The Impact of the Branched-Chain Ketoacid Dehydrogenase Complex on Amino Acid Homeostasis in Arabidopsis

Abstract

The branched-chain amino acids (BCAAs) Leu, Ile, and Val are among nine essential amino acids that must be obtained from the diet of humans and other animals, and can be nutritionally limiting in plant foods. Despite genetic evidence of its importance in regulating seed amino acid levels, the full BCAA catabolic network is not completely understood in plants, and limited information is available regarding its regulation. In this study, transcript coexpression analyses revealed positive correlations among BCAA catabolism genes in stress, development, diurnal/circadian, and light data sets. A core subset of BCAA catabolism genes, including those encoding putative branched-chain ketoacid dehydrogenase subunits, is highly expressed during the night in plants on a diel cycle and in prolonged darkness. Mutants defective in these subunits accumulate higher levels of BCAAs in mature seeds, providing genetic evidence for their function in BCAA catabolism. In addition, prolonged dark treatment caused the mutants to undergo senescence early and overaccumulate leaf BCAAs. These results extend the previous evidence that BCAAs can be catabolized and serve as respiratory substrates at multiple steps. Moreover, comparison of amino acid profiles between mature seeds and darktreated leaves revealed differences in amino acid accumulation when BCAA catabolism is perturbed. Together, these results demonstrate the consequences of blocking BCAA catabolism during both normal growth conditions and under energy-limited conditions.The branched-chain amino acids (BCAAs) Leu, Ile, and Val are among nine amino acids essential for humans and other animals because they cannot be synthesized de novo (Harper et al., 1984). Plants synthesize BCAAs and are the main source of these essential nutrients in the diets of humans and agriculturally important animals. In addition to their nutritional value, BCAAs and BCAA-derived metabolites such as glucosinolates, fatty acids, and acyl sugars contribute to plant growth, development, defense, and flavor (Mikkelsen and Halkier, 2003;Taylor et al., 2004;Ishizaki et al., 2005;Slocombe et al., 2008;Araújo et al., 2010;Ding et al., 2012;Kochevenko et al., 2012).The BCAA biosynthetic pathway and its regulation have been investigated in Arabidopsis (Arabidopsis thaliana) and other plants for the past two decades, in large part because of the commercial importance of herbicides that inhibit acetohydroxy acid synthase, which is the committing enzyme of BCAA biosynthesis (Singh and Shaner, 1995;Aubert et al., 1997;Singh, 1999;McCourt et al., 2006;Tan et al., 2006;Binder, 2010;Chen et al., 2010;Yu et al., 2010). Strong correlations between the levels of free BCAAs were found in wildtype Arabidopsis seeds and tomato (Solanum lycopersicum) fruits Lu et al., 2008), which suggests coregulation of biosynthesis and/or degradation. This presumably is due, at least in part, to the fact that they share four common biosynthetic enzymes and three catabolic steps. However, despite long-term interest in the desirability of optim...

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

“…In plants, BCAAs and their derivatives contribute to growth, defense, and the production of food flavor components (Kang et al, 2006;Yoshikawa et al, 1995;Zeier, 2013;Kimball and Jefferson, 2006;Gonda et al, 2010;Galili et al, 2016). In addition, BCAA catabolism provides an alternative source of energy in plants under long-term dark treatment conditions (Peng et al, 2015;Kochevenko et al, 2012). Moreover, acetohydroxyacid synthase (AHAS), the committed enzyme of Val biosynthesis, is the target of four classes of commercial herbicides, and numerous plant herbicide resistance AHAS alleles have been reported (Jander et al, 2003).…”

“…Biosynthesis and catabolism both play roles in BCAA regulation in plants (Galili et al, 2016). Arabidopsis mutants blocked in BCAA degradation accumulate BCAAs in seeds of plants grown under standard environmental conditions (Angelovici et al, 2013;Gu et al, 2010;Lu et al, 2011) and in leaves of dark-treated plants (Peng et al, 2015;Araújo et al, 2010), implicating BCAA catabolism in seed BCAA homeostasis and as an alternative energy source under energy-deprived conditions. These catabolic enzymes are coregulated at steady state mRNA level under diel and extended darkness conditions as well as during seed development, consistent with the mutant results (Peng et al, 2015;Uygun et al, 2016).…”

“…Arabidopsis mutants blocked in BCAA degradation accumulate BCAAs in seeds of plants grown under standard environmental conditions (Angelovici et al, 2013;Gu et al, 2010;Lu et al, 2011) and in leaves of dark-treated plants (Peng et al, 2015;Araújo et al, 2010), implicating BCAA catabolism in seed BCAA homeostasis and as an alternative energy source under energy-deprived conditions. These catabolic enzymes are coregulated at steady state mRNA level under diel and extended darkness conditions as well as during seed development, consistent with the mutant results (Peng et al, 2015;Uygun et al, 2016). While less is known about the importance of genetic regulation of BCAA biosynthetic enzymes, the activities of committed enzymes of the BCAA network are sensitive to in vitro regulation by amino acid products (Less and Galili, 2008;Pratelli and Pilot, 2014).…”

Network-Guided GWAS Improves Identification of Genes Affecting Free Amino Acids

“…At high concentrations, this inhibition can lead to starvation of a downstream metabolic product, Met, which inhibits plant growth (Bright et al, 1982;Rognes et al, 1983;Heremans and Jacobs, 1995). In addition, several amino acid catabolic products, such as those emanating from branched-chain amino acids (BCAAs) or Lys degradation pathways, can be channeled toward cellular energy production under both standard and stress growth conditions (Angelovici et al, 2009(Angelovici et al, , 2011 Araújo et al, 2010;Peng et al, 2015).Reconstructions of seed metabolic networks in several model species and tissues have offered important insights into these underlying metabolic interactions and regulation Toubiana et al, 2013Toubiana et al, , 2015. For example, a correlation-based network metabolic [ reconstruction of FAA levels revealed that individual FAAs are strongly correlated during seed development compared with leaf or fruit development (Toubiana et al, 2012), which suggests a much tighter interaction of this metabolic network in seeds.…”

“…At high concentrations, this inhibition can lead to starvation of a downstream metabolic product, Met, which inhibits plant growth (Bright et al, 1982;Rognes et al, 1983;Heremans and Jacobs, 1995). In addition, several amino acid catabolic products, such as those emanating from branched-chain amino acids (BCAAs) or Lys degradation pathways, can be channeled toward cellular energy production under both standard and stress growth conditions (Angelovici et al, 2009(Angelovici et al, , 2011Araújo et al, 2010;Peng et al, 2015).…”