Perturbation of Cytokinin and Ethylene-signalling Pathways Explain the Strong Rooting Phenotype Exhibited by Arabidopsis Expressing the Schizosaccharomyces Pombe mitotic inducer, cdc25.

Background Entry into mitosis is regulated by cyclin dependent kinases that in turn are phosphoregulated. In most eukaryotes, phosphoregulation is through WEE1 kinase and CDC25 phosphatase. In higher plants a homologous CDC25 gene is unconfirmed and hence the mitotic inducer Schizosaccharomyces pombe (Sp) cdc25 has been used as a tool in transgenic plants to probe cell cycle function. Expression of Spcdc25 in tobacco BY-2 cells accelerates entry into mitosis and depletes cytokinins; in whole plants it stimulates lateral root production. Here we show, for the first time, that alterations to cytokinin and ethylene signaling explain the rooting phenotype elicited by Spcdc25 expression in Arabidopsis. Results Expressing Spcdc25 in Arabidopsis results in increased formation of lateral and adventitious roots, a reduction of primary root width and more isodiametric cells in the root apical meristem (RAM) compared with wild type. Furthermore it stimulates root morphogenesis from hypocotyls when cultured on two way grids of increasing auxin and cytokinin concentrations. Microarray analysis of seedling roots expressing Spcdc25 reveals that expression of 167 genes is changed by > 2-fold. As well as genes related to stress responses and defence, these include 19 genes related to transcriptional regulation and signaling. Amongst these was the up-regulation of genes associated with ethylene synthesis and signaling. Seedlings expressing Spcdc25 produced 2-fold more ethylene than WT and exhibited a significant reduction in hypocotyl length both in darkness or when exposed to 10 ppm ethylene. Furthermore in Spcdc25 expressing plants, the cytokinin receptor AHK3 was down-regulated, and endogenous levels of iPA were reduced whereas endogeous IAA concentrations in the roots increased. Conclusions We suggest that the reduction in root width and change to a more isodiametric cell phenotype in the RAM in Spcdc25 expressing plants is a response to ethylene over-production. The increased rooting phenotype in Spcdc25 expressing plants is due to an increase in the ratio of endogenous auxin to cytokinin that is known to stimulate an increased rate of lateral root production. Overall, our data reveal important cross talk between cell division and plant growth regulators leading to developmental changes.

Effect of CDC25 and WEE1 on Plant Cell Cycle and Morphogenesis

The cell cycle comprise the four phases of, G1, S-phase, G2 and mitosis. Two critical transitions are G1/S and G2/M; the latter is regulated by WEE1 kinase and CDC25 phosphatases. The scope of this thesis was to investigate the regulation of the G2/M transition of the cell cycle by WEE1 and CDC25, and how these genes interface with plant growth regulators in Arabidopsis thaliana. In Arabidopsis roots, the frequency of lateral roots was found to be increased by ectopic expression of Schizosaccharomyces pombe (Sp)cdc25e and reduced by Arath;WEE1 expression. I examined the effect of Arath;WEE1 and Spcdc25 on induction of shoots and roots in Arabidopsis hypocotyls in vitro. Hypocotyl explants from two over-expressing WEE1 lines , three T-DNA insertion lines and two expressing cdc25 (Spcdc25e) lines together with wild type (WT) were cultured on two-way gradients of kinetin (Kin) and naphthyl acetic acid (NAA). Below a threshold concentration of NAA (100 ng ml-1), WEE1 repressed morphogenesis in vitro, whereas at all NAA/Kin combinations Spcdc25 promoted morphogenesis (particularly root formation) over and above that in WT. Loss of function wee1-1 cultures were very similar to WT. Quantitative data indicated a significant increase in the frequency of root formation in Spcdc25e cultures compared with WT particularly at low Kin concentrations, and WEE1oe’s repressive effect was overcome by NAA but not Kin. In conclusion, WEE1 has a repressive effect on morphogenesis in vitro that can be overcome by auxin whereas Spcd25 by-passes a cytokinin requirement for the induction of morphogenesis in vitro. The role of CDC25 and WEE1 in DNA damage responses was also analysed. Two over-expressing Arath;CDC25 lines and T-DNA mutants showed no difference to WT either in standard conditions or zeocin-supplemented treatments. However, root length was longer in Arath;CDC25oe lines treated with hydroxyurea (HU) and lateral root number was increased compared to WT. This suggests a differential response of Arath;CDC25oe in the DNA replication (HU-induced) and DNA damage (zeocin-induced) checkpoints (Chapter 5). Finally the roles of WEE1 and CDC25 in cell cycle regulation were examined using tobacco TBY-2 cell cultures expressing Arath;WEE1, Nicotiana tabacum (Nicta)WEE1 or Arath;CDC25. Whilst Nicta;WEE1 lengthened G2 of the cell cycle, Arath;WEE1 had an unusual effect of shortening G2 phase and Arath;CDC25 had no observable effect (Chapter 6).

Arabidopsis T-DNA Insertional Lines For CDC25 Are Hypersensitive to Hydroxyurea But Not to Zeocin or Salt Stress.

Background and Aims In yeasts and animals, cyclin-dependent kinases are key regulators of cell cycle progression and are negatively and positively regulated by WEE1 kinase and CDC25 phosphatase, respectively. In higher plants a full-length orthologue of CDC25 has not been isolated but a shorter gene with homology only to the C-terminal catalytic domain is present. The Arabidopis thaliana;CDC25 can act as a phosphatase in vitro. Since in arabidopsis, WEE1 plays an important role in the DNA damage/DNA replication checkpoints, the role of Arath;CDC25 in conditions that induce these checkpoints or induce abiotic stress was tested. Methods arath;cdc25 T-DNA insertion lines, Arath;CDC25 over-expressing lines and wild type were challenged with hydroxyurea (HU) and zeocin, substances that stall DNA replication and damage DNA, respectively, together with an abiotic stressor, NaCl. A molecular and phenotypic assessment was made of all genotypes Key Results There was a null phenotypic response to perturbation of Arath;CDC25 expression under control conditions. However, compared with wild type, the arath;cdc25 T-DNA insertion lines were hypersensitive to HU, whereas the Arath;CDC25 over-expressing lines were relatively insensitive. In particular, the over-expressing lines consistently outgrew the T-DNA insertion lines and wild type when challenged with HU. All genotypes were equally sensitive to zeocin and NaCl. Conclusions Arath;CDC25 plays a role in overcoming stress imposed by HU, an agent know to induce the DNA replication checkpoint in arabidopsis. However, it could not enhance tolerance to either a zeocin treatment, known to induce DNA damage, or salinity stress.

Complex Interplay Between Determinants of Pacing and Performance During 20 km Cycle Time Trials

Purpose This study examined the determinants of pacing strategy and performance during self paced maximal exercise. Methods Eight well trained cyclists completed two 20 km time trials. Power output, RPE, positive and negative affect, and iEMG activity of the active musculature were recorded every 0.5km, confidence in achieving pre-exercise goals was assessed every 5 km, and blood lactate and pH were measured post-exercise. Differences in all parameters were assessed between fastest (FAST) and slowest (SLOW) trials performed. Results Mean power output was significantly higher during the initial 90% of FAST, but not the final 10%, and blood lactate concentration was significantly higher and pH significantly lower following FAST. Mean iEMG activity was significantly higher throughout SLOW. RPE was similar throughout both trials, but participants had significantly more positive affect and less negative affect throughout FAST. Participants grew less confident in their ability to achieve their goals throughout SLOW. Conclusions The results suggest that affect may be the primary psychological regulator of pacing strategy and that higher levels of positivity and lower levels of negativity may have been associated with a more aggressive strategy during FAST. Although the exact mechanisms through which affect acts to influence performance are unclear, it may determine the degree of physiological disruption that can be tolerated, or be reflective of peripheral physiological status in relation to the still to be completed exercise task.

Gene Dosage Effect of WEE1 on Growth and Morphogenesis from Arabidopsis Hypocotyl Explants.

Background and Aims How plant cell-cycle genes interface with development is unclear. Preliminary evidence from our laboratory suggested that over-expression of the cell cycle checkpoint gene, WEE1, repressed growth and development. Here the hypothesis is tested that the level of WEE1 has a dosage effect on growth and development in Arabidospis thaliana. To do this, a comparison was made of the development of gain- and loss-of-function WEE1 arabidopsis lines both in vivo and in vitro. Methods Hypocotyl explants from an over-expressing Arath;WEE1 line (WEE1oe), two T-DNA insertion lines (wee1-1 and wee1-4) and wild type (WT) were cultured on two-way combinations of kinetin and naphthyl acetic acid. Root growth and meristematic cell size were also examined. Key Results Quantitative data indicated a repressive effect in WEE1oe and a significant increase in morphogenetic capacity in the two T-DNA insertion lines compared with WT. Compared with WT, WEE1oe seedlings exhibited a slower cell-doubling time in the root apical meristem and a shortened primary root, with fewer laterals, whereas there were no consistent differences in the insertion lines compared with WT. However, significantly fewer adventitious roots were recorded for WEE1oe and significantly more for the insertion mutant wee1-1. Compared with WT there was a significant increase in meristem cell size in WEE1oe for all three ground tissues but for wee1-1 only cortical cell size was reduced. Conclusions There is a gene dosage effect of WEE1 on morphogenesis from hypocotyls both in vitro and in vivo.

Can the Revised UK Code Direct Practice?

The Nursing and Midwifery Council, the United Kingdom regulator of nursing and midwifery has recently revised its professional code of practice. This paper begins by arguing that a professional code must be capable of sustaining close reading and of action guidance. Using four exemplar clauses it is argued that the new revised code does not met this purpose. First, I show that in setting out requirements for consent and documentation, the meaning of the relevant clause has changed significantly during the editing process so that a literal reading of the final document bears little relation to established professional practice. Second, I argue that the clause concerning the nature of professional relationships has also been altered during the editing process so that it is inconsistent with other professional groups and established accounts of the professional nurse-patient relationship. Third, I argue that the clause concerning disclosure of confidential information, which survived revision and editing with its meaning intact is nevertheless factually incorrect and inconsistent with UK law and authoritative guidance. Finally, fourth, I argue that use of the word ‘inappropriate’ is inappropriate as it amounts to meaningless circularity, discussed in relation to a clause on expressing personal beliefs. Taken together, these examples demonstrate that the Code is seriously flawed and does not fulfil its purpose. One way that simple prescriptive clauses in the Code can be usefully understood is through the provision of detailed guidance. I argue that the NMC has changed its position on its view of the value of guidance and has significantly reduced the amount of written guidance and advice is provides. The paper concludes by arguing that in order to meet its action directing function, further clarifying revision and the provision of detailed guidance is required.