Robert J. Herbert

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.

Plant WEE1 kinase is cell cycle regulated and removed at mitosis via the 26S proteasome machinery

In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 INTERACTING PARTNER 1 (SKIP1). Furthermore, the AtWEE1–green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1–YFPC (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1–YFPN negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome.

Activation of latent origins of DNA replication in florally determined shoot meristems of long-day and short-day plants: Silene coeli-rosa and Pharbitis nil

Abstract. Replicon spacing was measured during the S-phase of the cell cycle in shoot meristems of Silene coeli-rosa L., a long-day (LD) plant, and Pharbitis nil Chois, a short-day (SD) plant to examine the hypothesis that activation of latent origins of DNA replication is a feature of floral determination. Silene coeli-rosa was germinated and grown in SD for 28 d and then exposed to either a florally inductive combination of 7 LD + 2 SD, the last day of which coincides with determination of the sepal and stamen whorls, or was germinated and grown in 37 non-inductive SD. Pharbitis nil was germinated and grown in continuous light (CL) for 5 d and then given either 48 h of inductive darkness followed by 1 d of CL, the last day of which coincides with determination of the sepal, petal and stamen whorls, or given one of two independent non-inductive treatments: 48 h dark interrupted by red light (R) + 1 d of CL, or 8 d of CL. Following these treatments, each batch of plants was exposed to tritiated [methyl-3H]thymidine for 30, 60, 90 or 120 min. Apical domes were dissected, nuclei lysed and prepared as fibre autoradiographs from which replicon size was recorded. In S. coeli-rosa, replicon size was in the range 10–15 μm in SD (non-inductive) and 0–5 μm in LD (inductive) while in P. nil it was 10–15 μm in the 48 h dark interrupted by R, 5–10 μm in CL (both non-inductive) but was reduced to 0–5 μm in the 48 h dark treatment (inductive). Therefore, the recruitment of additional initiation points for DNA replication occurred in both a LD and a SD plant immediately before the appearance of floral organs. The data are consistent in showing that a shortening of S-phase, which is a characteristic feature of florally determined shoot meristems for both species, is brought about by the activation of latent origins of DNA replication.

Perturbation of cytokinin and ethylene-signalling pathways explain the strong rooting phenotype exhibited by Arabidopsis expressing the Schizosaccharomyces pombe mitotic inducer, cdc25

BackgroundEntry 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.ResultsExpressing 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.ConclusionsWe 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.

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 (WEE1(oe)), 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 WEE1(oe) and a significant increase in morphogenetic capacity in the two T-DNA insertion lines compared with WT. Compared with WT, WEE1(oe) 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 WEE1(oe) and significantly more for the insertion mutant wee1-1. Compared with WT there was a significant increase in meristem cell size in WEE1(oe) 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.

Plant WEE1 Kinase Interacts with a 14-3-3 Protein, GF14ω but a Mutation of WEE1 at S485 Alters Their Spatial Interaction

In animals, 14-3-3 proteins bind two cell cycle proteins WEE1 and CDC25 stabilising their phosphorylated state. We report here for the first time interactions between WEE1 and 14-3-3 proteins both in vitro and in vivo in plants. The Arabidopsis 14-3-3 family partitions into either an Epsilon or Non-Epsilon group. In a yeast 2-hybrid screen Arabidopsis WEE1 interacted with the Non-Epsilon group. Subsequently, we focussed on Non-Epsilon GF14

Multitrait analysis of fresh-cut cantaloupe melon enables discrimination between storage times and temperatures and identifies potential markers for quality assessments

Fresh-cut cantaloupe melon is valued for its aroma but is highly perishable. Temperature of storage (typically 0-5°C) is critical for maintaining fresh-cut melon quality, but often reaches 10°C during transportation and in retail outlets. A comparison amongst 0, 5 and 10°C storage temperatures for fresh-cut melon over 14days reveals that storage at 0°C is optimal for avoiding increases in microbial load and loss of vitamin C especially at later time points. However, higher temperatures maintain better the balance of esters (acetate versus non-acetate) and phenolic content. The whole volatile organic compound (VOC) profile can be used to discriminate both time and temperature effects especially at earlier time points. Potential VOC markers for changes in vitamin C from day 0 to day 6 of storage (3-methyl butane nitrile) and temperature (limonene) are identified through a multi-trait analysis.

Regulation of cell division in the shoot apex

The aim of this paper is to explore how changes in the plane, and in the rate, of cell division affect primordium initiation in the shoot apex. Leaf initiation is characterised by a change in the plane of cell division in incipient primordia or changes in the rate of cell division, or both. We refer to published work on the preprophase band (PPB) and the phragmosome to indicate how planes of cell division are predicted in plant cells and argue that the presence of F-actin in PPBs may be a potential substrate for key regulatory cell cycle protein kinases. However the key signalling molecules that may cause a repositioning of PPBs during leaf initiation are unknown. We also attempt to make a link between homeotic, or organ identity genes and the regulation of cell size during floral morphogenesis. Here, emphasis is placed on a model by EM Lord proposing that homeotic genes have heterochronic function. In keeping with a timer mechanism would be genes which regulate cell size at division. The timing mechanism could act first, through homeotic genes determining where and when flower primordia are initiated and second, through genes which cause cell size to alter in cells which are determined as a particular floral primordium.