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PubMed2006

Extraction of wheat endosperm proteins for proteome analysis.

Total protein extracts of wheat endosperm are widely used for the analysis of the highly abundant gliadins and glutenins. In this review, the most popular total endosperm extraction methods are compared for their effectiveness in proteome coverage. A drawback of total endosperm extracts is that the enormous dynamic range of protein abundance limits the detection, quantification, and identification of low abundance proteins. Protein fractionation is invaluable for improving proteome coverage, because it reduces sample complexity while enriching for specific classes of less abundant proteins. A wide array of techniques is available for isolating protein subpopulations. Sequential extraction is a method particularly suited for subfractionation of wheat endosperm proteins, because it takes advantage of the specific solubility properties of the different classes of endosperm proteins. This method effectively separates the highly abundant gliadins and glutenins from the much less abundant albumins and globulins. Subcellular fractionation of tissue homogenates is a classical technique for isolating membranes and organelles for functional analysis. This approach is suitable for defining the biochemical processes associated with amyloplasts, specialized organelles in the endosperm that function in the synthesis and storage of starch. Subproteome fractionation, when combined with 2-DE and protein identification, provides a powerful approach for defining endosperm protein composition and providing new insights into cellular functions.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2002

Identification of diploid endosperm in an early angiosperm lineage.

In flowering plants, the developmental and genetic basis for the establishment of an embryo-nourishing tissue differs from all other lineages of seed plants. Among extant nonflowering seed plants (conifers, cycads, Ginkgo, Gnetales), a maternally derived haploid tissue (female gametophyte) is responsible for the acquisition of nutrients from the maternal diploid plant, and the ultimate provisioning of the embryo. In flowering plants, a second fertilization event, contemporaneous with the fusion of sperm and egg to yield a zygote, initiates a genetically biparental and typically triploid embryo-nourishing tissue called endosperm. For over a century, triploid biparental endosperm has been viewed as the ancestral condition in extant flowering plants. Here we report diploid biparental endosperm in Nuphar polysepalum, a basal angiosperm. We show that diploid endosperms are common among early angiosperm lineages and may represent the ancestral condition among flowering plants. If diploid endosperm is plesiomorphic, the triploid endosperms of the vast majority of flowering plants must have evolved from a diploid condition through the developmental modification of the unique fertilization process that initiates endosperm.

Diploidy↗

PubMed1995

Maternal-specific demethylation and expression of specific alleles of zein genes in the endosperm of Zea mays L.

Zeins constitute 60-70% of maize endosperm protein. Zein genes are specifically transcribed in the endosperm, and a correlation has been established between tissue-specific expression and demethylation. Three inbred lines and their reciprocal crosses were analysed to assess for allele-specific differences in methylation, transcription and translation. DNAs from endosperm, embryo and seedling tissues analysed by cleavage with methylation-sensitive restriction enzymes and Southern blot hybridization with zein cDNA and genomic sequences show that specific demethylation of zein sequences occurs only in endosperm and is restricted to the maternal complements. Steady-state transcript accumulation of zein mRNA assessed by RNase protection assay reveals qualitative and quantitative differences among endosperm RNAs of the inbreds and of their reciprocal hybrids. Moreover, two-dimensional gel electrophoresis of zein proteins identified polypeptides that are maternally imprinted in reciprocal crosses. These results indicate that endosperm-specific expression of specific zein alleles may occur via parental imprinting and disclose a possible role of methylation in regulating the expression of genes differently contributed in the endosperm by the maternal and paternal genomes.

Alleles↗

PubMed2005

Grain growth and endosperm cell size under high night temperatures in rice (Oryza sativa L.).

BACKGROUND AND AIMS: High night temperatures are more harmful to grain weight in rice than high day temperatures. Grain growth rate and growth duration were investigated to determine which was the cause of the decrease in final grain weight under high night temperatures. Endosperm cell number and cell sizes were also examined to determine which might cause the decrease in final grain weight. METHODS: Rice plants were grown outdoors in plastic pots and moved at heading time to three temperature-controlled glasshouses under high night temperature (HNT; 22/34 degrees C), high day temperature (HDT; 34/22 degrees C) and control conditions (CONT; 22/22 degrees C). Grains were sampled periodically, and the time-course of grain growth was divided into rate and duration by logistic regression analysis. Endosperm cell numbers and cell sizes were analysed by digitalized hand-tracing images of endosperm cross-sections. KEY RESULTS: The duration of grain growth was reduced by high temperature both day and night. However, the rate of grain growth was lower in HNT than in HDT. The number of cells in endosperm cross-sections in HNT was similar to that in HDT, and higher than that in CONT. The average cell area was smaller in HNT than in either CONT or HDT. The differences in average cell areas between HNT and HDT were greater at distances 60-80 % from the central point of endosperm towards the endosperm surface. CONCLUSIONS: The results show that HNT compared with HDT reduced the final grain weight by a reduction in grain growth rate in the early or middle stages of grain filling, and also reduced cell size midway between the central point and the surface of endosperm.

Biomass↗

PubMed2002

Statistical methods for dissecting triploid endosperm traits using molecular markers. An autogamous model.

The endosperm, a result of double fertilization in flowering plants, is a triploid tissue whose genetic composition is more complex than diploid tissue. We present a new maximum-likelihood-based statistical method for mapping quantitative trait loci (QTL) underlying endosperm traits in an autogamous plant. Genetic mapping of quantitative endosperm traits is qualitatively different from traits for other plant organs because the endosperm displays complicated trisomic inheritance and represents a younger generation than its mother plant. Our endosperm mapping method is based on two different experimental designs: (1) a one-stage design in which marker information is derived from the maternal genome and (2) a two-stage hierarchical design in which marker information is derived from both the maternal and offspring genomes (embryos). Under the one-stage design, the position and additive effect of a putative QTL can be well estimated, but the estimates of the dominant and epistatic effects are upward biased and imprecise. The two-stage hierarchical design, which extracts more genetic information from the material, typically improves the accuracy and precision of the dominant and epistatic effects for an endosperm trait. We discuss the effects on the estimation of QTL parameters of different sampling strategies under the two-stage hierarchical design. Our method will be broadly useful in mapping endosperm traits for many agriculturally important crop plants and also make it possible to study the genetic significance of double fertilization in the evolution of higher plants.

Chromosome Mapping↗

PubMed2004

The seeds of Lotus japonicus lines transformed with sense, antisense, and sense/antisense galactomannan galactosyltransferase constructs have structurally altered galactomannans in their endosperm cell walls.

Galactomannan biosynthesis in legume seed endosperms involves two Golgi membrane-bound glycosyltransferases, mannan synthase and galactomannan galactosyltransferase (GMGT). GMGT specificity is an important factor regulating the distribution and amount of (1-->6)-alpha-galactose (Gal) substitution of the (1-->4)-beta-linked mannan backbone. The model legume Lotus japonicus is shown now to have endospermic seeds with endosperm cell walls that contain a high-Gal galactomannan (mannose [Man]/Gal = 1.2-1.3). Galactomannan biosynthesis in developing L. japonicus endosperms has been mapped, and a cDNA encoding a functional GMGT has been obtained from L. japonicus endosperms during galactomannan deposition. L. japonicus has been transformed with sense, antisense, and sense/antisense ("hairpin loop") constructs of the GMGT cDNA. Some of the sense, antisense, and sense/antisense transgenic lines exhibited galactomannans with altered (higher) Man/Gal values in their (T(1) generation) seeds, at frequencies that were consistent with posttranscriptional silencing of GMGT. For T(1) generation individuals, transgene inheritance was correlated with galactomannan composition and amount in the endosperm. All the azygous individuals had unchanged galactomannans, whereas those that had inherited a GMGT transgene exhibited a range of Man/Gal values, up to about 6 in some lines. For Man/Gal values up to 4, the results were consistent with lowered Gal substitution of a constant amount of mannan backbone. Further lowering of Gal substitution was accompanied by a slight decrease in the amount of mannan backbone. Microsomal membranes prepared from the developing T(2) generation endosperms of transgenic lines showed reduced GMGT activity relative to mannan synthase. The results demonstrate structural modification of a plant cell wall polysaccharide by designed regulation of a Golgi-bound glycosyltransferase.

Amino Acid Sequence↗

PubMed1994

The regulation of gene expression in transformed maize aleurone and endosperm protoplasts. Analysis of promoter activity, intron enhancement, and mRNA untranslated regions on expression.

Gene expression in the aleurone and endosperm is highly regulated during both seed development and germination. Studies of alpha-amylase expression in the aleurone of barley (Hordeum vulgare) have generated the current paradigm for hormonal control of gene expression in germinating cereal grain. Gene expression studies in both the aleurone and endosperm tissues of maize (Zea mays) seed have been hampered because of a lack of an efficient transformation system. We report here the rapid isolation of protoplasts from maize aleurone and endosperm tissue, their transformation using polyethylene glycol or electroporation, and the regulation of gene expression in these cells. Adh1 promoter activity was reduced relative to the 35S promoter in aleurone and endosperm protoplasts compared to Black Mexican Sweet suspension cells in which it was nearly as strong as the 35S promoter. Intron-mediated stimulation of expression was substantially higher in transformed aleurone or endosperm protoplasts than in cell-suspension culture protoplasts, and the data suggest that the effect of an intron may be affected by cell type. To examine cytoplasmic regulation, the 5' and 3' untranslated regions from a barley alpha-amylase were fused to the firefly luciferase-coding region, and their effect on translation and mRNA stability was examined following the delivery of in vitro synthesized mRNA to aleurone and endosperm protoplasts. The alpha-amylase untranslated regions regulated translational efficiency in a tissue-specific manner, increasing translation in aleurone or endosperm protoplasts but not in maize or carrot cell-suspension protoplasts, in animal cells, or in in vitro translation lysates.

Animals↗

PubMed1984

A debranching enzyme deficiency in endosperms of the sugary-1 mutants of maize.

Many of the sugary-1 mutants of maize (Zea mays L.) have the highly branched water-soluble polysaccharide, phytoglycogen, in quantities equal to or greater than starch as an endosperm storage product in mature seeds. We find that all sugary mutants investigated are deficient in debranching enzyme [alpha-(1, 6)-glucosidase] activity in endosperm tissue 23 days postpollination and suggest that this deficiency is the primary biochemical lesion leading to phytoglycogen accumulation in sugary endosperms. This would indicate that the amylopectin component of starch depends on an equilibrium between the activities of branching enzymes introducing alpha-1,6 branch points into the linear alpha-1,4 glucans and debranching enzymes. The debranching enzyme activities from nonsugary endosperms can be separated into three peaks on a hydroxyapatite column. The sugary endosperm extracts lack one of these peaks of activity while the other two fractions have much reduced activity. The embryos of developing seeds (23 days after pollination) from both sugary and nonsugary genotypes have equivalent debranching activity. The debranching enzyme activity of developing endosperms is proportional to the number of copies (0 to 3) of the nonmutant (Su) allele present suggesting that the Su allele may be the structural gene for this debranching enzyme, although this is not definitive. This identification of debranching enzyme activity as being the biochemical lesion in sugary endosperms is consistent with several previous observations on the mutant.

Journal Article↗

PubMed1987

In Vitro Sugar Transport in Zea mays L. Kernels : I. Characteristics of Sugar Absorption and Metabolism by Developing Maize Endosperm.

Short-term transport studies were conducted using excised whole Zea mays kernels incubated in buffered solutions containing radiolabeled sugars. Following incubation, endosperms were removed and rates of net (14)C-sugar uptake were determined. Endogenous sugar gradients of the kernel were estimated by measuring sugar concentrations in cell sap collected from the pedicel and endosperm. A sugar concentration gradient from the pedicel to the endosperm was found. Uptake rates of (14)C-labeled glucose, fructose, and sucrose were linear over the concentration range of 2 to 200 millimolar. At sugar concentrations greater than 50 millimolar, hexose uptake exceeded sucrose uptake. Metabolic inhibitor studies using carbonylcyanide-m-chlorophenylhydrazone, sodium cyanide, and dinitrophenol and estimates of Q(10) suggest that the transport of sugars into the developing maize endosperm is a passive process. Sucrose was hydrolyzed to glucose and fructose during uptake and in the endosperm was either reconverted to sucrose or incorporated into insoluble matter. These data suggest that the conversion of sucrose to glucose and fructose may play a role in sugar absorption by endosperm. Our data do not indicate that sugars are absorbed actively. Sugar uptake by the endosperm may be regulated by the capacity for sugar utilization (i.e. starch synthesis).

Journal Article↗

PubMed1999

Imprinting of the MEDEA polycomb gene in the Arabidopsis endosperm.

In flowering plants, two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus that replicates to generate the endosperm, which is a tissue that supports embryo development. MEDEA (MEA) encodes an Arabidopsis SET domain Polycomb protein. Inheritance of a maternal loss-of-function mea allele results in embryo abortion and prolonged endosperm production, irrespective of the genotype of the paternal allele. Thus, only the maternal wild-type MEA allele is required for proper embryo and endosperm development. To understand the molecular mechanism responsible for the parent-of-origin effects of mea mutations on seed development, we compared the expression of maternal and paternal MEA alleles in the progeny of crosses between two Arabidopsis ecotypes. Only the maternal MEA mRNA was detected in the endosperm from seeds at the torpedo stage and later. By contrast, expression of both maternal and paternal MEA alleles was observed in the embryo from seeds at the torpedo stage and later, in seedling, leaf, stem, and root. Thus, MEA is an imprinted gene that displays parent-of-origin-dependent monoallelic expression specifically in the endosperm. These results suggest that the embryo abortion observed in mutant mea seeds is due, at least in part, to a defect in endosperm function. Silencing of the paternal MEA allele in the endosperm and the phenotype of mutant mea seeds supports the parental conflict theory for the evolution of imprinting in plants and mammals.

Alleles↗

PubMed2007

Endosperm formation in aposporous Crataegus (Rosaceae, Spiraeoideae, tribe Pyreae): parallels to Ranunculaceae and Poaceae.

Apomixis in Crataegus is primarily aposporous and requires pollination. The embryo sac is of the Polygonum type. A combination of meiotically unreduced embryo sacs with apparently reduced pollen would violate the usual requirement for a 2 : 1 ratio of maternal to paternal contributions to the endosperm. We therefore investigated the origin of endosperm in seeds of sexual diploids and apomictic polyploids of the sister genera Crataegus and Mespilus. Flow-cytometric DNA measurements from embryo and endosperm in mature seeds were converted to ploidy levels using leaf-tissue information. The diploids had triploid endosperm. In c. 60% of seed from polyploids, one sperm apparently contributes to the endosperm, while 25% or more may involve two sperm. Additional results suggest that trinucleate central cells also occur. Fertilization of meiotically unreduced eggs is indicated. The ratio of maternal to paternal contributions to the endosperm in these apomictic Crataegus is not constrained to 2 : 1. They thus resemble some Sorbus (Pyreae) and very distantly related Ranunculus (Ranunculaceae). It is suggested that Paspalum (Poaceae) may have similarly flexible endosperm ploidy levels.

Crataegus↗

PubMed2000

Positional cues specify and maintain aleurone cell fate in maize endosperm development.

A genetic analysis of maize aleurone development was conducted. Cell lineage was examined by simultaneously marking cells with C1 for anthocyanin pigmentation in the aleurone and wx1 for amylose synthesis in the starchy endosperm. The aleurone and starchy endosperm share a common lineage throughout development indicating that positional cues specify aleurone fate. Mutants in dek1 block aleurone formation at an early stage and cause peripheral endosperm cells to develop as starchy endosperm. Revertant sectors of a transposon-induced dek1 allele showed that peripheral endosperm cells remain competent to differentiate as aleurone cells until late in development. Ds-induced chromosome breakage was used to generate Dek1 loss-of-function sectors. Events occurring until late development caused aleurone cells to switch fate to starchy endosperm indicating that cell fate is not fixed. Thus, positional cues are required to specify and maintain aleurone fate and Dek1 function is required to respond to these cues. An analysis of additional mutants that disrupt aleurone differentiation suggests a hierarchy of gene functions to specify aleurone cell fate and then control aleurone differentiation. These mutants disrupt aleurone differentiation in reproducible patterns suggesting a relationship to endosperm pattern formation.

Amylose↗

PubMed2004

Programmed cell death in wheat during starchy endosperm development.

The starchy endosperm of wheat underwent programmed cell death (PCD) during its development. It showed a ladder of DNA fragments in multiples of 180-200 bp at specific stages when analyzed by agarose gel electrophoresis. The onset of internucleosomal fragmentation of the genome in wheat endosperm could be accelerated by ethylene. While treatment with abscisic acid (ABA) did not delay the onset of DNA fragmentation, but reduced its extent in developing wheat endosperm. During PCD, beside some ultrastructural changes common to other animal and plant apoptotic cells, the starchy endosperm cells also showed some peculiar ultrastructural features. For example, the chromatins in the nucleoplasm were densely aggregated, only a few condensed chromatins abutted onto the inner surface of the nuclear envelope; the nucleus was the first to suffer from degeneration; during nuclear disintegration a large number of organelles could still be observed in the cytoplasm; after nuclear disintegration, the cell remained alive, and synthesis and accumulation of starches and proteins were carried out as usual in the cytoplasm until the starchy endosperm cell died when it was filled with reserves; no formation of apoptotic bodies could be found and the dead starchy endosperm cell served as a reservoir. The PCD of starchy endosperm cells in wheat was a special form of PCD.

Abscisic Acid↗

PubMed2026

Polycomb protein ZmEMF1a restricts endosperm proliferation and directs differentiation via stage-specific H2Aub1 and H3K27me3 landscapes in maize.

Polycomb group (PcG) proteins serve as pivotal epigenetic repressors that govern the transcriptional programs underlying cell growth and differentiation. However, their functional roles in maize endosperm remain largely unexplored. Here, we characterize the recessive maize small-kernel mutant sks1, which exhibits persistent endosperm cell hyperproliferation and compromised cell expansion during grain filling. Map-based cloning reveals that SKS1 encodes ZmEMF1a, a PcG protein that physically interacts with subunits of both PRC1 and PRC2. Integrated ChIP-seq and RNA-seq analyses were performed to investigate its epigenetic regulatory functions. ZmEMF1a orchestrates a stage-specific epigenetic regulatory program: it predominantly mediates H3K27me3 deposition at 6 d after pollination (DAP), while coordinately regulating the deposition of both H3K27me3 and H2Aub1 at 10 DAP. Loss of ZmEMF1a leads to ectopic hyperproliferation of differentiated endosperm tissues, specifically the basal endosperm transfer layer (BETL) and aleurone (AL), as well as elevated vitamin B content in the endosperm. Collectively, these findings establish ZmEMF1a as an epigenetic regulator that balances endosperm proliferation, cell fate specification, and nutrient accumulation through stage-specific histone modifications, thereby offering promising targets for enhancing maize yield and nutritional quality.

H2Aub1↗

PubMed2005

Proteome analysis of embryo and endosperm from germinating tomato seeds.

Proteome analysis of embryo and endosperm tissues from germinating tomato seed was conducted using 1-DE, 2-DE, and MS. Mobilization of the most abundant proteins, which showed similar profiles in the two tissues, occurred first in the endosperm. CBB R-250 staining of 2-DE gels revealed 352 and 369 major protein spots in the embryo and endosperm, respectively, at 0 h. Of these, 75 major spots were selected, excised, in-gel digested with trypsin, and analyzed by MALDI-TOF-MS and/or LC-ESI-Q/TOF-MS/MS. Peptide MS and MS/MS data were searched against publicly available protein and EST databases, and 47 proteins identified. Embryo-specific proteins included a BAC19.13 homologue, whereas four proteins specific to the endosperm were tomato mosaic virus coat proteins related to defense mechanisms. The most abundant proteins both in the embryo and endosperm were seed storage proteins, i.e., legumins (11 spots), vicilins (11 spots), albumin (2 spots). Housekeeping enzymes, actin-binding profilin, defense-related protein kinases, nonspecific lipid transfer protein, and proteins involved in general metabolism were also identified. The roles of some of the proteins identified in the embryo and endosperm are discussed in relation to seed germination in tomato.

Chromatography, Liquid↗

PubMed2001

Cell fate specification in the cereal endosperm.

While superficially simple, endosperm development is a complex, dynamic process. Cereal endosperms contain three major cell types: starchy endosperm, transfer cells and aleurone. The localized accumulation of the END1 transcript in the syncitial endosperm suggests that signals from the maternal placental tissue specify transfer cell type early. Aleurone fate is plastic and requires the continual input of positional cues to maintain cell identity. Starchy endosperm appears to be the default cell type. Mutant patterns suggest that a regulatory hierarchy integrates endosperm development. Requirements for gametic imprinting, maternal : paternal genome ratios and putative chromatin modeling factors indicate the importance of genomic control.

Cell Differentiation↗

PubMed1993

The origin of lysine-containing proteins in opaque-2 maize endosperm.

The reduction of zein synthesis in the maize (Zea mays L.) opaque-2 mutant is associated with an increased percentage of lysine in the endosperm protein. When expressed on an endosperm basis, we found that W64A opaque-2 contains 490 micrograms of lysine compared with 350 micrograms in W64A normal. SDS-PAGE analysis of endosperm proteins indicated that several non-zein proteins are more abundant in the mutant than in normal genotype. To determine the subcellular origin of these proteins, we separated an endosperm homogenate from developing kernels by sucrose density gradient centrifugation and used marker enzyme assays and immunoblot analyses to identify cellular components. Amino acid analysis of proteins in the gradient fractions showed that the majority of the lysine occurs in soluble proteins at the top of the gradient. To identify these proteins, we prepared a complex antiserum against the entire soluble protein fraction and used it to immunoscreen an endosperm cDNA expression library. Sequence analysis of clones identified mRNAs involved in carbohydrate metabolism, amino acid biosynthesis, and protein synthesis. RNA dot blot hybridization analysis with these clones revealed significant variation in the levels of transcripts between normal and opaque-2 endosperm, but we identified several mRNAs that are elevated in opaque-2 and that may encode proteins responsible for the enhanced lysine content.

Cloning, Molecular↗

PubMed2006

Analysis of the wheat endosperm transcriptome.

Among the cereals, wheat is the most widely grown geographically and is part of the staple diet in much of the world. Understanding how the cereal endosperm develops and functions will help generate better tools to manipulate grain qualities important to end-users. We used a genomics approach to identify and characterize genes that are expressed in the wheat endosperm. We analyzed the 17,949 publicly available wheat endosperm EST sequences to identify genes involved in the biological processes that occur within this tissue. Clustering and assembly of the ESTs resulted in the identification of 6,187 tentative unique genes, 2,358 of which formed contigs and 3,829 remained as singletons. A BLAST similarity search against the NCBI non-redundant sequence database revealed abundant messages for storage proteins, putative defense proteins, and proteins involved in starch and sucrose metabolism. The level of abundance of the putatively identified genes reflects the physiology of the developing endosperm. Half of the identified genes have unknown functions. Approximately 61% of the endosperm ESTs has been tentatively mapped in the hexaploid wheat genome. Using microarrays for global RNA profiling, we identified endosperm genes that are specifically up regulated in the developing grain.

Expressed Sequence Tags↗