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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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Related in: MedlinePlus

Expression of cell cycle and stomatal reporters in FAMALGK plants (FAMAp:FAMALGK;fama) and amiRBR (FAMAp:amiRBR) mutants and examples of timelapse images for SPCH and MUTE markers.(A) GUS staining of transcriptional reporters for cell cycle genes CDKB1;1 and CDC6 (rows) in WT Col (left column), amiRBR (middle), and FAMALGK plants (right column). Note that the pattern and levels of expression differ between the RBR knockdown line (amiRBR) and when the interaction between FAMA and RBR is disrupted (FAMALGK). In the amiRBR line, CDKB1;1 and CDC6 are strongly expressed in both GCs, each of which displays ectopic cell divisions (outline in left guard cell and arrowheads in the right guard cell). Broad expression of CDKB1;1 and CDC6 is consistent with RBR's function as a direct repressor of the transcription factor E2F and its cell cycle target genes required for the G1 to S-phase transition (Gutzat et al., 2012). In FAMALGK plants, however, CDKB1;1 and CDC6 are restricted to only some stomatal cell divisions (stars mark new GCs and arrows mark amplifying ACDs). Expression of CDKB1;1 and CDC6 are likely consequences of regulated cell divisions as the mutant GCs progress through the stomatal lineage. (B) Confocal images of stomatal lineage reporters in GCs of amiRBR. Cell outlines are visualized with propidium iodide (purple). Arrowheads correspond to ectopic cell divisions. Of the reporters tested, SPCH and MUTE are weakly and infrequently seen (<20% of GCs) and only in GCs with many ectopic divisions (green asterisks). TMM and EPF2, however, were not detectable. Small blue disks visible in these cells are chloroplasts. Scale bar, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.03271.007
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fig2s1: Expression of cell cycle and stomatal reporters in FAMALGK plants (FAMAp:FAMALGK;fama) and amiRBR (FAMAp:amiRBR) mutants and examples of timelapse images for SPCH and MUTE markers.(A) GUS staining of transcriptional reporters for cell cycle genes CDKB1;1 and CDC6 (rows) in WT Col (left column), amiRBR (middle), and FAMALGK plants (right column). Note that the pattern and levels of expression differ between the RBR knockdown line (amiRBR) and when the interaction between FAMA and RBR is disrupted (FAMALGK). In the amiRBR line, CDKB1;1 and CDC6 are strongly expressed in both GCs, each of which displays ectopic cell divisions (outline in left guard cell and arrowheads in the right guard cell). Broad expression of CDKB1;1 and CDC6 is consistent with RBR's function as a direct repressor of the transcription factor E2F and its cell cycle target genes required for the G1 to S-phase transition (Gutzat et al., 2012). In FAMALGK plants, however, CDKB1;1 and CDC6 are restricted to only some stomatal cell divisions (stars mark new GCs and arrows mark amplifying ACDs). Expression of CDKB1;1 and CDC6 are likely consequences of regulated cell divisions as the mutant GCs progress through the stomatal lineage. (B) Confocal images of stomatal lineage reporters in GCs of amiRBR. Cell outlines are visualized with propidium iodide (purple). Arrowheads correspond to ectopic cell divisions. Of the reporters tested, SPCH and MUTE are weakly and infrequently seen (<20% of GCs) and only in GCs with many ectopic divisions (green asterisks). TMM and EPF2, however, were not detectable. Small blue disks visible in these cells are chloroplasts. Scale bar, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.03271.007

Mentions: Phenotypes conferred by FAMALGK and by manipulating RBR in the late stomatal lineage both involved increased cell division, but were not identical. To improve phenotypic resolution, we characterized the expression patterns of cell fate and cell cycle markers in FAMALGK and FAMAp:amiRBR plants (Figure 2 and Figure 2—figure supplement 1). This detailed analysis revealed clear phenotypic differences between reducing RBR levels in GMCs and reducing RBR's interaction with FAMA (Figure 2—figure supplement 1A–B). Notably, the FAMALGK phenotype results, not from chaotic or uncontrolled divisions and fate changes, but rather an orderly reiteration of stomatal lineage progression. This manifested itself as a progressive increase in phenotypic severity with age (Figure 1L) and by the appearance of stomatal lineage markers in patterns suggesting that the GCs reverted to MMC identity and proceeded through the intermediate stages of the pathway normally (Figure 2). Expression of stomatal-promoting transcription factors (SPCH, MUTE, FAMA, Figure 2A–C and Figure 2—figure supplement 2), stomatal-restricting signaling elements (TMM, EPF1, EPF2, Figure 3A–D), and general division reporters (CDKA1;1, Figure 2B–C) followed the normal temporal patterns, and ectopic GC divisions appeared to follow early lineage division rules. For example, when a ‘reprogrammed’ GC produced two stomata, they were separated by a non-stomatal cell, indicating that spacing divisions occurred. Distinct cell orientations characteristic of amplifying divisions were also visible (Figure 2B and Figure 2—figure supplement 3). Further evidence for normal asymmetric divisions is polarized localization of BASL (Dong et al., 2009) in the larger daughter of a GC division (Figure 3E). Based on the lack of expression of stomatal lineage markers (Figure 2D), we interpret the lobed GCs we observe at low, but significant, frequencies in FAMALGK plants (Figure 1K, inset, and Figure 1L) as cells that are transdifferentiating into an epidermal pavement cell identity.10.7554/eLife.03271.006Figure 2.Disruption of FAMA-RBR interaction leads to failure of terminal differentiation and reiteration of stomatal lineage divisions and gene expression programs.


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)

Expression of cell cycle and stomatal reporters in FAMALGK plants (FAMAp:FAMALGK;fama) and amiRBR (FAMAp:amiRBR) mutants and examples of timelapse images for SPCH and MUTE markers.(A) GUS staining of transcriptional reporters for cell cycle genes CDKB1;1 and CDC6 (rows) in WT Col (left column), amiRBR (middle), and FAMALGK plants (right column). Note that the pattern and levels of expression differ between the RBR knockdown line (amiRBR) and when the interaction between FAMA and RBR is disrupted (FAMALGK). In the amiRBR line, CDKB1;1 and CDC6 are strongly expressed in both GCs, each of which displays ectopic cell divisions (outline in left guard cell and arrowheads in the right guard cell). Broad expression of CDKB1;1 and CDC6 is consistent with RBR's function as a direct repressor of the transcription factor E2F and its cell cycle target genes required for the G1 to S-phase transition (Gutzat et al., 2012). In FAMALGK plants, however, CDKB1;1 and CDC6 are restricted to only some stomatal cell divisions (stars mark new GCs and arrows mark amplifying ACDs). Expression of CDKB1;1 and CDC6 are likely consequences of regulated cell divisions as the mutant GCs progress through the stomatal lineage. (B) Confocal images of stomatal lineage reporters in GCs of amiRBR. Cell outlines are visualized with propidium iodide (purple). Arrowheads correspond to ectopic cell divisions. Of the reporters tested, SPCH and MUTE are weakly and infrequently seen (<20% of GCs) and only in GCs with many ectopic divisions (green asterisks). TMM and EPF2, however, were not detectable. Small blue disks visible in these cells are chloroplasts. Scale bar, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.03271.007
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fig2s1: Expression of cell cycle and stomatal reporters in FAMALGK plants (FAMAp:FAMALGK;fama) and amiRBR (FAMAp:amiRBR) mutants and examples of timelapse images for SPCH and MUTE markers.(A) GUS staining of transcriptional reporters for cell cycle genes CDKB1;1 and CDC6 (rows) in WT Col (left column), amiRBR (middle), and FAMALGK plants (right column). Note that the pattern and levels of expression differ between the RBR knockdown line (amiRBR) and when the interaction between FAMA and RBR is disrupted (FAMALGK). In the amiRBR line, CDKB1;1 and CDC6 are strongly expressed in both GCs, each of which displays ectopic cell divisions (outline in left guard cell and arrowheads in the right guard cell). Broad expression of CDKB1;1 and CDC6 is consistent with RBR's function as a direct repressor of the transcription factor E2F and its cell cycle target genes required for the G1 to S-phase transition (Gutzat et al., 2012). In FAMALGK plants, however, CDKB1;1 and CDC6 are restricted to only some stomatal cell divisions (stars mark new GCs and arrows mark amplifying ACDs). Expression of CDKB1;1 and CDC6 are likely consequences of regulated cell divisions as the mutant GCs progress through the stomatal lineage. (B) Confocal images of stomatal lineage reporters in GCs of amiRBR. Cell outlines are visualized with propidium iodide (purple). Arrowheads correspond to ectopic cell divisions. Of the reporters tested, SPCH and MUTE are weakly and infrequently seen (<20% of GCs) and only in GCs with many ectopic divisions (green asterisks). TMM and EPF2, however, were not detectable. Small blue disks visible in these cells are chloroplasts. Scale bar, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.03271.007
Mentions: Phenotypes conferred by FAMALGK and by manipulating RBR in the late stomatal lineage both involved increased cell division, but were not identical. To improve phenotypic resolution, we characterized the expression patterns of cell fate and cell cycle markers in FAMALGK and FAMAp:amiRBR plants (Figure 2 and Figure 2—figure supplement 1). This detailed analysis revealed clear phenotypic differences between reducing RBR levels in GMCs and reducing RBR's interaction with FAMA (Figure 2—figure supplement 1A–B). Notably, the FAMALGK phenotype results, not from chaotic or uncontrolled divisions and fate changes, but rather an orderly reiteration of stomatal lineage progression. This manifested itself as a progressive increase in phenotypic severity with age (Figure 1L) and by the appearance of stomatal lineage markers in patterns suggesting that the GCs reverted to MMC identity and proceeded through the intermediate stages of the pathway normally (Figure 2). Expression of stomatal-promoting transcription factors (SPCH, MUTE, FAMA, Figure 2A–C and Figure 2—figure supplement 2), stomatal-restricting signaling elements (TMM, EPF1, EPF2, Figure 3A–D), and general division reporters (CDKA1;1, Figure 2B–C) followed the normal temporal patterns, and ectopic GC divisions appeared to follow early lineage division rules. For example, when a ‘reprogrammed’ GC produced two stomata, they were separated by a non-stomatal cell, indicating that spacing divisions occurred. Distinct cell orientations characteristic of amplifying divisions were also visible (Figure 2B and Figure 2—figure supplement 3). Further evidence for normal asymmetric divisions is polarized localization of BASL (Dong et al., 2009) in the larger daughter of a GC division (Figure 3E). Based on the lack of expression of stomatal lineage markers (Figure 2D), we interpret the lobed GCs we observe at low, but significant, frequencies in FAMALGK plants (Figure 1K, inset, and Figure 1L) as cells that are transdifferentiating into an epidermal pavement cell identity.10.7554/eLife.03271.006Figure 2.Disruption of FAMA-RBR interaction leads to failure of terminal differentiation and reiteration of stomatal lineage divisions and gene expression programs.

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
Related in: MedlinePlus