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.

Metabolic profiling of tryptophan-overproducing rice calli that express a feedback-insensitive alpha subunit of anthranilate synthase.

The profile of aromatic metabolites in calli was compared between wild-type rice (Oryza sativa cv. Nipponbare) and tryptophan-overproducing transgenic rice lines that express a gene (OASA1D) for a feedback-insensitive alpha subunit of anthranilate synthase. Metabolic profiling by high-performance liquid chromatography coupled with photodiode array detection of ultraviolet absorbance revealed a total of 71 peaks in both wild-type and transgenic calli. Only a limited effect on the pattern of major aromatic compounds was observed in tryptophan-accumulating transgenic rice lines, with the exception of an approximately 80-fold increase in the amount of tryptophan. Expression of OASA1D induced relatively small changes in several minor metabolites. One of the minor metabolites whose abundance was increased by OASA1D expression was purified and identified as a previously unknown indole-alkaloid glucoside. The levels of free and conjugated forms of indole-3-acetic acid (IAA), a plant hormone derived from the tryptophan biosynthetic pathway, were determined separately by liquid chromatography and tandem mass spectrometry (LC-MS/MS). The amounts of both free IAA and its conjugates were increased in the transgenic calli, suggesting that the activity of anthranilate synthase or the concentration of tryptophan (or both) is an important determinant of IAA biosynthesis.

Metabolic flux analysis of the phenylpropanoid pathway in elicitor-treated potato tuber tissue.

The effects of beta-1,3-oligosaccharide elicitor on the metabolism of phenylpropanoids in potato tuber were analyzed quantitatively, by monitoring the time-dependent changes in the levels of seven compounds. The elicitor treatment caused an increase in the pool size of octopamine and tyramine amides (N-p-coumaroyloctopamine, N-feruloyloctopamine, N-p-coumaroyltyramine and N-feruloyltyramine), as well as a decrease in that of chlorogenic acid and putrescine amides (caffeoylputrescine and feruloylputrescine). An analysis of metabolic flux using stable isotope labeling and liquid chromatography-spectrometry (LC-MS) detection clearly demonstrated that the changes in the pool size of these compounds were correlated with the changes in their flux for biosynthesis (Jin) upon elicitor treatment. The increase in Jin in the cases of octopamine and tyramine amides was accompanied by an increase in flux for the transformation (Jout), indicating a rapid turnover of these compounds in the elicitor-treated tuber tissue. The result of the flux analysis indicated that the actual activation of the biosynthesis of octopamine and tyramine amides after the elicitor treatment was greater than that estimated from the changes in their levels in the potato tissue. These findings suggest that these amide compounds and their metabolic derivatives play an important role in the defense-related metabolism of phenylpropanoids in potato.

Determination of potato glycoalkaloids using high-pressure liquid chromatography-electrospray ionisation/mass spectrometry.

A method for quantifying two toxic glycoalkaloids, alpha-solanine and alpha-chaconine, in potato (Solanum tuberosum) tuber tissue was developed using HPLC-electrospray ionisation (ESI)/MS. Potato samples were extracted with 5% aqueous acetic acid, and the extracts were subjected directly to HPLC-ESI/MS after filtration. By determining the intensities of the protonated molecules of alpha-solanine (m/z 868) and alpha-chaconine (m/z 852) using selected ion monitoring (positive ion mode), a sensitive assay was attained with detection limits of 38 and 14 ppb for the two glycoalkaloids, respectively. The high sensitivity and selectivity of MS detection effectively reduced the time of analysis thus enabling a high throughput assay of glycoalkaloids in potato tubers.

Metabolic flux analysis of the phenylpropanoid pathway in wound-healing potato tuber tissue using stable isotope-labeled tracer and LC-MS spectroscopy.

The metabolic flux of two phenylpropanoid metabolites, N-p-coumaroyloctopamine (p-CO) and chlorogenic acid (CGA), in the wound-healing potato tuber tissue was quantitatively analyzed by a newly developed method based upon the tracer experiment using stable isotope-labeled compounds and LC-MS. Tuber disks were treated with aqueous solution of L-phenyl-d(5)-alanine, and the change in the ratio of stable isotope-labeled compound to non-labeled (isotope abundance) was monitored for p-CO and CGA in the tissue extract by LC-MS. The time-dependent change in the isotope abundance of each metabolite was fitted to an equation that was derived from the formation and conversion kinetics of each compound. Good correlations were obtained between the observed and calculated isotope abundances for both p-CO and CGA. The rates of p-CO formation and conversion (i.e. fluxes) were 1.15 and 0.96 nmol (g FW)(-1) h(-1), respectively, and for CGA, the rates 4.63 and 0.42 nmol (g FW)(-1) h(-1), respectively. This analysis enabled a direct comparison of the biosynthetic activity between these two compounds.