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PubMed1990

Crossreactivity of IgE antibodies from sera of subjects allergic to both ryegrass pollen and wheat endosperm proteins: evidence for common allergenic determinants.

Positive RAST (greater than 5% radioactive uptakes) to wheat endosperm proteins were found in approximately one-quarter of subjects who had both a positive skin prick and RAST (greater than 10% radioactive uptake) to ryegrass pollen proteins. Immunoblotting of proteins electrophoretically transferred to nitrocellulose membrane after SDS-PAGE of ryegrass pollen and wheat endosperm proteins confirmed the crossreactive properties of the sera identified by RAST testing. Immunoadsorption of serum IgE onto nitrocellulose membrane, to which ryegrass pollen or wheat endosperm proteins had been adsorbed, removed IgE from crossreactive sera reactive to both ryegrass pollen and wheat endosperm proteins. Elution of the adsorbed IgE from the nitrocellulose membrane after immunoadsorption and probing blotted strips of both ryegrass pollen and wheat endosperm proteins supported the results obtained from the immunoadsorption experiments. This data provides evidence that the crossreactivity of IgE antibodies in sera reacting with both ryegrass pollen and wheat endosperm proteins involves common or related determinants and has implications for the clinical management of these allergic subjects.

Blotting, Western↗

PubMed1998

Barley BLZ1: a bZIP transcriptional activator that interacts with endosperm-specific gene promoters.

A cDNA encoding a bZIP transcription factor was obtained from barley endosperm and used to identify the corresponding gene from a genomic library. The gene, designated Blz1, contained six exons and five introns, plus a 442 nt-long 5'-untranslated leader sequence, and was located on chromosome 5H. The Blz1 mRNA was detected early in endosperm development and was also expressed in roots and leaves. The BLZ1 protein was a potent transcriptional activator in a yeast system; 85% of its activity was associated with the first 203 amino acid residues at the N-terminus, which included two acidic regions. Presumptive involvement of Blz1 in the regulation of gene expression in endosperm was ascertained by the DNA-binding properties of BLZ1 in electrophoretic mobility shift assays (EMSA) and by transient expression in barley developing endosperms, using, as effectors, Blz1 in both sense and anti-sense orientations. In the co-bombardment experiments, the beta-glucuronidase (GUS) reported gene responded to Blz1 if under the control of the endosperm-specific ltr1 promoter or under a synthetic promoter containing the endosperm box of gene Hor2. Sucrose synthase promoters Ss1 and Ss2 and synthetic promoters containing mutated sequences of Hor2 were unaffected in trans by Blz1.

Amino Acid Sequence↗

PubMed2006

Transgenic rice lines expressing maize C1 and R-S regulatory genes produce various flavonoids in the endosperm.

Flavonoids, compounds that possess diverse health-promoting benefits, are lacking in the endosperm of rice. Therefore, to develop transgenic lines that produce flavonoids, we transformed a white rice cultivar, Oryza sativa japonica cv. Hwa-Young, with maize C1 and R-S regulatory genes. Expression of these transgenes was restricted to the endosperm using the promoter of a rice prolamin gene. The pericarp of the C1/R-S homozygous lines became dark brown in accordance with their maternal genotype, whereas the endosperm turned chalky, similar to the opaque kernel phenotype. Analysis via high-performance liquid chromatography (HPLC) revealed that numerous kinds of flavonoids were produced in these transgenic kernels. To identify individual flavonoids, the number of HPLC peaks was reduced through moderate acid hydrolysis, followed by ethyl acetate partitioning. Amongst the major flavonoids, dihydroquercetin (taxifolin), dihydroisorhamnetin (3'-O-methyl taxifolin) and 3'-O-methyl quercetin were identified through liquid chromatography/mass spectrometry/mass spectrometry and nuclear magnetic resonance analyses. Fluorescence labelling with diphenylboric acid showed that the flavonoids were highly concentrated in the cells of four to five outer endosperm layers. More importantly, a high fluorescence signal was present in the cytosol of the inner endosperm layers. However, the overall signal in the inner layers was significantly lower because starch granules and protein bodies occupied most of the cytosolic space. Our estimate of the total flavonoid content in the transgenic kernels suggests that C1/R-S rice has the potential to be developed further as a novel variety that can produce various flavonoids in its endosperm.

Chromatography, High Pressure Liquid↗

PubMed2001

Mitotic-cycle time and the development of embryo and endosperm in compatible and incompatible crosses in tuber-bearing Solanum species.

To understand the relationship between early seed development and the EBN (endosperm balance number) hypothesis, the embryo and endosperm growth rates in crosses among Solanum spp. with the same and different EBNs were analyzed. For the embryo, the differences in the mean cell-doubling time (MCDT) between the compatible species 2x Solanum gourlayi and Solanum acaule was 3.9 h, whereas the incompatible species Solanum commersonii had MCDT differences of 10 and 13.9 h with 2x S. gourlayi and S. acaule, respectively. The embryo growth rates of the 2EBN species S. acaule and S. gourlayi were almost twice as fast as that of the 1EBN species S. commersonii. Nuclei of variable sizes were observed in the endosperms resulting from incompatible crosses. The author discusses the possibility that the collapse of the endosperm in inter-EBN crosses could be caused by differences in the MCDT between the parents that produce hybrid endosperms with high levels of DNA synthesis and transcription activity, resulting in increased nuclear size. A model is proposed to explain the formation of enlarged endosperm nuclei in incompatible crosses in the genus Solanum.

Cell Cycle↗

PubMed2005

Chromosome organization in wheat endosperm and embryo.

We have analysed the chromosome organization in endosperm and embryo of bread wheat (Triticum aestivum L.), in order to compare these tissues with developing anthers, in which the centromeres associate, and the developing root xylem vessel cells, in which the chromosomes endoreduplicate to become polytene and associate via their centromeres. Both endosperm and embryo showed a typical Rabl configuration and a degree of non-homologous centromere association and the endosperm also showed extensive telomere association. Wheat endosperm is initially triploid and during its development a percentage of the nuclei increase their DNA content to 6C and 12C. 6C nuclei showed twice as many centromeres as 3C nuclei and the centromere number increased further in 12C nuclei. The higher the C-content of a nucleus the more the telomeres associated in endosperm. The vast majority of 12C nuclei showed six rye chromosome arms, although a few showed three associated groups of rye chromosome arms. This means that during endosperm development wheat nuclei show both polyploidization and polytenization.

Base Sequence↗

PubMed2006

Maternal gametophytic baseless1 is required for development of the central cell and early endosperm patterning in maize (Zea mays).

In angiosperms, double fertilization of an egg cell and a central cell with two sperm cells results in the formation of a seed containing a diploid embryo and a triploid endosperm. The extent to which the embryo sac controls postfertilization events in the seed is unknown. The novel gametophytic maternal-effect maize mutation, baseless1 (bsl1) affects central cell development within the embryo sac, frequently by altering the position of the two polar nuclei. Despite this irregularity, fertilization is as efficient as in wild type. The spatial expression of basal endosperm-specific transcripts is altered in free-nuclear and cellular mutant endosperms. At later stages of seed development, bsl1 predominantly affects development of the basal endosperm transfer layer (BETL). When bsl1/+ diploid plants were pollinated by wild-type tetraploid plants, the BETL abnormalities observed in bsl1/bsl1/+/+ tetraploid endosperms were diverse and of variable severity. Moreover, the frequency of kernels with severely perturbed BETL development correlated with the percentage of severely affected bsl1 central cells. Therefore, BSL1 is likely required in the central cell before fertilization for correct BETL patterning to occur. These findings provide new genetic evidence that a maternal gametophytic component is necessary for correct endosperm patterning.

Gametogenesis↗

PubMed2000

The second step of the biphasic endosperm cap weakening that mediates tomato (Lycopersicon esculentum) seed germination is under control of ABA.

The role of abscisic acid (ABA) in the weakening of the endosperm cap prior to radicle protrusion in tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds was studied. The endosperm cap weakened substantially in both water and ABA during the first 38 h of imbibition. After 38 h the force required for endosperm cap puncturing was arrested at 0.35 N in ABA, whereas in water a further decrease occurred until the radicle protruded. During the first 2 d of imbibition endo-beta-mannanase activity was correlated with the decrease in required puncture force and with the appearance of ice-crystal-induced porosity in the cell walls as observed by scanning electron microscopy. Prolonged incubation in ABA resulted in the loss of endo-beta-mannanase activity and the loss of ice-crystal-induced porosity, but not in a reversion of the required puncture force. ABA also had a distinct but minor effect on the growth potential of the embryo. However, endosperm cap resistance played the limiting role in the completion of germination. It was concluded that (a) endosperm cap weakening is a biphasic process and (b) inhibition of germination by ABA is through the second step in the endosperm cap weakening process.

Abscisic Acid↗

PubMed1977

[Patterns in the cellular organization of ovule growth in the pine, Pinus silvestris L. I. Growth processes in endosperm].

The dynamics of growth processes was studied in the endosperm of Pinus silvestris during the year of fertilization. The growth of endosperm is described by an diauxic curve where the period of delay corresponds, by time, to fertilization. The growth of tissue proceeds, mainly, at the expense of cell proliferation. The mitotic activity is preserved in the endosperm till the beginning of formation of reserve substances in the cells; cell division and elongation occur simultaneously. The unfavourable environmental conditions influence the mitotic activity of cells only. The size of fully grown cells is relatively constant and somewhat decreases later, during maturation. One can distinguish rather early those regions in the endosperm where the cell size is 2-3 times less than in the rest tissue; the size of cells and nuclei in these regions suffers only insignificant changes during the growth. The large nuclei are observed in the beginning of the cell endosperm formation and, then, their size decreases 1.5-2 times; the second increase in nuclear size in the endosperm is observed during the period prior to the formation of reserve substances.

Animals↗

PubMed1996

Inhibition of UDP-glucose: protein transglucosylase by a maize endosperm protein factor.

UDP-glucose: protein transglucosylase (UPTG, EC 2.4.1.112) catalyzes the first step of protein-bound alpha-glucan synthesis in potato tuber and developing maize endosperm. The presence of a non-dialyzable, heat labile protein responsible for low levels of UPTG activity in developing maize endosperm was investigated. UPTG activity in 5-day old maize seedlings and potato tuber solubilized preparations was also reduced by the endosperm preparation. FPLC-Mono Q column chromatography of developing maize endosperm was effective in separating the inhibitor protein (IP) from UPTG. After gel filtration on Superose 12, IP yielded a major polypeptide of about 80 kDa on SDS-PAGE. IP was purified by gel filtration on Superose 12 and preparative SDS-PAGE, and specific antibodies were prepared. Polyclonal antibodies reacted specifically with an 80 kDa polypeptide of developing maize endosperm on Western blot. They also recognized a similar band in 5-day old maize seedlings, but not in potato tubers. The identification of a factor that regulates the level of UPTG activity in developing maize endosperm may help to elucidate the functional role of the enzyme in the initiation of starch synthesis during seed development.

Enzyme Inhibitors↗

PubMed1991

Binding of an endosperm-specific nuclear protein to a maize beta-zein gene correlates with zein transcriptional activity.

Promoter regions of alpha- and beta-zein genes were analyzed for binding of nuclear proteins from developing endosperm and seedling tissue of maize. Using a band-shift assay, we identified two distinct protein factors, alpha-1 and beta-1, that interacted specifically with alpha- and beta-zein gene promoter regions, respectively. Alpha-1 was present in nuclei from both endosperm and seedling tissue, whereas beta-1 was found only in nuclei from developing endosperm tissue. Mixing of nuclear extracts demonstrated that seedling tissue contained undetectable amounts of beta-1, rather than having an inhibitor for formation of the beta-1/DNA complex. Chemical footprinting analysis localized the beta-1 recognition site to a 22 bp sequence flanked by CCAT and TATA boxes. The apparent molecular mass of beta-1 was determined to be 29 kDa by southwestern blotting. Based on in vitro binding assays, the greatest concentration of the beta-1 in endosperm nuclei is at 16 days after pollination, which coincides with the time of highest transcriptional activity of the beta-zein gene. These results suggest that beta-1 may act as a tissue-specific, trans-acting regulator of the expression of the beta-zein gene in developing maize endosperm.

Base Sequence↗

PubMed1984

Zein synthesis in the embryo and endosperm of maize mutants.

Zein synthesis in the developing (22 and 50 days postpollination) endosperm and embryo of maize (Zea mays L.) double mutants, brittle-1; opaque-2 and brittle-2;opaque-2, were compared and correlated with sucrose concentration and RNase activity in order to test the hypothesis that high sucrose concentrations may prevent the interaction between zein polyribosomes and endoplasmic reticulum and make the zein mRNAs more susceptible to hydrolysis by high RNase activity, resulting in a severe reduction in zein synthesis. The double-mutant combinations of opaque-2 with each of the starch-deficient mutants, brittle-1 and brittle-2, maintained not only a high sucrose concentration in the endosperm but also a higher RNase activity than either one of the single mutants alone. Consequently, these double mutants severely suppressed the synthesis of two major zein components in their endosperms. In contrast to the endosperm system, embryos of the double mutants produced amounts of zein (and electrophoretic patterns) similar to that of the opaque-2 embryo, and their embryos contained levels of sucrose and RNase activity comparable to that of the o2 and normal control. These results are consistent with the notion that a posttranscriptional degradation of zein mRNAs by RNase, rather than a specific transcriptional block, is involved in the endosperm to suppress zein synthesis in these double mutants.

Amino Acids↗

PubMed1975

[Investigation on fat-quality of embryo, endosperm and crush fractions of maize kernel (author's transl)].

Investigations were conducted on the composition of fatty acids in the fat of the embryo, the endosperm and several crush fractions of the maize kernel. The following results were obtained: 1. The embryo fat contained relatively lower contents of palmitin-, stearin- and linolenic acid, on the other hand higher contents of oleic- and linoleic acid in comparism to endosperm fat. 2. This result was identical to each one of the seven varieties examined. 3. The examined varieties presented clear differences both in the absolute contents of single fatty acids in the embryo and in the endosperm, as well as in the relationship between the components in the germ and in the endosperm. 4. The differences observed between the germ- and the endosperm fat in the crush fractions in some cases were clearly visible, while in other cases were not traceable.

Fatty Acids↗

PubMed1996

Hordein promoter methylation and transcriptional activity in wild-type and mutant barley endosperm.

B- and C-hordein gene transcription is severely reduced in the endosperm of the regulatory barley mutant lys3a, and this is correlated with persistent hypermethylation of the promoters. In contrast, D-hordein is expressed at normal levels in the mutant. To confirm the connection between methylation and transcriptional activity, a genomic D-hordein clone was isolated and sequenced. The nucleotide composition of the promoter region revealed a CpG island and methylation analysis, using bisulphite treatment of genomic DNA, confirmed that the D-hordein promoter is unmethylated in endosperm and leaf tissue. Immunocytochemical studies localized D-hordein to the reticular component of protein bodies in both the wild-type Bomi and lys3a. Transient expression of GUS reporter gene constructs in barley endosperm, following transfection by particle bombardment revealed the D-hordein promotors. Comparison of transient expression in Bomi and lys3a endosperm demonstrated that the activities of the unmethylated D-hordein and the Hor1-14 C-hordein promoters were equivalent, while the activities in the mutant of the Horl-17 C-hordein and the Hor2-4 B-hordein promoters were reduced two- and tenfold, respectively. Methylation of plasmids in vitro prior to expression severely inhibited B- and D-hordein promoter activities. Based on these observations two categories of promoters for endosperm-specific expression of storage proteins are recognized and a model involving methylation and modulation of chromatin structure in the regulation by the Lys3 gene is presented.

Base Sequence↗

PubMed2002

Cloning of cDNA for UDP-glucose pyrophosphorylase and the expression of mRNA in rice endosperm.

Rice endosperm UDP-glucose pyrophosphorylase (UGPase) cDNA clones were isolated by screening a lambda ZAP II library prepared from poly (A(+)) RNA of japonica rice (cv Sasanishiki) endosperm with a probe of potato UGPase cDNA. One cDNA clone, possessing about 1,700 nucleotides, contained the complete open reading frame of rice UGPase. At the nucleotide-sequence level, the UGPase cDNA of rice endosperm had high homology with the UGPase cDNA of barley endosperm (84%) and potato tuber (71%). The calculated molecular weight (50 kDa) agrees with the value determined by SDS-PAGE (51 kDa). At the amino-acid sequence level, rice UGPase has high homology with the UGPase of barley (92%) and potato (85%). The enzyme contained conserved sequence elements which are thought to be involved in substrate binding and catalytic activity. A Southern-blot analysis indicated that the gene existed as a single copy. Expression of the enzyme in rice endosperm examined by Northern-blot analysis was high at 10-15 days after heading.

Journal Article↗

PubMed2004

Nuclear fusions contribute to polyploidization of the gigantic nuclei in the chalazal endosperm of Arabidopsis.

Somatic polyploidization is recognized as a means to increase gene expression levels in highly active metabolic cells. The most common mechanisms are endoreplication, endomitosis and cell fusion. In animals and plants the nuclei of multinucleate cells are usually prevented from fusing. Here, we report that the nuclei from the syncytial cyst of the chalazal endosperm of Arabidopsis thaliana (L.) Heynh. are polyploid with some intermediate ploidy levels that cannot be attributed to endoreplication, suggesting nuclear fusion. Analysis of isolated nuclei, together with fluorescent in situ hybridization (FISH), revealed that nuclei from the chalazal endosperm are two or three times bigger than the nuclei from the peripheral endosperm and have a corresponding increase in ploidy. Together with the consistent observation of adjoined nuclei, we propose that nuclear fusion contributes, at least in part, to the process of polyploidization in the chalazal endosperm. Confocal analysis of intact seeds further suggested that free nuclei from the peripheral endosperm get incorporated into the chalazal cyst and likely participate in nuclear fusions.

Arabidopsis↗

PubMed2006

Temporal and spatial appearance of wall polysaccharides during cellularization of barley (Hordeum vulgare) endosperm.

Barley endosperm begins development as a syncytium where numerous nuclei line the perimeter of a large vacuolated central cell. Between 3 and 6 days after pollination (DAP) the multinucleate syncytium is cellularized by the centripetal synthesis of cell walls at the interfaces of nuclear cytoplasmic domains between individual nuclei. Here we report the temporal and spatial appearance of key polysaccharides in the cell walls of early developing endosperm of barley, prior to aleurone differentiation. Flowering spikes of barley plants grown under controlled glasshouse conditions were hand-pollinated and the developing grains collected from 3 to 8 DAP. Barley endosperm development was followed at the light and electron microscope levels with monoclonal antibodies specific for (1-->3)-beta-D: -glucan (callose), (1-->3,1-->4)-beta-D: -glucan, hetero-(1-->4)-beta-D: -mannans, arabino-(1-->4)-beta-D: -xylans, arabinogalactan-proteins (AGPs) and with the enzyme, cellobiohydrolase II, to detect (1-->4)-beta-D: -glucan (cellulose). Callose and cellulose were present in the first formed cell walls between 3 and 4 DAP. However, the presence of callose in the endosperm walls was transient and at 6 DAP was only detected in collars surrounding plasmodesmata. (1-->3,1-->4)-beta-D: -Glucan was not deposited in the developing cell walls until approximately 5 DAP and hetero-(1-->4)-beta-D: -mannans followed at 6 DAP. Deposition of AGPs and arabinoxylan in the wall began at 7 and 8 DAP, respectively. For arabinoxylans, there is a possibility that they are deposited earlier in a highly substituted form that is inaccessible to the antibody. Arabinoxylan and heteromannan were also detected in Golgi and associated vesicles in the cytoplasm. In contrast, (1-->3,1-->4)-beta-D: -glucan was not detected in the cytoplasm in endosperm cells; similar results were obtained for coleoptile and suspension cultured cells.

Cell Wall↗

PubMed1998

Genetic evidence that the two isozymes of sucrose synthase present in developing maize endosperm are critical, one for cell wall integrity and the other for starch biosynthesis.

In maize, two paralogous genes, Sh1 and Sus1, encode two biochemically similar isozymes of sucrose synthase, SS1 and SS2, respectively. Previous studies have attributed the mild starch deficiency of the shrunken1 (sh1) endosperm to the loss of the SS1 isozyme in the mutant. Here we describe the first mutation in the sucrose synthase1 (Sus1) gene, sus1-1, and the isolation of a double recessive genotype, sh1 sus1-1. Combined data from diverse studies, including Northern and Western analyses, RT-PCR and genomic PCR, cloning and sequencing data for the 3' region, show that the mutant sus1-1 gene has a complex pattern of expression, albeit at much reduced levels as compared to the Sus1 gene. Endosperm sucrose synthase activity in sh1 sus1-1 was barely 0.5% of the total activity in the Sh1 Sus1 genotype. Significantly, comparative analyses of Sh1 Sus1, sh1 Sus1 and sh1 sus1-1 genotypes have, for the first time, allowed us to dissect the relative contributions of each isozyme to endosperm development. Starch contents in endosperm of the three related genotypes were 100, 78 and 53%, respectively. Anatomical analyses, which confirmed the previously described early cell degeneration phenotype unique to the sh1 Sus1 endosperm, revealed no detectable difference between the two sh1 genotypes. We conclude that the SS1 isozyme plays the dominant role in providing the substrate for cellulose biosynthesis, whereas the SS2 protein is needed mainly for generating precursors for starch biosynthesis.

Base Sequence↗

PubMed2004

Vacuolar H+-translocating inorganic pyrophosphatase (Vpp1) marks partial aleurone cell fate in cereal endosperm development.

Cereal endosperm is a model system for cell fate determination in plants. In wild-type plants the outermost endosperm cells adopt aleurone cell fate, while all underlying cells display starchy endosperm cell fate. Mutant analysis showed that cell fate is determined by position rather than lineage. To further characterise the precise cell fate of the outermost cells, we performed a differential screen and isolated the novel marker gene Vpp1 . It encodes a vacuolar H+-translocating inorganic pyrophosphatase (V-PPase) and is mainly expressed in kernels, leaves and tassels. In kernels, its expression is restricted to the aleurone layer with the maximum of expression shifting from the adaxial to the abaxial side during early stages. Together with three other marker genes Vpp1 was then used to analyse the cell fate of the outermost cells in Dap3 , Dap7 , cr4 and dek1 mutants, all of which have aberrant aleurone layers. In the Dap3 and Dap7 mutants the Vpp1 and Ltp2 markers but not the A1 and Zein markers were expressed in patches without aleurone indicating that the outermost cells had some but not all features of aleurone cells and did not simply adopt starchy endosperm cell fate. A similar result was obtained in the cr4 mutant, although Ltp2 expression was less generalised. In other Dap7 patches characterised by multiple aleurone-like cell layers the expression of Vpp1 and Ltp2 confirmed the aleurone cell fate of the cells in the additional cell layers. The analysis of dek1 mutants confirmed the starchy endosperm cell fate of the majority but not all outermost cells. Based on these data we propose a model suggesting a stepwise commitment to aleurone cell fate. Sequential steps are marked by the expression of Vpp1 , the expression of Ltp2 , the acquisition of a regular shape and thick walls and finally pigmentation coupled with A1 expression.

Cell Lineage↗