The Rice Endosperm ADP-Glucose Pyrophosphorylase Large Subunit is Essential for Optimal Catalysis and Allosteric Regulation of the Heterotetrameric Enzyme

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.

Analysis of the rice ADPglucose transporter (OsBT1) indicates the presence of regulatory processes in the amyloplast stroma that control ADPglucose flux into starch

“…The shriveled grain phenotype of shr3 is similar to that seen for the mutant lines EM541 (shr1) and EM22 (shr2), which also produce shrunken seeds due to the lack of large (L2) and small (S2b) subunits, respectively, of the endosperm cytosolic AGPase (Kawagoe et al, 2005;Tuncel et al, 2014). To determine whether EM1093 contained a mutation in one of the structural genes for the major cytoplasmic AGPase, the EM1093 line was crossed with EM541 (Tuncel et al, 2014) or EM22 (Kawagoe et al, 2005), marker lines for the shr1 and shr2 loci, respectively.…”

“…1A). This phenotype is likely caused by a defect in one of the major enzymes involved in starch biosynthesis (Kawagoe et al, 2005;Tuncel et al, 2014). Starch levels were significantly decreased around 80% in shr3 seeds compared with the wild-type (Fig.…”

“…To determine whether EM1093 contained a mutation in one of the structural genes for the major cytoplasmic AGPase, the EM1093 line was crossed with EM541 (Tuncel et al, 2014) or EM22 (Kawagoe et al, 2005), marker lines for the shr1 and shr2 loci, respectively. F1 seeds derived from both the EM1093 3 EM541 and EM1093 3 EM22 crosses were normal in appearance, whereas F2 seeds demonstrated a ratio of wild-type to shrunken phenotype that fitted well to the expected 9:7 ratio of independent gene inheritance ( Table I).…”

Effects of organic fertilizer applications on starch changes in tobacco (Nicotiana tabacumL.) leaves during maturation

“…The first step is catalyzed by ADPase, a heterotetramer composed of small and large subunits. AGPase synthesizes substrate committed to starch synthesis (Emes et al 2003;Rosti et al 2006;Tuncel et al 2014). The second step is the actual polymerization of starch, catalyzed by DBE, SBE and SS.…”

WB1, a Regulator of Endosperm Development in Rice, Is Identified by a Modified MutMap Method

“…With the improvement of living standards, there is increasing demand for high-quality rice, with greater quality of exterior, eating, and processing. Quality of rice appearance and yield are negatively affected by abnormally developed endosperm, which leads to grains with decreased weight and floury endosperm [2,3,4,5,6,7,8], shrunken endosperm [9,10,11,12,13], and great chalkiness [14,15]. Therefore, elucidating the mechanisms of endosperm development will be conducive to cultivating rice varieties with better appearance and higher yield.…”