p 34cdc2 kinase homologue in the preprophase band

Summary Immunofluorescence microscopy with a monoclonal antibody raised against the PSTAIR sequence, which corresponds to a peptide conserved in the p 34^cdc2 protein kinase throughout the phylogenetic scale including higher plants, was used to study the intracellular localization of p 34^cdc2 during the cell cycle in onion root tip cells. Although p 34^cdc2 was evenly distributed in the cytoplasm throughout the cell cycle, a more intense staining was observed in the cortical region, where the preprophase band of microtubules (MTs) was located. Double staining with the PSTAIR and plant tubulin antibodies showed that the width of p 34^cdc2 band was narrower than that of MT band. These data raise the interesting question regarding the possible role of p 34^cdc2 protein kinase in determining the division site in plant cells.     

Cold-sensitive mutants of p34cdc2 that suppress a mitotic catastrophe phenotype in fission yeast.

Summary The p34^cdc2 protein kinase plays a central role in the regulation of the eukaryotic cell cycle, being required both in late G1 for the commitment to S-phase and in late G2 for the initiation of mitosis. p34^cdc2 also determines the precise timing of entry into mitosis in fission yeast, where a number of gene produts that regulate p34^cdc2 activity have been identified and characterised. To investigate further the mitotic role of p34^cdc2 in this organism we have isolated new cold-sensitive p34^cdc2 mutants. These are defective only in their G2 function and are extragenic suppressors of the lethal premature entry into mitosis brought about by mutating the mitotic inhibitor p107^wee1 and overproducing the mitotic activator p80^cdc25. One of the mutant proteins p34^cdc2-E8 is only functional in the absence of p107^wee1, and all the mutant strains have reduced histone H1 kinase activity in vitro. Each mutant allele has been cloned and sequenced, and the lesions responsible for the cold-sensitive phenotypes identified. All the mutations were found to map to regions that are conserved between the fission yeast p34^cdc2 and functional homologues from higher eukaryotes.     

Molecular cloning and nucleotide sequence cDNA encoding nucleoside diphosphate kinase of rice (Oryza sativa L.)

We isolated a rice cDNA encoding nucleoside diphosphate kinase (NDK, EC 2.7.4.6). The deduced amino acid sequence of the rice NDK shows highest homology to spinach NDK-I. The rice NDK gene exhibits a strong codon bias (73.8% GC) in the third position of the codon. DNA blot analysis indicated that at least single NDK gene is present in rice genome.     

Levels of p34cdc2-like protein in dividing,differentiating and dedifferentiating cells of carrot

P34^cdc2 is a key cell-cycle protein in fission yeast that is necessary for progress in the cell cycle from the G1 to the S phase and from G2 through mitosis. Homologues of p34^cdc2 have been found in all eukaryotes that have been investigated. Levels of p34^cdc2-like protein were studied by quantitative Western blotting in developing cotyledons of Daucus carota L. (carrot) seedlings, in expiants from the same seedlings transferred to tissueculture media with and without 2,4-dichlorophenoxyacetic acid (2,4-D), and in nutrient-starved suspension cultures derived from carrot callus. During the cessation of cell division, which accompanies development of the cotyledon to maturity, there was a 16-fold decline in the level of the p34^cdc2-like protein. Auxin-stimulated dedifferentiation in excised tissue from mature cotyledons was accompanied by restoration of the level of p34^cdc2-like protein, and the responding cells formed a callus. These data support our earlier proposition, based upon evidence from wheat leaf, that changes in the level of p34^cdc2-like protein act in the switch between cycling and differentiation. Persisting high levels of p34^cdc2-like protein in suspension cultures, when division was stopped by nutrient limitation, indicated that decline of the protein was not an inevitable consequence of the cessation of division. Decline of p34^cdc2 in differentiation may therefore be a regulated process that determines exit from the cell cycle and the converse increase in p34^cdc2 may be a regulated process controlling dedifferentiation and resumption of cell division.Abbreviations BrdUrd 5-bromodeoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - kDa kilodalton - MS Murashige and Skoog (1962) J.R.G. gratefully acknowledges the support of a National Research Fellowship from the Australian Government during the time this work was done.     

Expression of functional proteins of cDNA encoding rice nucleoside diphosphate kinase (NDK) in Escherichia coli and organ-related alteration of NDK activities during rice seed germination (Oryza sativa L.)

The GST (glutathione S-transferase)-NDK (nucleoside diphosphate kinase) fusion protein was expressed in Escherichia coli. The GST-NDK protein was capable of transferring -phosphate from ATP to nucleoside diphosphates such as GDP, CDP, TDP and UDP. Western blot analysis using anti-NDK antibody indicated that NDK in endosperm gradually decreased during 36 h of imbibition. On the contrary, NDK in embryo increased during the same period. NDK activities in both tissues were in accord with these observations. Whereas the NDK protein in roots of rice seedlings during 7 days of imbibition remained constant, in shoots it declined after 5 days of imbibition. Thus, NDK may play a significant role in the cellular event modulated by adenylate energy charge level.     

Cloning and characterization of two genes encoding sulfate transporters from rice (Oryza sativa L.)*

Two genes were isolated from a rice genomic library and the coding region of their corresponding cDNAs generated by RT-PCR. These single copy genes, designated ORYsa;Sultr1;1 and ORYsa;Sultr4;1, encode putative sulfate transporters. Both genes encode proteins with predicted topologies and signature sequences of the H⁺/SO₄^2- symporter family of transporters and exhibit a high degree of homology to other plant sulfate transporters. ORYsa;Sultr1;1 is expressed in roots with levels of expression being strongly enhanced by sulfate starvation. In situ hybridization experiments revealed that ORYsa;Sultr1;1 expression is localized to the main absorptive region of roots. This gene probably encodes a transporter that is responsible for uptake of sulfate from the soil solution. In contrast, ORYsa;Sultr4;1 was expressed in both roots and shoots and was unresponsive to the sulfur status of the plant. The sequence of ORYsa;Sultr4;1 contains a possible plastid-targeting transit peptide which may indicate a role in transport of sulfate to sites of sulfate reduction in plastids. The role of the transporter encoded by ORYsa;Sultr4;1 is likely to be significantly different fromORYsa;Sultr1;1. These are the first reports of isolation of genes encoding sulfate transporters from rice and provide a basis for further studies involving sulfate transport.     

Isolation of gametes and central cells from Oryza sativa L.

In vitro fertilization system of higher plants has been well established using maize gametes and central cells, which can produce embryos and endosperms. In the present study, procedures for isolating gametes and central cells from rice (Oryza sativa L. cv. Nipponbare), a model plant, are reported with the goal of establishing rice in vitro fertilization system. Egg cells and central cells were isolated by manual manipulation of enzyme-treated unpollinated ovules, and an alternative direct isolation method for egg cells that does not use enzymatic treatment was also established. Fluorescent visualization of the granular structures in the cytoplasm of isolated egg cells and the nucleoli in two polar nuclei of isolated central cells suggest that these cells are reliable gametes and central cells. For sperm cell isolation, the contents of rice pollen grains were released by osmotic pressure-induced bursting of the grains. In addition, electrofusion with isolated gametes was successfully conducted.     

Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase

In excised pith parenchyma from Nicotiana tabacum L. cv. Wisconsin Havana 38, auxin (naphthalene-1-acetic acid) together with cytokinin (6-benzylaminopurine) induced a greater than 40-fold increase in a p34^cdc2-like protein, recoverable in the p13^suc1-binding fraction, that had high H1 histone kinase activity, but enzyme induced without cytokinin was inactive. In suspension-cultured N. plumbaginifolia Viv., cytokinin (kinetin) was stringently required only in late G2 phase of the cell division cycle (cdc) and cells lacking kinetin arrested in G2 phase with inactive p34^cdc2-like H1 histone kinase. Control of the Cdc2 kinase by inhibitory tyrosine phosphorylation was indicated by high phosphotyrosine in the inactive enzyme of arrested pith and suspension cells. Yeast cdc25 phosphatase, which is specific for removal of phosphate from tyrosine at the active site of p34^cdc2 enzyme, was expressed in bacteria and caused extensive in-vitro activation of p13^suc1-purified enzyme from pith and suspension cells cultured without cytokinin. Cytokinin stimulated the removal of phosphate, activation of the enzyme and rapid synchronous entry into mitosis. Therefore, plants can control cell division by tyrosine phosphorylation of Cdc2 but differ from somatic animal cells in coupling this mitotic control to hormonal signals.Abbreviations BAP 6-benzylaminopurine - BrdUrd 5-bromo-2-deoxyuridine - cdc cell division cycle - Cdc25 cdc phospho-protein phosphatase - CKI cyclin dependent kinase inhibitor - 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6 diamidino-2-phenylindole - GST-cdc25 glutathione sulfur transferase-truncated cdc25 fusion - MS Murashige and Skoog (1962) - NAA naphthalene-1-acetic acid - p34^cdc2 34-kDa product of the cdc2 gene     

Molecular Cloning and Expression of a cDNA Encoding Lon Protease from rice (Oryza sativa)

The ATP-dependent Lon protease is a highly conserved enzyme that is present in archeae, eubacteria, and eukaryotes, and plays an important role in intracellular protein degradation. We have isolated a Lon protease gene, OsLon1, from Oryza sativa. The cDNA contained a 2,655 bp ORF. Comparative analysis showed that OsLon1 shared significant similarity with the previously reported Lon proteases from maize, Arabidopsis, human, and bacteria. Tissue expression pattern analysis revealed that OsLon1 was highly expressed in young leaves, mature leaves, and leaf sheaths but only weakly in young roots, mature roots, and young panicles. The OsLon1 gene was successfully expressed in E. coli and the detected protein size, about 120 kDa, matched the expected molecular mass of the His-tagged OsLon1 protein.     

The identification of a Caenorhabditis elegans homolog of p34cdc2 kinase

We have identified a Caenorhabditis elegans homolog of p34^cdc2 kinase. The C. elegans homolog, ncc-1, is -60% identical to p34^cdc2 of Homo sapiens. When expressed from a constitutive yeast promoter, ncc-1 is capable of complementing a conditional lethal mutation in the CDC28 gene of Saccharomyces cerevisiae, indicating that this C. elegans homolog can properly regulate the cell cycle.     

Genetic analysis of human p34CDC2 function in fission yeast

The p34^cdc2 protein kinase plays a key role in the control of the mitotic cell cycle of fission yeast, being required for both entry into S-phase and for entry into mitosis in the mitotic cell cycle, as well as for the initiation of the second meiotic nuclear division. In recent years, structural and functional homologues of p34^cdc2, as well as several of the proteins that interact with and regulate p34^cdc2 function in fission yeast, have been identified in a wide range of higher eukaryotic cell types, suggesting that the control mechanisms uncovered in this simple eukaryote are likely to be well conserved across evolution. Here we describe the construction and characterisation of a fission yeast strain in which the endogenous p34^cdc2 protein is entirely absent and is replaced by its human functional homologue p34^CDC2, We have used this strain to analyse aspects of the function of the human p34^CDC2 protein genetically. We show that the function of the human p34^CDC2 protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80^cdc25 that it responds to altered levels of both the mitotic inhibitor p107²³³¹ and the p34^cdc2-binding protein p13^suc1, and is lethal in combination with the mutant B-type cyclin p56^cdc13-117. In addition, we demonstrate that the human p34^CDC2 protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34^CDC2 proteins in fission yeast.     

Protein phosphatase 2A: identification in Oryza sativa of the gene encoding the regulatory A subunit

A 2225 bp cDNA, designated RPA1, was isolated from an Oryza sativa cDNA library. Analysis revealed a 1761 bp coding sequence with 15 non-identical repeat units. The ORF encoded the A regulatory subunit of protein phosphatase 2A (PP2A-A) as ascertained by complementation of the yeast tpd3 mutant defective in this gene. The corresponding genomic DNA from a rice genome BAC library revealed that the gene contains eleven introns. The rice genome contains only a single copy of this gene as judged by Southern blot analysis. The PP2A protein is highly conserved in nature; the rice protein shows 88% amino acid identity with its counterparts in Arabidopsis or Nicotiana tabacum.     

Isolation, characterization and expression of cDNA clones encoding a mitochondrial malate translocator from Panicum miliaceum L.

Three cDNA clones that hybridize to a partial rice cDNA that show similarity to bovine mitochondrial 2-oxoglutarate/malate translocator were isolated from leaves of Panicum miliaceum L. (proso millet), an NAD-malic enzyme-type C₄ plant. The nucleotide sequences of the clones resemble each other, and some of the isolated cDNAs contained extra sequences that seemed to be introns. The predicted proteins encoded by the cDNAs have 302 amino acids and molecular weights of 32211 and 32150. The hydrophobic profile of the amino acid sequence predicted the existence of six transmembrane -helices that is a common property of members in the mitochondrial transporter family. The predicted amino acid sequence showed the highest similarity with that of the 2-oxoglutarate/malate translocator from mammalian mitochondria. An expression plasmid containing the coding region of the cDNAs was used to over-express recombinant protein with a C-terminal histidine tag Escherichia coli, which was affinity-purified. The antibody against the recombinant protein cross-reacted with proteins of 31–32 kDa in the membrane fraction from P. miliaceum mitochondria, but not with the chloroplast fraction. The recombinant protein reconstituted in liposomes efficiently transported malate, citrate, and 2-oxoglutarate.     

Cell-cycle dependency of radiosensitivity and mutagenesis in fertilized egg cells of rice,Oryza sativa L.

Summary In order to examine changes in survival and mutation rates during a cell cycle in higher plant, fertilized egg cells of rice were irradiated with X-rays at 2 h intervals for the first 36 h after pollination, i.e., at different phases of the first and second cell cycles. The most sensitive phase in lethality was late G₁ to early S, followed by late G₂ to M, which were more sensitive than the other phases. In both M₁ and M₂ generations, sterile plants appeared most frequently when fertilized egg cells were irradiated at G₂ and M phases. Different kinds of mutated characters gave rise to the respective maximum mutation rates at different phases of a cell cycle: namely, albino and viridis were efficiently induced at early G₁, xantha at early S, short-culm mutant at mid G₂, heading-date mutant at M to early G₁. The present study suggests the possibility that the differential mutation spectrums concerning agronomic traits are obtained by selecting the time of irradiation after pollination.     

Plant regeneration from rice (Oryza sativa L.) embryogenic suspension cells cryopreserved by vitrification

Summary Rice cells were precultured for 10 d in medium containing 60 g/L sucrose and subsequently for 1 d in medium supplemented with 0. 4 M sorbitol. After loading with 25%PVS2 at 22°C for 10 min and dehydration in 100%PVS2 at 0°C for 7. 5 min,they were plunged into liquid nitrogen directly. Survival was 45. 0 ±5.1% (based on the reduction of triphenyl tetrazolium chloride)following warming and unloading. For regrowth, cells were plated on semi-solid medium replenished with 40%(w/v) starch for 2d prior to reculture. Cell suspensions were reestablished and plants were regenerated from recovered cells. Twenty eight plants set seeds in the greenhouse.Abbreviations PVS plant vitrification solution - P preculture - LN liquid nitrogen - TTC triphenyl tetrazolium chloride - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - EG ethylene glycol - BSA bovine serum albumin