Amy M. Clore

EF-1[alpha] Is Associated with a Cytoskeletal Network Surrounding Protein Bodies in Maize Endosperm Cells.

By using indirect immunofluorescence and confocal microscopy, we documented changes in the distribution of elongation factor-1[alpha] (EF-1[alpha]), actin, and microtubules during the development of maize endosperm cells. In older interphase cells actively forming starch grains and protein bodies, the protein bodies are enmeshed in EF-1[alpha] and actin and are found juxtaposed with a multidirectional array of microtubules. Actin and EF-1[alpha] appear to exist in a complex, because we observed that the two are colocalized, and treatment with cytochalasin D resulted in the redistribution of EF-1[alpa]. These data suggest that EF-1[alpha] and actin are associated in maize endosperm cells and may help to explain the basis of the correlation we found between the concentration of EF-1[alpha] and lysine content. The data also support the hypothesis that the cytoskeleton plays a role in storage protein deposition. The distributions of EF-1[alpha] actin, and microtubules change during development. We observed that in young cells before the accumulation of starch and storage protein, EF-1[alpha], actin, and microtubules are found mainly in the cell cortex or in association with nuclei.

Characterization of maize elongation factor 1A and its relationship to protein quality in the endosperm.

The protein synthesis elongation factor 1A (eEF1A) is a multifunctional protein in eukaryotic cells. In maize (Zea mays L.) endosperm eEF1A co-localizes with actin around protein bodies, and its accumulation is highly correlated with the protein-bound lysine (Lys) content. We purified eEF1A from maize kernels by ammonium sulfate precipitation, ion-exchange, and chromatofocusing. The identity of the purified protein was confirmed by microsequencing of an endoproteinase glutamic acid-C fragment and by its ability to bundle actin. Using purified eEF1A as a standard, we found that this protein contributes 0.4% of the total protein in W64A+ endosperm and approximately 1% of the protein in W64Ao2. Because eEF1A contains 10% Lys, it accounts for 2.2% of the total Lys in W64A+ and 2.3% of the Lys in W64Ao2. However, its concentration predicts 90% of the Lys found in endosperm proteins of both genotypes, indicating that eEF1A is a key component of the group of proteins that determines the nutritional quality of the grain. This notion is further supported by the fact that in floury2, another high-Lys mutant, the content of eEF1A increases with the dosage of the floury2 gene. These data provide the biochemical basis for further investigation of the relationship between eEF1A content and the nutritional quality of cereals.

Maize early endosperm growth and development: From fertilization through cell type differentiation

UNLABELLED • PREMISE OF THE STUDY Given the worldwide economic importance of maize endosperm, it is surprising that its development is not the most comprehensively studied of the cereals. We present detailed morphometric and cytological descriptions of endosperm development in the maize inbred line B73, for which the genome has been sequenced, and compare its growth with four diverse Nested Association Mapping (NAM) founder lines.• METHODS The first 12 d of B73 endosperm development were described using semithin sections of plastic-embedded kernels and confocal microscopy. Longitudinal sections were used to compare endosperm length, thickness, and area.• KEY RESULTS Morphometric comparison between Arizona- and Michigan-grown B73 showed a common pattern. Early endosperm development was divided into four stages: coenocytic, cellularization through alveolation, cellularization through partitioning, and differentiation. We observed tightly synchronous nuclear divisions in the coenocyte, elucidated that the onset of cellularization was coincident with endosperm size, and identified a previously undefined cell type (basal intermediate zone, BIZ). NAM founders with small mature kernels had larger endosperms (0-6 d after pollination) than lines with large mature kernels.• CONCLUSIONS Our B73-specific model of early endosperm growth links developmental events to relative endosperm size, while accounting for diverse growing conditions. Maize endosperm cellularizes through alveolation, then random partitioning of the central vacuole. This unique cellularization feature of maize contrasts with the smaller endosperms of Arabidopsis, barley, and rice that strictly cellularize through repeated alveolation. NAM analysis revealed differences in endosperm size during early development, which potentially relates to differences in timing of cellularization across diverse lines of maize.

Actin in Protein Synthesis and Protein Body Formation

We review the recent evidence that plant cells, like animal cells, have mRNA associated with their cytoskeleton, and that much of this mRNA is present in polyribosomes. One of these cell types is the maize endosperm cell which contains storage proteins residing inside the lumen of the endoplasmic reticulum (ER). Data from both wild type and mutant maize endosperm support the proposition that the cytoskeleton is the scaffold upon which both polyribosomes (especially those synthesizing zein) and membranes adhere. While much of the data implies a major role for the actin cytoskeleton, additional roles are suggested for the microtubule system and for elongation factor, eEF1, which might help connect the two cytoskeleton networks. Results from rice endosperm have been especially beneficial to understanding the role of the cytoskeleton, insofar as rice has two entirely different kinds of protein bodies, the cereal-type and the legume-type. The former derive directly from the ER, store prolamines, and are closely associated with the cytoskeleton, while the latter arise from the coalescence of ER-derived vesicles, store glutelins, but are not associated with the cytoskeleton. The roles of the cytoskeleton appear to be in the segregation of specific mRNAs to specific sub-cellular locations, the enhanced translation of these mRNAs, and perhaps the accumulation of these water-insoluble storage proteins within the protein body.

Cereal grass pulvini: Agronomically significant models for studying gravitropism signaling and tissue polarity

Cereal grass pulvini have emerged as model systems that are not only valuable for the study of gravitropism, but are also of agricultural and economic significance. The pulvini are regions of tissue that are apical to each node and collectively return a reoriented stem to a more vertical position. They have proven to be useful for the study of gravisensing and response and are also providing clues about the establishment of polarity across tissues. This review will first highlight the agronomic significance of these stem regions and their benefits for use as model systems and provide a brief historical overview. A detailed discussion of the literature focusing on cell signaling and early changes in gene expression will follow, culminating in a temporal framework outlining events in the signaling and early growth phases of gravitropism in this tissue. Changes in cell wall composition and gene expression that occur well into the growth phase will be touched upon briefly. Finally, some ongoing research involving both maize and wheat pulvini will be introduced along with prospects for future investigations.

Protein quality and its potential relationship to the cytoskeleton in maize endosperm

Summary Multiple strategies are being employed world wide in an attempt to improve the nutritional value of seeds. Genetic engineering is being investigated as a means of improving the amino acid contents and digestibility of seed proteins. Additionally, naturally occurring mutations have been identified which increase the levels of essential amino acids such as lysine. Unfortunately, these mutations often lead to soft, chalky endosperms with increased susceptibility to insect pathogens. One such mutant in maize is called opaque2 ( o 2). «Modifier» genes have been found which, when introduced into o2 mutants, ameliorate the negative features of the mutation. Indeed, the modifiers may prove an effective means of generating maize with high protein quality despite the complexity of introducing multiple modifier loci into elite germplasm. We have been identifying and characterizing lysine-rich proteins in o2 in order to understand the pleiotropic effect by which this mutant increases the lysine content of the grain. The level of one protein, EF-1α, was found to be uniformly increased in o2 mutants. Furthermore, it was found to be highly correlated with the lysine content of the grain. In an effort to learn the basis for this correlation, we used immunocytochemistry and confocal microscopy to determine the subcellular location of this protein during endosperm development, and to identify proteins/structures with which it associates. We found that EF-1α is part of an elaborate cytoskeletal network associated with protein bodies. We hypothesize that this cytoskeleton might be involved in protein body formation. Further, we surmise that other lysine-rich proteins may exist in association with this cytoskeletal complex and that this complex may be increased in the endosperm cells of higher lysine genotypes.

Genetic Manipulation for the Production of High Lysine Corn

Cereals provide 50% of the dietary protein for humans and can comprise 70% of the protein intake for people in developing countries (Deutscher, 1978). It is expected that the demand for cereal grains will increase dramatically in the future, as a consequence of the expanding human population, which could double by the year 2030 (Mann, 1997). Unfortunately, most cereals do not provide a nutritionally balanced source of protein (FAO/WHO, 1985). The abundant proteins they contain, the storage proteins or proclaims, are devoid of several amino acids essential for monogastric animals. The most limiting of these is lysine (Nelson, 1969). There are several naturally occurring mutants in cereals, such as opaque2 (o2) and floury2 (fl2) in maize (Nelson, 1969), that decrease prolamin (zein) synthesis and increase the lysine content of the endosperm, but the pleiotropic effect by which these mutations increase the synthesis of lysine-containing proteins is not understood.