Functional Analysis of the Plant Chromosomal Passenger Complex

Abstract

The Aurora B kinase, encoded by the AURORA 3 (AUR3) gene in Arabidopsis (Arabidopsis thaliana), is a key regulator of cell division in all eukaryotes. Aurora B has at least two central functions during cell division; it is essential for the correct, i.e. balanced, segregation of chromosomes in mitosis and meiosis by controlling kinetochore function, and it acts at the division plane, where it is necessary to complete cytokinesis. To accomplish these two spatially distinct functions, Aurora B in animals is guided to its sites of action by Borealin, inner centromere protein (INCENP), and Survivin, which, together with Aurora B, form the chromosome passenger complex (CPC). However, besides Aurora homologs, only a candidate gene with restricted homology to INCENP has been described in Arabidopsis, raising the question of whether a full complement of the CPC exists in plants and how Aurora homologs are targeted subcellularly. Here, we have identified and functionally characterized a Borealin homolog, BOREALIN RELATED (BORR), in Arabidopsis. Together with detailed localization studies including the putative Arabidopsis INCENP homolog, these results support the existence of a CPC in plants.

Figure 1.

BORR gene structure in plants. A, Protein sequence of BORR in Arabidopsis. Lowercase letters indicate predictions of the N-terminal helix in BORR (a) and alignment of the most conserved sequence region of the protein (b). Arrowheads in b indicate the conserved CDK consensus sites. B, Phylogenetic analysis of the Borealin family in yeast, animals, and plants. The tree was constructed using MEGA X by the neighbor-joining method.

Figure 2.

BORR is required for seed development. A, Wild-type or heterozygous borr mutant plants pollinated with pollen from wild-type or heterozygous borr mutants. Black and gray arrowheads indicate tiny white ovules (unfertilized ovules/early arrested seeds) and collapsed brown seeds or seeds without green embryo (late aborted seeds), respectively. Scale bar = 1 mm. B, Proportions of the seed phenotypes shown in A. Total number (n) of seeds and ovules from five hand-pollinated siliques are shown in each cross. C, Seeds and ovules from the heterozygous borr siliques cleared in chloral hydrate solution. At the globular embryo stage, developing seeds with abnormal embryos, developing seeds with no embryo, and unfertilized ovules were observed. Scale bar = 50 μm. D, Classification of the embryo phenotype for each cross. Total number (n) of developing seeds and unfertilized ovules from six to eight hand-pollinated pistils are shown.

Figure 3.

Phenotypical analysis of amiRNA-mediated BORR knockdown plants. A, Sequence alignments of amiRNAs and their target sites on BORR mRNA. B, Relative expression level of BORR in the amiBORR plants was confirmed by RT-qPCR analysis with three biological replicates. C, Nine-day-old wild-type and amiBORR seedlings. Scale bar = 1 cm. D, Root growth measurements of wild-type and amiBORR plants. Four-day-old seedlings were transferred to a Murashige and Skoog plate and root lengths were measured for 5 d. Asterisks indicated significance using Student's t test (**P < 0.01). Error bars indicate the SD (n = 10). E, Confocal images of 7-d-old wild-type and amiBORR1-1 roots stained with propidium iodide. amiBORR1-1 root mutations were categorized as Mild and Severe based on the phenotype. Arrowheads indicate the boundary between the dividing region and the elongation region of the root. Regions marked by white and yellow dotted boxes are shown in close-up under Columella region and Dividing region, respectively. Scale bars =50 μm. F and G, Root meristem size (F) and number of meristematic cortex cells (G). Asterisks indicated significance using Student's t test (**P < 0.01). Error bars indicate the SD (n = 15). H, Representative images of normally distributed and lagging chromosomes in 7-d-old wild-type and amiBORR1-1 root cells. Microtubules and centromeres were visualized by TagRFP:TUA5 and GFP:CENH3, respectively. The arrowhead indicates a lagging chromosome. Scale bar = 5 μm. I, Frequency of lagging chromosomes in anaphase in H. Error bars indicate the SD (n = 50). J, Representative images of AUR3 accumulation levels at kinetochores in 7-d-old wild-type and amiBORR#1-1 root cells. Microtubules and AUR3 were visualized by TagRFP:TUA5 and AUR3:GFP, respectively. Scale bar = 5 μm. K, AUR3 signal intensity at kinetochores in J. Forty AUR3-GFP signals at kinetochores from 10 cells were measured. The center line indicates the median, the box represents the interquartile range, error bars were determined as 1.5× the interquartile range, and the circle represents an outlier. Asterisks indicated significance using Student's t test (**P < 0.01). Error bars indicate the SD. a.u., Arbitrary units.

Figure 4.

Interaction among the CPC components. A, Interaction among the CPC components as revealed by Y2H assays. B, Interaction between AUR3 and various regions of INCENP or IN-box-mutated INCENP. Monomeric GFP (mGFP) was used as a negative control. Each strain was spotted on SD plates without Trp and Leu (−TL; control media) or without Trp, Leu, and His (−TLH; selection media) and photographed after incubation at 30°C for 2 d. AD, GAL4-activation domain; BD, GAL4-DNA binding domain. C, IP of INCENP with BORR. 7-d-old Arabidopsis seedlings expressing BORR:RFP and mGFP:INCENP or BORR:RFP and mGFP were used for IP with an anti-GFP antibody. Both input and IP fraction were subjected to immunoblotting with an anti-RFP antibody. The asterisk indicates a nonspecific band.

Figure 5.

Expression and subcellular localization of CPC components. A to F, Expression patterns of BORR:GUS in the shoot (A), root (B), lateral roots (C), inflorescence (D), ovules (E), and young flower buds (F). Plants used in A to C were 10 d old and those in D to F were 4 weeks old. Three independent transgenic lines were analyzed and representative images are shown. Scale bars = 500 μm. G, Subcellular localization of GFP:INCENP, BORR:GFP, and AUR3:GFP during the cell cycle. Each reporter line was crossed with TagRFP:TUA5-expressing plants to visualize microtubule structures. Scale bar = 10 μm. H, Colocalization of the CPC components during the cell cycle. Scale bar = 10 μm. For live imaging in G and H, root tips of 5-d-old seedlings were used.

Figure 6.

CPC localizes to inner centromeres. Localization of the CPC from prophase to immediately before anaphase onset. Inner kinetochores and CPC complexes are visualized by TagRFP:CENH3 and BORR:GFP, respectively. For live cell imaging, root tips of 5-d-old seedlings were used. The blue bar indicates the position where the line profiles were obtained. Scale bar = 10 μm.

Figure 7.

Subcellular localization of CPC components in meiosis. A and B, Subcellular localization of BORR:GFP (A) and GFP:INCENP (B) during meiosis. Each reporter line was crossed with TagRFP:TUA5-expressing plants to visualize the microtubule structures. Scale bars = 10 μm. For live imaging of A and B, flower buds of 1-month-old plants were used.