Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-017-1022-6) contains supplementary material, which is available to authorized users.

Vitamin B6 and Its Role in Cell Metabolism and Physiology

“…The second example of a possible regulatory mechanism controlling PLP synthase activity is related to an acetolactate synthase (ALS), which has been found to interact with an Arabidopsis PDX1 protein in yeast-2-hybrid experiments [ 60 ]. ALS is the first enzyme in the branched-chain amino acid (BCAA) synthesis pathway, which leads to the production of leucine, isoleucine, and valine, for example.…”

“…VitB 6 is an essential co-factor for branched-chain amino acid transaminase (BCAT), the last step of BCAA synthesis [ 61 ]. Given that the results of the yeast-2-hybrid approach can be verified in planta, this finding could represent a novel connection between BCAA and vitB 6 biosynthesis [ 60 ]. As BCAAs are not only synthesized in plants, but also in various other organisms including fungi, bacteria, and archaea, this potential regulatory interplay may have broader implications.…”

Comparative acetylomic analysis reveals differentially acetylated proteins regulating anther and pollen development in kenaf cytoplasmic male sterility line

“…SCPs have also been implicated in the autolysis of cellular constituents during PCD, in brassinosteroid signaling, regulating cell elongation and seed development (Domínguez and Cejudo , Li et al , Bienert et al ). Branched‐chain amino‐acid aminotransferase (Unigene_052140; 3.24) catalyzes the synthesis or degradation of the branched chain amino acids leucine, isoleucine and valine, and thus plays important roles in various BPs (Dezfulian et al ). There were also many acetylated proteins detected in the present study that were involved in other processes, such as fatty acid metabolism, auxin transport, but we have refrained from discussing them in detail here due to the space limitations.…”

Peroxisomes: versatile organelles with diverse roles in plants

“…Arabidopsis peroxisomal 3-hydroxyisobutyryl (HIBYL)-CoA hydrolase 1 (CHY1), and probably its homologs CHY1H1 and CHY1H2 as well, can catabolize valine and possibly other BCAAs (Zolman et al, 2001;Lingner et al, 2011). Although BCAA synthesis is known to occur in chloroplasts, ALS-interacting protein 1 (AIP1) and AIP3 are dual localized to peroxisomes and chloroplasts and interact with acetolactate synthase (ALS), the first enzyme in BCAA synthesis, indicating that peroxisomes may have a role in BCAA synthesis besides degradation (Dezfulian et al, 2017). Based on known activity of its bacterial and mammalian homologs, it has been speculated that Arabidopsis sarcosine oxidase (SOX) oxidizes secondary or tertiary amino acids (Goyer et al, 2004).…”