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.

[1]  K. Turksen,et al.  Isolation and characterization , 2006 .

[2]  A. Hanson,et al.  Plant Metabolic Engineering—Entering the S Curve , 2002 .

[3]  B. Séve,et al.  Physiological roles of tryptophan in pig nutrition. , 1999, Advances in experimental medicine and biology.

[4]  G. M. Paulsen,et al.  Relationship of indoleacetic acid and tryptophan to dormancy and preharvest sprouting of wheat. , 2003, Functional plant biology : FPB.

[5]  S. Kaeppler,et al.  Genetic instability of plant tissue cultures: breakdown of normal controls. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Xiaohong Zhu,et al.  Increased Lysine Synthesis Coupled with a Knockout of Its Catabolism Synergistically Boosts Lysine Content and Also Transregulates the Metabolism of Other Amino Acids in Arabidopsis Seeds Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.009647 , 2003, The Plant Cell Online.

[7]  B. Eggum,et al.  Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a sunflower seed albumin gene. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[8]  C. Town,et al.  Isolation and Characterization of a Mutant of Arabidopsis thaliana Resistant to α-Methyltryptophan , 1992 .

[9]  J. Widholm,et al.  Targeting a nuclear anthranilate synthase alpha-subunit gene to the tobacco plastid genome results in enhanced tryptophan biosynthesis. Return of a gene to its pre-endosymbiotic origin. , 2001, Plant physiology.

[10]  B. Larkins,et al.  Genetic analysis of amino acid accumulation in opaque-2 maize endosperm. , 2001, Plant physiology.

[11]  P. Quail,et al.  Non-systemic expression of a stress-responsive maize polyubiquitin gene (Ubi-1) in transgenic rice plants , 1994, Plant Molecular Biology.

[12]  J. Widholm Relation between auxin autotrophy and tryptophan accumulation in cultured plant cells , 2004, Planta.

[13]  H. Pitkow,et al.  A review of physiological and metabolic effects of essential amino acids , 2005, Amino Acids.

[14]  N. Brisson,et al.  Redirection of tryptophan leads to production of low indole glucosinolate canola. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[15]  J. Ludwig-Müller,et al.  Indole-3-butyric acid in Arabidopsis thaliana , 1993, Plant Growth Regulation.

[16]  B. Larkins,et al.  Aspartate kinase 2. A candidate gene of a quantitative trait locus influencing free amino acid content in maize endosperm. , 2001, Plant physiology.

[17]  J. Widholm,et al.  Increasing tryptophan synthesis in a forage legume Astragalus sinicus by expressing the tobacco feedback-insensitive anthranilate synthase (ASA2) gene. , 2000, Plant physiology.

[18]  D. D. Mueller,et al.  Identification of l-Tryptophan as an Endogenous Inhibitor of Embryo Germination in White Wheat. , 1988, Plant physiology.

[19]  Board on Agriculture,et al.  Nutrient requirements of swine , 1964 .

[20]  B. Séve,et al.  Interactive effects of dietary levels of tryptophan and protein on voluntary feed intake and growth performance in pigs, in relation to plasma free amino acids and hypothalamic serotonin. , 1992, Journal of animal science.

[21]  H. Miyagawa,et al.  Quantification of Indole-3-Acetic Acid and Amino Acid Conjugates in Rice by Liquid Chromatography–Electrospray Ionization–Tandem Mass Spectrometry , 2005, Bioscience, biotechnology, and biochemistry.

[22]  K. Wakasa,et al.  Efficient Transformation of Suspension-cultured Rice Cells Mediated by Agrobacterium tumefaciens , 2001 .

[23]  Z. R. Sung,et al.  Relationship of indole-3-acetic acid and tryptophan concentrations in normal and 5-methyltryptophan-resistant cell lines of wild carrots , 2004, Planta.

[24]  K. Eder,et al.  Studies on the tryptophan requirement of piglets , 2001, Archiv fur Tierernahrung.

[25]  N. Brisson,et al.  Creation of a Metabolic Sink for Tryptophan Alters the Phenylpropanoid Pathway and the Susceptibility of Potato to Phytophthora infestans. , 1995, The Plant cell.

[26]  S. C. Falco,et al.  Transgenic Canola and Soybean Seeds with Increased Lysine , 1995, Bio/Technology.

[27]  E. Krebbers,et al.  Gene discovery and product development for grain quality traits. , 1999, Science.

[28]  F. Salamini,et al.  Plant biotechnology and breeding: allied for years to come. , 2003, Trends in plant science.

[29]  K. Ishihara,et al.  Science of the rice plant , 1993 .

[30]  T. Komari,et al.  Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. , 1994, The Plant journal : for cell and molecular biology.

[31]  H. Nemoto,et al.  New Rice Varieties for Whole Crop Silage Use in Japan , 2003 .

[32]  G. Glevarec,et al.  Genetic and Physiological Analysis of Germination Efficiency in Maize in Relation to Nitrogen Metabolism Reveals the Importance of Cytosolic Glutamine Synthetase , 2002, Plant Physiology.

[33]  R. Bandurski,et al.  Concentration of Indole-3-acetic Acid and Its Derivatives in Plants. , 1977, Plant physiology.

[34]  J. Slovin,et al.  Selection and Characterization of [alpha]-Methyltryptophan-Resistant Lines of Lemna gibba Showing a Rapid Rate of Indole-3-Acetic Acid Turnover , 1995, Plant physiology.

[35]  H. Miyagawa,et al.  Metabolic profiling of tryptophan-overproducing rice calli that express a feedback-insensitive alpha subunit of anthranilate synthase. , 2005, Plant & cell physiology.

[36]  J. Messing,et al.  Increasing maize seed methionine by mRNA stability. , 2002, The Plant journal : for cell and molecular biology.

[37]  K. Wakasa,et al.  Use of a feedback-insensitive α subunit of anthranilate synthase as a selectable marker for transformation of rice and potato , 2005, Molecular Breeding.

[38]  K. Wakasa,et al.  Characterization of rice anthranilate synthase alpha-subunit genes OASA1 and OASA2. Tryptophan accumulation in transgenic rice expressing a feedback-insensitive mutant of OASA1. , 2001, Plant physiology.

[39]  J. Widholm,et al.  A 5-methyltryptophan resistant rice mutant, MTR1, selected in tissue culture , 1987, Theoretical and Applied Genetics.

[40]  D. Rondeau,et al.  Physiological and molecular aspects of aspartate-derived amino acid metabolism during germination and post-germination growth in two maize genotypes differing in germination efficiency. , 2006, Journal of experimental botany.

[41]  G. Fink,et al.  A Myb homologue, ATR1, activates tryptophan gene expression in Arabidopsis. , 1998, Proceedings of the National Academy of Sciences of the United States of America.