A novel Arabidopsis gene TONSOKU is required for proper cell arrangement in root and shoot apical meristems

Root apical meristem (RAM) and shoot apical meristem (SAM) are vital for the correct development of the plant. The direction, frequency, and timing of cell division must be tightly controlled in meristems. Here, we isolated new Arabidopsis mutants with shorter roots and fasciated stems. In the tonsoku (tsk) mutant, disorganized RAM and SAM formation resulted from the frequent loss of proper alignment of the cell division plane. Irregular cell division also occurred in the tsk embryo, and the size of cells in meristems and embryo in tsk mutant was larger than in the wild type. In the enlarged SAM of the tsk mutant, multiple centers of cells expressing WUSCHEL (WUS) were observed. In addition, expression of SCARECROW (SCR) in the quiescent center (QC) disappeared in the disorganized RAM of tsk mutant. These results suggest that disorganized cell arrangements in the tsk mutants result in disturbed positional information required for the determination of cell identity. The TSK gene was found to encode a protein with 1311 amino acids that possesses two types of protein-protein interaction motif, leucine-glycine-asparagine (LGN) repeats and leucine-rich repeats (LRRs). LGN repeats are present in animal proteins involved in asymmetric cell division, suggesting the possible involvement of TSK in cytokinesis. On the other hand, the localization of the TSK-GFP (green fluorescent protein) fusion protein in nuclei of tobacco BY-2 cells and phenotypic similarity of tsk mutants to other fasciated mutants suggest that the tsk mutation may cause disorganized cell arrangements through defects in genome maintenance.     

An Arabidopsis protein with a novel calcium-binding repeat sequence interacts with TONSOKU/MGOUN3/BRUSHY1 involved in meristem maintenance

TONSOKU(TSK)/MGOUN3/BRUSHY1 from Arabidopsis thaliana, which plays an important role in the maintenance of meristem organization, contains an LGN repeat motif similar to that found in animal proteins involved in asymmetric cell division. One protein that interacts with the LGN motif of TSK in a yeast two-hybrid screen, TSK-associating protein 1 (TSA1), contains a 10-fold repeat of a unique 41 amino acid sequence. The repeat sequence, with a glutamic acid-phenylalanine-glutamic acid (EFE) conserved core sequence, is enriched with acidic amino acids. TSA1 also contains an N-terminal putative signal peptide and it interacts with the LGN motif of TSK through a C-terminal region separated from the EFE repeats by a putative membrane-spanning region. The recombinant protein consisting of EFE repeats was rich in alpha-helical structure and possessed Ca2+-binding activity. Unlike nuclear localization of TSK, the TSA1 fused with green fluorescent protein (GFP) expressed in tobacco BY-2 cells was localized in small cytoplasmic vesicles during interphase. However, cellular localization of both TSA1-GFP and GFP-TSK changed dynamically during mitosis. In particular, both GFP-TSK and TSA1-GFP were concentrated in limited areas that are close to the ends of spindle microtubules ahead of separating chromatids. These results are discussed in terms of the possible involvement of TSK and TSA1 in mitosis.     

Indomethacin-induced G1/S phase arrest of the plant cell cycle

In animal systems, indomethacin inhibits cAMP production via a prostaglandin-adenylyl cyclase pathway. To examine the possibility that a similar mechanism occurs in plants, the effect of indomethacin on the cell cycle of a tobacco bright yellow 2 (TBY-2) cell suspension was studied. Application of indomethacin during mitosis did not interfere with the M/G1 progression in synchronized BY-2 cells but it inhibited cAMP production at the beginning of the G1 phase and arrested the cell cycle progression at G1/S. These observations are discussed in relation to the putative involvement of cAMP biosynthesis in the cell cycle progression in TBY-2 cells.     

Crosstalk between elicitor-induced cell death and cell cycle regulation in tobacco BY-2 cells

The molecular links between cell cycle control and the regulation of programmed cell death are largely unknown in plants. Here we studied the relationship between the cell cycle and elicitor-induced cell death using synchronized tobacco BY-2 cells. Flow cytometry and fluorescence microscopy of nuclear DNA, and RNA gel-blot analyses of cell cycle-related genes revealed that the proteinaceous elicitor cryptogein induced cell cycle arrest at the G1 or G2 phase before the induction of cell death. Furthermore, the patterns of cell death induction and defence-related genes were different in different phases of the cell cycle. Constitutive treatment with cryptogein induced cell cycle arrest and cell death at the G1 or G2 phase. With transient treatment for 2 h, cell cycle arrest and cell death were only induced by treatment with the elicitor during the S or G1 phase. By contrast, the elicitor-induced production of reactive oxygen species was observed during all phases of the cell cycle. These results indicate that although recognition of the elicitor signal is cell cycle-independent, the induction of cell cycle arrest and cell death depends on the phase of the cell cycle.     

Tobacco BY-2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycle

The mitotic inducer gene from Schizosaccharomyces pombe, Spcdc25, was used as a tool to investigate regulation of G2/M in higher plants using the BY-2 (Nicotiana tabacum) cell line as a model. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size through a shortening of the G2 phase. The cells often formed isodiametric double files both in BY-2 cells and in cell suspensions derived from 35S::Spcdc25 tobacco plants. In Spcdc25-expressing cells, the tobacco cyclin-dependent kinase, NtCDKB1, showed high activity in early S phase, S/G2 and early M phase, whereas in empty vector cells CDKB1 activity was transiently high in early S phase but thereafter remained lower. Spcdc25-expressing cells also bypassed a block on G2/M imposed by the cytokinin biosynthetic inhibitor lovastatin (LVS). Surprisingly, cytokinins were at remarkably low levels in Spcdc25-expressing cells compared with the empty vector, explaining why these cells retained mitotic competence despite the presence of LVS. In conclusion, synchronised Spcdc25-expressing BY-2 cells divided prematurely at a small cell size, and they exhibited premature, but sustained, CDKB1 activity even though endogenous cytokinins were virtually undetectable.     

Auxin induction of cell cycle regulated activity of tobacco telomerase.

Telomerase activity was measured at each phase of the cell cycle in synchronized tobacco (Nicotiana tabacum) BY-2 cells in suspension culture with the use of the telomeric repeat amplification protocol assay. The activity was low or undetectable at most phases of the cell cycle but showed a marked increase at early S phase. The induction of telomerase activity was not affected by the S phase blockers aphidicolin (which inhibits DNA polymerase alpha) or hydroxyurea (which inhibits ribonucleotide reductase), but it was prevented by olomoucine, an inhibitor of Cdc2/Cdk2 kinases that blocks G(1)-S cell cycle transition. These results suggest that the induction of telomerase activity is not directly coupled to DNA replication by conventional DNA polymerases, but rather is triggered by the entry of cells into S phase. Various analogs of the plant hormone auxin, including indole-3-acetic acid, alpha-naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid, potentiated the increase in telomerase activity at early S phase; the growth-inactive analog 2,3-dichlorophenoxyacetic acid, however, had no such effect. Potentiation by indole-3-acetic acid of the induction of telomerase activity was dose dependent. Together, these data indicate that telomerase activity in tobacco cells is regulated in a cell cycle-dependent manner, and that the increase in activity at S phase is specifically inducible by auxin.     

Effects of mevinolin on cell cycle progression and viability of tobacco BY-2 cells

Mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, was used to study the importance of mevalonic acid (MVA) for cell cycle progression of tobacco (Nicotiana tabacum L.) BY-2 cells. After treatment with 5 microM mevinolin, the cell cycle progression was completely blocked and two cell populations accumulated (80% in phase G0/G1 and 20% in G2/M). The arrest could be released by subsequent addition of MVA. Effects were compared to those caused by aphidicolin, an inhibitor of alpha-like DNA polymerases that blocks cell cycle at the entry of the S phase. The 80% proportion of mevinolin-treated TBY-2 cells was clearly arrested before the aphidicolin-inducible block. By the aid of a double-blocking technique, it was shown that the mevinolin-induced cell arrest of highly synchronized cells was due to interaction with a control point located at the mitotic telophase/entry G1 phase. Depending on the developmental stage, mevinolin induced rapid cell death in a considerable percentage of cells. Mevinolin treatment led to a partial synchronization, as shown by the increase in mitotic index. The following decrease was correlated with the above-mentioned induction of cell death.     

Localization of green fluorescent protein fusions with the seven Arabidopsis vacuolar sorting receptors to prevacuolar compartments in tobacco BY-2 cells

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis (Arabidopsis thaliana) contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here, we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1-7) based on the previously proven hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to green fluorescent protein (GFP) and TMD/CT sequences (sp-GFP-TMD/CT) of the seven individual AtVSR. Transgenic tobacco BY-2 cell lines expressing these seven sp-GFP-TMD-CT fusions all exhibited typical punctate signals colocalizing with VSR proteins by confocal immunofluorescence. In addition, wortmannin caused the GFP-marked prevacuolar organelles to form small vacuoles, and VSR antibodies labeled these enlarged MVBs in transgenic BY-2 cells. Wortmannin also caused VSR-marked PVCs to vacuolate in other cell types, including Arabidopsis, rice (Oryza sativa), pea (Pisum sativum), and mung bean (Vigna radiata). Therefore, the seven AtVSRs are localized to MVBs in tobacco BY-2 cells, and wortmannin-induced vacuolation of PVCs is a general response in plants.     

Expression of the telomeric repeat binding factor gene NgTRF1 is closely coordinated with the cell division program in tobacco BY-2 suspension culture cells

Telomeres are vital for preserving chromosome integrity during cell division. Several genes encoding potential telomere-binding proteins have recently been identified in higher plants, but nothing is known about their function or regulation during cell division. In this study, we have isolated and characterized a cDNA clone, pNgTRF1, encoding a putative double-stranded telomeric repeat binding factor of Nicotiana glutinosa, a diploid tobacco plant. The predicted protein sequence of NgTRF1 (Mr = 75,000) contains a single Myb-like domain with significant homology to a corresponding motif in human TRF1/Pin2 and TRF2. Gel retardation assays revealed that bacterially expressed full-length NgTRF1 was able to form a specific complex only with probes containing three or more contiguous telomeric TTTAGGG repeats. The Myb-like domain of NgTRF1 is essential, but not sufficient, to bind the telomeric repeat sequence. The glutamine-rich extreme C-terminal region, which does not exist in animal proteins, was additionally required to form a specific telomere-protein complex. The dissociation constant (Kd) of the Myb motif plus the glutamine-rich domain of NgTRF1 to the two-telomeric repeat sequence was evaluated to be 4.5 +/- 0.2 x 10-9 m, which is comparable to that of the Myb domain of human TRF1. Expression analysis showed that NgTRF1 gene activity was inversely correlated with the cell division capacity of tobacco root cells and during the 9-day culture period of BY-2 suspension cells, while telomerase activity was positively correlated with cell division. In synchronized BY-2 cells, NgTRF1 was selectively expressed in G1 phase, whereas telomerase activity peaked in S phase. These findings suggest that telomerase activity and NgTRF1 expression are differentially regulated in an opposing fashion during growth and cell division in tobacco plants. The possible physiological functions of NgTRF1 in tobacco cells are also discussed.     

Protein farnesyltransferase in plants: molecular characterization and involvement in cell cycle control.

Farnesylation is required for membrane targeting, protein-protein interactions, and the biological activity of key regulatory proteins, such as Ras small GTPases and protein kinases in a wide range of eukaryotes. In this report, we describe the molecular identification of a plant protein farnesyltransferase (FTase) and evidence for its role in the control of the cell cycle in plants. A pea gene encoding a homolog of the FTase beta subunit was previously cloned using a polymerase chain reaction-based strategy. A similar approach was used to clone a pea gene encoding a homolog of the FTase alpha subunit. The biochemical function of the pea FTase homologs was demonstrated by the reconstitution of FTase enzyme activity using FTase fusion proteins coexpressed in Escherichia coll. RNA gel blot analyses showed that levels of FTase mRNAs are generally higher in tissues, such as those of nodules, that are active in cell division. The relationship of FTase to cell division was further analyzed during the growth of suspension-cultured tobacco BY-2 cells. A biphasic fluctuation of FTase enzyme activity preceded corresponding changes in mitotic activity at the early log phase of cell growth. Moreover, manumycin, a specific inhibitor of FTase, was effective in inhibiting mitosis and growth in these cells. Using synchronized BY-2 cells, manumycin completely blocked mitosis when added at the early S phase but not when added at the G2 phase. These data suggest that FTase is required for the plant cell cycle, perhaps by modulating the progression through the S phase and the transition from G1 to the S phase.     

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.     

Dehydroascorbate influences the plant cell cycle through a glutathione-independent reduction mechanism

Glutathione is generally accepted as the principal electron donor for dehydroascorbate (DHA) reduction. Moreover, both glutathione and DHA affect cell cycle progression in plant cells. But other mechanisms for DHA reduction have been proposed. To investigate the connection between DHA and glutathione, we have evaluated cellular ascorbate and glutathione concentrations and their redox status after addition of dehydroascorbate to medium of tobacco (Nicotiana tabacum) L. cv Bright Yellow-2 (BY-2) cells. Addition of 1 mm DHA did not change the endogenous glutathione concentration. Total glutathione depletion of BY-2 cells was achieved after 24-h incubation with 1 mm of the glutathione biosynthesis inhibitor l-buthionine sulfoximine. Even in these cells devoid of glutathione, complete uptake and internal reduction of 1 mm DHA was observed within 6 h, although the initial reduction rate was slower. Addition of DHA to a synchronized BY-2 culture, or depleting its glutathione content, had a synergistic effect on cell cycle progression. Moreover, increased intracellular glutathione concentrations did not prevent exogenous DHA from inducing a cell cycle shift. It is therefore concluded that, together with a glutathione-driven DHA reduction, a glutathione-independent pathway for DHA reduction exists in vivo, and that both compounds act independently in growth control.     

Endogenous cytokinin oscillations control cell cycle progression of tobacco BY-2 cells

The significance of cytokinins for the progression of the cell cycle is well known. Cytokinins contribute to the control of the expression of D-cyclins and other cell cycle genes, but knowledge as to how they affect the progression of the cell cycle is still limited. Highly synchronized tobacco BY-2 cells with clearly defined cell cycle stages were employed to determine cytokinin patterns in detail throughout the entire cycle. Concentrations of trans-zeatin, and of some other cytokinins, oscillated during the course of the cell cycle, increasing substantially at all four phase transitions and decreasing again to a minimum value during the course of each subsequent phase. Addition of exogenous cytokinins or inhibition of cytokinin biosynthesis promoted the progression of the cell cycle when the effects of these manipulations intensified the endogenous fluctuations, whereas the progression of the cycle was retarded when the amplitude of the fluctuations was decreased. The results show that the attainment of low concentrations of cytokinins is as important as the transient increases in concentration for a controlled progression from one phase of the cell cycle to the next. Cytokinin oxidase/dehydrogenase activity also showed fluctuations during the course of the cell cycle, the timing of which could at least partly explain oscillations of cytokinin levels. The activities of the enzyme were sufficient to account for the rates of cytokinin disappearance observed subsequent to a phase transition.     

Crosstalk between auxin, cytokinins, and sugars in the plant cell cycle

Plant meristems are utilization sinks, in which cell division activity governs sink strength. However, the molecular mechanisms by which cell division activity and sink strength are adjusted to a plant's developmental program in its environmental setting are not well understood. Mitogenic hormonal as well as metabolic signals drive and modulate the cell cycle, but a coherent idea of how this is accomplished, is still missing. Auxin and cytokinins are known as endogenous mitogens whose concentrations and timing, however, can be externally affected. Although the sites and mechanisms of signal interaction in cell cycle control have not yet been unravelled, crosstalk of sugar and phytohormone signals could be localized to several biochemical levels. At the expression level of cell cycle control genes, like cyclins, Cdks, and others, synergistic but also antagonistic interactions could be demonstrated. Another level of crosstalk is that of signal generation or modulation. Cytokinins affect the activity of extracellular invertases and hexose-uptake carriers and thus impinge on an intracellular sugar signal. With tobacco BY-2 cells, a coordinated control of cell cycle activity at both regulatory levels could be shown. Comparison of the results obtained with the root cell-representing BY-2 cells with literature data from shoot tissues or green cell cultures of Arabidopsis and Chenopodium suggests opposed and tissue-specific regulatory patterns of mitogenic signals and signal crosstalk in root and shoot meristems.     

Dynamic organization of vacuolar and microtubule structures during cell cycle progression in synchronized tobacco BY-2 cells

In higher plant cells, vacuoles show considerable diversity in their shapes and functions. The roles of vacuoles in the storage, osmoregulation, digestion and secretory pathway are well established; however, their functions in cell morphogenesis and cell division are still unclear. To observe the dynamic changes of vacuoles in living plant cells, we attempted to visualize the vacuolar membrane (VM) by pulse-labeling tobacco BY-2 cells with a styryl fluorescent dye, FM4-64. By time-sequence observations using confocal laser scanning microscopy (CLSM), we could follow the dynamics of vacuolar structures throughout the cell cycle in living higher plant cells. We also confirmed the dynamic changes of VM structures by the observation using transgenic BY-2 cells expressing GFP-AtVam3p fusion protein (BY-GV). Furthermore, by using transgenic BY-2 cells that stably express a GFP-tubulin fusion protein [BY-GT16, Kumagai et al. (2001) Plant Cell Physiol. 42: 723], we could study the relationship between the dynamics of vacuoles and microtubules. From these observations, we identified, for the first time, some remarkable events: (1) at the late G(2) phase, tubular structures of the vacuolar membrane developed in the central region of the cell, probably in the premitotic cytoplasmic band (phragmosome), surrounding the mitotic apparatus; (2) from anaphase to telophase, these tubular structures invaded the region of the phragmoplast within which the cell plate was being formed; (3) at the early G(1) phase, some of the tubular structures expanded rapidly between the cell plate and daughter nuclei, and subsequently developed into large vacuoles at interphase.     

The RING Finger Protein Nt RCP1 Is Involved in the Floral Transition in Tobacco(Nicotiana tabacum)

The transition from the vegetative phase to the reproductive phase is a major developmental process in flowering plants.The underlying mechanism controlling this cellular process remains a research focus in the field of plant molecular biology.In the present work,we identified a gene encoding the C3H2C3-type RING finger protein Nt RCP1 from tobacco BY-2 cells.Enzymatic analysis demonstrated that Nt RCP1 is a functional E3 ubiquitin ligase.In tobacco plants,expression level of Nt RCP1 was higher in the reproductive shoot apices than in the vegetative ones.Nt RCP1-overexpressing plants underwent a more rapid transition from the vegetative to the reproductive phase and flowered markedly earlier than the wild-type control.Histological analysis revealed that the shoot apical meristem of Nt RCP1-overexpressing plants initiated inflorescence primordia precociously compared to the wild-type plant due to accelerated cell division.Overexpression of Nt RCP1 in BY-2 suspension cells promoted cell division,which was a consequence of the shortened G2 phase in the cell cycle.Together,our data suggest that Nt RCP1 may act as a regulator of the phase transition,possibly through its role in cell cycle regulation,during vegetative/reproductive development in tobacco plant.