Search

ZnO nanowalls were grown in situ on the surface of porous reduced graphene oxide (PG) films using spray, thermal reduction and facile solution method in this work. The products were characterized by powder X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), Raman spectroscopy. The results showed that the highly developed interconnected 3-D ZnO nanowall networks were anchored homogeneously on the surface of PG films to construct 3D ZnO/PG hybrid nanocomposites. This 3D hybrid nanostructure provided many channels for gas diffusion. The fabricated sensor based on ZnO/PG composites showed good photo sensing response (7.4) to 365 nm UV light and an enhanced gas sensitivity (35.31) to 50 ppm NO2 with irradiation of UV light of 1.2 mW/cm2 in the air at room temperature, which was 2.24 fold higher than that of pure ZnO, and the response-recovery times were (∼37s, and 2s) when exposed to 50 ppm NO2. The optoelectronic gas sensing mechanisms of ZnO/PG composites were proposed in detail to understand the effect of UV irradiation in the NO2 detection process.

Three kinds of poly (3-alkylthiophene), such as poly (3-butylthiophene) (P3BT), poly (3-hexylthiophene) (P3HT) and poly (3-dodecylthiophene) (P3DDT) were prepared by the Grignard Metathesis Method (GRIM). Their chemical structures are characterized by FTIR,1H-NMR and GPC.1H-NMR result shows that regioregular structure of these polymers is relatively higher. The GPC results show that the molecular weight of the polymers is 8.42×104, 1.06×105and 1.21×104g/mol with molecular weight distributions of 1.41, 2.18 and 1.18, respectively. The DSC result shows that the glass transition temperature of three kinds of polymers is 120.8, 146, 118 °C, respectively. UV absorption spectrum and fluorescence spectroscopy of three polymers in THF solution show that the maximum absorption wavelength is 448 nm, 470 nm, 390 nm, and the maximum emission wavelength is 531, 535, 527 nm with the band gap of 2.06, 1.86, 2.21 eV, respectively.

In order to study the infrared spectral characteristics of side-chain liquid crystal polymer, an infrared monomer containing a permanent dipole azobenzene group spaced from the methacryloyl moiety by a polymethylene segment was synthesized in three steps starting from 4-cyanoaniline. The monomer was polymerized with a push-pull azobenzene monomer, (-)-menthy1 methacrylate in the presence of azobisisobutyronitrile (AIBN) as the radical initiator. Structurally similar amphiphilic polymers were obtained by polymerization of analogous methacrylates in which the polymethylene spacers was replaced by oligo (oxyethy1ene) segments. The obtained polymer was characterized by Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible spectro photometry (UV-vis), Hot Stage Polarized Microscopy (HS-POM), Differential Scanning Calorimetry (DSC) and Thermo Gravimetric Analyzer (TGA). Its infrared emissivity at different temperatures was tested by Bruker-V70 infrared analyzer. The results showed that the azobenzene poly (methyl methacrylate) (azo-PMMA) has high glass transition temperature(about 40°C), good thermal stability(divied in 294°C) and low infrared emission under 70%.

In present study, ANSYS finite element method was utilized to analyze the modal of simply supported beam and then the study on damage identification was performed based on modal strain energy method. The comparative analysis of results obtained by modal strain energy changes rate method and modal strain energy index demonstrates that modal strain energy index is an effective damage identification parameter.

Earthquake damages to the tall and towering structures are usually very serious, but these dangers are often hidden. The paper uses the finite element software ANSYS to set up the overall modeling of the TC6010 tower crane which is produced by a company, and carry on the modal analysis based on it. The paper obtains the data of the displacement and the speed response of the complete machine after exerting the Ninghe Tianjin seismic wave, and has the preliminary analysis to the result. The results show that the tower crane is a low-frequency vibrating system and easy to cause the structure resonance, the crane tower may damage in the boom firstly under the action of seismic wave.

Ion Beam Figure (IBF) is believed to be one of the most effective technics that can fabricate lens with nano or even sub-nano accuracy. For different sizes of IBF removal functions, the correct effects in different spatial frequency range are different. Power Spectral Density (PSD) curve can describe surface errors in full spatial frequency range, so it is a very convenient way to evaluate the quality of lens' surface. In this paper, firstly, the principles of IBF and PSD are introduced briefly; Secondly, IBF removal functions with sizes from 2 mm to 15 mm are generated. A lens with surface error more than PV value 400nm is simulated with different sizes of IBF removal functions by Lucy-Richardson algorithm. Finally, experiments are done by IBF plant. A lens is fabricated by different sizes of removal functions and the fabricate results are tested by interferometer precisely and calculated to PSD curves. By the comparison of these curves, the IBF fabricate effects with different removal sizes are analyzed, which show that the smaller the removal size, the better the removal effect in higher spatial frequency range, but in the meantime, it will take a much longer time. Also the reasons of the difference between theory simulation and actual fabrication result are taken into account, and several influence factors are analyzed. (2013) Trans Tech Publications, Switzerland.

With high-rise construction increasing in city, it demands more and higher-performance tower cranes. Damages by wind to the tall and towering structures are usually very serious, but these dangers are often hidden.The paper uses the finite element software ANSYS to set up the overall modeling of the TC6010 tower crane which is produced by a company, and carry on the FEM analysis effected by wind based on it . The paper obtains the data of the displacement response of the complete machine after exerting the wind wave, and has the preliminary analysis to the result. The results show that the tower crane is a low-frequency vibrating system and easy to cause the structure resonance, the crane tower may damage in the boom firstly under the action of wind load.

Gene families often show degrees of differences in terms of exon–intron structures depending on their distinct evolutionary histories. Comparative analysis of gene structures is important for understanding their evolutionary and functional relationships within plant species. Here, we present a comparative genomics database named PIECE (http://wheat.pw.usda.gov/piece) for Plant Intron and Exon Comparison and Evolution studies. The database contains all the annotated genes extracted from 25 sequenced plant genomes. These genes were classified based on Pfam motifs. Phylogenetic trees were pre-constructed for each gene category. PIECE provides a user-friendly interface for different types of searches and a graphical viewer for displaying a gene structure pattern diagram linked to the resulting bootstrapped dendrogram for each gene family. The gene structure evolution of orthologous gene groups was determined using the GLOOME, Exalign and GECA software programs that can be accessed within the database. PIECE also provides a web server version of the software, GSDraw, for drawing schematic diagrams of gene structures. PIECE is a powerful tool for comparing gene sequences and provides valuable insights into the evolution of gene structure in plant genomes.

Rapid evolution of powdery mildew resistance gene MlIW170 orthologous genomic regions in wheat subgenomes. Wheat is one of the most important staple grain crops in the world and also an excellent model for plant ploidy evolution research with different ploidy levels from diploid to hexaploid. Powdery mildew disease caused by Blumeria graminis f.sp. tritici can result in significant loss in both grain yield and quality in wheat. In this study, the wheat powdery mildew resistance gene MlIW170 locus located at the Triticum dicoccoides chromosome 2B short arm was further characterized by constructing and sequencing a BAC-based physical map contig covering a 0.3 cM genetic distance region (880 kb) and developing additional markers to delineate the resistance gene within a 0.16 cM genetic interval (372 kb). Comparative analyses of the T. dicoccoides 2BS region with the orthologous Aegilops tauschii 2DS region showed great gene colinearity, including the structure organization of both types of RGA1/2-like and RPS2-like resistance genes. Comparative analyses with the orthologous regions from Brachypodium and rice genomes revealed considerable dynamic evolutionary changes that have re-shaped this MlIW170 region in the wheat genome, resulting in a high number of non-syntenic genes including resistance-related genes. This result might reflect the rapid evolution in R-gene regions. Phylogenetic analysis on these resistance-related gene sequences indicated the duplication of these genes in the MlIW170 region, occurred before the separation of the wheat B and D genomes.

Two subtractive cDNA libraries were developed to study genes associated with bud dormancy (reverse library) and initiation of shoot growth (forward library) in leafy spurge. To identify unique sequences represented in each library, 15744 clones were screened to reduce the level of redundancy within both libraries. A total of 516 unique sequences were obtained from 2304 minimally redundant clones. Radioactive probes developed from RNAs extracted from crown buds of either intact (para-dormant control) or a series of growth-induced (2 h, 2, and 4 d after decapitation) plants were used to identify differentially expressed genes by macroarray analysis. Semi-quantitative RT-PCR was used to confirm results obtained by macroarray analysis and to determine the expression profiles for other transcripts identified within the subtractive libraries. Selected clones were also used to examine gene expression in crown buds after growth induction and/or during normal seasonal growth. In this study, four distinct patterns of gene expression were observed during the transition from para-dormancy to growth-induction. Many of the differentially regulated genes identified have unknown or hypothetical functions while others are known to play important roles in molecular functions. Gene ontology analysis identified a greater proportion of genes involved with catalytic activity in the forward library while the reverse library had a greater proportion of genes involved in DNA/RNA binding.

Systems biology analysis of protein modules is important for understanding the functional relationships between proteins in the interactome. Here, we present a comprehensive database named AIM for Arabidopsis (Arabidopsis thaliana) interactome modules. The database contains almost 250 000 modules that were generated using multiple analysis methods and integration of microarray expression data. All the modules in AIM are well annotated using multiple gene function knowledge databases. AIM provides a user-friendly interface for different types of searches and offers a powerful graphical viewer for displaying module networks linked to the enrichment annotation terms. Both interactive Venn diagram and power graph viewer are integrated into the database for easy comparison of modules. In addition, predicted interologs from other plant species (homologous proteins from different species that share a conserved interaction module) are available for each Arabidopsis module. AIM is a powerful systems biology platform for obtaining valuable insights into the function of proteins in Arabidopsis and other plants using the modules of the Arabidopsis interactome. Database URL: http://probes.pw.usda.gov/AIM

The Pti4, Pti5, and Pti6 proteins from tomato were identified based on their interaction with the product of the Pto disease resistance gene, a Ser-Thr protein kinase. They belong to the ethylene-response factor (ERF) family of plantunique transcription factors and bind specifically to the GCC-box cis element present in the promoters of many pathogenesis-related ( PR ) genes. Here, we show that these tomato ERFs are localized to the nucleus and function in vivo as transcription activators that regulate the expression of GCC box‐containing PR genes. Expression of Pti4 , Pti5 , or Pti 6 in Arabidopsis activated the expression of the salicylic acid‐regulated genes PR1 and PR2 . Expression of jasmonic acid‐ and ethylene-regulated genes, such as PR3 , PR4 , PDF1.2 , and Thi2.1 , was affected differently by each of the three tomato ERFs, with Arabidopsis -Pti4 plants having very high levels of PDF1.2 transcripts. Exogenous application of salicylic acid to Arabidopsis- Pti4 plants suppressed the increased expression of PDF1.2 but further stimulated PR1 expression. Arabidopsis plants expressing Pti4 displayed increased resistance to the fungal pathogen Erysiphe orontii and increased tolerance to the bacterial pathogen Pseudomonas syringae pv tomato . These results indicate that Pti4, Pti5, and Pti6 activate the expression of a wide array of PR genes and play important and distinct roles in plant defense.

The importance of two putative Zn2+-binding (Asp347, Glu429) and two catalytic (Arg431, Lys354) residues in the tomato leucine aminopeptidase (LAP-A) function was tested. The impact of substitutions at these positions, corresponding to the bovine LAP residues Asp255, Glu334, Arg336, and Lys262, was evaluated in His6–LAP-A fusion proteins expressed in Escherichia coli. Sixty-five percent of the mutant His6–LAP-A proteins were unstable or had complete or partial defects in hexamer assembly or stability. The activity of hexameric His6–LAP-As on Xaa-Leu and Leu-Xaa dipeptides was tested. Most substitutions of Lys354 (a catalytic residue) resulted in His6–LAP-As that cleaved dipeptides at slower rates. The Glu429 mutants (a Zn2+-binding residue) had more diverse phenotypes. Some mutations abolished activity and others retained partial or complete activity. The E429D His6–LAP-A enzyme had Km and kcat values similar to the wild-type His6–LAP-A. One catalytic (Arg431) and one Zn-binding (Asp347) residue were essential for His6–LAP-A activity, as most R431 and D347 mutant His6–LAP-As did not hydrolyze dipeptides. The R431K His6–LAP-A that retained the positive charge had partial activity as reflected in the 4.8-fold decrease in kcat. Surprisingly, while the D347E mutant (that retained a negative charge at position 347) was inactive, the D347R mutant that introduced a positive charge retained partial activity. A model to explain these data is proposed.

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is one of the most important wheat diseases in the world. In this study, a single dominant powdery mildew resistance gene MlIW172 was identified in the IW172 wild emmer accession and mapped to the distal region of chromosome arm 7AL (bin7AL-16-0.86-0.90) via molecular marker analysis. MlIW172 was closely linked with the RFLP probe Xpsr680-derived STS marker Xmag2185 and the EST markers BE405531 and BE637476. This suggested that MlIW172 might be allelic to the Pm1 locus or a new locus closely linked to Pm1. By screening genomic BAC library of durum wheat cv. Langdon and 7AL-specific BAC library of hexaploid wheat cv. Chinese Spring, and after analyzing genome scaffolds of Triticum urartu containing the marker sequences, additional markers were developed to construct a fine genetic linkage map on the MlIW172 locus region and to delineate the resistance gene within a 0.48 cM interval. Comparative genetics analyses using ESTs and RFLP probe sequences flanking the MlIW172 region against other grass species revealed a general co-linearity in this region with the orthologous genomic regions of rice chromosome 6, Brachypodium chromosome 1, and sorghum chromosome 10. However, orthologous resistance gene-like RGA sequences were only present in wheat and Brachypodium. The BAC contigs and sequence scaffolds that we have developed provide a framework for the physical mapping and map-based cloning of MlIW172.

Wounding of tomato leaves results in the accumulation of an exoprotease called leucine aminopeptidase (LAP-A). While the expression of LapA genes are well characterized, the specificity of the LAP-A enzyme has not been studied. The LAP-A preprotein and mature polypeptide were overexpressed in Escherichia coli. PreLAP-A was not processed and was inactive accumulating in inclusion bodies. In contrast, 55-kDa mature LAP-A subunits assembled into an active, 357-kDa enzyme in E. coli. LAP-A from E. coli cultures was purified to apparent homogeneity and characterized relative to its animal (porcine LAP) and prokaryotic (E. coli PepA) homologues. Similar to the porcine and E. coli enzymes, the tomato LAP-A had high temperature and pH optima. Mn2+ was a strong activator for all three enzymes, while chelators, zinc ion, and the slow-binding aminopeptidase inhibitors (amastatin and bestatin) strongly inhibited activities of all three LAPs. The substrate specificities of porcine, E. coli and tomato LAPs were determined using amino-acid-p-nitroanilide and -beta-naphthylamide substrates. The tomato LAP-A preferentially hydrolyzed substrates with N-terminal Leu, Met and Arg residues. LAP-A had substantially lower levels of activity on other chromogenic substrates. Several differences in substrate specificities for the animal, plant and prokaryotic enzymes were noted.

LapARNAs, proteins, and activities increased in response to systemin, methyl jasmonate, abscisic acid (ABA), ethylene, water deficit, and salinity in tomato (Lycopersicon esculentum). Salicylic acid inhibited wound-induced increases of LapA RNAs. Experiments using the ABA-deficient flacca mutant indicated that ABA was essential for wound and systemin induction ofLapA, and ABA and systemin acted synergistically to induce LapA gene expression. In contrast,pin2 (proteinase inhibitor 2) was not dependent on exogenous ABA. Whereas both LapA and le4(L. esculentumdehydrin) were up-regulated by increases in ABA, salinity, and water deficit, only LapAwas regulated by octadecanoid pathway signals. Comparison ofLapA expression with that of thePR-1 (pathogenesis-related 1) andGluB (basic β-1,3-glucanase) genes indicated that these PR protein genes were modulated by a systemin-independent jasmonic acid-signaling pathway. These studies showed that at least four signaling pathways were utilized during tomato wound and defense responses. Analysis of the expression of aLapA1:GUS gene in transgenic plants indicated that theLapA1 promoter was active during floral and fruit development and was used during vegetative growth only in response to wounding, Pseudomonas syringae pv tomatoinfection, or wound signals. This comprehensive understanding of the regulation of LapA genes indicated that this regulatory program is distinct from the wound-induced pin2, ABA-responsive le4, and PR protein genes.

An important recent advance in the field of plant–microbe interactions has been the cloning of genes that confer resistance to specific viruses, bacteria, fungi or nematodes. Disease resistance (R) genes encode proteins with predicted structural motifs consistent with them having roles in signal recognition and transduction. The future challenge is to understand howRgene products specifically perceive defence–eliciting signals from the pathogen and transduce those signals to pathways that lead to the activation of plant defence responses. In tomatoes, the Pto kinase (product of thePto Rgene) confers resistance to strains of the bacterial speck pathogen,Pseudomonas syringaepv.tomato, that carry the corresponding avirulence geneavrPto. Resistance to bacterial speck disease is initiated by a mechanism involving the physical interaction of the Pto kinase and the AvrPto protein. This recognition event initiates signalling events that lead to defence responses including an oxidative burst, the hypersensitive response and expression of pathogenesis–related genes. Pto–interacting (Pti) proteins have been identified that appear to act downstream of the Pto kinase and our current studies are directed at elucidating the roles of these components.

BackgroundHessian fly (Mayetiola destructor) is one of the most destructive pests of wheat. The genes encoding 12-oxo-phytodienoic acid reductase (OPR) and lipoxygenase (LOX) play critical roles in insect resistance pathways in higher plants, but little is known about genes controlling resistance to Hessian fly in wheat.ResultsIn this study, 154 F6:8recombinant inbred lines (RILs) generated from a cross between two cultivars, ‘Jagger’ and ‘2174’ of hexaploid wheat (2n = 6 × =42; AABBDD), were used to map genes associated with resistance to Hessian fly. Two QTLs were identified. The first one was a major QTL on chromosome 1A (QHf.osu-1A), which explained 70% of the total phenotypic variation. The resistant allele at this locus in cultivar 2174 could be orthologous to one or more of the previously mapped resistance genes (H9,H10,H11,H16, andH17) in tetraploid wheat. The second QTL was a minor QTL on chromosome 2A (QHf.osu-2A), which accounted for 18% of the total phenotypic variation. The resistant allele at this locus in 2174 is collinear to anYr17-containing-fragment translocated from chromosome 2N ofTriticum ventricosum(2n = 4 × =28; DDNN) in Jagger. Genetic mapping results showed that twoOPRgenes,TaOPR1-AandTaOPR2-A, were tightly associated withQHf.osu-1AandQHf.osu-2A,respectively. AnotherOPRgene and threeLOXgenes were mapped but not associated with Hessian fly resistance in the segregating population.ConclusionsThis study has located two major QTLs/genes in bread wheat that can be directly used in wheat breeding programs and has also provided insights for the genetic association and disassociation of Hessian fly resistance withOPRandLOXgenes in wheat.

Leucine aminopeptidase (LAP) mRNAs are induced in response to mechanical wounding, pathogen infection, and insect infestation (V. Pautot, F.M. Holzer, B. Reisch, L.L. Walling [1993] Proc Natl Acad Sci USA 90: 9906-9910). Polyclonal antibodies to a glutathione S-transferase-LAP fusion protein and affinity-purified antibodies recognizing LAP antigenic determinants detected four classes of polypeptides in tomato (Lycopersicon esculentum) leaves. All four classes had multiple polypeptides in two-dimensional polyacrylamide gel electrophoresis immunoblots. Although antigenically related to the wound-induced tomato LAP proteins, the 77- and 66-kD LAP-like proteins accumulated in both healthy and wounded leaves. Two classes of 55-kD polypeptides with distinctive isoelectric points were designated as plant LAPs; only the acidic LAP proteins accumulated to high levels after mechanical wounding or Pseudomonas syringae pv tomato infection of tomato leaves. The temporal accumulation of LAP mRNAs was correlated with the increase in acidic LAP protein subunits. A slow-migrating LAP activity was detected using a native gel assay after wounding. The molecular mass of the native wound-induced LAP enzyme was 353 kD. The 55-kD acidic LAP proteins were associated with induced LAP activity, whereas the neutral LAPs and the LAP-like proteins were not associated with this exopeptidase. A second, fast-migrating aminopeptidase was detected in both healthy and wounded tomato leaves. Cell fractionation experiments revealed that wound-induced LAP is a soluble enzyme.

For Lens that will be used in photo lithography systems, not only Low Spatial Frequency (LSF) errors, but also Middle and High Spatial Frequency (M/HSF) errors should be corrected to very low level. Ion Beam Figuring (IBF) is believed to be one of the most effective ways that can fabricate lens to especially high surface accuracy; In fact, it has also certain effects on removing errors in higher spatial frequency range. Firstly, the principle of IBF was introduced; Then, Power Spectral Density (PSD) is introduced, which is used to evaluate IBF’s corrective effects on errors in different spatial frequency ranges; Finally, by the use of IBF machine, a convex lens with diameter 90mm was fabricated, and its surface error was improved from PVr value15nm to less than 10nm, measured by Fizeau phase shift interferometer precisely. Also, M/HSF errors were measured before and after IBF fabrication, by White Light Interferometer and Atom Force Microscope separately. The initial and final Power Spectrum Density (PSD) curves of the lens were generated and compared with each other, which shows that IBF technic has really good ability to remove LSF errors, and can also reduce M/HSF errors greatly in the meantime. The results help to evaluate the corrective effects of IBF technic on errors in the full spatial frequency range.

Lymphocyte function associated antigen-1 (CD11a/CD18, LFA-1) plays an important role in the structure of the immunological synapse and is required for efficient lysis of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. To study the activation mode of LFA-1 on the NK cell surface, optical tweezers were used in the work. As an emerging technology, optical tweezers are widely used to manipulate microscopic objects and measure the forces of molecular interactions in the field of biological research. In our study, a new platform was constructed to study the single molecular behavior of receptor on cell surface using optical tweezers. Based on the platform, the interaction between an NK cell and a polystyrene microsphere coated with anti-LFA-1 antibody was observed. The result confirmed that the adhesion forces between an NK cell and a polystyrene bead were time-dependent. According to our findings, we propose that anti-LFA-1 antibody may cause the clustering of LFA-1 on NK cell surface.

The central importance of protein phosphorylation in plant defense responses has been demonstrated by the isolation of several disease-resistance genes that encode protein kinases. In addition, there are many reports of changes in protein phosphorylation accompanying plant responses to pathogens. In contrast, little is known about the role of protein dephosphorylation in regulating plant defenses. We report that expression of the LePP2Ac1 gene, which encodes a catalytic subunit of the heterotrimeric protein phosphatase 2A (PP2Ac), is rapidly induced in resistant tomato leaves upon inoculation with an avirulent strain of Pseudomonas syringae pv. tomato. By analysis of PP2Ac gene sequences from several plant species, we found that PP2Ac genes cluster into two subfamilies, with LePP2Ac1 belonging to subfamily I. Virus-induced gene silencing (VIGS) in Nicotiana benthamiana was used to suppress expression of genes from subfamily I and not from subfamily II. The PP2Ac-silenced plants had greatly decreased PP2A activity, constitutively expressed pathogenesis-related (PR) genes, and developed localized cell death in stems and leaves. In addition, the plants were more resistant to a virulent strain of P. syringae pv. tabaci and showed an accelerated hypersensitive response (HR) to effector proteins from both P. syringae and the fungal pathogen, Cladosporium fulvum. Thus, catalytic subunits of PP2Ac subfamily I act as negative regulators of plant defense responses likely by de-sensitizing protein phosphorylation cascades.

The importance of two putative Zn2+-binding (Asp347, Glu429) and two catalytic (Arg431, Lys354) residues in the tomato leucine aminopeptidase (LAP-A) function was tested. The impact of substitutions at these positions, corresponding to the bovine LAP residues Asp255, Glu334, Arg336, and Lys262, was evaluated in His6-LAP-A fusion proteins expressed in Escherichia coli. Sixty-five percent of the mutant His6-LAP-A proteins were unstable or had complete or partial defects in hexamer assembly or stability. The activity of hexameric His6-LAP-As on Xaa-Leu and Leu-Xaa dipeptides was tested. Most substitutions of Lys354 (a catalytic residue) resulted in His6-LAP-As that cleaved dipeptides at slower rates. The Glu429 mutants (a Zn2+-binding residue) had more diverse phenotypes. Some mutations abolished activity and others retained partial or complete activity. The E429D His6-LAP-A enzyme had Km and kcat values similar to the wild-type His6-LAP-A. One catalytic (Arg431) and one Zn-binding (Asp347) residue were essential for His6-LAP-A activity, as most R431 and D347 mutant His6-LAP-As did not hydrolyze dipeptides. The R431K His6-LAP-A that retained the positive charge had partial activity as reflected in the 4.8-fold decrease in kcat. Surprisingly, while the D347E mutant (that retained a negative charge at position 347) was inactive, the D347R mutant that introduced a positive charge retained partial activity. A model to explain these data is proposed.

The tomato Pti4 gene encodes a transcription factor that was identified on the basis of its specific interaction with the product of the Pto disease resistance gene in a yeast two-hybrid system. We show here that the Pti4 protein specifically binds the GCC-box cis element, which is present in the promoter region of many pathogenesis-related (PR) genes. Expression of the Pti4 gene in tomato leaves was rapidly induced by ethylene and by infection with Pseudomonas syringae pv tomato, and this induction preceded expression of GCC-box-containing PR genes. Although salicylic acid also induced Pti4 gene expression, it did not induce GCC-box PR genes. Rather, salicylic acid antagonized ethylene-mediated expression of GCC-box PR genes. We demonstrate that the Pti4 protein is specifically phosphorylated by the Pto kinase and that this phosphorylation enhances binding of Pti4 to the GCC box. In addition, induced overexpression of Pto and Pti4 in tomato leaves resulted in a concomitant increase in GCC-box PR genes. Our results support a model in which phosphorylation of the Pti4 protein by the Pto kinase enhances the ability of Pti4 to activate expression of GCC-box PR genes in tomato.

International audience

Leucine aminopeptidase (LAP) is induced by wounding and bacterial pathogen infection in tomato. DNA blot analysis of XbaI-digested lambdalap genomic clones demonstrated that LapA1 and LapA2 cDNAs were encoded by two different LapA genes in the tomato genome. The coding and untranslated regions of LapA1 and LapA2 mRNAs shared more than 93% identity. The deduced amino acid sequences of LapA cDNA clones and in vitro translation of LapA1 mRNA indicated that LAP-A was synthesized as a 60-kDa precursor protein. The processing of a 60-kDa preLAP-A into the mature 55-kDa LAP-A was demonstrated in vivo by expression of the full-length LapA1 cDNA in insect cells. Sequencing of a single LAP-A form isolated from a two-dimensional polyacrylamide gel indicated that LAP-A proteins had two different N termini that were separated by two residues. The LAP-A presequence had features similar to chloroplast transit peptides. Comparison of LAP-A levels in chloroplast and total protein extracts from methyl jasmonate-treated leaves indicated that a small proportion of the LAP-A proteins was detected in the plastids. Inspection of the LAP-A presequence indicated the presence of a dibasic protease (Kex2/furin) processing site motif 6-8 residues upstream from the LAP-A N termini. Its potential role in LAP-A precursor biogenesis is discussed.

A cofactor-independent phosphoglycerate mutase (PGAM-i) was isolated to homogeneity from monocotyledonous Lilium longiflorum Thunb. Two-dimensional (2D) polyacrylamide gel electrophoresis resolved three PGAM-i forms. This enzyme was originally identified by cross-reactivity to antibodies affinity-purified from 2D gels using human vitronectin (VN). Antibody produced against a denatured protein spot from a 2D gel did not recognize VN protein, but partial protein and DNA sequencing showed similarity of the former protein to maize PGAM-i. Immunoblots from roots, styles, leaves, and anthers showed the presence of PGAM-i in all tissues examined; it was isolated predominantly from the soluble cell fraction, with some present in the insoluble cell fraction. Immunoelectron microscopy demonstrated its localization in the cytoplasm and plastids in root cells near the apical meristem. In addition, immunogold labeling detected signals from the nucleus. The immunohistochemical localization of the enzyme in the nucleus, as well as in the cytosol and plastids, indicates that lily PGAM-i might have multiple functions in the cell.

Peptidases have a central role in the degradation of proteins by hydrolyzing peptide bonds. Based on the reaction catalyzed, these enzymes can be classified as endopeptidases, aminopeptidases or carboxypeptidases. Relative to the endoproteases and carboxypeptidases, the three types of exopeptidases that act at free N-termini of proteins or peptides are less well characterized. Aminopeptidases, dipeptidyl peptidases and tripeptidyl peptidases cleave peptides or proteins and liberate single amino acid residues, dipeptides or tripeptides, respectively. Dipeptidases and tripeptidases are aminopeptidases that preferentially act on dipeptides and tripeptides. While all three classes of N-terminal exopeptidases have been described in animals, only aminopeptidases have been described in plants.

Vitronectin (VN) is a substrate adhesion molecule, an extracellular matrix glycoprotein that facilitates cell adhesion and cell movement in animals. We have reported the cross-reactivity of a 55-kD protein in plants with rabbit anti-human VN antibodies and the presence of VN-like sequences in plant genomes using a human VN cDNA probe. We have extended these studies by using human VN riboprobes to detect VN-like mRNAs in lily (Lilium longiflorum L.) and soybean. In both species, two mRNAs were detected. We have also identified a new cross-reactive protein (41 kD) using a different preparation of human VN antiserum. In lily roots five 41-kD isoforms were observed, whereas only three of these isoforms accumulated in leaves. Monospecific antibodies prepared against the plant proteins cross-reacted with the human VN protein and vice versa. We have purified the 41-kD protein using two-dimensional gel electrophoresis, and amino acid composition analysis indicates that it is similar in composition to human VN.