Tissue-Specific Control of the Endocycle by the Anaphase Promoting Complex/Cyclosome Inhibitors UVI4 and DEL1

Abstract

The endocycle represents a modified mitotic cell cycle that in plants is often coupled to cell enlargement and differentiation. Endocycle onset is controlled by activity of the Anaphase Promoting Complex/Cyclosome (APC/C), a multisubunit E3 ubiquitin ligase targeting cell-cycle factors for destruction. CELL CYCLE SWITCH52 (CCS52) proteins represent rate-limiting activator subunits of the APC/C. In Arabidopsis (Arabidopsis thaliana), mutations in either CCS52A1 or CCS52A2 activators result in a delayed endocycle onset, whereas their overexpression triggers increased DNA ploidy levels. Here, the relative contribution of the APC/C^CCS52A1 and APC/C^CCS52A2 complexes to different developmental processes was studied through analysis of their negative regulators, being the ULTRAVIOLET-B-INSENSITIVE4 protein and the DP-E2F-Like1 transcriptional repressor, respectively. Our data illustrate cooperative activity of the APC/C^CCS52A1 and APC/C^CCS52A2 complexes during root and trichome development, but functional interdependency during leaf development. Furthermore, we found APC/C^CCS52A1 activity to control CCS52A2 expression. We conclude that interdependency of CCS52A-controlled APC/C activity is controlled in a tissue-specific manner.

Figure 1.

Endoreplication phenotypes of the ccs52a1-1 DEL1^OE double mutant. A, Flow cytometric analysis of wild-type (Col-0), ccs52a1-1, DEL1^OE, and ccs52a1-1 DEL1^OE three-week-old first true leaves. Data are representative for the mean (n = 3). B, Scanning electron microscope images of wild-type (Col-0), ccs52a1-1, DEL1^OE, and ccs52a1-1 DEL1^OE trichomes. Images are representative for the mean (n = 3). Bars = 300 μm. C, Epifluorescence images of DAPI-stained wild-type, ccs52a1-1, DEL1^OE, and ccs52a1-1 DEL1^OE trichome nuclei. Images are representative for the mean (n > 13). Bars = 10 μm.

Figure 2.

Endoreplication phenotypes of the uvi4 ccs52a2-1 double mutant. A, Flow cytometric analysis of wild-type (Col-0), uvi4, ccs52a2-1, and uvi4 ccs52a2-1 three-week-old first true leaves. Data are representative for the mean (n = 3). B, Scanning electron microscope images of wild-type (Col-0), uvi4, ccs52a2-1, and uvi4 ccs52a2-1 trichomes. Images are representative for the mean. Bars = 300 μm. C, Epifluorescence images of DAPI-stained wild-type, uvi4, ccs52a2-1, and uvi4 ccs52a2-1 trichome nuclei. Images are representative for the mean (n > 8). Bars = 10 μm.

Figure 3.

UVI4 and DEL1 independently control trichome ploidy levels. A and B, Expression pattern of UVI4 and DEL1 in the root meristem (A) and leaves (B). Bars = 0.1 mm. C, Scanning electron microscope images of wild-type (Col-0), uvi4, del1-1, and uvi4 del-1 trichomes. Images are representative for the mean. Bars = 300 μm. D, Epifluorescence images of DAPI-stained wild-type, uvi4, del-1, and uvi4 del-1 trichome nuclei. Images are representative for the mean (n > 8). Bars = 10 μm.

Figure 4.

UVI4 and DEL1 independently control cell cycle exit in the root. A, Representative confocal microscopy images of wild-type (Col-0), uvi4, del1-1, and uvi4 del1-1 one-week-old root meristems stained with propidium iodide. Arrowheads indicate the meristem size based on the cortical cell length. Bar = 50 μm. B, Number of meristematic cortex cells for lines presented in (A). Data represent mean ± se (n > 8, *P < 0.05, Student’s t test). C, Flow cytometric analysis of wild-type (Col-0), uvi4, del1-1, and uvi4 del1-1 one-week-old mutant roots. Data are representative for the mean.

Figure 5.

UVI4 and DEL1 control leaf ploidy levels through a common mechanism. A, Images of three-week-old wild-type (Col-0), uvi4, del1-1, and uvi4 del1-1 rosettes. Bar = 1 cm. B, Average first true leaf sizes of three-week-old wild-type (Col-0), uvi4, del1-1, and uvi4 del1-1 plants (in mm²). Data represent mean ± se (n = 45, *P < 0.05, Student’s t test). C and D, Average abaxial epidermal cell size (× 1.000 μm²) and average cell number (× 1.000), respectively, of first leaves of three-week-old lines presented in (B). Data represent mean ± se (n = 18, *P < 0.05, Student’s t test). E, Flow cytometric analysis of wild-type (Col-0), uvi4, del1-1, and uvi4 del1-1 three-week-old first true leaves. Data are representative for the mean (n = 3).

Figure 6.

CCS52A1 activity affects CCS52A2 expression. A, Relative expression levels of CCS52A1 and CCS52A2 in first true leaves from wild-type (Col-0), uvi4, del1-1, and uvi4 del1-1. First true leaves were harvested at stage 1.04. The relative expression levels for wild-type leaves were arbitrarily set to 1. Data represent mean ± se (n = 3, *P < 0.05, Student’s t test). B, Increased CCS52A2 transcript levels in CCS52A1^OE first true leaves compared to the wild type. First true leaves were harvested at stage 1.04. The relative expression levels for wild-type leaves were arbitrarily set to 1. Data represent mean ± se (n = 3, *P < 0.05, Student’s t test).