Functional identification of cytokinesis-related genes from tobacco BY-2 cells

The molecular mechanisms controlling cytokinesis in plant cell division cycle remains largely unknown. In this study, a functional approach was taken to identify genes that may play roles in cytokinesis in tobacco BY-2 cells, using fission yeast as the host organism. A total of 22 BY-2 genes that perturbed the terminal stage of cell division when ectopically expressed in yeast cells were isolated, among which, several encode for uncharacterized genes. Additionally, RT-PCR analysis indicated that four of the isolated genes were expressed in a cell cycle-dependent manner. Our results demonstrate that fission yeast system can be efficiently used to identify the genes that may function, either positively or negatively, in the regulation of cytokinesis. More importantly, the candidate genes we have isolated in this work can provide useful information for unraveling the regulators controlling cell separation at the late stage of BY-2 cell division. Yi Yu and Hai-Yun Wang contributed equally to this work.     

Polyamine metabolism during the growth cycle of tobacco BY-2 cells.

We studied polyamine (PA) biosynthesis, oxidation and conjugation in asynchronously dividing cells of tobacco BY-2 cell suspension culture (Nicotiana tabacum L.) during 7-day growth cycle. We analyzed the levels of free and conjugated PAs and the activities of biosynthetic and catabolic enzymes during the subculture interval. The contents of free spermidine and spermine started to increase after the inoculation into the fresh medium, positively correlated with the mitotic activity of BY-2 cells and reached their maxima at the beginning of exponential phase on day 3. On the contrary, the endogenous level of free Put showed a transient decline in the lag-phase, and then increased till the end of exponential phase (day 5). The time-course of the content of PCA-soluble conjugates showed a trend similar to that of the free PAs. The inoculation of BY-2 cells into the fresh medium resulted in a sharp increase in the activities of ornithine decarboxylase (ODC, EC 4.1.1.17) and S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50). Arginine decarboxylase (ADC; EC 4.1.1.19) activity remained low during the whole subculture interval. The rise of diamine oxidase (DAO; EC 1.4.3.6) in the first day after subculture coincided with the decrease in free Put level. De novo synthesis of PAs in BY-2 cells after inoculation into the fresh medium and the participation of both PA conjugation with hydroxycinnamic acids and Put oxidative degradation in maintaining of free PA levels during the growth cycle are discussed.     

Sites of microtubule reorganization in tobacco BY-2 cells during cell-cycle progression

Summary The sites of microtubule (MT) reorganization were examined in synchronized tobacco BY-2 cells. The MTs of these cells were completely destroyed by a combined cold and drug treatment at 0 °C with 100 M propyzamide for 3 h. After the cells were washed and cultured at 30 °C, the reorganization of MTs was observed in detail. Sites for MT reorganization at each stage of the cell cycle were identified on the cell cortex and nuclei, the mitotic apparatus, the nuclei (or the nuclei and cell cortex), and the cell cortex in the S-G₂ phase, M phase, M/G₁ interface, and g₁ phase, respectively. The polypeptide synthesis elongation factor (EF)-1 is co-localized with these sites of MT reorganization. At some stages, microfilaments (MFs) were found to be involved in the reorganization of MTs. Based on these results, the mode of MT reorganization during cell cycle progression is discussed.Abbreviations EF-1 elongation factor 1 - MAP microtubule-associated protein - MF microfilament - MIs mitotic indices - MT microtubule     

Displacement of the mitotic apparatuses by centrifugation reveals cortical actin organization during cytokinesis in cultured tobacco BY-2 cells

In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global–local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global–local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.     

Role of peroxynitrite in the responses induced by heat stress in tobacco BY-2 cultured cells

Temperatures above the optimum are sensed as heat stress (HS) by all living organisms and represent one of the major environmental challenges for plants. Plants can cope with HS by activating specific defense mechanisms to minimize damage and ensure cellular functionality. One of the most common effects of HS is the overproduction of reactive oxygen and nitrogen species (ROS and RNS). The role of ROS and RNS in the regulation of many plant physiological processes is well established. On the contrary, in plants very little is known about the physiological role of peroxynitrite (ONOO^?), the RNS species generated by the interaction between NO and O₂^?. In this work, the role of ONOO^? on some of the stress responses induced by HS in tobacco BY-2 cultured cells has been investigated by measuring these responses both in the presence and in the absence of 2,6,8-trihydroxypurine (urate), a specific scavenger of ONOO^?. The obtained results suggest a potential role for ONOO^? in some of the responses induced by HS in tobacco cultured cells. In particular, ONOO^? seems implicated in a form of cell death showing apoptotic features and in the regulation of the levels of proteins involved in the response to stress.     

SCAMPs highlight the developing cell plate during cytokinesis in tobacco BY-2 cells

We previously demonstrated that rice (Oryza sativa) SECRETORY CARRIER MEMBRANE PROTEIN1 (OsSCAMP1)-yellow fluorescent protein in transgenic tobacco (Nicotiana tabacum) Bright Yellow-2 cells locates to the plasma membrane and to motile punctate structures, which represent the trans-Golgi network/early endosome and are tubular-vesicular in nature. Here, we now show that SCAMPs are diverted to the cell plate during cytokinesis dividing Bright Yellow-2 cells. As cells progress from metaphase to cytokinesis, punctate OsSCAMP1-labeled structures begin to collect in the future division plane. Together with the internalized endosomal marker FM4-64, they then become incorporated into the cell plate as it forms and expands. This was confirmed by immunogold electron microscopy. We also monitored for the Golgi apparatus and the prevacuolar compartment (PVC)/multivesicular body. Golgi stacks tend to accumulate in the vicinity of the division plane, but the signals are clearly separate to the cell plate. The situation with the PVC (labeled by green fluorescent protein-BP-80) is not so clear. Punctate BP-80 signals are seen at the advancing periphery of the cell plate, which was confirmed by immunogold electron microscopy. Specific but weak labeling was observed in the cell plate, but no evidence for a fusion of the PVC/multivesicular body with the cell plate could be obtained. Our data, therefore, support the notion that cell plate formation is mainly a secretory process involving mass incorporation of domains of the trans-Golgi network/early endosome membrane. We regard the involvement of multivesicular late endosomes in this process to be equivocal.     

Differential expression of alpha- and beta-globin genes during differentiation of cultured erythroleukemic cells.

Murine erythroleukemic cells induced to differentiate in vitro with dimethylsulfoxide provide a model for events involved in the regulated expression of the globin genes. Here we examine alpha- and beta-globin gene expression in such cells which contain no detectable globin RNA prior to induction. To quantitate alpha- and beta-globin RNAs in cellular RNA samples by molecular hybridization techniques, highly radioactive complementary DNAs were synthesized using mouse alpha- and beta-globin RNAs purified by formamide gel electrophoresis. Maximally induced erythroleukemic cells and mouse reticulocytes contain nearly equal relative amounts of alpha- and beta-globin RNA. During the period in which globin RNA accumulates in differentiating erythroleukemic cells, however, alpha- and beta-globin RNAs are not present in equivalent amounts. alphaRNA is present in substantial excess (alpha/beta ratio 3.7) early in induction, and the alpha/beta RNA ratio progressively approaches 1 as differentiation proceeds further. These observations directly suggest that the alpha- and beta-globin genes are differentially expressed during cellular differentiation and raise questions as to how relative expression of globin genes is controlled during normal development.     

Radial microtubule organization by histone H1 on nuclei of cultured tobacco BY-2 cells

In acentriolar higher plant cells, the surface of the nucleus acts as a microtubule-organizing center, substituting for the centrosome. However, the protein factors responsible for this microtubule organization are unknown. The nuclear surfaces of cultured tobacco BY-2 cells possess particles that generate microtubules. We attempted to isolate the proteins in these particles to determine their role in microtubule organization. When incubated with plant or mammalian tubulin, some, but not all, of the isolated nuclei generated abundant microtubules radially from their surfaces. The substance to induce the formation of radial microtubules was confirmed by SDS-PAGE to be a protein with apparent molecular mass of 38 kDa. Partial analysis of the amino acid sequences of the peptide fragments suggested it was a histone H1-related protein. Cloning and cDNA sequence analysis confirmed this and revealed that when the recombinant protein was incubated with tubulin, it could organize microtubules as well as the 38-kDa protein. Histone H1 and tubulin formed complexes immediately, even on ice, and then clusters of these structures were formed. These clusters generated radial microtubules. This microtubule-organizing property was confined to histone H1; all other core histones failed to act as organizers. On immunoblot analysis, rabbit antibodies raised against the 38-kDa protein cross-reacted with histone H1 proteins from tobacco BY-2 cells. These antibodies virtually abolished the ability of the nucleus to organize radial microtubules. Indirect immunofluorescence showed that the antigen was distributed at the nuclear plasm and particularly at nuclear periphery independently from DNA.     

Arabidopsis ASYMMETRIC LEAVES2 protein required for leaf morphogenesis consistently forms speckles during mitosis of tobacco BY-2 cells via signals in its specific sequence

Leaf primordia with high division and developmental competencies are generated around the periphery of stem cells at the shoot apex. Arabidopsis ASYMMETRIC-LEAVES2 (AS2) protein plays a key role in the regulation of many genes responsible for flat symmetric leaf formation. The AS2 gene, expressed in leaf primordia, encodes a plant-specific nuclear protein containing an AS2/LOB domain with cysteine repeats (C-motif). AS2 proteins are present in speckles in and around the nucleoli, and in the nucleoplasm of some leaf epidermal cells. We used the tobacco cultured cell line BY-2 expressing the AS2-fused yellow fluorescent protein to examine subnuclear localization of AS2 in dividing cells. AS2 mainly localized to speckles (designated AS2 bodies) in cells undergoing mitosis and distributed in a pairwise manner during the separation of sets of daughter chromosomes. Few interphase cells contained AS2 bodies. Deletion analyses showed that a short stretch of the AS2 amino-terminal sequence and the C-motif play negative and positive roles, respectively, in localizing AS2 to the bodies. These results suggest that AS2 bodies function to properly distribute AS2 to daughter cells during cell division in leaf primordia; and this process is controlled at least partially by signals encoded by the AS2 sequence itself.     

Differential interaction of branch-specific inhibitors of isoprenoid biosynthesis with cell cycle progression in tobacco BY-2 cells

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGR), which catalyzes the formation of mevalonic acid (MVA), can be specifically blocked by mevinolin. Inhibition of HMGR in vivo leads to an arrest in cell cycle progression in tobacco ( Nicotiana tabacum L. Bright Yellow 2) cells. As MVA in plants is the common precursor of a myriad of isoprenoid products synthesized in the cytosol and mitochondria, it is difficult to identify among such MVA-dependent molecules those whose lack may lead to cell cycle arrest. In an attempt to do so, branch-specific inhibitors of the cytosolic isoprenoid pathway downstream from MVA were used to study their capacity to block cell cycle progression. The effects of squalestatin (sterol biosynthesis inhibitor), chaetomellic acid A and patulin (protein prenyltransferase (PT) inhibitors) and tunicamycin (inhibitor of dolichol-dependent protein glycosyl transferase, thus mimicking the effect of an absence of dolichol) were compared to those induced by mevinolin. In this way, squalestatin and chaetomellic acid were identified as behaving like true cell cycle inhibitors, in that they led to a specific arrest in the cell cycle. However, they did not exactly mimic the mevinolin-induced effects. Patulin proved to be of high general toxicity, which suggests that it may affect other reactions besides blockage of protein isoprenylation. Finally, tunicamycin efficiently blocked growth of cell suspension cultures, but did not arrest the cells in a specific phase of the cell cycle. Results are discussed in the context of a better understanding of the essential implication of isoprenoids in plant cell cycle progression.     

Early steps in the oxidative burst induced by cadmium in cultured tobacco cells (BY-2 line)

The rapid generation of H(2)O(2) by Cd(2+)-treated plant cells was investigated in cultured tobacco (Nicotiana tabacum L.) BY-2 cells. The starting point for the generation of H(2)O(2) has been located at the cell plasma membrane using cytochemical methods. Treatment of the cells with diphenyleneiodonium (DPI) and imidazol, both inhibitors of the neutrophil NADPH oxidase, prevented the generation of H(2)O(2) induced by Cd(2+). These data suggest the involvement of an NADPH oxidase-like enzyme leading to H(2)O(2) production through O(2)(*-) dismutation by superoxide dismutase enzymes. To investigate the implication of Ca(2+) channels in a Cd(2+)-induced oxidative burst, different inhibitors of Ca(2+) channels were used. Only La(3+) totally inhibited the generation of H(2)O(2) induced by Cd(2+). However, verapamil and nifedipine, inhibitors of Ca(2+) channels, were not effective. Calmodulin or a Ca(2+)-dependent protein kinase is also implicated in the signal transduction sequence, based on the results obtained with two types of calmodulin antagonists, fluphenazine and N-(-6-amino-hexyl)-5-chloro-1-naphthalenesulphonamide (W-7) and staurosporine, an inhibitor of protein kinases. However, neomycin, an inhibitor of the phosphoinositide cycle, did not inhibit the generation of H(2)O(2) induced by Cd(2+), suggesting mainly an induction of the oxidative burst mediated by calmodulin and/or calmodulin-dependent proteins.     

Vacuolar and cytoskeletal dynamics during elicitor-induced programmed cell death in tobacco BY-2 cells

Responses of plant cells to environmental stresses often involve morphological changes, differentiation and redistribution of various organelles and cytoskeletal network. Tobacco BY-2 cells provide excellent model system for in vivo imaging of these intracellular events. Treatment of the cell cycle-synchronized BY-2 cells with a proteinaceous oomycete elicitor, cryptogein, induces highly synchronous programmed cell death (PCD) and provide a model system to characterize vacuolar and cytoskeletal dynamics during the PCD. Sequential observation revealed dynamic reorganization of the vacuole and actin microfilaments during the execution of the PCD. We further characterized the effects cryptogein on mitotic microtubule organization in cell cycle-synchronized cells. Cryptogein treatment at S phase inhibited formation of the preprophase band, a cortical microtubule band that predicts the cell division site. Cortical microtubules kept their random orientation till their disruption that gradually occurred during the execution of the PCD twelve hours after the cryptogein treatment. Possible molecular mechanisms and physiological roles of the dynamic behavior of the organelles and cytoskeletal network in the pathogenic signal-induced PCD are discussed.Key words: actin microfilament, cell cycle, cryptogein, microtubules, nuclei, programmed cell death, tobacco BY-2 cells, vacuoles     

Glucocorticoid-induced expression of a foreign gene by the GVG system in transformed tobacco BY-2 cells

A glucocorticoid-induced target gene expression system was used to control the expression of the uidA gene, whose product was beta-glucuronidase (GUS), in tobacco BY-2 cell suspension culture. This targeting system showed quick, sensitive, and reversible response to dexamethazone (DEX), an artificial glucocorticoid hormone. Addition of DEX greatly and quickly enhanced uidA gene expression, whose level was as high as that under the control of the CaMV 35S promoter whereas in the absence of DEX, the GUS specific activity was suppressed to be as low as that of nontransformed BY-2 cells. The dilution of DEX decreased GUS specific activity showing that the concentration of DEX plays a major role in controlling the expression level of the target. The use of the glucocorticoid-induced system in plant cell suspension culture was demonstrated to precisely control target gene expression.     

ARP2 and ARP3 are localized to sites of actin filament nucleation in tobacco BY-2 cells

Summary. Complete depolymerization of actin filaments (AFs) at low temperature (0 °C) is followed by the formation of transient actin structures at 25 °C in tobacco BY-2 cells (Nicotiana tabacum L.). Using antibodies against fission yeast actin-related proteins (ARP2 and ARP3), we show here that transient actin structures (dots, dotted filaments, rods) colocalize with epitopes stained by these antibodies and thus are likely to represent sites of actin filament nucleation (SANs). In contrast to the cold-induced disassembly of AFs, no transient actin structures were detectable during recovery of AFs from latrunculin B-induced depolymerization. However, the staining pattern obtained with ARP antibodies in latrunculin B-treated cells was similar to that in controls and cold-treated cells. This suggests that, in addition to the complete depolymerization of AFs, disruption of other cellular structures is needed for the formation of transient actin structures during the early phase of recovery from cold treatment. Correspondence and reprints: Department of Plant Physiology, Faculty of Science, Charles University, Viničná 5, 128 44 Prague 2, Czech Republic.     

Myosin XI-dependent formation of tubular structures from endoplasmic reticulum isolated from tobacco cultured BY-2 cells

The reticular network of the endoplasmic reticulum (ER) consists of tubular and lamellar elements and is arranged in the cortical region of plant cells. This network constantly shows shape change and remodeling motion. Tubular ER structures were formed when GTP was added to the ER vesicles isolated from tobacco (Nicotiana tabacum) cultured BY-2 cells expressing ER-localized green fluorescent protein. The hydrolysis of GTP during ER tubule formation was higher than that under conditions in which ER tubule formation was not induced. Furthermore, a shearing force, such as the flow of liquid, was needed for the elongation/extension of the ER tubule. The shearing force was assumed to correspond to the force generated by the actomyosin system in vivo. To confirm this hypothesis, the S12 fraction was prepared, which contained both cytosol and microsome fractions, including two classes of myosins, XI (175-kD myosin) and VIII (BY-2 myosin VIII-1), and ER-localized green fluorescent protein vesicles. The ER tubules and their mesh-like structures were arranged in the S12 fraction efficiently by the addition of ATP, GTP, and exogenous filamentous actin. The tubule formation was significantly inhibited by the depletion of 175-kD myosin from the S12 fraction but not BY-2 myosin VIII-1. Furthermore, a recombinant carboxyl-terminal tail region of 175-kD myosin also suppressed ER tubule formation. The tips of tubules moved along filamentous actin during tubule elongation. These results indicated that the motive force generated by the actomyosin system contributes to the formation of ER tubules, suggesting that myosin XI is responsible not only for the transport of ER in cytoplasm but also for the reticular organization of cortical ER.     

Amyloplast formation in cultured tobacco cells. III Determination of the timing of gene expression necessary for starch accumulation

When BY-2 cultured tobacco (Nicotiana tabacum L.) cells were transferred to auxin-depleted culture medium containing cytokinin (benzyladenine, 1 mg/l), the starch content per cell started increasing from 18 h of culture and amyloplasts had formed by 48 h. Pulse-treatment of the cells with actinomycin D and cycloheximide for the first 12 h (or longer) of culture significantly decreased the cellular starch content after 48 h, whereas the starch content did not decrease significantly when the cells were released from the inhibition within 6 h. This suggests that nuclear gene expression necessary for amyloplast formation begins 6–12 h after the transfer. Immunoblotting analysis of the accumulation of ADP-glucose starch glycosyl transferase (starch synthase) supported this inference. Received: 21 August 1998/ Revision received: 3 September 1998/ Accepted: 2 October 1998