Two distinct biosynthetic pathways for Phe in plants have been proposed: conversion of prephenate to Phe via phenylpyruvate or arogenate. The reactions catalyzed by prephenate dehydratase (PDT) and arogenate dehydratase (ADT) contribute to these respective pathways. The Mtr1 mutant of rice (Oryza sativa) manifests accumulation of Phe, Trp, and several phenylpropanoids, suggesting a link between the synthesis of Phe and Trp. Here, we show that the Mtr1 mutant gene (mtr1-D) encodes a form of rice PDT with a point mutation in the putative allosteric regulatory region of the protein. Transformed callus lines expressing mtr1-D exhibited all the characteristics of Mtr1 callus tissue. Biochemical analysis revealed that rice PDT possesses both PDT and ADT activities, with a preference for arogenate as substrate, suggesting that it functions primarily as an ADT. The wild-type enzyme is feedback regulated by Phe, whereas the mutant enzyme showed a reduced feedback sensitivity, resulting in Phe accumulation. In addition, these observations indicate that rice PDT is critical for regulating the size of the Phe pool in plant cells. Feeding external Phe to wild-type callus tissue and seedlings resulted in Trp accumulation, demonstrating a connection between Phe accumulation and Trp pool size.

/pdf/mutation-of-a-rice-gene-encoding-a-phenylalanine-709mh9kfaf.pdf

Mutation of a rice gene encoding a phenylalanine biosynthetic enzyme results in accumulation of phenylalanine and tryptophan.

A cDNA clone that encodes a feedback-insensitive anthranilate synthase (AS), ASA2 , isolated from a 5-methyl-tryptophan (Trp) (5MT)-resistant tobacco cell line under the control of the constitutive cauliflower mosaic virus 35S promoter, was introduced into the forage legume Astragalus sinicus by Agrobacterium rhizogenes with kanamycin selection. The 35S- ASA2 gene was expressed constitutively as demonstrated by northern-blot hybridization analyses and the presence of feedback-insensitive AS. Hairy root lines transformed with 35S- ASA2 grew in concentrations of up to 100 μm 5MT, whereas the controls were completely inhibited by 15 μm 5MT. Expression of the feedback-insensitive ASA2 resulted in a 1.3- to 5.5-fold increase in free Trp. Kinetic studies of the AS activity demonstrate the Trp feedback alterations and indicate that the ASA2 α-subunit can interact with the native A. sinicus β-subunit to form an active enzyme. The ASA2 transcript and high free Trp were also detected in the leaves, stems, and roots of plants regenerated from the transformed hairy roots. Thus, we show for the first time that ASA2 can be used to transform plants of a different species to increase the levels of the essential amino acid Trp and impart 5MT resistance.

http://www.plantphysiol.org/content/plantphysiol/123/3/1069.full.pdf

Increasing tryptophan synthesis in a forage legume Astragalus sinicus by expressing the tobacco feedback-insensitive anthranilate synthase (ASA2) gene.

A 5-methyltryptophan resistant Nicotiana anthranilate synthase genes are disclosed, in particular the ASA2 gene of Nicotiana tabacum . Constructs containing the genes are also disclosed. This genes are useful for transforming plant cells and producing transformed plants and progenies which, compared to untransformed plant cells and plants, are more resistant to tryptophan analogs and/or produce increased levels of tryptophan. The transformed seeds are also disclosed. The genes are also useful as selectable markers. The amino acid sequence of ASA2, purified ASA2, and recombinant ASA2 are also disclosed.

Nucleotide sequences encoding anthranilate synthase

Aromatic amino acids function as building blocks of proteins and as precursors for secondary metabolism. To obtain plants that accumulate tryptophan (Trp) and phenylalanine (Phe), we modified the biosynthetic pathways for these amino acids in rice and dicot species. By introducing a gene encoding a feedback-insensitive anthranilate synthase (AS) alpha subunit, we successfully obtained transgenic plants that over-accumulated Trp. In addition, we found mutant calli that accumulated Phe and Trp at high concentrations. The causal gene (mtr1-D) encoded an arogenate dehydratase (ADT)/prephenate dehydratase (PDT) that catalyzes the final reaction in Phe biosynthesis. The wild-type enzyme was sensitive to feedback inhibition by Phe, but the mutant enzyme encoded by mtr1-D was relatively insensitive. Further, detailed analysis of downstream secondary metabolism from Trp in rice revealed that the Trp pathway, by producing serotonin, is involved in the defense response against pathogenic infection. Based on these findings we propose that the reactions catalyzed by AS and ADT are critical regulatory points in the biosynthesis of Trp and Phe, respectively. In addition, detailed characterization of transgenic lines that accumulate these aromatic amino acids provided new insights into the regulation of downstream secondary metabolism, translocation of aromatic amino acids, and effects of accumulation of aromatic amino acids on various agronomic traits.

Metabolic engineering of the tryptophan and phenylalanine biosynthetic pathways in rice

A selectable marker, the ASA2 gene of Nicotiana tabacum, is provided for transforming plant cells. The ASA2 promoter sequence is also provided which is capable of directing tissue culture specific transcription of a downstream structural gene. Also disclosed are truncated forms of the ASA2 promoter which are capable of directing high level constitutive transcription of downstream structural genes. Constructs containing the above genes and promoters are also disclosed.

For selectable markers and promoters for plant tissue culture transformation

Despite the fact that rice is one of the easily manipulated cereals in tissue culture whereby plants can be readily regenerated from anther cultures (Niizeki and Oono 1968), from callus cultures (Nishi et al. 1968), and from protoplasts prepared from suspension cultures (Fujimura et al. 1985), only a very limited amount of selection for resistance to amino acids or amino acid analogs has been reported. One would think that rice could be improved in several ways by selecting for amino acid analog resistance since most resistant mutants overproduce the corresponding natural amino acid.

J. M. Widholm

Papers

Rice Mutants Resistant to Amino Acids and Amino Acid Analogs