A new class of N-hydroxycinnamoyltransferases. Purification,cloning, and expression of a barley agmatine coumaroyltransferase (EC 2.3.1.64)

Agmatine coumaroyltransferase (ACT), which catalyzes the first step in the biosynthesis of antifungal hydroxycinnamoylagmatine derivatives, was purified to apparent homogeneity from 3-day-old etiolated barley (Hordeum vulgare L.) seedlings. The enzyme was highly specific for agmatine as acyl acceptor and had the highest specificity for p-coumaroyl-CoA among various acyl donors with a specific activity of 29.7 nanokatal x mg(-1) protein. Barley ACT was found to be a single polypeptide chain of 48 kDa with a pI of 5.20 as determined by isoelectric focusing. The 15 N-terminal amino acid residues were identified by micro-sequencing of the native protein and were used to clone a full-length barley ACT cDNA that predicted a protein of 439 amino acid residues. The sequence was devoid of N-terminal signal peptide, suggesting a cytosolic localization of barley ACT. Recombinant ACT produced and affinity-purified from Escherichia coli had a specific activity of 189 nanokatal x mg(-1) protein, thus confirming the identity of the purified native protein. A partial cDNA sequence for ACT was obtained from wheat that predicted a protein of 353 amino acid residues and had 95% sequence identity to barley ACT. Two motifs in the amino acid sequence reveal that barley ACT represents a new class of N-hydroxycinnamoyltransferases belonging to the transferase superfamily. The barley ACT is unique in producing the precursor of hordatine, a proven antifungal factor that may be directed toward Blumeria graminis.     

Monoclonal antibodies to barley aleurain and homologs from other plants

Barley aleurain is contained within a specific type of vacuole characterized by acidic pH and the presence of other hydrolytic enzymes. The aleurain-containing vacuole is distinct from protein storage vacuoles, and anti-aleurain antibodies serve as markers for this organelle in barley cells. Aleurain is a unique type of cysteine protease, and other plant species have genes for homologs whose sequences are highly conserved, but little is known about these homologs at the protein level. Seven monoclonal antibodies to barley aleurain were isolated, which bind to and define aleurain homologues in Arabidopsis, Petunia , and tobacco cell extracts. Interestingly, in addition to 29–32 kDa aleurain homologs, Petunia extracts contain a protein of ∼50 kDa and tobacco extracts a protein of ∼40 kDa that are recognized by multiple different mono-clonal antibodies, indicating an unexpected diversity to the aleurain protein family. Among the group of antibodies are some that efficiently immunoprecipitate metabolically labeled aleurain from barley cell extracts, and some that efficiently label aleurain in immunofluorescence assays using root tip cells. These antibodies should be useful for plant cell biologists who study vacuole biogenesis and function and sorting of proteins to specific vacuolar compartments, in barley as well as other plant species.     

Common occurrence of homologues of petunia glycine-rich protein-1 among plants

The presence of specific glycine-rich proteins (GRP) related to petunia GRP1 (ptGRP1) was examined in three species of monocots (wheat, barley and maize) and five species of dicots (rape, turnip, soybean, crabapple and tomato). Protein blot analysis showed that anti-ptGRP1 antibody cross-reacted with a single different polypeptide in all species except maize. The molecular mass of these polypeptides ranged from 14 to 55 kDa. Tissue-print immunoblots of rape petioles and stems showed that the rape ptGRP1 homologue, like ptGRP1, is primarily located in the vascular tissue, and that its expression decreases with developmental age of the tissue. In barley, the ptGRP1 homologue is found in leaf vascular bundles, and may also be present in the surrounding bundle sheaths. Unlike the dicots examined, expression of the protein did not appear to decrease significantly with developmental age.     

Zinc stress induces changes in apoplasmic protein content and polypeptide composition of barley primary leaves

Excessive zinc in the rooting medium, and consequently increasingzinc content in the shoot of barley (Hordeum vulgare) seedlings,strongly affected the protein content and the polypeptide compositionof the leaf apoplasm. In contrast, only small changes In polypeptide composition (in the IEF range of pH 4–8 and between14 and 80 kDa) were detected In mesophyll and epidermis protoplasts.The zinc stress-induced changes In apoplasmic protein contentwere analysed in detail. The apoplasmic protein content Increasedby more than 3-fold when the zinc concentration in the mediumwas increased from 0.02 to about 200 µmol l^–1; athigher zinc concentrations no further changes occurred. Theincrease in apoplasmic protein content was due both to a generalincrease in abundance of the most dominant apoplasmic polypeptidesand to a pronounced increase in the abundance of specific polypeptidesas monitored in electrophoretic separations or by measuringthe activity of apoplasmic enzymes. The largest induction wasseen for four apoplasmic polypeptides of molecular masses 16,23, 27, and 28 kDa with as yet unknown function. Key words: Apoplasm, barley, leaf, peroxidases, potypeptide, zinc stress     

Sucrose-phosphate synthase from wheat. Characterization of peptides by immunoblotting analysis

The structure of sucrose-phosphate synthase (SPS: EC 2.4.1.14) from wheat ( Triticum aestivum L. cv. San Agustin was studied using antibodies prepared against the enzyme purified from wheat germ. The antibodies revealed the presence of 55 and 35 kDa polypeptides in wheat germ, endosperm, embryos and whole seed, while in whole wheat leaf, a 90 kDa was detected. It is not clear whether the 35 and 55 kDa polypeptide are truly subunits of SPS or they are the product of protease action, more active in non-photosynthetic tissues than in leaves. The antibodies from wheat germ clearly recognized polypeptides in leaf protein preparations from other plants (barley, soybean, maize) and, weakly in others (peanut, tobacco). It did not recognize any polypeptide in spinach and mustard leaf extracts. In the case of maize leaf, a peptide of higher molecular mass (116 kDa) than the wheat ones was revealed. The results may indicate the presence of different polypeptide compositions for sucrose-phosphate synthase, and suggest the existence of at least two types of this enzyme.     

大麦6H染色体特异性标记的筛选和鉴定

从大麦、小麦和小麦－大麦６Ｈ染色体附加系ＲＡＰＤ分析筛选出对６Ｈ染色体特异的２个ＲＡＰＤ标记,转换为特异性ＰＣＲ标记,利用标记对不同植物材料进行ＰＣＲ扩增鉴定。表明凡含有大麦６Ｈ染色体的材料（Ｂｅｔｚｅｓ、Ｉｇｒｉ、ＣＳ６Ｈ附加系）均能扩增出特异带;而不含６Ｈ染色体的材料,包括小科、黑麦、长穗偃麦草、中间偃麦草、簇毛麦以及含有其他大麦染色体的小麦附加系均不主增出特异带。可见,２对ＰＣＲ引物具有大麦     

The barley scutellar peptide transporter: biochemical characterization and localization to the plasma membrane

Thiol-affinity labelling was used to identify and characterize components of the peptide transport system in the barley (Hordeum vulgare) scutellar epithelium. SDS-PAGE and 2D-PAGE in conjunction with fluorography were used to study derivatized proteins. Membrane proteins of 42 kDa and 66 kDa were identified using a strategy devised to label substrate protectable protein with the thiol specific reagent [14C] N-ethylmaleimide (NEM). The scutellar plasma membrane is the anticipated site of transporters involved in the mobilization of endosperm storage reserves in the germinating barley grain. The subcellular localization of these proteins to the plasma membrane was demonstrated by thiol-affinity labelling of high purity plasma membrane vesicles isolated from barley scutellar tissue. A peptide transporter, HvPTR1, specific to the barley scutellum has recently been cloned in this laboratory. A 66 kDa protein, comparable to the predicted molecular mass of HvPTR1, was identified by [14C]NEM labelling studies of Xenopus laevis oocytes expressing HvPTR1 cRNA, but not water injected controls. Peptide antiserum raised to HvPTR1 also cross-reacted with a 66 kDa membrane protein in barley scutellar tissue. This confirms that the 66 kDa protein identified here by thiol-affinity labelling studies is the barley scutellum peptide transporter HvPTR1, and demonstrates that this protein is localized to the plasma membrane of scutellar epithelial cells during germination.     

A 33 kDa molecular marker of sperm acrosome differentiation and maturation in the tammar wallaby (Macropus eugenii)

This study was undertaken to identify potential molecular markers of acrosomal biogenesis and post-testicular maturation in marsupials, using the tammar wallaby as a model species. A two-step sperm extraction procedure yielded two protein extracts of apparent acrosomal origin and a tail extract. The extracts were analysed by SDS-PAGE under reducing conditions. Several prominent polypeptide bands (45, 38 and 33 kDa) appeared common to both acrosomal extracts. Antiserum raised against the 33 kDa polypeptide from the inner acrosomal membrane matrix (IAMM) extract showed immunoreactivity with 45, 38 and 33 kDa polypeptides in both acrosomal extracts, indicating that the 33 kDa polypeptide was related to the proteins in the 45 and 38 kDa bands. Therefore, the antiserum was used as a molecular probe. Indirect immuno-fluorescence indicated that the acrosome was the major location of the 33 kDa polypeptide. This contention was confirmed by ultrastructural study: immunogold labelling indicated that the 33 kDa polypeptide associated with acrosomal matrix components throughout acrosomal development in the testes and throughout post-testicular maturation in the epididymis. The label clearly delineated the changing morphology of the maturing marsupial acrosome. This is the first study to use immunocytochemical techniques to chart testicular and post-testicular development of any sperm organelle in a marsupial. As a result of this study, a 33 kDa molecular marker of marsupial acrosome differentiation and maturation has been identified. It may be possible to chart similar events in other marsupial species and identify opportunities for manipulating fertility.     

Biochemical characterization of Thermotoga maritima endoglucanase Cel74 with and without a carbohydrate binding module (CBM)

The genome of the hyperthermophilic bacterium Thermotoga maritima (Tm) encodes at least eight glycoside hydrolases with putative signal peptides; the biochemical characteristics of seven of these have been reported previously. The eighth, Tm Cel74, is encoded by an open reading frame of 2124 bp corresponding to a polypeptide of 79 kDa with a signal peptide at the amino-terminus. The gene (lacking the signal peptide) encoding Tm Cel74 was expressed as a 77 kDa monomeric polypeptide in Escherichia coli and found to be optimally active at pH 6, 90 degrees C, with a melting temperature of approximately 105 degrees C. The cel74 gene was previously found to be induced during T. maritima growth on a variety of polysaccharides, including barley glucan, carboxymethyl cellulose (CMC), glucomannan, galactomannan and starch. However, while Tm Cel74 was most active towards barley glucan and to a lesser extent CMC, glucomannan and tamarind (xyloglucan), no activity was detected on other glycans, including galactomannan, laminarin and starch. Also, Tm Cel74 did not contain a carbohydrate binding module (CBM), versions of which have been identified in the amino acid sequences of other family 74 enzymes. As such, a CBM associated with a chitinase in another hyperthermophile, Pyrococcus furiosus, was used to create a fusion protein that was active on crystalline cellulose; Tm Cel74 lacked activity on this substrate. Based on the cleavage pattern determined for Tm Cel74 on glucan-based substrates, this enzyme likely initiates recruitment of carbohydrate carbon and energy sources by creating oligosaccharides that are transported into the cell for further processing.     

Towards Positional Isolation of Three Quantitative Trait Loci Conferring Resistance to Powdery Mildew in Two Spanish Barley Landraces

Three quantitative trait loci (QTL) conferring broad spectrum resistance to powdery mildew, caused by the fungus Blumeria graminis f. sp. hordei, were previously identified on chromosomes 7HS, 7HL and 6HL in the Spanish barley landrace-derived lines SBCC097 and SBCC145. In the present work, a genome-wide putative linear gene index of barley (Genome Zipper) and the first draft of the physical, genetic and functional sequence of the barley genome were used to go one step further in the shortening and explicit demarcation on the barley genome of these regions conferring resistance to powdery mildew as well as in the identification of candidate genes. First, a comparative analysis of the target regions to the barley Genome Zippers of chromosomes 7H and 6H allowed the development of 25 new gene-based molecular markers, which slightly better delimit the QTL intervals. These new markers provided the framework for anchoring of genetic and physical maps, figuring out the outline of the barley genome at the target regions in SBCC097 and SBCC145. The outermost flanking markers of QTLs on 7HS, 7HL and 6HL defined a physical area of 4 Mb, 3.7 Mb and 3.2 Mb, respectively. In total, 21, 10 and 16 genes on 7HS, 7HL and 6HL, respectively, could be interpreted as potential candidates to explain the resistance to powdery mildew, as they encode proteins of related functions with respect to the known pathogen defense-related processes. The majority of these were annotated as belonging to the NBS-LRR class or protein kinase family.     

Redox changes accompanying storage protein mobilization in moist chilled and warm incubated walnut kernels prior to germination

Alterations in the redox state of storage proteins and the associated proteolytic processes were investigated in moist-chilled and warm-incubated walnut (Juglans regia L.) kernels prior to germination. The kernel total protein labeling with a thiol-specific fluorochrome i.e. monobromobimane (mBBr) revealed more reduction of 29–32 kDa putative glutelins, while in the soluble proteins, both putative glutelins and 41, 55 and 58 kDa globulins contained reduced disulfide bonds during mobilization. Thus, the in vivo more reduced disulfide bonds of storage proteins corresponds to greater solubility. After the in vitro reduction of walnut kernel proteins pre-treated by N-ethyl maleimide (NEM) with dithioerythrethiol (DTT) and bacterial thioredoxin, the 58 kDa putative globulin and a 6 kDa putative albumin were identified as disulfide proteins. Thioredoxin stimulated the reduction of the H₂O₂-oxidized 6 kDa polypeptide, but not the 58 kDa polypeptide by DTT. The solubility of 6 kDa putative albumin, 58 and 19–24 kDa putative globulins and glutelins, respectively, were increased by DTT. The in vitro specific mobilization of the 58 kDa polypeptide that occurred at pH 5.0 by the kernel endogenous protease was sensitive to the serine-protease inhibitor phenylmethylsulfonyl fluoride (PMSF) and stimulated by DTT. The specific degradation of the 58 kDa polypeptide might be achieved through thioredoxin-mediated activation of a serine protease and/or reductive unfolding of its 58 kDa polypeptide substrate. As redox changes in storage proteins occurred equally in both moist chilled and warm incubated walnut kernels, the regulatory functions of thioredoxins in promoting seed germination may be due to other germination related processes.     

Mapping quantitative trait loci associated with barley net blotch resistance

Net blotch of barley, caused by Pyrenophora teres Drechs., is an important foliar disease worldwide. Deployment of resistant cultivars is the most economic and eco-friendly control method. This report describes mapping of quantitative trait loci (QTL) associated with net blotch resistance in a doubled-haploid (DH) barley population using diversity arrays technology (DArT) markers. One hundred and fifty DH lines from the cross CDC Dolly (susceptible)/TR251 (resistant) were screened as seedlings in controlled environments with net-form net blotch (NFNB) isolates WRS858 and WRS1607 and spot-form net blotch (SFNB) isolate WRS857. The population was also screened at the adult-plant stage for NFNB resistance in the field in 2005 and 2006. A high-density genetic linkage map of 90 DH lines was constructed using 457 DArT and 11 SSR markers. A major NFNB seedling resistance QTL, designated QRpt6, was mapped to chromosome 6H for isolates WRS858 and WRS1607. QRpt6 was associated with adult-plant resistance in the 2005 and 2006 field trials. Additional QTL for NFNB seedling resistance to the more virulent isolate WRS858 were identified on chromosomes 2H, 4H, and 5H. A seedling resistance QTL (QRpts4) for the SFNB isolate WRS857 was detected on chromosome 4H as was a significant QTL (QRpt7) on chromosome 7H. Three QTL (QRpt6, QRpts4, QRpt7) were associated with resistance to both net blotch forms and lines with one or more of these demonstrated improved resistance. Simple sequence repeat (SSR) markers tightly linked to QRpt6 and QRpts4 were identified and validated in an unrelated barley population. The major 6H QTL, QRpt6, may provide adequate NFNB field resistance in western Canada and could be routinely selected for using molecular markers in a practical breeding program.     

Evidence for different kinases in thylakoid protein phosphorylation.

Thylakoid protein phosphorylation was facilitated in darkness by using the ferredoxin-NADPH system. CoCl2 and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) were potent inhibitors of LHCP (light-harvesting chlorophyll-binding protein) phosphorylation, but 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea and atrazine had no significant effect. Differential effects on phosphorylation of the 9 kDa polypeptide and LHCP were observed in darkness with DBMIB and certain other inhibitors specific for Photosystem-II electron transport. Similarly, during illumination of intact chloroplasts or of the reconstituted chloroplast system, a differential action of bicarbonate was observed on the relative phosphorylation of the two proteins. The degree of phosphorylation of the 9 kDa polypeptide was increased in the presence of bicarbonate compared with its absence, whereas that of LHCP was relatively unchanged. Changes in the degree of phosphorylation of the 32 kDa polypeptide in these experiments did not correlate consistently with changes in phosphorylation of either LHCP or the 9 kDa polypeptide, although changes in the 32 kDa polypeptide more often paralleled phosphorylation of the 9 kDa polypeptide rather than the phosphorylation of LHCP. These observations suggest that the protein kinase that phosphorylates LHCP is distinct from that which phosphorylates the 9 kDa polypeptide.     

Identification of an endothelial cell surface protein that binds plasminogen

To identify and characterize endothelial cell surface components that bind plasminogen, we used ligand-blotting to study binding of plasminogen to sodium dodecyl sulphate solubilized extracts of human umbilical vein endothelial cells. It was observed that glu-plasminogen bound predominantly to a 45 kDa endothelial cell polypeptide. The interaction of labelled glu-plasminogen with this polypeptide was reversible and specific as the binding could be inhibited by both excess cold lysine and unlabelled glu-plasminogen but not by unrelated proteins. Binding of glu-plasminogen to cell extracts prepared from endothelial cells that had been pretreated with proteinase K was significantly reduced indicating that the 45 kDa polypeptide is a cell-surface protein. The cell-surface localization of the 45 kDa polypeptide was also indicated by the positive interaction of glu-plasminogen with membrane fractions of endothelial cells. Lys-plasminogen also interacted with the 45 kDa polypeptide in a specific manner and reversibility experiments indicated that lysplasminogen could also displace the bound glu-plasminogen. Since binding of plasminogen to the 45 kDa endothelial cell surface polypeptide was very similar to plasminogen binding to intact endothelial cells, we propose that the 45 kDa protein represents one of the major receptors for plasminogen on human endothelial cells.     

A cDNA clone from barley encoding the precursor from the photosystem I polypeptide PSI-G: Sequence similarity to PSI-K

A cDNA clone encoding the photosystem I subunit, PSI-G was isolated from barley using an oligonucleotide specifying a partial amino acid sequence from a 9 kDa polypeptide of barley photosystem I. The 724 bp sequence contains an open reading frame encoding a precursor polypeptide of 15 107 kDa. Import studies using the in vitro expressed barley PsaG cDNA clone demonstrate that PSI-G migrates with an apparent molecular mass of 9 kDa on SDS-polyacrylamide gels together with PSI-C (subunit-VII). The previous assignment of the gene product of PsaG from spinach as subunit V (Steppuhn J, Hermans J, Nechushtai R, Ljungberg U, Thümmler F, Lottspeich F, Herrmann RG, FEBS Lett 237: 218–224, 1988) needs to be re-examined. The expression of the psaG gene is light-induced similar to other barley photosystem I genes. A significant sequence similarity to PSI-K from Chlamydomonas reinhardtii was discovered when a gene database was searched with the barley PSI-G amino acid sequence. Extensive sequence similarity between the nuclear-encoded photosystem I subunits has not previously been found. The observed sequence similarity between PSI-G and PSI-K suggests a symmetric location of these subunits in the photosystem I complex. The hydropathy plot of the barley PSI-G polypeptide indicates two membrane-spanning regions which are also found at the corresponding locations in the PSI-K polypeptide. PSI-G and PSI-K probably have evolved from a gene duplication of an ancestral gene.     

The multidrug resistance protein is photoaffinity labeled by a quinoline-based drug at multiple sites

Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRP has been shown to mediate the transport of cytotoxic natural products, in addition to glutathione-, glucuronidate-, and sulfate-conjugated cell metabolites. However, the mechanism of MRP drug binding and transport is at present not clear. In this study, we have used a photoreactive quinoline-based drug, N-(hydrocinchonidin-8'-yl)-4-azido-2-hydroxybenzamide (IACI), to show the photoaffinity labeling of the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labeling of the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled protein as the MRP was confirmed by immunoprecipitation with the monoclonal antibody QCRL-1. Furthermore, a molar excess of leukotriene C(4), doxorubicin, colchicine, and other quinoline-based drugs, including MK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACI accumulation in MRP-expressing cells which was reversed by depleting ATP levels in H69/AR cells. Mild digestion of the purified IACI-photolabeled MRP with trypsin showed two large polypeptides ( approximately 111 and approximately 85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody epitopes corresponds to the C-terminal half of the MRP (amino acids approximately 900-1531) containing the third multiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptide which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the MRP sequence (amino acids 1-900) containing the MSD1 and MSD2 plus the first nucleotide binding domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and approximately 6 plus 4 kDa photolabeled peptides, respectively. In addition, resolution of the exhaustively digested IACI-photolabeled MRP by HPLC showed two major and one minor radiolabeled peaks that eluted late in the gradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI to the MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localize to the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studying MRP-drug interactions.     

Functional analysis of a human A(1) adenosine receptor/green fluorescent protein/G(i1)alpha fusion protein following stable expression in CHO cells

The nuclear lamina protein, lamin A is produced by proteolytic cleavage of a 74 kDa precursor protein, prelamin A. The conversion of this precursor to mature lamin A is mediated by a specific endoprotease, prelamin A endoprotease. Subnuclear fractionation indicates that the prelamin A endoprotease is localized at the nuclear membrane. The enzyme appears to be an integral membrane protein, as it can only be removed from the nuclear envelope with detergent. It is effectively solubilized by the detergent n-octyl-beta-D-glucopyranoside and can be partially-purified (approximately 1200-fold) by size exclusion and cation exchange (Mono S) chromatography. Prelamin A endoprotease from HeLa cells was eluted from Mono S with 0.3 M sodium chloride as a single peak of activity. SDS-PAGE analysis of this prelamin A endoprotease preparation shows that it contains one major polypeptide at 65 kDa and smaller amounts of a second 68 kDa polypeptide. Inhibition of the enzyme activity in this preparation by specific serine protease inhibitors is consistent with the enzyme being a serine protease.     

Identification of the Product of ndhA Gene as a Thylakoid Protein Synthesized in Response to Photooxidative Treatment

A 76 amino acid sequence of NDH-A (the protein encoded by plastidndhA gene) from barley (Hordeum vulgare L.) was expressed asa fusion protein with rß-galactosidase in E. coli.The corresponding antibody generated in rabbits was used toinvestigate localization, expression and synthesis in vitroof NDH-A. NDH-A was identified as a 35 kDa polypeptide localizedin thylakoid membrane. Western blots shows a large increasein NDH-A levels when barley leaves were incubated under photooxidativeconditions, which was more pronounced in mature-senescent leavesthan in young leaves. Immunoprecipitation of the [³⁵S]methioninelabelled proteins, synthesized in vitro by isolated chloroplasts,demonstrated the synthesis in chloroplasts of the NDH-A 35 kDapolypeptide when barley leaves had been incubated under photooxidativeconditions. The results indicate that ndh genes may be involvedin the protection of chloroplasts against photooxidative stress,particularly in mature-senescent leaves. (Received November 13, 1995; Accepted February 5, 1996)