Maize starchy endosperm mutants have kernel phenotypes that include a brittle texture, susceptibility to insect pests, and inferior functional characteristics of products made from their flour. At least 18 such mutants have been identified, but only in the cases of opaque2 ( o2 ) and floury2 ( fl2 ), which affect different aspects of storage protein synthesis, is the molecular basis of the mutation known. To better understand the relationship between the phenotypes of these mutants and their biochemical bases, we characterized the protein and amino acid composition, as well as the mRNA transcript profiles, of nearly isogenic inbred lines of W64A o1 , o2 , o5 , o9 , o11 , Mucuronate (

Endosperm is a filial structure resulting from a second fertilization event in angiosperms. As an absorptive storage organ, endosperm plays an essential role in support of embryo development and seedling germination. The accumulation of carbohydrate and protein storage products in cereal endosperm provides humanity with a major portion of its food, feed, and renewable resources. Little is known regarding the regulatory gene networks controlling endosperm proliferation and differentiation. As a first step toward understanding these networks, we profiled all mRNAs in the maize kernel and endosperm at eight successive stages during the first 12 d after pollination. Analysis of these gene sets identified temporal programs of gene expression, including hundreds of transcriptionfactor genes. We found a close correlation of the sequentially expressed gene sets with distinct cellular and metabolic programs in distinct compartments of the developing endosperm. The results constitute a preliminary atlas of spatiotemporal patterns of endosperm gene expression in support of future efforts for understanding the underlying mechanisms that control seed yield and quality.mRNA localization | time series

The maize (Zea mays) floury1 (fl1) mutant was first reported almost 100 years ago, but its molecular identity has remained unknown. We report the cloning of Fl1, which encodes a novel zein protein body membrane protein with three predicted transmembrane domains and a C-terminal plant-specific domain of unknown function (DUF593). In wild-type endosperm, the FL1 protein accumulates at a high level during the period of zein synthesis and protein body development and declines to a low level at kernel maturity. Immunogold labeling showed that FL1 resides in the endoplasmic reticulum surrounding the protein body. Zein protein bodies in fl1 mutants are of normal size, shape, and abundance. However, mutant protein bodies ectopically accumulate 22-kD a-zeins in the g-zein-rich periphery and center of the core, rather than their normal discrete location in a ring at outer edge of the core. The 19-kD a-zein is uniformly distributed throughout the core in wildtype protein bodies, and this distribution is unaffected in fl1 mutants. Pairwise yeast two-hybrid experiments showed that FL1 DUF593 interacts with the 22-kD a-zein. Results of these studies suggest that FL1 participates in protein body formation by facilitating the localization of 22-kD a-zein and that this is essential for the formation of vitreous endosperm.

Defective endosperm* (De*)-B30 is a dominant maize (Zea mays) mutation that depresses zein synthesis in the developing endosperm. The mutant kernels have an opaque, starchy phenotype, malformed zein protein bodies, and highly increased levels of binding protein and other chaperone proteins in the endosperm. Immunoblotting revealed a novel ␣-zein protein in De*-B30 that migrates between the 22-and 19-kD ␣-zein bands. Because the De*-B30 mutation maps in a cluster of 19-kD ␣-zein genes, we characterized cDNA clones encoding these proteins from a developing endosperm library. This led to the identification of a 19-kD ␣-zein cDNA in which proline replaces serine at the 15th position of the signal peptide. Although the corresponding gene does not appear to be highly expressed in De*-B30, it was found to be tightly linked with the mutant phenotype in a segregating F 2 population. Furthermore, when the protein was synthesized in yeast cells, the signal peptide appeared to be less efficiently processed than when serine replaced proline. To test whether this gene is responsible for the De*-B30 mutation, transgenic maize plants expressing this sequence were created. T 1 seeds originating from the transformants manifested an opaque kernel phenotype with enhanced levels of binding protein in the endosperm, similar to De*-B30. These results are consistent with the hypothesis that the De*-B30 mutation causes a defective signal peptide in a 19-kD ␣-zein protein.A number of mutations have been identified that affect storage protein synthesis in maize (Zea mays) endosperm (for review, see Coleman and Larkins, 1998). The mutant kernels typically have a starchy endosperm texture and low density, both of which are thought to be associated with the reduced synthesis of the prolamin storage proteins, or zeins. When placed on a light box, endosperm of these mutants does not transmit light, i.e. it is opaque, whereas that of normal, wild-type kernels is vitreous and translucent. Recessive mutations that produce such an opaque kernel phenotype are traditionally classified as "opaque" (e.g. opaque1-15), whereas semidominant opaque mutations are called "floury" (e.g. floury1-3). There are, in addition, several dominant opaque mutations, defective endosperm* (De*)-B30 and Mucuronate (Mc;Soave et al., 1979;Soave and Salamini, 1984). The basis of the relationship between an opaque kernel phenotype and zein synthesis is not well understood. Although several of these mutants have a reduced zein content and show altered expression of specific types of zein genes, the degree to which zein synthesis is reduced among them is variable (Hunter et al., 2002). For example, in o1, zein synthesis is barely affected. Generally, these mutations result in increased expression of genes associated with physiological stress and the unfolded protein response (UPR; Kaufman, 1999) during endosperm development.Because of their influence on zein synthesis and the nature of their pleiotropic effects on other genes expressed in the endosperm, the genetic defects re...

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