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. 2006 Jan;140(1):67-80.
doi: 10.1104/pp.105.071027. Epub 2005 Dec 16.

Cell Type-Specific Role of the retinoblastoma/E2F Pathway During Arabidopsis Leaf Development

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Cell Type-Specific Role of the retinoblastoma/E2F Pathway During Arabidopsis Leaf Development

Bénédicte Desvoyes et al. Plant Physiol. .
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Abstract

Organogenesis in plants is almost entirely a postembryonic process. This unique feature implies a strict coupling of cell proliferation and differentiation, including cell division, arrest, cell cycle reactivation, endoreplication, and differentiation. The plant retinoblastoma-related (RBR) protein modulates the activity of E2F transcription factors to restrict cell proliferation. Arabidopsis contains a single RBR gene, and its loss of function precludes gamete formation and early development. To determine the relevance of the RBR/E2F pathway during organogenesis, outside its involvement in cell division, we have used an inducible system to inactivate RBR function and release E2F activity. Here, we have focused on leaves where cell proliferation and differentiation are temporally and developmentally regulated. Our results reveal that RBR restricts cell division early during leaf development when cell proliferation predominates, while it regulates endocycle occurrence at later stages. Moreover, shortly after leaving the cell cycle, most of leaf epidermal pavement cells retain the ability to reenter the cell cycle and proliferate, but maintain epidermal cell fate. On the contrary, mesophyll cells in the inner layers do not respond in this way to RBR loss of activity. We conclude that there exists a distinct response of different cells to RBR inactivation in terms of maintaining the balance between cell division and endoreplication during Arabidopsis (Arabidopsis thaliana) leaf development.

Figures

Figure 1.
Figure 1.
Expression of the geminivirus RBR-binding RepA proteins in transgenic plants. A, Details of the constructs used to express the RepAwt and RepAE198K coding sequences in the Dex-inducible system (Aoyama and Chua, 1997). B, Western-blot analysis of RepA protein 7 h after induction with different concentrations of Dex (0–20 μm) in 10-d-old seedlings. Ten micrograms of protein were loaded in each lane. The bottom section is a region of the Coomassie-stained gel (molecular mass approximately 55 kD) used as loading control. C, Detection of RepAwt and RepAE198K after Dex treatment (20 μm, 20 h) in 10-d-old seedlings (20 μg protein per lane). The bottom section is the loading control as in B. D, Time course of RepAwt expression in 10-d-old seedlings (10 μg) after treatment with 20 μm Dex. The bottom section is the loading control as in B. Induction of RepAE198K protein followed a similar pattern. E, Stability of RepAwt protein after Dex treatment (1 μm). The bottom section is the loading control as in B. F, Detection of RepAwt in cross sections of leaves 3/4, 1 d after Dex treatment of control- and RepAwt-expressing plants to show that the viral protein accumulates in the epidermis as well as in the internal cell layers. Bars correspond to 50 μm. G, Phenotype at the rosette stage (18-d-old seedlings) of control (transformed with the empty vector) and transgenic plants expressing RepAwt or RepAE198K, 5 d after spraying with 1 μm Dex. The right sections show, from left to right, the two cotyledons and the first and second pairs of the leaves at the same magnification.
Figure 2.
Figure 2.
Disruption of RBR/E2F complexes in yeast and in planta. A, Effect of RepAwt and RepAE198K proteins on RBR/E2F interaction shown by yeast three-hybrid assay. Yeast cells expressing AtRBR-Gal4 DNA-binding domain (BD-RBR) were cotransformed, as indicated, with plasmids expressing the different AtE2F fused to the Gal4 activation domain (AD-E2F) or with the empty vector (AD), and with a vector (TFT) expressing RepAwt and RepAE198K proteins. All transformants grew normally in plates containing His (data not shown). The top sections show the growth of different transformants in selective medium (without His) at different dilutions. Galactosidase activity is shown in the bottom section and is expressed as Miller units. Data correspond to two independent experiments, which were carried out in triplicate. B, Detection of E2F DNA-binding activity in plant extracts by EMSA. Total protein extracts (15 μg) of the indicated transgenic plants, with or without Dex treatment, were incubated with a 32P-labeled double-stranded oligonucleotide containing a consensus E2F site (Ramirez-Parra and Gutierrez, 2000). Arrow points to E2F-DNA complexes. A 100-fold excess of the unlabeled probe was added as competitor (right section). Relative E2F DNA-binding activity (bottom section) was quantified using a GS-710 Calibrated Image densitometer (Bio-Rad). The analysis is based on three independent experiments. Bars show the sds. C, Expression levels of each of the six Arabidopsis E2F genes determined by real-time RT-PCR analysis in extracts of 10-d-old seedlings of controls (plants transformed with an empty vector) or transgenics expressing RepAwt and RepAE198K proteins. The analysis was carried out 7 h after treatment with Dex (20 μm). Values were first normalized to the amount of actin (AtACT2) and then made relative to the mRNA amount in the control.
Figure 3.
Figure 3.
Inactivation of RBR up-regulates the expression of a subset of E2F target genes and induces ectopic cell division. A, Expression level of the indicated E2F target genes determined by real-time RT-PCR analysis in extracts of 10-d-old seedlings of controls (plants transformed with an empty vector) or transgenics expressing RepAwt and RepAE198K proteins. The analysis was carried out 7 h after treatment with Dex (20 μm). Values were first normalized to the amount of Actin (AtACT2) and then made relative to the mRNA amount in the control. B to E, Control and transgenic plants were crossed with the cyclin B1;1-GUS marker lines (Colon-Carmona et al., 1999). Cyclin B1;1-GUS activity was detected 3 d after treating 16-d-old plants with Dex (1 μm) in the controls (B and D) and plants expressing RepAwt protein (C and E). The aerial parts around the shoot tip (B and C) and a region of leaves 1/2 (D and E) are shown. The inset in B shows a higher magnification of a growing leaf primordium showing scattered cyclin B1;1-GUS positive cells. Bars represent 50 μm.
Figure 4.
Figure 4.
Inactivation of RBR induces endoreplication in late developing leaves. Nuclear DNA ploidy distribution of control, RepAwt, and RepAE198K transgenic plants before (13 das; A and D), 2 d (15 das; B and E), and 5 d (18 das; C and F) after treatment with Dex (1 μm). Flow cytometry measurements were carried out in the first (nos. 1/2; A–C) and second (nos. 3/4; D–F) pairs of leaves. The appearance of the developmental stage of representative RepAwt-expressing plants at the time of flow cytometry analysis is also included (middle sections).
Figure 5.
Figure 5.
Inactivation of RBR alters trichome morphology. Control, RepAwt, and RepAE198K transgenic plants were sprayed with 1 μm Dex 13 d after sowing and analyzed 5 d after treatment. A, Examples of trichomes with three to five branches under light microscopy (top sections) or fluorescence microcopy after DAPI staining (bottom sections). In the latter, the arrowheads point to the nuclei. Branch number distribution of trichomes in the first pair of leaves (nos. 1/2), n > 100 in at least eight leaves (B), and in the second pair of leaves (nos. 3/4), n > 350 in at least six leaves (C). Numbers represent the percentage of each class. Asterisks indicate statistically significant differences relative to the control (at least, P < 0.05).
Figure 6.
Figure 6.
Microscopic analysis of leaf epidermis of control, RepAwt, and RepAE198K transgenic plants. A to C, DAPI staining of the adaxial epidermis (leaf nos. 3/4). Note the large increase in the number of nuclei in the RepAwt plants. D to F, Cryo-scanning electron microscopy of the adaxial epidermis (nos. 3/4). Note the irregular surface of the leaf due to hyperplasia of the epidermis. D′ to F′, Close-up of D to F to show details of the clusters of small cells. G to I, DAPI staining of the adaxial epidermis (leaf nos. 1/2). Note that clusters of small cells do not appear. J to L, Light microscopy of the adaxial epidermis (leaf nos. 1/2). Note the absence of clusters of small cells, but instead the presence of ectopic cell wall dividing fully expanded pavement cells (arrows). Bars in all sections correspond to 50 μm. The study was carried out in the middle region of the leaf blade of 18-d-old plants 5 d after treatment with Dex (1 μm).
Figure 7.
Figure 7.
Microscopic analysis and cell distribution in the leaves of control, RepAwt, and RepAE198K transgenic plants. A to C, Cross sections of leaf number 3. Sections below (A′ to C′) are higher magnification of A to C. Arrow in B indicates anticlinal division in the adaxial epidermis. Arrowheads in B′ and B′′; indicate periclinal and anticlinal divisions, respectively. Bars correspond to 50 μm. D, Size of adaxial epidermal and mesophyll cells (leaf nos. 3/4), n > 600 cells. E, Cell density of adaxial epidermal and mesophyll cells (leaf nos. 3/4). F, Cell size distribution of adaxial epidermal cells (leaf nos. 3/4). The study was carried out in the middle region of the leaf blade of 18-d-old plants, 5 d after treatment with Dex (1 μm).

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