Abstract
BackgroundBranched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets. Acetolactate synthase (ALS) is the first enzyme in the BCAA synthesis pathway. Although the functional contribution of ALS to BCAA biosynthesis has been extensively characterized, a comprehensive understanding of the regulation of this pathway at the molecular level is still lacking.ResultsTo characterize the regulatory processes governing ALS activity we utilized several complementary approaches. Using the ALS catalytic protein subunit as bait we performed a yeast two-hybrid (Y2H) screen which resulted in the identification of a set of interacting proteins, two of which (denoted as ALS-INTERACTING PROTEIN1 and 3 [AIP1 and AIP3, respectively]) were found to be evolutionarily conserved orthologues of bacterial feedback-regulatory proteins and therefore implicated in the regulation of ALS activity. To investigate the molecular role AIPs might play in BCAA synthesis in Arabidopsis thaliana, we examined the functional contribution of aip1 and aip3 knockout alleles to plant patterning and development and BCAA synthesis under various growth conditions. Loss-of-function genetic backgrounds involving these two genes exhibited differential aberrant growth responses in valine-, isoleucine-, and sodium chloride-supplemented media. While BCAA synthesis is believed to be localized to the chloroplast, both AIP1 and AIP3 were found to localize to the peroxisome in addition to the chloroplast. Analysis of free amino acid pools in the mutant backgrounds revealed that they differ in the absolute amount of individual BCAAs accumulated and exhibit elevated levels of BCAAs in leaf tissues. Despite the phenotypic differences observed in aip1 and aip3 backgrounds, functional redundancy between these loci was suggested by the finding that aip1/aip3 double knockout mutants are severely developmentally compromised.ConclusionsTaken together the data suggests that the two regulatory proteins, in conjunction with ALS, have overlapping but distinct functions in BCAA synthesis, and also play a role in pathways unrelated to BCAA synthesis such as sodium-ion homeostasis, extending to broader aspects of patterning and development.
Highlights
Branched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets
Two out of the eight classes, AIP1 (AT2G31810) and AIP3/ VAT1 (AT5G16290), showed high deduced protein similarity to prokaryotic and eukaryotic regulatory Acetolactate synthase (ALS) subunits (Additional file 1: Figure S1), suggesting that both these putative regulatory subunits associate with the ALS catalytic subunit in planta
In the absence of information pertaining to the binding affinities of the regulatory subunits with the catalytic subunit, we assessed the qualitative binding stability of both proteins using the indirect measure of Yeast two-hybrid (Y2H) signal intensity in yeast
Summary
Branched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets. The de novo synthesis of BCAAs has been an historical object of attention for two main reasons: this pathway is a known target for at least five independent classes of inhibitory herbicides [2] and, secondly, animals lack the necessary genes encoding enzymes for BCAA synthesis requiring that this class of essential amino acids be obtained via dietary intake These features have stimulated interest in the genetic manipulation of plant BCAA metabolism both for the generation of herbicide-tolerant crops as well as enhanced nutritional value through increased levels of storage BCAAs [3, 4]. While ALS enzymatic activity has been shown to be potently feedback-inhibited by Val, Ile, and Leu in vitro [9], the current literature lacks an in vivo-based assessment of this proposed model
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