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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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Validation of primers for the stem cell markers FUS3, LEC1, STM and WOX9.RT-PCR reactions for RNA extracted from immature siliques of Arabidopsis. Target size of the amplified products is indicated in Supplementary file 1. Lanes: molecular weight DNA ladder (MW), independent RNA samples (1, 2, 3), negative controls (−). Gel was stained with ethidium bromide.DOI:http://dx.doi.org/10.7554/eLife.03271.013
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fig5s1: Validation of primers for the stem cell markers FUS3, LEC1, STM and WOX9.RT-PCR reactions for RNA extracted from immature siliques of Arabidopsis. Target size of the amplified products is indicated in Supplementary file 1. Lanes: molecular weight DNA ladder (MW), independent RNA samples (1, 2, 3), negative controls (−). Gel was stained with ethidium bromide.DOI:http://dx.doi.org/10.7554/eLife.03271.013

Mentions: Expression of early stomatal markers indicated that FAMALGK GCs re-acquired stomatal precursor identities, but did these cells return to an even earlier stem-cell or embryonic identity? Moreover, was a change in identity tied to failure of FAMALGK to activate its normal downstream targets? We addressed these questions by monitoring gene expression in isolated 12-dpg cotyledons of WT (Col) and FAMALGK plants (Figure 5A). Analysis of genes shown in Figures 2 and 3 to be inappropriately up-regulated in FAMALGK verified that a qRT-PCR-based approach could accurately assess gene expression changes (Figure 5A, bracket indicating stomatal precursor genes). There was a dramatic increase in expression levels for the stomatal precursor genes, but variable change in expression of mature GC genes, consistent with a situation in which the overproduction of GCs through repeated re-entry is balanced by the loss of identity of older GCs (Figure 5A, mature GC genes). FAMALGK was also still capable of up-regulating several, but not all, of the direct targets reported in (Hachez et al., 2011) (Figure 5A, FAMA direct targets). When expression of shoot meristem (SHOOT MERISTEMLESS, STM), root meristem (WUS HOMEOBOX, WOX5) or embryo genes (WOX9, WOX2, FUSCA3 (FUS3), LEC1) (Breuninger et al., 2008; De Smet et al., 2010) was monitored in FAMALGK plants, however, we found no evidence that cells were being reprogrammed into embryonic or other stem-cell-like fates (Figure 5A and Figure 5—figure supplement 1). Taken together, the gene expression data indicate that disruption of FAMA-RBR interaction via the FAMALGK modification leads to a stomatal lineage-specific loss of terminal commitment.10.7554/eLife.03271.012Figure 5.Terminal differentiation of guard cells may be mediated by FAMA-guided recruitment of RBR to suppress stomatal regulatory genes.


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)

Validation of primers for the stem cell markers FUS3, LEC1, STM and WOX9.RT-PCR reactions for RNA extracted from immature siliques of Arabidopsis. Target size of the amplified products is indicated in Supplementary file 1. Lanes: molecular weight DNA ladder (MW), independent RNA samples (1, 2, 3), negative controls (−). Gel was stained with ethidium bromide.DOI:http://dx.doi.org/10.7554/eLife.03271.013
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225492&req=5

fig5s1: Validation of primers for the stem cell markers FUS3, LEC1, STM and WOX9.RT-PCR reactions for RNA extracted from immature siliques of Arabidopsis. Target size of the amplified products is indicated in Supplementary file 1. Lanes: molecular weight DNA ladder (MW), independent RNA samples (1, 2, 3), negative controls (−). Gel was stained with ethidium bromide.DOI:http://dx.doi.org/10.7554/eLife.03271.013
Mentions: Expression of early stomatal markers indicated that FAMALGK GCs re-acquired stomatal precursor identities, but did these cells return to an even earlier stem-cell or embryonic identity? Moreover, was a change in identity tied to failure of FAMALGK to activate its normal downstream targets? We addressed these questions by monitoring gene expression in isolated 12-dpg cotyledons of WT (Col) and FAMALGK plants (Figure 5A). Analysis of genes shown in Figures 2 and 3 to be inappropriately up-regulated in FAMALGK verified that a qRT-PCR-based approach could accurately assess gene expression changes (Figure 5A, bracket indicating stomatal precursor genes). There was a dramatic increase in expression levels for the stomatal precursor genes, but variable change in expression of mature GC genes, consistent with a situation in which the overproduction of GCs through repeated re-entry is balanced by the loss of identity of older GCs (Figure 5A, mature GC genes). FAMALGK was also still capable of up-regulating several, but not all, of the direct targets reported in (Hachez et al., 2011) (Figure 5A, FAMA direct targets). When expression of shoot meristem (SHOOT MERISTEMLESS, STM), root meristem (WUS HOMEOBOX, WOX5) or embryo genes (WOX9, WOX2, FUSCA3 (FUS3), LEC1) (Breuninger et al., 2008; De Smet et al., 2010) was monitored in FAMALGK plants, however, we found no evidence that cells were being reprogrammed into embryonic or other stem-cell-like fates (Figure 5A and Figure 5—figure supplement 1). Taken together, the gene expression data indicate that disruption of FAMA-RBR interaction via the FAMALGK modification leads to a stomatal lineage-specific loss of terminal commitment.10.7554/eLife.03271.012Figure 5.Terminal differentiation of guard cells may be mediated by FAMA-guided recruitment of RBR to suppress stomatal regulatory genes.

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