ADP-glucose pyrophosphorylase is located in the plastid in developing tomato fruit

Abstract

The subcellular location of activity and protein of ADP-glucose pyrophosphorylase (AGPase) in developing tomato (Lycopersicon esculentum) fruit was determined following a report that the enzyme might be present inside and outside the plastids in this organ. Plastids prepared from crude homogenates of columella and pericarp, the starch-accumulating tissues of developing fruit, contained 8% to 18% of the total activity of enzymes known to be confined to plastids, and 0.2% to 0.5% of the total activity of enzymes known to be confined to the cytosol. The proportion of the total activity of AGPase in the plastids was the same as that of the enzymes known to be confined to the plastid. When samples of plastid and total homogenate fractions were subjected to immunoblotting with an antiserum raised to AGPase, most or all of the protein detected was plastidial. Taken as a whole, these data provide strong evidence that AGPase is confined to the plastids in developing tomato fruit.

Figure 1

Starch content of developing tomato fruit. Starch contents of columellar plus placental tissue (left) and pericarp tissue (right) were measured during the phase of net starch accumulation. Each data point represents a measurement on a sample made up of tissue from at least four fruit. Values are milligrams per gram of fresh weight. Rates of starch synthesis presented in the text were calculated from the difference in starch content between tissue from fruit at 4 and 12 DPP.

Figure 2

Immunoblots of fractions from plastid preparations. Samples of homogenate (H), supernatant (S and S'), and pellet (P) fractions from plastid preparations were loaded onto a 7.5% (w/v) SDS-polyacrylamide gel, subjected to electrophoresis, and electroblotted onto nitrocellulose. For each tissue, sample sizes for lanes H, S, and P were adjusted so that on a given gel each lane contained the same activity of the plastid marker enzyme alkaline pyrophosphatase. Sample sizes for lanes S' were adjusted so that these lanes contained the same activity of the cytosolic marker enzyme alcohol dehydrogenase as the adjacent lanes P. AGPase is the band of approximately 50 kD. Checks on the nature of other bands on the blot are described in “Materials and Methods.” A, Immunoblot developed with antiserum raised against the BT2 protein of maize (small subunit of cytosolic AGPase), at a dilution of 1/500. Each lane contained an AGPase activity of approximately 12.5 nmol min⁻¹. On the right are prestained molecular mass markers, the masses of which are indicated in kilodaltons. B, Immunoblot developed with antiserum raised against AGPase from spinach leaf, at a dilution of 1/6,667. For pericarp, lanes P and S contained an AGPase activity of 39 nmol min⁻¹, and lane S' contained an AGPase activity of 0.23 nmol min⁻¹. For columella, lanes P and S contained an AGPase activity of 17 nmol min⁻¹, and lane S' contained an AGPase activity of 0.41 nmol min⁻¹. On the right are prestained molecular mass markers, the masses of which are indicated in kilodaltons.