Cadmium induced mitochondrial redox changes in germinating pea seed

Mitochondria play an essential role in producing the energy required for seedling growth following imbibition. Heavy metals, such as cadmium impair mitochondrial functioning in part by altering redox regulation. The activities of two protein redox systems present in mitochondria, thioredoxin (Trx) and glutaredoxin (Grx), were analysed in the cotyledons and embryo of pea (Pisum sativum L.) germinating seeds exposed to toxic Cd concentration. Compared to controls, Cd-treated germinating seeds showed a decrease in total soluble protein content, but an increase in –SH content. Under Cd stress conditions, Grx and glutathione reductase (GR) activities as well as glutathione (GSH) concentrations decreased both in cotyledons and the embryo. Similar results were obtained with the Trx system: Trx and NADPH-dependent thioredoxin reductase (NTR) activities were not stimulated, whereas total NAD(P) contents diminished in the embryo. However, Cd enhanced the levels of all components of the Trx system in the cotyledons. On the other hand, Cd caused a significant increase in oxidative stress parameters such as the redox ratio of coenzymes (oxidized to reduced forms) and NAD(P)H oxidase activities. These results indicate that Cd induces differential redox responses on different seed tissues. We suggest that neither Grx system nor Trx one may improve the redox status of mitochondrial thiols in the embryo of germinating pea seeds exposed to Cd toxicity, but in the cotyledons the contribution of Trx/NTR/NADPH can be established in despite the vulnerability of the coenzyme pools due to enzymatic oxidation.     

Peptide uptake in germinating barley embryos involves a dithiol-dependent transport protein

An active transport system for small peptides occurs in the scutellar membrane of germinating barley and serves to move the products of partial hydrolysis of storage proteins from the endosperm into the growing embryo. Transport of peptides, but not amino acids or glucose, is inhibited by the thiol reagents, N-ethylmaleimide and p-chloromercuribenzene sulphonic acid (PCMBS). Peptide substrates protect against PCMBS inactivation. The dithiol-specific reagent, phenylarsine oxide (PAO) also inhibits. The reducing agent, dithiothreitol, reverses the inactivation caused by PCMBS and PAO. We conclude that the peptide transport system contains a redox-sensitive, dithiol-dependent protein.     

Thioredoxin targets in Arabidopsis roots

The importance of redox‐regulation in Arabidopsis thaliana roots has been investigated through the identification of the proteins interacting with thioredoxin (TRX), an ubiquitous thiol‐disulfide reductase. We have applied a proteomic approach based on affinity chromatography on a monocysteinic mutant of plastidial y‐type TRX used as a bait to trap putative partners in a crude extract of root proteins. Seventy‐two proteins have been identified, functioning mainly in metabolism, detoxification and response to stress, protein processing and signal transduction. This study allowed us to isolate 24 putative new targets and to propose the mevalonic acid‐dependent biosynthesis of isoprenoids as a new redox‐mediated process. The redox‐regulation of phenylpropanoid biosynthesis is also suggested, three enzymes of this pathway being retained on the column. We also provided experimental evidence that phenylammonia‐lyase was enzymatically more active when reduced by TRXy in root crude extract. Among the high number of partners involved in defense against stress we isolated from the column, we focused on plastidial monodehydroascorbate reductase and showed that its activity was dramatically increased in vitro in the presence of DTT‐reduced TRXy1 in root crude extracts. Our data strongly suggest that TRXy1 could be the physiological regulator of monodehydroascorbate reductase in root plastids.     

Selenium-containing peroxidases of germinating barley

Germinating barley grown on an artificial medium was exposed to⁷⁵Se-selenite for 8 d. Then the leaves were homogenized and proteins were separated by means of Sephadex G-150 filtration, followed by DEAE-Sepharose chromatography. Each fraction collected was assayed for total protein, radioactivity, and peroxidase activity. In barley leaves, three protein peaks (peaks no. I, II, and III) with peroxidase activity could be separated by Sephadex G 150 filtration. Each fraction was then further separated on DEAE-Sepharose chromatography. Thus, peaks I and II were resolved by DEAE-Sepharose into one major and two minor peaks of radioactivity. However, only the major peak showed peroxidase activity. Peak III was resolved from the gel filtration on the DEAE-sepharose into one major and four minor peaks of radioactivity. The major and three of the minor radioactivity peaks contained peroxidase activity. The protein fractions were separated by polyacrylamide gel electrophoresis. The molecular weights of separated proteins were estimated by means of molecular markers, and⁷⁵Se radioactivity was evaluated by autoradiography. Thus, gel filtration peak I contained four bands with mol wts of 128, 116, 100, and 89 kDa. Of these, the 89 kDa protein contained selenium. Peak II contained three protein bands, with mol wts 79.4, 59.6, and 59.9. The 59.6 band was a selenoprotein. Peak III contained four protein bands (and some very weak bands). The four major bands had mol wts of 38.6, 31.6, 30.2, and 29.2 kDa. The last mentioned band was a selenoprotein.     

trxS基因对大麦发芽籽粒中蛋白质降解的影响

对转trxS基因大麦籽粒发芽过程中蛋白酶活性、不同蛋白组分含量和贮藏蛋白SDS-PAGE图谱的变化进行了研究。结果表明：与对照相比,转基因籽粒中的蛋白酶活性提高;清蛋白、球蛋白、醇溶蛋白和谷蛋白含量低于对照。SDS-PAGE图谱也表明,转基因籽粒中贮藏蛋白降解快于对照。     

Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase

Ribonucleotide reductase(RNR), the rate-limitingenzyme in DNA synthesis, catalyzes reduction of thedifferent ribonucleotides to their corresponding deoxyri-bonucleotides. The crucial role of RNR in DNA synthesishas made it an important target for the development ofantiviral and anticancer drugs. Taking account of the re-cent developments in this field of research, this reviewfocuses on the role of thioredoxin and glutaredoxin sys-tems in the redox reactions of the RNR catalysis.     

Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase Sengupta R,Holmgren A简

Ribonucleotide reductase (RNR), the rate-limiting enzyme in DNA synthesis, catalyzes reduction of the different ribonucleotides to their corresponding deoxyribonucleotides. The crucial role of RNR in DNA synthesis has made it an important target for the development of antiviral and anticancer drugs. Taking account of the recent developments in this field of research, this review focuses on the role of thioredoxin and glutaredoxin systems in the redox reactions of the RNR catalysis.     

Peptide transport by germinating barley embryos

Glycylsarcosine, a dipeptide which is resistant to peptidase activity, was accumulated intact against a concentration gradient by germinating embryos of Hordeum vulgare L., var. Maris Otter, Winter. This is the first clear evidence for the presence of a dipeptide transport system involved in the movement of protein reserves across the scutellum from the endosperm to the embryo during germination.Abbreviations gly-sar glycylsarcosine - TCA trichloroacetic acid     

Glutamine synthetase isozymes in germinating barley seeds

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of ammonia assimilation in higher plants. In the present study the subunit composition and localization of GS in germinating barley ( Hordeum vulgare ) seed have been clarified. Analysis of the GS polypeptide composition by immunoblotting revealed two different polypeptides. A and B, with a molecular mass of 42 and 40 kDa, respectively. In the scutellum subunit A was already present in the ungerminated seed and remained unchanged, whereas subunit B appeared on day 2 and increased about 5-fold during germination. Polypeptide B also appeared later during germination in the aleurone layer, roots and weakly in the etiolated shoots. By immunogold microscopy, GS was detected in the scutellum and the aleurone layer of barley seeds during germination. Subcellular localization of GS on ultrathin cryosections showed that a cytosolic isozyme was present in the scutellum. Our study confirms that only a cytosolic GS is expressed in barley seed, and its subunit composition changes during germination.     

Turnover of starch-bound lysophosphatidylcholine in germinating barley

Free and starch-bound lysophosphatidylcholine (LPC) in germinating barley was isolated and quantified during the first 8 days of germination. During the first 4 days the starch-bound LPC remained at a relatively constant level (ca 0.4,μmol/seed) and then declined during the next 2 days to ca 0.1 μmol/seed. There appeared to be no further loss of this starch-bound lipid on further germination. The decrease in the content of starch-bound LPC is not due to the action of phospholipase C and/or D on the starch lipid because there was no corresponding accumulation of starchbound lysophosphatidic acid or monoacylglycerol. The free LPC remained relatively constant at 0.02 to 0.04 μmol/seed during the entire germination period indicating that the LPC released from the starch during days 5 and 6 is further metabolized. Amylase activity was also measured in the germinating seed and increased 20-fold between days 2 and 4 which just precedes the rapid decline in starch-bound LPC. The starch content of the seed however declined to ca one third of the original value by day 5. LPC represents 65–70 % of the starch-bound lipid phosphorus in the dry seed. Through days 5 and 6 when the loss of LPC is most rapid there is no marked change in this percentage. After 8 days, however, the LPC is only ca 30%. of the starch-bound lipid phosphorus. Ofthe two major populations of starch-bound LPC, the one bearing a linoleyl group appears to decline more rapidly during days 4–6 than does that carrying a palmitoyl group. The role of starch-bound LPC in barley development and germination is discussed.     

Localization and activity of various peptidases in germinating barley

Summary Germinating barley grains contain at least eight different peptidases: three carboxypeptidase (pH optima 4.8, 5.2, and 5.7), three aminopeptidases which act on aminoacyl--naphthylamides (pH opitima in the hydrolysis of di- and tripeptides at pH 5.8–6.5), and two peptidases which hydrolyse Ala-Gly and Leu-Tyr optimally at pH 7.8 and 8.6 respectively. We have determined the activities of these enzymes in the different tissues of non-germinated grains and followed the changes in the activities during a 5-day germination at 16°C.The aleurone layers contain high activities of all three groups of peptidases; there are no changes in the activities of the five aminopeptidases on germination, while the carboxypeptidases exhibit a small increase of activity. The starchy endosperms contain high carboxypeptidase activities, which increase during germination, but are totally devoid of the five aminopeptidases.All the peptidases exhibit high activities in the scutella; the carboxypeptidases and the enzymes acting on Ala-Gly and Leu-Tyr increase in activity during germination, while the naphthylamidase activities remain constant.The three peptidase groups occur in the seedling as well, but compared to the other tissues the carboxypeptidase activities are very small and the naphthylamidase activities are very high. The last-named enzymes seem to be characteristic for growing tissues.The starchy endosperm contains about two thirds of the total reserve proteins of the grain. Its internal pH during germination is 5.0–5.2, a value at which all the carboxypeptidases are highly active. As these enzymes are present in high concentrations in this tissue, it is probable that they have a central role in the mobilization of the reserve proteins during germination. The high peptidase activities of the scutellum, on the other hand, suggest that some of the hydrolysis products are absorbed as peptides and these are further hydrolysed to amino acids in this tissue.Abbreviations used DTT dithiothreitol - GA₃ gibberellic acid - -NA -naphthylamide - TNBS 2,4,6-trinitrobenzene sulphonic acid - Z- N-carbobenzoxy     

Transport-related amino acid metabolism in germinating barley grains

When eight [¹⁴C]-labelled amino acids were separately injected into the endosperm of germinating (4 days at 20°C) barley (Hordeum vulgare L. cv. Himalaya) grains, the label was rapidly taken up by the scutellum and further transported to the shoot and roots. Some of the amino acids (leucine, lysine and asparagine) were transported in an intact form through the scutellum to the seedling, whilst glutamic acid and aspartic acid were largely converted to glutamine in the scutellum. Proline was mainly transported unchanged, but a small part of the label appeared in glutamine. Arginine was mostly broken down in the scutellum, possibly providing ammonia for the synthesis of glutamine. During further transport in the seedling there was a partial transfer of label from glutamine to asparagine, particularly in the shoot. None of the amino acids used supplied carbon for the synthesis of sucrose, glucose or fructose. Glutamine synthetase activity was particularly high in the scutellum during the period of rapid amino acid transport.     

Inactivation of a MAPK-like protein kinase and activation of a MBP kinase in germinating barley embryos

We provide evidence for involvement of two different 45 kDa protein kinases in rehydration and germination of barley embryos. In dry embryos, a myelin basic protein (MBP) phosphorylating kinase was detected, which could be immunoprecipitated with an anti-MAPK (mitogen-activated protein kinase) antibody. Rehydration of the embryo induced a decrease in activity of this 45 kDa MAPK-like protein kinase. In addition, activity of a MBP kinase of the same molecular weight was subsequently found to be induced. This second MBP kinase activity could not be immunoprecipitated with the anti-MAPK antibody and was induced only in germinating embryos, not in dormant embryos.     

Stereospecificity of peptide transport by germinating barley embryos

The stereospecific requirements for peptide transport in the scutellum of germinating barley (Hordeum vulgare) embryos are described. Replacement of an L-amino acid residue in a peptide by its D-stereoisomer decreases the affinity of the peptide for the transport site, leading to a reduction in transport. Substitution of a second D-residue reduces affinity still further. The extent to which transport is inhibited depends upon the position of the D-residue in the primary sequence, with D-residues at the C-terminus of the peptide having the greatest effect. Competition between D- and L-peptides indicates that they both enter via the same transport system. Although D-amino acids can be accumulated when presented as a peptide, these same D-residues are not transported when supplied as the free amino acids. L-Leu-D-leu is accumulated intact against a concentration gradient, indicating the operation of an active transport mechanism that can function without the involvement of peptidase activity.     

The role of alpha-glucosidase in germinating barley grains

The importance of α-glucosidase in the endosperm starch metabolism of barley (Hordeum vulgare) seedlings is poorly understood. The enzyme converts maltose to glucose (Glc), but in vitro studies indicate that it can also attack starch granules. To discover its role in vivo, we took complementary chemical-genetic and reverse-genetic approaches. We identified iminosugar inhibitors of a recombinant form of an α-glucosidase previously discovered in barley endosperm (ALPHA-GLUCOSIDASE97 [HvAGL97]), and applied four of them to germinating grains. All four decreased the Glc-to-maltose ratio in the endosperm 10 d after imbibition, implying inhibition of maltase activity. Three of the four inhibitors also reduced starch degradation and seedling growth, but the fourth did not affect these parameters. Inhibition of starch degradation was apparently not due to inhibition of amylases. Inhibition of seedling growth was primarily a direct effect of the inhibitors on roots and coleoptiles rather than an indirect effect of the inhibition of endosperm metabolism. It may reflect inhibition of glycoprotein-processing glucosidases in these organs. In transgenic seedlings carrying an RNA interference silencing cassette for HvAgl97, α-glucosidase activity was reduced by up to 50%. There was a large decrease in the Glc-to-maltose ratio in these lines but no effect on starch degradation or seedling growth. Our results suggest that the α-glucosidase HvAGL97 is the major endosperm enzyme catalyzing the conversion of maltose to Glc but is not required for starch degradation. However, the effects of three glucosidase inhibitors on starch degradation in the endosperm indicate the existence of unidentified glucosidase(s) required for this process.