Hisako Igarashi

A new class of microtubule-associated proteins in plants

In plants there are three microtubule arrays involved in cellular morphogenesis that have no equivalent in animal cells. In animals, microtubules are decorated by another class of proteins – the structural MAPS – which serve to stabilize microtubules and assist in their organization. The best-studied members of this class in plants are the MAP-65 proteins that can be purified together with plant microtubules after several cycles of polymerization and depolymerization. Here we identify three similar MAP-65 complementary DNAs representing a small gene family named NtMAP65-1, which encode a new set of proteins, collectively called NtMAP65-1. We show that NtMAP65-1 protein localizes to areas of overlapping microtubules, indicating that it may function in the behaviour of antiparallel microtubules in the mitotic spindle and the cytokinetic phragmoplast.

The plant cytoskeleton: recent advances in the study of the plant microtubule-associated proteins MAP-65, MAP-190 and the Xenopus MAP215-like protein, MOR1

The microtubule cytoskeleton is a dynamic filamentous structure involved in many key processes in plant cell morphogenesis including nuclear and cell division, deposition of cell wall, cell expansion, organelle movement and secretion. The principal microtubule protein is tubulin, which associates to form the wall of the tubule. In addition, various associated proteins bind microtubules either to anchor, cross-link or regulate the microtubule network within cells. Biochemical, molecular biological and genetic approaches are being successfully used to identify these microtubule-associated proteins (MAPs) in plants, and we describe recent progress on three of these proteins.

VASCULAR-RELATED NAC-DOMAIN6 and VASCULAR-RELATED NAC-DOMAIN7 Effectively Induce Transdifferentiation into Xylem Vessel Elements under Control of an Induction System

We previously showed that the VASCULAR-RELATED NAC-DOMAIN6 (VND6) and VND7 genes, which encode NAM/ATAF/CUC domain protein transcription factors, act as key regulators of xylem vessel differentiation. Here, we report a glucocorticoid-mediated posttranslational induction system of VND6 and VND7. In this system, VND6 or VND7 is expressed as a fused protein with the activation domain of the herpes virus VP16 protein and hormone-binding domain of the animal glucocorticoid receptor, and the proteins activity is induced by treatment with dexamethasone (DEX), a glucocorticoid derivative. Upon DEX treatment, transgenic Arabidopsis (Arabidopsis thaliana) plants carrying the chimeric gene exhibited transdifferentiation of various types of cells into xylem vessel elements, and the plants died. Many genes involved in xylem vessel differentiation, such as secondary wall biosynthesis and programmed cell death, were up-regulated in these plants after DEX treatment. Chemical analysis showed that xylan, a major hemicellulose component of the dicot secondary cell wall, was increased in the transgenic plants after DEX treatment. This induction system worked in poplar (Populus tremula × tremuloides) trees and in suspension cultures of cells from Arabidopsis and tobacco (Nicotiana tabacum); more than 90% of the tobacco BY-2 cells expressing VND7-VP16-GR transdifferentiated into xylem vessel elements after DEX treatment. These data demonstrate that the induction systems controlling VND6 and VND7 activities can be used as powerful tools for understanding xylem cell differentiation.

Dynamic interaction of NtMAP65-1a with microtubules in vivo

Plant microtubules are intrinsically more dynamic than those from animals. We know little about the dynamics of the interaction of plant microtubule-associated proteins (MAPs) with microtubules. Here, we have used tobacco and Arabidopsis MAPs with relative molecular mass 65 kDa (NtMAP65-1a and AtMAP65-1), to study their interaction with microtubules in vivo. Using fluorescence recovery after photobleaching we report that the turnover of both NtMAP65-1a and AtMAP65-1 bound to microtubules is four- to fivefold faster than microtubule treadmilling (13 seconds compared with 56 seconds, respectively) and that the replacement of NtMAP65-1a on microtubules is by random association rather than by translocation along microtubules. MAP65 will only bind polymerised microtubules and not its component tubulin dimers. The turnover of NtMAP65-1a and AtMAP65-1 on microtubules is similar in the interphase cortical array, the preprophase band and the phragmoplast, strongly suggesting that their role in these arrays is the same. NtMAP65-1a and AtMAP65-1 are not observed to bind microtubules in the metaphase spindle and their rate of recovery is consistent with their cytoplasmic localisation. In addition, the dramatic reappearance of NtMAP65-1a on microtubules at the spindle midzone in anaphase B suggests that NtMAP65-1a is controlled post-translationally. We conclude that the dynamic properties of these MAPs in vivo taken together with the fact that they have been shown not to effect microtubule polymerisation in vitro, makes them ideally suited to a role in crossbridging microtubules that need to retain spatial organisation in rapidly reorganising microtubule arrays.

Clathrin is involved in organization of mitotic spindle and phragmoplast as well as in endocytosis in tobacco cell cultures

Summary.We previously identified a 175 kDa polypeptide in Lilium longiflorum germinating pollen using a monoclonal antibody raised against myosin II heavy chain from Physarum polycephalum. In the present study, the equivalent polypeptide was also found in cultured tobacco BY-2 cells. Analysis of the amino acid sequences revealed that the 175 kDa polypeptide is clathrin heavy chain and not myosin heavy chain. After staining of BY-2 cells, punctate clathrin signals were distributed throughout the cytoplasm at interphase. During mitosis and cytokinesis, clathrin began to accumulate in the spindle and the phragmoplast and then was intensely concentrated in the cell plate. Expression of the C-terminal region of clathrin heavy chain, in which light chain binding and trimerization domains reside, induced the suppression of endocytosis and the formation of an aberrant spindle, phragmoplast, and cell plate, the likely cause of the observed multinucleate cells. These data strongly suggest that clathrin is intimately involved in the formation of the spindle and phragmoplast, as well as in endocytosis.

An isoform of Arabidopsis myosin XI interacts with small GTPases in its C-terminal tail region

Myosin XI, a class of myosins expressed in plants is believed to be responsible for cytoplasmic streaming and the translocation of organelles and vesicles. To gain further insight into the translocation of organelles and vesicles by myosin XI, an isoform of Arabidopsis myosin XI, MYA2, was chosen and its role in peroxisome targeting was examined. Using the yeast two-hybrid screening method, two small GTPases, AtRabD1 and AtRabC2a, were identified as factors that interact with the C-terminal tail region of MYA2. Both recombinant AtRabs tagged with His bound to the recombinant C-terminal tail region of MYA2 tagged with GST in a GTP-dependent manner. Furthermore, AtRabC2a was localized on peroxisomes, when its CFP-tagged form was expressed transiently in protoplasts prepared from Arabidopsis leaf tissue. It is suggested that MYA2 targets the peroxisome through an interaction with AtRabC2a.

Presence of aquaporin and V-ATPase on the contractile vacuole of Amoeba proteus.

Background information. The results of water permeability measurements suggest the presence of an AQP (aquaporin) in the membrane of the CV (contractile vacuole) in Amoeba proteus [Nishihara, Shimmen and Sonobe ( 2004 ) Cell Struct. Funct. 29, 85–90].

The Putative RNA-Processing Protein, THO2, is a Microtubule-Associated Protein in Tobacco

THO2 is a component of the THO-TREX (transcription and export factor) complex that participates in mRNA metabolism and export from the nucleus in yeast and animal cells. Here we report that tobacco putative THO2-related protein (NtTHO2) is a microtubule-associated protein, which directly binds to microtubules in vitro and co-localizes with cortical microtubules in vivo. We purified endogenous NtTHO2 by cycles of microtubule polymerization-depolymerization from crude extracts of tobacco BY-2 miniprotoplasts. Purified NtTHO2 sedimented with microtubules in vitro. Immunofluorescence revealed that NtTHO2 was present in both the nucleus and cytoplasm. In interphase, cytoplasmic NtTHO2 was localized along cortical microtubules. In the mitotic phase, NtTHO2 was localized to the mitotic spindle but not to either the preprophase band or the phragmoplast. In mature cells of seedling roots, and in BY-2 cells in which proliferation was stopped by removing 2,4-D, NtTHO2 staining was confined mainly to the nucleolus. These results suggest that NtTHO2 is a multifunctional protein that participates in mRNA metabolism, and also functions within the cortical microtubules and mitotic spindle.

Acropetal leaflet initiation of Eschscholzia californica is achieved by constant spacing of leaflets and differential growth of leaf

In compound leaves, leaflet primordia are initiated directionally along the lateral sides. Our understanding of the molecular basis of leaflet initiation has improved, but the regulatory mechanisms underlying spatio-temporal patterns remain unclear. In this study, we investigated the mechanisms of acropetal (from the base to the tip) progression of leaflet initiation in Eschscholzia californica. We established an ultraviolet-laser ablation system to manipulate compound-leaf development. Local ablation at the leaflet incipient site generated leaves with asymmetric morphology. In the majority of cases, leaflets that were initiated on the ablated sides shifted apically. Finite time-course observation revealed that the timing of leaflet initiation was delayed, but the distance from the leaf tip did not decrease. These results were suggestive of the local spacing mechanism in leaflet initiation, whereby the distance from the leaf tip and adjacent pre-existing leaflet determines the position of leaflet initiation. To understand how such a local patterning mechanism generates a global pattern of successive leaflet initiation, we assessed the growth rate gradient along the apical–basal axis. Our time-course analysis revealed differential growth rates along the apical–basal axis of the leaf, which can explain the acropetal progression of leaflet initiation. We propose that a leaflet is initiated at a site where the distances from pre-existing leaflets and the leaf tip are sufficient. Furthermore, the differential growth rate may be a developmental factor underlying the directionality of leaflet initiation.

Arabidopsis Pol II-Dependent in Vitro Transcription System Reveals Role of Chromatin for Light-Inducible rbcS Gene Transcription

Light-inducible in vitro transcription from an Arabidopsis gene observed only from chromatin but not from naked DNA defines higher order of DNA structure. In vitro transcription is an essential tool to study the molecular mechanisms of transcription. For over a decade, we have developed an in vitro transcription system from tobacco (Nicotiana tabacum)-cultured cells (BY-2), and this system supported the basic activities of the three RNA polymerases (Pol I, Pol II, and Pol III). However, it was not suitable to study photosynthetic genes, because BY-2 cells have lost their photosynthetic activity. Therefore, Arabidopsis (Arabidopsis thaliana) in vitro transcription systems were developed from green and etiolated suspension cells. Sufficient in vitro Pol II activity was detected after the minor modification of the nuclear soluble extracts preparation method; removal of vacuoles from protoplasts and L-ascorbic acid supplementation in the extraction buffer were particularly effective. Surprisingly, all four Arabidopsis Rubisco small subunit (rbcS-1A, rbcS-1B, rbcS-2B, and rbcS-3B) gene members were in vitro transcribed from the naked DNA templates without any light-dependent manner. However, clear light-inducible transcriptions were observed using chromatin template of rbcS-1A gene, which was prepared with a human nucleosome assembly protein 1 (hNAP1) and HeLa histones. This suggested that a key determinant of light-dependency through the rbcS gene transcription was a higher order of DNA structure (i.e. chromatin).