Alleviation of Changes in Protein Metabolism in NaCl-Stressed Wheat Seedlings by Thiamine

NaCl-stress induced a pronounced suppression in growth of wheat seedlings. The most abundant amino acids (cysteine, arginine, methionine) constituting about 55 % of total free amino acid content in control wheat were reduced in 100 mM NaCl-treated plants. However, valine, isoleucine, aspartic acid and proline accumulated in response to NaCl stress and NaCl-treated wheat seedlings showed 1.6 fold increase in total free amino acids compared to the control. Addition of 2 [micro ]M thiamine alleviated the effects of NaCl on the amino acid composition and the amount of total free amino acids decreased to that in the control. Content of 26 kDa protein increased in NaCl-treated plants, stimulation was more pronounced in roots than in shoots. In contrast, the contents of 13 and 20 kDa proteins decreased. After addition of thiamine, the 24 kDa protein, which disappeared with NaCl treatment, has been initiated again. Moreover, thiamine treatment stimulated the accumulation of the 20 kDa protein.     

Hyperosmotically induced accumulation of a phosphorylated p38-like MAPK involved in protoplast volume regulation of plasmolyzed wheat root cells

A 46 kDa protein resembling immunochemically to the mammalian dually phosphorylated p38-MAPK was detected in wheat root cells under hyperosmotic conditions, using Western blot analysis. This protein accumulated in a time- and dose-dependent fashion and exhibited pharmacological sensitivity similar to the activated p38-MAPK. The application of a highly specific p38-MAPK inhibitor revealed that the p38-like MAPK is probably implicated in hyperosmotically induced tubulin cytoskeleton reorganization as well as in protoplast volume regulation and osmotic tolerance of wheat root cells. As far as we know, the p38-MAPK has not been previously reported in higher plants.     

Synthesis of soluble heat shock proteins in seminal root tissues of some cultivated and wild wheat genotypes

Effect of heat stress on the synthesis of soluble heat shock proteins (HSPs) and the regrowth in seminal roots of three cultivated and three wild wheat genotypes was examined. In regrowth experiments, 2-d-old etiolated seedlings were exposed to 23 (control), 32, 35, 37 and 38 degrees C for 24 h, and 35 and 37 degrees C (24 h) followed by 50 degrees C (1 h). The lengths of the seminal roots generally decreased significantly at the end of 48 and 72 h recovery growth periods at 35, 37 and 38 degrees C temperature treatments compared with control. Genotypic variability was significant level at all temperature treatments for the seminal root length. Also, genotypic differences for the number of seminal roots were determined among the wheat cultivars and between the wild wheat species and the wheat cultivars at all temperature treatments; but genotypic differences among wild wheat species were only detected at 37-->50 degrees C treatment. Acquired thermotolerance for the seminal root length is over 50% at 37-->50 degrees C treatment. The genotypic variability of soluble heat shock proteins in seminal root tissues were analyzed by two-dimensional electrophoresis (2-DE). Total number of low molecular weight (LMW) HSPs was more than intermediate-(IMW) and high- (HMW) HSPs at high temperature treatments. The most of LMW HSPs which were generally of acidic character ranged between 14.2-30.7 kDa. The genotypes had both common (43 HSP spots between at least two genotypes and 23 HSP spots between 37 and 37-->50 degrees C) and genotype-specific (72 HSP spots) LMW HSPs.     

Changes in protein quantities of phosphoenolpyruvate carboxylase and Rubisco activase in various wheat genotypes

In early seedlings of wheat genotypes two isoforms of Rubisco activase with molecular weights of 42 and 46 kDa are expressed. Amounts of both isoforms significantly increase in early seedlings of the durum wheat genotype Barakatli-95 exposed to salt stress. But at the beginning of the tillering stage, the changes in quantities of both RCA isoforms are different in durum and bread wheat genotypes subjected to a 3-day drought stress. In the leaves of the early seedlings of the studied wheat genotypes exposed to drought stress quantities of PEPC subunits increase compared to the control but they remain relatively stable in early roots and germinating seeds. However, quantities of its subunits decrease sharply in roots and germinating seeds of early seedlings under the influence of 100 mM NaCl. In flag leaves and ear elements of the Barakatli-95 genotype grown under normal water supply conditions protein quantities of PEPC subunits change differently depending on time. Changes in protein quantities of RCA, PEPC and Rubisco enzymes have been studied comparatively in ear elements and flag leaves after the fourth day of anthesis.     

The identification and characterization of collagen receptors involved in HeLa cell-substratum adhesion

Four proteins of molecular mass 102, 87, 45, and 38 kDa were isolated from plasma membrane preparations by affinity chromatography. The 102-, 87-, and 38-kDa proteins were shown to be collagen receptors involved in the adhesion of HeLa cells to a gelatin substratum. All four proteins were eluted by high salt from affinity columns made of either types I or IV collagen or type I gelatin. Generally, a total of six major proteins were found in the high salt eluates, although the relative amounts of each varied among experiments. Immunoprecipitation, immunoblotting, and limited peptide mapping indicated that the 102-kDa protein was most sensitive to proteolysis leading to the formation of proteins of molecular mass 58 and 54 kDa. Even in the presence of a mixture of protease inhibitors the 58-kDa fragment was usually the more abundant species. Lectin binding indicated that the 102-, 87-, and 38-kDa proteins contain carbohydrate. Phase-partitioning with Triton X-114 and the need to solubilize the proteins in Triton X-100 indicated that the 102-, 87-, 45-, and 38-kDa proteins have a hydrophobic domain. The 87-kDa protein partitioned exclusively with the detergent-rich phase, suggesting that it is the most hydrophobic. Cell surface labeling with 125I indicated that the four proteins have an extracellular domain. Four criteria were used to determine which of the four proteins are collagen receptors mediating cell-substrate adhesion: 1) during HeLa cell adhesion, proteins with Mr values similar to all four proteins or their peptide fragments were cross-linked to a gelatin substratum derivatized with a photoactivatable probe; 2) a pentapeptide containing the Arg-Gly-Asp cell recognition sequence eluted the same four proteins as those found by high salt elution of collagen affinity columns; 3) monospecific antibodies to the 102-, 87-, and 38-kDa proteins, but not the 45-kDa protein, inhibited the spreading of HeLa cells on a gelatin substratum; 4) monospecific antibodies to the 102-, 87-, and 38-kDa proteins, but not the 45-kDa protein, bound to culture dishes substituted for gelatin in mediating the spreading of HeLa cells. Taken together, the data suggest that the 102-, 87-, and 38-kDa proteins are collagen receptors involved in HeLa cell adhesion. Although the 45-kDa protein has two of the characteristics of a collagen receptor defined here, it does not fit the criteria for one involved in cell-substratum adhesion.     

N-terminal sequencing of photosystem II low-molecular-mass proteins. 5 and 4.1 kDa components of the O2-evolving core complex from higher plants

High resolution gel electrophoresis in the low-molecular-mass region combined with electroblotting using polyvinylidene difluoride membranes enabled us to sequence the low-molecular-mass proteins of photosystem II membrane fragments from spinach and wheat. The determined N-terminal sequences, all showing considerable homology between the two plants, involved two newly determined sequences for the 4.1 kDa protein and one for the 5 kDa proteins. The sequence of the 4.1 kDa protein did not match any part of the chloroplast DNA sequence from tobacco or liverwort, suggesting that it is encoded by the nuclear genome. In contrast, the sequence of the 5 kDa protein matched ORF38, which is located just downstream of psbE and psbF in the chloroplast DNA and is assumed to be co-transcribed with them. These two components were associated with the O2-evolving core complex. Sequences of other low-molecular-mass proteins confirmed the previous identification as photosystem II components.     

Changes of phospholipase A2 inhibitory activity in the K+-sensitive actin gelation factor during the differentiation of myeloid leukemia cells

The phospholipase A2 inhibitory activity of a 38 kDa K+-sensitive actin gelation factor in a murine leukemia cell line (M1) was examined. A specific antibody against 38 kDa protein was found to cross-react with 37 kDa protein (lipocortin) in rat peritoneal exudates. Although the native 38 kDa protein from M1 cells did not block phospholipase A2 activity, pretreatment with alkaline phosphatase produced a form that did inhibit this enzyme. However, a purified 38 kDa protein from differentiated M1 cells blocked phospholipase A2 activity without pretreatment with alkaline phosphatase. Phospholipase A2 inhibitory activity of the 38 kDa protein was not altered by addition of actin. These findings suggest that the phospholipase A2 inhibitory of our 38 kDa protein was induced during differentiation. We also proposed that our 38 kDa protein has the same epitope as lipocortin.     

Identification and biochemical characterization of 20S proteasome in wheat roots under salt stress

In order to gain a better understanding of proteases state in wheat roots under salt stress, the roots of wheat (Triticum aestivum L., H6756) exposed to 300 mM NaCl were investigated at different days after treatment (DAT). The results showed that the content of soluble proteins decreased continuously, but the activity of total proteases increased gradually, and five root endopeptidase isoenzymes (RE1-5) were detected by natural gradient polyacrylamide gel electrophoresis (GPAGE) with supplement of gelatin as a substrate. Among five REs, RE1 was not only detected in salt-treated roots, but also obviously possessed the higher activity, whereas its protein amount decreased gradually during the salt treatment process. The biochemical characters of RE1 were examined afterwards. The results showed that its molecular mass was about 740 kDa and its optimal temperature was about 40°C. But the optimal pH was 5 to substrate gelatin or 7–8 to substrate casein. Its activity was obviously inhibited by 10 mM PMSF and 20 μM MG115, and RE1 was further confirmed as 20S proteasome by Western blotting. The results in this study suggested that 20S proteasome of wheat roots might be an important protease accumulated in roots in response to salt stress.     

Isolation of a Zn-binding protein mediating cell adhesion from common carp

Extraordinarily high concentrations of Zn (300-500 microg/[g fresh tissue]) are often found in the digestive tract tissue of common carp Cyprinus carpio, and most of the Zn is bound to membrane protein located on plasma membranes that are attached to basal laminae. To isolate the Zn-binding protein, the basolateral plasma membranes were separated from the extracellular matrix by treating the nuclei/cell debris fraction of the tissue with collagenase type IV and Arg-Gly-Asp (RGD) peptide. The Zn-binding protein was isolated from the separated plasma membranes by immobilized metal affinity chromatography and affinity chromatography on laminin-Sepharose. A 43 kDa protein was bound by the laminin-Sepharose and specifically eluted with tirofiban (a mimic of RGD). Affinity chromatography on wheat germ agglutinin and concanavalin A-Sepharose showed that the 43 kDa protein is a glycoprotein. The 43 kDa protein was labelled with 65Zn and became incorporated into liposomes at a high efficiency. Liposomes containing this protein were bound to laminin-Sepharose or reconstituted basement membrane. We propose that the Zn-binding protein is a cell surface receptor involved in the adhesion of cells to laminin.     

Identification,purification, and partial characterization of vitellin from the eggs of Eurygaster integriceps putton (heteroptera,pentatomidae)

A single vitellin was identified in the eggs of the wheat bug, Eurygaster integriceps, and purified by a two-step procedure including ion exchange and gel permeation chromatography. Its nondenatured molecular mass is estimated to be 385 kDa. Under reducing conditions—SDS-PAGE—the wheat bug vitellin gives five polypeptides at 110 kDa, 80 kDa, 69 kDa, 53 kDa, and 38 kDa. Its amino acid composition is characterized by high content of aspartic acid/asparagine and glutamic acid/glutamine and low content of methionine and histidine. The lipid moiety (5.65% by weight) includes diacylglycerols, cholesterol, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin. Carbohydrate content amounts to 4.54% by weight. A part of the wheat bug vitellin is dimerized during the course of deposition into the yolk granules. © 1995 Wiley-Liss, Inc.     

Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface

Biofilm formation on a polymer surface which involves initial attachment and accumulation in multilayered cell clusters (intercellular adhesion) is proposed to be the major pathogenicity factor in Staphylococcus epidermidis foreign-body-associated infections. We have characterized two distinct classes of biofilm-negative Tn917 mutants in S. epidermidis affected in initial attachment (class A) or intercellular adhesion (class B). mut1 (class A mutant) lacks five surface-associated proteins with molecular masses of 120, 60, 52, 45 and 38 kDa and could be complemented by transformation with a 16.4 kb wild-type DNA fragment. The complemented mutant was able to attach to a polystyrene surface, to form a biofilm, and produced all of the proteins missing from mut1. Subcloning experiments revealed that the 60 kDa protein is sufficient for initial attachment. Immunofluorescence microscopy using an antiserum raised against the 60 kDa protein showed that this protein is located at the cell surface. DNA-sequence analysis of the complementing region revealed a single open reading frame which consists of 4005 nucleotides and encodes a deduced protein of 1335 amino acids with a predicted molecular mass of 148 kDa. The amino acid sequence exhibits a high similarity (61% identical amino acids) to the atl gene product of Staphylococcus aureus, which represents the major autolysin; therefore the open reading frame was designated atlE. By analogy with the S. aureus autolysin, AtlE is composed of two bacteriolytically active domains, a 60 kDa amidase and a 52 kDa glucosaminidase domain, generated by proteolytic processing. The 120 kDa protein missing from mut1 presumably represents the unprocessed amidase and glucosaminidase domain after proteolytic cleavage of the signal- and propeptide. The 45 and 38 kDa proteins are probably the degradation products of the 60 and 52 kDa proteins, respectively. Additionally, AtlE was found to exhibit vitronectin-binding activity, indicating that AtlE plays a role in binding of the cells not only to a naked polystyrene surface during early stages of adherence, but also to plasma protein-coated polymer surfaces during later stages of adherence. Our findings provide evidence for a new function of an autolysin (AtlE) in mediating the attachment of bacterial cells to a polymer surface, representing the prerequisite for biofilm formation.     

Two novel monoclonal antibodies to fibronectin that recognize the Hep II and CS-1 regions respectively: their differential effect on lymphocyte adhesion.

We have obtained two new mAbs to the carboxy-terminal region of fibronectin, namely P3D4 and P1F11, and have studied their binding sites and their ability to block lymphocyte adhesion to fibronectin. ELISA and Western blot analyses showed that P3D4 reacts with both fibronectin chains and both Hep II-containing fragments (58 kDa and 38 kDa). P1F11, raised against the synthetic peptide CS-1, reacted with the 38 kDa fragment and with a 190 kDa fragment derived from the A chain of fibronectin. P1F11 did not react with the 58 kDa fragment thus clearly establishing that 58 kDa comes from the B chain of fibronectin and lacks the CS-1 sequence. mAbs P3D4 and P1F11 were used to evaluate the contribution of the Hep II and CS-1 sites in cell attachment to fibronectin. P3D4 effectively inhibited B cell adhesion to 38 kDa, 58 kDa and fibronectin; P1F11 however produced only limited inhibition, suggesting that lymphocyte interaction with Hep II may modulate further binding to the CS-1 site.     

Occurrence and characterization of a UDP-glucose:hydroxamic acid glucosyltransferase isolated from wheat (Triticum aestivum) seedlings

Cyclic hydroxamic acid glucosides are present at high concentrations immediately after germination in wheat (Triticum aestivum L.). Changes in the activity of UDP-Glucose:cyclic hydroxamic acid glucosyltransferase (EC 2.4.1.-) in wheat were investigated using the cyclic hydroxamic acids 2.4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and its 7-methoxy derivative (DIMBOA) as sugar acceptors. Glucosyltransferase activity on both substrates was detected in dry seeds, with activity increasing after imbibition, peaking in shoots and roots 36-48 hours after imbibition and decreasing thereafter. The transience of glucosyltransferase activity was concurrent with the transient occurrence of the hydroxamic acid glucosides [Nakagawa E., Amano T., Hirai N., and Iwamura H. (1995) Phytochemistry 38, 1349-1354], suggesting that glucosyltransferases regulate the accumulation of hydroxamic acid glucosides in wheat seedlings. Two peaks in activity of UDP-Glucose:DIMBOA glucosyltransferase were detected using a Mono Q column, indicating the presence of at least two isozymes of this glucosyltransferase. The enzyme in the major peak was purified about 1500-fold and shown to be in a monomeric form with a molecular mass of 47 or 49 kDa. The enzyme reacted strongly with DIMBOA, less so with DIBOA. The enzyme of the minor peak on the Mono Q chromatogram, which was also a monomeric enzyme with a molecular mass of 47 kDa, showed similar substrate specificity to that of the major peak enzyme.     

<Emphasis Type="Bold">Role of microRNAs and their target genes in salinity response in plant</Emphasis>s

We examined the effects of 2,4-epibrassinolide (EBR) application on photosynthesis, antioxidant enzyme activity, and Rubisco activase (RCA) gene expression in wheat (Triticum aestivum L.) seedlings under a combination of drought and heat stress. The net photosynthetic rates (P_n) of wheat seedlings decreased significantly, the photosynthetic capability was inhibited, and the activities of superoxide (SOD), peroxidase (POD), catalase (CAT), and RCA as well as the initial and total activity of Rubisco declined under the combined stress. These decreases and inhibitory effects were significantly ameliorated by exogenous EBR application. Three subunits (45–46, 41–42, and 38–39 kDa) of RCA were observed in wheat seedlings. The abundances of the 38–39 kDa and 41–42 kDa subunits were significantly lower in plants subjected to stressful conditions than in unstressed plants. Interestingly, a marked increase in 45–46 kDa RCA was observed under heat or heat combined with drought stress. The abundance of 38–39 kDa RCA in seedlings exposed to heat, drought, or their combination was significantly enhanced by EBR pretreatment, which paralleled the changes in initial Rubisco activity and P_n, but was not consistent with observed mRNA abundance. These results indicated that the larger subunit of RCA (45–46 kDa), which is more thermostable and increased in response to moderate heat stress, and the smaller isoform (38–39 kDa) of RCA may play important roles in maintaining the photosynthetic capability by EBR under stress conditions.     

A mycorrhiza-responsive protein in wheat roots

A small protein, designated Myk15, was found to be strongly induced in wheat ( Triticum aestivum) roots colonized by the arbuscular mycorrhizal fungus Glomus intraradices. This protein, which is most abundant in root fractions characterized by strong mycorrhizal colonization, has been characterized using two-dimensional polyacrylamide gel electrophoresis and microsequencing. It has an apparent molecular mass of 15 kDa and an isoelectric point of 4.5. The N-terminal sequence has high similarity to a peptide sequence deduced from an expressed sequence tag (EST) clone derived from Medicago truncatula roots colonized by G. intraradices. This EST clone is predicted to code for a protein with a similar size and isoelectric point as Myk15. The N-terminus of the deduced M. truncatula protein contains a highly hydrophobic stretch of 24 amino acid residues preceding the region with high similarity to the Myk15 N-terminus. This hydrophobic stretch is predicted to form a transmembrane alpha-helix and may correspond to a cleavable targeting domain.     

Presence of guanine nucleotide-binding proteins in Catharanthus roseus transformed roots

Biochemical analysis revealed the presence of GTP-binding proteins (G-proteins) in Catharanthus roseus hairy root cultures. In a microsomal fraction, several proteins, with molecular masses of 17, 21, 38, 42, 65, and 79 kDa were substrates for ADP-ribosylation by cholera toxin. Antisera raised against a conserved amino-acid sequence (GTSNSGKSTIVKQMK) of mammalian G α subunits recognized three proteins of 42, 50, and 79 kDa. Incubation of nitrocellulose blots with [ α -³²P]-GTP also indicated the presence of several proteins (17, 21, 50, and 79 kDa) that could bind GTP. In this system, we previously identified a phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC, EC 3.1.4.11) activity. As the activation of PLC by G-proteins was described, we decided to see whether, in our system, G-protein activators, such as guanosine 5- o -(3-thiotriphosphate) (GTP Γ S) and sodium fluoride ions, were able to regulate PLC activity in C. roseus transformed roots. Our results show that these agents regulated PLC activity in an inhibitory fashion and that this effect is dose-dependent. GTP was ineffective in producing either stimulation or inhibition of PLC activity. Our results demonstrate that non-hydrolyzable guanine nucleotides and fluoride ions exert an inhibitory effect on membrane PLC activity. In summary, a set of proteins of 17, 21, 38, 42, 50, and 79 kDa present in C. roseus transformed roots possessed at least two of the three main characteristics of a GTP-binding protein, and one of these proteins may be involved in the regulation of PLC activity in C. roseus transformed roots.     

HEp-2 adhesion and the expression of a 94 kDa outer-membrane protein by strains of Escherichia coli belonging to enteropathogenic serogroups

Sixty strains of Escherichia coli belonging to enteropathogenic serogroups (EPEC) were examined for the ability to adhere to HEp-2 cells, the possession of the genes encoding EPEC adherence factor (EAF) and the ability to express an outer-membrane protein (OMP) of 94 kDa thought to be involved in bacterial adhesion to eukaryotic cells. An absolute correlation was found between HEp-2 adhesion and the possession of the genes encoding EAF. An OMP of 94 kDa was observed in the SDS-PAGE profile of most adhesive strains. In some strains this protein was prone to proteolytic degradation. An antiserum raised to a HEp-2 adhesive strain of EPEC did not react with the 94 kDa OMP of all EPEC which were EAF-positive and HEp-2 adhesive, indicating some interstrain antigenic variation of this protein. Although this 94 kDa protein was surface-exposed, specific antibodies binding to the 94 kDa protein in situ in the outer membrane did not interfere with adhesion of EPEC to HEp-2 cells. Therefore, these studies question the value of this protein as a potential vaccine component.