and RETINOBLASTOMA-RELATED module.

Matos JL, Lau OS, Hachez C, Cruz-Ramírez A, Scheres B, Bergmann DC - Elife (2014)

Generation of transgenic lines expressing Myc-tagged RBR driven by FAMA promoter, in vivo immunoprecipitation of the RBR-Myc protein and phenotypic analysis of the transgenic lines.(A) Western detection of RBR-Myc in transgenic plants harboring the FAMAp:RBR-MYC construct in either Col (lines 1–4) or FAMALGK (lines 1–3) backgrounds. Total protein was extracted from 5-dpg seedlings of the indicated genotypes and probed with α-Myc antibody. Recombinant RBR-Myc has a calculated M.W. of 128.5 kDa. (B) In vivo pull-down assay of stomatal lineage expressed RBR-MYC from transgenic plants. Total soluble protein from Col and FAMAp:RBR-MYC (in Col) was incubated with an anti-Myc antibody. Precipitated samples were probed with the same antibody in Western analysis. IB: Immunoblot, IP: Immunoprecipitation. (C–F) Confirmation that expression of FAMAp:RBR-MYC does not alter guard cell development. (C–F) Confocal images of 6-dpg cotyledons and DIC images of 12-dpg cotyledons of wild type and FAMAp:RBR-MYC plants. Transgenic plants harboring the FAMAp:RBR-MYC construct (E–F) do not exhibit changes in GC divisions (neither fewer, nor more divisions are found) and are indistinguishable from wild type (C–D) at 6 and 12-dpg. Cell outlines (purple) in confocal images were visualized with propidium iodide. All images are at the same magnification. Scale bar, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.03271.014
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fig5s2: Generation of transgenic lines expressing Myc-tagged RBR driven by FAMA promoter, in vivo immunoprecipitation of the RBR-Myc protein and phenotypic analysis of the transgenic lines.(A) Western detection of RBR-Myc in transgenic plants harboring the FAMAp:RBR-MYC construct in either Col (lines 1–4) or FAMALGK (lines 1–3) backgrounds. Total protein was extracted from 5-dpg seedlings of the indicated genotypes and probed with α-Myc antibody. Recombinant RBR-Myc has a calculated M.W. of 128.5 kDa. (B) In vivo pull-down assay of stomatal lineage expressed RBR-MYC from transgenic plants. Total soluble protein from Col and FAMAp:RBR-MYC (in Col) was incubated with an anti-Myc antibody. Precipitated samples were probed with the same antibody in Western analysis. IB: Immunoblot, IP: Immunoprecipitation. (C–F) Confirmation that expression of FAMAp:RBR-MYC does not alter guard cell development. (C–F) Confocal images of 6-dpg cotyledons and DIC images of 12-dpg cotyledons of wild type and FAMAp:RBR-MYC plants. Transgenic plants harboring the FAMAp:RBR-MYC construct (E–F) do not exhibit changes in GC divisions (neither fewer, nor more divisions are found) and are indistinguishable from wild type (C–D) at 6 and 12-dpg. Cell outlines (purple) in confocal images were visualized with propidium iodide. All images are at the same magnification. Scale bar, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.03271.014
Mentions: By what molecular mechanism might this specific loss of commitment take place? Analysis of chromatin states in maturing leaves revealed H3K27me3 (a chromatin mark associated with transcriptional repression) in the genomic regions of SPCH, MUTE, FAMA, EPF1 and other stomatal genes (Lafos et al., 2011), and a recent report showed that manipulation of a member of the POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) can alter developmental regulation of H3K27me3 deposition at SPCH and MUTE loci (Lee et al., 2014b). Animal and plant Rb/RBR proteins can serve as interaction bridges between chromatin modifying enzymes and specific genomic contexts (Burkhart and Sage, 2008; Gutzat et al., 2012) and RBR was previously found to be associated with SPCH regulatory regions via ChIP in whole seedlings (Weimer et al., 2012). Therefore it is plausible that, as the final master regulator bHLH in the stomatal pathway, FAMA (with RBR) ensures terminal differentiation of GCs by facilitating stable repression of early stomatal lineage genes. To test this model, we assayed the co-association of FAMA and RBR with regulatory regions of three key stomatal lineage genes that have significant H3K27me3 coverage (SPCH, FAMA and EPF1) and, as specificity controls, two cell cycle genes known to be RBR targets (Nowack et al., 2012). Because RBR is essential and expressed in nearly all cells, to accurately assay its role as a potential partner of FAMA in the stomatal lineage, we generated a Myc-tagged version of RBR expressed under the FAMA promoter (FAMAp:RBR-MYC). We confirmed that expression of this transgene did not alter stomatal development and that we could effectively immunoprecipitate it from plants (Figure 5—figure supplement 2). In ChIP assays, SPCH, FAMA and EPF1 were all targets of FAMA and of stomatal lineage-expressed RBR (Figure 5B–C and Figure 5—figure supplement 3). Further dissection of the binding regions at SPCH and EPF1 indicates that FAMA and RBR are enriched in the same pattern, suggesting that they bind as part of the same complex (Figure 5—figure supplement 4). We then tested the key prediction of our model–that association of RBR with a stomatal target gene is dependent on its interaction with FAMA. ChIPs of FAMAp:RBR-MYC in a FAMALGK background showed that RBR enrichment at the promoters of SPCH and FAMA, but not of the general RBR target gene PCNA, was significantly reduced (Figure 5D and Figure 5—figure supplement 4), consistent with our recruitment model. RBR enrichment at the promoter of a negative regulator of stomatal development, EPF1, was more variable in our assays, sometimes showing little change (Figure 5D), but failing to associate with RBR in other replicates (Figure 5—figure supplement 3C).

Bottom Line: In the Arabidopsis stomatal lineage, a transient self-renewing phase creates precursors that differentiate into one of two epidermal cell types, guard cells or pavement cells.We found that irreversible differentiation of guard cells involves RETINOBLASTOMA-RELATED (RBR) recruitment to regulatory regions of master regulators of stomatal initiation, facilitated through interaction with a terminal stomatal lineage transcription factor, FAMA.Disrupting physical interactions between FAMA and RBR preferentially reveals the role of RBR in enforcing fate commitment over its role in cell-cycle control in this developmental context.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Stanford University, Stanford, United States.

ABSTRACT
The presumed totipotency of plant cells leads to questions about how specific stem cell lineages and terminal fates could be established. In the Arabidopsis stomatal lineage, a transient self-renewing phase creates precursors that differentiate into one of two epidermal cell types, guard cells or pavement cells. We found that irreversible differentiation of guard cells involves RETINOBLASTOMA-RELATED (RBR) recruitment to regulatory regions of master regulators of stomatal initiation, facilitated through interaction with a terminal stomatal lineage transcription factor, FAMA. Disrupting physical interactions between FAMA and RBR preferentially reveals the role of RBR in enforcing fate commitment over its role in cell-cycle control in this developmental context. Analysis of the phenotypes linked to the modulation of FAMA and RBR sheds new light on the way iterative divisions and terminal differentiation are coordinately regulated in a plant stem-cell lineage.

Show MeSH