Cloning of the beta-amylase gene from Bacillus cereus and characteristics of the primary structure of the enzyme.

The gene encoding the beta-amylase of Bacillus cereus BQ10-S1 (SpoII) was cloned into Escherichia coli JM 109. A sequenced DNA fragment of 2,001 bp contains the beta-amylase gene. The N-terminal sequences (AVNGKG MNPDYKAYLMAPLKKI), the C-terminal sequences (SHTSSW), and the amino acid sequences of the five regions in the beta-amylase molecules were determined. The mature beta-amylase contains 514 amino acid residues with a molecular mass of 57,885 Da. The amino acid sequence homology with those of known beta-amylases was 52.7% for Bacillus polymyxa, 52.0% for Bacillus circulans, 43.4% for Clostridium thermosulfurogenes, 31.8% for Arabidopsis thaliana, 31.5% for barley, 29.9% for sweet potato, and 28.9% for soybean. Ten well-conserved regions were found between the N terminus and the area around residue 430, but the C-terminal region of 90 residues has no similarity with those of the plant beta-amylases. The homology search revealed that this C-terminal region has homology with C-terminal regions of the beta-amylase from C. thermosulfurogenes, some bacterial alpha-amylases, cyclodextrin glucanotransferase, and glucoamylase. Some of these sequences are known as the raw-starch-binding domain. These results suggest that B. cereus beta-amylase has an extra domain which has raw-starch-binding ability and that the domain has considerable sequence homology with those of other amylases or related enzymes from a wide variety of microorganisms.     

Sugar-inducible expression of a gene for beta-amylase in Arabidopsis thaliana.

The levels of beta-amylase activity and of the mRNA for beta-amylase in rosette leaves of Arabidopsis thaliana (L.) Heynh. increased significantly, with the concomitant accumulation of starch, when whole plants or excised mature leaves were supplied with sucrose. A supply of glucose or fructose, but not of mannitol or sorbitol, to plants also induced the expression of the gene for beta-amylase, and the induction occurred not only in rosette leaves but also in roots, stems, and bracts. These results suggest that the gene for beta-amylase of Arabidopsis is subject to regulation by a carbohydrate metabolic signal, and expression of the gene in various tissues may be regulated by the carbon partitioning and sink-source interactions in the whole plant. The sugar-inducible expression of the gene in Arabidopsis was severely repressed in the absence of light. The sugar-inducible expression in the light was not inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea or by chloramphenicol, but it was inhibited by cycloheximide. These results suggest that a light-induced signal and de novo synthesis of proteins in the cytoplasm are involved in the regulation. A fusion gene composed of the 5' upstream region of the gene for beta-amylase from Arabidopsis and the coding sequence of beta-glucuronidase showed the sugar-inducible expression in a light-dependent manner in rosette leaves of transgenic Arabidopsis.     

The phenylalanine ammonia-lyase gene family in Arabidopsis thaliana

Phenylpropanoid derivatives are a complex class of secondary metabolites that have many important roles in plants during normal growth and in responses to environmental stress. Phenylalanine ammonialyase (PAL) catalyzes the first step in the biosynthesis of phenylpropanoids, and is usually encoded by a multi-gene family. Genomic clones for three Arabidopsis thaliana PAL genes containing the entire protein-coding region and upstream and downstream sequences have been obtained and completely sequenced. Two A. thaliana PAL genes (PAL1 and PAL2) are structurally similar to PAL genes that have been cloned from other plant species, with a single intron at a conserved position, and a long highly conserved second exon. Previously identified promoter motifs plus several additional sequence motifs were found in the promoter regions of PAL1 and PAL2. Expression of PAL1 and PAL2 is both qualitatively and quantitatively similar in different plant organs and under various inductive conditions. A third A. thaliana PAL gene, PAL3, differs significantly from PAL1 and PAL2 and other sequenced plant PAL genes. PAL3 contains an additional intron, and its deduced amino acid sequence is less homologous to other PAL proteins. The PAL3 promoter region lacks several sequence motifs conserved between A. thaliana PAL1 and PAL2, as well as motifs described in other genes involved in phenylpropanoid metabolism. A. thaliana PAL3 was expressed at very low levels under the conditions examined.     

Enhanced seed phytosterol accumulation through expression of a modified HMG-CoA reductase

The regulation of phytosterol biosynthesis in seeds is of interest to biotechnologists because of the efficacy of dietary phytosterols in reducing blood cholesterol in humans. Mevalonate synthesis via 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) is a key step in phytosterol biosynthesis. HMG-CoA reductase is inactivated by phosphorylation by SNF1-related protein kinase 1 (SnRK1). With the aim of increasing seed phytosterol levels, transgenic tobacco plants were produced expressing a full-length Arabidopsis (Arabidopsis thaliana) HMG-CoA reductase gene (HMG1) coding sequence, a modified HMG1 sequence encoding a protein lacking the target serine residue for phosphorylation by SnRK1, or a chimaeric sequence encoding the N-terminal domain of the Arabidopsis HMG1 enzyme fused with the catalytic domain of yeast HMG-CoA reductase, which lacks an SnRK1 target site. All three transgenes (35S-AtHMG1, 35S-AtHMG1m and 35S-AtScHMG1) were under the control of a cauliflower mosaic virus 35S RNA promoter. Levels of seed phytosterols were up to 2.44-fold higher in plants transformed with the 35S-AtHMG1m gene than in the wild-type, and were significantly higher than in plants expressing 35S-AtHMG1 or 35S-AtScHMG1. In contrast, levels of phytosterols in leaves of plants transformed with the 35S-AtHMG1m gene were unchanged, suggesting that regulation of HMG-CoA reductase by SnRK1 is an important factor in seeds but not in leaves. A total of 11 independent transgenic lines expressing 35S-AtHMG1m or 35S-AtScHMG1 also showed an altered flower phenotype, comprising a compact floret, prolonged flowering, short, pale petals, a protruding style, short stamens, late anther development, little or no pollen production, premature flower abscission and poor seed set. Because of this phenotype, the modified HMG-CoA reductase gene would have to be expressed seed specifically if it were to be engineered into a crop plant for biotechnological purposes.     

Isolation and Characterization of a Bacillus cereus Mutant Strain Hyperproductive of Exo-beta-Amylase

Starting with a strain of Bacillus cereus excreting about 40-fold more beta-amylase than does the original wild-type strain, we isolated, after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, a strain designated BQ10-S1 SpoIII which showed under optimal conditions a further 5.5-fold increase in beta-amylase activity. The amylase production of this strain was observed to increase in the presence of 0.5% glucose or 1% maltose and, more markedly, in the presence of 2% soluble starch in the culture medium. The enzyme produced by this strain was immunologically identical to the wild-type enzyme, suggesting that either the copy number of the gene or the efficiency of enzyme synthesis from it, or both, are altered in this strain.     

Identification and characterization of a phloem-specific beta-amylase.

A monoclonal antibody, RS 5, was raised by injecting sieve elements isolated from tissue cultures of Streptanthus tortuosus (Brassicacae) into BALB/c mice and screening resultant hybridoma supernatants for the labeling of phloem using immunofluorescence microscopy. The RS 5 monoclonal antibody identifies a 57-kD protein on immunoblots, which is present in phloem-forming tissue cultures of S. tortuosus but is absent in cultures that lack phloem. Purified 57-kD protein of S. tortuosus is demonstrated to be a phloem-specific beta-amylase. Partial peptide sequences of the 57-kD protein of S. tortuosus are shown to be 96% identical with the corresponding portions of a deduced sequence reported for a major form of beta-amylase in Arabidopsis thaliana. The RS 5 antibody cross-reacts with the major form of A. thaliana beta-amylase on immunoblots, and the antibody also binds to the sieve elements of A. thaliana using immunofluorescence microscopy. The results suggest that the major form of A. thaliana beta-amylase is a phloem-specific enzyme.     

Increased sucrose level and altered nitrogen metabolism in Arabidopsis thaliana transgenic plants expressing antisense chloroplastic fructose-1,6-bisphosphatase

The pea chloroplastic fructose-1,6-bisphosphatase (FBPase) antisense construct reduced the endogenous level of expression of the corresponding Arabidopsis thaliana gene. The reduction of foliar FBPase activity in the transformants T(2) and T(3) generation ranged from 20% to 42%, and correlated with lower levels of FBPase protein. FBPase antisense plants displayed different phenotypes with a clear increase in leaf fresh weight. Measurements of photosynthesis revealed a higher carbon-assimilation rate. Decreased FBPase activity boosted the foliar carbohydrate contents, with a shift in the sucrose:starch ratio, which reached a maximum of 0.99 when the activity loss was 41%. Nitrate reductase activity decreased simultaneously with an increase in glutamine synthetase activity, which could be explained in terms of ammonium assimilation regulation by sugar content. These results suggest the role of FBPase as a key enzyme in CO(2) assimilation, and also in co-ordinating carbon and nitrogen metabolism.     

The impact of PEPC phosphorylation on growth and development of Arabidopsis thaliana: Molecular and physiological characterization of PEPC kinase mutants

Two phosphoenolpyruvate carboxylase (PEPC) kinase genes (PPCk1 and PPCk2) are present in the Arabidopsis genome; only PPCk1 is expressed in rosette leaves. Homozygous lines of two independent PPCk1 T-DNA-insertional mutants showed very little (dln1), or no (csi8) light-induced PEPC phosphorylation and a clear retard in growth under our greenhouse conditions. A mass-spectrometry-based analysis revealed significant changes in metabolite profiles. However, the anaplerotic pathway initiated by PEPC was only moderately altered. These data establish the PPCk1 gene product as responsible for leaf PEPC phosphorylation in planta and show that the absence of PEPC phosphorylation has pleiotropic consequences on plant metabolism.     

Cloning and characterization of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in melon (Cucumis melo L. reticulatus)

We have isolated a cDNA for Cm-HMGR, encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in melon (Cucumis melo L. reticulatus; Genbank Accession No. AB021862). Cm-HMGR encodes a polypeptide of 588 amino acids that contains two transmembrane domains and a catalytic domain. Database searches revealed that Cm-HMGR shows homology to HMG1 (63.7%) and HMG2 (70.3%) of tomato, to HMG1 (77.2%) and HMG2 (69.4%) of Arabidopsis thaliana, and to HMGR of tobacco (72.6%). Functional expression in a HMG-CoA reductase-deficient mutant yeast showed that Cm-HMGR products mediate the synthesis of mevalonate. Northern analysis revealed that the level of Cm-HMGR mRNA in the fruit increased after pollination and markedly decreased at the end of fruit enlargement. During ripening, Cm-HMGR mRNA levels increased markedly in the fruit. In parallel with mRNA expression, Cm-HMGR activity increased after pollination, whereas no Cm-HMGR activity was detectable during fruit ripening. Our results suggest that Cm-HMGR is important during early post-pollination development of the fruit in melon.     

Immunogold localization of nodule-enhanced phosphoenolpyruvate carboxylase in alfalfa

Phosphoenolpyruvate carboxylase (PEPC; EC 4-1-1-31) plays a paramount role in providing carbon for synthesis of malate and aspartate in alfalfa (Medicago sativa L.) root nodules. PEPC protein and activity levels are highly enhanced in N₂-fixing alfalfa nodules. To ascertain the relationship between the cellular location of PEPC and root nodule metabolism, enzyme localization was evaluated by immunogold cytochemistry using alfalfa nodule PEPC antibodies. Gold labelling patterns in effective nodules showed that PEPC is a cytosolic enzyme and is distributed relatively equally in infected and uninfected cells of the nodule symbiotic zone. A high amount of labelling was also observed in pericycle cells of the nodule vascular system. Labelling was also detected within inner cortical cells, but the density was reduced by 60%. When Lotus corniculatus was transformed with a chimeric gene consisting of the 5′-upstream region of the PEPC gene fused to β-glucuronidase (GUS), GUS staining in nodules was consistent with immunogold localization patterns. The occurrence of PEPC in both infected and uninfected cells of the symbiotic zone of effective nodules coupled to the reduced amounts in ineffective nodules suggests a direct role for this enzyme in supporting N₂-fixation. PEPC localization in the uninfected, interstitial cells of the symbiotic zone indicates that these cells may also have a role in nodule carbon metabolism. Moreover, the association of PEPC with the nodule vascular system implies a role for the enzyme in the transport of assimilates to and from the shoot.     

香格里拉水韭磷酸烯醇式丙酮酸羧化酶基因(PEPC)的克隆及其表达载体构建

以中国特有植物香格里拉水韭（Isoetes shangrilaensis X.Liu）为材料,通过转录组测序数据分析筛选出磷酸烯醇式丙酮酸羧化酶基因（IsPEPC）,根据该基因序列,从香格里拉水韭cDNA中克隆获得磷酸烯醇式丙酮酸羧化酶（PEPCase）的编码基因IsPEPC,并将此基因插入pCAMBIA-2300-N-eGFP及pMD质粒载体上,再采用农杆菌介导的花序浸染法将2个重组载体分开转入野生型拟南芥（Arabidopsis thaliana（L.）Heynh.）中。结果显示：IsPEPC基因蛋白编码序列长度为2928 bp,编码975个氨基酸;同源性检索分析结果表明,该蛋白与其近源物种江南卷柏（Selaginella moellendorffii Hieron.）的PEPC基因蛋白序列同源性为79.8%。对转基因的T₁代拟南芥通过抗性筛选并在gDNA水平上阳性鉴定,初步鉴定得到pC2300-N-eGFP-IsPEPC转基因株系26个和pMD-IsPEPC转基因株系32个。     

The Arabidopsis phenylalanine ammonia lyase gene family: kinetic characterization of the four PAL isoforms

In Arabidopsis thaliana, four genes have been annotated as provisionally encoding PAL. In this study, recombinant native AtPAL1, 2, and 4 were demonstrated to be catalytically competent for l-phenylalanine deamination, whereas AtPAL3, obtained as a N-terminal His-tagged protein, was of very low activity and only detectable at high substrate concentrations. All four PALs displayed similar pH optima, but not temperature optima; AtPAL3 had a lower temperature optimum than the other three isoforms. AtPAL1, 2 and 4 had similar K(m) values (64-71 microM) for l-Phe, with AtPAL2 apparently being slightly more catalytically efficacious due to decreased K(m) and higher k(cat) values, relative to the others. As anticipated, PAL activities with l-tyrosine were either low (AtPAL1, 2, and 4) or undetectable (AtPAL3), thereby establishing that l-Phe is the true physiological substrate. This detailed knowledge of the kinetic and functional properties of the various PAL isoforms now provides the necessary biochemical foundation required for the systematic investigation and dissection of the organization of the PAL metabolic network/gene circuitry involved in numerous aspects of phenylpropanoid metabolism in A. thaliana spanning various cell types, tissues and organs.     

Glutathionylation of cytosolic glyceraldehyde-3-phosphate dehydrogenase from the model plant Arabidopsis thaliana is reversed by both glutaredoxins and thioredoxins in vitro

Plants contain both cytosolic and chloroplastic GAPDHs (glyceraldehyde-3-phosphate dehydrogenases). In Arabidopsis thaliana, cytosolic GAPDH is involved in the glycolytic pathway and is represented by two differentially expressed isoforms (GapC1 and GapC2) that are 98% identical in amino acid sequence. In the present study we show that GapC1 is a phosphorylating NAD-specific GAPDH with enzymatic activity strictly dependent on Cys(149). Catalytic Cys(149) is the only solvent-exposed cysteine of the protein and its thiol is relatively acidic (pK(a)=5.7). This property makes GapC1 sensitive to oxidation by H(2)O(2), which appears to inhibit enzyme activity by converting the thiolate of Cys(149) (-S-) into irreversible oxidized forms (-SO(2)(-) and -SO(3)(-)) via a labile sulfenate intermediate (-SO(-)). GSH (reduced glutathione) prevents this irreversible process by reacting with Cys(149) sulfenates to give rise to a mixed disulfide (Cys(149)-SSG), as demonstrated by both MS and biotinylated GSH. Glutathionylated GapC1 can be fully reactivated either by cytosolic glutaredoxin, via a GSH-dependent monothiol mechanism, or, less efficiently, by cytosolic thioredoxins physiologically reduced by NADPH:thioredoxin reductase. The potential relevance of these findings is discussed in the light of the multiple functions of GAPDH in eukaryotic cells (e.g. glycolysis, control of gene expression and apoptosis) that appear to be influenced by the redox state of the catalytic Cys(149).     

Palmitate and oleate induction of acylation stimulating protein resistance in 3T3-L1 adipocytes and preadipocytes

Acylation stimulating protein (ASP) stimulates triglyceride synthesis and glucose transport via its receptor C5L2. The aims were (i) to evaluate ASP response under insulin-resistant conditions and (ii) to identify mechanisms of ASP resistance using 3T3-L1 adipocytes and preadipocytes. Overnight incubation with palmitate (PAL) or oleate (OLE) induced dose-dependent inhibition of ASP-stimulated glucose transport in adipocytes (198 +/- 18% +ASP, 100 +/- 4% basal, 131 +/- 14% + ASP + 1 mmol/L PAL) and preadipocytes (287 +/- 21% + ASP, 100 +/- 4% basal, 109 +/- 13% + ASP + 1 mmol/L PAL). In adipocytes, dose-dependent maximal C5L2 mRNA decreases were -41 +/- 15% and -82 +/- 2%, with decreased cell-surface C5L2 of -55 +/- 12% and -39 +/- 9% (1 mmol/L PAL and OLE, respectively) with no change in preadipocytes. Adipocytes treated with PAL or OLE evidenced inhibition of ASP stimulation of G proteins: Gbeta (-50%), Galphaq/11 (-50%) and protein kinase C: PKCalpha-P (-52%), PKCzeta-P (-43%). Fatty acid-induced ASP resistance via C5L2 may contribute to altered adipose tissue function and obesity/insulin resistance phenotype in humans.