Azetidine-2-carboxylate Research Articles

Genes and Enzymes of Azetidine-2-Carboxylate Metabolism: Detoxification and Assimilation of an Antibiotic

l-(-)-Azetidine-2-carboxylate (AC) is a toxic, natural product analog of l-proline. This study revealed the genes and biochemical strategy employed by Pseudomonas sp. strain A2C to detoxify and assimilate AC as its sole nitrogen source. The gene region from Pseudomonas sp. strain A2C required for detoxification was cloned into Escherichia coli and sequenced. The 7.0-kb region contained eight identifiable genes. Four encoded putative transporters or permeases for gamma-amino acids or drugs. Another gene encoded a homolog of 2-haloacid dehalogenase (HAD). The encoded protein, denoted l-azetidine-2-carboxylate hydrolase (AC hydrolase), was highly overexpressed by subcloning. The AC hydrolase was shown to catalyze azetidine ring opening with the production of 2-hydroxy-4-aminobutyrate. AC hydrolase was further demonstrated to be a new hydrolytic member of the HAD superfamily by showing loss of activity upon changing aspartate-12, the conserved active site nucleophile in this family, to an alanine residue. The presence of a gene encoding a potential export chaperone protein, CsaA, adjacent to the AC hydrolase gene suggested that AC hydrolase might be found inside the periplasm in the native Pseudomonas strain. Periplasmic and cytoplasmic cell fractions from Pseudomonas sp. strain A2C were prepared. A higher specific activity for AC hydrolysis was found in the periplasmic fraction. Protein mass spectrometry further identified AC hydrolase and known periplasmic marker proteins in the periplasmic fraction. A model was proposed in which AC is hydrolyzed in the periplasm and the product of that reaction is transported into and further metabolized in the cytoplasm.

In Vitro Selection of High Phenolic and Rosmarinic Acid Clonal Lines of Oregano

ABSTRACT Oregano, Origanum vulgare, is a source of essential oils and phenolic metabolites, such as rosmarinic acid, which exhibits anti-oxidant and antimicrobial activity. This suggests that the plant material could be useful in food preservation, and in nutraceutical and cosmetic applications. Due to natural cross-pollination, however, oregano has substantial plant-to-plant variation and therefore a considerable genetic inconsistency in the type and level of phytochemicals produced. In this study, clonal lines developed from a population of heterozygous seeds using tissue culture were screened for desirable traits (multiple shoot formation and enhanced biomass, and rosmarinic acid and phenolic acid production) based on tolerance of the clonal lines to Pseudomonas and azetidine-2-carboxylate (A2C). Of the 20 clonal lines isolated, two over-expressed phenolic acids in response to both Pseudomonas and A2C.

Production of mutants with high cold tolerance in spring canola (Brassica napus)

A major factor affecting spring canola (Brassica napus) production in Canada is killing frosts during seedling development in the spring and seed maturation in the fall. The objective of this study was to explore the possibility of producing spring canola lines with mutations that have altered biochemical pathways that increase cold tolerance. The approach was to generate UV point mutations in cultured microspores followed by chemical in vitro selection of individual mutant microspores or embryos resulting in measurable alterations to various biochemical pathways with ele- vated levels of key defense signaling molecules such as, salicylic acid (SA), p-Fluoro-D,L-Phenyl Alanine (FPA), and jasmonic acid (JA). In addition, since proline (Pro) is known to protect plant tissues in the cold-induced osmotic stress pathway, mutants that overproduce Pro were selected in vitro by using three Pro analogues: hydroxyproline (HP), azetidine-2-carboxylate (A2C); and, 3,4-dehydro-D,L-proline (DP). Of the 329 in vitro selected mutant embryos produced, 74 were identified with significant cold tolerance compared to their donor parents through indoor freezer tests at -6� C, and 19 had better winter field survival than winter canola checks. All chemically selected mutant doubled haploids with increased cold tolerance compared well with parent lines for all seed quality and agronomic parameters.

Glucose Deprivation Induces Heme Oxygenase-1 Gene Expression by a Pathway Independent of the Unfolded Protein Response

Nutrients such as glucose regulate the expression of genes that are involved in plasma membrane transport, metabolic functions, and protein trafficking in the endoplasmic reticulum. Depletion of nutrients results in cellular stress, which evokes adaptive and protective responses, one of which is the induction of heme oxygenase-1 (HO-1), a 32-kDa endoplasmic reticulum enzyme that catalyzes the rate-limiting step in heme degradation. Incubation of HepG2 human hepatoma cells in glucose-free medium resulted in an increased HO-1 mRNA content, reaching a maximum of approximately 25-fold over control cells after 12 h. The glucose-dependent induction of HO-1 mRNA was concentration-dependent (k(12) approximately 0.5 mm) and was attenuated by fructose, galactose, mannose, and 2-deoxyglucose, but not by the non-metabolizable glucose analog, 3-O-methylglucose. Tunicamycin, thapsigargin, or azetidine 2-carboxylate, each of which activates the unfolded protein response pathway, did not induce HO-1 mRNA expression, whereas glucose-regulated protein 78 mRNA was increased. These results demonstrate that glucose availability regulates transcription of the HO-1 gene via a pathway that is different from the unfolded protein response. The induction of HO-1 may serve as a protective response in hypoglycemic circumstances and underscores the importance of understanding nutrient control of the HO-1 gene.

SOMATIC EMBRYOGENESIS IN ANISE (PIMPINELLA ANISUM L.): THE EFFECT OF PROLINE ON EMBRYOGENIC CALLUS FORMATION AND ABA ON ADVANCED EMBRYO DEVELOPMENT

Somatic embryos of anise (Pimpinella anisum) are an excellent system to investigate the biosynthesis of food-related phenolic metabolites like anethol which is linked to differentiation. Somatic embryogenesis in anise is a two part system. The first is a 2,4-D (2,4-dichlorophenoxy acetic acid)-induced embryogenic callus formation which requires optimal levels of endogenous auxin and cytokinin levels. The second is the advanced embryo development stage in the absence of 2,4-D which requires abscisic acid to stimulate embryo growth. Our work has shown that proline and the precursors of proline (ornithine and arginine), in combination with the proline analog, azetidine-2-carboxylate (A2C) significantly stimulated 2,4-D-induced embryogenic callus formation and subsequent somatic embryo development in hormone-free medium. Further, abscisic acid stimulated somatic embryo development in anise in the second stage. Total phenolic assays done on embryogenic tissue during 2,4-D-induced embryogenic callus, formation, and during abscisic acid-mediated embryo development showed that levels were highest in embryogenic tissue induced in the presence of proline plus A2C. Total phenolic levels of developing embryos in the absence of 2,4-D and in the presence of abscisic acid were higher than that of the control. A hypothesis for further investigation is proposed based on the potential role of the proline-linked pentose phosphate pathway in stimulating the biosynthesis of purines and phenolic metabolites during anise somatic embryogenesis.

Screening of high biomass and phenolic producing clonal lines of spearmint in tissue culture using pseudomonas and azetidine‐2 carboxylate.

Rosmarinic acid (RA) and related phenolics are natural antioxidants found as secondary metabolites in spearmint (Mentha spicata). These phenolic‐secondary metabolites have diverse food processing and nutraceutical applications. Since natural cross‐pollination results in plant to plant variation in the level of phenolic metabolites, tissue culture‐based techniques are essential to isolate elite antioxidant‐producing clonal lines. The objective of this research is to develop tissue culture‐based selection techniques to isolate high rosmarinic acid and phenolic‐producing clonal lines from a heterogenous bulk seed population of spearmint. Multiplied clonal shoots of each line were screened for tolerance to azetidine‐2‐carboxylate (A2C). Individual shoot apex of each line were also screened for Pseudomonas tolerance. Rosmarinic acid and total phenolics were assayed in all treated clonal lines and compared to uninoculated/untreated shoot expiants of corresponding line. The Pseudomonas and A2C treatment strategy allowed the rapid tissue culture‐based screening of potentially high phenolic antioxidant‐producing clonal lines of spearmint for future field and greenhouse evaluation. Targeted elite lines had combinations of Pseudomonas tolerance with no loss in biomass in response to the bacterium and enhanced levels of total phenolics and rosmarinic acid in response to A2C.

Regulation of Benzyladenine-Induced in Vitro Shoot Organogenesis and Endogenous Proline in Melon (Cucumis melo L.) by Exogenous Proline, Ornithine, and Proline Analogues

Melon (Cucumis melo L.) is an excellent system to study the role of proline in regulating benzyladenine-induced shoot organogenesis, which is also an important system in our laboratory for investigating flavor and antioxidant metabolite synthesis in differentiated tissues. We have hypothesized that proline stimulated in vitro shoot organogenesis through the activation of the proposed proline-linked pentose phosphate pathway, which may regulate endogenous cytokinin and auxin biosynthesis. To further investigate this hypothesis, it is essential to first confirm whether benzyladenine-induced shoot organogenesis can be stimulated by enhancing proline synthesis through the use of proline analogues and ornithine (proline precursor). In this study, benzyladenine-induced shoot organogenesis was substantially stimulated by exogenous proline and/or ornithine in combination with proline analogues such as azetidine-2-carboxylate (A2C) and hydroxyproline (HP). Among these treatments, proline in combination with A2C or HP showed the highest stimulation. The stimulation of organogenesis correlated closely with increased levels of endogenous proline content, thereby strengthening the hypothesis that proline-linked activation of purine and aromatic metabolism via the stimulation of pentose phosphate pathway may be important for in vitro organogenesis in melon.

Further Studies on Substrates Inducing Metacyclogenesis in Trypanosoma cruzi

Epimastigotes of Trypanosoma cruzi, Peru strain, incubated in Contreras' artificial triatomine urine transformed into metacyclic trypomastigotes when 10 mM L-glutamine, L-asparagine or D-fructose was added to the medium. Metacyclogenesis with these substrates was comparable to the percent metacyclic morphotype formation induced by L-proline and significantly greater than that stimulated by 10 mM D-glucose. Sodium acetate (10 mM) increased transformation induced by L-proline, and L-hydroxyproline (10 mM) increased transformation induced by D-fructose. Phosphoenolpyruvate (10 mM) inhibited L-proline-induced metacyclic trypomastigote stage formation. Three antimetabolites, azetidine 2-carboxylate (5 mM), malonic acid (1 mM), and desthiobiotin (5 mM), completely inhibited D-fructose-induced but not L-proline-induced transformation. The Costa Rica, Y, and CL strains of T. cruzi showed different patterns of percent metacyclogenesis with substrates that induce transformation in the Peru strain.

Expression of glyphosate resistance in carrot somatic hybrid cells through the transfer of an amplified 5-enolpyruvylshikimic acid-3-phosphate synthase gene

A Daucus carota cell line selected as resistant to N-(phosphonomethyl)-glycine (glyphosate) was found to have increased levels of 5-enolpyruvylshikimic acid-3-phosphate synthase (EPSPS) activity of 5.5 times over wild-type carrot and an EPSPS protein level increase of 8.7 times as confirmed by Western hybridization analysis. Southern blot hybridization using a petunia EPSPS probe showed increases in the number of copies of EPSPS genes in the glyphosate-resistant line which correlated with the higher levels of the EPSPS enzyme. The mechanism of resistance to glyphosate is therefore due to amplification of the EPSPS gene. To examine the stability of the amplified genes, cloned lines selected as doubly resistant to Dl-5-methyltryptophan (5MT) and azetidine-2-carboxylate (A2C) were fused with the amplified EPSPS glyphosate-resistant cell line. Somatic hybrids expressed resistances to 5MT in a semidominant fashion while A2C and glyphosate resistance was expressed as dominant, or semi-dominant traits, in a line-specific manner. The hybrid lines possessed additive chromosome numbers of the parental lines used and no double minute chromosomes were observed. The glyphosate-resistant parental line and most somatic hybrids retained the amplified levels of EPSPS in the absence of selection pressure over a 3-year period.

Selection of a Nicotiana plumbaginifolia universal hybridizer and its use in intergeneric somatic hybrid formation

A Nicotiana plumbaginifolia cell strain carrying a positive (dominant) trait, resistance to azetidine-2-carboxylate (A2C), was selected in strain NX1 which lacked nitrate reductase activity (a negative or recessive trait). This universal hybridizer strain, denoted NXAr, was fused with dextran to a Daucus carota strain, PR, which carried glyphosate (GLP) resistance. A large number of hybrids were selected in a medium with NO 3 - as the sole nitrogen source and A2C as inhibitor, conditions which prevent the growth of both parents. When the selected colonies were then tested for GLP resistance, 93% carried this trait. In addition the hybrid nature was indicated by additive chromosome numbers, both A2C and GLP resistance in suspension cultures, intermediate nitrate reductase activity and the presence of banding patterns for three isozymes which match those of the parents. Southern hybridization analysis using an enolpyruvylshikimic acid-3-phosphate synthase (EPSPS) probe, pMON 6145, also showed the presence of the gene from both parents in the hybrid strains based on restriction length polymorphisms. The PR strain contains increased levels of EPSPS which confers GLPr due to gene amplification. Since the universal hybridizer can be used as a fusion partner with any wild-type line many protoplast fusion studies can be carried out easily.

Dominant expression of an amino-acid uptake deficiency in carrot somatic fusion hybrids

Somatic hybrids were selected previously by their ability to grow in medium containing normally inhibitory levels of the two amino acid analogs aminoethylcysteine (AEC) and 5-methyltryptophan (5MT) following fusion of protoplasts from a cell strain resistant to AEC with protoplasts resistant to 5MT. The hybrid nature of the selected clones was shown by several criteria including the presence of another resistance, azetidine-2-carboxylate (A2C), carried by one of the parental strains which was not selected for in the initial hybrid selection scheme. The characterization presented here shows that the AEC resistance in the parental strain, as well as the two somatic hybrids, was due to decreased AEC uptake. Also the 5MT resistance in the hybrids, as in the parent was caused by a feedback altered form of the tryptophan biosynthetic control enzyme, anthranilate synthase which leads to increases in free tryptophan. The A2C resistance was caused by the accumulation of free proline by a mechanism which has not been studied. These studies confirm that AEC resistance caused by decreased uptake can be expressed dominantly in protoplast fusion hybrids.

Hybrid Shoot Formation from Fused Daucus carota and D. capillifolius Protoplasts

Summary Protoplasts from line C123, a 5-methyltryptophan (5MT) and azetidine-2-carboxylate (A2C) resistant cell line of Daucus carota that has lost the ability to regenerate whole plants, were fused using dextran with protoplasts of suspension cultured D. capillifolius. Somatic hybrids were selected initially by culture in 5MT levels which inhibit the growth of D. capillifolius and then for green pigmentation and embryoid formation. The fusion and selection produced plants, lacking roots, with leaves showing intermediate morphology between those of the parents. The chromosome numbers of embryoid root tips and suspension cultures were near 36 which is the sum of the parents. Analysis of four isozymes following electrophoresis showed that malate dehydrogenase and peroxidase resembled the carrot parent while leucine aminopeptidase was like D. capillifolius. The superoxide dismutase pattern was like that expected from a hybrid. Callus initiated from the hybrid plants showed resistance to both 5MT and A2C with the resistance being expressed as semi-dominant and dominant characters, respectively. The feedback control characteristics of anthranilate synthase correlate with the 5MT resistance in the parental and hybrid lines. These results demonstrate that amino acid analog resistance and plant regeneration ability can be used as complementing markers to select for fusion hybrids since both can be expressed in the hybrids.