Dimerization and protease resistance: New insight into the function of PR-1

The group 1 pathogenesis-related (PR-1) proteins have long been considered hallmarks of hypersensitive response/defense pathways in plants, but their biochemical functions are still obscure despite resolution of the NMR/X-ray structures of several PR-1-like proteins, including P14a (the prototype PR-1). We report here the characterization of two basic PR-1 proteins (PR-1-1 and PR-1-5) recently identified from hexaploid wheat (Triticum aestivum). Both proteins were expressed in Pichia pastoris as a single major species of ∼15 kDa. Sequence identity of the expressed PR-1 proteins was verified by MALDI-TOF/TOF analysis. Accumulation of the native PR-1-5 protein in pathogen-challenged wheat was confirmed by protein gel blot analysis. Low-temperature SDS-PAGE and yeast two-hybrid assays revealed that PR-1-1 exists primarily as a monomer whereas PR-1-5 forms homodimers. Both PR-1 proteins are resistant to proteases compared to bovine serum albumin, but PR-1-1 shows resistance mainly to subtilisin and protease K (serine proteases) whereas PR-1-5 shows resistance to subtilisin, protease K and papain (a cysteine protease). Site-specific mutations at the five putative active sites in the PR-1 domain all affected dimerization, with the mutations at Glu-72 and Glu-102 (in the PR-1-5 numeration) also diminishing protease resistance. Sequence analysis revealed that the Glu-72 and Glu-102 residues are located in motif-like sequences that are conserved in both PR-1 and the human apoptosis-related caspase proteins. These findings prompt us to examine the function of PR-1 for a role in protease-mediated programmed cell death pathways in plants.     

Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products

In Escherichia coli, the uptake and phosphorylation of glucose is carried out mainly by the phosphotransferase system (PTS). Despite the efficiency of glucose transport by PTS, the required consumption of 1 mol of phosphoenolpyruvate (PEP) for each mol of internalized glucose represents a drawback for some biotechnological applications where PEP is a precursor of the desired product. For this reason, there is considerable interest in the generation of strains that can transport glucose efficiently by a non-PTS mechanism. The purpose of this work was to study the effect of different gene expression levels, of galactose permease (GalP) and glucokinase (Glk), on glucose internalization and phosphorylation in a E. coli PTS(-) strain. The W3110 PTS(-), designated VH32, showed limited growth on glucose with a specific growth rate (mu) of 0.03 h(-1). A low copy plasmid family was constructed containing E. coli galP and glk genes, individually or combined, under the control of a trc-derived promoter set. This plasmid family was used to transform the VH32 strain, each plasmid having different levels of expression of galP and glk. Experiments in minimal medium with glucose showed that expression of only galP under the control of a wild-type trc promoter resulted in a mu of 0.55 h(-1), corresponding to 89% of the mu measured for W3110 (0.62 h(-1)). In contrast, no increase in specific growth rate (mu) was observed in VH32 with a plasmid expressing only glk from the same promoter. Strains transformed with part of the plasmid family, containing both galP and glk genes, showed a mu value similar to that of W3110. Fermentor experiments with the VH32 strain harboring plasmids pv1Glk1GalP, pv4Glk5GalP, and pv5Glk5GalP showed that specific acetate productivity was twofold higher than in W3110. Introduction of plasmid pLOI1594, coding for pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis, to strain VH32 carrying one of the plasmids with galP and glk caused a twofold increase in ethanol productivity over strain W3110, also containing pLOI1594.     

马齿苋叶片PEP羧化酶的分子特性

马齿苋叶片PEPCase由四个相同的亚基组成,亚基分子量为83kD。远紫外CD光谱分析表明,此酶含有36.6％α—螺旋结构。马齿苋叶片PEPCase可被G6P激活,但不能被Gly、Ser激活。G6P可防止酶的尿素变性和枯草杆菌蛋白酶的作用。这种保护效应与G6P诱导的酶构象变化有关。 从酶对低温、高温及尿素的反应来看,马齿苋叶片PEPCase的稳定性高于高粱叶片PEP—Case,两者的免疫特性和电泳特性亦不同。     

Glycine Activating Enzyme in the Posterior Silkgland of Silkworm

1. Aminoacyl tRNA synthetase was extracted from the silkgland of silkworm (Bombyxmori Linné) and fractionated on a DEAE-cellulose column. Activities were estimated by ATP-PP_i exchange reaction as well as glycyl tRNA formation.

2. Two peaks, A and B, having ATP-PP_i exchange activity were found in the separated fractions, respectively. There was also observed a marked difference between the both peaks with respect to the pH optimum and activity dependence on MgCl₂ concentration.

3. Peak A showed no activity of glycyl tRNA formation. Only a part of peak B coincided with the activity of glycyl tRNA formation. The activities of both the ATP-PP_i exchange reaction and glycyl tRNA formation were found to be dependent on MgCl₂ concentration, and the optimum concentration was different between two peaks.

4. It also seemed to exist two peaks of activities, a and b, in glycyl tRNA formation which could be separated with a DEAE-cellulose column.     

Regulation of enzymes involved in C4 photosynthesis and the antioxidant metabolism by UV-B radiation in Egeria densa,a submersed aquatic species

Egeria densa, a submersed aquatic species, was exposed to different treatments under UV-B radiation, and the response of phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme (NADP-ME) was determined. Exposure to UV-B radiation for 4 h per day over 7–16 days caused an increase in both enzymes, together with an increase in the activity of some isoforms of several enzymes involved in the antioxidant metabolism, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD). The content of chlorophylls and carotenoids was considerably decreased, suggesting that degradation or repression of the synthesis of these molecules may be occurring after UV-B exposure. Reactive oxygen species (ROS) were also required for UV-B induction of PEPC and NADP-ME, as the addition of ascorbic acid before UV-B treatment prevented the induction of these enzymes, while salicylic acid was not effective in inducing NADP-ME but increased the expression of the lower molecular mass isoform of PEPC. On the other hand, damage to the photosynthetic machinery may be occurring after exposure to UV-B radiation for 8 per day over 1–2 days, as indicated by a decrease in the levels of Rubisco, PEPC and NADP-ME. Some of the enzymes involved in the antioxidant metabolism, such as CAT and APX, were also sensitive to continuous exposure, evidenced by a decrease in their activity. In this way, in E. densa, several enzymes involved in different metabolic pathways showed a distinct response, depending on the UV-B treatment. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inheritance of two foreign genes co-introduced intoPetunia hybrida by direct gene transfer

In previous work, transformedPetunia hybrida plants were obtained by direct gene transfer, using two different genes on separate plasmids (NPT II gene and a cDNA of PEPC from green sorghum leaves). In this study, we have analysed the sexual transmission of the acquired genes by genetic crossing analysis of 2 of the transgenic petunias. The ploïdies of the two clones were determined by flow cytometric analysis showing that one was 2n and the other 4n. Self and back crosses show that the kanamycin character was inherited as a single dominant trait, and that the two clones were heterozygotes for this character. Therefore, the 4n clone probably arises from an endoploidization followed by a transformation event. Southern blot analyses show that all of the resistant progenies which were analysed harboured the kanamycin gene, and expressed the phosphorylation activity in vitro. The DNA of several progenies were also tested for the presence of co-transformed PEPC cDNA sequence. All of the kanamycin-resistant progenies tested contained the second coding sequence, indicating that the two foreign genes might be genetically co-inherited in the transgenic plants. The way in which the two genes are integrated into the genome is discussed.Abbreviations NPT neomycin phosphotransferase - PEG polyethyleneglycol - PEPC phosphoenolpyruvate carboxylase     

Cloning,sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum

A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined. It was inserted into a bacterial expression vector and a PEPC^- Escherichia coli mutant could be complemented by the cDNA construct. A functional fusion protein could be synthesized in E. coli. The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes.