Maize VKS1 Regulates Mitosis and Cytokinesis During Early Endosperm Development

Abstract

Cell number is a critical factor that determines kernel size in maize (Zea mays). Rapid mitotic divisions in early endosperm development produce most of the cells that make up the starchy endosperm; however, the mechanisms underlying early endosperm development remain largely unknown. We isolated a maize mutant that shows a varied-kernel-size phenotype (vks1). Vks1 encodes ZmKIN11, which belongs to the kinesin-14 subfamily and is predominantly expressed in early endosperm development. VKS1 dynamically localizes to the nucleus and microtubules and plays key roles in the migration of free nuclei in the coenocyte as well as in mitosis and cytokinesis in early mitotic divisions. Absence of VKS1 has relatively minor effects on plants but causes deformities in spindle assembly, sister chromatid separation, and phragmoplast formation in early endosperm development, thereby resulting in reduced cell proliferation. Severities of aberrant mitosis and cytokinesis within individual vks1 endosperms differ, thereby resulting in varied kernel sizes. Our discovery highlights VKS1 as a central regulator of mitosis in early maize endosperm development and provides a potential approach for future yield improvement.

Figure 1.

Nonuniform Small-Kernel Phenotype of vks1. (A) to (D) Ear phenotypes of A619, vks1, A619 × vks1, and vks1 × A619. Three mutant seeds on a homozygous vks1 ear representing mild, medium, and severe developmental phenotypes are indicated by an asterisk, a rhombus, and an arrowhead, respectively. Bar = 1 cm. (E) Seed phenotypes of A619, vks1, A619 × vks1, and vks1 × A619. The vks1 seeds from (B) with mild, medium, and severe phenotypes are indicated by an asterisk, a rhombus, and an arrowhead, respectively. Bar = 1 cm. (F) Seed weight variation of single kernels in A619, vks1, A619 × vks1, and vks1 × A619. The number beside each column indicates the coefficient of variation. **, P < 0.01 as determined by Student’s t test. (G) and (H) Semithin sections of A619 (G) and vks1 (H) endosperms at 4 DAP. Bars = 100 μm. Em, Embryo; En, Endosperm; N, Nucellus. (I) and (J) Semithin sections of A619 (I) and vks1 (J) endosperms at 6 DAP. Bars = 100 μm.Em, Embryo; En, Endosperm. (K) and (L) Sections of A619 (K) and vks1 (L) endosperms at 18 DAP. Bars = 1 mm.Em, Embryo; En, Endosperm. (M) Cell numbers of A619 and vks1 endosperms at 4 and 6 DAP. **, P < 0.01 asd determined by Student’s t test. (N) Relative endosperm area at 4, 6, and 18 DAP. The ratio of the area of the endosperm to that of the whole seed in A619 and vks1 is shown The ratio in A619 was set as 1. **, P < 0.01 as determined by Student’s t test.

Figure 2.

Map-Based Cloning and Genetic Confirmation of vks1. (A) Mapping by sequencing of vks1. Ratios of SNP index (A619/−) between different pools of segregating phenotypic classes (top panel, normal phenotype in pool A: A619/A619 and A619/−; middle panel, vks1 phenotype in pool B: −/−; and bottom panel, SNP index between the top and middle panels) are shown for a region near the left terminal end of chromosome 7. (B) The vks1 mapping interval. An SNP in the eighth exon of Zm00001d018624 results in a CGA-to-TGA transition in vks1 mutants. UTR, untranslated region. (C) Schematic representation of Vks1 gene structure with the mutant alleles indicated. Red rectangles and black lines indicate exons and introns, respectively. The black triangles indicate the mutation sites in vks1-1, vks1-2, and vks1-4,5,6 alleles, and the white triangle indicates the Mu insertion in the vks1-3 allele. (D) Immunoblotting analysis of VKS1 in seeds of A619 and vks1-1. ACTIN was used as an internal control. The sizes of proteins are indicated beside the gels. (E) Expression levels of Vks1 in seeds of B73 and vks1-2 at 2 DAP. (F) Immunoblotting analysis of VKS1 in seeds of B73 and vks1-2 at 2 DAP. ACTIN was used as an internal control. The sizes of proteins are indicated beside the gels. (G) The kernel phenotype of vks1-2. Left panel, a self-pollinated vks1-2/+ ear segregating vks1 (as indicated by an asterisk) and normal (as indicated by a dot and a triangle) seeds. Right panel, all small kernels were determined to be homozygous for vks1-2, while normal kernels bore one (as indicated by triangles) or two wild-type (as indicated by dots) alleles. Bar = 1 cm (H) Allelic test of vks1-1 and vks1-2 alleles. Left panel, a representative ear from a homozygous vks1-1 plant pollinated by vks1-2/+ pollen segregating vks1 and normal seeds. Right panel, all small kernels were determined to be the genotype vks1-1/vks1-2, while all normal seeds were vks1-1/+. Bar = 1 cm.

Figure 3.

The Gene Expression Pattern of VKS1 and Its Protein Subcellular Localization. (A) RT-qPCR analysis of Vks1 during seed development. All expression levels are normalized to Actin. The expression level of Vks1 at 0 DAP is set to 1. Error bars represent sd from three biological replicates. (B) to (G) RNA in situ hybridization of Vks1 in seeds at 2 and 3 DAP. Positive signals (shown in red) are clearly restricted to the coenocyte at 2 DAP ([B] and [D]) and endosperm cells engaging in cellularization at 3 DAP ([C] and [E]). When hybridized with sense probes, no signal is observed in the sections of seeds at 2 DAP (F) and 3 DAP (G). Bars = 50 μm. (H) Schematic diagrams of constructs for subcellular localization. VKS1^FL, VKS1^△T, VKS1^△M, VKS1^△TM, and VKS1^△TCC represent the full-length VKS1 protein and fragments lacking the tail, the motor, both the tail and motor domains, and both the tail and CC domains, respectively. aa, amino acids. (I) Subcellular localization of VKS1^FL-GFP. The nucleus is stained with DAPI. (J) Localization of VKS1^FL-GFP in the nucleus. The nucleus is stained with DAPI. (K) Subcellular localization of VKS1^△T-GFP. The nucleus is stained with DAPI. (L) Colocalization of VKS1^△T-GFP and mCherry-TUB6. (M) Subcellular localization of VKS1^△M-GFP. The nucleus is stained with DAPI. (N) Colocalization of VKS1^△TM-GFP and mCherry-TUB6. (O) and (P) Subcellular localization of VKS1^△TCC-GFP and GFP-VKS1^△TCC. The nucleus is stained with DAPI. For (I) to (P), bars = 10 μm.

Figure 4.

Phenotypes of the Coenocyte in the Wild Type and vks1. (A) and (B) Semithin sections of A619 and vks1 seeds at the coenocytic stage (2 DAP). Arrows indicate the free nuclei evenly distributed along the peripheral region of the cell in the A619 wild type (A), while nuclei are not visible in vks1 (B). Bars = 50 μm. (C) and (D) Colocalization of VKS1 and MTs in the coenocyte as shown by immunofluorescence assays in the A619 wild type (C) and vks1 (D). Bars = 20 μm.

Figure 5.

Dynamic Localization of VKS1 during Mitosis. Localization of the VKS1 protein in early endosperm cells during mitosis was assayed by immunofluorescence. VKS1 signaling is colored green, and the nucleus is colored blue. Bar = 10 μm.

Figure 6.

Early Endosperm Mitotic Divisions in the Wild Type and vks1. MT arrays were visualized by immunostaining with anti-α-tubulin during mitosis in wild-type and vks1 endosperm cells at 6 DAP. MTs are colored green, and nuclei or chromosomes are colored blue. Phases are as follows: interphase (A); prometaphase (B); metaphase (C); anaphase (D); anaphase/telophase (E); and telophase (F). Bar = 10 μm.

Figure 7.

The Phenotypes of Endosperm Cells and Nuclei in A619 and vks1 during Early Development. (A) and (B) Endosperm cells undergoing mitosis and cytokinesis in A619 (A) and vks1 (B) at 6 DAP. Bar = 25 μm. (C) to (E) Nuclei of endosperm cells during mitosis in A619 (C) and vks1 ([D] and [E]) at 6 DAP. (D) shows a mild phenotype and (E) shows a severe phenotype of vks1. The nuclei stained by DAPI are in blue. Bar = 50 μm. (F) Frequency of aberrant nuclei in endosperms of A619 and vks1 at 6 DAP. More than 4000 nuclei of A619 and 2600 nuclei of vks1 from 20 microscope fields were observed. The mean value of the ratios of aberrant nuclei to total nuclei in each field of A619 or vks1 was used to represent the frequency of aberrant nuclei in endosperm. **, P < 0.01 as determined by Student’s t test.

Figure 8.

Proposed Model for VKS1 Function and the Varied-Kernel-Size Phenotype. Rapid mitotic divisions in early endosperm development produce most of the cells that make up the starchy endosperm. Vks1 is predominantly expressed in early endosperm development and plays key roles in critical events of mitosis and cytokinesis. VKS1 dynamically colocalizes with MTs during interphase, mitosis, and cytokinesis. A lack of VKS1 results in substantial defects in spindle assembly, sister chromatid separation, phragmoplast formation, and eventually cell division. Therefore, while normal mitosis maintains rapid cell division to build up the endosperm structure and function in the wild type, aberrant mitosis may frequently cause repression in cell division, thereby leading to reduced cell numbers in the vks1 endosperm. Perhaps as a consequence of varying numbers of affected cells, the kernel sizes of individual vks1 seeds display dramatic variation.