A cytosolic ADP-glucose pyrophosphorylase is a feature of graminaceous endosperms, but not of other starch-storing organs

Abstract

The occurrence of an extra-plastidial isoform of ADP-glucose (Glc) pyrophosphorylase (AGPase) among starch-storing organs was investigated in two ways. First, the possibility that an extra-plastidial isoform arose during the domestication of cereals was studied by comparing the intracellular distribution of enzyme activity and protein in developing endosperm of noncultivated Hordeum species with that previously reported for cultivated barley (Hordeum vulgare). As in cultivated barley, the AGPase of H. vulgare subsp. spontaneum and Hordeum murinum endosperm is accounted for by a major extra-plastidial and a minor plastidial isoform. Second, the ratio of ADP-Glc to UDP-Glc was used as an indication of the intracellular location of the AGPase activity in a wide range of starch-synthesizing organs. The ratio is expected to be high in organs in which UDP-Glc and ADP-Glc are synthesized primarily in the cytosol, because the reactions catalyzed by AGPase and UDP-Glc pyrophosphorylase will be coupled and close to equilibrium. This study revealed that ADP-Glc contents and the ratio of ADP-Glc to UDP-Glc were higher in developing graminaceous endosperms than in any other starch-storing organs. Taken as a whole the results indicate that an extra-plastidial AGPase is important in ADP-Glc synthesis in graminaceous endosperms, but not in other starch-storing organs.

Figure 1

Subcellular distribution of AGPase activity in barley endosperm determined by the ratio method. Five samples of amyloplast-enriched pellet, each contaminated to a different extent with cytosol, were obtained from a single homogenate of endosperm of H. murinum or H. spontaneum as follows. A sample of the homogenate was taken for assays as described below, then the remainder was divided into four or five samples, each of which was centrifuged identically to produce pellets and supernatant fractions. The supernatant fractions were pooled together. The pellets were resuspended in 1 mL of material that consisted entirely of the supernatant fraction, or of a mixture of the supernatant fraction and AIM, or entirely of AIM. After rupture of the plastids and centrifugation, each of these four or five samples was assayed for a cytosolic marker enzyme, ADH or PFP, for the plastidial marker enzyme alkaline pyrophosphatase (APPase), and for AGPase. The distribution of AGPase activity was determined from the slopes of the plots of (AGPase activity)/(plastid marker enzyme activity) versus (cytosolic marker enzyme activity)/(plastid marker enzyme activity) for these samples (as shown), according to the following assumption: Total AGPase activity = C₁ + C₂(CMA/PMA), where C₁ = pAGPase/PMA and C₂ = cAGPase/CMA. PMA, Plastid marker enzyme activity in the unfractionated homogenate; CMA, cytosolic marker enzyme activity in the unfractionated homogenate; pAGPase, plastidial AGPase activity; cAGPase, cytosolic AGPase activity. Top graph, H. spontaneum. Bottom graph, H. murinum. Data from the graphs were as follows: H. spontaneum, y = 1.73x + 0.143 (hence, C₁ = 0.143 and C₂ = 1.73), CMA/PMA = 0.097; and H. murinum, y = 0.220x + 0.025 (hence, C₁ = 0.025 and C₂ = 0.220), CMA/PMA = 0.560.

Figure 2

Detection of AGPase small subunits by immunoblotting of fractions from amyloplast preparations. Homogenate, pellet, and supernatant fractions were prepared from endosperms of H. spontaneum and H. murinum as described in “Materials and Methods.” They were subjected to SDS-PAGE and blotted onto nitrocellulose membranes. Blots were developed with antiserum against the major form of the small subunit of AGPase from maize endosperm (the BRITTLE2 gene product), at a dilution of 1/15,000. To obtain the pellet fractions shown in B, samples of pellets were concentrated by freeze drying. Blots were repeated on several amyloplast preparations from each species. Typical, representative examples are shown. H, Homogenate; P, pellet; S, supernatant. A, Lanes all contain the same activity of the plastidial marker enzyme alkaline pyrophosphatase. Top, H. spontaneum. Bottom, H. murinum. B, Lanes contain the same activity of the cytosolic marker enzyme ADH. Samples are from a single plastid preparation from H. spontaneum.