Depletion of KNL2 Results in Altered Expression of Genes Involved in Regulation of the Cell Cycle, Transcription, and Development in Arabidopsis

Abstract

Centromeres contain specialized nucleosomes at which histone H3 is partially replaced by the centromeric histone H3 variant cenH3 that is required for the assembly, maintenance, and proper function of kinetochores during mitotic and meiotic divisions. Previously, we identified a KINETOCHORE NULL 2 (KNL2) of Arabidopsis thaliana that is involved in the licensing of centromeres for the cenH3 recruitment. We also demonstrated that a knockout mutant for KNL2 shows mitotic and meiotic defects, slower development, reduced growth rate, and fertility. To analyze an effect of KNL2 mutation on global gene transcription of Arabidopsis, we performed RNA-sequencing experiments using seedling and flower bud tissues of knl2 and wild-type plants. The transcriptome data analysis revealed a high number of differentially expressed genes (DEGs) in knl2 plants. The set was enriched in genes involved in the regulation of the cell cycle, transcription, development, and DNA damage repair. In addition to comprehensive information regarding the effects of KNL2 mutation on the global gene expression, physiological changes in plants are also presented, which provides an integrated understanding of the critical role played by KNL2 in plant growth and development.

Keywords: Arabidopsis; KNL2; RNA-seq; centromere; kinetochores.

Figure 1

Differential gene expression. Venn diagram showing the number of highly differentially expressed genes (DEGs) (2-fold cutoff, false discovery rate (FDR) corrected p-value < 0.05) obtained by comparing knl2 versus wild-type ecotype Columbia-0 (Col-0) genes in seedlings and knl2 versus Col-0 genes in flower buds.

Figure 2

Transposable elements and their amount identified in the knl2 mutant line. Information is given about transposons up- and downregulated in the knl2 seedlings and knl2 flower buds.

Figure 3

Effect of KNL2 depletion on the root development. (A) Representative phenotypes of wild-type (WT) and knl2 plants germinated and grown for seven days on ½ Murashige and Skoog (MS) medium. Bar = 10 mm. (B) Quantitative data for plants described in (A). Data are the means ± standard errors, n = 20. Student’s t-test, p < 0.01.

Figure 4

Sensitivity of knl2 plants to DNA damage. (A) Representative phenotypes of wild-type (WT) and knl2 plants grown for 14 days on mitomycin C (MMC)-containing media. Bar = 10 mm. (B) Quantitative data for plants grown as described in (A). Error bars represent the standard deviation between the means of three biological replicates, each represented by 15 to 20 plants. Letters above the bars indicate statistically significantly different groups in ANOVA (p-value ≤ 0.05) and post hoc Tukey‘s test. (C) Analysis of propidium iodide (PI) uptake (dark sectors) in roots of WT and knl2 plants. Four-day-old plants were treated with the specified concentration of MMC for 24 h prior to analysis. Bar = 50 µm.

Figure 5

Differentially expressed genes encoding transcription factors in the knl2 mutant line. Expression in knl2 flower buds and knl2 seedlings is depicted as F and S, respectively. Red and blue represent up- and downregulated differentially expressed genes, respectively. All values are log₂ transformed. The gradient color illustrates the expression value.

Figure 6

Effect of KNL2 depletion on the flowering time. (A) At early growth stages, no obvious phenotypical differences between wild-type (WT) and the knl2 mutant were observed. (B) The flowering time of the knl2 mutant delayed by 10–14 days compared to Arabidopsis wild-type. Seeds of the knl2 mutant and wild-type were germinated under short-day conditions, 8 h light/20° C and 16 h dark/18 °C, for two weeks and then plants were transferred to the cultivation room with long-day conditions, 16 h light/20 °C and 8 h dark/18 °C.

Figure 7

qRT–PCR validation of RNA-seq results. Eight genes were selected from the genes differentially expressed in seedlings according to the RNA-seq data analysis. Detailed annotation of the selected genes is presented in Table S2 (Supplementary Materials). *At5g02520 corresponds to KNL2. Error bars indicate the standard error of three biological replicates in qRT-PCR.