Activation of glyoxalase I during the cell division cycle and its homology with auxin regulated genes

In several plant systems increase in glyoxalase I activity has been correlated with cell proliferation. Cell cycle studies of tobacco protoplasts indicate a rise in glyoxalase I activity prior to G₂/M phase. Further, synthetic auxin, NAA, induced glyoxalase I activity and cell division significantly. This induction was specific in response to auxin only. Cytokinins alone do not induce cell division or increase enzyme activity. Analysis of glyoxalase I cDNA sequence from soybean shows significant homology with auxin inducible genes particularly Nt107 and limited but strong similarity with identified plant mitotic cyclins, implicating glyoxalase I in possible relationship with certain cell division regulating factors.     

Uncoupling of chloroplast reproductive events from cell cycle division processes by 5-fluorodeoxyuridine in the algaScenedesmus quadricauda

Summary FdUrd (5-fluorodeoxyuridine), a specific inhibitor of thymidylate synthase, was used to study the relationship between reproductive processes in chloroplast and nucleocytoplasmic compartments of the chlorococcal algaScenedesmus quadricauda. The courses of DNA replication and nuclear division in both the compartments were followed in populations synchronised by the alternation of light and dark periods. DAPI-staining of DNA-containing structures was used for their visualisation and quantification. In contrast with cellular reproductive events, those in chloroplasts were not substantially affected by the presence of FdUrd (25 g/ml). It was shown that FdUrd specifically blocked nucDNA replication but not ptDNA replication. Thus, cells which had attained commitment to ptDNA replication, fission of pt-nuclei and chloroplast kinesis triggered and terminated these processes while the corresponding cellular processes were blocked. The courses of reproductive processes in chloroplasts were also substantially unaffected in cells grown in the presence of FdUrd for the whole cell cycle. This provided evidence that attainment of commitment to and termination of the entire sequence of reproductive events, including chloroplast fission, were controlled by different mechanisms than the reproductive processes in the nucleocytoplasmic compartment.Abbreviations DAPI 4,6-diamidino-2-phenylindole - ptDNA DNA of chloroplast nuclei - nucDNA DNA in cell nuclei - FdUrd 5-fluorodeoxyuridine     

atToc159 is a selective transit peptide receptor for the import of nucleus-encoded chloroplast proteins

The members of the Toc159 family of GTPases act as the primary receptors for the import of nucleus-encoded preproteins into plastids. Toc159, the most abundant member of this family in chloroplasts, is required for chloroplast biogenesis (Bauer, J., K. Chen, A. Hiltbunner, E. Wehrli, M. Eugster, D. Schnell, and F. Kessler. 2000. Nature. 403:203-207) and has been shown to covalently cross-link to bound preproteins at the chloroplast surface (Ma, Y., A. Kouranov, S. LaSala, and D.J. Schnell. 1996. J. Cell Biol. 134:1-13; Perry, S.E., and K. Keegstra. 1994. Plant Cell. 6:93-105). These reports led to the hypothesis that Toc159 functions as a selective import receptor for preproteins that are required for chloroplast development. In this report, we provide evidence that Toc159 is required for the import of several highly expressed photosynthetic preproteins in vivo. Furthermore, we demonstrate that the cytoplasmic and recombinant forms of soluble Toc159 bind directly and selectively to the transit peptides of these representative photosynthetic preproteins, but not representative constitutively expressed plastid preproteins. These data support the function of Toc159 as a selective import receptor for the targeting of a set of preproteins required for chloroplast biogenesis.     

The Cytokinin-hypersensitive genes of Arabidopsis negatively regulate the cytokinin-signaling pathway for cell division and chloroplast development

We isolated Arabidopsis thaliana mutants that respond more sensitively than the wild type to cytokinins. The calli produced from the mutants exhibit typical cytokinin responses, including rapid proliferation and chloroplast development in response to lower levels of cytokinins than in the wild type. The mutations are recessive and belong to two complementation groups designated ckh1 and ckh2 for cytokinin-hypersensitive. CKH1 and CKH2 were mapped to the top of chromosome I and the middle of chromosome II, respectively. The cytokinin levels in these mutants were not increased. We speculate that the CKH1 and CKH2 gene products negatively regulate the signaling pathway leading from cytokinin perception to cell proliferation and chloroplast development.     

Expression of the RAD1 and RAD3 genes of Saccharomyces cerevisiae is not affected by DNA damage or during the cell division cycle

Summary The RAD1 and RAD3 genes of Saccharomyces cerevisiae are required for excision repair of UV damaged DNA. In addition, the RAD3 gene is essential since rad3 deletions are recessive lethals. We have examined the induction of the RAD1 and RAD3 genes by DNA damage and during the cell division cycle. We have made fusions of the RAD1 and RAD3 genes with the Escherichia coli lacZ gene encoding -galactosidase. -galactosidase activity was measured in a Rad⁺ yeast strain containing the RAD1-lacZ or the RAD3-lacZ fusion, either in a multicopy replicating plasmid or as a single copy integrant resulting from transformation with an integrating plasmid which transforms yeast by homologous recombination in the yeast genome. No induction of -galactosidase activity occurred after ultraviolet light (UV) or 4-nitroquinoline-1-oxide (NQO) treatment. Haploid cells of mating type a were synchronized by treatment with factor and -galactosidase activity was determined during different cell cycle stages. No change in -galactosidase activity was observed in the strain containing the RAD1-lacZ or the RAD3-lacZ fusion integrated in the yeast genome.     

Protein feature based identification of cell cycle regulated proteins in yeast

DNA microarrays have been used extensively to identify cell cycle regulated genes in yeast; however, the overlap in the genes identified is surprisingly small. We show that certain protein features can be used to distinguish cell cycle regulated genes from other genes with high confidence (features include protein phosphorylation, glycosylation, subcellular location and instability/degradation). We demonstrate that co-expressed, periodic genes encode proteins which share combinations of features, and provide an overview of the proteome dynamics during the cycle. A large set of novel putative cell cycle regulated proteins were identified, many of which have no known function.     

In vivo robustness analysis of cell division cycle genes in Saccharomyces cerevisiae

Intracellular biochemical parameters, such as the expression level of gene products, are considered to be optimized so that a biological system, including the parameters, works effectively. Those parameters should have some permissible range so that the systems have robustness against perturbations, such as noise in gene expression. However, little is known about the permissible range in real cells because there has been no experimental technique to test it. In this study, we developed a genetic screening method, named “genetic tug-of-war” (gTOW) that evaluates upper limit copy numbers of genes in a model eukaryote Saccharomyces cerevisiae, and we applied it for 30 cell-cycle related genes (CDC genes). The experiment provided unique quantitative data that could be used to argue the system-level properties of the cell cycle such as robustness and fragility. The data were used to evaluate the current computational model, and refinements to the model were suggested.     

Accumulation, activity and localization of cell cycle regulatory proteins and the chloroplast division protein FtsZ in the alga Scenedesmus quadricauda under inhibition of nuclear DNA replication

Synchronized cultures of the green alga Scenedesmus quadricauda were grown in the absence (untreated cultures) or in the presence (FdUrd-treated cultures) of 5-fluorodeoxyuridine, the specific inhibitor of nuclear DNA replication. The attainment of commitment points, at which the cells become committed to nuclear DNA replication, mitosis and cellular division, and the course of committed processes themselves were determined for cell cycle characterization. FdUrd-treated cultures showed nearly unaffected growth and attainment of the commitment points, while DNA replication(s), nuclear division(s) and protoplast fission(s) were blocked. Interestingly, the FdUrd-treated cells possessed a very high mitotic histone H1 kinase activity in the absence of any nuclear division(s). Compared with the untreated cultures, the kinase activity as well as mitotic cyclin B accumulation increased continuously to high values without any oscillation. Division of chloroplasts was not blocked but occurred delayed and over a longer time span than in the untreated culture. The FtsZ protein level in the FdUrd-treated culture did not exceed the level in the untreated culture, but rather, in contrast to the untreated culture, remained elevated. FtsZ structures were both localized around pyrenoids and spread inside of the chloroplast in the form of spots and mini-rings. The abundance and localization of the FtsZ protein were comparable in untreated and FdUrd-treated cells until the end of the untreated cell cycle. However, in the inhibitor-treated culture, the signal did not decrease and was localized in intense spots surrounding the chloroplast/cell perimeter; this was in agreement with both the elevated protein level and persisting chloroplast division.     

Translation and stability of proteins encoded by the plastid psbA and psbB genes are regulated by a nuclear gene during light-induced chloroplast development in barley

We have characterized a nuclear mutant of barley, viridis-115, lacking photosystem II (PSII) activity and compared it to wild-type seedlings during light-induced chloroplast development. Chloroplasts isolated from wild-type and viridis-115 seedlings illuminated for 1 h synthesized similar polypeptides and had similar protein composition. After 16 h of illumination, however, mutant plastids exhibited reduced ability to radiolabel D1, CP47, and several low Mr membrane polypeptides, and by 72 h, synthesis of these proteins was undetectable. Immunoblot analysis showed that plastids of dark-grown wild-type barley lacked several PSII proteins (D1, D2, CP47, and CP43) and that 16 h of illumination resulted in the accumulation of these polypeptides. In contrast, these polypeptides did not accumulate in illuminated viridis-115 seedlings, although mutant plastids accumulated two PSII proteins that participate in oxygen evolution, oxygen-evolving enhancers 1 and 3. Northern analysis showed that the levels of psbA and psbB mRNA in mutant plastids were equal to or greater than levels in wild-type plastids throughout the developmental period examined here. These results indicate that the nuclear mutation present in viridis-115 affects the translation and stability of the chloroplast-encoded D1 and CP47 polypeptides and that its influence is expressed after the onset of light-induced chloroplast development.     

Chloroplast targeting of chloroplast division FtsZ2 proteins in Arabidopsis

Plant nuclear genomes encode chloroplast division proteins homologous to the eubacterial cell division protein FtsZ. In higher plants, FtsZ genes constitute a small gene family that consists of two subgroups, FtsZ1 and FtsZ2. It was previously hypothesized that members of one family (FtsZ1) targeted chloroplasts, while members of the other family (FtsZ2) localized in the cytoplasm. We determined the full-length cDNA sequences of two FtsZ2 genes from Arabidopsis thaliana (AtFtsZ2-1 and AtFtsZ2-2) and found that the genes encode polypeptides of 478 and 473 amino acids, respectively, and both contain N-terminal extensions beyond what have previously been predicted. The N-terminal regions of both AtFtsZ2-1 and AtFtsZ2-2 were expressed as green fluorescent protein (GFP) fusions under the cauliflower mosaic virus 35S promoter in bombarded tobacco cells. Confocal laser scanning microscopy revealed both fusions exclusively localized to chloroplasts, demonstrating that the N-terminal regions function as chloroplast-targeting signals in vivo. Thus, FtsZ2 proteins function within chloroplasts.