Recent progress in living cell imaging of plant cytoskeleton and vacuole using fluorescent-protein transgenic lines and three-dimensional imaging.

In higher-plant cells, microtubules, actin microfilaments, and vacuoles play important roles in a variety of cellular events, including cell division, morphogenesis, and cell differentiation. These intracellular structures undergo dynamic changes in their shapes and functions during cell division and differentiation, and to analyse these sequential structural changes, the vital labelling technique, using the green-fluorescent protein or other fluorescent proteins, has commonly been used to follow the localisation and translocation of specific proteins. To visualise microtubules, actin filaments, and vacuoles, several strategies are available for selecting the appropriate fluorescent-protein fusion partner: microtubule-binding proteins, tubulin, and plus-end-tracking proteins are most suitable for microtubule labelling; the actin binding domain of mouse talin and plant fimbrin for actin microfilament visualisation; and the tonoplast-intrinsic proteins and syntaxin-related proteins for vacuolar imaging. In addition, three-dimensional reconstruction methods are indispensable for localising the widely distributed organelles within the cell. The maximum intensity projection method is suitable for cytoskeletal structures, while contour-based surface modelling possesses many advantages for vacuolar membranes. In this article, we summarise the recent progress in living cell imaging of the plant cytoskeleton and vacuoles using various fusions with green-fluorescent proteins and three-dimensional imaging techniques.

Development and disintegration of phragmoplasts in living cultured cells of a GFP::TUA6 transgenic Arabidopsis thaliana plant.

Cultured suspension cells of Arabidopsis thaliana that stably express a green-fluorescent protein-alpha-tubulin 6 fusion protein were used to follow the development and disintegration of phragmoplasts. The development and disintegration of phragmoplasts in the living cultured cells could be successively observed by detecting the green-fluorescent protein fluorescence of the microtubules. In the early telophase spindle, where two kinetochore groups and two daughter chromosome groups had completely separated from one another, fluorescence appeared in the interzone between the two chromosome groups. The fluorescent region was gradually condensed at the previous equator and increased in fluorescence intensity, and finally it formed the initial phragmoplast. The initial phragmoplast moved from the cell center towards the cell periphery, and it lost fluorescence at its center and became double rings in shape. The expansion orientation of the phragmoplast was not always the same as that of the future new cell wall before it came in contact with the cell wall. The phragmoplast did not usually come in contact with the cell wall simultaneously with its entire length. A portion of the phragmoplast which was earlier in contact with the cell wall disappeared earlier than other portions of the phragmoplast. The duration of contact between any portions of the phragmoplast and the plasma membrane of the cell wall was 15-30 min. The fluorescence intensity of the cytoplasm did not seem to be elevated by the disintegration of the strongly fluorescent phragmoplast.

Roles of actin-depleted zone and preprophase band in determining the division site of higher-plant cells, a tobacco BY-2 cell line expressing GFP-tubulin.

The mode of cytokinesis, especially in determining the site of cell division, is not well understood in higher-plant cells. The division site appears to be predicted by the preprophase band of microtubules that develop with the phragmosome, an intracellular structure of the cytoplasm suspending the nucleus and the mitotic apparatus in the center. As the preprophase band disappears during mitosis, it is thought to leave some form of "memory" on the plasma membrane to guide the growth of the new cell plate at cytokinesis. However, the intrinsic nature of this "memory" remains to be clarified. In addition to microtubules, microfilaments also dynamically change forms during cell cycle transition from the late G2 to the early G1 phase. We have studied the relationships between microtubules and microfilaments in tobacco BY-2 cells and transgenic BY-2 cells expressing a fusion protein of green-fluorescent protein and tubulin. At the late G2 phase, microfilaments colocalize with the preprophase band of microtubules. However, an actin-depleted zone which appears at late prometaphase is observed around the chromosomes, especially at metaphase, but also throughout anaphase. To study the functions of the actin-depleted zone, we disrupted the microfilament structures with bistheonellide A, a novel macrolide that depolymerizes microfilaments very rapidly even at low concentrations. The division planes became disorganized when the drug was added to synchronized BY-2 cells before the appearance of the actin-depleted zone. In contrast, the division planes appeared smooth, as in control cells, when the drug was added after the appearance of the actin-depleted zone. These results suggest that the actin-depleted zone may participate in the demarcation of the division site at the final stage of cell division in higher plants.

Fate of nascent microtubules organized at the M/G1 interface, as visualized by synchronized tobacco BY-2 cells stably expressing GFP-tubulin: time-sequence observations of the reorganization of cortical microtubules in living plant cells.

Transgenic BY-2 cells stably expressing a GFP (green fluorescent protein)-tubulin fusion protein (BY-GT16) were subcultured in a modified Linsmaier and Skoog medium. The BY-GT16 cells could be synchronized by aphidicolin and the dynamics of their microtubules (MTs) were monitored by the confocal laser scanning microscopy (CLSM). We have succeeded in investigating the mode of reorganization of cortical MTs at the M/G1 interface. The cortical MTs were initially organized in the perinuclear regions and then they elongated to reach the cell cortex, forming the bright spots there. Subsequently, the first cortical MTs rapidly elongated from the spots and they were oriented parallel to the long axis towards the distal end of the cells. Around the time when the tips of the parallel MTs reached the distal end, the formation of transverse cortical MTs followed in the cortex near the division site, as we had previously suggested [Hasezawa and Nagata (1991) Bot. Acta 104: 206, Nagata et al. (1994) Planta 193: 567]. It was confirmed in independent observations that the appearance of the parallel MTs was followed by the appearance of the transverse MTs in each cell. We found that the transverse MTs spread through the whole cell cortex within about 20-30 min, while the parallel MTs disappeared. The significance of these observations on the mode of cortical MT organization is discussed.

Changes in tubulin protein expression in guard cells of Vicia faba L. accompanied with dynamic organization of microtubules during the diurnal cycle.

We previously reported that the organization of microtubules (MTs) in guard cells of Vicia faba L. shows dynamic diurnal changes [Fukuda et al. (1998) Plant Cell Physiol. 39: 80]. Here, we report a method to directly extract total proteins from guard cells to investigate the biochemical changes in guard cells of Vicia faba L. during the diurnal cycle. Electrophoretic profiles of total proteins of guard cells showed distinct patterns with the time of extraction. Immunoblot analysis also demonstrated changes in alpha-tubulin and beta-tubulin contents with the diurnal cycle. Both tubulins were abundant at 6:00 h and 12:00 h but were almost undetectable at 24:00 h. Although treatment with either actinomycin D or cycloheximide at 18:00 h inhibited neither radial organization of cortical MTs nor stomatal opening, that at 6:00 h inhibited both. These results suggest that the dynamic diurnal changes in the organization of MTs in guard cells and stomatal movement of Vicia faba L. may be, at least partly, regulated by de novo synthesis and decomposition of tubulin molecules in guard cells.

Time-sequence observations of microtubule dynamics throughout mitosis in living cell suspensions of stable transgenic Arabidopsis--direct evidence for the origin of cortical microtubules at M/G1 interface.

Transgenic Arabidopsis thaliana, stably expressing a GFP-TUA6 fusion protein, were subcultured in B5 medium supplemented with 2,4-D and BA. In the cell suspensions, the microtubular changes in the mitotic cells could be monitored by time-sequence observations using a time-lapse system of fluorescence microscopy. We have succeeded in following the microtubule (MT) dynamics in living cells throughout mitosis, from the late G2 phase to early G1 phase, and found that, at the M/G1 interface, the cortical MTs were firstly reorganized in the perinuclear regions and then in the cortex, as we had previously suggested (Hasezawa and Nagata 1991, Nagata et al. 1994). The significance of this observation on the origin of cortical MTs is discussed.

Putative involvement of a 49 kDa protein in microtubule assembly in vitro.

In higher plant cells, thus far only a few molecules have been inferred to be involved in microtubule organizing centers (MTOCs). Examination of a 49 kDa tobacco protein, homologous to a 51 kDa protein involved in sea urchin MTOCs, showed that it also accumulated at the putative MTOC sites in tobacco BY-2 cells. In this report, we show that the 49 kDa protein is likely to play a significant role in microtubule organization in vitro. We have established a system prepared from BY-2 cells, capable of organizing microtubules in vitro. The fraction, which was partially purified from homogenized miniprotoplasts (evacuolated protoplasts) by salt extraction and subsequent ion exchange chromatography, contained many particles of diameters about 1 micron after desalting by dialysis. When this fraction was incubated with purified porcine brain tubulin, microtubules were elongated radially from the particles and organized into structures similar to the asters observed in animal cells, and therefore also termed "asters" here. Since we could hardly detect BY-2 tubulin molecules in this fraction, the microtubules in "asters" seemed to be solely composed of the added porcine tubulin. Tubulin molecules were newly polymerized at the ends of the microtubules distal to the particles, and the elongation rate of microtubules was more similar to the reported rate of the plus-ends than that of the minus-ends in vitro. By fluorescence microscopy, the 49 kDa protein was shown to be located at the particles. Thus, its location at the centers of the "asters" suggests that the protein plays a role in microtubule organization in vitro.

Role of cortical microtubules in the orientation of cellulose microfibril deposition in higher-plant cells.

Cortical microtubules (MTs) have been implicated in the morphogenesis of plant cells by regulating the orientation of newly deposited cellulose microfibrils (CMFs). However, the role of MTs in oriented CMF deposition is still unclear. We have investigated the mechanism of CMF deposition with cultured tobacco protoplasts derived from taxol-treated BY-2 cells (taxol protoplasts). The BY-2 protoplasts regenerated patches of beta-l,3-glucan (callose) and fibrils of beta-l,4-glucan (cellulose). Taxol protoplasts possessed the same ordered MT arrays as material cells and regenerated CMFs with patterns almost coincidental with MTs. Electron microscopy revealed that, on the surface of cultured taxol protoplasts, each CMF bundle appeared to be deposited on each cortical MT. These results suggest that MTs may attach directly to the cellulose-synthesizing complexes, by some form of linkage, and regulate the movement of these complexes in higher-plant cells.

Dynamic organization of microtubules in guard cells of Vicia faba L. with diurnal cycle.

Stomatal movement is regulated by changes in the volume of guard cells, thought to be mainly controlled by an osmo-regulatory system. In the present study, we examined the additional involvement of cytoskeletal events in the regulation of stomatal movement. Microtubules (MTs) in guard cells of Vicia faba L., grown under sunlight, were observed during the day and night by immunofluorescence microscopy. Cortical MTs began to be organized in a radial array at dawn and increased in numbers in the morning following the increase in the stomatal aperture size. Thereafter, MTs became localized near the nucleus and began to be destroyed from the evening to midnight, following the decrease in stomatal aperture size. These diurnal changes in MT organization were observed even two days after transfer from natural light condition to total darkness, and were accompanied by corresponding changes in stomatal aperture. The increase in stomatal aperture size in the early morning was inhibited by 50 microM propyzamide, which destroys cortical MTs in guard cells, whereas the decrease in aperture size in the evening was suppressed by 10 microM taxol, which stabilizes cortical MTs. These results suggest that radially-organized cortical MTs of guard cells may control diurnal stomatal movement.

Cell cycle-dependent accumulation of a kinesin-like protein, KatB/C in synchronized tobacco BY-2 cells.

Immunoblot analysis with antibodies prepared against highly purified recombinant truncated kinesin-like proteins, KatB(5-249) and KatC(207-754), encoded by the katB and katC genes of Arabidopsis thaliana revealed the presence of a kinesin-like polypeptide, termed KatB/C, in cultured tobacco BY-2 cells. The KatB/C polypeptide cosedimented with microtubules in the presence of a nonhydrolyzable ATP analogue and was released from microtubules in the presence of ATP, both of which are characteristics of kinesin proteins. The amount of KatB/C polypeptide in synchronous BY-2 cells increased during M phase of the cell cycle. Microtubule-based structures present in cells at M phase, such as the spindle and phragmoplast, may be the site of action of the KatB/C protein.

Expression of the auxin-regulated parA gene in transgenic tobacco and nuclear localization of its gene products.

An auxin-regulated gene, parA, comprises a gene family consisting of a handful genes which respond to various signals. Although Droog et al. (Plant Mol. Biol, 1993, 21, 965-972) postulated that the parA-related genes belong to the family of a cytoplasmic enzyme, glutathione S-transferase (GST), we detected a low level of GST activity in the parA products, whose value was below 1/30 of that of parB products encoding tobacco (Nicotiana tabacum L.) GST. Immunofluorescence studies using an antibody against parA protein revealed that the subcellular location of parA protein is the nucleus in cultured tobacco mesophyll protoplasts, while conventional GSTs' including the parB product were primarily located in the cytoplasm. Confocal laser scanning microscopy of tobacco BY-2 cells showed that the parA product was confined to the nucleus, but was excluded from the nucleolus. In addition, exon/intron organization of the parA family was appreciably different from that of conventional GSTs including parB. Furthermore, the parA protein is much more similar to a 24-kDa protein of Escherichia coli that is reported to bind to RNA polymerase. These different characteristics of parA compared with to the conventional GSTs, indicate that parA protein would have distinct functions, such as involvement in transcription, rather than functioning as a conventional GST. Transgenic tobacco plants that carried the parA promoter fused to a beta-glucuronidase gene were used to show that the parA gene is tissue-specific and also under developmental control.

Genes involved in the dedifferentiation of plant cells.

Since the initial process of culturing tobacco mesophyll protoplasts can be considered as a model system of dedifferentiation of higher plants, the mode of expression of genes induced by auxin, a key factor in inducing dedifferentiation, has been analyzed during the regaining of meristematic activity of quiescent and differentiated tobacco mesophyll. By differential screening we have isolated three auxin-regulated genes, which we named parA, parB and parC. parA and parC, which belong to the same gene family, were supposed to play a role in transcriptional regulation upon induction by auxin, while parB encoded glutathione S-transferase. Although it was supposed that the expression of these par genes should play a pivotal role in regaining the meristematic activity of the differentiated tobacco mesophyll cells, a possibility that other less abundantly expressing genes would have been neglected in these studies has not been excluded. On the other hand, the search for genes which would be involved in maintaining cell division activity in the dedifferentiated plant cells allowed us to isolate a few genes. One of these genes, designated arcA, belonged to a beta subunit-like protein of heterotrimeric G proteins. The significance of the involvement of this gene product in maintaining the meristematic activity of plant cells cultured in vitro has been discussed.

Further evidence for the transformation ofVinca rosea protoplasts byAgrobacterium tumefaciens spheroplasts.

Protoplasts ofVinca rosea were transformed by spheroplasts ofAgrobacterium tumefaciens harboring nopalinetype Ti plasmids according to the procedure of Hasezawa et al. (1981). These transformants frequently differentiated tracheids, but further differentiation to teratomata has not so far been observed. Transformation was confirmed by the improved detection of nopaline synthase, where the sensitivity and specificity of the enzyme reaction was increased by employing(14)C-α-ketoglutaric acid and(3)H-arginine as substrates. The nopaline synthase activity was identified by the comigration of these two radioisotopes in the cnromatogram. Furthermore, the T-DNA structure of one of these transformants was examined by Southern hybridization according to Thomashow et al. (1980) and compared with that ofVinca rosea crown gall.

Introduction of Escherichia coli cells and spheroplasts into Vinca protoplasts.

In the presence of 10% polyvinyl alcohol (PVA), Escherichia coli cells or spheroplasts can be easily introduced into Vinca protoplasts by endocytosis. Uptake proceeded quite rapidly; bacterial cells or spheroplasts were found within the cytoplasm of Vinca protoplasts after 10 min of incubation with PVA.