Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells.

Transient transformation with Agrobacterium is a widespread tool allowing rapid expression analyses in plants. However, the available methods generate expression in interphase and do not allow the routine analysis of dividing cells. Here, we present a transient transformation method (termed 'TAMBY2') to enable cell biological studies in interphase and cell division. Agrobacterium-mediated transient gene expression in tobacco BY-2 was analysed by Western blotting and quantitative fluorescence microscopy. Time-lapse microscopy of cytoskeletal markers was employed to monitor cell division. Double-labelling in interphase and mitosis enabled localization studies. We found that the transient transformation efficiency was highest when BY-2/Agrobacterium co-cultivation was performed on solid medium. Transformants produced in this way divided at high frequency. We demonstrated the utility of the method by defining the behaviour of a previously uncharacterized microtubule motor, KinG, throughout the cell cycle. Our analyses demonstrated that TAMBY2 provides a flexible tool for the transient transformation of BY-2 with Agrobacterium. Fluorescence double-labelling showed that KinG localizes to microtubules and to F-actin. In interphase, KinG accumulates on microtubule lagging ends, suggesting a minus-end-directed function in vivo. Time-lapse studies of cell division showed that GFP-KinG strongly labels preprophase band and phragmoplast, but not the metaphase spindle.

Control of protein translation by phosphorylation of the mRNA 5'-cap-binding complex.

Initiation of mRNA translation is a key regulatory step in the control of gene expression. Microarray analysis indicates that total mRNA levels do not always reflect protein levels, since mRNA association with polyribosomes is necessary for protein synthesis. Phosphorylation of translation initiation factors offers a cost-effective and rapid way to adapt to physiological and environmental changes, and there is increasing evidence that many of these factors are subject to multiple regulatory phosphorylation events. The present article focuses on the nature of reversible phosphorylation and the function of the 5'-cap-binding complex in plants.

Dynamic distribution of BIMG(PP1) in living hyphae of Aspergillus indicates a novel role in septum formation.

Mutation of bimG, the major protein phosphatase 1 gene in Aspergillus nidulans, causes multiple cell cycle and hyphal growth defects that are associated with overphosphorylation of subcellular components. We have used functional translational fusions with the green fluorescent protein (GFP) to show that BIMG has at least four discrete locations within growing hyphae. Three of these locations, the hyphal tip, the spindle pole body and the nucleus, correlate with previously known requirements for bimG(PP1) in mitosis and hyphal growth and are highly dynamic. BIMG-GFP in the hyphal tip seemed to be associated with the plasma membrane and formed a collar of fluorescence within the apical dome. The distribution of nuclear BIMG-GFP varied depending on nutritional conditions; on poor medium, it concentrated more in the nucleolus than in the nucleoplasm, whereas on rich medium, it was more evenly distributed between the two nuclear regions. The association of BIMG-GFP with developing septa was transient, and we present evidence that BIMG phosphatase plays a direct role in septum formation, distinct from its role in mitosis. We conclude that, by being physically present at several sites, the BIMG phosphatase has roles in multiple cellular processes.

Cellular basis of shoot apical meristem development.

Shoot apical meristems are composed of proliferating, embryonic type cells, that generate tissues and organs throughout the life of the plant. This review covers the cell biology of the higher plant shoot apical meristem (SAM). The first section describes the molecular basis of plant cell growth and division. The genetic mechanisms, that operate in meristem function and the identification of several key regulators of meristem behavior are described in the second section, and intercellular communication and coordination of cellular behavior in the third part. Finally, we discuss some recent results that indicate interaction between the cellular regulators, such as the cell cycle control genes and developmental regulators.

G2/M-phase-specific transcription during the plant cell cycle is mediated by c-Myb-like transcription factors.

Plant B-type cyclin genes are expressed specifically in late G2- and M-phases during the cell cycle. Their promoters contain a common cis-acting element, called the MSA (M-specific activator) element, that is necessary and sufficient for periodic promoter activation. This motif also is present in the tobacco kinesin-like protein gene NACK1, which is expressed with timing similar to that of B-type cyclin genes. In this study, we show that G2/M-phase-specific activation of the NACK1 promoter also is regulated by the MSA element, suggesting that a defined set of G2/M-phase-specific genes are coregulated by an MSA-mediated mechanism. In a search for MSA binding factors by yeast one-hybrid screening, we identified three different Myb-like proteins that interact specifically with the MSA sequence. Unlike the majority of plant Myb-like proteins, these Myb proteins, NtmybA1, NtmybA2, and NtmybB, have three imperfect repeats in the DNA binding domain, as in animal c-Myb proteins. During the cell cycle, the level of NtmybB mRNA did not change significantly, whereas the levels of NtmybA1 and A2 mRNAs fluctuated and peaked at M-phase, when B-type cyclin genes were maximally induced. In transient expression assays, NtmybA1 and A2 activated the MSA-containing promoters, whereas NtmybB repressed them. Furthermore, expression of NtmybB repressed the transcriptional activation mediated by NtmybA2. Our data show that a group of plant Myb proteins that are structurally similar to animal c-Myb proteins have unexpected roles in G2/M-phase by modulating the expression of B-type cyclin genes and may regulate a suite of coexpressed genes.

Cell cycle regulation of cyclin-dependent kinases in tobacco cultivar Bright Yellow-2 cells.

Plants possess two major classes of cyclin-dependent kinases (CDK) with cyclin-binding motifs PSTAIRE (CDK-a) and PPTA/TLRE (CDK-b). Tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells are the most highly synchronizable plant culture, but no detailed analysis of CDK activities has been reported in this system. Here we describe isolation of new PPTALRE CDKs (Nicta;CdkB1) from Bright Yellow-2 cells and present detailed analysis of the mRNA, protein and kinase activity levels of CdkB1, and the PSTAIRE CDKA during the growth and cell cycles. CdkA and CdkB1 transcripts are more abundant in exponential than in stationary phase cells, but the two genes show strikingly different regulation during the cell cycle. CdkA mRNA and protein accumulate during G1 in cells re-entering the cell cycle, and immunoprecipitated histone H1 kinase activity increases at the G1/S boundary. Aphidicolin synchronized cells show the highest CDKA-associated histone H1 kinase activity during S-G2 phases, although CdkA mRNA and protein levels are not significantly regulated. In contrast, CdkB1 transcripts are present at very low levels until S phase and CDKB1 protein and kinase activity is almost undetectable in G1. CdkB1 mRNA accumulates through S until M phase and its associated kinase activity peaks at the G2/M boundary, confirming that transcription of PPTALRE CDKs is cell cycle regulated. We suggest that CDKA kinase activity likely plays roles at the G1/S phase boundary, during S phase, and at the G2/M phase transition, and that CDKB1 kinase activity is present only at G2/M.

Microinjection reveals cell-to-cell movement of green fluorescent protein in cells of maize coleoptiles.

During the evaluation of dual-purpose plant/fungal expression systems, we found that green fluorescent protein (GFP) has the ability to move from cell to cell in the epidermis of Zea mays L. cv. Mutator coleoptiles as well as into underlying cortical cells. Movement of GFP was observed both when DNA encoding GFP and bacterially expressed GFP were microinjected into epidermal cells. This suggests that GFP is capable of cell-to-cell movement. From experiments using dextrans of known molecular weight linked to fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate, we estimate that the plasmodesmata of these cells have a size exclusion limit < 4.4 kDa. Cell-to-cell GFP movement did not occur when GFP was altered to include a nucleus- or endoplasmic reticulum-retention sequence. The fact that these transcripts differ from that of cytoplasmic GFP by a small number of nucleotides suggests that the transcripts are not capable of movement, but movement of nucleic acid cannot be excluded. Since GFP is widely used to study cell-to-cell movement and to localize the expression of transgenes, caution should be exercised when interpreting results where GFP expression is used for localization.

A type 2A protein phosphatase gene from Aspergillus nidulans is involved in hyphal morphogenesis.

A PCR-based approach, using degenerate oligonucleotide primers, was used to isolate fragments of two genes encoding type 2A protein phosphatases from the filamentous fungus, Aspergillus nidulans. The complete genomic sequence of one of these genes, pphA, was isolated and characterised. The pphA gene was predicted to encode a 329-residue protein which is about 85% identical to mammalian protein phosphatase 2A. Ectopic expression of the wild-type pphA+ gene slightly inhibited growth in some transformants; but a mutant form of pphA, in which R259 was mutated to Q, led to slow growth, delayed germ tube emergence and mitotic defects at low temperature. These results indicate that the pphA+ gene plays an important role in hyphal growth.

The Arabidopsis D-type cyclins CycD2 and CycD3 both interact in vivo with the PSTAIRE cyclin-dependent kinase Cdc2a but are differentially controlled.

D-type cyclins (CycD) play key roles in linking the Arabidopsis cell cycle to extracellular and developmental signals, but little is known of their regulation at the post-transcriptional level or of their cyclin-dependent kinase (CDK) partners. Using new antisera to CycD2 and CycD3, we demonstrate that the CDK partner of these Arabidopsis cyclins is the PSTAIRE-containing CDK Cdc2a. Previous analysis has shown that transcript levels of CycD2 and CycD3 are regulated in response to sucrose levels and that both their mRNA levels and kinase activity are induced with different kinetics during the G(1) phase of cells reentering the division cycle from quiescence. Here we analyze the protein levels and kinase activity of CycD2 and CycD3. We show that CycD3 protein and kinase activity parallel the abundance of its mRNA and that CycD3 protein is rapidly lost from cells in stationary phase or following sucrose removal. In contrast to both CycD3 and the regulation of its own mRNA levels, CycD2 protein is present at constant levels. CycD2 kinase activity is regulated by sequestration of CycD2 protein in a form inaccessible to immunoprecipitation and probably not complexed to Cdc2a.

A glucoamylase::GFP gene fusion to study protein secretion by individual hyphae of Aspergillus niger.

Although Aspergillus niger is used as a host for heterologous protein production, yields are generally lower than those obtained for homologous proteins. Mechanisms of protein secretion and the secretory pathway in filamentous fungi are poorly characterised, although there is evidence to suggest that secretion occurs by a mechanism similar to that in other eukaryotes, but with proteins destined for secretion being directed to the hyphal tip. We report on a method using a glucoamylase: GFP gene fusion which allows us for the first time to monitor, in vivo, protein secretion in A. niger at the single hyphal level. A synthetic green fluorescent protein (sGFP(S65T)) was fused to truncated A. niger glucoamylase (GLA:499). Southern blot analysis of transformants confirmed that the gene fusion had successfully integrated into the A. niger genome. Confocal and fluorescence microscopy revealed that the GLA::GFP fusion protein is fluorescent in A. niger and appears to be directed to the hyphal tip. In young mycelia, hyphal cell wall fluorescence is apparent and immunogold labelling of GFP confirmed that GFP was partially localised within the hyphal cell wall. Using Western blotting, extracellular GLA::GFP was detected only in culture filtrates of young mycelia grown in a soya milk medium. The actin inhibitor latrunculin B was used to disrupt the secretion process, and its effects on the distribution of GLA::GFP were monitored.

The expression of D-cyclin genes defines distinct developmental zones in snapdragon apical meristems and is locally regulated by the Cycloidea gene.

Three D-cyclin genes are expressed in the apical meristems of snapdragon (Antirrhinum majus). The cyclin D1 and D3b genes are expressed throughout meristems, whereas cyclin D3a is restricted to the peripheral region of the meristem, especially the organ primordia. During floral development, cyclin D3b expression is: (a) locally modulated in the cells immediately surrounding the base of organ primordia, defining a zone between lateral organs that may act as a developmental boundary; (b) locally modulated in the ventral petals during petal folding; and (c) is specifically repressed in the dorsal stamen by the cycloidea gene. Expression of both cyclin D3 genes is reduced prior to the cessation of cell cycle activity, as judged by histone H4 expression. Expression of all three D-cyclin genes is modulated by factors that regulate plant growth, particularly sucrose and cytokinin. These observations may provide a molecular basis for understanding the local regulation of cell proliferation during plant growth and development.

sodVIC is an alpha-COP-related gene which is essential for establishing and maintaining polarized growth in Aspergillus nidulans.

Strains of Aspergillus nidulans carrying the conditional-lethal mutation sodVIC1 (stabilization of disomy) are defective in nuclear division and hyphal extension. The mutation affects both the establishment and maintenance of polar growth, since mutant spores do not germinate at restrictive temperature and preexisting hyphae stop growing upon upshift. The defect is reversible within the first 3-4 h at restrictive temperature but longer periods of incubation are lethal due to cell lysis and morphological abnormalities. There is no evidence for a specific cell cycle lesion, suggesting the existence of a feedback mechanism whereby hyphal extension is coordinated with nuclear partitioning. The sodVIC gene has been cloned from a chromosome VI-specific cosmid library and its product exhibits strong homology to the alpha-COP subunit of the coatomer complex involved in the secretory pathway in yeast and higher organisms. Molecular disruption of the gene is lethal, indicating that SodVIC is essential for growth in A. nidulans.

Cloning and characterization of the unusual cyclin gene from an amphidiploid of Nicotiana glauca-Nicotiana langsdorffii hybrid.

Cyclins play an important role in the regulation of cell cycle progression in eukaryotic cells. As an aid to understanding the molecular nature of unregulated cell proliferation, a cDNA clone encoding a cyclin gene, GTcyc, was identified from genetic tumors. The clone contained 1095 bp including a 24 base poly(A) tail. GTcyc is an unusual cyclin gene, distantly related to mammalian cyclin D genes having 21-25% identity within the cyclin box. Northern blots showed that the genetic tumors express high levels of GTcyc relative to non-tumor hybrid tissues. Southern analysis suggests that GTcyc may be contained one or two families in genetic tumors.

The maize retinoblastoma protein homologue ZmRb-1 is regulated during leaf development and displays conserved interactions with G1/S regulators and plant cyclin D (CycD) proteins.

Recent discoveries of plant retinoblastoma (Rb) protein homologues and D-type cyclins suggest that control of the onset of cell division in plants may have stronger parallels with mammalian G1/S controls than with yeasts. In mammals, the Rb protein interacts specifically with D-type cyclins and regulates cell proliferation by binding and inhibiting E2F transcription factors. However, the developmental role of Rb in plants and its potential interaction with cell cycle regulators is unknown. We show that the maize Rb homologue ZmRb-1 is temporally and spatially regulated during maize leaf development. ZmRb-1 is highly expressed in differentiating cells, but almost undetectable in proliferating cells. In vitro, both ZmRb-1 and human Rb bind all classes of plant D-type cyclins with the involvement of a conserved N-terminal Leu-x-Cys-x-Glu (LxCxE) Rb-interaction motif. This binding is strongly reduced by mutation of the conserved Cys-470 of ZmRb-1. ZmRb-1 binds human and Drosophila E2F, and inhibits transcriptional activation of human E2F. We also show that ZmRb-1 is a good in vitro substrate for all human G1/S protein kinases. The functional conservation of proteins that control the G1/S transition in mammals and plants points to the existence of plant E2F homologues. We conclude that evolution of Rb and cyclin D proteins occurred after separation of the fungi from the higher eukaryotic lineage, but preceded the divergence of plant and animal kingdoms.

Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans.

Green fluorescent protein (GFP) is a useful reporter to follow the in vivo behaviour of proteins, but the wild-type gfp gene does not function in many organisms, including many plants and filamentous fungi. We show that codon-modified forms of gfp, produced for use in plants, function effectively in Aspergillus nidulans both as gene expression reporters and as vital reporters for protein location. To demonstrate the use of these modified gfps as reporter genes we have used fluorescence to follow ethanol-induced GFP expression from the alcA promoter. Translational fusions with the modified gfp were used to follow protein location in living cells; plant ER-retention signals targeted GFP to the endoplasmic reticulum, whereas fusion to the GAL4 DNA-binding domain targeted it to the nucleus. Nuclear-targeted GFP allowed real-time observation of nuclear movement and division. These modified gfp genes should provide useful markers to follow gene expression, organelle behaviour and protein trafficking in real time.

Plant cyclins: a unified nomenclature for plant A-, B- and D-type cyclins based on sequence organization.

The comparative analysis of a large number of plant cyclins of the A/B family has recently revealed that plants possess two distinct B-type groups and three distinct A-type groups of cyclins. Despite earlier uncertainties, this large-scale comparative analysis has allowed an unequivocal definition of plant cyclins into either A or B classes. We present here the most important results obtained in this study, and extend them to the case of plant D-type cyclins, in which three groups are identified. For each of the plant cyclin groups, consensus sequences have been established and a new, rational, plant-wide naming system is proposed in accordance with the guidelines of the Commission on Plant Gene Nomenclature. This nomenclature is based on the animal system indicating cyclin classes by an upper-case roman letter, and distinct groups within these classes by an arabic numeral suffix. The naming of plant cyclin classes is chosen to indicate homology to their closest animal class. The revised nomenclature of all described plant cyclins is presented, with their classification into groups CycA1, CycA2, CycA3, CycB1, CycB2, CycD1, CycD2 and CycD3.

Distinct classes of cdc2-related genes are differentially expressed during the cell division cycle in plants.

cdc2 and several related genes encode the catalytic subunits of cyclin-dependent kinases, which have been implicated in a number of cellular processes, including control of cell division. As a first step in exploring their function in plants, we isolated four cdc2-related genes from Antirrhinum. Two genes, cdc2a and cdc2b, encode proteins that contain a perfectly conserved PSTAIRE motif characteristic of cdc2 homologs, whereas the products of the two remaining genes, cdc2c and cdc2d, appear to represent a new subclass of proteins that have so far only been identified in plants. Transcripts of these novel genes were localized in isolated cells dispersed throughout actively dividing regions of the inflorescence. This localization is consistent with accumulation that is specific to particular phases of the cell cycle. Correlating cell labeling with nuclear condensation and double-labeling experiments using cdc2 and histone H4 as probes indicated that cdc2c transcripts accumulate during S phase as well as during the G2 and M transition, whereas cdc2d expression was specific to the G2 and M phases. All cells labeled with cdc2d also contained cdc2c label, Indicating that expression of cdc2d completely overlapped with that of cdc2c. Transcripts of cdc2a and cdc2b were detected in all cells within actively dividing regions, but at levels that were only slightly higher than those observed in nondividing areas. These transcripts did not appear to accumulate in a cell cycle-specific fashion. The genes cdc2a and cdc2b were able to partially complement a yeast cdc2 mutation, although all four genes appeared to interfere with the sizing mechanism of yeast cells. We propose that plants contain at least two classes of cdc2-related genes that differ in structure, expression, and perhaps function.

A temperature-sensitive splicing mutation in the bimG gene of Aspergillus produces an N-terminal fragment which interferes with type 1 protein phosphatase function.

Progression through anaphase requires high levels of type 1 protein phosphatase (PP1) activity in a variety of eukaryotes, including Aspergillus nidulans. A conditional lethal, temperature-sensitive mutant in one of the Aspergillus PP1 genes, bimG, prevents the normal completion of anaphase when cells are grown at restrictive temperature and this has been shown to be due to a reduction in type 1 phosphatase activity. We show that the bimG11 allele is recessive to the wild-type allele in heterozygous diploids, implying that the mutation is due to loss of function at restrictive temperature, but molecular disruption of the wild-type bimG gene shows that the gene is not essential and has no discernable phenotype under laboratory conditions. Sequence comparison of wild-type and mutant alleles reveals a single base pair difference between the two genes, within the 5' splicing site of the second intron. We demonstrate that the conditional lethal phenotype of bimG11 strains is due to impaired splicing of the mutant mRNA and that this leads to the production of a truncated protein comprising an intact N-subdomain and a modified C-terminus. Over-expression of this truncated form of PP1 in a wild-type haploid produces a lethal phenotype and reduced PP1 activity, supporting the idea that a toxic interfering protein is produced. PP1, therefore, may have at least two spatially separated sites, both of which are required for function. Temperature-sensitive splicing mutations may provide a novel means of engineering conditional versions of other proteins, particularly other phosphatases.

A nimA-like protein kinase transcript is highly expressed in meristems of Antirrhinum majus.

In plants, cell proliferation occurs mostly within meristems but a significant amount also occurs at other well-defined sites during specific stages of development. We have developed molecular markers to follow the location and progress of cell division within multicellular plant organs and thereby gain some insight into how cell division might be regulated during morphogenesis. As in other eukaryotes, cell division in plants is regulated by a highly conserved set of protein kinases and phosphatases. The molecular information available on these molecules from other eukaryotes has allowed the design of strategies by which plant homologues can be isolated. In this report we describe the identification of a nimA-like gene from Antirrhinum majus and describe the pattern in which its transcript is expressed. Comparison of the pattern of AmnimA gene expression with that of genes which are expressed in a cell cycle-dependent manner suggests that this gene is expressed in actively dividing tissues but expression is not specific to any particular phase of the cell cycle nor specific to any particular tissue type.

A novel Arabidopsis type 1 protein phosphatase is highly expressed in male and female tissues and functionally complements a conditional cell cycle mutant of Aspergillus.

Type 1 protein phosphatases are very highly conserved throughout eukaryotes where they regulate a number of key metabolic and morphogenetic processes. A cDNA, AtPP1bg, representing a new member of the type 1 protein phosphatase gene family in Arabidopsis has been isolated on the basis of hybridization with the Aspergillus bimG protein phosphatase gene. The AtPP1bg gene potentially encodes a 37 kDa protein very closely related to PP1 but with divergent N- and C-termini. The predicted amino acid sequence shows 71% identity to the ORF of the bimG gene. When expressed in Aspergillus under the alcA promoter, this phosphatase complements the temperature-sensitive bimG11 mutation allowing nearly normal vegetative growth at 37 degrees C (but not at 42 degrees C). Notably, the plant PP1 does not support morphogenesis (conidiation) at 37 degrees C. This may indicate that conidophore formation has particular phosphatase requirement(s) which the plant PP1 cannot supply. The pattern of expression of the AtPP1bg transcript has been studied during development of the plant. In situ hybridization of Arabidopsis with antisense probes shows that this phosphatase gene is expressed at a low level throughout the plant, but is strongly upregulated within developing flowers, especially in the tapetum, the developing and mature pollen and in the ovaries. This implies that the AtPP1bg either has a specialized role in the formation of these organs, or that there is an increased requirement for protein phosphatase 1 at these stages. It was found that the level of AtPP1bg transcript, as judged by the relative intensity of staining in different cells within the floral meristems, did not vary during the cell cycle.