Genetic analysis of pathway regulation for enhancing branched‐chain amino acid biosynthesis in plants

Abstract

SUMMARYThe branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that play critical roles in animal growth and development. Animals cannot synthesize these amino acids and must obtain them from their diet. Plants are the ultimate source of these essential nutrients, and they synthesize BCAAs through a conserved pathway that is inhibited by its end products. This feedback inhibition has prevented scientists from engineering plants that accumulate high levels of BCAAs by simply over-expressing the respective biosynthetic genes. To identify components critical for this feedback regulation, we performed a genetic screen for Arabidopsis mutants that exhibit enhanced resistance to BCAAs. Multiple dominant allelic mutations in the VALINE-TOLERANT 1 (VAT1) gene were identified that conferred plant resistance to valine inhibition. Map-based cloning revealed that VAT1 encodes a regulatory subunit of acetohydroxy acid synthase (AHAS), the first committed enzyme in the BCAA biosynthesis pathway. The VAT1 gene is highly expressed in young, rapidly growing tissues. When reconstituted with the catalytic subunit in vitro, the vat1 mutantcontaining AHAS holoenzyme exhibits increased resistance to valine. Importantly, transgenic plants expressing the mutated vat1 gene exhibit valine tolerance and accumulate higher levels of BCAAs. Our studies not only uncovered regulatory characteristics of plant AHAS, but also identified a method to enhance BCAA accumulation in crop plants that will significantly enhance the nutritional value of food and feed.

A Regulatory Hierarchy of the Arabidopsis Branched-Chain Amino Acid Metabolic Network

“…Leu biosynthesis branches off before the final transamination step of Val biosynthesis. In Arabidopsis, the three committed enzymes in BCAA biosynthesis, threonine deaminase (referred to in this work as OMR1 for L-Omethylthreonine resistant 1), AHAS, and IPMS, are feedback regulated, as demonstrated in vitro for Arabidopsis (Mourad and King, 1995;de Kraker et al, 2007;Chen et al, 2010;Lee and Duggleby, 2001). OMR1 activity is inhibited by Ile, and Val antagonizes this inhibition (Halgand et al, 2002;Garcia and Mourad, 2004).…”

Genome-Wide Analysis of Branched-Chain Amino Acid Levels inArabidopsisSeeds

“…BCATs catalyze reversible reactions that convert 2-oxo acids into their corresponding amino acids and vice versa (Diebold et al, 2002;Schuster and Binder, 2005;Binder, 2010), and this class of enzymes has been studied biochemically and through transgenic approaches in several plant species (Campbell et al, 2001;Diebold et al, 2002;Schuster and Binder, 2005;Knill et al, 2008;Gao et al, 2009;Binder, 2010;Chen et al, 2010;Gonda et al, 2010;Maloney et al, 2010;Fernie, 2011, 2012;Kochevenko et al, 2012a,b). These studies have defined roles for individual BCAT family members in amino acid and glucosinolate metabolism, hormone signaling, and volatile production (Binder et al, 2007;Knill et al, 2008;Gao et al, 2009;Binder, 2010).…”

Essential Role of Tissue-Specific Proline Synthesis and Catabolism in Growth and Redox Balance at Low Water Potential

“…g-Aminobutyrate (GABA) also could not restore root elongation (see below), and the Pro-related amino acids Glu, Gln, Arg, and Orn were strongly inhibitory to root elongation. Inhibition of plant growth by exogenous amino acids has been observed in a number of studies (Bonner and Jensen, 1997;Chen et al, 2010), and this is likely related to feedback inhibition or disturbance of metabolic equilibrium caused by applied amino acids or disruption of metabolite transport. Such observations also illustrate the uniqueness of Pro in promoting growth at low c w .…”