ACCUMULATION OF GLIADIN AND ADPGLUCOSE PYROPHOSPHORYLASE MESSENGER RNAS

The developmental accumulation pattern of messenger RNA transcripts and polypeptides for wheat gliadins and ADPglucose pyrophosphorylase was determined using cDNA and antibody probes. Gliadin mRNA was detected on Northern and RNA dot blots at 3 days after flowering, it increased 100-fold by 10 days and decreased subsequent to 14 days. The abundant mRNAs encoding a/,#and -y-type gliadins and mRNA for ADPglucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis, accumulated coordinately. Despite the coordinate accumulation of their mRNA transcripts, the accumulation of gliadin and ADPglucose pyrophosphorylase polypeptides, as determined by Western blot, differed significantly. The time at which gliadin and ADPglucose pyrophosphorylase mRNAs began accumulating was also the time when the overall pattern of gene expression, as seen by two-dimensional gel electrophoresis of in vitro translation products, changed most significantly. However, the accumulation of a number of other mRNAs or polypeptides having unknown function occurred at other times during endosperm development. The pattern of expression in the earliest stages ofdevelopment was strikingly similar to that of coleoptile, another rapidly growing, nonphotosynthetic tissue. Thus, the pattern of gene expression reflects the program of development observed cytologically. Wheat endosperm is a relatively homogeneous tissue and a good model system for studying developmental gene regulation in plants. The cytological events of endosperm development are divided into three distinct phases (3, 5). In the first few DAF2 endosperm tissue is formed as a multinucleated syncytium, followed by cellularization and multiple rounds of mitotic division. During the middle phase, between about 10 to 25 DAF, large amounts of storage protein, starch, and other seed reserves accumulate. The late phase (26-40 DAF) is a period ofdehydration and seed maturation. The wheat seed storage proteins that accumulate during the middle phase consist primarily of the alcohol-soluble gliadins, which are encoded by a family of at least 40 genes (27) on separate genetic loci located on two different chromosomes (21). Although gliadin mRNAs accumulate early in the middle phase of endosperm development (8), it is not clear whether the individual genes are coordinately or differentially expressed. We have recently characterized the sequence diversity of the a/fland y' Supported by Grants PCM-8215772 and DCB-8502244 from the National Science Foundation. Scientific paper No. 7041, Project 0590, of the College of Agriculture Research Center, Washington State University. 2Abbreviations: DAF, days after flowering; kb, kilobases. type gliadin gene subfamilies which encode most of the gliadin polypeptides (19). Individual genes within each subfamily are highly homologous, but can be resolved into additional homology groups by the presence of unique restriction sites, crosshybridization analysis at high stringency, and direct DNA sequence comparison. With probes specific for various homology groups within these subfamilies in hand, a study of the developmental regulation ofthis large gene family has now been initiated. Starch also accumulates at approximately the same time as the gliadins during the middle phase ofendosperm development (26). ADPglucose pyrophosphorylase, a key regulatory enzyme in starch biosynthesis (23), has a much higher activity in endosperm than other wheat tissues (26) and therefore provides a useful developmental marker in addition to the gliadins. Before attempting to answer the fundamental question of what cisand trans-acting factors determine the tissue and developmental specificity of gene expression in wheat endosperm, it is important to determine the temporal pattern of expression of the different endosperm-specific genes. In this study we have quantified the developmental accumulation of protein and mRNA for a/fland y-type gliadins as well as for ADPglucose pyrophosphorylase. The timing of accumulation of these mRNAs and polypeptides was compared to the overall pattern of mRNA accumulation as revealed by twodimensional gel electrophoresis of in vitro translation products. The temporal pattern of mRNA and polypeptide accumulation conforms to the cytological picture of early rapid cell proliferation followed by a shift to the accumulation of storage reserves. MATERIALS AND METHODS Plant Material. Triticum aestivum L. var. Cheyenne was germinated, vernalized for 6 weeks at 7C, grown in a greenhouse. Heads at various stages after anthesis were collected and stored at -800C. Seeds were separated from pericarp and other maternal tissue under liquid N2. Three day seed was purified further by removing seeds retained on a No. 8 Tyler screen (2.4 mm pores). Isolation and Gel Electrophoresis of Endosperm Proteins. Gliadins were purified by 70% ethanol extraction followed by electrophoresis on pH 3.1 aluminum lactate (ICN Pharmaceuticals) polyacrylamide gels (15). The two predominant Coomassiestained bands corresponding to the a/fland y-gliadins were excised and the gel slices were washed with 100 mM Tris-HCl (pH 8), macerated, and soak-eluted at 37°C with 1% SDS, 0.05 M Tris-HCl (pH 7.9), 0.1 mM EDTA, 0.15 M NaCl. Eluted protein was precipitated with five volumes of cold acetone and sedimented at 28,000g for 15 min. The pellet was dried in vacuo and resuspended in 1% SDS to 1 mg/ml. Alcohol extracted proteins were analyzed using a two-dimensional gel system with the first dimension being a 20 mm sodium lactate gel (pH 3.1) (25). Thin strips were cut from the first dimension gels, equilibrated in SDS sample buffer, and applied to SDS polyacrylamide gels (12).