By screening two rice (Oryza sativa L.) seed cDNA libraries, recombinant cDNA clones encoding the rice prolamine seed storage protein were isolated. Based on cross-hybridization and restriction enzyme map analyses, these clones can be divided into two homology classes. All clones contain a single open reading frame encoding a putative rice prolamine precursor (molecular weight = 17,200) possessing a typical 14-amino acid signal peptide. The deduced primary structures of both types of prolamine polypeptides are devoid of repetitive sequences, a feature prevalent in other cereal prolamines. Clones of these two homology classes diverge mainly by insertions/deletions of short nucleotide stretches and point mutations. An isolated genomic clone about 15.5 kilobases in length displays a highly conserved 2.5-kilobase EcoRI fragment, repeated in tandem four times, each containing the prolamine coding sequence. Close homology is exhibited by the coding segments of the genomic and cDNA sequences, although the 5' ends of the untranslated regions are widely divergent. The sequence heterogeneity displayed by these genomic and cDNA clones and large gene copy number (-80-100 copies/haploid genome) indicate that the rice prolamines are encoded by a complex multigene family.Prolamines, typified by their solubility in alcohol solutions, are the major seed storage proteins in most of the cereals. These proteins accumulate during endosperm development and serve as a source of nitrogen, carbon, and sulfur for the young developing seedling (12,26). The rice prolamines have molecular sizes of about 12 to 17 kD and, as seen for other cereal prolamines, contain a high mole percentage of glutamine residues and low levels oflysine, histidine, cysteine, and methionine (18,22). They are initially synthesized around 10 DAF (17, 31) and are deposited in protein bodies formed by direct dilation of the RER lumen (13). SDS-PAGE analysis of in vitro translation products purified by immunoprecipitation using a rice prolamine antibody revealed the synthesis of a 16 kD precursor form presumably containing a single peptide (14,32).Recently, we showed (21) that the rice prolamines are immunologically distinct from other cereal prolamines. DNA sequence analysis of a single near full length prolamine cDNA clone revealed that the derived primary sequence of the rice prolamine did not exhibit significant homology to prolamines from the major cereals ( 1). poly(A)+RNA and its ligation to lambda gt 11 arms or to plasmid vector were performed as described by Huynh et al. (9) and Heidecker and Messing (6), respectively. Lambda gt 11 recombinants were plated on host strain Y1090 at a density of 3 x I04 plaque-forming units/90-mm plate and screened with a partially purified rice prolamine antiserum as described (9). Screening of the cDNA library with a radiolabeled cDNA insert was performed by an established procedure as described ( 19). Dot Blot Hybridization. Selected plasmids containing prolamine cDNA inserts were immobilized onto Zeta-Probe membrane ...

Developing wheat (Triticum aestivum L.) endosperm was examined using ultrathin sections prepared from tissues harvested at 5, 9, 16 and 25 d after flowering. Protein bodies were evident by 9 d and displayed a variety of membranous structures and inclusions. The Golgi apparatus was a prominent organelle at all stages, and by 9 d was associated with small electron-dense inclusions. By immunocytochemical techniques, gliadin (wheat prolamine) was localized within these vesicles and in homogeneous regions of protein bodies, but not in the lumen of the rough endoplasmic reticulum. The protein bodies appear to enlarge by fusion of smaller protein bodies resulting in larger, irregular-shaped organelles. The affinity of the Golgi-derived vesicles for gliadin-specific probes during the period of maximal storage-protein synthesis and deposition indicates that this organelle includes the bulk, if not all, of the gliadin produced. The involvement of the Golgi apparatus in the packaging of gliadins into protein bodies indicates a pathway which differs from the mode of prolamine deposition in other cereals such as maize, rice and sorghum, and resembles the mechanism employed for the storage of rice glutelin and legume globulins.

The subcellular localization of ADPglucose pyrophosphorylase, a key regulatory enzyme in starch biosynthesis, was determined in developing potato tuber cells by immunocytochemical localization techniques at the light microscopy level. Specific labeling of ADPglucose pyrophosphorylase by either immunofluorescence or immunogold followed by silver enhancement was detected only in the amyloplasts and indicates that this enzyme is located exclusively in the amyloplasts in developing potato tuber cells. Labeling occurred on the starch grains and, in some instances, specific labeling patterns were evident which may be related to sites active in starch deposition.

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