Brian A. Larkins

Changes in the zein composition of protein bodies during maize endosperm development.

Zeins, the seed storage proteins of maize, are synthesized during endosperm development by membrane-bound polyribosomes and transported into the lumen of the endoplasmic reticulum, where they assemble into protein bodies. To better understand the distribution of the various zeins throughout the endosperm, and within protein bodies, we used immunolocalization techniques with light and electron microscopy to study endosperm tissue at 14 days and 18 days after pollination. Protein bodies increase in size with distance from the aleurone layer of the developing endosperm; this reflects a process of cell maturation. The protein bodies within the subaleurone cell layer are the smallest and contain little or no alpha-zein; beta-zein and gamma-zein are distributed throughout these small protein bodies. The protein bodies in cells farther away from the aleurone layer are progressively larger, and immunostaining for alpha-zein occurs over locules in the central region of these protein bodies. In the interior of the largest protein bodies, the locules of alpha-zein are fused. Concomitant with the appearance of alpha-zein in the central regions of the protein bodies, most of the beta- and gamma-zeins become peripheral. These observations are consistent with a model in which specific zeins interact to assemble the storage proteins into a protein body.

The opaque-2 mutation of maize differentially reduces zein gene transcription.

Zeins, the storage proteins of maize seed, are encoded by a large multigene family that is regulated developmentally and expressed in a tissue-specific manner during endosperm development. The synthesis of these proteins is affected by mutations, such as opaque-2, that cause a reduction in the accumulation of zein proteins and mRNAs. We used nuclear run-on transcription assays to analyze the expression of zein genes in developing normal and opaque-2 endosperms and to map the origin of these transcripts with respect to the coding and noncoding regions of the genes. These analyses demonstrate that zein gene expression is regulated transcriptionally and posttranscriptionally in developing endosperm. Transcription of genes encoding alpha-zeins is inhibited significantly in opaque-2 mutants, with expression of those encoding the M(r) 22,000 proteins being almost totally blocked. Other gene subfamilies were affected but to a lesser extent.

Interaction of the opaque-2 gene with starch-forming mutant genes on the synthesis of zein in maize endosperm

The combination of opaque-2 with starch-modified or starch-deficient mutants produced a cumulative and synergistic effect, respectively, in regulating zein synthesis. The double mutant, brittle-2 opaque-2, which almost completely prevented the synthesis of Z1 and Z2, had high RNase activity. The possible involvement of RNase in effecting zein synthesis is discussed.

Molecular Genetic Approaches to Maize Improvement

Part I. Introduction 1 Biotechnology and maize improvement - an overview by Robb Fraley 2 Maize tissue culture, transformation and regeneration - Todd Jones Part II. Transgenic traits 3 Insect resistance - Graham Head 4 Reduced phytic acid - Victor Raboy 5 Stress tolerance - John Mullet 6 Nitrogen use efficiency - Steve Moose 7 Protein nutritional quality and digestibility - Al Kriz 8 Over-expression of novel proteins - Elisabeth Hood 9 Global regulations of transgenic crops - Bruce Chassy Part III. Breeding and genetics 10 Doubled haploids - Ming Chang 11 Transposon tagging and reverse genetics - Mark Settles 12 EMS mutagenesis and TILLING - Cliff Weil 13 Association mapping - Ed Buckler 14 Genetic resources - Marty Sachs Part IV. The corn genome 15 Structure - Jo Messing 16 Molecular markers - Pat Schnable 17 Chromosome analysis - Kelly Dawe 18 Expression profiling - David Galbraith Part V. Molecular biology and physiological studies 19 Storage proteins - Brian Larkins 20 Starch - Curt Hannah 21 Lipids - Jill Deikman 22 Chloroplasts - Arnold Bendich Part VI. Biomass and energy 23 -Biomass and energy - Mike Ladisch

Maize Storage Proteins: Characterization and Biosynthesis

Developing seeds provide plant molecular biologists with useful model systems for studying the physiological and genetic mechanisms regulating the synthesis of specific plant proteins, i.e. seed storage proteins. These studies have perhaps even greater significance considering the importance of seed proteins in human and livestock nutrition. Maize storage proteins are interesting from both these aspects, since maize is an economically important crop and mutations affecting both the quantitative and qualitative synthesis of maize storage proteins have been identified (Mertz et al., 1964; Nelson et al., 1965). Our research in the last several years has been devoted to the characterization of these storage proteins and the reactions regulating their biosynthesis.

The zein proteins of maize endosperm

Abstract The storage protein in maize seed is composed of a group of alcohol-soluble polypeptides called zeins. These proteins are synthesized and deposited in the endosperm during seed development and are metabolized during seed germination.

Immunolocalization of avenin and globulin storage proteins in developing endosperm of Avena sativa L.

The seed storage proteins of oats (Avena sativa L.) are synthesized and assembled into vacuolar protein bodies in developing endosperm tissue. We used double-label immunolocalization to study the distribution of these proteins within protein bodies of the starchy endosperm. When sections of developing oat endosperm sampled 8 d after anthesis were stained with uranyl acetate and lead citrate, the vacuolar protein bodies consisted of light-staining regions which were usually surrounded by a darker-staining matrix. Immunogold staining of this tissue demonstrated a distinct segregation of proteins within protein bodies; globulins were localized in the dark-staining regions and prolamines were localized in the light-staining regions. We observed two additional components of vacuolar protein bodies: a membranous component which was often appressed to the outside of the globulin, and a granular, dark-staining region which resembled tightly clustered ribosomes. Neither antibody immunostained the membranous component, but the granular region was lightly labelled with the anti-globulin antibody. Anti-globulin immunostaining was also observed adjacent to cell walls and appeared to be associated with plasmodesmata. Immunostaining for both antigens was also observed within the rough endoplasmic reticulum. Based on the immunostaining patterns, the prolamine proteins appeared to aggregate within the rough endoplasmic reticulum while most of the globulin appeared to aggregate in the vacuole.

In vitro synthesis of zein-like protein by maize polyribosomes.

Abstract Free and membrane-bound polyribosomes were isolated in an undegraded form from developing maize kernels. Translation of the membrane-bound polyribosomes in vitro produced one main radioactive protein. This protein was soluble in 70% ethanol and had the same mobility in electrophoresis on sodium dodecyl sulfate-gels as a zein standard. The ratio of [14C] leucine to [14C] lysine incorporated into the 70% ethanol extractable protein was similar to the mole fraction ratio of these amino acids in zein. The zein-like protein may represent as much as 50% of the total protein synthesized by the membrane-bound polyribosomes.

Comparative analysis of polypeptides synthesized in vivo and in vitro by two strains of barley stripe mosaic virus.

Proteins synthesized in a wheat germ system containing RNA from the Type or ND18 strain of barley stripe mosaic virus (BSMV), or polyribosomes from Type or ND18-infected barley, were compared with each other and with polypeptides from Type and ND18-infected plants. Polypeptides with apparent molecular weights (Mr) of 25, 67, and 120 x 10(3) that were induced in infected barley were synthesized in vitro by polyribosomes from infected plants or RNA from purified BSMV. The 25,000 Mr polypeptide was identified as BSMV coat protein by coelectrophoresis with coat protein from purified virions, by immunoprecipitation, and by absence of [35S]methionine incorporation into the protein. Strain-specific differences were observed in two additional translation products synthesized in both wheat germ and reticulocyte lysate systems containing Type of ND18 RNA. RNAs from both strains directed synthesis of 71 and 82 x 10(3) Mr polypeptides. However, ND18 RNA directed synthesis of a larger amount of the 71,000 Mr polypeptide, whereas with Type RNA the 82,000 Mr polypeptide predominated. These two proteins may be processed in vivo because reduced synthesis of both polypeptides in wheat germ extracts containing polyribosomes from infected plants was correlated with increased synthesis of the 67,000 Mr polypeptide. Several additional polypeptides synthesized in the wheat germ system were probably premature termination products because their synthesis in the reticulocyte lysate was greatly reduced.

Deletion of DNA sequences flanking an Mr 19,000 zein gene reduces its transcriptional activity in heterologous plant tissues.

SummaryAnalysis of a series of clones containing deletions in the 5′ noncoding sequence of a gene encoding an Mr 19 000 zein allowed identification of a region required for maximal transcription. Transcriptional activity was assayed in two heterologous plant systems. In one system, the Ti plasmid was used to introduce the modified zein genes into the sunflower genome. In the other system, electroporation was used to transform carrot protoplasts with plasmids containing the zein genes. For the electrophoration experiments, the 5′ noncoding sequences from the zein clones were linked to the protein coding sequence of chloramphenicol acetyl transferase. The results showed that an upstream sequence, delimited by nucleotides-337 and-125 with respect to the mRNA cap site, is required for maximal transcription of the gene. In contrast, very low levels of transcription were directed by constructs that contained 125 bp of 5′ noncoding sequence that included the CAAT and TATA boxes, suggesting that the additional sequences (-337 to-125) further 5′ exert a quantitative effect on transcription. Examination of the additional 5′ sequences showed five regions that share homology with the SV40 enhancer core sequence.