CDKG1 Is Required for Meiotic and Somatic Recombination Intermediate Processing in Arabidopsis

Abstract

The Arabidopsis (Arabidopsis thaliana) cyclin-dependent kinase G1 (CDKG1) is necessary for recombination and synapsis during male meiosis at high ambient temperature. In the cdkg1-1 mutant, synapsis is impaired and there is a dramatic reduction in the number of class I crossovers, resulting in univalents at metaphase I and pollen sterility. Here, we demonstrate that CDKG1 is necessary for the processing of recombination intermediates in the canonical ZMM recombination pathway and that loss of CDKG1 results in increased class II crossovers. While synapsis and events associated with class I crossovers are severely compromised in a cdkg1-1 mutant, they can be restored by increasing the number of recombination intermediates in the double cdkg1-1 fancm-1 mutant. Despite this, recombination intermediates are not correctly resolved, leading to the formation of chromosome aggregates at metaphase I. Our results show that CDKG1 acts early in the recombination process and is necessary to stabilize recombination intermediates. Finally, we show that the effect on recombination is not restricted to meiosis and that CDKG1 is also required for normal levels of DNA damage-induced homologous recombination in somatic tissues.

Figure 1.

The cdkg1-1 Mutation Increases Bivalent Formation in the msh5-2 Mutant Background. (A) DAPI-stained metaphase I spreads. Bar = 2 µm. (B) Ratio of bivalent to univalent pairs present at metaphase I. Error bars represent average ± sd, and n indicates the number of metaphases counted for each genotype. Asterisk indicates that the bivalent number in the cdkg1-1 msh5-2 mutant is significantly different from the single msh5-2 mutant for P < 0.001, two-tailed t test. (C) Fertility counts in the wild type (Col-0) and indicated mutants. Graphs show mean and interquartile range as well as the actual seed counts. For each genotype, at least 30 siliques from three independent plants were counted. Superscript letters indicate the significance groups for P < 0.001 calculated using ANOVA, with post hoc pairwise t tests using nonpooled sd and Bonferroni correction.

Figure 2.

Bivalent Distribution Comparisons for Simulated and Experimentally Observed Meioses. Bivalent distributions for observed (pink) and simulated (blue) meioses for cdkg1-1, msh5-2, and cdkg1-1 msh5-2. For simulations, the number of class I (CI) COs was fixed based on experimental observations, and the number of class II (CII) COs varied from 0.5 to 7 per meiosis. Bivalent distributions of best fit simulations are shown (*). In addition, bivalent distributions for cdkg1-1 and cdkg1-1 msh5-2 are compared with those from simulated meiosis with 1.16 CII COs (the best fit value for msh5-2), and the msh5-2 bivalent distribution is compared with simulated meiosis with 3.41 CII COs (the best fit for cdkg1-1 msh5-2). P-values are Bonferroni-corrected values derived from two-sample Kolmogorov–Smirnov tests.

Figure 3.

The Meiotic Phenotype of the Double cdkg1-1 mus81-2 Mutant Is Similar to the Single cdkg1-1 Mutant. (A) DAPI-stained metaphase I spreads of the indicated mutants. Bar = 2 µm. (B) Ratio of bivalent to univalent pairs present at metaphase I. Error bars represent average ± sd, and n indicates the number of metaphases counted for each genotype. (C) Fertility counts in the indicated mutants. Graphs show mean and interquartile range as well as the actual seed counts. For each genotype at least 30 siliques from three independent plants were counted. Superscript letters indicate the significance groups for P < 0.001 calculated using ANOVA, with post hoc pairwise t tests using nonpooled sd and Bonferroni correction. (D) Number of MLH1 foci observed in the indicated mutants. Graphs show mean and interquartile range as well as the actual foci counts. Superscript letters indicate the significance groups for P < 0.001 calculated using ANOVA, with post hoc pairwise t tests using nonpooled sd and Bonferroni correction. (E) Immunolocalization of the class I CO marker protein MLH1 in the indicated mutants. The axial element protein ASY1 is labeled in red, the central element protein ZYP1 in gray, and MLH1 foci in green (top panels). The MLH1 channel (green) is also shown separately (bottom panels). Images represent maximum projections of Z-stacks. Bar = 2 µm.

Figure 4.

Chromosome Synapsis Is Restored in the cdkg1-1 fancm-1 Double Mutant. (A) Immunolocalization of the axial element protein ASY1 (red) and the central element protein ZYP1 (gray) in pachytene nuclei of Col-0, cdkg1-1, fancm-1, and cdkg-1-1 fancm-1 mutant plants. DNA is counterstained with DAPI (blue), and a merge of all channels is shown. Images represent maximum projections of Z-stacks. Bar = 2 µm. (B) Three-dimensional reconstruction of a whole nucleus and individual bivalents from a cdkg1-1 pachytene-like nucleus and a cdkg1-1 fancm-1 pachytene nucleus. The nuclei were processed using Imaris software, and each bivalent pair was isolated and false colored. Unpaired regions are marked by the presence of ASY1 (red) and paired regions by the presence of ZYP1 (gray). Bar = 2 µm.

Figure 5.

Class I CO Formation Is Restored in the cdkg1-1 fancm-1 Double Mutant. (A) Immunolocalization of the class I CO marker proteins (green) MLH1 (top panel) and HEI10 (bottom panel) in diplotene nuclei of Col-0, cdkg1-1, fancm-1, and cdkg-1-1 fancm-1 mutant plants. DNA is counterstained with DAPI (blue). Images represent maximum projections of Z-stacks. Bar = 2 µm. (B) and (C) Number of MLH1 (B) and HEI10 (C) foci observed in the indicated mutants. Graphs show mean and interquartile range as well as the actual foci counts. Superscript letters indicate the significance groups for P < 0.001, calculated using ANOVA, with post hoc pairwise t tests using nonpooled sd and Bonferroni correction.

Figure 6.

CDKG1 Is Necessary for the Resolution of Recombination Intermediates in the fancm-1 Mutant. (A) DAPI-stained chromosome spreads of different meiotic stages in the cdkg1-1, fancm-1, and cdkg1-1 fancm-1 mutant as indicated. While in the single mutant five bivalents are observed at metaphase I, in the double mutant chromosome aggregates are present at metaphase I and chromosome bridges observed at anaphase I. Bar = 2 µm. (B) Ratio of bivalent, univalent pairs and chromosome aggregates present at metaphase I. Error bars represent average ± sd, and n indicates the number of metaphases counted for each genotype. (C) Fertility counts in the indicated mutants. Graphs show mean and interquartile range as well as the actual seed counts. For each genotype at least 30 siliques from three independent plants were counted. Superscript letters indicate the significance groups for P < 0.001, calculated using ANOVA, with post hoc pairwise t tests using nonpooled sd and Bonferroni correction.

Figure 7.

In the Absence of CDKG1, the fancm-1 Mutation Is Not Able to Rescue the msh5-2 Phenotype. (A) DAPI-stained metaphase I spreads. Bar = 2 µm. (B) Ratio of bivalent, univalent pairs and chromosome aggregates present at metaphase I. Error bars represent average ± sd, and n indicates the number of metaphases counted for each genotype. (C) Fertility counts in the indicated mutants. Graphs show mean and interquartile range as well as the actual seed counts. For each genotype at least 30 siliques from three independent plants were counted. Superscript letters indicate the significance groups for P < 0.001, calculated using ANOVA, with post hoc pairwise t tests using nonpooled sd and Bonferroni correction.

Figure 8.

Rates of Somatic HR Are Reduced in the cdkg1-1 Mutant. (A) Spontaneous recombination rates in Col-0 and the cdkg1-1 mutant. Graphs represent averages ± sd for 10 plants. Asterisks indicate values that are significantly different for P < 0.001, using two-tailed t test. (B) and (C) Recombination rates for Col-0 and the cdkg1-1 mutant in the presence of 7 µM bleomycin (B) or 5 µM cisplatin (C). Graphs represent averages ± sd for 10 plants. (D) Percentage of root growth in media containing 0.25 µM bleomycin for Col-0, cdkg1-1, and sog1-1 seedlings compared with growth in media with no bleomycin. Graphs represent averages ± sd for thirty 8-d-old seedlings. Superscript letters indicate the significance groups for P < 0.001 calculated using ANOVA, with post hoc pairwise t tests using nonpooled sd and Bonferroni correction. (E) Expression of the HR repair pathway gene RAD51 in Col-0, the cdkg1-1 mutant, and atm-1 control 8-d-old seedlings, treated or not with 10 µM bleomycin for 3 h, as determined by qPCR.

Figure 9.

A Model for the Impact of cdkg1-1 and msh5-2 Mutations on the Class I (CI) CO, Class II (CII) CO, and Non-crossover (NCO) Pathways in Arabidopsis. Arrow thickness relates to the proportion of the initial DSBs going through each pathway. Numbers indicate the number of DSBs channeled to each pathway. DSBs: ∼200 DMC1/RAD51/γH2AX foci are observed in early meiotic prophase (Choi et al., 2013; Girard et al., 2015; Séguéla-Arnaud et al., 2015; Xue et al., 2018). ZMM intermediates: ∼100 MSH4/MSH5/HEI10 foci are observed in the wild-type leptotene/zygotene (Higgins et al., 2004, 2008a; Chelysheva et al., 2012). In cdkg1-1, we observe ∼20 HEI10 foci in leptotene/zygotene. CI, class I CO.