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.

Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, a-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zeinrich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals.The cereal endosperm consists of three main cell types: an inner mass of starchy endosperm cells, one to three layers of epidermal aleurone cells, and the transfer cells that contact the maternal vascular tissue (Olsen, 2004). The starchy endosperm accounts for 80 to 90% of the grain weight and contains large amounts of storage proteins and starch. These cells undergo programmed cell death during maturation. Aleurone cells are rich in protein storage vacuoles (PSVs), minerals, and lipid bodies and remain alive during seed development. It is assumed that the breakdown of proteins localized to PSVs in aleurone cells provides an essential source of the amino acids necessary for the synthesis of hydrolytic enzymes required for mobilizing food stored in the starchy endosperm (Filner and Varner, 1967;Jacobsen et al., 1988;Bethke et al., 1998).Besides its biological relevance as a model to study plant development, cell differentiation, and programmed cell death, the cereal endosperm is very important in terms of its nutritional value. Cereal grains contain less protein than do legume seeds, but because cereals are produced and consumed in much larger quantities, they are the main source of protein for the nutrition of humans and livestock (Shewry and Halford, 2002). The major storage proteins in maize (Zea mays) kernels are the alcoholsoluble prolamins, a type of storage proteins present only in grasses. Maize prolamins, referred to as zeins, are divided into different types: a-, b-, g-, and d-zeins (Coleman and Larkins, 1999) that differ in amino acid composition and structural properties (Shewry and Halford, 2002)...

Essential amino acids like lysine and tryptophan are deficient in corn meal because of the abundance of zein storage proteins that lack these amino acids. A natural mutant, opaque 2 (o2) causes reduction of zeins, an increase of nonzein proteins, and as a consequence, a doubling of lysine levels. However, o2's soft inferior kernels precluded its commercial use. Breeders subsequently overcame kernel softness, selecting several quantitative loci (QTLs), called o2 modifiers, without losing the high-lysine trait. These maize lines are known as "quality protein maize" (QPM). One of the QTLs is linked to the 27-kDa γ-zein locus on chromosome 7S. Moreover, QPM lines have 2-to 3-fold higher levels of the 27-kDa γ-zein, but the physiological significance of this increase is not known. Because the 27-and 16-kDa γ-zein genes are highly conserved in DNA sequence, we introduced a dominant RNAi transgene into a QPM line (CM105Mo2) to eliminate expression of them both. Elimination of γ-zeins disrupts endosperm modification by o2 modifiers, indicating their hypostatic action to γ-zeins. Abnormalities in protein body structure and their interaction with starch granules in the F1 with Mo2/+; o2/o2; γRNAi/+ genotype suggests that γ-zeins are essential for restoring protein body density and starch grain interaction in QPM. To eliminate pleiotropic effects caused by o2, the 22-kDa α-zein, γ-zein, and β-zein RNAis were stacked, resulting in protein bodies forming as honeycomb-like structures. We are unique in presenting clear demonstration that γ-zeins play a mechanistic role in QPM, providing a previously unexplored rationale for molecular breeding.electron microscopy | stacking of RNAi events | storage organs | opaque phenotype | kernel hardness G rain hardness is a key agronomic trait in maize (Zea mays L.)because it provides resistance to damage during harvesting and marketing, as well as to insect and fungal damage. Kernel texture is determined by the relative amounts of hard (vitreous) and soft (opaque) endosperm and there is a positive correlation between zein storage proteins and kernel vitreousness (1). Zeins are a heterogeneous mixture of alcohol-soluble proteins, falling into four classes based on their structure (α-, β-, γ-, and δ-zeins) (2). The zeins extracted with the Osborne method (3) are classified as z1 (19-and 22-kDa α-zeins) and the cross-linked z2 group (50-, 27-, and 16-kDa γ-zeins, 15-kDa β-zein, and 18-and 10-kDa δ-zeins) (4, 5). Zeins are deposited in rough endoplasmic reticulum-delimited protein bodies (PBs) in endosperm cells from around 10 d after pollination (DAP) (6, 7). Alpha-and δ-zeins are mainly stored in the center of PBs, and γ-and β-zeins are deposited in the peripheral region (8). The Cys-rich γ-and β-zeins have redundant function in the stabilization of PB morphology (9). The translucency (vitreousness) of the mature kernel is influenced by PB composition and the spatial organization of α-, β-, γ-, and δ-zeins (10-16).Because zeins are essentially devoid of lysine and tryptophan, their high-lev...

Background: Gene knockouts are a critical resource for functional genomics. In Arabidopsis, comprehensive knockout collections were generated by amplifying and sequencing genomic DNA flanking insertion mutants. These Flanking Sequence Tags (FSTs) map each mutant to a specific locus within the genome. In maize, FSTs have been generated using DNA transposons. Transposable elements can generate unstable insertions that are difficult to analyze for simple knockout phenotypes. Transposons can also generate somatic insertions that fail to segregate in subsequent generations.

BackgroundSorghum is an important cereal crop, which requires large quantities of nitrogen fertilizer for achieving commercial yields. Identification of the genes responsible for low-N tolerance in sorghum will facilitate understanding of the molecular mechanisms of low-N tolerance, and also facilitate the genetic improvement of sorghum through marker-assisted selection or gene transformation. In this study we compared the transcriptomes of root tissues from seven sorghum genotypes having differential response to low-N stress.ResultsIllumina RNA-sequencing detected several common differentially expressed genes (DEGs) between four low-N tolerant sorghum genotypes (San Chi San, China17, KS78 and high-NUE bulk) and three sensitive genotypes (CK60, BTx623 and low-NUE bulk). In sensitive genotypes, N-stress increased the abundance of DEG transcripts associated with stress responses including oxidative stress and stimuli were abundant. The tolerant genotypes adapt to N deficiency by producing greater root mass for efficient uptake of nutrients. In tolerant genotypes, higher abundance of transcripts related to high affinity nitrate transporters (NRT2.2, NRT2.3, NRT2.5, and NRT2.6) and lysine histidine transporter 1 (LHT1), may suggest an improved uptake efficiency of inorganic and organic forms of nitrogen. Higher abundance of SEC14 cytosolic factor family protein transcript in tolerant genotypes could lead to increased membrane stability and tolerance to N-stress.ConclusionsComparison of transcriptomes between N-stress tolerant and sensitive genotypes revealed several common DEG transcripts. Some of these DEGs were evaluated further by comparing the transcriptomes of genotypes grown under full N. The DEG transcripts showed higher expression in tolerant genotypes could be used for transgenic over-expression in sensitive genotypes of sorghum and related crops for increased tolerance to N-stress, which results in increased nitrogen use efficiency for sustainable agriculture.

Prolamin storage proteins are the main repository for nitrogen in the endosperm of cereal seeds. These stable proteins accumulate at massive levels due to the high level expression from extensively duplicated genes in endoreduplicated cells. Such abundant accumulation is achieved through efficient packaging in endoplasmic reticulum localized protein bodies in a process that is not completely understood. Prolamins are also a key determinant of hard kernel texture in the mature seed; an essential characteristic of cereal grains like maize. However, deficiencies of key essential amino acids in prolamins result in relatively poor grain protein quality. The inverse relationship between prolamin accumulation and protein quality has fueled an interest in understanding the role of prolamins and other proteins in endosperm maturation. This article reviews recent technological advances that have enabled dissection of overlapping and non-redundant roles of prolamins, particularly the maize zeins. This has come through molecular characterization of mutants first identified many decades ago, selective down-regulation of specific zein genes or entire zein gene families, and most recently through combining deletion mutagenesis with current methods in genome and transcriptome profiling. Works aimed at understanding prolamin deposition and function as well as creating novel variants with improved nutritional and digestibility characteristics, are reported.

Quality protein maize (QPM) was created by selecting genetic modifiers that convert the starchy endosperm of an opaque2 (o2) mutant to a hard, vitreous phenotype. Genetic analysis has shown that there are multiple, unlinked o2 modifiers (Opm), but their identity and mode of action are unknown. Using two independently developed QPM lines, we mapped several major Opm QTLs to chromosomes 1, 7 and 9. A microarray hybridization performed with RNA obtained from true breeding o2 progeny with vitreous and opaque kernel phenotypes identified a small group of differentially expressed genes, some of which map at or near the Opm QTLs. Several of the genes are associated with ethylene and ABA signaling and suggest a potential linkage of o2 endosperm modification with programmed cell death.

Zeins, the maize (Zea mays) prolamin storage proteins, accumulate at very high levels in developing endosperm in endoplasmic reticulum membrane-bound protein bodies. Products of the multigene a-zein families and the single-gene g-zein family are arranged in the central hydrophobic core and the cross-linked protein body periphery, respectively, but little is known of the specific roles of family members in protein body formation. Here, we used RNA interference suppression of different zein subclasses to abolish vitreous endosperm formation through a variety of effects on protein body density, size, and morphology. We showed that the 27-kilodalton (kD) g-zein controls protein body initiation but is not involved in protein body filling. Conversely, other g-zein family members function more in protein body expansion and not in protein body initiation. Reduction in both 19-and 22-kD a-zein subfamilies severely restricted protein body expansion but did not induce morphological abnormalities, which result from reduction of only the 22-kD a-zein class. Concomitant reduction of all zein classes resulted in severe reduction in protein body number but normal protein body size and morphology.