LITTLE NUCLEI genes affecting nuclear morphology in Arabidopsis thaliana

Abstract

Efforts to understand nuclear organization in plant cells have received little assistance from the better-studied animal nuclei, because plant proteomes do not contain recognizable counterparts to the key animal proteins involved in nuclear organization, such as lamin nuclear intermediate filament proteins. Previous studies identified a plant-specific insoluble nuclear protein in carrot (Daucus carota), called Nuclear Matrix Constituent Protein1 (NMCP1), which contains extensive coiled-coil domains and localizes to the nuclear periphery. Here, we describe a genetic characterization of two NMCP1-related nuclear proteins in Arabidopsis thaliana, LITTLE NUCLEI1 (LINC1) and LINC2. Disruption of either gene caused a reduction in nuclear size and altered nuclear morphology. Moreover, combining linc1 and linc2 mutations had an additive effect on nuclear size and morphology but a synergistic effect on chromocenter number (reduction) and whole-plant morphology (dwarfing). The reduction in nuclear size in the linc1 linc2 double mutant was not accompanied by a corresponding change in endopolyploidy. Rather, the density of DNA packaging at all endopolyploid levels in the linc1 linc2 mutants was increased significantly. Our results indicate that the LINC coiled-coil proteins are important determinants of plant nuclear structure.

Figure 1.

The LINC Protein Family. (A) The protein organization of Dc NMCP1 is compared with that of Arabidopsis LINC1 to LINC4. Putative coiled-coiled domains are shown in black, and the lengths of the proteins are shown (number of amino acids [a.a.]). (B) An unrooted phylogenetic distance tree showing the probable relationships among 12 homologs of Dc NMCP1. (C) Selected regions of the multiple sequence alignment used to construct the phylogenetic tree shown in (B) are detailed.

Figure 2.

Nuclear Localization of LINC1 and LINC2. Localization of LINC1-YFP and LINC2-YFP fusion proteins in transgenic linc1-1 linc2-1 Arabidopsis root cells expressing either a LINC1-YFP or LINC2-YFP transgene under the control of the constitutive 35S viral promoter. (A) and (B) YFP signals from transgenic LINC1-YFP roots at the root meristem (A) and from a differentiated cell (B). (C) and (D) YFP signals from transgenic LINC2-YFP roots from the meristem (C) and from a differentiated cell (D). LINC1-YFP is localized predominantly at the nuclear periphery, and LINC2-YFP is localized diffusely throughout the nucleoplasm and is partially excluded from the nucleolus. Bars = 5 μm.

Figure 3.

Morphological Phenotypes of linc1 and linc2 Mutants. Representative plants from an F2 family segregating linc1-1 and linc2-1. Genotypes are indicated above the plants. Only linc1-1 linc2-1 plants have an obvious whole-plant phenotype.

Figure 4.

Endopolyploidy Measurements in linc1 and linc2 Mutants. Nuclear flow cytometry analysis of wild-type and linc mutant nuclei. (A) Representative flow cytometry histogram plots of nuclei isolated from 4-week-old leaf tissue. From left are data for the wild type, linc1-1, linc2-1, and linc1-1 linc2-1. (B) Aggregate data from individual histograms of independent wild-type (white columns; n = 4) and linc1-1 linc2-1 mutant (black columns; n = 12) samples.

Figure 5.

Nuclear Morphology Changes in linc1 and linc2 Mutants. (A) and (B) Epifluorescence images of representative DAPI-stained nuclei isolated from 4-week-old leaf tissue from wild-type (A) or linc1-1 linc2-1 (B) plants. Bars = 5 μm. (C) and (D) Confocal projection images of propidium iodide–stained nuclei in whole tissue anther filament cells from wild-type (C) or linc1-1 linc2-1 (D) plants. Bars = 10 μm. (E) Histogram of nuclear areas (μm²) as measured from DAPI-stained nuclei isolated from 4-week-old leaves. Blue, wild type (n = 518); red, linc1-1 (n = 526); yellow, linc2-1 (n = 479); green, linc1-1 linc2-1 (n = 657). (F) Average nuclear volume (μm³) ± se of propidium iodide–stained nuclei within anther filament tissue. Blue, wild type (n = 14); green, linc1-1 linc2-1 (n = 30). (G) Histogram of nuclear circularity indices (4πA/P²) as measured from DAPI-stained nuclei isolated from 4-week-old leaves. Blue, wild type (n = 518); red, linc1-1 (n = 526); yellow, linc2-1 (n = 479); green, linc1-1 linc2-1 (n = 657). (H) Bar graph displaying the average number of chromocenters ± se observed in propidium iodide–stained nuclei isolated from 2-week-old root tissue. Blue, wild type (n = 150); red, linc1-1 (n = 116); yellow, linc2-1 (n = 117); green, linc1-1 linc2-1 (n = 220).

Figure 6.

Increased DNA Density in linc1-1 linc2-1 Mutants. Scatterplot of nuclear area (μm²; x axis) versus total DAPI signal per nucleus (y axis). Nuclei were isolated from 4-week-old leaf tissue. Open diamonds, wild type; closed diamonds, linc1-1 linc2-1.