SIAMESE cooperates with the CDH1-like protein CCS52A1 to establish endoreplication in Arabidopsis thaliana trichomes

Abstract

Endoreplication, also known as endoreduplication, is a phyogenetically widespread modified version of the cell cycle in which DNA replication is not followed by cell division. The SIAMESE (SIM) gene of Arabidopsis thaliana encodes the founding member of a novel class of plant-specific cyclin-dependent kinase (CDK) inhibitors and is a key regulator of endoreplication during the development of trichomes (shoot epidermal hairs). Here, we have identified mutations in the CCS52A1 gene as genetic modifiers of the multicellular trichome phenotype of sim mutants. Loss-of-function ccs52A1 mutations dramatically enhance the multicellularity of sim mutants trichomes in double mutants, whereas overexpression of CCS52A1 completely suppresses the sim mutant phenotype. CCS52A1 encodes a CDH1/FZR-like protein, a class of proteins that function as activators of the anaphase-promoting complex. Unicellular ccs52A1 trichomes become multicellular upon overexpression of B-type cyclin, consistent with repression of the accumulation of mitotic cyclins in the developing trichome by CCS52A1. As these M-phase-specific cyclins are known to accumulate in sim mutant trichomes, our data suggest that CCS52A1 and SIM cooperate in repressing accumulation of mitotic cyclins to establish the trichome endocycle. Comparison with endoreplication pathways in Drosophila and mammals indicates that while these organisms all use similar components to initiate endoreplication, the components are deployed differently in each organism.

Figure 1.—

Identification of a genetic enhancer of the sim multicellular phenotype. (A) Scanning electron micrograph (SEM) of wild-type unicellular Col-0 trichome. (B) SEM of a sim-1 mutant trichome consisting of three cells. Arrows indicate cell junctions. (C) SEM of a ccs52a1-3 sim-1 double-mutant trichome initiation site (TIS). All cells (at least 11) extending from the epidermis would be considered part of the same TIS. Arrows indicate two cell junctions. (D) Cross section through a ccs52a1-3 sim-1 TIS. Arrows point to two of the eight cells visible. (E) SEM of a ccs52a1-3 single-mutant trichome. (F) SEM of a ccs52a1-T1 T-DNA insertion mutant trichome. Scale bars in A, B, C, and E are 100 μm; in D and F, they are 50 μm.

Figure 2.—

Identification of the CCS52A1 gene as the genetic enhancer. (A) Structure of the AT4G22910 locus, showing the location of the ccs52a1 mutations referred to in the text. Solid boxes are exons. (B) Wild-type Col-0 trichomes. (C) ccs52a1 trichomes. (D) Complementation of ccs52a1-3 by proGL2:CCS52A1. (E) Suppression of ccs52a1-3 phenotype by proGL2:CCS52A2. Scale bars are 4 mm in B and C, and 2 mm in D and E.

Figure 3.—

Effect of ccs52a1 and ccs52a2 mutations on endoreplication in trichomes. (A) DNA content of DAPI-stained trichome nuclei of the indicated genotypes measured in situ, presented as relative fluorescence units (RFU). Data were normalized to an assumed wild-type mean of 32C for Col trichomes. Data are presented as box plots, where the box encompasses the 25th through the 75th percentile of the data, the line within the box is the median (50th percentile), and the error bars represent the 5th (bottom bar) and 95th (top bar) percentiles. All pairwise comparisons are significant at P < 0.05 except ccs52a1-3 vs. ccs52a1-T2 and Col-0 vs. ccs52a2 T-DNA. (B) Nuclear DNA content of Col-0 and ccs52a1-3 21-day-old cotyledons. (C) Nuclear DNA content of Col-0 and ccs52a1-3 21-day-old leaves. For B and C, DNA content was determined by flow cytometry.

Figure 4.—

Expression of either CCS52A1 or CCS52A2 in trichomes from the GL2 promoter results in increased trichome branching and suppression of the multicellular trichome phenotype of sim. (A) Wild-type Col-0 trichome. (B) proGL2:CCS52A1 trichome. (C) proGL2:CCS52A2 trichome. (D) sim-1 multicellular trichome. (E) sim-1 proGL2:CCS52A1 trichome. (F) sim-1 proGL2:CCS52A2 trichome. All images are SEMs. Scale bars, 100 μm.

Figure 5.—

Expression of either CCS52A1 or CCS52A2 in trichomes from the GL2 promoter increases the level of endoreplication. DNA content of DAPI-stained trichome nuclei of the indicated genotypes measured in situ, presented as RFU, normalized to an assumed wild-type mean of 32C for Col-0 trichomes. Data are presented as box plots, where the box encompasses the 25th through the 75th percentile of the data, the line within the box is the median (50th percentile), and the error bars represent the 5th (bottom bar) and 95th (top bar) percentiles. (A) Col-0 wild-type and four independent proGL2:CCS52A1 transgenic lines. Pairwise comparisons of Col-0 with each of the four transgenic lines were significant at P < 0.05. (B) Col-0 wild-type and four independent proGL2:CCS52A2 lines. Pairwise comparisons of Col-0 with each of the four transgenic lines were significant at P < 0.05.

Figure 6.—

Expression of B-type cyclins in ccs52a1 mutant trichomes results in multicellular trichomes. (A) Wild-type Col-0 trichome. (B) ccs52a1-3 trichome. (C) proGL2:CYCB1;1 trichome. (D) ccs52a1-3 proGL2:CYCB1;1 multicellular trichome. (E) proGL2:CYCB1;2 trichome (F) ccs52a1-3 proGL2:CYCB1;2 multicellular trichome.

Figure 7.—

Expression of B-type cyclins in ccs52a1-3 mutant trichomes strongly inhibits endoreplication. DNA content of DAPI-stained trichome nuclei of the indicated genotypes measured in situ, presented as RFU, normalized to an assumed wild-type mean of 32C for Col-0 trichomes. The CYCB1;1 and CYCB1;2 transgenes were expressed from the GL2 promoter. Data are presented as box plots, where the box encompasses the 25th through the 75th percentile of the data, the line within the box is the median (50th percentile), and the error bars represent the 5th (bottom bar) and 95th (top bar) percentiles. Two-way comparisons of CYCB1;1 vs. ccs52a1-3 CYCB1;1 and CYCB1;2 vs. ccs52a1-3 CYCB1;2 were significant at P < 0.05.