No abstract

Several histone variants are posttranslationally phosphorylated. Little is known about phosphorylation of the centromere-specific histone 3 (CENH3) variant in plants. We show that CENH3 of Arabidopsis thaliana is phosphorylated in vitro by Aurora3, predominantly at serine 65. Interaction of Aurora3 and CENH3 was found by immunoprecipitation (IP) in A. thaliana and by bimolecular fluorescence complementation. Western blotting with an anti-CENH3 pS65 antibody showed that CENH3 pS65 is more abundant in flower buds than elsewhere in the plant. Substitution of serine 65 by either alanine or aspartic acid resulted in a range of phenotypic abnormalities, especially in reproductive tissues. We conclude that Aurora3 phosphorylates CENH3 at S65 and that this post-translational modification is required for the proper development of the floral meristem.

Condensins are best known for their role in shaping chromosomes. Other functions such as organizing interphase chromatin and transcriptional control have been reported in yeasts and animals, but little is known about their function in plants. To elucidate the specific composition of condensin complexes and the expression of CAP-D2 (condensin I) and CAP-D3 (condensin II), we performed biochemical analyses in Arabidopsis. The role of CAP-D3 in interphase chromatin organization and function was evaluated using cytogenetic and transcriptome analysis in cap-d3 T-DNA insertion mutants. CAP-D2 and CAP-D3 are highly expressed in mitotically active tissues. In silico and pulldown experiments indicate that both CAP-D proteins interact with the other condensin I and II subunits. In cap-d3 mutants, an association of heterochromatic sequences occurs, but the nuclear size and the general histone and DNA methylation patterns remain unchanged. Also, CAP-D3 influences the expression of genes affecting the response to water, chemicals, and stress. The expression and composition of the condensin complexes in Arabidopsis are similar to those in other higher eukaryotes. We propose a model for the CAP-D3 function during interphase in which CAP-D3 localizes in euchromatin loops to stiffen them and consequently separates centromeric regions and 45S rDNA repeats.

Condensins are best known for their role in shaping chromosomes. However, other functions as organizing interphase chromatin and transcriptional control have been reported in yeasts and animals. Yeasts encode one condensin complex, while higher eukaryotes have two of them (condensin I and II). Both, condensin I and II, are conserved in Arabidopsis thaliana, but so far little is known about their function. Here we show that the A. thaliana CAP-D2 (condensin I) and CAP-D3 (condensin II) subunits are highly expressed in mitotically active tissues. In silico and pull-down experiments indicate that both CAP-D proteins interact with the other condensin I and II subunits. Our data suggest that the expression, localization and composition of the condensin complexes in A. thaliana are similar as in other higher eukaryotes. Previous experiments showed that the lack of A. thaliana CAP-D3 leads to centromere association during interphase. To study the function of CAP-D3 in chromatin organization more in detail we compared the nuclear distribution of rDNA, of centromeric chromocenters and of different epigenetic marks, as well as the nuclear size between wild-type and cap-d3 mutants. In these mutants an association of heterochromatic sequences occurs, but nuclear size and the general methylation and acetylation patterns remain unchanged. In addition, transcriptome analyses revealed a moderate influence of CAP-D3 on general transcription, but a stronger one on transcription of stress-related genes. We propose a model for the CAP-D3 function during interphase, where CAP-D3 localizes in euchromatin loops to stiff them, and consequently separates centromeric regions and 45S rDNA repeats.