Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydrodipicolinate synthase

In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance the accumulation of the corresponding amino acids, we have generated transgenic barley plants that constitutively express mutant Escherichia coli genes encoding lysine feed-back insensitive forms of AK and DHPS. As a result, leaves of primary transformants (T₀) exhibited a 14-fold increase of free lysine and an 8-fold increase in free methionine. In mature seeds of the DHPS transgenics, there was a 2-fold increase in free lysine, arginine and asparagine and a 50% reduction in free proline, while no changes were observed in the seeds of the two AK transgenic lines analysed. When compared to that of control seeds, no differences were observed in the composition of total amino acids. The introduced genes were inherited in the T₁ generation where enzymic activities revealed a 2.3-fold increase of AK activity and a 4.0–9.5-fold increase for DHPS. T₁ seeds of DHPS transformants showed the same changes in free amino acids as observed in T₀ seeds. It is concluded that the aspartate family pathway may be genetically engineered by the introduction of genes coding for feed-back-insensitive enzymes, preferentially giving elevated levels of lysine and methionine.     

Lysine biosynthesis and nitrogen metabolism in quinoa (Chenopodium quinoa): study of enzymes and nitrogen-containing compounds.

Aspartate kinase (AK, EC 2.7.2.4), homoserine dehydrogenase (HSDH, EC 1.1.1.3) and dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) were isolated and partially purified from immature Chenopodium quinoa Willd seeds. Enzyme activities were studied in the presence of the aspartate-derived amino acids lysine, threonine and methionine and also the lysine analogue S-2-aminoethyl-l-cysteine (AEC), at 1 mM and 5 mM. The results confirmed the existence of, at least, two AK isoenzymes, one inhibited by lysine and the other inhibited by threonine, the latter being predominant in quinoa seeds. HSDH activity was also shown to be partially inhibited by threonine, whereas some of the activity was resistant to the inhibitory effect, indicating the presence of two isoenzymes, one resistant and another sensitive to threonine inhibition. Only one DHDPS isoenzyme highly sensitive to lysine inhibition was detected. The results suggest that the high concentration of lysine observed in quinoa seeds is possibly due to a combined effect of increased lysine synthesis and accumulation in the soluble form and/or as protein lysine. Nitrogen assimilation was also investigated and based on nitrate content, nitrate reductase activity, amino acid distribution and ureide content, the leaves were identified as the predominant site of nitrate reduction in this plant species. The amino acid profile analysis in leaves and roots also indicated an important role of soluble glutamine as a nitrogen transporting compound.     

Selection of Lysine Plus Threonine-Resistant Mutant of Maize

Resistance to certain amino acids or amino acid analogs can lead to overproduce specific 'free amino acids. By selection-Mutagenic treatment-Selection, lysine plus threonine-resistant mutant (RLT) was obtained from tissue culture of maize, W77-R3019V The resistance of RLT was 20 times higher than that of wild type. The levels of all free aspartate family amino acids in RLT were higher than those in wild type. Especially, threonine was 20 times higher. The resistance was inheritable and segregation in progenies, RLT1 and F1, was approximate to 3:1 and 1:1 resistant/sensitive ratio, respectively. The resistance was inherited as a single dominant or semidominant nuclear gene. In RLT2 embryo cultures, the resistance and free threonine levels in resistant callus were 20 and 23 times higher than those in sensitive one, respectively. In the homozygous seeds of RLT2, the levels of free threonine, arginine, lysine, methionine and isoleucine were 11, 8, 5, 5 and 3 times higher than those of wild type.     

Lysine and threonine metabolism are subject to complex patterns of regulation in Arabidopsis

To study the regulation of lysine and threonine metabolism in plants, we have transformed Arabidopsis thaliana with chimeric genes encoding the two bacterial enzymes dihydrodipicolinate synthase (DHPS) and aspartate kinase (AK). These bacterial enzymes are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts. Transgenic plants expressing the bacterial DHPS overproduced lysine, but lysine levels were quite variable within and between transgenic genotypes and there was no direct correlation between the levels of free lysine and the activity of DHPS. The most lysine-overproducing plants also exhibited abnormal phenotypes. However, these phenotypes were detected only at early stages of plant growth, while at later stages, new buds emerged that looked completely normal and set seeds. Wild-type plants exhibited relatively high levels of free threonine, suggesting that in Arabidopsis AK regulation may be more relaxed than in other plants. This was also supported by the fact that expression of the bacterial AK did not cause any dramatic elevation in this amino acid. Yet, the relaxed regulation of threonine synthesis in Arabidopsis was not simply due to a reduced sensitivity of the endogenous AK to feedback inhibition by lysine and threonine because growth of wild-type plants, but not of transgenic plants expressing the bacterial AK, was arrested in media containing these two amino acids. The present results, combined with previous studies from our laboratory, suggest that the regulation of lysine and threonine metabolism is highly variable among plant species and is subject to complex biochemical, physiological and environmental controls. The suitability of these transgenic Arabidopsis plants for molecular and genetic dissection of lysine and threonine metabolism is also discussed.     

Lysine-insensitive aspartate kinase in two threonine-overproducing mutants of maize

Aspartate kinase (AK; EC 2.7.2.A) catalyzes the first reaction in the biosynthesis pathway for aspartate-derived amino acids in plants. Aspartate kinase was purified from wildtype and two maize (Zea mays L.) genotypes carrying unlinked dominant mutations,Ask LT19 andAsk2 -LT20, that conferred overproduction of threonine, lysine, methionine and isoleucine. The objective of this investigation was to characterize the AKs from mutant and wildtype plants to determine their role in regulating the synthesis of aspartate-derived amino acids in maize. Kernels of the homozygousAsk2 mutant exhibited 174-, 10-, 13- and 2-fold increases in, in this sequence, free threonine, lysine, methionine and isoleucine, compared to wildtype. In wildtype maize, AK was allosterically feedback-inhibited by lysine with 10 μMl-lysine required for 50% inhibition. In contrast, AK purified from the isogenic heterozygousAsk and homozygousAsk2 mutants required 25 and 760 μM lysine for 50% inhibition, respectively, indicating thatAsk andAsk2 were separate structural loci for lysine-regulated AK subunits in maize. Further characterization of purified AK from the homozygous mutantAsk2 line indicated altered substrate and lysine inhibition kinetics. The apparent Hill coefficient was 0.7 for the mutantAsk2 AK compared with 1.6 for the wildtype enzyme, indicating that the mutant allele conferred the loss of a lysinebinding site to the mutant AK. Lysine appeared to be a linear noncompetitive inhibitor ofAsk2 AK with respect to MgATP and an uncompetitive inhibitor with respect to aspartate compared to S-parabolic, I parabolic noncompetitive inhibition of wildtype AK. Reduced lysine sensitivity of theAsk2 gene product appeared to reduce the lysine inhibition of all of the AK activity detected in homozygousAsk2 plants, indicating that maize AK is a heteromeric enzyme consisting of the two lysine-sensitive polypeptides derived from theAsk andAsk2 structural genes. Scientific paper No. 17419, Minnesota Agricultural Experiment Station projects No. 0302-4813-56 and No. 0302-4818-32 This research was supported in part by the U.S. Depatment of Agriculture Competitive Research Grants Office grant 86-CRCR-1-2019. The authors are grateful to Charles Grissom for providing the computer programs in an IBM-PC format.     

Enzymes of lysine metabolism from Coix lacryma-jobi seeds

Lysine, threonine, methionine and isoleucine are synthesized through the aspartate metabolic pathway. The concentrations of soluble lysine and threonine in cereal seeds are very low. Coix lacryma-jobi (coix) is a maize-related grass and the enzymological aspects of the aspartate metabolic pathway are completely unknown. In order to obtain information on lysine metabolism in this plant species, two enzymes involved in the biosynthesis of these amino acids (aspartate kinase 〚AK, EC 2.7.2.4〛 and homoserine dehydrogenase 〚HSDH, EC 1.1.1.3〛) and two enzymes involved in lysine degradation (lysine 2-oxoglutarate reductase 〚LOR, EC 1.5.1.8〛 and saccharopine dehydrogenase 〚SDH, EC 1.5.1.9〛) were isolated and partially characterized in coix seeds. AK activity was inhibited by threonine and lysine separately, suggesting the presence of two isoenzymes, one sensitive to lysine and the other sensitive to threonine, with the latter corresponding to approximately 60% of the total AK activity. In contrast to previous results from other plant species, the threonine-sensitive AK eluted from an ion exchange chromatography column at higher KCl concentration than the lysine-sensitive form. The HSDH activity extracted from the seeds was partially inhibited by threonine, indicating the presence of threonine-sensitive and threonine-resistant isoenzymes. LOR and SDH activities were detected only in the endosperm tissue and co-purified on an anion exchange chromatography column, suggesting that the two activities may be linked on a single bifunctional polypeptide, as observed for other plant species. One single SDH activity band was observed on non-denaturing PAGE gels. The K_m for saccharopine of SDH was determined as 0.143 mM and the K_m for NAD as 0.531 mM. Although SDH activity was shown to be stable, LOR, AK and HSDH were extremely unstable, under all buffer systems tested.     

Increasing lysine levels in pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millsp) seeds through genetic engineering

In higher plants the essential amino acids lysine, threonine, methionine and isoleucine are synthesised through a branched pathway starting from aspartate. The key enzyme of lysine biosynthesis in this pathway—dihydrodipicolinate synthase (DHDPS)—is feedback-inhibited by lysine. The dhdps-r1 gene from a mutant Nicotiana sylvestris, which encodes a DHDPS enzyme insensitive to feedback inhibition, was used to improve the lysine content in pigeonpea seeds. The dhdps-r1 coding region driven by a phaseolin or an Arabidopsis 2S2 promoter was successfully overexpressed in the seeds of pigeonpea by using Agrobacterium transformation and particle bombardment. In 11 lines analysed, a 2- to 6-fold enhanced DHDPS activity in immature seeds at a late stage of maturation was found in comparison to wild type. The overexpression of dhdps-r1 led to an enhanced content of free lysine in the seeds of pigeonpea from 1.6 to 8.5 times compared with wild type. However, this was not reflected in an increase in total seed lysine content. This might be explained by a temporal discrepancy between maximal expression of dhdps-r1 and the rate of amino acid incorporation into storage proteins. Assays of the lysine degradative enzyme lysine-ketoglutarate reductase in these seeds showed no co-ordinated regulation of lysine biosynthesis and catabolism during seed maturation. All transgenic plants were fertile and produced morphologically normal seeds.     

Threonine accumulation in the seeds of a barley mutant with an altered aspartate kinase

Barley (Hordeum vulgare L.) mutants altered in the regulation of synthesis of aspartate-derived amino acids were sought by screening embryos for growth on a medium containing lysine plus threonine. One mutant, Rothamsted 2501, was selected with good growth. From the segregation of resistance in the following generations, it was concluded that the resistance was conferred by a dominant gene, Lt1. No homozygous Lt1/Lt1 fertile plants have been recovered. Partially purified aspartate kinase preparations from resistant and sensitive plants were separated on DEAE-cellulose chromatography into three peaks of activity (I, II, III) and the feedback regulatory properties of these peaks determined. These peaks are considered to be three isozymic forms of aspartate kinase, one predominantly sensitive to threonine and two sensitive to lysine or lysine plus S-adenosyl methionine. The feedback characteristics of one of the peaks of aspartate kinase activity from resistant plants were changed such that lysine was half-maximally inhibitory at 10 rather than 0.4mm. Increases in the concentrations of the free pools of threonine (4×) and methionine (2×) were measured in young plants grown on a basal medium. Threonine in the soluble fraction of mature seeds from resistant plants was increased from 0.8 to 9.6% of the total threonine content. The total content of both threonine and methionine of the seeds was increased by 6% compared with grain of similar nitrogen content.S.E.R. acknowledges the receipt of a Council of Europe Scholarship through The British Council. Part of this was also supported by EEC Grant 473.     

Seed-specific expression of a bacterial desensitized aspartate kinase increases the production of seed threonine and methionine in transgenic tobacco

In order to study the regulation of threonine and methionine synthesis in plant seeds, tobacco plants were transformed with a chimeric gene containing the coding DNA sequence of a mutant lysC gene from Escherichia coli fused to a promoter from a phaseolin seed storage protein gene. The bacterial mutant lysC gene codes for aspartate kinase (AK) which is desensitized to feedback inhibition by lysine and threonine. Increased AK activity, compared with control non-transformed plants, was detected in seeds but not in leaves, roots and flowers of the transgenic plants. This expression was accompanied by a significant increase in the levels of free threonine and methionine in the seed. The level of these amino acids also correlated positively with the levels of the bacterial enzyme. No alteration in plant phenotype and 'average seed weight' was observed in any of the transgenic plants, indicating that plant growth and seed development were normal. This study demonstrates, for the first time, that the threonine and methionine biosynthetic pathways are active in plant seeds. Thus, targeting of the production of favorable biosynthetic enzymes to plant seeds may represent a desirable molecular approach for production of crop plants with a more balanced nutritional quality.     

A Mutant of Arabidopsis thaliana lpar;L.) Heynh. with Modified Control of Aspartate Kinase by Threonine

Mutagenesis and subsequent selection of Arabidopsis thaliana plantlets on a growth inhibitory concentration of lysine has led to the isolation of lysine-resistant mutants. The ability to grow on 2 m M lysine has been used to isolate mutants that may contain an aspartate kinase with altered regulatory-feedback properties. One of these mutants (RL 4) was characterized by a relative enhancement of soluble lysine. The recessive monogenic nuclear transmission of the resistance trait was established. It was associated with an aspartate kinase less sensitive to feedback inhibition by threonine. Two mutants (RLT 40 and RL 4) in Arabidopsis, characterized by an altered regulation of aspartate kinase, were crossed to assess the effects of the simultaneous presence of these different aspartate kinase forms. A double mutant (RLT40 × RL4) was isolated and characterized by two feedback-desensitized isozymes of aspartate kinase to, respectively, lysine and threonine but no threonine and/or lysine overproduction was observed. Genetical analysis of this unique double aspartate kinase mutant indicated that both mutations were located on chromosome 2, but their loci (ak1and ak2) were found to be unlinked.     

Lysine and threonine biosynthesis in sorghum seeds: characterisation of aspartate kinase and homoserine dehydrogenase isoenzymes

Aspartate kinase (AK, EC 2.7.2.4) and homoserine dehydrogenase (HSDH, EC 1.1.1.3) have been partially purified and characterised from immature sorghum seeds. Two peaks of AK activity were eluted by anion‐exchange chromatography [diethylaminoethyl (DEAE)‐Sephacel] with 183 and 262 mM KCl, and both activities were inhibited by lysine. Similarly, two peaks of HSDH activity were eluted with 145 and 183 mM KCl; the enzyme activity in the first peak in elution order was shown to be resistant to threonine inhibition, whereas the second was sensitive to threonine inhibition. However, following gel filtration chromatography (Sephacryl S‐200), one peak of AK activity co‐eluted with HSDH and both activities were sensitive to threonine inhibition, suggesting the presence of a bifunctional threonine‐sensitive AK–HSDH isoenzyme with a molecular mass estimated as 167 kDa. The activities of AK and HSDH were studied in the presence of lysine, threonine, methionine, valine, calcium, ethylene glycol bis(2‐aminoethylether)‐N,N,N′N′‐tetraacetic acid, calmodulin, S‐adenosylmethionine (SAM), S‐2‐aminoethyl‐l ‐cysteine (AEC) and increasing concentrations of KCl. AK was shown to be inhibited by threonine and lysine, confirming the existence of two isoenzymes, one sensitive to threonine and the other sensitive to lysine, the latter being predominant in sorghum seeds. Methionine, SAM plus lysine and AEC also inhibited AK activity; however, increasing KCl concentrations and calcium did not produce any significant effect on AK activity, indicating that calcium does not play a role in AK regulation in sorghum seeds. HSDH also exhibited some inhibition by threonine, but the majority of the activity was not inhibited, thus indicating the existence of a threonine‐sensitive isoenzyme and a second predominant threonine‐insensitive isoenzyme. Valine and SAM plus threonine also inhibited HSDH; however, increasing concentrations of KCl and calcium had no inhibitory effect.     

Isolation of auxotrophic mutants from acetobacter methanolicus MB 58

The lysine content of the biomass of the acidophilic facultatively methylotrophic bacterium Acetobacter methanolicus MB 58 was increased by genetic manipulations. A homoserine auxotroph, MB 58.196, and a threonine auxotroph, MB 58.195, were obtained from Acetobacter methanolicus MB 58 by N-methyl-N′-nitro-N-nitrosoguanidine treatment. Investigations of enzyme activities revealed that the homoserine auxotroph lacks homoserine dehydrogenase activity, and the threonine auxotroph lacks homoserine kinase activity. Concerning the lysine-producing ability, only the homoserine auxotrophic mutant accumulates lysine in the intracellular pool. The intracellular lysine content of this mutant was increased 40-fold. An excretion of amino acids into the medium was not detected. A homoserine resistant mutant, MB 58.196.10, isolated from MB 58.196 by UV-irradiation, was able to excrete lysine. About 95% of free lysine were found in the culture medium. Altogether, the free lysine concentration was increased 800-fold in comparison to the wild-type strain. By these genetic manipulations the total lysine concentration of MB 58.196 was increased to 2.7% and of MB 58.196.10 to 56% in comparison to the wild-type strain.     

Two Feedback-Insensitive Enzymes of the Aspartate Pathway in Nicotiana sylvestris

Lysine and threonine overproducer mutants in Nicotiana sylvestris, characterized by an altered regulation of, respectively, dihydrodipicolinate synthase and aspartate kinase activities, were crossed to assess the effects of the simultaneous presence of these genes on the biosynthesis of aspartate-derived amino acids. The monogenic dominant behavior of both resistance traits was confirmed, and their loci were found to be unlinked. Study of the inhibition properties of dihydrodipicolinate synthase and aspartate kinase activities in RAEC-1 × RLT 70 confirmed the heterozygote state of both mutations, because only half of their lysine-sensitive activity could still be inhibited by this negative effector. Analysis of the free amino acid pool during the growth of the double mutant revealed a major free lysine overproduction reaching up to 50% of the total pool, whereas the other aspartate-derived amino acids remained equally or even less abundant than in the wild type. An abnormal phenotype was clearly associated with such high levels of lysine accumulation, which points out the possible role of this amino acid in the developmental features of the plant. Comparison of the amino acid content, free and total (free + protein-bound), between the wild type, the two mutants, and the double mutant obtained by crossing them brings new insights on the regulation of the aspartate pathway, and on its implications in relationship to plant nutritional value improvement.     

Effect of Pyruvate Kinase Deficiency on l-Lysine Productivities of Mutants with Feedback-resistant Aspartokinases

The cultural conditions were investigated for a Brevibacterium flavum mutant, No. 2–190, with a low level of citrate synthase (CS) and with feedback-resistant phosphoenoipyruvate (PEP) carboxylase and aspartokinase (AK). The productivity was increased from 28 to 38 g/1 (as the HC1 salt) with a medium containing 10% glucose. From this strain, pyruvate kinase (PK)-defective mutants were derived and selected as to the inability to grow on ribose. Among them, strain Kl-18 showed higher lysine productivity than the parent under all cultural conditions tested, and produced 43 g/1 of lysine, at maximum. A lysine-producing mutant, No. 536–4, with a feedback- resistant AK was derived from PK-defective strain KH-21 which had low CS activity and a feedback-resistant PEP carboxylase. The mutant was isolated by a new selection method, that is, on the basis of resistance to α-amino-ß-hydroxyvaleric acid, a threonine analogue plus lysine. In this strain, HD had been altered so as to become feedback-resistant at the same time, resulting in the byproduction of threonine and isoleucine. The total amount of these aspartate family amino acids was higher on molar basis than that of lysine produced by strain No. 2–190.     

Metabolic engineering and profiling of rice with increased lysine

Lysine (Lys) is the first limiting essential amino acid in rice, a stable food for half of the world population. Efforts, including genetic engineering, have not achieved a desirable level of Lys in rice. Here, we genetically engineered rice to increase Lys levels by expressing bacterial lysine feedback‐insensitive aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS) to enhance Lys biosynthesis; through RNA interference of rice lysine ketoglutaric acid reductase/saccharopine dehydropine dehydrogenase (LKR/SDH) to down‐regulate its catabolism; and by combined expression of AK and DHPS and interference of LKR/SDH to achieve both metabolic effects. In these transgenic plants, free Lys levels increased up to ~12‐fold in leaves and ~60‐fold in seeds, substantially greater than the 2.5‐fold increase in transgenic rice seeds reported by the only previous related study. To better understand the metabolic regulation of Lys accumulation in rice, metabolomic methods were employed to analyse the changes in metabolites of the Lys biosynthesis and catabolism pathways in leaves and seeds at different stages. Free Lys accumulation was mainly regulated by its biosynthesis in leaves and to a greater extent by catabolism in seeds. The transgenic plants did not show observable changes in plant growth and seed germination nor large changes in levels of asparagine (Asn) and glutamine (Gln) in leaves, which are the major amino acids transported into seeds. Although Lys was highly accumulated in leaves of certain transgenic lines, a corresponding higher Lys accumulation was not observed in seeds, suggesting that free Lys transport from leaves into seeds did not occur.     

Genetic and amino-acid analysis of two maize threonine-overproducing,lysine-insensitive aspartate kinase mutants

The aspartate-derived amino-acid pathway leads to the production of the essential amino-acids lysine, methionine, threonine and isoleucine. Aspartate kinase (AK) is the first enzyme in this pathway and exists in isoforms that are feedback inhibited by lysine and threonine. Two maize (Zea mays L.) threonine-overproducing, lysine-insensitive AK mutants (Ask1-LT19 and Ask2-LT20) were previously isolated. The present study was conducted to determine the map location of Ask2 and to examine the amino-acid profiles of the Ask mutants. The threonine-overproducing trait conferred by Ask2-LT20 was mapped to the long arm of chromosome 2. Both mutants exhibited increased free threonine concentrations (nmol/mg dry weight) over wild-type. The percent free threonine increased from approximately 2% in wild-type kernels to 37–54% of the total free amino-acid pool in homozygous mutant kernels. Free methionine concentrations also increased significantly in homozygous mutants. Free lysine concentrations were increased but to a much lesser extent than threonine or methionine. In contrast to previous studies, free aspartate concentrations were observed to decrease, indicating a possible limiting factor in threonine synthesis. Total (free plus protein-bound) amino-acid analyses demonstrated a consistent, significant increase in threonine, methionine and lysine concentrations in the homozygous mutants. Significant increases in protein-bound (total minus free) threonine, methionine and lysine were observed in the Ask mutants, indicating adequate protein sinks to incorporate the increased free amino-acid concentrations. Total amino-acid contents (nmol/kernel) were approximately the same for mutant and wild-type kernels. In five inbred lines both Ask mutations conferred the threonine-overproducing phenotype, indicating high expressivity in different genetic backgrounds. These analyses are discussed in the context of the regulation of the aspartate-derived amino-acid pathway.     

Identification of a monofunctional aspartate kinase gene of Arabidopsis thaliana with spatially and temporally regulated expression

We screened a gene trap library of Arabidopsis thaliana and isolated a line in which a gene encoding a homologue of monofunctional aspartate kinase was trapped by the reporter gene. Aspartate kinase (AK) is a key enzyme in the biosynthsis of aspartate family amino acids such as lysine, threonine, isoleucine, and methionine. In plants, two types of AK are known: one is AK which is sensitive to feedback inhibition by threonine and carries both AK and homoserine dehydrogenase (HSD) activities. The other one is monofunctional, sensitive to lysine and synergistically S-adenosylmethionine, and has only AK activity. We concluded that the trapped gene encoded a monofunctional aspartate kinase and designated as AK-lys3, because it lacked the HSD domain and had an amino acid sequence highly similar to those of the monofunctional aspartate kinases ofA. thaliana. AK-lys3 was highly expressed in xylem of leaves and hypocotyls and stele of roots. Significant expression of this gene was also observed in trichomes after bolting. Slight expression of AK-lys3 was detected in vascular bundles and mesophyll cells of cauline leaves, inflorescence stems, sepals, petals, and stigmas. These results indicated that this aspartate kinase gene was not expressed uniformly but in a spatially specific manner.     

Concerted regulation of lysine and threonine synthesis in tobacco plants expressing bacterial feedback-insensitive aspartate kinase and dihydrodipicolinate synthase

The essential amino acids lysine and threonine are synthesized in higher plants by two separate branches of a common pathway. This pathway is primarily regulated by three key enzymes, namely aspartate kinase (AK), dihydrodipicolinate synthase (DHPS) and homoserine dehydrogenase (HSD), but how these enzymes operate in concert is as yet unknown. Addressing this issue, we have expressed in transgenic tobacco plants high levels of bacterial AK and DHPS, which are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts. Such expression of the bacterial DHPS by itself resulted in a substantial overproduction of lysine, whereas plants expressing only the bacterial AK overproduced threonine. When both bacterial enzymes were expressed in the same plant, the level of free lysine exceeded by far the level obtained by the bacterial DHPS alone. This increase, however, was accompanied by a significant reduction in threonine accumulation compared to plants expressing the bacterial AK alone. Our results suggested that in tobacco plants the synthesis of both lysine and threonine is under a concerted regulation exerted by AK, DHPS, and possibly also by HSD. We propose that the balance between lysine and threonine synthesis is determined by competition between DHPS and HSD on limiting amounts of their common substrate 3-aspartic semialdehyde, whose level, in turn, is determined primarily by the activity of AK. The potential of this molecular approach to increase the nutritional quality of plants is discussed.     

Lysine metabolism in a barley mutant resistant to S(2-aminoethyl)cysteine

Lysine and S(2-aminoethyl)cysteine (AEC) metabolism were investigated in normal barley (Hordeum vulgare L. cv. Bomi) and a hemozygous recessive AEC-resistant mutant (R906). Feedback regulation of lysine and threonine synthesis from [¹⁴C] acetate was unimpaired in plants of the mutant 3 d after germination. Seeds of Bomi and R906 contained similar total amounts of lysine, threonine, methionine and isoleucine. Concentrations of these amino acids in the soluble fraction of plants grown 6 d without AEC were also similar. The concentration of AEC in R906 plants was less than in the parent variety when both were grown in the presence of 0.25 mM AEC for 6 d. The uptake of [³H]AEC and [³H]lysine by roots of R906 was, respectively, 33% and 32% of that by Bomi roots whereas the uptake of these compounds into the scutellum was the same in both the mutant and its parent. The uptake of [³H]leucine and its incorporation into proteins was also the same in Bomi and R906 plants. These results suggest that a transport system specific for lysine and AEC but not leucine is altered or lost in roots of the mutant R906. AEC is incorporated into protein and this could be the reason for inhibition of growth rather than action as a false-feedback inhibitor of lysine biosynthesis.Abbreviations AEC S(2-aminoethyl)cysteine - LYS lysine - THR threonine     

Regulation of lysine synthesis in transgenic potato plants expressing a bacterial dihydrodipicolinate synthase in their chloroplasts

The essential amino acid lysine is synthesized in higher plants by a complex pathway that is predominantly regulated by feedback inhibition of two enzymes, namely aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). Although DHPS is thought to play a major role in this regulation, the relative importance of AK is not known. In order to study this regulation, we have expressed in the chloroplasts of transgenic potato plants a DHPS derived from Escherichia coli at a level 50-fold above the endogenous DHPS. The bacterial enzyme is much less sensitive to lysine inhibition than its potato counterpart. DHPS activity in leaves, roots and tubers of the transgenic plants was considerably higher and more resistant to lysine inhibition than in control untransformed plants. Furthermore, this activity was accompanied by a significant increase in level of free lysine in all three tissues. Yet, the extent of lysine overproduction in potato leaves was significantly lower than that previously reported in leaves of transgenic plants expressing the same bacterial enzyme, suggesting that in potato, AK may also play a major regulatory role in lysine biosynthesis. Indeed, the elevated level of free lysine in the transgenic potato plants was shown to inhibit the lysine-sensitive AK activity in vivo. Our results support previous reports showing that DHPS is the major rate-limiting enzyme for lysine synthesis in higher plants, but they suggest that additional plant-specific regulatory factors are also involved.