Arabidopsis CROWDED NUCLEI (CRWN) proteins are required for nuclear size control and heterochromatin organization

Abstract

Background: Plant nuclei superficially resemble animal and fungal nuclei, but the machinery and processes that underlie nuclear organization in these eukaryotic lineages appear to be evolutionarily distinct. Among the candidates for nuclear architectural elements in plants are coiled-coil proteins in the NMCP (Nuclear Matrix Constituent Protein) family. Using genetic and cytological approaches, we dissect the function of the four NMCP family proteins in Arabidopsis encoded by the CRWN genes, which were originally named LINC (LITTLE NUCLEI).

Results: CRWN proteins are essential for viability as evidenced by the inability to recover mutants that have disruptions in all four CRWN genes. Mutants deficient in different combinations of the four CRWN paralogs exhibit altered nuclear organization, including reduced nuclear size, aberrant nuclear shape and abnormal spatial organization of constitutive heterochromatin. Our results demonstrate functional diversification among CRWN paralogs; CRWN1 plays the predominant role in control of nuclear size and shape followed by CRWN4. Proper chromocenter organization is most sensitive to the deficiency of CRWN4. The reduction in nuclear volume in crwn mutants in the absence of a commensurate reduction in endoreduplication levels leads to an increase in average nuclear DNA density.

Conclusions: Our findings indicate that CRWN proteins are important architectural components of plant nuclei that play diverse roles in both heterochromatin organization and the control of nuclear morphology.

Figure 1

Phylogenetic relationships among CRWN proteins. A maximum likelihood tree of CRWN homologs constructed from an alignment of amino acid sequences that correspond to the coiled-coil domains. Bootstrap values (of 1000 replicates) are indicated on each branch. The A. thaliana CRWN proteins are indicated in bold, and homologs are labeled with the genus name and an assigned number (Additional file 1: Table S1). Two major clades are marked by blue and green; the yellow inset oval indicates a subgroup of CRWN4-like proteins from dicots that lack the conserved C-terminal domain (Additional file 2).

Figure 2

Whole plant phenotypes of crwn mutants. Leaf rosette structure of one month-old plants imaged just after initiation of flowering. Representative plants for the various genotypes are compared to a wild type (WT) Columbia plant. All plants were grown in parallel and photographed at the same magnification; the diameter of a WT rosette at flowering is ca. 8 cm.

Figure 3

Nuclear phenotypes of crwn mutants. Images of representative DAPI-stained adult leaf cell nuclei from wild type and the twelve viable crwn mutants. The top row contains the wild type control and crwn2, crwn3 and crwn2 crwn3 mutants with normal nuclear morphology phenotypes. The middle row shows nuclear phenotypes of crwn1 and double mutants containing a crwn1 mutation. The bottom row displays nuclear phenotypes from crwn4 mutants, as well as higher-order mutants containing a crwn4 mutation. The arrowheads highlight thin projections from crwn4 nuclei. A 5 μm size bar is shown in the upper left inset; all images are shown at the same magnification.

Figure 4

The effects of crwn mutations on nuclear size and nuclear DNA density in leaf cells. (A) Developmentally matched rosette leaves from approximately one month-old plants were fixed, cells isolated and stained with DAPI, and nuclei imaged using epifluorescence microscopy. The average areas of randomly-selected individual nuclei (n = 32–108) were determined for each genotype. Error bars indicated standard error of the mean. (B) Nuclear area measurements from panel A (see also, Additional file 4) were converted to relative values and were plotted against average endopolyploidy level for each genotype expressed as a fraction of the wild-type value. The average endopolyploidy levels (Additional file 4) were measured by flow cytometry using corresponding leaf samples. The diagonal dashed line indicates the expected linear relationship between nuclear area and endopolyploid level observed in wild-type plants. Note that panel A measures nuclear area but the isolated nuclei are flattened under a coverslip to a uniform thickness (see Additional file 6) and therefore nuclear area is proportional to and therefore a proxy for nuclear volume. The numbers next to the symbols indicate the corresponding crwn genotype. Data points above the dashed line indicate an elevated nuclear DNA density relative to wild type. The solid line corresponds to a nuclear DNA density four-times that of wild type.

Figure 5

Average leaf guard cell nuclear sizes in crwn mutants. Two week-old plants were harvested, fixed, stained with DAPI, and leaf guard cell nuclei were imaged using confocal laser scanning microscopy. The area of randomly-selected individual nuclei (n = 19–29) were determined for each genotype. Error bars indicated standard error of the mean.

Figure 6

Chromocenter morphology changes in crwn mutants. (A) Nuclei were imaged from developmentally matched rosette leaves from approximately one month-old plants, cells isolated and stained with DAPI, and nuclei imaged using epifluorescence microscopy. The area and chromocenter number of randomly-selected individual nuclei (n = 47–132) were determined for each genotype, and chromocenter number was plotted against nuclear area. A linear regression line showing the relationship between chromocenter number and nuclear size (as a proxy for endopolyploidy level) was plotted for each genotype. (B) Nuclei were imaged fully expanded rosette leaves from approximately one month-old plants and the chromocenter Aggregation Index (AI) was plotted against the total chromocenter area (μm²).

Figure 7

Chromocenter organization is altered in crwn1 crwn2 and crwn4 mutants. (A and B) Fluorescence in situ hybridization of representative small and large nuclei from wild type, crwn1 crwn2 and crwn4 cells prepared from leaves of adult plants after bolting. Blue: DAPI, pink: 180-bp centromere repeats, green: 5S RNA genes, bar, 5 μm. (C) The distribution of nuclei based on the number of centromere repeat clusters in different genotypes. Each circle represents an individual nucleus in the FISH experiment. The color indicates the level of decondensation of the centromere signal. (D) The distribution of nuclei based on the number of 5S RNA repeat clusters in different genotypes. Each circle represents an individual nucleus. The color indicates the level of dispersion of the 5S repeat signal.