Regulatory dephosphorylation of CDK at G₂/M in plants: yeast mitotic phosphatase cdc25 induces cytokinin-like effects in transgenic tobacco morphogenesis

Background

During the last three decades, the cell cycle and its control by cyclin-dependent kinases (CDKs) have been extensively studied in eukaryotes. This endeavour has produced an overall picture that basic mechanisms seem to be largely conserved among all eukaryotes. The intricate regulation of CDK activities includes, among others, CDK activation by CDC25 phosphatase at G₂/M. In plants, however, studies of this regulation have lagged behind as a plant Cdc25 homologue or other unrelated phosphatase active at G₂/M have not yet been identified.

Scope

Failure to identify a plant mitotic CDK activatory phosphatase led to characterization of the effects of alien cdc25 gene expression in plants. Tobacco, expressing the Schizosaccharomyces pombe mitotic activator gene, Spcdc25, exhibited morphological, developmental and biochemical changes when compared with wild type (WT) and, importantly, increased CDK dephosphorylation at G₂/M. Besides changes in leaf shape, internode length and root development, in day-neutral tobacco there was dramatically earlier onset of flowering with a disturbed acropetal floral capacity gradient typical of WT. In vitro, de novo organ formation revealed substantially earlier and more abundant formation of shoot primordia on Spcdc25 tobacco stem segments grown on shoot-inducing media when compared with WT. Moreover, in contrast to WT, stem segments from transgenic plants formed shoots even without application of exogenous growth regulator. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size due to a shortening of the G₂ phase together with high activity of cyclin-dependent kinase, NtCDKB1, in early S-phase, S/G₂ and early M-phase. Spcdc25-expressing tobacco (‘Samsun’) cell suspension cultures showed a clustered, more circular, cell phenotype compared with chains of elongated WT cells, and increased content of starch and soluble sugars. Taken together, Spcdc25 expression had cytokinin-like effects on the characteristics studied, although determination of endogenous cytokinin levels revealed a dramatic decrease in Spcdc25 transgenics.

Conclusions

The data gained using the plants expressing yeast mitotic activator, Spcdc25, clearly argue for the existence and importance of activatory dephosphorylation at G₂/M transition and its interaction with cytokinin signalling in plants. The observed cytokinin-like effects of Spcdc25 expression are consistent with the concept of interaction between cell cycle regulators and phytohormones during plant development. The G₂/M control of the plant cell cycle, however, remains an elusive issue as doubts persist about the mode of activatory dephosphorylation, which in other eukaryotes is provided by Cdc25 phosphatase serving as a final all-or-nothing mitosis regulator.     

Tobacco BY-2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycle

The mitotic inducer gene from Schizosaccharomyces pombe, Spcdc25, was used as a tool to investigate regulation of G2/M in higher plants using the BY-2 (Nicotiana tabacum) cell line as a model. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size through a shortening of the G2 phase. The cells often formed isodiametric double files both in BY-2 cells and in cell suspensions derived from 35S::Spcdc25 tobacco plants. In Spcdc25-expressing cells, the tobacco cyclin-dependent kinase, NtCDKB1, showed high activity in early S phase, S/G2 and early M phase, whereas in empty vector cells CDKB1 activity was transiently high in early S phase but thereafter remained lower. Spcdc25-expressing cells also bypassed a block on G2/M imposed by the cytokinin biosynthetic inhibitor lovastatin (LVS). Surprisingly, cytokinins were at remarkably low levels in Spcdc25-expressing cells compared with the empty vector, explaining why these cells retained mitotic competence despite the presence of LVS. In conclusion, synchronised Spcdc25-expressing BY-2 cells divided prematurely at a small cell size, and they exhibited premature, but sustained, CDKB1 activity even though endogenous cytokinins were virtually undetectable.     

Expression of the fission yeast cell cycle regulator cdc25 induces de novo shoot formation in tobacco: evidence of a cytokinin-like effect by this mitotic activator.

During the last decade, the cell cycle and its control by cyclin-dependent kinases (CDKs) has been extensively studied in eukaryotes. The regulation of CDK activity includes, among others, its activation by Cdc25 phosphatase at G2/M. However, within the plant kingdom studies of this regulation have lagged behind and a plant cdc25 homologue has not been identified yet. Here, we report on the effects of transformation of tobacco (Nicotiana tabacum L., cv. Samsun) with fission yeast (Schizosaccharomyces pombe) cdc25 (Spcdc25) on de novo plant organ formation, a process dependent on rate and orientation of cell division. On shoot-inducing medium (low 1-naphthylacetic acid (NAA), high 6-benzylaminopurine (BAP)) the number of shoots formed on internode segments cultured from transgenic plants was substantially higher than in the non-transformed controls. Anatomical observations indicated that the shoot formation process was accelerated but with no changes in the quality and sequence of shoot development. Surprisingly, and in contrast to the controls, when on root-inducing medium (high NAA, low BAP) cultured segments from transgenic plants failed to initiate hardly any roots. Instead, they continued to form shoots at low frequencies. Moreover, in marked contrast to the controls, stem segments from transgenic plants were able to form shoots even without the addition of exogenous growth regulators to the medium. The results indicate that Spcdc25 expression in culture tobacco stem segments mimicked the developmental effects caused by an exogenous hormone balance shifted towards cytokinins. The observed cytokinin-like effects of Spcdc25 transformation are consistent with the concept of an interaction between cell cycle regulators and phytohormones during plant development.     

A yeast mitotic activator sensitises the shoot apical meristem to become floral in day-neutral tobacco

True day-neutral (DN) plants flower regardless of day-length and yet they flower at characteristic stages. DN Nicotiana tabacum cv. Samsun, makes about forty nodes before flowering. The question still persists whether flowering starts because leaves become physiologically able to export sufficient floral stimulus or the shoot apical meristem (SAM) acquires developmental competence to interpret its arrival. This question was addressed using tobacco expressing the Schizosaccharomyces pombe cell cycle gene, Spcdc25, as a tool. Spcdc25 expression induces early flowering and we tested a hypothesis that this phenotype arises because of premature floral competence of the SAM. Scions of vegetative Spcdc25 plants were grafted onto stocks of vegetative WT together with converse grafts and flowering onset followed (as the time since sowing and number of leaves formed till flowering). Spcdc25 plants flowered significantly earlier with fewer leaves, and, unlike WT, also formed flowers from axillary buds. Scions from vegetative Spcdc25 plants also flowered precociously when grafted to vegetative WT stocks. However, in a WT scion to Spcdc25 stock, the plants flowered at the same time as WT. SAMs from young vegetative Spcdc25 plants were elongated (increase in SAM convexity determined by tracing a circumference of SAM sections) with a pronounced meristem surface cell layers compared with WT. Presumably, Spcdc25 SAMs were competent for flowering earlier than WT and responded to florigenic signal produced even in young vegetative WT plants. Precocious reproductive competence in Spcdc25 SAMs comprised a pronounced mantle, a trait of prefloral SAMs. Hence, we propose that true DN plants export florigenic signal since early developmental stages but the SAM has to acquire competence to respond to the floral stimulus.     

The fission yeast mitotic activator cdc25 and sucrose induce early flowering synergistically in the day-neutral Nicotiana tabacum cv. Samsun

Here, the tobacco (Nicotiana tabacum) day-neutral (DN) cv. Samsun transformed with the Schizosaccharomyces pombe mitotic activator gene Spcdc25 was used to study the onset of flowering. Wild type (WT) and cdc25 plants were grown from seeds in vitro until they were 20 cm high. Apical and basal nodes were then subcultured repeatedly and the regenerated plants were used to document time to flowering and the number of leaves formed before flowering. Three sucrose treatments (3, 5 or 7% (weight/volume)) were used and measurements of leaf endogenous soluble carbohydrates were performed. In the 3% treatment, cdc25 plants flowered but WT plants did not. The higher sucrose treatments enabled WT flowering; two-thirds of the plants flowered at 5%, while all plants flowered at 7% sucrose. However, in all treatments, cdc25 plants exhibited significantly earlier flowering and fewer leaves compared with wild type. Remarkably, a typical acropetal flowering gradient in WT plants did not occur in cdc25 plants. In cdc25 leaves, there were significantly higher amounts of endogenous sugars with a higher proportion of sucrose compared with WT. Our data demonstrate that Spcdc25 expression and sucrose act synergistically to induce precocious flowering.     

Assessment of lovastatin application as tool in probing cytokinin-mediated cell cycle regulation

Lovastatin, a potent inhibitor of the mevalonate pathway, has been used in plant cell cycle studies to eliminate the cytosolic cytokinin biosynthesis. However, several implications can blur the results, as cytokinins may be alternatively formed from isopentenylpyrophosphate produced by the plastid 1-deoxy-xylulose 5-phosphate pathway and because the endogenous cytokinin levels oscillate considerably in the course of a cell cycle. In the work presented here, short- and long-term effects of lovastatin on suspension- cultured Nicotiana tabacum (L.) BY-2 cells were differentiated. The short-term experiments revealed a fast action of lovastatin, resulting in a significantly, though not completely, decreased content of endogenous cytokinins that became visible already after 10 min and was most pronounced after 30 min. But the impact of lovastatin on cell cycle progression depended also on the phase of the cell cycle at which it was administered. Lowering of the cytokinin level during the early S phase, when the endogenous cytokinin levels increased, delayed the S/G2 transition, whereas the same treatment in the late S phase, when the cellular cytokinin concentrations had already started to decrease, promoted it. Incubation periods longer than 48 h resulted in about 50% loss of viable of the cells and also in a reduced capability of division of the survivors. These cells later on resumed cell division. A second treatment with lovastatin of that culture again killed about 50% of the cells, but the surviving cells showed faster re-growth. In conclusion, lovastatin appears as a useful inhibitor of cytokinin biosynthesis in short-term studies, but its use in long-term experiments may create complex effects and therefore requires substantial caution.     

Fruit set and cytokinin-like activity in the xylem sap of sweet cherry (Prunus avium) as affected by rootstock

Using a trace-enrichment procedure involving solvent partitioning and high pressure liquid chromatography, followed by bioassay, two cytokinin-like substances believed to be zeatin and zeation riboside were detected in the xylem sap of sweet cherry ( Prunus avium L.). The levels of cytokinin varied between rootstocks with the higher yielding and fruit setting rootstocks producing the highest cytokinin levels. Most of the cytokinin activity occurred in the spring during bloom and early fruit set. A relationship between cytokinin level in the xylem sap and fruit set is proposed.     

Inhibition of cellular proliferation by the Wilms' tumor suppressor WT1 is associated with suppression of insulin-like growth factor I receptor gene expression.

We have investigated the regulation of the insulin-like growth factor I receptor (IGF-I-R) gene promoter by the Wilms' tumor suppressor WT1 in intact cells. The levels of endogenous IGF-I-R mRNA and the activity of IGF-I-R gene promoter fragments in luciferase reporter constructs were found to be significantly higher in G401 cells (a Wilms' tumor-derived cell line lacking detectable WT1 mRNA) than in 293 cells (a human embryonic kidney cell line which expresses significant levels of WT1 mRNA). To study whether WT1 could suppress the expression of the endogenous IGF-I-R gene, WT1-negative G401 cells were stably transfected with a WT1 expression vector. Expression of WT1 mRNA in G401 cells resulted in a significant decrease in the rate of cellular proliferation, which was associated with a reduction in the levels of IGF-I-R mRNA, promoter activity, and ligand binding and with a reduction in IGF-I-stimulated cellular proliferation, thymidine incorporation, and anchorage-independent growth. These data suggest that a major aspect of the action of the WT1 tumor suppressor is the repression of IGF-I-R gene expression.     

Exogenous auxin and cytokinin dependent activation of CDKs and cell division in leaf protoplast-derived cells of alfalfa

Alfalfa leaf protoplast cultures were used to study the role ofexogenously supplied auxin and cytokinin on the level and activity ofCdc2-related protein kinases and progression through the first celldivision cycle after re-activation of cell division. Among the threealfalfa Cdc2-related kinases studied, the Cdc2MsA/B kinase (PSTAIRE)showed only significant activity during the first four days ofprotoplast culture while the Cdc2MsD (PPTALRE) and Cdc2MsF kinases(PPTTLRE) exhibited only low or undetectable activity, respectively,during this period. Although the Cdc2MsA/B protein could be detectedin leaves and freshly isolated protoplasts in variable amounts, thekinase was never active in these cells. The kinase protein disappearedfrom protoplast-derived cells at the beginning (8h) of culture but itssynthesis re-commenced dependent on the presence of exogenous auxin butnot cytokinin. The cytokinin response of alfalfa protoplast-derivedcells varied significantly in different experiments although cytokininwas always required for completion of the first cell division cycle.Frequently both auxin and cytokinin was required for DNA replication asnot more than 5% of cells could incorporate BrdU into their DNAduring three days and significant Cdc2MsA/B activity could not bedetected in the absence of exogenous cytokinin. In other protoplastpopulations, the Cdc2MsA/B kinase was activated by auxin alone andallowed the protoplast-derived cells to enther the S-phase at a similarrate observed in parallel cultures with both auxin and cytokinin. Evenin these cultures, however, ca. 95% of the protoplast-derivedcells were arrested before mitosis without exogenous cytokinin supplywhich could be correlated with decreasing Cdc2MsA/B activity. Theseobservations suggest, that although cytokinin is required for bothG0-G1/S and G2/M cell cycle transitions, in certain cultures theG1/S requirement is overcome by some unknown factors (e.g.conditions of explants; endogenous cytokinins etc.). Furthermore, ourexperiments indicate, that the roles of cytokinin are related to thepost-translational regulation of the Cdc2MsA/B kinase complex atboth cell cycle transition points in alfalfa leaf protoplast-derivedcells. Finally, as a marker for the transition from the differentiated(G0) stage to the activated (G1) stage, we suggest using the parametersof nuclear morphology (size and ratio ofnucleus/nucleolus).     

Polyoma virus middle t antigen: a tumor progression factor.

Polyoma virus (PyV) deletion mutant dl23 (affecting both large T and middle t but not small t antigens) was used to study transformation of 3T3 rat cells. This mutant generated stable transformants in the agar assay at a frequency similar to that of wild-type virus (WT). However, WT-induced transformants were detected 3 weeks after infection, whereas those induced by the mutant could not be detected until 6 to 8 weeks after infection. In this respect, dl23 PyV behaved like WT simian virus 40 (SV40). Cells transformed by WT SV40 or by dl23 PyV were similar in all their transformed properties. Those transformed by WT PyV were different from the others on the basis of morphology, cell adhesion to the substrate, release of protease activity, efficiency of doubling in agar, growth rate, and time required for tumor formation. Saturation density, the ability to grow in agar, the serum requirement for cloning, and the ability to grow on a cell monolayer were similar for all transformants. Middle t antigen enhanced membrane alterations and growth rate of the transformed cells, shortening the time required for tumor formation in rats.     

Expression of fission yeast cdc25 driven by the wheat ADP-glucose pyrophosphorylase large subunit promoter reduces pollen viability and prevents transmission of the transgene in wheat

Cell number was to be measured in wheat (Triticum aestivum) endosperm expressing Spcdc25 (a fission yeast cell-cycle regulator) controlled by a supposedly endosperm-specific promoter, AGP2 (from the large subunit of ADP glucose pyrophosphorylase). Wheat was transformed by biolistics either with AGP2::GUS or AGP2::Spcdc25. PCR and RT-PCR checked integration and expression of the transgene, respectively. In cv. Chinese Spring, AGP2::GUS was unexpectedly expressed in carpels and pollen, as well as endosperm. In cv. Cadenza, three AGP2::Spcdc25 plants, AGP2::Spcdc25.1, .2 and .3, were generated. Spcdc25 expression was detected in mature leaves of AGP2::Spcdc25.1/.3 which exhibited abnormal spikes, 50% pollen viability and low seed set per plant; both were small compared with the nonexpressing and normal AGP2::Spcdc25.2. Spcdc25 was not transmitted to the T(1) in AGP2::Spcdc25.1 or .3, which developed normally. Spcdc25 was PCR-positive in AGP2::Spcdc25.2, using primers for a central portion, but not with primers for the 5' end, of the ORF, indicating a rearrangement; Spcdc25 was not expressed in either T(0) or T(1). The AGP2 promoter is not tissue-specific and Spcdc25 expression disrupted reproduction.     

Increase of endogenous zeatin riboside by introduction of the ipt gene in wild type and the lateral suppressor mutant of tomato

We studied axillary meristem formation of the lateral suppressor (ls) mutant of tomato after elevating the endogenous cytokinin levels through introduction of the isopentenyltransferase (ipt) gene from Agrobacterium tumefaciens. Growth and development of several transformants were examined during in vitro culture. Transformants exhibited phenotypes varying in severity and were divided into four classes. A number of the ipt transformants had a normal phenotype, as non-transformed plants. Others showed a mild to severe ‘cytokinin-like’ phenotype. Transformants with a mild phenotype exhibited reduced internode length and reduced root development. Transformants with a severe phenotype showed even shorter internodes, loss of apical dominance, reduction of leaf size, production of callus at the basis of the shoots and absence of root development or development of green non-branching roots. The severity of the phenotype correlated well with the level of ipt gene expression, as measured by northern analysis. Transformants with a severe phenotype also exhibited increased levels of zeatin riboside, but zeatin levels were not elevated. The increase in endogenous zeatin riboside levels in the ls mutant did not restore axillary meristem formation, but sometimes bulbous structures were formed in the initially ‘empty’ leaf axils. Several adventitious meristems and shoots developed from below the surface of these structures. It is concluded that a reduced level of cytokinins in the ls mutant shoots is not responsible for the absence of axillary meristem formation.     

SHP-2 phosphatase regulates DNA damage-induced apoptosis and G2/M arrest in catalytically dependent and independent manners, respectively

SHP-2, a tyrosine phosphatase implicated in diverse signaling pathways induced by growth factors and cytokines, is also involved in DNA damage-triggered signaling and cellular responses. We previously demonstrated that SHP-2 played an important role in DNA damage-induced apoptosis and G2/M cell cycle checkpoint. In the present studies, we have provided evidence that SHP-2 functions in DNA damage apoptosis and G2/M arrest in catalytically dependent and independent manners, respectively. Mutant embryonic fibroblasts with the Exon 3 deletion mutation in SHP-2 showed decreased apoptosis and diminished G2/M arrest in response to cisplatin treatment. Wild type (WT), but not catalytically inactive mutant SHP-2 (SHP-2 C459S), rescued the apoptotic response of the mutant cells. Interestingly, both WT and SHP-2 C459S efficiently restored the G2/M arrest response. Furthermore, inhibition of the catalytic activity of endogenous SHP-2 in WT cells by overexpression of SHP-2 C459S greatly decreased cell death but not G2/M arrest induced by cisplatin. Biochemical analyses revealed that activation of c-Abl kinase was decreased in SHP-2 C459S-overexpressing cells. However, DNA damage-induced translocation of Cdc25C from the nucleus to the cytoplasm was fully restored in both WT and SHP-2 C459S "rescued" cells. Additionally, we demonstrated that the role of SHP-2 in DNA damage-induced cellular responses was independent of the tumor suppressor p53. Embryonic stem cells with the SHP-2 deletion mutation showed markedly decreased sensitivity to cisplatin-induced apoptosis, attributed to impaired induction of p73 but not p53. In agreement with these results, DNA damage-induced apoptosis and G2/M arrest were also decreased in SHP-2/p53 double mutant embryonic fibroblasts. Collectively, these studies have further defined the mechanisms by which SHP-2 phosphatase regulates DNA damage responses.     

蟾毒灵可抑制人红白血病细胞增殖并下调肾母细胞瘤1基因表达

已有研究证实蟾毒灵具有抑制肿瘤细胞增殖及诱导细胞凋亡的作用,在白血病治疗中疗效显著,然而其机制尚未阐明。本研究试图探讨蟾毒灵对人红系白血病(HEL)细胞增殖,肾母细胞瘤基因1 (Wilms'tumor 1 gene, WT1)甲基化的影响及其可能的作用机制。本研究采用不同浓度的蟾毒灵处理HEL细胞,观察细胞形态、增殖情况和细胞周期,采用RT-PCR、Western blotting和免疫细胞化学法检测WT1的mRNA和蛋白表达水平,并用甲基化特异性分析WT1的DNA甲基化和DNA甲基转移酶3a (DNMT3a)的蛋白表达水平。研究结果表明,蟾毒灵对HEL细胞的增殖抑制作用呈剂量依赖性,抑制率为23.13%～84.62%。在蟾毒灵处理的HEL细胞中观察到典型的凋亡形态特征;细胞周期增殖指数由75.45降至49.67;WT1 mRNA及其蛋白表达水平随着蟾毒灵剂量的增加而逐渐降低,同时WT1基因的甲基化状态由未甲基化状态变为部分或完全甲基化状态。而蟾毒灵处理后DNMT3a蛋白的表达水平逐渐增加,呈剂量依赖性。我们的研究初步说明蟾毒灵不仅能显著抑制HEL细胞增殖,阻滞G0/G1期细胞周期,还能诱导细胞凋亡,下调WT1的表达水平。     

DNA-mediated transformation of N-acetylglucosaminyltransferase I activity into an enzyme deficient cell line

N-acetylglucosaminyltransferase I (GlcNAc-TI) catalyzes the first reaction in the conversion of ASN-linked cell surface oligosaccharides from a mannose-terminating structure to more complex carbohydrate structures. The mutant Chinese hamster ovary (CHO) cell line, Lec1, is deficient in this enzyme and, therefore, shows increased sensitivity to the lectin, Concanavalin A, which binds to the mannose-terminating oligosaccharides that accumulate on Lec1 cell surface glycoproteins. Spontaneous revertants of the Lec1 phenotype have never been observed. We report here the isolation of stable revertants of Lec1 cells to the parental CHO cell lectin-resistance phenotype after DNA-mediated transformation with human DNA. Both primary and secondary transformants express varying levels of GlcNAc-TI enzyme activity which was stable even when the cells were cultured in nonselective conditions. Human alu repeat DNA sequences are present in the primary transformants, but these sequences could not be detected in the secondary transformants.     

Effects of temperature and gibberellin on the activity of endogenous cytokinin-like substances in leaves of Begonia x cheimantha

Changes in activity of endogenous cytokinin-like substances were examined in intact plants and excised leaves of Begonia x chemantha Everett cv. Prinsesse Astrid (Christimas Begonia) by means of the tobacco callus bioassay. Cytokinin activity in the leaves of intact plants was higher in plants grown at 18°C than in those grown at 21° or 24°C. In excised leaves, an increase in cytokinin activity was observed during the first 4 days following leaf detachment. However, after the seventh day cylokinin activity decreased again. This decrease was more profound in leaves exposed to 24°C than in those exposed to 18°C.
Treatment of detached leaves with gibberellic acid (2.8 m M ) caused an increase in measurable cytokinin activity. This increase was more profound in the zones of activity which correspond with zeatin glucosides on paper and Sephadex LH-20 chromatography. Additional zones of activity appeared after Sephadex chromatography. These were of a more slow moving nature with elution volumes corresponding to N^b-(Δ²-isopentenyl)adenine and its derivaties. Water-treated control leaves had higher activity in the regions corresponding to zeatin and zeatin riboside.     

Phenotypic conversion of SV40-immortalized human diploid fibroblasts to senescing cells by introduction of an antisense gene for SV40-T antigen.

Normal human lung fibroblast diploid cells, WI-38, become senescent after a definite number of divisions. VA-13 is a line of immortalized cells established by transformation of WI-38 cells by SV40 virus. To determine whether SV40 large T (SV40-T) antigen is essential for this immortalization of WI-38 cells we introduced an antisense gene for T antigen into VA-13. Two morphologically different types of antisense transformant (VA-AS5-8 and VA-AS37-8) were obtained. In both antisense transformants the expression of T antigen was reduced by more than 70% as compared to that in the parent cells. The morphology of the antisense transformants indicated a partial conversion to the senescent phenotype of WI-38. The relative number of cells in the S phase of the antisense transformants was decreased as compared to that in cultures of VA-13 and about 50% of cells were at G1/0. The doubling time of the transformants was prolonged to close to the doubling time of WI-38. The level of expression of retinoblastoma protein (pRB) complexed with SV40-T antigen of the antisense transformants was significantly decreased although the level of total pRB was much higher than that in VA-13. The pRB was present exclusively in the underphosphorylated form. Thus, the decreased level of formation of the complex between SV40-T and pRB or the underphosphorylation of pRB may explain the suppression of growth of antisense transformants. Together, these results show that an antisense gene for SV40-T antigen can efficiently block the cell proliferation and the cell immortalization of VA-13 cells.