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1.  Plant WEE1 kinase is cell cycle regulated and removed at mitosis via the 26S proteasome machinery 
Journal of Experimental Botany  2013;64(7):2093-2106.
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.
doi:10.1093/jxb/ert066
PMCID: PMC3638832  PMID: 23536609
Arabidopsis thaliana; bimolecular fluorescence complementation (BiFC); BY-2 cell line; CDKA/B; cell cycle; F-box; green fluorescent protein (GFP); mitosis; Nicotiana tabacum; 26S proteasome SKIP1; WEE1.
2.  Gene dosage effect of WEE1 on growth and morphogenesis from arabidopsis hypocotyl explants 
Annals of Botany  2012;110(8):1631-1639.
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.
doi:10.1093/aob/mcs223
PMCID: PMC3503502  PMID: 23065633
Arabidopsis thaliana; cell cycle; development; growth; hypocotyl; tissue culture; WEE1
3.  Differential spatial expression of A- and B-type CDKs, and distribution of auxins and cytokinins in the open transverse root apical meristem of Cucurbita maxima 
Annals of Botany  2010;107(7):1223-1234.
Background and Aims
Aside from those on Arabidopsis, very few studies have focused on spatial expression of cyclin-dependent kinases (CDKs) in root apical meristems (RAMs), and, indeed, none has been undertaken for open meristems. The extent of interfacing between cell cycle genes and plant growth regulators is also an increasingly important issue in plant cell cycle studies. Here spatial expression/localization of an A-type and B-type CDK, auxin and cytokinins are reported in relation to the hitherto unexplored anatomy of RAMs of Cucurbita maxima.
Methods
Median longitudinal sections were cut from 1-cm-long primary root tips of C. maxima. Full-length A-type CDKs and a B-type CDK were cloned from C. maxima using degenerate primers, probes of which were localized on sections of RAMs using in situ hybridization. Isopentenyladenine (iPA), trans-zeatin (t-Z) and indole-3yl-acetic acid (IAA) were identified on sections by immunolocalization.
Key Results
The C. cucurbita RAM conformed to an open transverse (OT) meristem typified by an absence of a clear boundary between the eumeristem and root cap columella, but with a distinctive longitudinally thickened epidermis. Cucma;CDKA;1 expression was detected strongly in the longitudinally thickened epidermis, a tissue with mitotic competence that contributes cells radially to the root cap of OT meristems. Cucma;CDKB2 was expressed mainly in proliferative regions of the RAM and in lateral root primordia. iPA and t-Z were mainly distributed in differentiated cells whilst IAA was distributed more uniformly in all tissues of the RAM.
Conclusions
Cucma;CDKA;1 was expressed most strongly in cells that have proliferative competence whereas Cucma;CDKB2 was confined mainly to mitotic cells. iPA and t-Z marked differentiated cells in the RAM, consistent with the known effect of cytokinins in promoting differentiation in root systems. iPA/t-Z were distributed in a converse pattern to Cucma;CDKB2 expression whereas IAA was detected in most cells in the RAM regardless of their proliferative potential.
doi:10.1093/aob/mcq127
PMCID: PMC3091794  PMID: 20601387
Auxin; cytokinins; CDKs; Cucurbita maxima; root apical meristems
4.  Arabidopsis T-DNA insertional lines for CDC25 are hypersensitive to hydroxyurea but not to zeocin or salt stress 
Annals of Botany  2010;107(7):1183-1192.
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.
doi:10.1093/aob/mcq142
PMCID: PMC3091795  PMID: 20647223
Arabidopsis thaliana; cell-cycle checkpoints; hydroxyurea; root growth; NaCl; zeocin
5.  A commentary on the G2/M transition of the plant cell cycle 
Annals of Botany  2011;107(7):1065-1070.
Background
The complex events of mitosis rely on precise timing and on immaculate preparation for their success, but the G2/M transition in the plant cell cycle is currently steeped in controversy and alternative models.
Scope
In this brief review, the regulation of the G2/M transition in plants is commented on. The extent to which the G2/M transition is phosphoregulated by WEE1 kinase and CDC25 phosphatase, as exemplified in yeasts and animals, is discussed together with an alternative model that excludes these proteins from this transition. Arabidopsis T-DNA insertional lines for WEE1 and CDC25 that develop normally prompted the latter model. An argument is then presented that environmental stress is the norm for higher plants in temperate conditions. If so, the repressive role that WEE1 has under checkpoint conditions might be part of the normal cell cycle for many proliferative plant cells. Arabidopsis CDC25 can function as either a phosphatase or an arsenate reductase and recent evidence suggests that cdc25 knockouts are hypersensitive to hydroxyurea, a drug that induces the DNA-replication checkpoint. That other data show a null response of these knockouts to hydroxyurea leads to an airing of the controversy surrounding the enigmatic plant CDC25 at the G2/M transition.
doi:10.1093/aob/mcr055
PMCID: PMC3091806  PMID: 21558458
Arabidopsis thaliana; cell-cycle checkpoints; CDC25 phosphatase; cyclin-dependent kinases; G2/M; WEE1 kinase
6.  Perturbation of cytokinin and ethylene-signalling pathways explain the strong rooting phenotype exhibited by Arabidopsis expressing the Schizosaccharomyces pombe mitotic inducer, cdc25 
BMC Plant Biology  2012;12:45.
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.
doi:10.1186/1471-2229-12-45
PMCID: PMC3362767  PMID: 22452972
8.  A Strong Nucleotypic Effect on the Cell Cycle Regardless of Ploidy Level 
Annals of Botany  2008;101(6):747-757.
Background and Aims
In published studies, positive relationships between nucleotype and the duration of the mitotic cell cycle in angiosperms have been reported but the highest number of species analyzed was approx. 60. Here an analysis is presented of DNA C-values and cell cycle times in root apical meristems of angiosperms comprising 110 measurements, including monocots and eudicots within a set temperature range, and encompassing an approx. 290-fold variation in DNA C-values.
Methods
Data for 110 published cell cycle times of seedlings grown at temperatures between 20–25 °C were compared with DNA C-values (58 values for monocots and 52 for eudicots). Regression analyses were undertaken for all species, and separately for monocots and eudicots, diploids and polyploids, and annuals and perennials. Cell cycle times were plotted against the nuclear DNA C-values.
Key Results
A positive relationship was observed between DNA C-value and cell cycle time for all species and for eudicots and monocots separately, regardless of the presence or absence of polyploid values. In this sample, among 52 eudicots the maximum cell cycle length was 18 h, whereas the 58 monocot values ranged from 8–120 h. There was a striking additional increase in cell cycle duration in perennial monocots with C-values greater than 25 pg. Indeed, the most powerful relationship between DNA C-value and cell cycle time and the widest range of cell cycle times was in perennials regardless of ploidy level.
Conclusions
DNA replication is identified as a rate limiting step in the cell cycle, the flexibility of DNA replication is explored, and we speculate on how the licensing of initiation points of DNA replication may be a responsive component of the positive nucleotypic effect of C-value on the duration of the mitotic cell cycle.
doi:10.1093/aob/mcn038
PMCID: PMC2710215  PMID: 18339642
Cell cycle times; DNA C-value; nucleotype; ploidy level
9.  Replication Origins in XenopusEgg Extract Are 5–15 Kilobases Apart and Are Activated in Clusters That Fire at Different Times 
The Journal of Cell Biology  2001;152(1):15-26.
When Xenopus eggs and egg extracts replicate DNA, replication origins are positioned randomly with respect to DNA sequence. However, a completely random distribution of origins would generate some unacceptably large interorigin distances. We have investigated the distribution of replication origins in Xenopus sperm nuclei replicating in Xenopus egg extract. Replicating DNA was labeled with [3H]thymidine or bromodeoxyuridine and the geometry of labeled sites on spread DNA was examined. Most origins were spaced 5–15 kb apart. This regular distribution provides an explanation for how complete chromosome replication can be ensured although origins are positioned randomly with respect to DNA sequence. Origins were grouped into small clusters (typically containing 5–10 replicons) that fired at approximately the same time, with different clusters being activated at different times in S phase. This suggests that a temporal program of origin firing similar to that seen in somatic cells also exists in the Xenopus embryo. When the quantity of origin recognition complexes (ORCs) on the chromatin was restricted, the average interorigin distance increased, and the number of origins in each cluster decreased. This suggests that the binding of ORCs to chromatin determines the regular spacing of origins in this system.
PMCID: PMC2193667  PMID: 11149917
replication origin; Xenopus; S phase; ORC; origin clusters

Results 1-9 (9)