The subunit structures of soluble and chromatin-bound RNA polymerase II from soybean

DNA-dependent RNA polymerase II is present in two forms, IIa and IIb, in germinating soybean. Form IIa is the dominant form of the enzyme in ungerminated embryos and appears to be a soluble enzyme. Form IIb increases in amount as germination progresses and is tightly bound to the chromatin template. The subunit structures of soybean RNA polymerases IIa and IIb are identical except for the molecular weights of their largest subunits which are 200,000 daltons and 170,000 daltons for IIa and IIb, respectively. The enzymes have seven common subunits: 142,000, 42,000, 26,000, 20,000, 16,000, 15,000, and 14,000 daltons.     

Long-lived Messenger RNA and its Relationship to Protein Synthesis During Germination of Pea (Pisum sativum L.) Seeds

Synthesis of both protein and RNA is initiated very early ingermination in the embryo axes of pea seeds. The early RNA synthesisinvolves all three types, although there is some evidence forpreferential synthesis of mRNA in the first few hours afterthe onset of imbibition. In addition to newly synthesized mRNA,the embryo axis also contains long-lived mRNA. The amount ofthis long-lived mRNA declines markedly during the first 20 hof germination. Synthesis of both protein and RNA is initiated very early ingermination in the embryo axes of pea seeds. The early RNA synthesisinvolves all three types, although there is some evidence forpreferential synthesis of mRNA in the first few hours afterthe onset of imbibition. In addition to newly synthesized mRNA,the embryo axis also contains long-lived mRNA. The amount ofthis long-lived mRNA declines markedly during the first 20 hof germination. Results from in vitro and in vivo protein synthesis experimentsand from studies of polysome formation suggest that much ofthe long-lived mRNA present in the embryo axis does not directprotein synthesis. The increase in the rate of protein synthesisduring germination is thus dependent on recruitment of newlysynthesized mRNA molecules. Pea, Pisum sativum L., germination, mRNA, protein synthesis     

RNA synthesis in germinating embryonic axes of soybean and wheat

The rate of synthesis of RNA during early germination of wheat and soybean embryos was investigated by ascertaining the incorporation of radioactive uridine into RNA. In wheat embryos, where the lag period preceding rapid growth is 5.5 hours, there is a 2-fold increase in RNA synthesis between 1.5 and 5.5 hours, with half of the increase occurring by 3.5 hours. In soybean axes, where the lag period is 9.5 hours, the increased rate of RNA synthesis is 5.5-fold between 1.5 and 9.5 hours, with three fourths of this increase occurring after 4 hours.     

Phosphorylation in vitro and in vivo of the wheat embryo RNA polymerase II

One of the large subunits (220 000 daltons) of the wheat embryo RNA polymerase II was demonstrated to undergo phosphorylation with [gamma-32P]ATP in a reaction catalysed by a homologous protein kinase preparation. The same subunit was also observed to be phosphorylated in vivo, at the onset of germination. The phosphorylation resulted in a moderate increase of the RNA polymerase activity.     

PVA和PEG(6000)预处理对大豆胚轴生长、蛋白质合成和ATP含量的影响

在低温吸胀阶段,经PVA(聚乙烯醇)和PEG(聚乙二醇6000)预处理的大豆胚轴蛋白质合成和ATP含量均比对照高。在萌发阶段,胚轴生长增快,蛋白质合成明显加快,ATP迅速被消耗,而对照胚轴则相反。试验结果表明,预处理大豆种子萌发和生长与其蛋白质合成、ATP水平和消耗能力有密切关系。     

Mobilization of storage proteins in soybean seed (Glycine max L.) during germination and seedling growth

During germination and early growth of the seedling, storage proteins are degraded by proteases. Currently, limited information is available on the degradation of storage proteins in the soybean during germination. In this study, a combined two-dimensional gel electrophoresis and mass spectrometry approach was utilized to determine the proteome profile of soybean seeds (Glycine max L.; Eunhakong). Comparative analysis showed that the temporal profiles of protein expression are dramatically changed during the seed germination and seedling growth. More than 80% of the proteins identified were subunits of glycinin and β-conglycinin, two major storage proteins. Most subunits of these proteins were degraded almost completely at a different rate by 120h, and the degradation products were accumulated or degraded further. Interestingly, the acidic subunits of glycinin were rapidly degraded, but no obvious change in the basic chains. Of the five acidic subunits, the degradation of G2 subunit was not apparently affected by at least 96h but the levels decreased rapidly after that, while no newly appearing intermediate was detected upon the degradation of G4 subunit. On the other hand, the degradation of β-conglycinin during storage protein mobilization appeared to be similar to that of glycinin but at a faster rate. Both α and α' subunits of β-conglycinin largely disappeared by 96h, while the β subunits degraded at the slowest rate. These results suggest that mobilization of subunits of the storage proteins is differentially regulated for seed germination and seedling growth. The present proteomic analysis will facilitate future studies addressing the complex biochemical events taking place during soybean seed germination.     

Dehydration Injury in Germinating Soybean (Glycine max L. Merr.) Seeds

The sensitivity of soybean (Glycine max L. Merr. cv Maple Arrow) seeds to dehydration changed during germination. Seeds were tolerant of dehydration to 10% moisture if dried at 6 hours of imbibition, but were susceptible to dehydration injury if dried at 36 hours of imbibition. Dehydration injury appeared as loss of germination, slower growth rates of isolated axes, hypocotyl and root curling, and altered membrane permeability. Increased electrolyte leakage due to dehydration treatment was observed only from isolated axes but not from cotyledons, suggesting that cotyledons are more tolerant of dehydration. The transition from a dehydration-tolerant to a dehydration-susceptible state coincided with radicle elongation. However, the prevention of cell elongation by osmotic treatment in polyethylene glycol (−6 bars) or imbibition in 20 micrograms per milliliter cycloheximide did not prevent the loss of dehydration tolerance suggesting that neither cell elongation nor cytoplasmic protein synthesis was responsible for the change in sensitivity of soybean seeds to dehydration. Furthermore, the rate of dehydration or rate of rehydration did not alter the response to the dehydration stress.     

菜用大豆种子活力与DNA,RNA及蛋白质合成的关系

菜用大豆种子随着其活力的下降,对DNA,RNA和蛋白质前体的吸收,以及合成这些大分子的能力都明显下降,已丧失合成DNA和蛋白质能力的失活种子,仍能进行微弱的RNA合成。高活力种子在吸胀初期DNA合成速率较低,然后增加,至16h达高峰;RNA的合成速率在吸胀一开始就很高,在整个吸胀过程中均保持较高水平;蛋白质的合成速率则在开始较高,并随着吸胀过程呈增强趋势。     

Protein synthesis and phospholipids in soybean axes in response to imbibitional chilling

The responses of two cultivars of soybean (Merr.) to a chilling treatment (4 C for first hour of imbibition) were compared. The germination of cv. Biloxi was unaffected by the treatment, while the germination of cv. Fiskeby was reduced. The phospholipid fatty acids of dry axes of the two cultivars were very similar, and, thus, could not be correlated with their responses to chilling. The fatty acid composition of chilling-tolerant Biloxi did not change over a subsequent 23-hour warm incubation, but there was a marked reduction in the unsaturated fatty acids of chilling-sensitive Fiskeby after 12 hours, which may be a symptom of deterioration. Protein synthesis in both cultivars was reduced by the chilling treatment. Redrying of Biloxi axes up to 18 hours after the onset of imbibition had no effect on their germination upon rehydration. Germination of Fiskeby axes was reduced by redrying after 8 hours of imbibition. After 7 months of dry storage of intact seeds, the sensitivity of the axes to chilling was retested. Biloxi axes had become chilling-sensitive, while the germination of Fiskeby axes was reduced to zero by the chilling treatment. A hypothesis is presented that imbibitional chilling sensitivity is an indication of reduced vigor, axes with a high vigor can tolerate the stress, while those without cannot.     

大豆种子吸胀冷害抗性与其质体膜脂不饱和度正相关

吸胀冷害是干种子在吸胀阶段遭受低温造成不萌发的现象,结果可能造成农作物损失严重。虽然吸胀过程中细胞膜的修复是关键事件,而且细胞膜在响应水分和温度胁迫中扮演重要角色,但是种子吸胀过程中膜变化的过程,特别是膜流动性变化过程研究较少。本文比较了吸胀冷害耐受型（LX）和敏感型（R5）两个大豆品种在吸胀冷害过程中膜脂不饱和度（double bond index, DBI）的变化,结果发现,LX和R5在常温（25℃）吸胀时变化趋势一致,质体膜脂DBI升高,质体外膜脂中磷脂酰甘油(phosphatidylglycerol, PG)分子DBI下降。LX和R5在低温（4℃）吸胀时DBI变化有很大差异,低温吸胀仅仅延缓了耐受型LX中质体膜脂DBI的升高,但是敏感性R5质体膜脂DBI不仅没有升高反而下降。用浓度33%的聚乙二醇 (polyethylene glycol, PEG)引发没有直接引起DBI变化,但是所引起的细微而显著的变化可能为萌发做好准备。PEG引发处理后的R5在吸胀冷害后第二和第三阶段质体膜脂DBI迅速增加,这个增加模式与LX的DBI增加相似。结果表明,吸胀冷害延缓或者阻滞了质体膜脂不饱和度的升高,大豆种子的吸胀冷害抗性与质体膜脂不饱和度正相关,提高质体膜质DBI可以提高吸胀冷害抗性。     

ADP-ribosylation of DNA-dependent RNA polymerase of Escherichia coli by an NAD+: protein ADP-ribosyltransferase from bacteriophage T4.

A protein from bacteriophage T4 responsible for the alteration of host DNA-dependent RNA polymerase and absent in T4 alt- phage was purified from T4 phage and enriched from T4-infected cells. It is injected during infection together with the known internal proteins. It has a molecular weight of about 70000 and catalyses the release of nicotinamide and the transfer of the ADP-ribosyl moiety from NAD+ to arginyl residues of various proteins including itself. RNA polymerase from Escherichia coli accepts ADP-ribosyl residues in all four subunits; the alpha subunit reacts with very high specificity. Only half of the alpha subunits are labelled, 45% with one, 5% with two residues. The main product shows the same electrophoretic mobility as alpha subunits altered or modified in vivo. The alpha subunit in modified RNA polymerase is no acceptor.     

Oxidative Stress Affects [alpha]- Tocopherol Content in Soybean Embryonic Axes upon Imbibition and following Germination

The content of [alpha]-tocopherol ([alpha]T) in isolated soybean (Glycine max, var Hood) embryonic axes was measured upon germination. Dry, high-vigor axes contained 1.2 [plus or minus] 0.1, nmol/axis and after an increase during the initial 6 h of imbibition, there was a decline to 1.0 [plus or minus] 0.1 nmol/axis at 24 h of incubation. Incubation in the presence of the redox-cycling agent paraquat (4 mM) for 24 h increased the [alpha]T content to 1.9 [plus or minus] 0.2 nmol/axis. When the incubation medium was supplemented with 500 [mu]M Fe-EDTA over 24 h, the content of [alpha]T increased to 1.8 [plus or minus] 0.1 nmol/axis. Isolated axes from soybean seeds stored for 56 months contained 6.5 [plus or minus] 0.3 nmol of [alpha]T/axis after 24 h of imbibition as compared to 1.0 [plus or minus] 0.1 nmol of [alpha]T/axis in axes from soybean seeds stored for 8 months. In all of these experimental situations, oxidant production as assessed in vivo by a fluorometric assay was increased by 4 mM paraquat (8-fold), 500 [mu]M iron (2-fold), and 56 months of storage (4-fold) after 24 h of imbibition. The data presented here suggest that the cellular content of [alpha]T is physiologically adjusted as a response to conditions of oxidative stress.     

RNA polymerase II subunit composition, stoichiometry, and phosphorylation.

RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme.     

Endogenous abscisic acid during the germination of chick-pea seeds

Over the past twenty years many studies have been undertaken to elucidate the regulation of seed germination. Abscisic acid (ABA) and the gibberellins (GAs) are the hormones proposed to control this process, the first by inhibiting and the second by inducing germination. It has been proposed that a high water potential increases the growth potential of the embryo, presumably permitting the production or activation by GA of the cell wall hydrolases and thus decreasing the yield threshold of the endosperm close to the radicle tip. A low water potential, e.g., imbibition in an osmoticum. imposes a stress on cell metabolism, by reducing the turgor of the radicle cells, and there is a decrease in growth potential. Exogenous ABA also causes a decline in growth potential of the radicle: however, the actions of low water potential in preventing germination are not mediated through an increase in ABA in the seeds. In the present paper an attempt is made to asses the role of ABA and polyethylene glycol (PEG) in the germination of chick-pea (Cicer arietinum L.) seeds. The endogenous ABA of chick-pea seeds was purified by reversed-phase HPLC and quantified by GC-ECD. The variations in the ABA levels in the embryonic axes and the cotyledons were studied during 120 h. of imbibition. The highest ABA level in the embryome axes was found at 18 h. coinciding with an increase in fresh weight and a high germination percentage. ABA was not detected in the cotyledons during incubation which probably indicates that the hormone is more involved in the active growth of the embryonic axes itself than in the mobilization process of the reserves. When seeds were treated with different PEG-cycles. PEG delayed germination, reduced the fresh weight of embryonic axes, and retarded the onset of ABA synthesis. It is concluded that endogenous ABA is related to the onset of germination and the growth of the embryonic axis. In addition, there is no correlation among the different PEG-cycles and the level of ABA and germination. Germination was related more to the water conditions inside the embryo's cells than to ABA levels.     

Phosphorylation of rat C6 glioma cell DNA-dependent RNA polymerase II in vivo. Identification of phosphorylated subunits and modulation of phosphorylation by isoproterenol and N6,O2'-dibutyryl cyclic AMP

Evidence is presented that isoproterenol treatment of rat C6 glioma cells, under conditions that increase glioma cell cAMP levels, causes the phosphorylative modification of several RNA polymerase II subunits. RNA polymerase II in control and isoproterenol-stimulated 32Pi-labeled confluent glioma cells was immunoprecipitated from ribonuclease-treated nuclear extracts with hen anti-calf RNA polymerase II antiserum conjugated to Sepharose. The immunoprecipitated RNA polymerase II was analyzed for 32P-labeled subunits by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Using this technique, we have shown that isoproterenol causes a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 214,000, 180,000, 140,000, 35,000, 28,000, and 16,500 daltons. Phosphate incorporation occurred exclusively on serine in all of the six subunits. About 0.5-2 mol of phosphate/mol of RNA polymerase II subunit were incorporated. Dibutyryl cAMP (10(-3)M) mimics the stimulatory action of isoproterenol and mediates increased phosphate incorporation into the six subunits. (RS)-propranolol (10(-4)M) prevents the isoproterenol-mediated phosphorylative changes. These data indicate that isoproterenol, via cAMP, mediates a transient structural modification of RNA polymerase II subunits in rat C6 glioma cells which may possibly lead to a modulation of RNA polymerase II function(s).     

Cloning, expression and characterisation of a Family B ATP-dependent phosphofructokinase activity from the hyperthermophilic crenarachaeon Aeropyrum pernix

Protein interactions among RNA polymerase small subunits from the archaeon Methanococcus jannaschii were investigated using affinity pulldown assays in pairwise and higher-order combinations. In the most extensive study of archaeal RNA polymerase subunit interactions to date, including 37 pairs of proteins, 10 ternary combinations, and three quaternary combinations, we found evidence for pairwise interactions of subunit D with subunits L and N, and a ternary complex of subunits D, L and N. No other small subunit interactions occurred. These results are consistent with interactions observed in a crystal structure of eukaryotic RNA polymerase II and support a common archaeal/eukaryal RNA polymerase architecture. We further propose that subunit E" is not an integral member of archaeal RNA polymerases. Finally, we discuss the relative accuracy of the various methods that have been used to predict protein-protein interactions in RNA polymerase.