Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron-and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels.

As part of an in vivo study of carbohydrate metabolism during development of Zea mays L. kernels, quantities of nucleotides and nucleotide sugars were measured in endosperm extracts from normal, the single-mutant genotypes shrunken-7 (shl), shrunken-2 (shz), and brittle-7 (btl), and the multiple-mutant genotypes shlbtl, shzbtl, and shlsh2btl. Results showed that b t l kernels accumulated more than 13 times as much adenosine 5' diphosphoglucose (ADP-Clc) as normal kernels. Activity of starch synthase in btl endosperm was equal to that in endosperm extracts from normal kernels. Thus the ADP-Clc accumulation in btl endosperm cells was not d u e t o a deficiency in starch synthase. ADP-Clc content in extracts of s h l b t l endosperms was similar t o that in btl, but i n extracts of the sh2btl mutant kernels ADP-Clc content was much reduced compared to b t l (about 3 times higher than that in normal). Endosperm extracts from shlsh2bt7, kernels that are deficient i n both ADP-Clc pyrophosphorylase (ACPase) and sucrose synthase, had quantities of ADP-Clc much lower than in normal kernels. These results clearly indicate that AGPase is the predominant enzyme responsible for the in vivo synthesis of ADP-Clc in b t l mutant kernels, but SUC synthase may also contribute to the synthesis of ADP-Clc i n kernels deficient in ACPase.A number of maize (Zea mays L.) endosperm mutants that affect the quantity and quality of carbohydrates in the endosperm, as well as kernel development and morphology, have been identified and extensively studied (Shannon and Garwood, 1984). Mutants such as waxy (wx), amylose extender (ne), and sugary-l affect the quantity and branching characteristic of kernel polysaccharides (Shannon and Garwood, 1984). Severa1 other mutants involve a defect in the metabolism of sugars, resulting in the accumulation of Suc and the reduction in starch. For example, skl kernels are deficient in the major Suc synthase enzyme (Chourey and Nelson, 1976), and sh2 and brittle-2 mutant kernels are both deficient in AGPase (Preiss, 1991). The genetic lesion of b t l , another high-sugarllow-starch mutant, is not as well defined. Early screening of developing b t l kernels for the activity of various enzymes of carbohydrate metabolism and starch biosynthesis failed to identify a specific enzyme lesion. Developing b t l kernels, compared to normal, have been reported to be low in a starch granule-bound phospho-oligosaccharide synthase (Pan and Nelson, 1985) and starch debranching enzyme but twice as high in AGPase (Doehlert and Kuo, 1990). Sullivan et al. (1991) reported the isolation and analysis of the B t l gene from maize and showed that the protein with greatest similarity to the Btl-encoded protein is a yeast adenylate translocator. Li et al. (1992) showed that the in vitro translated B t l gene product could be imported into chloroplasts, where it was processed and localized to the inner envelope membrane. We have isolated amyloplast membranes from normal and b t l kernels and showed that the four most abundant amylo...