Purification and characterization of a XIP-type endoxylanase inhibitor from Rice (Oryza sativa)

A rice XIP-type inhibitor was purified by affinity chromatography with an immobilized Aspergillus aculeatus family 10 endoxylanase. Rice XIP is a monomeric protein, with a molecular mass of ca. 32?kDa and a pI of ca. 5.6. Its N-terminal amino acid sequence was identical to that of a rice chitinase homologue, demonstrating the difficulty when using sequence information to differentiate between endoxylanase inhibitors and (putative) chitinases in rice. Rice XIP inhibited different endoxylanases to a varying degree. In particular, it most strongly inhibited family 10 endoxylanases from A. niger and A. oryzae, while several family 11 enzymes from Bacillus subtilis, A. niger and Trichoderma sp. were not sensitive to inhibition. The above mentioned A. aculeatus endoxylanase was not inhibited either, although gel permeation chromatography revealed that it complexed rice XIP in a 1:1 molar stoichiometric ratio.     

Purification and characterization of lipoxygenase from aromatic and non-aromatic rice (Oryza sativa L.)

Lipoxygenase (Lox) has been extensively purified from aromatic (Bas-370) and non-aromatic (Pusa-834) rice varieties. Crude isolates of Lox from the aromatic varieties (Bas-370 and PB-1) showed higher specific activity (4-fold) when compared to non-aromatic varieties (Pusa-677 and Pusa-834). The activity was optimum at pH 8.0 in all four varieties. Anionic PAGE of Lox from three days old seedlings revealed one extra band (Rm 0.48) in aromatic varieties, besides the presence of a major band (Rm 0.28) in all the four varieties. Elution profile of Lox from Bas-370 and Pusa-834 on DEAE-cellulose column showed three distinct peaks (L-1, L-2 and L-3), L-2 being the major fraction in both the varieties. SDS-PAGE of purified L-2 from Bas-370 showed a single band of molecular mass -88 kDa.     

Purification and characterization of a novel 7-kDa non-specific lipid transfer protein-2 from rice (Oryza sativa)

A novel 7-kDa non-specific lipid transfer protein-2 (nsLTP2) has been isolated from rice (Oryza sativa) seeds. In contrast to nsLTP1s, few nsLTP2s have been purified and characterized. Complete amino acid sequence of rice nsLTP2 was determined by N-terminal Edman degradation of the intact protein as well as the peptide fragments resulted from trypsin digestions. Rice nsLTP2 consists of 69 amino acid residues with eight conserved cysteines forming four disulfide bonds. The secondary structure of rice nsLTP2 is predominantly alpha-helical as determined by circular dichroism spectroscopy. Cysteine pairings of nsLTP2 have one miss match at Cys(35)-X-Cys(37) motif compared to nsLTP1. Primary structure analysis of various plant nsLTP2s revealed an interesting conservation of sequence features among nsLTP2 family.     

Isolation and characterization of a jacalin-related mannose-binding lectin from salt-stressed rice (Oryza sativa) plants

A novel plant lectin was isolated from salt-stressed rice (Oryzasativa L.) plants and partially characterized. The lectin occurs as a natural mixture of two closely related isoforms consisting of two identical non-covalently linked subunits of 15 kDa. Both isoforms are best inhibited by mannose and exhibit potent mitogenic activity towards T-lymphocytes. Biochemical analyses and sequence comparisons further revealed that the rice lectins belong to the subgroup of mannose-binding jacalin-related lectins. In addition, it could be demonstrated that the lectins described here correspond to the protein products of previously described salt-stress-induced genes. Our results not only identify the rice lectin as a stress protein but also highlight the possible importance of protein-carbohydrate interactions in stress responses in plants. Received: 27 July 1999 / Accepted: 11 November 1999     

Cloning and characterization of catalases from rice, Oryza sativa L

Catalase is the major H(2)O(2)-scavenging enzyme in all aerobic organisms. From the cDNA sequences of three rice (Oryza sativa L.) genes that encode for predicted catalases (OsCatA, OsCatB, and OsCatC), complete ORFs were subcloned into pET21a and expressed as (His)(6)-tagged proteins in Escherichia coli. The recombinant (His)(6)-polypeptides were enriched to apparent homogeneity and characterized. With H(2)O(2) as substrate, the highest catalase k(cat) value (20±1.71×10(-3) min(-1)) was found in recombinant OsCatB. The optimum temperatures for catalase activity were 30 °C for OsCatA and OsCatC and 25 °C for OsCatB, while the pH optima were 8.0, 7.5, and 7.0 for OsCatA, OsCatB, and OsCatC respectively. All the catalases were inhibited by sodium azide, β-mercaptoethanol, and potassium cyanide, but only weakly by 3-amino-1,2,4-triazole. The various catalases exhibited different catalase activities in the presence of different salts at different concentrations, OsCatC showing higher salt inhibitory effects than the two other OsCats.     

Purification and partial characterization of pyruvate decarboxylase from Oryza sativa L

Pyruvate decarboxylase(PyrDC) was purified from rice bran to a specific activity of 1 mu kat/mg and partially characterized. The holoenzyme is a tetramer of two types of subunits with molecular masses 64 kDa and 62 kDa. Purified rice PyrDC exhibits positive cooperative kinetics with respect to pyruvate and functions with a significant lag phase. When compared to other plant PyrDC, the lag phase was shorter at low pyruvate concentrations and the S0.5 was smaller. The optimum pH (6.25) was also less acidic and the enzyme retained 30% of its maximal activity at neutral pH. In contrast to other plant PyrDC, rice PyrDC could be active at the onset of anoxia and would be activated by small changes in pyruvate concentration.     

Purification and characterization of carbonic anhydrase of rice (Oryza sativa L.) expressed in Escherichia coli

Rice carbonic anhydrase (CA) was successfully expressed as a glutathione-S-transferase (GST) fusion protein in an Escherichia coli expression system. The optimal induction concentration of IPTG and growth temperature was found to be 1.0mM and 28 degrees C. To obtain milligram amounts of homogeneous active recombinant proteins, 150mM NaCl and Mg-ATP solution were used during the purification procedures. After improving the conditions of expression and the purification procedures, final yield of recombinant proteins was 1.3mg/g wet cell weight after enzymatic cleavage of the GST tag, and the molecular weight was about 29kDa. The purified protein had CO(2) hydration activity, and had no detectable esterase activity in vitro. Addition of zinc improved the CO(2) hydration activity of the rice CA produced by E. coli. The effects of acetazolamide (AZ) and the anions N3-, NO3-, I(-), Br(-), and Cl(-) on CO(2) hydration activity of CA were studied. AZ and N3- were found to be strong inhibitors of rice CA. The inhibitory activity of AZ and ions was in the order AZ>N3->NO3->I(-)>Br(-)>Cl(-).     

Purification and characterization of extracellular beta-galactosidase secreted by supension cultured rice (Oryza sativa L.) cells

A beta-galactosidase was purified 1300-fold by lactosyl-Sepharose 4B and Sephacryl S-200 column chromatographies from the cultured medium of a rice-cell suspension. The purified enzyme appeared as 47 kD and 40 kD polypeptides on SDS-PAGE and had a specific activity of 65.1 units/mg. Optimum activity was observed at pH 3.5 and 60 degrees C. The enzyme released galactose from galactoxyloglucan and pectic galactans.     

Purification and characterization of a neutral endoxylanase from Aspergillus nidulans

Abstract A neutral endoxylanase from a culture filtrate of Aspergillus nidulans grown on oat spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on SDS-PAGE with a molecular mass of 22,000 and had an isoelectric point of 6.4. The enzyme was a non-debranching endoxylanase highly specific for xylans and completely free from cellulolytic activity. The xylanase showed an optimum activity at pH 5.5 and 62°C and had a K m of 4.2 mg oat spelt xylan per ml and a V max of 710 μmol min⁻¹ (mg protein)⁻¹.     

Separation and characterization of proteins in rice (Oryza sativa) suspension cultured cells

Proteins extracted from suspension cultured cells of rice were separated by two-dimensional polyacrylamide gel electrophoresis. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane and 103 electroblotted proteins were analyzed. The N-terminal amino-acid sequences of 20 out of 103 proteins were determined in this manner. N-terminal regions of the remaining proteins could not be sequenced and they were inferred to have a blocking group at the N-terminus. Internal amino-acid sequences of 32 proteins were determined by sequence analysis of peptides obtained by Cleveland peptide mapping. The amino-acid sequences determined here were compared with those of known plant and animal proteins. Furthermore, the concanavalin A-peroxidase method was used to determine which of the 103 proteins were glycosylated, and in vitro and in vivo phosphorylation was carried out to identify some of the phosphorylated proteins. Using this experimental approach, we could identify the major proteins involved in growth and development of rice cell suspension cultures and discuss on the physiological function of some of these identified proteins including the calcium binding protein, superoxide dismutase and rice ascorbate peroxidase. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genomewide survey and characterization of metacaspase gene family in rice (Oryza sativa)

Metacaspases (MCs), which are cysteine-dependent proteases found in plants, fungi, and protozoa, may be involved in programmed cell death processes, being distant relatives of metazoan caspases. In this study, we analysed the structures, phylogenetic relationship, genome localizations, expression patterns and domestic selections of eight MC genes identified in rice (OsMC). Alignment analysis of the corresponding protein sequences suggested OsMC proteins can be classified into two subtypes. The expression profiles of eight OsMC genes were analysed in 27 tissues covering the whole life cycle of rice. There are four OsMC genes uniquely expressed in mature tissues, indicating that these genes might play certain roles in senescence. Under abiotic and biotic stresses, four OsMC genes were expressed with treatments of one or more of Magnaporthe oryzae (M. oryzae) infected, pest damaged, cold stress and drought stress, indicating they might be involved in plant defense. In addition, gene trees and genetic diversity (π) were performed to measure whether candidate genes were selected during rice domestication. The results suggested that all the type I genes could not be domestication genes. However, two of five type II OsMC genes showed strong evidence for selective sweep, suggesting that these genes might be involved in cultivated rice domestication. These results provide a foundation for future functional genomic studies of this family in rice.     

Purification and physicochemical characterization of a recombinant phospholipid hydroperoxide glutathione peroxidase from Oryza sativa

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an unique antioxidant enzyme that directly reduces lipid hydroperoxides in biomembranes. In the present work, the entire encoding region for Oryza sativa PHGPx was expressed in Escherichia coli M15, and the purified fusion protein showed a single band with 21.0 kD and pI = 8.5 on SDS- and IFE-PAGE, respectively. Judging from CD and fluorescence spectroscopy, this protein is considered to have a well-ordered structure with 12.2% alpha-helix, 30.7% beta-sheet, 18.5% gamma-turn, and 38.5% random coil. The optimum pH and temperature of the enzyme activity were pH 9.3 and 27 degrees C. The enzyme exhibited the highest affinity and catalytical efficiency to phospholipid hydroperoxide employing GSH or Trx as electron donor. Moreover, the protein displayed higher GSH-dependent activity towards t-Butyl-OOH and H(2)O(2). These results show that OsPHGPx is an enzyme with broad specificity for hydroperoxide substrates and yielded significant insight into the physicochemical properties and the dynamics of OsPHGPx.     

杂草稻落粒粳的抗逆境特性研究

杂草稻落粒粳（Oryza sativa）发生在我国辽宁丹东.落粒粳植株明显高于当地大多数栽培品种,颖果呈中长型,成熟后容易掉粒;果壳稻草色或黄间黑灰色,小穗无芒或有芒,芒长4～12 cm;颖果千粒重235 g,种皮桔红色.落粒粳种子在13～38 ℃条件下的发芽率均大于88%,水层2.5～10 cm处理,落粒粳植株干重减少50%～69%.在幼苗期,落粒粳对无芒稗的各项影响因子均明显大于化感潜力品种I-kung-pao,表明落粒粳无化感作用.落粒粳可以忍耐0.5%的盐碱.     

Protein profile of rice (Oryza sativa) seeds

Seeds are the most important plant storage organ and play a central role in the life cycle of plants. Since little is known about the protein composition of rice (Oryza sativa) seeds, in this work we used proteomic methods to obtain a reference map of rice seed proteins and identify important molecules. Overall, 480 reproducible protein spots were detected by two-dimensional electrophoresis on pH 4–7 gels and 302 proteins were identified by MALDI-TOF MS and database searches. Together, these proteins represented 252 gene products and were classified into 12 functional categories, most of which were involved in metabolic pathways. Database searches combined with hydropathy plots and gene ontology analysis showed that most rice seed proteins were hydrophilic and were related to binding, catalytic, cellular or metabolic processes. These results expand our knowledge of the rice proteome and improve our understanding of the cellular biology of rice seeds.     

RNAi-mediated knockdown of the XIP-type endoxylanase inhibitor gene, OsXIP, has no effect on grain development and germination in rice

OsXIP (Oryza sativa xylanase inhibitor protein) is a XIP-type xylanase inhibitor which was identified as a protein encoded by a wound stress-responsive gene in rice. Although the OsXIP gene was specifically expressed in mature grains under basal conditions, recombinant OsXIP had no effect on rice endogenous xylanases, and OsXIP-suppressed transgenic rice plants did not exhibit any change in grain development and germination, suggesting that rice development may be independent of OsXIP. Analysis using an OsXIP-specific antibody revealed that OsXIP is markedly accumulated in apoplast in rice root cells by wounding. These results reinforced the possibility that OsXIP is involved in plant defense mechanisms against phytopathogens.     

Oxygen dynamics in submerged rice (Oryza sativa)

Complete submergence of plants prevents direct O(2) and CO(2) exchange with air. Underwater photosynthesis can result in marked diurnal changes in O(2) supply to submerged plants. Dynamics in pO(2) had not been measured directly for submerged rice (Oryza sativa), but in an earlier study, radial O(2) loss from roots showed an initial peak following shoot illumination. O(2) dynamics in shoots and roots of submerged rice were monitored during light and dark periods, using O(2) microelectrodes. Tissue sugar concentrations were also measured. On illumination of shoots of submerged rice, pO(2) increased rapidly and then declined slightly to a new quasi-steady state. An initial peak was evident first in the shoots and then in the roots, and was still observed when 20 mol m(-3) glucose was added to the medium to ensure substrate supply in roots. At the new quasi-steady state following illumination, sheath pO(2) was one order of magnitude higher than in darkness, enhancing also pO(2) in roots. The initial peak in pO(2) following illumination of submerged rice was likely to result from high initial rates of net photosynthesis, fuelled by CO(2) accumulated during the dark period. Nevertheless, since sugars decline with time in submerged rice, substrate limitation of respiration could also contribute to morning peaks in pO(2) after longer periods of submergence.     

A calcium- and phospholipid-dependent protein kinase from rice (Oryza sativa) leaves

Protein kinases in plants have not been examined in detail, but protein phosphorylation has been shown to be essential for regulating plant growth via the signal transduction system. A Ca²⁺- and phospholipid-dependent protein kinase, possibly involved in the intracellular signal transduction system from rice leaves, was partially purified by sequential chromatography on DE52, Phenyl Superose and Superose 12. This protein kinase phosphorylated the substrate, histone III-S, in the presence of Ca²⁺ and phosphatidylserine. The apparent molecular mass of the Ca²⁺- and phosphatidylserine-dependent protein kinase (Ca²⁺/PS PK), determined by phosphorylation in SDS-polyacrylamide gel containing histone III-S, was 50 kDa. The protein kinase differed from Ca²⁺-dependent protein kinase (CDPK) in rice leaves in that Ca²⁺/PS PK showed phospholipid dependency and the molecular mass of Ca²⁺/PS PK exceeded that of CDPK. Investigations were carried out on changes in Ca²⁺/PS PK and CDPK activity in the cytosolic and membrane fractions during germination. The maximum activity of Ca²⁺/PS PK in the cytosolic fraction was observed before imbibition and that of CDPK in the membrane fraction was noted at 6 days following imbibition. Protein kinases are likely to regulate plant growth through protein phosphorylation.