Herbicide-Resistant Mutations in Acetolactate Synthase Can Reduce Feedback Inhibition and Lead to Accumulation of Branched-Chain Amino Acids

Masaki Endo,Seiichi Toki,Tsutomu Shimizu,Tamaki Fujimori,Shuichi Yanagisawa

doi:10.4236/fns.2013.45067

Masaki Endo, Seiichi Toki + Show 3 more

Open Access

https://doi.org/10.4236/fns.2013.45067

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Abstract

The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that are critical for animal growth and development. Animals need to obtain BCAAs from their diet because they cannot synthesize them. Plants are the ultimate source of these amino acids. Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of BCAAs. The metabolic control of BCAA biosynthesis involves allosteric regulation of ALS by the end-products of the pathway, i.e., valine, leucine and isoleucine. ALS holoenzyme seems to consist of two large catalytic subunits and two small regulatory subunits. In a previous study, using homologous recombination dependent gene targeting we created rice plants in which W548Land S627I mutations were induced into the endogenous gene encoding the ALS catalytic subunit. These two amino acid substitutions conferred hypertolerance to the ALS-inhibiting herbicide bispyripac-sodium. In this study, we revealed that feedback regulation by valine and leucine was reduced by these two amino acid substitutions. Furthermore, in leaves and seeds of ALS mutants with W548Land/or S627I substitution, a 2- to 3-fold increase in BCAAs was detected. Our results suggest that the ALS catalytic subunit is also involved in feedback regulation of ALS, and that judicious modification of the regulatory and catalytic subunits of ALS-coding genes by gene targeting can lead to the efficient accumulation of BCAA in plants.

Highlights

Branched-chain amino acids (BCAAs) are essential amino acids that play important roles in protein anabolism and neurotransmitter biosynthesis in animals [1,2]
Protein extracts from wild-type and Acetolactate synthase (ALS) mutants [W548L/S627I (m)], in which W548L and S627I double mutations were introduced into the ALS catalytic subunit [16], as well as extracts of plants overexpressing ALS harboring the double mutation [W548L/ S627I] were examined for their sensitivities to valine and leucine
We succeeded in creating bispyripac-sodium hyper-resistant rice plants by introducing W548L and S627I mutations into the catalytic subunit of ALS by gene targeting [16]

Summary

Introduction

Branched-chain amino acids (BCAAs) are essential amino acids that play important roles in protein anabolism and neurotransmitter biosynthesis in animals [1,2]. Plant ALS is inhibited by all three BCAAs, with valine and leucine being potent [4]. As the key enzyme in BCAA biosynthesis and a primary target site of action for at least four structurally distinct classes of herbicides (sulfonylureas, imidazolinones, triazolopyrimidine sulfonamides and pyrimidinyl carboxy herbicides; for review, see [5]), ALS has been well characterized in certain organisms. Most ALS enzymes that have been characterized have both a catalytic subunit (65 kDa) and a smaller regulatory subunit, which varies in size between 9 and 54 kDa, depending on the species of origin. The plant ALS regulatory subunit has been suggested to be able to stabilize and enhance catalytic subunit [6,7,8]. The ALS regulatory subunit mediates end-product inhibition by BCAAs through a complex domain interaction, which in turn

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