• 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 fi rst 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 identifi ed a previously undefi ned 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-specifi c 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.

The maize floury2 mutation results in the formation of a soft, starchy endosperm with a reduced amount of prolamin (zein) proteins and twice the lysine content of the wild type. The mutation is semidominant and is associated with small, irregularly shaped protein bodies, elevated levels of a 70-kDa chaperone in the endoplasmic reticulum, and a novel 24-kDa polypeptide in the zein fraction. The 24-kDa polypeptide is a precursor of a 22-kDa ␣-zein protein that is not properly processed. The defect is due to an alanine-tovaline substitution at the C-terminal position of the signal peptide, which causes the protein to be anchored to the endoplasmic reticulum. We postulated that the phenotype associated with the floury2 mutation is caused by the accumulation of the 24-kDa ␣-zein protein. To test this hypothesis, we created transgenic maize plants that produce the mutant protein. We found that endosperm in seeds of these plants manifests the floury2 phenotype, thereby confirming that the mutant ␣-zein is the molecular basis of this mutation.Zeins are prolamin storage proteins that accumulate in the endosperm of maize (Zea mays L.) seeds. They are composed of four different types of polypeptides, classified as ␣-, ␤-, ␥-, and ␦-zeins (1). Accretions of zein proteins form spherical protein bodies within the lumen of the endoplasmic reticulum (ER), and there is a distinct spatial arrangement of these proteins within a protein body: ␤-and ␥-zeins are located on the periphery, whereas ␣-and ␦-zeins are found in the interior (2, 3). Collectively, the zein proteins are rich in glutamine and proline, but they lack lysine and tryptophan. Because zeins constitute such a large proportion of the total seed protein (60-70%), the amino acid composition of these proteins causes the grain to be of inferior nutritional quality for monogastric animals.Efforts to improve the protein quality of maize seed have focused on mutants in which zein synthesis is reduced and the lysine content is increased. The first ''high-lysine'' mutants to be identified were opaque2 (o2) and floury2 ( fl2) (4, 5). Unfortunately, the favorable nutritional quality of these mutants is offset by the inferior physical properties of their soft, starchy endosperms. It appears that the starchy endosperm of the o2 and fl2 mutants is caused by changes in the nature of their protein bodies. The o2 mutation affects a transcriptional activator of a subset of ␣-zein genes, leading to a reduction in ␣-zein protein synthesis and the formation of protein bodies that are significantly smaller than normal (6-9). The fl2 mutation, which is semidominant, causes a decrease in synthesis of all classes of zeins, and the resultant protein bodies are not only smaller than normal, but they are also asymmetrical and misshapen (10, 11). Another feature of fl2 endosperm is the overexpression of the ER-resident binding protein (BiP), which becomes deposited at the periphery of the mutant protein bodies (12-15).We have postulated that the phenotype associated with fl2 is caused by ...

Prolamin-containing protein bodies in maize endosperm are composed of four different polypeptides, the alpha-, beta-, gamma-, and delta-zeins. The spatial organization of zeins within the protein body, as well as interactions between them, suggests that the localized synthesis of gamma-zeins could initiate and target protein body formation at specific regions of the rough endoplasmic reticulum. To investigate this possibility, we analyzed the distribution of mRNAs encoding the 22-kD alpha-zein and the 27-kD gamma-zein proteins on cisternal and protein body rough endoplasmic reticulum membranes. In situ hybridization revealed similar frequencies of the mRNAs in both regions of the endoplasmic reticulum, indicating that the transcripts are distributed more or less randomly. This finding implies that zein protein interactions determine protein body assembly. To address this question, we expressed cDNAs encoding alpha-, beta-, gamma-, and delta-zeins in the yeast two-hybrid system. We found strong interactions among the 50-, 27-, and 16-kD gamma-zeins and the 15-kD beta-zein, consistent with their colocalization in developing protein bodies. Interactions between the 19- and 22-kD alpha-zeins were relatively weak, although each of them interacted strongly with the 10-kD delta-zein. Strong interactions were detected between the alpha- and delta-zeins and the 16-kD gamma-zein and the 15-kD beta-zein; however, the 50- and 27-kD gamma-zeins did not interact with the alpha- and delta-zein proteins. We identified domains within the 22-kD alpha-zein that bound preferentially the alpha- and delta-zeins and the beta- and gamma-zeins. Affinities between zeins generally were consistent with results from immunolocalization experiments, suggesting an important role for the 16-kD gamma-zein and the 15-kD beta-zein in the binding and assembly of alpha-zeins within the protein body.

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.