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

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ABSTRACT

Background: 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. 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.

Results: To 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.

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

No MeSH data available.


Related in: MedlinePlus

Protein amino acid profiles of two aip1-2 and aip3 mutants. Concentration of 20 free protein amino acids (nmol mg−1) in Col-0, aip1-2 and aip3-1 in leaves. Average values ± SD of three biological replicates are given. An asterisk indicates a significant difference from the wild type, determined by the Student’s t test (P < 0.05)
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Fig6: Protein amino acid profiles of two aip1-2 and aip3 mutants. Concentration of 20 free protein amino acids (nmol mg−1) in Col-0, aip1-2 and aip3-1 in leaves. Average values ± SD of three biological replicates are given. An asterisk indicates a significant difference from the wild type, determined by the Student’s t test (P < 0.05)

Mentions: Given our finding that the root growth of aip3-1 mutants was resistant to Val and Ile inhibition, we reasoned this might be the consequence of a loss of feedback inhibition by BCAAs on the biosynthesis pathway. If true, one would hypothesize that mutant aip1 and aip3 plants would exhibit elevated steady-state levels of BCAAs in plant tissues. To investigate this possibility, we measured the abundance of free amino acids in leaf tissues from 28-day-old aip1-2 and aip3-1 plant genetic lines using an LC-MS/MS-based approach (Fig. 6). The results revealed a modest increase in the abundance of Val and Leu in the leaves of aip3-1 background, and an approximate two-fold increase in the abundance of Val and Leu in the aip1-2 mutant. Taken together, the results suggest that, while AIP1 and AIP3 genes have overlapping functions, they have also acquired distinct roles in the regulation of BCAA synthesis.Fig. 6


Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development
Protein amino acid profiles of two aip1-2 and aip3 mutants. Concentration of 20 free protein amino acids (nmol mg−1) in Col-0, aip1-2 and aip3-1 in leaves. Average values ± SD of three biological replicates are given. An asterisk indicates a significant difference from the wild type, determined by the Student’s t test (P < 0.05)
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getmorefigures.php?uid=PMC5384131&req=5

Fig6: Protein amino acid profiles of two aip1-2 and aip3 mutants. Concentration of 20 free protein amino acids (nmol mg−1) in Col-0, aip1-2 and aip3-1 in leaves. Average values ± SD of three biological replicates are given. An asterisk indicates a significant difference from the wild type, determined by the Student’s t test (P < 0.05)
Mentions: Given our finding that the root growth of aip3-1 mutants was resistant to Val and Ile inhibition, we reasoned this might be the consequence of a loss of feedback inhibition by BCAAs on the biosynthesis pathway. If true, one would hypothesize that mutant aip1 and aip3 plants would exhibit elevated steady-state levels of BCAAs in plant tissues. To investigate this possibility, we measured the abundance of free amino acids in leaf tissues from 28-day-old aip1-2 and aip3-1 plant genetic lines using an LC-MS/MS-based approach (Fig. 6). The results revealed a modest increase in the abundance of Val and Leu in the leaves of aip3-1 background, and an approximate two-fold increase in the abundance of Val and Leu in the aip1-2 mutant. Taken together, the results suggest that, while AIP1 and AIP3 genes have overlapping functions, they have also acquired distinct roles in the regulation of BCAA synthesis.Fig. 6

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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. 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.

Results: To 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.

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

No MeSH data available.


Related in: MedlinePlus