Metabolomics for metabolically manipulated plants: effects of tryptophan overproduction

Atsushi Ishihara 0 1 2 3 Fumio Matsuda 0 1 2 3 Hisashi Miyagawa 0 1 2 3 Kyo Wakasa 0 1 2 3 0 K. Wakasa (&) Department of Agriculture, Tokyo University of Agriculture , 1737 Funako, Atsugi, Kanagawa 243-0034, Japan 1 A. Ishihara F. Matsuda H. Miyagawa Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University , Kyoto 606-8502, Japan 2 A. Ishihara F. Matsuda H. Miyagawa K. Wakasa CREST, Japan Science and Technology Agency , Tokyo 103-0027, Japan 3 Present Address: F. Matsuda Metabolome Research Group, RIKEN Plant Science Center , Yokohama, Kanagawa 230-0045, Japan 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) a 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. 1 Introduction Historically, plants have represented a critical nutritional and valuable life resource not only for human beings but for the domesticated animals they feed. Hence, enormous efforts are expended on improving the qualities and character of major crops. Whilst this improvement has relied on classical breeding techniques for most of the earlier historical period, recent advances in recombinant gene technology have made it possible to manipulate or improve the properties of plants in a relatively short time span. Genetic modification of crops through recombinant technology can be categorized by the following three goals. The first goal centers on endowing crops with properties typically absent in plants. For example, (1) crops producing insecticidal Bt toxins of bacterial origin have been developed to enhance herbivore resistance, and are now cultivated worldwide (Shelton et al. 2000); (2) a gene encoding an enzyme (5-enolpyruvylshikimate-3-phosphate synthase) that is insensitive to a specific herbicide is utilized to generate herbicide-resistant crops, thus alleviating weeding labor in the field (Dill 2005); (3) production of a biodegradable plastic polymer, poly-b-hydroxybutyrate (PBH), is undertaken by transforming plants with a series of bacterial genes involved in PBH production (Saruul et al. 2002). In a second goal, genetic modification is applied to improve the productivity of beneficial components that are present only in a trace amount. Targets include, not only essential amino acids and vitamins that are associated with the nutritional value of crops, but also secondary metabolites of pharmaceutical value (see sections below for further discussion on how understanding the regulation of biosynthesis of a given plant component is critical to efficacious enhancement of its production). Third, one can attempt to decrease the amount of harmful or wasteful components in plants. For example; (1) high levels of certain glucosinolates in Brassica can restrict their use as food and feed, and hence, reduction of their amounts by altering metabolic flux through their biosynthetic pathway is a goal (Chavadej et al. 1994); (2) modification of the content of lignin has attracted considerable attention, as lignin disturbs both the paper manufacturing process and digestion of feed crops in farm animals (Boudet andGrima-Pettenati 1996). For all three major goals, manipulation of metabolic function in a plant can cause changes in the activity, not only of the target pathway, but also of multiple pathways interacting directly or indirectly with the target pathway. Such interactions may involve, for example, competition for common precursors. When the activity of a target pathway is enhanced, this could result in the decrease in substrate supply to a competing pathway. On the other hand, suppression of a target pathway may result in increased substrate supply for a competing pathway. Any impact on feedback regulation through altered substrate levels is also likely to have pronounced and potentially more complicated effects on metabolite composition. In this review, we survey recent advances in plant metabolic engineering as a means to enhance the nutritional value of major crops. We also specifically discuss our recent successes in increasing the production of the essential amino acid, tryptophan (Trp), utilizing a mutant gene encoding the anthranilate synthase (AS) a subunit. The effects of activating Trp biosynthesis on the composition of the plant metabolome will be discussed in the context of our recent metabolite profiling analyses and to provide more general lessons for the future generation of genetically modified crops. 2 Increasing the nutritional value of crop plants by metabolic engineering Most animals, including humans, rely primarily on plants as a source of carbon. Plants, of course, can fix inorganic carbon to edible organic forms through photosynthesis. Enhancement of photosynthetic activity to produce and accumulate increased levels of carbohydrate has therefore represented one of the most important research subjects in metabolic engineering of crops (Matsuoka et al. 2001; Nunes-Nesi et al. 2005; Parry et al. 2007). Increasing seed oil content is also an issue of primary concern though, generally, breeding efforts are more typically directed to modifying fatty acid composition as a means to improve oil quality (Thelen and Ohlrogge 2002). Plants are also a valuable nitrogen source. For example, animals cannot synthesize many of the amino acids required for protein synthesis. Whilst carnivorous animals acquire amino acids and other organic nitrogen compounds by devouring their prey, herbivorous and omnivorous animals are dependent on plants for their supply of these essential amino acids. Yet plants do not always co (...truncated)