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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.


AIP3 plays a dominant role during BCAA growth inhibition. Primary root inhibition was measured in aip mutants and wild type plants on BCAA-supplemented media. Percent primary root inhibition of 5-day-old seedlings grown on Leu-, Val-, and Ile-supplemented media for 5 days. Error bars represent standard deviation (n = 6) for three biological replicates. An asterisk indicates a significant difference from the wild type (Col-0), determined by the Student’s t test (P < 0.05)
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Fig5: AIP3 plays a dominant role during BCAA growth inhibition. Primary root inhibition was measured in aip mutants and wild type plants on BCAA-supplemented media. Percent primary root inhibition of 5-day-old seedlings grown on Leu-, Val-, and Ile-supplemented media for 5 days. Error bars represent standard deviation (n = 6) for three biological replicates. An asterisk indicates a significant difference from the wild type (Col-0), determined by the Student’s t test (P < 0.05)

Mentions: The ALS regulatory subunits have been shown to have two functional roles in the holoenzyme complex: they act to stabilize the ALS complex thus contributing to the enhancement of ALS catalytic activity, and are also responsible for BCAA-mediated end-point inhibition of ALS catalytic activity, most likely via the highly-conserved ACT domain [5, 6]. To investigate whether the two regulatory subunits may have different effects on ALS activity, we examined the axenic growth of aip knockout mutants in media supplemented with exogenous BCAAs. The analyses revealed that the aip3-1 line was relatively insensitive to primary root inhibition by Val and Ile in comparison to the Col-0 wild-type (Fig. 5 & Additional file 1: Figure S4). Indeed, aip3-1 exhibited enhanced growth in comparison to the wild type and aip1-2 in media supplemented with 250 μM and 500 μM Val. However, aip1-2 exhibited a pattern of primary root inhibition similar to wild-type plants. Notably, there were no observable differences in primary root elongation inhibition between the two aip mutants and Col-0 among seedlings germinated on Leu-supplemented media (Fig. 5 & Additional file 1: Figure S4). These root elongation-inhibition patterns were reproducible across three independent experiments, and suggest that AIP3 plays a principal role in Val- and Ile-mediated feedback inhibition in Arabidopsis roots.Fig. 5


Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development
AIP3 plays a dominant role during BCAA growth inhibition. Primary root inhibition was measured in aip mutants and wild type plants on BCAA-supplemented media. Percent primary root inhibition of 5-day-old seedlings grown on Leu-, Val-, and Ile-supplemented media for 5 days. Error bars represent standard deviation (n = 6) for three biological replicates. An asterisk indicates a significant difference from the wild type (Col-0), determined by the Student’s t test (P < 0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5384131&req=5

Fig5: AIP3 plays a dominant role during BCAA growth inhibition. Primary root inhibition was measured in aip mutants and wild type plants on BCAA-supplemented media. Percent primary root inhibition of 5-day-old seedlings grown on Leu-, Val-, and Ile-supplemented media for 5 days. Error bars represent standard deviation (n = 6) for three biological replicates. An asterisk indicates a significant difference from the wild type (Col-0), determined by the Student’s t test (P < 0.05)
Mentions: The ALS regulatory subunits have been shown to have two functional roles in the holoenzyme complex: they act to stabilize the ALS complex thus contributing to the enhancement of ALS catalytic activity, and are also responsible for BCAA-mediated end-point inhibition of ALS catalytic activity, most likely via the highly-conserved ACT domain [5, 6]. To investigate whether the two regulatory subunits may have different effects on ALS activity, we examined the axenic growth of aip knockout mutants in media supplemented with exogenous BCAAs. The analyses revealed that the aip3-1 line was relatively insensitive to primary root inhibition by Val and Ile in comparison to the Col-0 wild-type (Fig. 5 & Additional file 1: Figure S4). Indeed, aip3-1 exhibited enhanced growth in comparison to the wild type and aip1-2 in media supplemented with 250 μM and 500 μM Val. However, aip1-2 exhibited a pattern of primary root inhibition similar to wild-type plants. Notably, there were no observable differences in primary root elongation inhibition between the two aip mutants and Col-0 among seedlings germinated on Leu-supplemented media (Fig. 5 & Additional file 1: Figure S4). These root elongation-inhibition patterns were reproducible across three independent experiments, and suggest that AIP3 plays a principal role in Val- and Ile-mediated feedback inhibition in Arabidopsis roots.Fig. 5

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.