Evaluating the microtubule cytoskeleton and its interacting proteins in monocots by mining the rice genome

Abstract

Background: Microtubules (MTs) are assembled by heterodimers of alpha- and beta-tubulins, which provide tracks for directional transport and frameworks for the spindle apparatus and the phragmoplast. MT nucleation and dynamics are regulated by components such as the gamma-tubulin complex which are conserved among eukaryotes, and other components which are unique to plants. Following remarkable progress made in the model plant Arabidopsis thaliana toward revealing key components regulating MT activities, the completed rice (Oryza sativa) genome has prompted a survey of the MT cytoskeleton in this important crop as a model for monocots.

Scope: The rice genome contains three alpha-tubulin genes, eight beta-tubulin genes and a single gamma-tubulin gene. A functional gamma-tubulin ring complex is expected to form in rice as genes encoding all components of the complex are present. Among proteins that interact with MTs, compared with A. thaliana, rice has more genes encoding some members such as the MAP65/Ase1p/PRC1 family, but fewer for the motor kinesins, the end-binding protein EB1 and the mitotic kinase Aurora. Although most known MT-interacting factors have apparent orthologues in rice, no orthologues of arabidopsis RIC1 and MAP18 have been identified in rice. Among all proteins surveyed here, only a few have had their functions characterized by genetic means in rice. Elucidating functions of proteins of the rice MT cytoskeleton, aided by recent technical advances made in this model monocot, will greatly advance our knowledge of how monocots employ their MTs to regulate their growth and form.

Fig. 1.

Neighbor–Joining tree of MAP65 family proteins. Phylogenetic relationships among the MAP65 members of rice (Os), A. thaliana (At), the budding yeast Saccharomyces cerevisiae (ScAse1p), the fission yeast Schizosaccharomyces pombe (SpAse1) and human (HsPRC1) are shown. For rice proteins, shown in this figure were IDs annotated by RGAP omitting LOC_. The AtMAP65 members were assigned according to published nomenclature (Hussey and Hawkins, 2001). The GenBank accession numbers for the others are: NP_116582 for ScAse1p, CAC21482 for SpAse1 and AAC02688 for HsPRC1. Bootstrap values at the branches represent the percentages obtained in 1000 replications. Only values >50 % are presented.

Fig. 2.

Immunolocalization of OsMAP65-3 in developing phragmoplasts. OsMAP65-3 was detected by an isoform specific antibody, MTs by an anti-α-tubulin antibody, and DNA by DAPI (4′,6-diamidino-2-phenylindole). Cells at an early and late stage of cytokinesis are shown. OsMAP65-3 appeared in a broader distribution pattern in the early phragmoplast, and then became more concentrated in the phragmoplast midline in the late phragmoplast. In the merged image, OsMAP65-3 was pseudocoloured in green, MTs in red and DNA in blue.

Fig. 3.

Immunolocalization of OsKinesin-12A in the phragmoplast. OsKinesin-12A was labelled with an antibody raised against a truncated version of the protein, MTs by an anti-α-tubulin antibody, and DNA by DAPI (4′,6-diamidino-2-phenylindole). OsKinesin-12A was pseudocolored in green, MTs in red, and DNA in blue. OsKinesin-12A exclusively decorated the plus end of phragmoplast MTs.

Fig. 4.

Neighbor–joining tree of members of the Kinesin-14 subfamily. Phylogenetic relationships among Kinesin-14 members of rice (Os), A. thaliana (At), and representatives from the budding yeast S. cerevisiae (ScKar3p), the filamentous fungus Aspergillus nidulans (AnKLPA), the fly Drosophila melanogaster (DmNCD) and human (HsCHO2) are presented. AtPAKRP2 was used as an outgroup kinesin. For rice proteins, shown in this figure were IDs annotated by RGAP omitting LOC_. Documented plant kinesins are listed with their abbreviated names following the locus calls. Among Kinesin-14 members, those having their motor domains located in the N-terminus, the C-terminus, and the middle are highlighted as N-terminal motor, C-terminal motor, internal motor, and KCHs as specialized internal motor kinesins. Bootstrap values at the branches represent the percentages obtained in 1000 replications.