Identification of Arabidopsis meiotic cyclins reveals functional diversification among plant cyclin genes

Abstract

Meiosis is a modified cell division in which a single S-phase is followed by two rounds of chromosome segregation resulting in the production of haploid gametes. The meiotic mode of chromosome segregation requires extensive remodeling of the basic cell cycle machinery and employment of unique regulatory mechanisms. Cyclin-dependent kinases (CDKs) and cyclins represent an ancient molecular module that drives and regulates cell cycle progression. The cyclin gene family has undergone a massive expansion in angiosperm plants, but only a few cyclins were thoroughly characterized. In this study we performed a systematic immunolocalization screen to identify Arabidopsis thaliana A- and B-type cyclins expressed in meiosis. Many of these cyclins exhibit cell-type-specific expression in vegetative tissues and distinct subcellular localization. We found six A-type cyclins and a single B-type cyclin (CYCB3;1) to be expressed in male meiosis. Mutant analysis revealed that these cyclins contribute to distinct meiosis-related processes. While A2 cyclins are important for chromosome segregation, CYCB3;1 prevents ectopic cell wall formation. We further show that cyclin SDS does not contain a D-box and is constitutively expressed throughout meiosis. Analysis of plants carrying cyclin SDS with an introduced D-box motif determined that, in addition to its function in recombination, SDS acts together with CYCB3;1 in suppressing unscheduled cell wall synthesis. Our phenotypic and expression data provide extensive evidence that multiplication of cyclins is in plants accompanied by functional diversification.

Figure 1. Immunolocalization of CYC:GUS proteins in PMCs.

DNA was counterstained with DAPI, microtubules were detected with anti-α-tubulin antibody. Bar = 10 µm.

Figure 2. Meiotic defects in cyca2;234 mutants.

(A) Pollen viability in cyca2;2 and cyca2;234 mutants determined by Alexander staining. Bars = 100 µm. (B) Meiotic chromosomes stained by DAPI in PMCs from cyca2;234 mutants. Bar = 10 µm.

Figure 3. Ectopic cell wall-like structures in cycb3;1 mutants.

(A–C) PMCs from cycb3;1 plants visualized by epifluorescence (left panel) and bright field (right panel) microscopy. DNA is counterstained with DAPI (white in A, red in B,C), microtubules (green in B,C) were detected with anti-α-tubulin antibody. (A) early anaphase I, (B) interkinesis, (C) metaphase II. Bars = 10 µm. (D,E) Section through anthers with PMCs in late meiosis I visualized by transmission scan electron microscopy. Arrows indicate ectopic cell wall-like structures, c – callose, Tp – tapetum cells. Bars represent 3 µm.

Figure 4. Cyclin SDS contributes to suppressing ectopic cell wall-like structures.

(A) PMCs from cycb3;1-1 sds mutants visualized by epifluorescence (left panel) and BF (right panel) microscopy. DNA is counterstained with DAPI. Bars = 10 µm. (B) Section through anthers with PMCs in early meiosis I. Arrows point to cell walls separating PMCs. Bars represent 5 µm. (C) Diagram of the SDS protein indicating D-box insertion. (D) Expression of the SDS:GUS and SDS-D-box:GUS constructs in inflorescence. (E) Immunolocalization of SDS:GUS and SDS-D-box:GUS proteins in PMCs. Bars = 10 µm. (F) Pollen viability determined by Alexander staining in sds mutatns complemented with SDS or SDS-D-box transgenes. Bars = 100 µm. (G) Frequency of meiocytes containing cell-wall like structure. At least 100 meiocytes were counted in each category.

Figure 5. Pollen development in sds cycb3;1 smg7 and sds cycb3;1 tdm1 mutants.

(A–C) Spreads of PMCs from sds cycb3;1 smg7 mutants. DNA was stained with DAPI. Besides meiocytes arrested at anaphase II (A), sds cycb3;1 smg7 mutants contained also PMCs with decondensed nuclei (B,C). Bars = 10 µm. (D) Pollen viability determined by Alexander staining in sds cycb3;1 smg7 and sds cycb3;1 tdm1 mutants. Bars = 100 µm.

Figure 6. Overview of Arabidopsis meiotic cyclins.

The diagram depicts expression of cyclins in the course of male meiosis and indicates their role in diverse meiotic processes.