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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.

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AIP1 and AIP3 localize to both the chloroplast and peroxisome. a Subcellular localization of C-terminal YFP-tagged fusions of ALS, AIP1, and AIP3 in N. benthamiana. ALS-, AIP1-, and AIP3-YFP were expressed in Nicotiana leaves and analyzed by confocal microscopy. The localization of the AIPs and ALS are visualized by yellow fluorescence. Red fluorescence: excited chlorophyll emission to mark plastid location. YFP-ALS arrowheads: stromal localization of C-terminal YFP-tagged fusion, YFP-ALS. AIP3-YFP arrowheads: localization of AIP3-YFP outside of chloroplasts. Scale Bar, 20 μm (b) Subcellular localization of ALS, AIP1, AIP3, N-terminal YFP tagged fusions in Nicotiana leaves along with a peroxisomal marker (Px-CFP). Red fluorescence: excited chlorophyll emission to mark plastid location. Arrowheads: co-localization of Px-CFP with AIP1-YFP and AIP3-YFP
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Fig3: AIP1 and AIP3 localize to both the chloroplast and peroxisome. a Subcellular localization of C-terminal YFP-tagged fusions of ALS, AIP1, and AIP3 in N. benthamiana. ALS-, AIP1-, and AIP3-YFP were expressed in Nicotiana leaves and analyzed by confocal microscopy. The localization of the AIPs and ALS are visualized by yellow fluorescence. Red fluorescence: excited chlorophyll emission to mark plastid location. YFP-ALS arrowheads: stromal localization of C-terminal YFP-tagged fusion, YFP-ALS. AIP3-YFP arrowheads: localization of AIP3-YFP outside of chloroplasts. Scale Bar, 20 μm (b) Subcellular localization of ALS, AIP1, AIP3, N-terminal YFP tagged fusions in Nicotiana leaves along with a peroxisomal marker (Px-CFP). Red fluorescence: excited chlorophyll emission to mark plastid location. Arrowheads: co-localization of Px-CFP with AIP1-YFP and AIP3-YFP

Mentions: AIP1 and AIP3 proteins share ~80% identity at the deduced amino acid level (Additional file 1: Figure S2). The two deduced protein sequences were most divergent within the N-terminal 85 amino acids where sub-cellular localization signaling peptides typically reside. Although it has been well established that the initial catalytic reaction in BCAA synthesis is mediated by ALS enzymes in the chloroplast, no experimental data currently exist to localize individual ALS regulatory subunits to the chloroplast. We therefore evaluated the sub-cellular localization profile of ALS catalytic and regulatory subunits expressed as functional fusions with the auto-fluorescent yellow fluorescent protein (YFP). Whereas the C-terminal-tagged YFP-fusion of both regulatory and catalytic subunits localized to the plastid, the AIP3-YFP fusion protein also localized as dense signal-foci of varying sizes outside of the plastid (white arrows; Fig. 3a). The observed pattern resembled that of peroxisome localization, which led us to ask whether AIP3 also localizes to the peroxisome. We therefore assessed the co-localization of AIP3-YFP with a CFP-tagged peroxisome (Px-CFP) marker protein [15]. The results revealed that the YFP and CFP signals co-localize, suggesting a dual localization for AIP3-YFP to both the plastid and the peroxisome (Fig. 3b). To assess whether the potential masking of the otherwise exposed N-terminal plastid localization signal would have an effect on the peroxisomal localization of these proteins, we generated the reciprocal set of N-terminal YFP fusion proteins and assessed their sub-cellular localization. All three N-terminal YFP-tagged proteins were found to be mostly excluded from the plastid, with ALS-YFP accumulating only in the cytoplasm (Fig. 3b). However, both YFP-AIP1 and YFP-AIP3 fusion proteins were not only excluded from the chloroplast but also readily co-localized with the Px-CFP peroxisomal marker (Fig. 3b). Although both AIP1 and AIP3 regulatory proteins possess an identifiable N-terminal plastid localization domain [16], neither protein was found to contain a canonical PTS1 or PTS2 peroxisomal localization signal [17]. However, upon closer analysis, potential “cryptic” PTS1 motifs are found in the C-terminal region of both AIP1 (amino acid sequence S-K-Y) and AIP3 (S-T-Y) proteins, which may be important given that the SKY motif was recently shown to confer peroxisomal localization to a typically plastid-localized glucose-6-phosphate dehydrogenase protein [18]. The results suggest that both AIP1 and AIP3 regulatory subunits localize to the chloroplast as well as the peroxisome. Given the presence of a cryptic PTS1 motif, together with C-terminal peroxisomal targeting following masking of the plastid transit peptide, the structural and experimental results support the suggestion of a dual subcellular localization of these two proteins that in itself may be an object of regulation during development [19].Fig. 3


Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development
AIP1 and AIP3 localize to both the chloroplast and peroxisome. a Subcellular localization of C-terminal YFP-tagged fusions of ALS, AIP1, and AIP3 in N. benthamiana. ALS-, AIP1-, and AIP3-YFP were expressed in Nicotiana leaves and analyzed by confocal microscopy. The localization of the AIPs and ALS are visualized by yellow fluorescence. Red fluorescence: excited chlorophyll emission to mark plastid location. YFP-ALS arrowheads: stromal localization of C-terminal YFP-tagged fusion, YFP-ALS. AIP3-YFP arrowheads: localization of AIP3-YFP outside of chloroplasts. Scale Bar, 20 μm (b) Subcellular localization of ALS, AIP1, AIP3, N-terminal YFP tagged fusions in Nicotiana leaves along with a peroxisomal marker (Px-CFP). Red fluorescence: excited chlorophyll emission to mark plastid location. Arrowheads: co-localization of Px-CFP with AIP1-YFP and AIP3-YFP
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Fig3: AIP1 and AIP3 localize to both the chloroplast and peroxisome. a Subcellular localization of C-terminal YFP-tagged fusions of ALS, AIP1, and AIP3 in N. benthamiana. ALS-, AIP1-, and AIP3-YFP were expressed in Nicotiana leaves and analyzed by confocal microscopy. The localization of the AIPs and ALS are visualized by yellow fluorescence. Red fluorescence: excited chlorophyll emission to mark plastid location. YFP-ALS arrowheads: stromal localization of C-terminal YFP-tagged fusion, YFP-ALS. AIP3-YFP arrowheads: localization of AIP3-YFP outside of chloroplasts. Scale Bar, 20 μm (b) Subcellular localization of ALS, AIP1, AIP3, N-terminal YFP tagged fusions in Nicotiana leaves along with a peroxisomal marker (Px-CFP). Red fluorescence: excited chlorophyll emission to mark plastid location. Arrowheads: co-localization of Px-CFP with AIP1-YFP and AIP3-YFP
Mentions: AIP1 and AIP3 proteins share ~80% identity at the deduced amino acid level (Additional file 1: Figure S2). The two deduced protein sequences were most divergent within the N-terminal 85 amino acids where sub-cellular localization signaling peptides typically reside. Although it has been well established that the initial catalytic reaction in BCAA synthesis is mediated by ALS enzymes in the chloroplast, no experimental data currently exist to localize individual ALS regulatory subunits to the chloroplast. We therefore evaluated the sub-cellular localization profile of ALS catalytic and regulatory subunits expressed as functional fusions with the auto-fluorescent yellow fluorescent protein (YFP). Whereas the C-terminal-tagged YFP-fusion of both regulatory and catalytic subunits localized to the plastid, the AIP3-YFP fusion protein also localized as dense signal-foci of varying sizes outside of the plastid (white arrows; Fig. 3a). The observed pattern resembled that of peroxisome localization, which led us to ask whether AIP3 also localizes to the peroxisome. We therefore assessed the co-localization of AIP3-YFP with a CFP-tagged peroxisome (Px-CFP) marker protein [15]. The results revealed that the YFP and CFP signals co-localize, suggesting a dual localization for AIP3-YFP to both the plastid and the peroxisome (Fig. 3b). To assess whether the potential masking of the otherwise exposed N-terminal plastid localization signal would have an effect on the peroxisomal localization of these proteins, we generated the reciprocal set of N-terminal YFP fusion proteins and assessed their sub-cellular localization. All three N-terminal YFP-tagged proteins were found to be mostly excluded from the plastid, with ALS-YFP accumulating only in the cytoplasm (Fig. 3b). However, both YFP-AIP1 and YFP-AIP3 fusion proteins were not only excluded from the chloroplast but also readily co-localized with the Px-CFP peroxisomal marker (Fig. 3b). Although both AIP1 and AIP3 regulatory proteins possess an identifiable N-terminal plastid localization domain [16], neither protein was found to contain a canonical PTS1 or PTS2 peroxisomal localization signal [17]. However, upon closer analysis, potential “cryptic” PTS1 motifs are found in the C-terminal region of both AIP1 (amino acid sequence S-K-Y) and AIP3 (S-T-Y) proteins, which may be important given that the SKY motif was recently shown to confer peroxisomal localization to a typically plastid-localized glucose-6-phosphate dehydrogenase protein [18]. The results suggest that both AIP1 and AIP3 regulatory subunits localize to the chloroplast as well as the peroxisome. Given the presence of a cryptic PTS1 motif, together with C-terminal peroxisomal targeting following masking of the plastid transit peptide, the structural and experimental results support the suggestion of a dual subcellular localization of these two proteins that in itself may be an object of regulation during development [19].Fig. 3

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