Dehydroascorbate reduction in plant mitochondria is coupled to the respiratory electron transfer chain

The reduction of dehydroascorbate (DHA) was investigated in plant mitochondria. Mitochondria isolated from Bright Yellow-2 tobacco cells were incubated with 1 m M of DHA, and the ascorbate generation was followed by high-performance liquid chromatography. Mitochondria showed clear ability to reduce DHA and to maintain a significant level of ascorbate. Ascorbate generation could be stimulated by the respiratory substrate succinate. The complex I substrate malate and the complex I inhibitor rotenone had no effect on the ascorbate generation from DHA. Similarly, the complex III inhibitor antimycin A, the alternative oxidase inhibitor salicylhydroxamic acid, and the uncoupling agent 2,4-dinitrophenol were ineffective on mitochondrial ascorbate generation both in the absence and in the presence of succinate. However, the competitive succinate dehydrogenase inhibitor malonate almost completely abolished the succinate-dependent increase in ascorbate production. The complex IV inhibitor KCN strongly stimulated ascorbate accumulation. These results together suggest that the mitochondrial respiratory chain of plant cells – presumably complex II – plays important role in the regeneration of ascorbate from its oxidized form, DHA.     

The plant cell cycle

The first aim of this paper is to review recent progress in identifying genes in plants homologous to cell division cycle (cdc) genes of fission yeast. In the latter, cdc genes are well-characterised. Arguably, most is known about cdc2 which encodes a 34 kDa protein kinase (p34^cdc2) that functions at the G2-M and G1-S transition points of the cell cycle. At G2-M, the p34^cdc2 protein kinase is regulated by a number of gene products that function in independent regulatory pathways. The cdc2 kinase is switched on by a phosphatase encoded by cdc25, and switched off by a protein kinase encoded by weel. p34 Must also bind with a cyclin protein to form maturation promoting factor before exhibiting protein kinase activity. In plants, homologues to p34^cdc2 have been identified in pea, wheat, Arabidopsis, alfalfa, maize and Chlamydomonas. They all exhibit the PSTAIRE motif, an absolutely conserved amino acid sequence in all functional homologues sequenced so far. As in animals, some plant species contain more than one cdc2 protein kinase gene. but in contrast to animals where one functions at G2-M and the other (CDK2 in humans and Egl in Xenopus) at G1-S, it is still unclear whether there are functional differences between the plant p34^cdc2 protein kinases. Again, whereas in animals cyclins are well characterised on the basis of sequence analysis, into class A, class B (G2-M) and CLN (G1 cyclins), cyclins isolated from several plant species cannot be so clearly characterised. The differences between plant and animal homologues to p34^cdc2 and cyclins raises the possibility that some of the regulatory controls of the plant genes may be different from those of their animal counterparts. The second aim of the paper is to review how planes of cell division and cell size are regulated at the molecular level. We focus on reports showing that p34^cdc2 binds to the preprophase band (ppb) in late G2 of the cell cycle. The binding of p34^cdc2 to ppbs may be important in regulating changes in directional growth but, more importantly, there is a requirement to understand what controls the positioning of ppbs. Thus, we highlight work resolving proteins such as the microtubule associated proteins (MAPs) and those mitogen activated protein kinases (MAP kinases), which act on, or bind to, mitotic microtubules. Plant homologues to MAP kinases have been identified in alfalfa. Finally, some consideration is given to cell size at division and how alterations in cell size can alter plant development. Transgenic tobacco plants expressing the fission yeast gene, cdc25, exhibited various perturbations of development and a reduced cell size at division. Hence, cdc25 affected the cell cycle (and as a consequence, cell size at division) and cdc25 expression was correlated with various alterations to development including precocious flowering and altered floral morphogenesis. Our view is that the cell cycle is a growth cycle in which a cell achieves an optimal size for division and that this size control has an important bearing on differentiation and development. Understanding how cell size is controlled, and how plant cdc genes are regulated, will be essential keys to ‘the cell cycle locks’, which when ‘opened’, will provide further clues about how the cell cycle is linked to plant development.     

The plant cell cycle

Molecular controls of the plant cell cycle must integrate environmental signals within developmental contexts. Recent advances highlight the fundamental conservation of underlying cell cycle mechanisms between animals and plants, overlaid by a rich molecular and regulatory diversity that is specific to plant systems. Here we review plant cell cycle regulators and their control.     

Hormonal control of the plant cell cycle

Plant organogenesis is essentially a post-embryonic process that requires a strict balance between cell proliferation and differentiation. This is subject to a complex regulatory network which, in some cases, depends on the action of a variety of plant hormones. Of these, auxins and cytokinins are those best documented as impinging directly on cell cycle control. However, increasing evidence is accumulating to indicate that other hormones also have an impact on cell cycle control by influencing the availability of cell cycle regulators. In this article, we review the results that point to the variety of situations in which cell cycle progression is controlled by phytohormones.     

The plant cell cycle in context

Biological scientists are eagerly confronting the challenge of understanding the regulatory mechanisms that control the cell division cycle in eukaryotes. New information will have major implications for the treatment of growth-related diseases and cancer in animals. In plants, cell division has a key role in root and shoot growth as well as in the development of vegetative storage organs and reproductive tissues such as flowers and seeds. Many of the strategies for crop improvement, especially those aimed at increasing yield, involve the manipulation of cell division. This review describes, in some detail, the current status of our understanding of the regulation of cell division in eukaryotes and especially in plants. It also features an outline of some preliminary attempts to exploit transgenesis for manipulation of plant cell division.     

Chemical inhibitors: a tool for plant cell cycle studies

Synchrony provides a large number of cells at defined points of the cell cycle. Highly synchronised cells are powerful and effective tools for molecular analyses and for studying the biochemical events of the cell cycle in plants. Usually, plant cell suspensions can be synchronised by chemical agents, which arrest the cell cycle by acting on the driving forces of the cell cycle engine such as cyclin-dependent kinase activity, enzymes involved in DNA synthesis or proteolysis of cell cycle regulators or by acting on the cell cycle apparatus (mitotic spindle). The specificity, reversibility and efficiency of each type of cell cycle inhibitor are described and related to their mode of action.     

Parental genomes are separated throughout the cell cycle in a plant hybrid

The positions of the genomes originating from each parent were analysed in root-tip nuclei of the mature, sexual F₁ hybrid plant Hordeum vulgare (barley) x Secale africanum (a wild rye). The two genomes of the hybrid were identified in both spread and sectioned material by non-radioactive DNA:DNA in situ hybridization using labelled total genomic DNA from one parent as a proble and unlabelled total genomic DNA from the other parent to block non-specific hybridization. Complete three-dimensional reconstructions of sets of labelled sections enabled detailed analysis of the position of the genomes at interphase. The parental genomes lay in various non-intermixed configurations, including lateral and concentric arrangements. On spread preparations, the two parental genomes were found to be spatially separated throughout the cell cycle; the genome originating from H. vulgare tended to be located more centrally than that from S. africanum. This results show that the nucleus is spatially organized above the level of the DNA and chromosome at the genome level.by M.F. Trendelenburg     

Epidermal growth factor induces cell cycle arrest and apoptosis of squamous carcinoma cells through reduction of cell adhesion

Most squamous epithelial cells are strictly anchorage-dependent cell types. We observed that epidermal growth factor (EGF) promoted the growth of A431 squamous carcinoma cells in suspension cultures but suppressed cell growth and induced apoptosis in monolayer cultures, suggesting that loss of adhesion is responsible for the effects observed in monolayer culture, before cell death. Consistent with this finding, we demonstrated that EGF reduced cell attachment, cell-cell interaction, and cell spreading. Treatment with EGF increased cell adhesion-regulated expression of p21 but suppressed expressions of cyclin A, D1, cdk2, and retinoblastoma protein (pRb), leading to cell cycle arrest and adhesion-regulated programmed cell death. To test directly whether promoting cell adhesion could reduce the effects of EGF, we grew cultures on plates coated with type II collagen. On these plates, cell adhesion was enhanced and EGF treatment had little effect on cell adhesion and apoptosis when cells were attached to the collagen. The collagen effects were dose dependent, and cell cycle and cell cycle-associated proteins were altered accordingly. Finally, when cultures were plated on bacterial Petri dishes, which completely disrupted cell attachment to substratum, the level of apoptosis was greatly higher and cell cycle was arrested as compared with monolayer cultures. Taken together, our results strongly suggest that the EGF-induced cell cycle arrest and apoptosis in monolayer cultures was the result of a decline in cell adhesion.     

Plant identity influences decomposition through more than one mechanism

Plant litter decomposition is a critical ecosystem process representing a major pathway for carbon flux, but little is known about how it is affected by changes in plant composition and diversity. Single plant functional groups (graminoids, legumes, non-leguminous forbs) were removed from a grassland in northern Canada to examine the impacts of functional group identity on decomposition. Removals were conducted within two different environmental contexts (fertilization and fungicide application) to examine the context-dependency of these identity effects. We examined two different mechanisms by which the loss of plant functional groups may impact decomposition: effects of the living plant community on the decomposition microenvironment, and changes in the species composition of the decomposing litter, as well as the interaction between these mechanisms. We show that the identity of the plant functional group removed affects decomposition through both mechanisms. Removal of both graminoids and forbs slowed decomposition through changes in the decomposition microenvironment. We found non-additive effects of litter mixing, with both the direction and identity of the functional group responsible depending on year; in 2004 graminoids positively influenced decomposition whereas in 2006 forbs negatively influenced decomposition rate. Although these two mechanisms act independently, their effects may be additive if both mechanisms are considered simultaneously. It is essential to understand the variety of mechanisms through which even a single ecosystem property is affected if we are to predict the future consequences of biodiversity loss.