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.     

Intracellular survival of apicomplexan parasites and host cell modification

The intracellular stages of apicomplexan parasites are known to extensively modify their host cells to ensure their own survival. Recently, considerable progress has been made in understanding the molecular details of these parasite-dependent effects for Plasmodium-, Toxoplasma- and Theileria-infected cells. We have begun to understand how Plasmodium liver stage parasites protect their host hepatocytes from apoptosis during parasite development and how they induce an ordered cell death at the end of the liver stage. Toxoplasma parasites are also known to regulate host cell survival pathways and it has been convincingly demonstrated that they block host cell major histocompatibility complex (MHC)-dependent antigen presentation of parasite epitopes to avoid cell-mediated immune responses. Theileria parasites are the masters of host cell modulation because their presence immortalises the infected cell. It is now accepted that multiple pathways are activated to induce Theileria-dependent host cell transformation. Although it is now known that similar host cell pathways are affected by the different parasites, the outcome for the infected cell varies considerably. Improved imaging techniques and new methods to control expression of parasite and host cell proteins will help us to analyse the molecular details of parasite-dependent host cell modifications.     

System-level feedbacks control cell cycle progression

Repetitive cell cycles, which are essential to the perpetuation of life, are orchestrated by an underlying biochemical reaction network centered around cyclin-dependent protein kinases (Cdks) and their regulatory subunits (cyclins). Oscillations of Cdk1/CycB activity between low and high levels during the cycle trigger DNA replication and mitosis in the correct order. Based on computational modeling, we proposed that the low and the high kinase activity states are alternative stable steady states of a bistable Cdk-control system. Bistability is a consequence of system-level feedback (positive and double-negative feedback signals) in the underlying control system. We have also argued that bistability underlies irreversible transitions between low and high Cdk activity states and thereby ensures directionality of cell cycle progression.     

Changes in cytokinins during initiation and development of potato tubers

Cytokinin-like activity was assayed in stolons and tubers of Solanum tuberosum L. ssp. andigena (Juz. et Buk.) Hawkes cv. 165 grown in pots under controlled environment conditions. The plants were allowed to tuberise without the application of environmental or other external stimuli. The soluble sugar and starch contents of stolon tips and tubers were measured. Starch accumulation was a precise indicator of tuber initiation. Cytokinin-like activity began to increase in tubers with a diameter greater than 7.5 mm and, as assessed on a per tuber basis, was greatest in the largest size-category analysed. However, expressed as a function of fresh and dry weight, activity was greatest in tubers of 15–20 mm in diameter. Increases in cytokinin-like activity occurred subsequent to tuber formation, indicating that the tuberisation stimulus is unlikely to be cytokinin-like in nature.     

Stimulation of cytokinins and chlorophyll synthesis in cucumber cotyledons by triadimefon

Triadimefon [1-(4-chlorophenoxy)-3,3-dimethyl-1-(1,2,4-triazol-1-yl)-2-butanone] changes the morphology and partitioning of dry matter in cucumber ( Cucumis sativus L. cv. National Pickling) seedlings. The dry weights, potassium and cytokinin levels in the cotyledons and roots of the treated seedlings were higher, whereas the hypocotyl weights were lower than the controls. When etiolated intact seedlings or cotyledons excised from triadimefon-pretreated dark-grown seedlings were exposed to light, chlorophyll synthesis in the pretreated cotyledons was stimulated. Triadimefon does not have cytokinin-like activity in the cucumber cotyledon greening bioassay, but appears to induce the plants to produce more cytokinims, probably by stimulating root growth. Hence it is proposed that the stimulation of chlorophyll production by triadimefon in cucumber cotyledons is mediated by maintaining high levels of potassium and cytokinins in the cotyledons.     

细胞分裂素对植物衰老的延缓作用

细胞分裂素是一类重要的植物激素,它可在一定程度上延缓植物的衰老。主要从3个方面综述了细胞分裂素与植物衰老之间的关系,即:(1)植物衰老过程中内源细胞分裂素含量变化;(2)外源细胞分裂素的影响;(3)转入与细胞分裂素的合成、降解相关的基因对植物衰老产生的影响。此外,还从细胞分裂素与糖、与脂质氧化反应以及与其它植物激素的关系方面探讨了细胞分裂素在延缓植物衰老中的作用机理。     

Phosphoinositides and their functions in apicomplexan parasites

Phosphoinositides are the phosphorylated derivatives of the structural membrane phospholipid phosphatidylinositol. Single or combined phosphorylation at the 3, 4 and 5 positions of the inositol ring gives rise to the seven different species of phosphoinositides. All are quantitatively minor components of cellular membranes but have been shown to have important functions in multiple cellular processes. Here we describe our current knowledge of phosphoinositide metabolism and functions in apicomplexan parasites, mainly focusing on Toxoplasma gondii and Plasmodium spp. Even though our understanding is still rudimentary, phosphoinositides have already shown their importance in parasite biology and revealed some very particular and parasite-specific functions. Not surprisingly, there is a strong potential for phosphoinositide synthesis to be exploited for future anti-parasitic drug development.     

A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites

Apicomplexan parasites constitute one of the most significant groups of pathogens infecting humans and animals. The liver stage sporozoites of Plasmodium spp. and tachyzoites of Toxoplasma gondii, the causative agents of malaria and toxoplasmosis, respectively, use a unique mode of locomotion termed gliding motility to invade host cells and cross cell substrates. This amoeboid-like movement uses a parasite adhesin from the thrombospondin-related anonymous protein (TRAP) family and a set of proteins linking the extracellular adhesin, via an actin-myosin motor, to the inner membrane complex. The Plasmodium blood stage merozoite, however, does not exhibit gliding motility. Here we show that homologues of the key proteins that make up the motor complex, including the recently identified glideosome-associated proteins 45 and 50 (GAP40 and GAP50), are present in P. falciparum merozoites and appear to function in erythrocyte invasion. Furthermore, we identify a merozoite TRAP homologue, termed MTRAP, a micronemal protein that shares key features with TRAP, including a thrombospondin repeat domain, a putative rhomboid-protease cleavage site, and a cytoplasmic tail that, in vitro, binds the actin-binding protein aldolase. Analysis of other parasite genomes shows that the components of this motor complex are conserved across diverse Apicomplexan genera. Conservation of the motor complex suggests that a common molecular mechanism underlies all Apicomplexan motility, which, given its unique properties, highlights a number of novel targets for drug intervention to treat major diseases of humans and livestock.     

Distribution and metabolism of root-applied cytokinins in Pisum sativum

When N ⁶ [8–¹⁴C] furfuryladenine was applied to the intact root system of Pisum sativum L. cv. Meteor seedlings it was almost completely metabolised to other compounds within 24 h. Of the total activity recovered from the plants 94.5% was retained in the root system itself. ¹⁴C was recovered in a number of ethanol-soluble compounds and in ribonucleic acid, deoxyribonucleic acid and protein fractions of roots, stems, leaves and axillary buds. In rapidly growing axillary buds released from apical dominance by removal of the shoot apex the combined nucleic acid fractions accounted for 63.3% of the total ¹⁴C recovered from these organs. Xylem exudate collected from decapitated plants 0 to 12 h after supplying N ⁵[8–¹⁴C]furfuryladenine to the roots consistently contained a single major ¹⁴C-labelled compound which, in three different solvent systems, had the same Rf values as a major endogenous cytokinin isolated from the xylem of unlabelled plants. The content of N ⁶ [8–¹⁴C] furfuryladenine itself in the xylem exudate was always low and in some experiments it could not be detected.
It is suggested that part of the label from N ⁶ [8- ¹⁴CJfurfuryladenine taken up by the intact root system may have become incorporated in an endogenous cylokinin before export to the shoot.     

水稻茎伸长生长与植物激素

赤霉素（GA）,生长素（IAA）,脱落酸（ABA）和乙烯影响水稻茎（或节间）的伸长,其中赤霉素与水稻茎伸长生长的关系最密切。GA1是植物体内刺激茎伸长的至关重要的赤霉素,GA3已作为最常用的外源激素诱导水稻的节间伸长。水稻茎秆的伸受激素浓度和敏感性的双重控制,激素浓度或敏感性任一方的改变都有可能导致株高的变异。赤霉素如此显著地促进茎的伸长可能与增加细胞分裂和促使细胞壁松弛有关。而生长素主要促进细胞伸长。植物激素促进水稻茎长的分子机理的研究已有较大的进展,预期这方面的研究和应用在未来几年内将有新的突破。     

Involvement of carbohydrates and cytokinins in pathogenicity ofHelminthosporium carbonum

Significant stimulation of the number of appressoria, penetration and colonization by conidia ofHelminthosporium carbonum occurred on decolorized maize leaves when exogenous carbohydrates and leaf leachates were added. Germination and germ tube length, however, did not exhibit appreciable differences on decolorized or non-decolorized maize leaves. Lower germination was recorded by leached conidia on decolorized leaves; while appressoria, penetration and colonization were absent. Addition of exogenous nutrients (sucrose>leaf leachates>yeast extract>glucose) enabled conidia to accomplish appressoria, penetration and colonization. Optimum levels for various nutrients observed were 2% (w/v) sucrose/glucose or 0.1% (w/v) yeast extract. Higher concentrations inhibited the infection stages of the pathogen. Depletion of host carbohydrates from green islands/infection sites adversely affect appressoria formation, penetration and colonization; and the loss of carbohydrates from the spore affects germination. Cytokinin-like activity at the infection site/green islands increased with the period of incubation of the host as compared to the surrounding tissue or tissue under water drops. The culture filtrate extracts ofH. carbonum recorded cytokinin-like activity which increased with growth of the fungus. TLC (thin layer chromatography) of cytokinin-like substances (tissue extract and culture filtrate) revealed major activity was confined to Rf zones 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. These substances increase at infection sites by virtue of which carbohydrates accumulate at these sites ensuring a continuous supply to the growing pathogen.     

Effects of Plant Hormones on Phloem Transport in Grapevines

The translocation of assimilates in grapevines, their nature and pathways as well as the possible role of hormones in the translocation processes are summarized in the light of the source-sink-relationship between vegetative growth and berry development.     

The influence of cytokinins on proliferation and polyphenol accumulation in shoot cultures of Scutellaria altissima L.

S. altissima shoots were cultivated on MS (Murashige and Skoog) solid medium supplemented with IAA (indole-3-acetic acid, 0.57 μM) and various concentrations of cytokinin: zeatin, kinetin, BAP (6-benzylaminopurine) (1, 2, 4, 8 μM) or TDZ (thidiazuron) (0.2, 0.5, 1 μM). The effects on shoot proliferation and their accumulation of pharmacologically valuable phenolic compounds (baicalin, wogonoside, luteolin, luteolin-7-O-glucoside, verbascoside) were evaluated. Ultra-high performance liquid chromatography (UHPLC) was used for quantitative analysis of the compounds accumulated in the plant biomass. The metabolites were identified by comparing their retention time, UV spectra and mass spectra with those of the standard compounds and published data. The highest metabolite contents were recorded in shoots treated with TDZ at a concentration of 1 μM; under these conditions, the total level of all evaluated flavones expressed as the sum of baicalin, wogonoside, luteolin and luteolin-7-O-glucoside (17.35 mg/g dry wt) was more than twice that of those grown on MS cytokinin-free medium (7.55 mg/g dry wt). Verbascoside accumulation was also stimulated by 1 μM TDZ; its level was about six times higher than that found on the control medium (6.2 mg/g dry wt vs. 1.03 mg/g dry wt). The highest number of shoots (5.5–6.4 per explant within five weeks) was achieved with 2–8 μM BAP.     

Modification of plant hormone levels and signaling as a tool in plant biotechnology

Plant hormones are signal molecules, present in trace quantities, that act as major regulators of plant growth and development. They are involved in a wide range of processes such as elongation, flowering, root formation and vascular differentiation. For many years, agriculturists have applied hormones to their crops to either increase the yield, or improve the quality of the commercial product. Nowadays, the knowledge of hormone biosynthesis, degradation and signaling pathways has allowed the utilization of biotechnological tools to further improve the main agricultural crops. Natural or artificial mutants, with impaired functioning of the corresponding genes, have been adopted because of their superior phenotype in specific agricultural traits. In addition, transgenic plants have been generated to regulate internal hormone levels, or their signaling pathways, resulting in some crops that have revolutionized agriculture.     

Inhibition by aphidicolin of cell cycle progression and DNA replication in sea urchin embryos.

We have recently found that aphidicolin, a tetracyclic diterpene-tetraol produced by several fungi, blocks DNA synthesis of sea urchin embryos by interfering with the activity of DNA polyermase alpha. These cells fail to proliferate in the presence of aphidicolin. In continuation of these studies, we determined the drug-sensitive stage in the first cell cycle of the sea urchin Clypeaster japonicus embryo. In continuous exposure to aphidicolin (2 micrograms/ml) from five minutes after fertilization, mitotic division of the embryo was completely suppressed. Embryos were exposed to the drug at progressively later intervals and their capability for cytokinesis was examined. Evidence was thereby obtained that aphidicolin acts at the S-period to inhibit DNA synthesis resulting in developmental arrest of the embryo.     

The origin recognition complex in silencing, cell cycle progression, and DNA replication.

This report describes the isolation of ORC5, the gene encoding the fifth largest subunit of the origin recognition complex, and the properties of mutants with a defective allele of ORC5. The orc5-1 mutation caused temperature-sensitive growth and, at the restrictive temperature, caused cell cycle arrest. At the permissive temperature, the orc5-1 mutation caused an elevated plasmid loss rate that could be suppressed by additional tandem origins of DNA replication. The sequence of ORC5 revealed a potential ATP binding site, making Orc5p a candidate for a subunit that mediates the ATP-dependent binding of ORC to origins. Genetic interactions among orc2-1 and orc5-1 and other cell cycle genes provided further evidence for a role for the origin recognition complex (ORC) in DNA replication. The silencing defect caused by orc5-1 strengthened previous connections between ORC and silencing, and combined with the phenotypes caused by orc2 mutations, suggested that the complex itself functions in both processes.     

Juvenile hormones inhibit murine cell cycle progression and expression of type c viruses

Summary Juvenile hormones (JH), congeners of retinoic acid, were examined for their capacity to inhibit cell cycle progression and chemically induced expression of endogenous xenotropic retrovirus in Kirsten sarcoma virus-transformed BALB (K-BALB) mouse cells. JHI, II, and III were found to inhibit induction of virus by 5-iododeoxyuridine (IUdR) and histidinol (Hdl) in a concentration-dependent fashion. Some inhibition of macromolecular synthesis was observed upon culture of the cells with JH; the most affected was RNA synthesis, which was reduced 27 to 40% within 4 h by the juvenoids. Epoxide hydrase (EH) activity, as determined by high-pressure liquid chromatography (HPLC), was present in amounts sufficient for the cells to convert the hormones metabolically to an ultimate form. A contact-inhibited K-BALB variant was synchronized by mitotic arrest and the cell cyclespecific effect of JHIII on virus induction during S phase was studied. JHIII added during G₁ phase, and followed by induction, inhibited virus expression 95 and 76% by IUdR and Hdl, respectively. Induction was inhibited only 35% when JHIII was added during S phase concomitantly with the inducers and no inhibition was observed when JHIII was added during G₂ phase followed by the inducers. JHIII added to synchronous cells in G₁ phase inhibited progression of cells into S phase and the onset of DNa synthesis. The results indicate that mouse fibroblasts have a juvenile hormone-sensitive restriction point in G₁ phase that might relate to the effects these hormones have on cell replication and differentiation. This work was supported under Contract NO-1-CO-75380 with the National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205.     

Drosophila Ctf4 is essential for efficient DNA replication and normal cell cycle progression

Background  

Proper coordination of the functions at the DNA replication fork is vital to the normal functioning of a cell. Specifically the precise coordination of helicase and polymerase activity is crucial for efficient passage though S phase. The Ctf4 protein has been shown to be a central member of the replication fork and links the replicative MCM helicase and DNA polymerase α primase. In addition, it has been implicated as a member of a complex that promotes replication fork stability, the Fork Protection Complex (FPC), and as being important for sister chromatid cohesion. As such, understanding the role of Ctf4 within the context of a multicellular organism will be integral to our understanding of its potential role in developmental and disease processes.     

Seed enhancement with cytokinins: changes in growth and grain yield in salt stressed wheat plants

Cytokinins are often considered abscisic acid (ABA) antagonists and auxins antagonists/synergists in various processes in plants. Seed enhancement (seed priming) with cytokinins is reported to increase plant salt tolerance. It was hypothesized that cytokinins could increase salt tolerance in wheat plants by interacting with other plant hormones, especially auxins and ABA. The present studies were therefore conducted to assess the effects of pre-sowing seed treatment with varying concentrations (100, 150 and 200 mg l⁻¹) of cytokinins (kinetin and benzylaminopurine (BAP)) on germination, growth, and concentrations of free endogenous auxins and ABA in two hexaploid spring wheat (Triticum aestivum L.) cultivars. The primed and non-primed seeds of MH-97 (salt-intolerant) and Inqlab-91 (salt-tolerant) were sown in both Petri dishes in a growth room and in the field after treatment with 15 dS m⁻¹ NaCl salinity. Both experiments were repeated during 2002 and 2003. Among priming agents, kinetin was effective in increasing germination rate in the salt-intolerant and early seedling growth in the salt-tolerant cultivar when compared with hydropriming under salt stress. Thus, during germination and early seedling growth, the cytokinin-priming induced effects were cultivar specific. In contrast, kinetin-priming showed a consistent promoting effect in the field and improved growth and grain yield in both cultivars under salt stress. The BAP-priming did not alleviate the inhibitory effects of salinity stress on the germination and early seedling growth in both cultivars. The increase in growth and grain yield in both cultivars was positively correlated with leaf indoleacetic acid concentration and negatively with ABA concentration under both saline and non-saline conditions. The decrease in ABA concentration in the plants raised from kinetin-primed seeds might reflect diminishing influence of salt stress. However, the possibility of involvement of other hormonal interactions is discussed.