Evaluation of the<Emphasis Type="Italic"> Escherichia coli</Emphasis> threonine deaminase gene as a selectable marker for plant transformation

The initial step in the synthesis of isoleucine (Ile) is the conversion of threonine to -ketobutyrate. This reaction is carried out by threonine deaminase (TD), which is feedback-regulated by Ile. Mutations in TD that manifest insensitivity to Ile feedback inhibition result in intracellular accumulation of Ile. Previous reports have shown that in planta expression of the wild-type Escherichia coli TD, ilvA, or an Ile-insensitive mutant designated ilvA-466, increased cellular concentrations of Ile. A structural analog of Ile, l-O-methylthreonine (OMT), is able to compete effectively with Ile during translation and induce cell death. It has been postulated that OMT could therefore be utilized as an effective selective agent in plant engineering studies. To test this concept, we designed two binary plasmids that harbored an nptII cassette and either the wild-type ilvA or mutant ilvA-466. The ilvA coding sequences were fused to a plastid transit peptide down stream of a modified 35S CaMV promoter. Tobacco transformations were set up implementing a selection protocol based on either kanamycin or OMT. The ilvA gene was effectively utilized as a selectable marker gene to identify tobacco transformants when coupled with OMT as the selection agent. However, the transformation efficiency was substantially lower than that observed with nptII using kanamycin as the selection agent. Moreover, in a subset of the ilvA transformants and in a majority of the ilvA-466 transgenic lines, a severe off-type was observed under greenhouse conditions that correlated with increased levels of expression of the ilvA transgene.Abbreviations ELISA enzyme-linked immunosorbent assay - Ile isoleucine - OMT l-O-methylthreonine - nptII neomycin phosphotransferase II - TD threonine deaminase     

L-O-Methylthreonine-Resistant Mutant of Arabidopsis Defective in Isoleucine Feedback Regulation

Threonine dehydratase/deaminase (TD), the first enzyme in the isoleucine biosynthetic pathway, is feedback inhibited by isoleucine. By screening M2 populations of ethyl methane sulfonate-treated Arabidopsis thaliana Columbia wild-type seeds, we isolated five independent mutants that were resistant to L-O-methylthreonine, an isoleucine structural analog. Growth in the mutants was 50- to 600-fold more resistant to L-O-methylthreonine than in the wild type. The resistance was due to a single, dominant nuclear gene that was denoted omr1 and was mapped to chromosome 3 in GM11b, the mutant line exhibiting the highest level of resistance. Biochemical characteristics (specific activities, Km, Vmax, and pH optimum) of TD in extracts from the wild type and GM11b were similar except for the inhibition constant of isoleucine, which was 50-fold higher in GM11b than in the wild type. Levels of free isoleucine were 20-fold higher in extracts from GM11b than in extracts from wild type. Therefore, isoleucine feedback insensitivity in GM11b is due to a mutant form of the TD enzyme encoded by omr1. The mutant allele omr1 of the line GM11b could provide a new selectable marker for plant genetic transformation.     

Involvement of the <Emphasis Type="Italic">Arabidopsis thaliana AtPMS1</Emphasis> gene in somatic repeat instability

Mismatch repair (MMR) genes participate in the maintenance of genome stability in all organisms. Based on its high degree of sequence conservation, it seems likely that the AtPMS1 gene of Arabidopsis thaliana is part of the MMR system in this model plant. To test this hypothesis, we aimed to disrupt AtPMS1 function by over-expressing mutated alleles expected to result in a dominant negative effect. To create one mutant allele we substituted two amino acids in the MutL-box, and for the other mutant allele we deleted 87 amino acids comprising the whole MutL-box. Contrary to published reports in some eukaryotes, transgenic plants expressing these alleles did not exhibit a decrease in fertility nor any other visible phenotype. To examine the impact of these mutations on microsatellite instability, the phenotype most often observed in organisms defective in MMR, reporter lines containing a uidA (GUS) gene inactivated by the insertion of a synthetic microsatellite (G₇ or G₁₆) were used. GUS gene function in these lines can be restored following the loss of one base or the gain of two bases in the repetitive tract. This results in the observation of blue sectors on a white background following histochemical staining. In a subset of the transformants, a significant increase (2- to 28-fold) in microsatellite instability was observed relative to wild-type. This report shows that MMR function can be disrupted via a dominant negative approach, and it is the first report to describe the phenotypic consequence of disrupting the function of a MutL homolog in plants.     

Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts

Summary Direct gene transfer has proved to be an efficient transformation method for arabidopsis thaliana, a member of the Brassicaceae. Transgenic Arabidopsis plants resistant to hygromycin B have been regenerated from mesophyll protoplasts treated with polyethylene glycol and plasmid DNA carrying the hygromycin phosphotransferase (HPT) gene under the control of the 35 S promoter of cauliflower mosaic virus. The transformation procedure reproducibly yields transformants at frequencies of approximately 1×10^-4 (based on the number of protoplasts treated) or 5% (based on the number of regenerating calli). DNA from plants regenerated from hygromycin resistant colonies was analysed by Southern blot hybridization demonstrating that the foreign gene is stably integrated into the plant chromosome. Genetic analysis of several hygromycin resistant plants showed that the HPT gene is transmitted to the progeny. Transformation experiments performed with a selectable and a non-selectable gene on separate plasmids resulted in a co-transformation rate of functionally active copies in about 25% of the transformants analysed. Hence this approach can be used to introduce non-selectable genes into the Arabidopsis genome.     

Threonine-74 Is a Key Site for the Activity of Clostridium perfringens Alpha-Toxin

A mutant toxin (MT) that abolished almost 99% of the hemolytic activity of alpha-toxin was isolated by random polymerase chain reaction (PCR) mutagenesis of the gene for Clostridium perfringens alpha-toxin. In the mutant toxin, the amino acids at Tyr (Y)-62, Thr (T)-74 and Ile (I)-345 were substituted with His, Ile and Met, respectively. Replacement of T-74 with Ile by site-directed mutagenesis resulted in the loss of hemolytic, phospholipase C and sphingomyelinase activities by 1/250-fold of that of the wild-type. The replacement of Y-62 with Ile or I-345 with Met alone did not affect the activities of the toxin. T74I mutant bound to sheep erythrocyte membranes and specifically bound [⁶⁵Zn]²⁺ in Tris-buffered saline, in the same manner as the wild-type, and contained 2 mol of zinc ions per mol of protein. These results suggest that the T-74 residue plays a key role in these biological activities of C. perfringens alpha-toxin.     

The influence of matrix attachment regions on transgene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> wild type and gene silencing mutants

Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a β-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chiMARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chiMARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Miguel F.C. De Bolle, Katleen M.J. Butaye Contributed equally to this work     

Identification,characterization and functional expression of a tyrosine ammonia-lyase and its mutants from the photosynthetic bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

A tyrosine ammonia-lyase (TAL) enzyme from the photosynthetic bacterium Rhodobacter sphaeroides (RsTAL) was identified, cloned and functionally expressed in Escherichia coli, where conversion of tyrosine to p-hydroxycinnamic acid (pHCA) was demonstrated. The RsTAL enzyme is implicated in production of pHCA, which serves as the cofactor for synthesis of the photoactive yellow protein (PYP) in photosynthetic bacteria. The wild type RsTAL enzyme, while accepting both tyrosine and phenylalanine as substrate, prefers tyrosine, but a serendipitous RsTAL mutant identified during PCR amplification of the RsTAL gene, demonstrates much higher preference for phenylalanine as substrate and deaminates it to produces cinnamic acid. Sequence analysis showed the presence of three mutations: Met4 → Ile, Ile325 → Val and Val409 → Met in this mutant. Sequence comparison with Rhodobacter capsulatus TAL (RcTAL) shows that Val409 is conserved between RcTAL and RsTAL. Two single mutants of RsTAL, Val409 → Met and Val 409 → Ile, generated by site-directed mutagenesis, demonstrate greater preference for phenylalanine compared to the wild type enzyme. Our studies illustrate that relatively minor changes in the primary structure of an ammonia-lyase enzyme can significantly affect its substrate specificity.     

The Inflorescence Stem Fibers of <Emphasis Type="Italic">Arabidopsis thaliana Revoluta</Emphasis> (<Emphasis Type="Italic">ifl1</Emphasis>) Mutant

Arabidopsis thaliana is gradually gaining significance as a model for wood and fiber formation.revolute/ifl1 is an important mutant in this respect. To better characterize the fiber system of therevolute/ifl1 mutant, we grew plants of two alleles (rev-9 in Israel andrev-1 in the USA) and examined the fiber system of the inflorescence stems using both brightfield and polarized light. Microscopic examination of sections of plants belonging to the two different alleles clearly revealed that, contrary to previous views, in 18 (13 in Israel and 5 in Ohio) out of 30 stems (20 in Israel and 10 in Ohio) the mutant produced the primary wavy fiber system of the inflorescence stems. Our findings are further supported by the fact that fibers are seen in the figures published in other studies of the mutant even when it was stated that there were no fibers. The impression of a total lack of the wavy band of fibers is in many cases just a result of poorly lignified secondary walls. This specific gene that reduces lignification in fibers is of great significance for biotechnological developments for the paper industry and thus for the global economy and ecology. We propose thatrevoluta, the first name given to this mutant (Talbert and others 1995), is more appropriate thanifl1. Online publication: 7 April 2005     

Transformation of a filamentous fungus<Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> using<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

AsAgrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast,Saccharomyces cerevisiae, a variety of fungi were subjected to theA. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. TheA. tumefaciens-mediated transformation of chestnut blight fungus,Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of theAspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1×10⁶ conidia ofC. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.     

Natural transformation of the thermophilic cyanobacterium<Emphasis Type="Italic"> Thermosynechococcus elongatus</Emphasis> BP-1: a simple and efficient method for gene transfer

Proteins derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, which performs plant-type oxygenic photosynthesis, are suitable for biochemical, biophysical and X-ray crystallographic studies. We found that T. elongatus displays natural transformation, and we established a simple and efficient protocol for transferring exogenous DNAs into the organisms genome. We obtained transformants directly on selective agar plates without having to amplify them prior to plating. We constructed several targeting vectors that enabled us to insert exogenous DNAs into specific sites without disrupting endogenous genes and operons. We also developed a new selectable marker gene for T. elongatus by optimizing the codons of the gene encoding a kanamycin nucleotidyltransferase derived from the thermophilic bacterium Bacillus stearothermophilus. This synthetic gene enabled us to select transformants as kanamycin-resistant colonies on agar plates at 52°C. Optimization of the conditions for natural transformation resulted in a transformation efficiency of up to 1.7×10³ transformants per g of DNA. The exogenous DNAs were integrated stably into the targeted sites of the T. elongatus genome via homologous recombination by double crossovers.Communicated by H. Ikeda     

Different influence of mutant alleles of the <Emphasis Type="Italic">APETALA1</Emphasis> gene on the development of reproductive organs in <Emphasis Type="Italic">abruptus</Emphasis> mutant flowers of the of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) Heynh.

The APETALA1 (AP1) gene of A. thaliana codes type II MADS protein with domains MADS, I, K, and C. The role of K- and C-domains in the functioning of AP1 protein is poorly investigated. The analysis of phenotypic manifestation of mutations disrupting the activity of various domains of the protein product allows one to obtain information on the function of domains and, thereby, on the structural-functional organization of the gene. We investigated the action of mutant alleles of the AP1 gene whose protein products are probably lacking the functionally active domains K (ap1-20), K- and C-domains (ap1-1 and ap1-6), and C-domain (ap1-3) on the flower morphology in abr mutant (the ABRUPTUS/PINOID gene allele). It was detected that, unlike the ap1-20 allele, the presence of ap1-3, ap1-6, and ap1-1 alleles results in reduction of a number of the generative organs in the flowers of the double mutants abr ap1-3, abr ap1-6, and abr ap1-1. It was suggested that C-domain of the AP1 protein prevents the alteration of determination of the type of reproductive organs when the AP1 gene ectropically expressed in the inner whorls of a flower in the abr mutant.     

Metabolomic and genetic analyses of flavonol synthesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> support the in vivo involvement of leucoanthocyanidin dioxygenase

Flavonol synthase (FLS) (EC-number 1.14.11.23), the enzyme that catalyses the conversion of flavonols into dihydroflavonols, is part of the flavonoid biosynthesis pathway. In Arabidopsis thaliana, this activity is thought to be encoded by several loci. In addition to the FLAVONOL SYNTHASE1 (FLS1) locus that has been confirmed by enzyme activity assays, loci displaying similarity of the deduced amino acid sequences to FLS1 have been identified. We studied the putative A. thaliana FLS gene family using a combination of genetic and metabolite analysis approaches. Although several of the FLS gene family members are expressed, only FLS1 appeared to influence flavonoid biosynthesis. Seedlings of an A. thaliana fls1 null mutant (fls1-2) show enhanced anthocyanin levels, drastic reduction in flavonol glycoside content and concomitant accumulation of glycosylated forms of dihydroflavonols, the substrate of the FLS reaction. By using a leucoanthocyanidin dioxygenase (ldox) fls1-2 double mutant, we present evidence that the remaining flavonol glycosides found in the fls1-2 mutant are synthesized in planta by the FLS-like side activity of the LDOX enzyme. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence database accession numbers: GenBank accession EU287457 and EU287459.     

Development of a homologous transformation system for Aspergillus niger based on the pyrG gene

Summary The development of a homologous transformation system for Aspergillus niger is described. The system is based on the use of an orotidine-5-phosphate decarboxylase deficient mutant (pyrG) and a vector, pAB4-1, which contains the functional A. niger pyrG gene as a selection marker. Transformation of the A. niger pyrG mutant with pAB4-1 resulted in the appearance of stable Pyr⁺ transformants at a frequency of 40 transformants per g of DNA. In 90% of these transformants integration had occurred at the resident pyrG locus, resulting either in replacement of the mutant allele by the wild-type allele (60%) or in insertion of one or two copies of the vector (40%). The A. niger pyrG mutant could also be transformed with the vector pDJB2 containing the pyr4 gene of Neurospora crassa, at a frequency of 2 transformants per g of DNA. Integration at the resident pyrG locus was not found with this vector. The vector pAB4-1 is also capable of transforming an Aspergillus nidulans pyrG mutant to Pyr⁺. The pyrG transformation system was used for the introduction of a non-selectable gene into A. niger.     

A novel method for the cloning of chromosomal mutations in a single step: Isolation of two mutant alleles of envZ, an osmoregulatory gene from Escherichia coli

Summary We have developed a simple, rapid and powerful method for the cloning of chromosomal mutations from total cellular DNA in a single step using a plasmid carrying the clined wild-type locus of interest and a convenient selectable marker such as antibiotic resistance. This method relies upon the ability of the cloned wild-type gene to form a heteroduplex with the mutant chromosomal locus. The plasmid from primary transformants can be screened rapidly by size; more than 50% of plasmids of the correct size contained the mutant locus. When this method was used to clone two chromosomal mutations in the envZ gene of Escherichia coli, a locus which encodes a membrane-bound sensory protein involved in the osmoregulation of outer membrane porin biosynthesis, more than 50% of the retransformants from the plasmids selected by size were found to exhibit the mutant phenotype. Preliminary characterization of these mutant alleles is discussed. This novel and powerful method should be generally applicable in any system where the cloned locus is available.This work was presented at the 86th Annual Meeting of the American Society for Microbiology, March 1986, Washingnton, D.C.     

Overexpression of a cyanobacterial phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase with diminished sensitivity to feedback inhibition in<Emphasis Type="Italic"> Arabidopsis</Emphasis> changes amino acid metabolism

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Synechococcus vulcanus (SvPEPC) is a unique enzyme, being almost insensitive to feedback inhibition at neutral pH. In order to assess its usefulness in metabolic engineering of plants, SvPEPC was expressed in Arabidopsis thaliana (L.) Heynh. under the control of the cauliflower mosaic virus 35S promoter. About one-third of the transformants of the T1 generation showed severe visible phenotypes such as leaf bleaching and were infertile when grown on soil. However, no such phenotype was observed with Arabidopsis transformed with Zea mays L. PEPC (ZmPEPC) for C4 photosynthesis, which is normally sensitive to a feedback inhibitor, l-malate. For the SvPEPC transformants of the T2 generation, which had been derived from fertile T1 transformants, three kinds of phenotype were observed when plants were grown on an agar medium containing sucrose: Type-I plants showed poor growth and a block in true leaf development; Type-II plants produced a few true leaves, which were partially bleached; Type-III plants were apparently normal. In Type-I plants, total PEPC activity was increased about 2-fold over the control plant but there was no such increase in Type-III plants. The phenotypes of Type-I plants were rescued when the sucrose-containing agar medium was supplemented with aromatic amino acids. Measurement of the free amino acid content in whole seedlings of Type-I transformants revealed that the levels of the aromatic amino acids Phe and Tyr were lowered significantly as compared with the control plants. In contrast, the levels of several amino acids of the aspartic and glutamic families, such as Asn, Gln and Arg, were markedly enhanced (4- to 8-fold per plant fresh weight). However, when the medium was supplemented with aromatic amino acids, the levels of Asn, Gln, and Arg decreased to levels slightly higher than those of control plants, accompanied by growth recovery. Taken together, it can be envisaged that SvPEPC is capable of efficiently exerting its activity in the plant cell environment so as to cause imbalance between aromatic and non-aromatic amino acid syntheses. The growth inhibition of Type-I plants was presumed to be primarily due to a decreased availability of phosphoenolpyruvate, one of the precursors for the shikimate pathway for the synthesis of aromatic amino acids and phenylpropanoids. The possible usefulness of SvPEPC as one of the key components for installing the C₄-like pathway is proposed.Abbreviations CaMV Cauliflower mosaic virus - GUS -Glucuronidase - Kan Kanamycin - 2-ME 2-Mercaptoethanol - MS/G medium 1/2 Murashige–Skoog and 1/2 Gamborg mixed medium - PEP Phosphoenolpyruvate - PEPC Phosphoenolpyruvate carboxylase - Sv Synechococcus vulcanus - ZmPEPC Maize PEPC involved in C₄ photosynthesis     

Generation of marker free salt tolerant transgenic plants of Arabidopsis thaliana using the gly I gene and cre gene under inducible promoters

Despite the advances in transgenesis, transformation technologies still rely on the introduction of a selectable marker gene to identify cells and tissues that have integrated the gene of interest in their genome. The continuous presence of the marker genes in the transgenics is often controversial as it can potentially have multiple undesirable impacts. The present study employed the self-excising Cre-loxP system to generate marker-free Arabidopsis thaliana expressing the agronomically important glyoxalase I (glyI) gene from Brassica juncea to confer salt stress tolerance. A binary vector was constructed wherein the salt-inducible rd29A promoter was used to drive the expression of the glyI gene and the transformants of A. thaliana were recovered using kanamycin resistance as the selectable marker. The neomycin phosphotransferase II (nptII) gene was flanked by the loxP sites followed by the introduction of a heat-inducible Cre-recombinase in between the loxP sites. The kanamycin-resistant transgenic lines of A. thaliana using this vector showed an ability to withstand stress imposed by 150 mM NaCl. The exposure of the T2 transgenic lines to a mild heat shock (37°C) resulted in the recovery of salt-tolerant, kanamycin-sensitive T3 progeny. Molecular analyses of the T3 transgenic lines following the heat shock treatment confirmed the excision of the nptII gene and the completion of their life cycle in the presence of 150 mM NaCl-induced stress.     

The Arabidopsis DNA mismatch repair gene <Emphasis Type="Italic">PMS1</Emphasis> restricts somatic recombination between homeologous sequences

The eukaryotic DNA mismatch repair (MMR) system contributes to maintaining the fidelity of genetic information by correcting replication errors and preventing illegitimate recombination events. This study aimed to examine the function(s) of the Arabidopsis thaliana PMS1 gene (AtPMS1), one of three homologs of the bacterial MutL gene in plants. Two independent mutant alleles (Atpms1-1 and Atpms1-2) were obtained and one of these (Atpms1-1) was studied in detail. The mutant exhibited a reduction in seed set and a bias against the transmission of the mutant allele. Somatic recombination, both homologous and homeologous, was examined using a set of reporter constructs. Homologous recombination remained unchanged in the mutant while homeologous recombination was between 1.7- and 4.8-fold higher than in the wild type. This increase in homeologous recombination frequency was not correlated with the degree of sequence divergence. In RNAi lines, a range of increases in homeologous recombination were observed with two lines showing a 3.3-fold and a 3.6-fold increase. These results indicate that the AtPMS1 gene contributes to an antirecombination activity aimed at restricting recombination between diverged sequences. Liangliang Li, Eric Dion contributed equally to this work.     

Identification of the OsOPR7 gene encoding 12-oxophytodienoate reductase involved in the biosynthesis of jasmonic acid in rice

Enzyme 12-oxophytodienoate (OPDA) reductase (EC1.3.1.42), which is involved in the biosynthesis of jasmonic acid (JA), catalyses the reduction of 10, 11-double bonds of OPDA to yield 3-oxo-2-(2′-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0). The rice OsOPR1 gene encodes OPDA reductase (OPR) converting (−)-cis-OPDA preferentially, rather than (+)-cis-OPDA, a natural precursor of JA. Here, we provide evidence that an OPR family gene in rice chromosome 8, designated OsOPR7, encodes the enzyme involved in the JA biosynthesis. Recombinant OsOPR7-His protein efficiently catalysed the reduction of both enantiomers of cis-OPDA, similar to the OPR3 protein in Arabidopsis thaliana (L.) Heynh. The expression of OsOPR7 mRNA was induced and reached maximum levels within 0.5 h of mechanical wounding and drought stress, and the endogenous JA level started to increase in accordance with the increase in OsOPR7 expression. The GFP-OsOPR7 fusion protein was detected exclusively in peroxisomes in onion epidermal cells. Furthermore, complementation analysis using an Arabidopsis opr3 mutant indicated that the OsOPR7 gene, but not OsOPR1, was able to complement the phenotypes of male sterility in the mutant caused by JA deficiency, and that JA production in the opr3 mutant was also restored by the expression of the OsOPR7 gene. We conclude that the OsOPR7 gene encodes the enzyme catalysing the reduction of natural (+)-cis-OPDA for the JA biosynthesis in rice. Tomoyuki Tani and Hiroyuki Sobajima have equally contributed to this work.     

Protein stability and mutations in the axial methionine loop of a minimal cytochrome<Emphasis Type="Italic"> c</Emphasis>

The minimal mono-heme ferricytochrome c from Bacillus pasteurii, containing 71 amino acids, has been further investigated through mutagenesis of different positions in the loop containing the iron ligand Met71. These mutations have been designed to sample different aspects of the loop structure, in order to obtain insights into the determinants of the stability of the iron(III) environment. In particular, positions 68, 72 and 75 have been essayed. Gln68 has been mutated to Lys to provide a suitable alternate ligand that can displace Met71 under denaturing conditions. Pro72 has been mutated to Gly and Ala to modify the range of allowed backbone conformations. Ile75, which is in van der Waals contact with Met71 and partly shields a long-lived water molecule in a protein cavity, has been substituted by Val and Ala to affect the network of inter-residue interactions around the metal site. The different contributions of the above amino acids to protein parameters such as structure, redox potential and the overall stability against unfolding with guanidinium hydrochloride are analyzed. While the structure remains essentially the same, the stability decreases with mutations. The comparison with mitochondrial c-type cytochromes is instructive.Abbreviations Bpcytc soluble fragment of cytochrome c₅₅₃ from Bacillus pasteurii - GdmCl guanidinium chloride - I75A Ile75 to Ala mutant - I75V Ile75 to Val mutant - P72A Pro72 to Ala mutant - P72G Pro72 to Gly mutant - Q68K Gln75 to Lys mutant - WT wild type     

Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli

Biosynthetic threonine deaminase (TD) is a key enzyme for the synthesis of isoleucine which is allosterically inhibited and activated by Ile and Val, respectively. The binding sites of Ile and Val and the mechanism of their regulations in TD are not clear, but essential for a rational design of efficient productive strain(s) for Ile and related amino acids. In this study, structure-based computational approach and site-directed mutagenesis were combined to identify the potential binding sites of Ile and Val in Escherichia coli TD. Our results demonstrated that each regulatory domain of the TD monomer possesses two nonequivalent effector-binding sites. The residues R362, E442, G445, A446, Y369, I460, and S461 only interact with Ile while E347, G350, and F352 are involved not only in the Ile binding but also in the Val binding. By further considering enzyme kinetic data, we propose a concentration-dependent mechanism of the allosteric regulation of TD by Ile and Val. For the construction of Ile overproducing strain, a novel TD mutant with double mutation of F352A/R362F was also created, which showed both higher activity and much stronger resistance to Ile inhibition comparing to those of wild-type enzyme. Overexpression of this mutant TD in E. coli JW3591 significantly increased the production of ketobutyrate and Ile in comparison to the reference strains overexpressing wild-type TD or the catabolic threonine deaminase (TdcB). This work builds a solid basis for the reengineering of TD and related microorganisms for Ile production.