High-level tryptophan accumulation in seeds of transgenic rice and its limited effects on agronomic traits and seed metabolite profile

Metabolic manipulation of plants to improve their nutritional quality is an important goal of plant biotechnology. Expression in rice (Oryza sativa L.) of a transgene (OASA1D) encoding a feedback-insensitive alpha subunit of rice anthranilate synthase results in the accumulation of tryptophan (Trp) in calli and leaves. It is shown here that the amount of free Trp in the seeds of such plants is increased by about two orders of magnitude compared with that in the seeds of wild-type plants. The total Trp content in the seeds of the transgenic plants was also increased. Two homozygous lines, HW1 and HW5, of OASA1D transgenic rice were generated for characterization of agronomic traits and aromatic metabolite profiling of seeds. The marked overproduction of Trp was stable in these lines under field conditions, although spikelet fertility and yield, as well as seed germination ability, were reduced compared with the wild type. These differences in agronomic traits were small, however, in HW5. In spite of the high Trp content in the seeds of the HW lines, metabolic profiling revealed no substantial changes in the amounts of other phenolic compounds. The amount of indole acetic acid was increased about 2-fold in the seeds of the transgenic lines. The establishment and characterization of these OASA1D transgenic lines have thus demonstrated the feasibility of increasing the Trp content in the seeds of rice (or of other crops) as a means of improving its nutritional value for human consumption or animal feed.     

Characterization of tryptophan-overproducing potato transgenic for a mutant rice anthranilate synthase α-subunit gene (<Emphasis Type="Italic">OASA1D</Emphasis>)

Potato plants (Solanum tuberosum cv. May Queen) transgenic for OASA1D, which encodes a point mutant of an -subunit of rice (Oryza sativa) anthranilate synthase (AS, EC 4.1.3.27), were generated in order to determine the effects of the mutant gene on levels of free tryptophan (Trp) and AS activity in this important crop. Expression of OASA1D in potato induced a 2- to 20-fold increase in the amount of free Trp. This increase was likely due to a reduction in the sensitivity of AS containing the mutant -subunit to feedback inhibition by Trp. Nontargeted metabolite profiling by high-performance liquid chromatography coupled with ultraviolet photodiode array detection as well as targeted profiling by liquid chromatography coupled with mass spectrometry revealed no marked changes in the levels of other metabolites, with the exception of indole-3-acetic acid (IAA), in the transgenic plants. The level of IAA in the upper part of the shoot was increased by a factor of 8.3–39, depending on the transgenic lines, with no detectable effect on plant growth or development. The effects of transformation thus appeared limited to the biosynthesis of Trp and IAA, with the overall metabolic network in potato being virtually unaffected. These results suggest that transformation with OASA1D may prove effective for the breeding of crops with an increased level of free Trp.     

Production of indole alkaloids by metabolic engineering of the tryptophan pathway in rice

Tryptophan decarboxylase (TDC) converts tryptophan (Trp) into tryptamine, consequently increasing the metabolic flow of tryptophan derivatives into the production of secondary metabolites such as indole alkaloids. We inserted an expression cassette containing OsTDC, a putative tryptophan decarboxylase gene from rice, into an expression plasmid vector containing OASA1D, the feedback‐resistant anthranilate synthase alpha‐subunit mutant (OASA1D). Overexpression of OASA1D has been reported to significantly increase Trp levels in rice. The co‐expression of OsTDC and OASA1D in rice calli led to almost complete depletion of the Trp pool and a consequent increase in the tryptamine pool. This indicates that TDC inactivity is a contributory factor for the accumulation of Trp in rice transgenics overexpressing OASA1D. Metabolic profiling of the calli expressing OsTDC and OASA1D revealed the accumulation of serotonin and serotonin‐derived indole compounds (potentially pharmacoactive β‐carbolines) that have not been reported from rice. Rice calli overexpressing OASA1D:OASA1D is a novel system for the production of significant amounts of pharmacologically useful indole alkaloids in rice.     

Manipulation of amino acid composition in soybean seeds by the combination of deregulated tryptophan biosynthesis and storage protein deficiency

The ability of genetic manipulation to yield greatly increased concentrations of free amino acids (FAAs) in seeds of soybean was evaluated by introduction of a feedback-insensitive mutant enzyme of tryptophan (Trp) biosynthesis into two transformation-competent breeding lines deficient in major seed storage proteins. The storage protein-deficient lines exhibited increased accumulation of certain other seed proteins as well as of FAAs including arginine (Arg) and asparagine in mature seeds. Introduction of the gene for a feedback-insensitive mutant of an α subunit of rice anthranilate synthase (OASA1D) into the two high-FAA breeding lines by particle bombardment resulted in a >10-fold increase in the level of free Trp in mature seeds compared with that in nontransgenic seeds. The amount of free Trp in these transgenic seeds was similar to that in OASA1D transgenic seeds of the wild-type cultivar Jack. The composition of total amino acids in seeds of the high-FAA breeding lines remained largely unaffected by the expression of OASA1D with the exception of an increase in the total Trp content. Our results therefore indicate that the extra nitrogen resource originating from storage protein deficiency was used exclusively for the synthesis of inherent alternative nitrogen reservoirs such as free Arg and not for deregulated Trp biosynthesis conferred by OASA1D. The intrinsic null mutations responsible for storage protein deficiency and the OASA1D transgene affecting Trp content were thus successfully combined and showed additive effects on the amino acid composition of soybean seeds.     

Evaluation of amino acid content and nutritional quality of transgenic soybean seeds with high-level tryptophan accumulation

Anthranilate synthase (AS) is a key regulatory enzyme in tryptophan (Trp) biosynthesis and is subject to feedback inhibition by Trp. The gene encoding a mutated feedback-resistant α subunit of rice AS (OASA1D) under the control of either a soybean glycinin gene promoter or the 35S promoter of cauliflower mosaic virus for seed-specific or constitutive expression, respectively, was introduced into soybean [Glycine max (L.) Merrill] by particle bombardment. A total of seven different transgenic lines that showed markedly increased accumulation of free Trp in their seeds were developed. The overproduction of free Trp was stably inherited in subsequent generations without any apparent detrimental effect on plant growth or reproduction. The total Trp content of transgenic seeds was also about twice that of nontransgenic seeds, whereas the amount of protein-bound Trp was not substantially affected by OASA1D expression. In spite of the marked increase in free Trp content, metabolic profiling by high-performance liquid chromatography coupled with mass spectrometry revealed little change in the amounts of other aromatic compounds in the transgenic seeds. We developed a rapid and feasible system based on farmed rainbow trout to evaluate the nutritional quality of a limited quantity of transgenic soybean seeds. Supplementation of fish food with OASA1D transgenic soybean seeds or with nontransgenic seeds plus crystalline Trp increased the growth rate of the farmed fish. These results indicate transformation with OASA1D is a reliable approach to improve the nutritional quality of soybean (or of other grain legumes) for human and animal food.     

Metabolic changes in Arabidopsis thaliana expressing the feedback-resistant anthranilate synthase alpha subunit gene OASA1D

Anthranilate synthase (AS) is a key enzyme in tryptophan (Trp) biosynthesis. Metabolic changes in transgenic Arabidopsis plants expressing the feedback-resistant anthranilate synthase alpha subunit gene OASA1D were investigated with respect to Trp synthesis and effects on secondary metabolism. The Trp content varied depending on the transgenic line, with some lines showing an approximately 200-fold increase. The levels of AS activity in crude extracts from the transgenic lines were comparable to those in the wild type. On the other hand, the enzyme prepared from the lines accumulating high levels of Trp showed a relaxed feedback sensitivity. The AS activity, determined in the presence of 50 microM L-Trp, correlated well with the amount of free Trp in the transgenic lines, indicating the important role of feedback inhibition in control of Trp pool size. In Arabidopsis, Trp is a precursor of multiple secondary metabolites, including indole glucosinolates and camalexin. The amount of indol-3-ylmethyl glucosinolate (I3 M) in rosette leaves of the high-Trp accumulating lines was 1.5- to 2.1-fold greater than that in wild type. The treatment of the leaves with jasmonic acid resulted in a more pronounced accumulation of I3 M in the high-Trp accumulating lines than in wild type. The induction of camalexin formation after the inoculation of Alternaria brassicicola was not affected by the accumulation of a large amount of Trp. The accumulation of constitutive phenylpropanoids and flavonoids was suppressed in high-Trp accumulating lines, while the amounts of Phe and Tyr increased, thereby indicating an interaction between the Trp branch and the Phe and Tyr branch in the shikimate pathway.     

Use of a feedback-insensitive α subunit of anthranilate synthase as a selectable marker for transformation of rice and potato

A selection system based on a mutant rice gene for a feedback-insensitive subunit of anthranilate synthase (OASA1D) was developed for the transformation of rice and potato. Expression of OASA1D conferred resistance to the tryptophan analog 5-methyltryptophan (5MT) in transformed cells of rice and potato. The selection system based on OASA1D and 5MT was associated with a high transformation efficiency, a short time frame for the generation of transgenic plants, simple culture procedures, and it was as effective as hygromycin B selection in rice (monocotyledon) and kanamycin selection in potato (dicotyledon). Transgenic rice and potato plants established by 5MT selection had normal morphology and accumulated tryptophan when OASA1D was expressed under the control of a constitutive promoter. These results demonstrate the efficacy of OASA1D as a selectable marker and they suggest that the 5MT selection system based on this gene will prove applicable to a wide range of plant species and culture procedures.     

In vitro reconstitution of rice anthranilate synthase: distinct functional properties of the alpha subunits OASA1 and OASA2

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of various indole compounds including tryptophan. AS consists of two subunits, alpha and beta, and converts chorismate to anthranilate. Two or more AS alpha-subunit genes have been identified and characterized in several land plants. Although alpha subunits of AS induced by elicitation have been suggested to play significant roles in secondary metabolism, the biochemical and precise functional properties of individual AS isozymes have remained unclear. We have previously identified and characterized two AS alpha-subunit genes (OASA1 and OASA2) in rice (Oryza sativa ). To provide further insight into the enzymatic functions of AS isozymes in rice, we have now isolated rice cDNAs encoding the AS beta subunits OASB1 and OASB2 and reconstituted AS isozymes in vitro with the wheat germ cell-free system for protein expression. Both OASB subunits conferred glutamine-dependent AS activity on either OASA1 or OASA2, indicating the absence of a marked functional difference between the two beta subunits in terms of amidotransferase activity. Furthermore, both OASA subunits required assembly with a beta subunit to achieve maximal enzymatic activity even with NH(4)(+) as the amino donor. The V (max) and K (i) for tryptophan of the OASA1-OASB1 isozyme with glutamine as the amino donor, however, were 2.4 and 7.5 times, respectively, those of OASA2-OASB1, suggesting that AS isozymes containing OASA1 possess a higher activity and are less sensitive to feedback inhibition than those containing OASA2. Our biochemical characterization of reconstituted AS isozymes has thus revealed distinct functional properties of these isozymes in rice.     

Metabolomics for metabolically manipulated plants: effects of tryptophan overproduction

Advances in molecular breeding technologies have enabled manipulation of the concentrations of specific plant components by modifying the genes that play a key role in their production. This has provided new opportunities to enhance the nutritional quality of major crops. However, given that metabolic pathways form a highly integrated network, any alteration in a given biosynthetic pathway is most likely to effect secondary and unpredicted changes in the metabolite profile of other pathways. Metabolomics technologies can contribute to the efficient detection of such unexpected effects caused by genetic modification. This has relevance not only from the perspective of safety evaluations of newly developed crops, but to basic science focused on uncovering hitherto unknown regulatory mechanisms associated with the biosynthesis and catabolism of primary and secondary metabolites in plants. In this review, recent advances in plant metabolic engineering for the overproduction of tryptophan (Trp), one of the essential amino acids, are described. In particular, the efficacy of a transgene OASA1D that encodes a mutant anthranilate synthase (AS) α subunit of rice in specifically elevating levels of Trp without marked secondary effects on the metabolite profile of rice is demonstrated. Related topics, such as regulation of Trp biosynthesis, possible interactions between the biosyntheses of Trp and other aromatic amino acids, and translocation of Trp in are discussed based on findings derived from metabolomic analyses of Trp-overproducing transgenic plants.     

Tiller formation in rice is altered by overexpression of OsIAGLU gene encoding an IAA-conjugating enzyme or exogenous treatment of free IAA

Optimization of plant architecture is important for cultivation and yield of cereal crops in the field. Tillering is an essential factor used to determine the overall architecture of cereal crops. It has long been recognized that the development of branching patterns is controlled by the level and distribution of auxin within a plant. To better understand the relationship between auxin levels and tillering in rice, we examined rice plants with increased or decreased levels of free IAA. To decrease IAA levels, we selected the rice IAA-glucose synthase gene (OsIAGLU) from the rice genome database based on high sequence homology with IAA-glucose synthase from maize (ZmIAGLU), which is known to generate IAAglucose conjugate from free IAA. The OsIAGLU gene driven by the Cauliflower Mosaic Virus 35S promoter was transformed into a rice cultivar to generate transgenic rice plants constitutively over-expressing this gene. The number of tillers and panicles significantly increased in the transgenic lines compared to the wild-type plants, while plant height and panicle length decreased. These results indicate that decreased levels of free IAA likely enhance tiller formation in rice. To increase levels of free IAA, we treated rice plants with three different concentrations of exogenous IAA (1 μM, 10 μM and 100 μM) twice a week by spraying. Exogenous IAA treatment at concentrations of 10 μM and 100 μM significantly reduced tiller number in three different rice cultivars. These results indicate that exogenously applied IAA inhibits shoot branching in rice. Overall, auxin tightly controls tiller formation in rice in a negative way.     

Free and Conjugated Indoleacetic Acid (IAA) Contents in Transgenic Tobacco Plants Expressing the iaaM and iaaH IAA Biosynthesis Genes from Agrobacterium tumefaciens

The Agrobacterium tumefaciens T-DNA gene iaaM was introduced by leaf-disc transformation into transgenic tobacco (Nicotiana tabacum) plants expressing the iaaH gene. Regenerated calli were screened for the presence of indole-3-acetamide (IAM), by gas chromatography-multiple ion monitoring-mass spectrometry, and IAM-containing calli were further analyzed for free and conjugated indoleacetic acid (IAA). It was found that transgenic calli on average contained twice as much free IAA and three times more conjugated IAA than calli from wild-type plants. About 40% of the transformed calli could be regenerated to plants. The distribution of free and conjugated IAA was measured in transformed plants with a normal phenotype and compared with equivalent wild-type plants. The IAA content of transgenic plants was only slightly increased, whereas IAA-conjugate levels were enhanced significantly. These data suggest that conjugation of IAA may serve as a regulatory mechanism, contributing to maintenance of steady-state IAA pool sizes during tobacco growth and development.     

Two anthranilate synthase genes in Arabidopsis: defense-related regulation of the tryptophan pathway.

Arabidopsis thaliana has two genes, ASA1 and ASA2, encoding the alpha subunit of anthranilate synthase, the enzyme catalyzing the first reaction in the tryptophan biosynthetic pathway. As a branchpoint enzyme in aromatic amino acid biosynthesis, anthranilate synthase has an important regulatory role. The sequences of the plant genes are homologous to their microbial counterparts. Both predicted proteins have putative chloroplast transit peptides at their amino termini and conserved amino acids involved in feedback inhibition by tryptophan. ASA1 and ASA2 cDNAs complement anthranilate synthase alpha subunit mutations in the yeast Saccharomyces cerevisiae and in Escherichia coli, confirming that both genes encode functional anthranilate synthase proteins. The distributions of ASA1 and ASA2 mRNAs in various parts of Arabidopsis plants are overlapping but nonidentical, and ASA1 mRNA is approximately 10 times more abundant in whole plants. Whereas ASA2 is expressed at a constitutive basal level, ASA1 is induced by wounding and bacterial pathogen infiltration, suggesting a novel role for ASA1 in the production of tryptophan pathway metabolites as part of an Arabidopsis defense response. Regulation of key steps in aromatic amino acid biosynthesis in Arabidopsis appears to involve differential expression of duplicated genes.     

Metabolic flux analysis in plants using dynamic labeling technique: application to tryptophan biosynthesis in cultured rice cells

The concept and methodology of using dynamic labeling for the MFA of plant metabolic pathways are described, based on a case study to develop a method for the MFA of the tryptophan biosynthetic pathway in cultured rice cells. Dynamic labeling traces the change in the labeling level of a metabolite in a metabolic pathway after the application of a stable isotope-labeled compound. In this study, [1-(13)C] l-serine was fed as a labeling precursor and the labeling level of Trp was determined by using the LC-MS/MS. The value of metabolic flux is determined by fitting a model describing the labeling dynamics of the pathway to the observed labeling data. The biosynthetic flux of Trp in rice suspension cultured cell was determined to be 6.0+/-1.1 nmol (gFWh)(-1). It is also demonstrated that an approximately sixfold increase in the biosynthetic flux of Trp in transgenic rice cells expressing the feedback-insensitive version of anthranilate synthase alpha-subunit gene (OASA1D) resulted in a 45-fold increase in the level of Trp. In this article, the basic workflow for the experiment is introduced and the details of the actual experimental procedures are explained. Future perspectives are also discussed by referring recent advances in the dynamic labeling approach.     

Selection and Characterization of [alpha]-Methyltryptophan-Resistant Lines of Lemna gibba Showing a Rapid Rate of Indole-3-Acetic Acid Turnover

Turnover rate is an important aspect of the regulation of plant processes by plant growth substances. To study turnover of indole-3-acetic acid (IAA), two [alpha]-methyltryptophan-resistant lines (MTR1 and MTR2) of Lemna gibba were generated by nitrosomethyl urea treatment of an inbred line derived from L. gibba G-3. In this report we describe: (a) the development of a selection system using this near isogenic line of L. gibba; (b) techniques for chemical mutation of the lines and selection for [alpha]-methyltryptophan resistance; and (c) the partial characterization of the selected lines. MTR lines contained 3-fold higher levels of anthranilate synthase activity. The enzyme in the MTR lines required higher levels of tryptophan for feedback inhibition. MTR lines also contained 8-fold higher levels of tryptophan, 3-fold higher levels of free IAA, and similar levels of total IAA compared to the inbred line. Turnover rates in the inbred and selected lines were calculated, using the first-order rate equation, based on the decrease over time in isotopic enrichment of I3C6-IAA introduced into L. gibba during a 1-h pulse period. Isotope enrichment in IAA was determined by using gas chromatography-mass spectrometry. Both MTR lines had an approximately 10-fold higher rate of IAA turnover than the parent inbred line.