RRC ID 54155
Author Tuncel A, Kawaguchi J, Ihara Y, Matsusaka H, Nishi A, Nakamura T, Kuhara S, Hirakawa H, Nakamura Y, Cakir B, Nagamine A, Okita TW, Hwang SK, Satoh H.
Title The rice endosperm ADP-glucose pyrophosphorylase large subunit is essential for optimal catalysis and allosteric regulation of the heterotetrameric enzyme.
Journal Plant Cell Physiol
Abstract Although an alternative pathway has been suggested, the prevailing view is that starch synthesis in cereal endosperm is controlled by the activity of the cytosolic isoform of ADPglucose pyrophosphorylase (AGPase). In rice, the cytosolic AGPase isoform is encoded by the OsAGPS2b and OsAGPL2 genes, which code for the small (S2b) and large (L2) subunits of the heterotetrameric enzyme, respectively. In this study, we isolated several allelic missense and nonsense OsAGPL2 mutants by N-methyl-N-nitrosourea (MNU) treatment of fertilized egg cells and by TILLING (Targeting Induced Local Lesions in Genomes). Interestingly, seeds from three of the missense mutants (two containing T139I and A171V) were severely shriveled and had seed weight and starch content comparable with the shriveled seeds from OsAGPL2 null mutants. Results from kinetic analysis of the purified recombinant enzymes revealed that the catalytic and allosteric regulatory properties of these mutant enzymes were significantly impaired. The missense heterotetramer enzymes and the S2b homotetramer had lower specific (catalytic) activities and affinities for the activator 3-phosphoglycerate (3-PGA). The missense heterotetramer enzymes showed more sensitivity to inhibition by the inhibitor inorganic phosphate (Pi) than the wild-type AGPase, while the S2b homotetramer was profoundly tolerant to Pi inhibition. Thus, our results provide definitive evidence that starch biosynthesis during rice endosperm development is controlled predominantly by the catalytic activity of the cytoplasmic AGPase and its allosteric regulation by the effectors. Moreover, our results show that the L2 subunit is essential for both catalysis and allosteric regulatory properties of the heterotetramer enzyme.
Volume 55(6)
Pages 1169-83
Published 2014-6-1
DOI 10.1093/pcp/pcu057
PII pcu057
PMID 24747952
MeSH Allosteric Regulation Amino Acid Sequence Catalysis Codon, Nonsense Endosperm / enzymology Endosperm / genetics Glucose-1-Phosphate Adenylyltransferase / genetics* Glucose-1-Phosphate Adenylyltransferase / isolation & purification Glucose-1-Phosphate Adenylyltransferase / metabolism Isoenzymes Kinetics Models, Structural Molecular Sequence Data Mutation, Missense Oryza / enzymology* Oryza / genetics Phenotype Plant Proteins / genetics Plant Proteins / isolation & purification Plant Proteins / metabolism Polymerization Recombinant Proteins Seeds / enzymology Seeds / genetics Sequence Alignment Starch / metabolism*
IF 4.062
Times Cited 24
Rice Induced mutation line