Endoreplication controls cell fate maintenance - PubMed (original) (raw)

Endoreplication controls cell fate maintenance

Jonathan Bramsiepe et al. PLoS Genet. 2010.

Abstract

Cell-fate specification is typically thought to precede and determine cell-cycle regulation during differentiation. Here we show that endoreplication, also known as endoreduplication, a specialized cell-cycle variant often associated with cell differentiation but also frequently occurring in malignant cells, plays a role in maintaining cell fate. For our study we have used Arabidopsis trichomes as a model system and have manipulated endoreplication levels via mutants of cell-cycle regulators and overexpression of cell-cycle inhibitors under a trichome-specific promoter. Strikingly, a reduction of endoreplication resulted in reduced trichome numbers and caused trichomes to lose their identity. Live observations of young Arabidopsis leaves revealed that dedifferentiating trichomes re-entered mitosis and were re-integrated into the epidermal pavement-cell layer, acquiring the typical characteristics of the surrounding epidermal cells. Conversely, when we promoted endoreplication in glabrous patterning mutants, trichome fate could be restored, demonstrating that endoreplication is an important determinant of cell identity. Our data lead to a new model of cell-fate control and tissue integrity during development by revealing a cell-fate quality control system at the tissue level.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Reduced trichome numbers on plants with altered cell-cycle control in trichomes.

Scanning electron micrographs of rosette leaf number 4 of Arabidopsis seedlings. (A) Columbia, (B) PROCPC:CYCD3;1, (C) PROGL2: ICK1/KRP1im, (D) CDKA;1T161D, (E) CDKA;1T14D/Y15E and (F) PROGL2:KRP1109 - CDKA;1T161D plants. (G) Quantification of trichome numbers of leaf 3 and 4 in comparison with Columbia. Col Columbia n = 31. sim siamese n = 18. ICK1/KRP1IM , PROGL2:GUS:YFP:KRP1109 n = 30. KRP1109 PROGL2:KRP1109 n = 32. D CDKA;1T161D n = 32. DE CDKA;1T14D/Y15E n = 32. D; ICK1/KRP1109 CDKA;1T161D; PROGL2:KRP1109 n = 26. Error bars: standard deviation. Scale bars: (A–F) 100 µm.

Figure 2

Figure 2. Analysis of DNA content in trichomes.

(A–P) Distribution of the trichome DNA contents in relative fluorescence units (RFU). Two RFUs roughly represent 2C, calibrated with wild-type and gl3 trichome nuclei. (A) Wild type. (B) glabra3 (gl3). (C) glabra2 (gl2). (D) PROGL2:ICK1/KRP1. (E) PROGL2:GUS:YFP:ICK1/KRP1109-191 (ICK1/KRP1im). (F) gl3 - ICK1/KRP1im. (G_) gl2 - ICK1/KRP1im_. (H) PROGL2:ICK1/KRP1109–191. (I) CDKA;1T161D (D). (J) gl3 - D. (K) gl2 - D. (L) D - PROGL2:ICK1/KRP1109–191. (M) CDKA;1T14D/Y15E (DE). (N) gl3 - DE. (O) gl2 - DE. (P) gl2 - gl3 - PROGL2:CCS52A1.

Figure 3

Figure 3. Genetic combinations of trichome patterning mutants with plants that have a reduced endoreplication levels in trichomes.

Scanning electron micrographs of rosette leaf number 4. (A) cpc try. (B) gl3. (C) PROGL2:KRP1109 in cpc try. (D) PROGL2:KRP1109 in gl3. (E) CDKA;1T161D in cpc try (F) CDKA;1T161D in gl3. (G) Quantification of trichome numbers of leaf 3 and 4 in comparison with wild type (Columbia). Error bars: standard deviation. Scale bars: (A–D) 50 µm; (E,F) 200 µm.

Figure 4

Figure 4. Analysis of early trichome initation.

Light micrographs of young rosette leaves. (A) Overview of a young leaf 4 in wild type. The trichome initiation zone was defined as the leaf region where trichomes emerge (indicated in blue) and only unbranched trichomes were counted. (B) Trichome initiation zone in wild type (Columbia). (C) Trichome initiation zone in CDKA;1T161D leaves. (D) Quantification of the number of trichome initiation sites (TIS) compared to Columbia. Error bars: standard deviation n≥8. Scale bars: (A) 100 µm; (B,C) 25 µm.

Figure 5

Figure 5. Morphology of aborted trichomes.

Scanning electron micrographs of rosette leaves of wild-type plants (A,B) and plants expressing PROGL2:ICK1/KRP1 (C–E). (A) Trichome development on a young wild-type leaf. (B) Emerging wild-type trichome. (C) Aborting trichome on a young leaf of PROGL2:ICK1/KRP1 plant. (D) Putative aborted trichome undergoing cell division. (E) Possible remnants of an aborting trichome that underwent repeated rounds of cell division. Putative division planes indicated in (E′). Scale bars: (A) 30 µm; (B–D) 10 µm.

Figure 6

Figure 6. Live-imaging of aborting trichomes.

Confocal-scanning micrographs of young rosette leaves; (A-C and D″-F″) overlay of propidium iodine channel and YFP/GFP channel; (D–F) Propidium iodine channel; (D′–F′) YFP/GFP channel. (A,B) Leaves of PROGL2:GFP expressing plants in wild-type background after 24 h (A) and 48 h (B). (C) Leaf of _gl3_- PROGL2:GUS:YFP:ICK1/KRP1109-191 with a putative trichome precursor cell (arrow head) after cell division without YFP fluorescence while an outgrowing, i.e. non-aborting, trichome (square) displays a strong YFP signal. (D) Young leaf of _gl3_- PROGL2:GUS:YFP:ICK1/KRP1109-191; young trichome cells can be identified by their increased size in comparison to the surrounding epidermal pavement cells and a bright fluorescent signal in the nucleus. Please note that some surrounding epidermal cells also display YFP fluorescence due to the low activity of the GL2 promoter in groups of epidermal cells encompassing the future trichome. (E) The same leaf as in (D) after 24 h and after 48 h (F). Three trichome initials are marked by a plus, an asterisks, a number sign (D″–F″). Please note yellow background fluorescence in E′/E″ (cell walls are marked) and F′/F″ (some cell walls and stomata) as a consequence of the high sensitivity in the detection procedure.

Figure 7

Figure 7. DNA content of young aborting trichomes.

Light micrographs of young rosette leaves stained with DAPI. (A) Columbia wild-type plants. (B) ICK1/KRP1im. (C) gl3 - ICK1/KRP1im. (D) Quantification of DNA contents of trichome nuclei in relative fluorescence units (RFU). The RFU are calibrated by dividing epidermal cells nuclei so that 2 RFU roughly represents 2C.

Figure 8

Figure 8. Partial rescue of trichome development of gl2 gl3 by PROGL2:CCS52A1 expression.

Scanning electron micrographs of rosette leaves (A–F) and light micrographs of GUS-stained leaves (G–I). (A) gl2. (B) gl2 - ICK1/KRP1im. (C) gl2 - DE. (D) gl2 - sim. (E) Mature leaves of gl2–gl3 double mutant are completely devoid of trichomes. (F) PROGL2:CCS52A1 expression promotes endoreplication in trichome precursor cells and causes the formation of trichome-like cells in gl2–gl3 double mutants. (G–I) Expression of the trichome marker PRONOK:GUS. (G) Wild type. (H) In gl2–gl3 mutants only a weak PRONOK:GUS activity can be detected in leaf margins. (I) The trichome-like cells in gl2–gl3-PROGL2:CCS52A1 plants show PRONOK:GUS activity. (J) Quantification of trichome number of leaf 3 and 4 in comparison with wild type.

Figure 9

Figure 9. New model of trichome development.

Trichome development appears to rely on two positive feed back loops. The first loop centers around the transcriptional activator GL3. The loop achieves the initial trichome pattern. Among other targets, GL3 directly activates GL2 and SIM that are involved in a second feed back loop that is important for trichome cell fate maintenance and entry into an endoreplication cycle. Please note that in both feedback loops, GL3 and GL2 are operating together with many more regulators, indicated here as other factors.

References

    1. Jakoby M, Schnittger A. Cell cycle and differentiation. Curr Opin Plant Biol. 2004;7:661–669. - PubMed
    1. Edgar BA, Orr-Weaver TL. Endoreplication cell cycles: more for less. Cell. 2001;105:297–306. - PubMed
    1. Kondorosi E, Roudier F, Gendreau E. Plant cell-size control: growing by ploidy? Curr Opin Plant Biol. 2000;3:488–492. - PubMed
    1. Sugimoto-Shirasu K, Roberts K. “Big it up”: endoreduplication and cell-size control in plants. Curr Opin Plant Biol. 2003;6:544–553. - PubMed
    1. Morgan DO. Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol. 1997;13:261–291. - PubMed

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