Showing papers on "Endosperm published in 1995"

The amylose content in rice endosperm is related to the post-transcriptional regulation of the waxy gene.

Abstract: The waxy (Wx) gene of rice encodes a granule-bound starch synthase (GBSS = waxy protein) required for the synthesis of amylose in endosperm. An analysis of Wx transcripts, Wx protein, and amylose content of 31 rice cultivars revealed that endosperm amylose and Wx protein contents are correlated with the ability of the cultivar to excise intron I from the leader sequence of the Wx transcript. Cultivars with high endosperm amylose content (group I) contain high levels of amylose, Wx protein, and the mature 2.3 kb Wx mRNA. Cultivars with intermediate amylose content (group II) produce substantial amounts of a large 3.3 kb Wx pre-mRNA, with intron I still present, in addition to the mature Wx mRNA, and intermediate levels of Wx protein. Glutinous rice (group III cultivars) contains no amylose, no Wx protein, and no mature Wx mRNA; only the incompletely spliced 3.3 kb Wx pre-mRNA is present in group III cultivars. Based on these results, it is hypothesized that the amylose content of rice endosperm is regulated at the level of Wx transcript processing, and, more specifically, at the stage of intron I excision from the Wx pre-mRNA.

Integrated control of seed maturation and germination programs by activator and repressor functions of Viviparous-1 of maize.

Abstract: The Viviparous-1 (VP1) transcriptional activator of maize is required for abscisic acid induction of maturation-specific genes late in seed development leading to acquisition of desiccation tolerance and arrest in embryo growth. Here, we show that VP1 also inhibits induction of the germination-specific alpha-amylase genes in aleurone cells of the developing seed and thereby appears to be involved in preventing precocious hydrolyzation of storage compounds accumulating in the endosperm. In developing seeds of the somatically instable vp1-m2 mutant, hydrolase activity was derepressed specifically in endosperm sectors underlying vp1 mutant aleurone. A barley alpha-amylase promoter-GUS reporter construct (Amy-GUS) was induced in developing vp1 mutant aleurone cells but not in wild-type aleurone cells. Moreover, transient expression of recombinant VP1 and vp1 mutant aleurone cells strongly inhibited expression of Amy-GUS and thus effectively complemented this aspect of the mutant phenotype. VP1 specifically repressed induction of Amy-GUS by the hormone gibberellic acid in aleurone of germinating barley seeds. Deletion of the acidic transcriptional activation domain of VP1 did not affect the inhibitory activity, indicating that VP1 has a discrete repressor function. Hence, physically combining activator and repressor functions in one protein may provide a mechanism to integrate the control of two normally consecutive developmental programs, seed maturation and seed germination.

Endoreduplication in maize endosperm: involvement of m phase--promoting factor inhibition and induction of s phase--related kinases.

Abstract: Endoreduplication is an endonuclear chromosome duplication that occurs in the absence of mitosis and in Zea mays (L.) is required for endosperm development. Induction of DNA synthesis during early stages of endosperm development is maintained by increasing the amount and activity of S phase-related protein kinases, which was demonstrated here by their ability to interact with human E2F or with the adenovirus E1A proteins. In addition it was shown that endoreduplicated endosperm cells contain an inhibitor that suppresses the activity of the M phase-promoting factor (MPF). These results demonstrate that in maize endosperm, endoreduplication proceeds as a result of two events, inhibition of MPF and induction of S phase-related protein kinases.

Plants and processes for obtaining them

Abstract: The invention relates to a transgenic or mutated plant having genomic material which alters the normal starch synthesis pathway within the plant. More specifically, the present invention relates to a plant having a genotype which creates new forms of starch in significant quantity. Particularly, the invention relates to grain having an embryo with a genotype heterozygous for two or more wild type genes (for example, Aa/Bb) and an endosperm having a genotype heterozygous for such genes (for example, AAa/BBb or AAa/bbB or aaA/BBb or aaA/bbB) and the starch produced therefrom.

Identification of multiple isoforms of soluble and granule-bound starch synthase in developing wheat endosperm

Abstract: We have investigated the nature and locations of isoforms of starch synthase in the developing endosperm of wheat (Triticum aestivum L.). There are three distinct granule-bound isoforms of 60 kDa (the Waxy gene product), 77 kDa and 100–105 kDa. One of these isoforms, the 77-kDa protein, is also present in the soluble fraction of the endosperm but it contributes only a small proportion of the total soluble activity. Most of the soluble activity is contributed by isoforms which are apparently not also granule-bound. The 60-kDa and 77kDa isoforms of wheat are antigenically related to isoforms of very similar size in the developing pea embryo, but the other isoforms in the endosperm appear to have no counterparts in the pea embryo. The significance of these results in terms of the diversity of isoforms of starch synthase and their locations is discussed.

Relationship between endosperm texture and the occurrence of friabilin and bound polar lipids on wheat starch

Abstract: Endosperm texture affects the milling and end-use properties of wheat grain. A better understanding of the physical-chemical mechanism and the genetic control of endosperm texture in wheat would aid in breeding, marketing, and utilization of grain. Here, we report on the relationship between endosperm texture and the occurrence of friabilin, a family of 15-kDa proteins, and hound glyco- and phospho-lipids on water-washed wheat starch. These two classes of bound polar lipids follow the same pattern of occurrence as friabilin: approximately equal levels in soft and hard wheat flour, much reduced levels in water-washed soft wheat starch compared to flour, and much reduced levels in hard wheat starch compared to soft. The type and quantity of these bound polar lipids is highly conserved among both soft and hard wheat starches. Further, these lipids are implicated in the interaction of friabilin with soft wheat starch. Propan-2-ol and water (90: 10), which is effective in removing bound polar lipids from starch, renders most friabilin components extractable with an aqueous salt solution. These results suggest that most friabilin components interact with starch through lipid-mediated hydrophobic interactions and ionic interactions. In addition, the results provide both an additional biochemical marker for grain softness and new insight into the possible physical-chemical mechanism and the genetic control of endosperm texture in wheat

Class I [beta]-1,3-Glucanases in the Endosperm of Tobacco during Germination

Abstract: Rupture of the seed coat and rupture of the endosperm are separate events in the germination of Nicotiana tabacum L. cv Havana 425 seeds. Treatment with 10–5 M abscisic acid (ABA) did not appreciably affect seed-coat rupture but greatly delayed subsequent endosperm rupture by more than 100 h and resulted in the formation of a novel structure consisting of the enlarging radicle with a sheath of greatly elongated endosperm tissue. Therefore, ABA appears to act primarily by delaying endosperm rupture and radicle emergence. Measurements of [beta]-1,3-glucanase activity, antigen content, and mRNA accumulation together with reporter gene experiments showed that induction of class I [beta]-1,3-glucanase genes begins just prior to the onset of endosperm rupture but after the completion of seed-coat rupture. This induction was localized exclusively in the micropylar region of the endosperm, where the radicle will penetrate. ABA treatment markedly inhibited the rate of [beta]-1,3-glucanase accumulation but did not delay the onset of induction. Independent of the ABA concentration used, onset of endosperm rupture was correlated with the same [beta]-1,3-glucanase content/seed. These results suggest that ABA-sensitive class I [beta]-1,3-glucanases promote radicle penetration of the endosperm, which is a key limiting step in tobacco seed germination.

Gibberellin Action in Germinated Cereal Grains

Abstract: The study of the response of cereal aleurone to gibberellin and abscisic acid (GA and ABA, respectively), particularly with reference to a-amylase synthesis, has made a significant contribution to our understanding of GA action in plant cells, especially as it relates to the control of protein synthesis, and also to the function of the endosperm during germination. While much of the work has been carried out using isolated aleurone from a single cultivar of barley (“Himalaya”), it seems so far that the principles which have emerged from this system can be applied to in vivo behaviour of barley and other cereal grains.

Cloning of a Coconut Endosperm cDNA Encoding a 1-Acyl-sn-Glycerol-3-Phosphate Acyltransferase That Accepts Medium-Chain-Length Substrates

Abstract: Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (HM Davies, DJ Hawkins, JS Nelsen [1995] Phytochemistry 39:989-996) Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 348 kD Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme

Accumulation of 15-Kilodalton Zein in Novel Protein Bodies in Transgenic Tobacco.

Abstract: Zeins, the seed storage proteins of maize, are a group of alcohol-soluble polypeptides of different molecular masses that share a similar amino acid composition but vary in their sulfur amino acid composition. They are synthesized on the rough endoplasmic reticulum (ER) in the endosperm and are stored in ER-derived protein bodies. Our goal is to balance the amino acid composition of the methionine-deficient forage legumes by expressing the sulfur amino acid-rich 15-kD zeins in their leaves. However, it is crucial to know whether this protein would be stable in nonseed tissues of transgenic plants. The major focus of this paper is to compare the accumulation pattern of the 15-kD zein protein with a vacuolar targeted seed protein, [beta]-phaseolin, in nonseed tissues and to determine the basis for its stability/instability. We have introduced the 15-kD zein and bean [beta]-phaseolin-coding sequences behind the 35S cauliflower mosaic virus promoter into tobacco (Nicotiana tabacum) and analyzed the protein's accumulation pattern in different tissues. Our results demonstrate that the 15-kD seed protein is stable not only in seeds but in all nonseed tissues tested, whereas the [beta]-phaseolin protein accumulated only in mid- and postmaturation seeds. Interestingly, zein accumulates in novel protein bodies both in the seeds and in nonseed tissues. We attribute the instability of the [beta]-phaseolin protein in nonseed tissues to the fact that it is targeted to protease-rich vacuoles. The stability of the 15-kD zein could be attributed to its retention in the ER or to the protease-resistant nature of the protein.

Molecular characterization of BET1, a gene expressed in the endosperm transfer cells of maize.

Abstract: A cDNA clone, BET1 (for basal endosperm transfer layer), was isolated from a cDNA bank prepared from 10-days after pollination (DAP) maize endosperm mRNA. BET1 mRNA was shown to encode a 7-kD cell wall polypeptide. Both the mRNA and protein were restricted in their distribution to the basal endosperm transfer layer and were not expressed elsewhere in the plant. BET1 expression commenced at 9 DAP, reached a maximum between 12 and 16 DAP, and declined after 16 DAP. The initial accumulation of the BET1 polypeptide reached a plateau by 16 DAP and declined thereafter, becoming undetectable by 20 DAP. The antibody raised against the BET1 protein reacted with a number of polypeptides of higher molecular mass than the BET1 monomer. Most of these were present in cytosolic fractions and were found in nonbasal cell endosperm extracts, but three species appeared to be basal cell specific. This result and the reactivity of exhaustively extracted cell wall material with the BET1 antibody suggest that a fraction of the protein is deposited in a covalently bound form in the extracellular matrix. We propose that the BET1 protein plays a role in the structural specialization of the transfer cells. In addition, BET1 provides a new molecular marker for the development of this endosperm domain.

The maize brittle 1 gene encodes amyloplast membrane polypeptides.

Abstract: A chimeric protein, formed of 56 amino acids from the carboxy terminus of the maize (Zea mays L.) wild-type Brittle1 (Bt1) protein fused to the glutathione-S-transferase gene, was synthesized in Escherichia coli, and used to raise antibodies. Following affinity purification, the antibodies recognized a set of 38- to 42-kDa proteins in endosperm from wild-type Bt1 plants, as well as from brittle2, shrunken2 and sugary1 plants, but not in mutant bt1 endosperm. Bt1 proteins were not detected with the preimmune antibodies. A low level of Bt1-specific proteins was detected at 10 d after pollination (DAP) and increased to a plateau at 16 DAP. At the same time, the ratio of slow- to fast-migrating forms of the protein decreased. During endosperm fractionation by differential centrifugation and membrane sedimentation in sucrose gradients, the Bt1 proteins co-purified with the carotenoid-containing plastid membranes. They were localized to amyloplasts by electron-microscopic immunocytochemistry; most of the signal was detected at the plastid periphery. These results are consistent with predictions made from the deduced amino-acid sequence and previous in-vitro experiments that the bt1 locus encodes amyloplast membrane proteins.

The Bean Seed Storage Protein [beta]-Phaseolin Is Synthesized, Processed, and Accumulated in the Vacuolar Type-II Protein Bodies of Transgenic Rice Endosperm.

Abstract: The seed storage protein [beta]-phaseolin of the common bean (Phaseolus vulgaris L.) was expressed in the endosperm of transgenic rice (Oryza sativa L.) plants. The 5.1- or 1.8-kb promoter fragment of the rice seed storage protein glutelin Gt1 gene was fused transcriptionally to either the genomic or cDNA coding sequence of the [beta]-phaseolin gene. The highest quantity of phaseolin estimated by enzyme-linked immunosorbent assay was 4.0% of the total endosperm protein in the transgenic rice seeds. The phaseolin trait was segregated as a single dominant trait with a positive gene dosage effect and was stably inherited through three successive generations. Both phaseolin genomic and cDNA coding sequences were used to synthesize four isoforms of mature phaseolin protein with apparent molecular masses of 51, 48, 47, and 45 kD. Enzyme deglycosylation experiments indicated that the 51-kD form contains high-mannose N-glycans; the 48- and 47-kD forms have further modified N-glycans; and the 45-kD form is a nonglycosylated protein. Immunolabeling studies using light and electron microscopy demonstrated that phaseolin accumulates primarily in the vacuolar type-II protein bodies located at the periphery of the endosperm near the aleurone layer. We discuss the implications of these results on nutritional improvement of rice grains.

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

Abstract: 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.

Endosperm-specific demethylation and activation of specific alleles of α-tubulin genes of Zea mays L.

Abstract: We have investigated the methylation status of the α-tubulin genes, and the degree of accumulation of their mRNAs in endosperm, embryo and seedling tissues of Zea mays L. We have found that many of the α-tubulin genes are differentially demethylated in the endosperm relative to the embryo and seedling. However, only for tubα2 and tubα4 could a correlation between DNA demethylation and increased RNA accumulation be detected. By analyzing the inbred lines W64A and A69Y and their reciprocal crosses, we have also identified in the endosperm two α-tubulin genes, tubα3 and tubα4, that are differentially demethylated if transmitted by the maternal germline, but that remain hypermethylated when transmitted by the paternal germline.

Elongation factor 1 alpha concentration is highly correlated with the lysine content of maize endosperm.

Abstract: Lysine is the most limiting essential amino acid in cereals, and for many years plant breeders have attempted to increase its concentration to improve the nutritional quality of these grains. The opaque2 mutation in maize doubles the lysine content in the endosperm, but the mechanism by which this occurs is unknown. We show that elongation factor 1 alpha (EF-1 alpha) is overexpressed in opaque2 endosperm compared with its normal counterpart and that there is a highly significant correlation between EF-1 alpha concentration and the total lysine content of the endosperm. This relationship is also true for two other cereals, sorghum and barley. It appears that genetic selection for genotypes with a high concentration of EF-1 alpha can significantly improve the nutritional quality of maize and other cereals.

Purification, Characterization, and Intracellular Localization of Glycosylated Protein Disulfide Isomerase from Wheat Grains

Abstract: Wheat (Triticum aestivum) storage proteins fold and assemble into complexes that are linked by intra- and intermolecular disulfide bonds, but it is not yet clear whether these processes are spontaneous or require the assistance of endoplasmic reticulum (ER)-resident enzymes and molecular chaperones. Aiming to unravel these processes, we have purified and characterized the enzyme protein disulfide isomerase (PDI) from wheat endosperm, as well as studied its developmental expression and intracellular localization. This ER-resident enzyme was previously shown to be involved in the formation of disulfide bonds in secretory proteins. Wheat PDI appears as a 60-kD glycoprotein and is among the most abundant proteins within the ER of developing grains. PDI is notably up-regulated in developing endosperm in comparison to embryos, leaves, and roots. In addition, the increase in PDI expression in grains appears at relatively early stages of development, preceding the onset of storage protein accumulation by several days. Subcellular localization analysis and immunogold labeling of electron micrographs showed that PDI is not only present in the lumen of the ER but is also co-localized with the storage proteins in the dense protein bodies. These observations are consistent with the hypothesis that PDI is involved in the assembly of wheat storage proteins within the ER.

Dynamics of maize endosperm development and DNA endoreduplication.

Abstract: Endosperm development in Zea mays is characterized by a period of intense mitotic activity followed by a period in which mitosis is essentially eliminated and the cell cycle becomes one of alternating S and G phases, leading to endoreduplication of the nuclear DNA. The endosperm represents a significant contribution to the grain yield of maize; thus, methods that facilitate the study of cellular kinetics may be useful in discerning cellular and molecular components of grain yield. Two mathematical models have been developed to describe the kinetics of endosperm growth. The first describes the kinetics of mitosis during endosperm development; the second describes the kinetics of DNA endoreduplication during endosperm development. The mitotic model is a modification of standard growth curves. The endoreduplication model is composed of six differential equations that represent the progression of nuclei from one DNA content to another during the endoreduplication process. Total nuclei number per endosperm and the number of 3C, 6C, 12C, 24C, 48C, and 96C nuclei per endosperm (C is the haploid DNA content per nucleus) for inbred W64A from 8 to 18 days after pollination were determined by flow cytometry. The results indicate that the change in number of nuclei expressed as a function of the number of days after pollination is the same from one yearly crop to another. These data were used in the model to determine the endosperm growth rate, the maximum nuclei number per endosperm, and transition rates from one C value to the next higher C value. The kinetics of endosperm development are reasonably well represented by the models. Thus, the models provide a means to quantify the complex pattern of endosperm development.

The involvement of Opaque 2 on β-prolamin gene regulation in maize and Coix suggests a more general role for this transcriptional activator

Abstract: The maize opaque 2 (o2) mutation is known to have numerous pleiotropic effects Some polypeptides have their expression depressed while others are enhanced The best characterized effects of the o2 mutation are those exerted on endosperm genes encoding the storage protein class of the 22 kDa α-zeins and the ribosome inactivating protein b-32 The Opaque 2 (O2) locus encodes a basic domain-leucine zipper DNA-binding factor, O2, which transcriptionally regulates these genes In the maize-related grass Coix lacryma-jobi, an O2-homologous protein regulates the 25 kDa α-coixin gene family We show in this paper that O2 transcriptionally regulates the structurally and developmentally different class of the β-prolamins A new O2-binding box was identified in β-prolamin genes from maize and Coix that, together with the boxes previously identified in other endosperm expressed genes, forms a curious collection of O2 cis elements This may have regulatory implications on the role of O2 in the mechanism that controls coordinated gene expression in the developing endosperm Considering that the O2 locus controls at least three distinct classes of genes in maize endosperm, we propose that the O2 protein may play a more general role in maize endosperm development than previously conceived

Molecular cloning, characterization and expression analysis of two catalase isozyme genes in barley.

Abstract: Clones representing two distinct barley catalase genes, Cat1 and Cat2, were found in a cDNA library prepared from seedling polysomal mRNA. Both clones were sequenced, and their deduced amino acid sequences were found to have high homology with maize and rice catalase genes. Cat1 had a 91% deduced amino acid sequence identity to CAT-1 of maize and 92% to CAT B of rice. Cat2 had 72 and 79% amino acid sequence identities to maize CAT-2 and-3 and 89% to CAT A of rice. Barley, maize or rice isozymes could be divided into two distinct groups by amino acid homologies, with one group homologous to the mitochondria-associated CAT-3 of maize and the other homologous to the maize peroxisomal/glyoxysomal CAT-1. Both barley CATs contained possible peroxisomal targeting signals, but neither had favorable mitochondrial targeting sequences. Cat1 mRNA occurred in whole endosperms (aleurones plus starchy endosperm), in isolated aleurones and in developing seeds, but Cat2 mRNA was virtually absent. Both mRNAs displayed different developmental expression patterns in scutella of germinating seeds. Cat2 mRNA predominated in etiolated seedling shoots and leaf blades. Barley genomic DNA contained two genes for Cat1 and one gene for Cat2. The Cat2 gene was mapped to the long arm of chromosome 4, 2.9 cM in telomeric orientation from the mlo locus conferring resistance to the powdery mildew fungus (Erysiphe graminis f.sp. hordei).

Identification of two opaque2 modifier loci in quality protein maize.

Abstract: Genetic modifiers of opaque2 convert the soft, starchy endosperm of opaque2 maize mutants to a hard, vitreous phenotype, while maintaining the enhanced lysine content of the grain. Genetic analysis of F2 segregating seeds from crosses of opaque2 by modified opaque2 genotypes indicated that the modifiers are complex traits that act codominantly. We developed two different segregating F2 populations and mapped the modifier loci by restriction fragment length polymorphism (RFLP) analysis. A relationship was found between formation of vitreous endosperm and the locus encoding the gamma-zein storage protein, which maps near the centromere of chromosome 7. Endosperm modification was consistently associated with the presence of two rather than one gamma-zein gene at this locus. A second modifier locus was mapped near the telomere of chromosome 7L.

Organismal duplication, inclusive fitness theory, and altruism: understanding the evolution of endosperm and the angiosperm reproductive syndrome.

Abstract: For almost a century, events relating to the evolutionary origin of endosperm, a unique embryo-nourishing tissue that is essential to the reproductive process in flowering plants, have remained a mystery. Integration of recent advances in phylogenetic reconstruction, comparative reproductive biology, and genetic theory can be used to elucidate the evolutionary events and forces associated with the establishment of endosperm. Endosperm is shown to be derived from one of two embryos formed during a rudimentary process of "double fertilization" that evolved in the ancestors of angiosperms. Acquisition of embryo-nourishing behavior (with accompanying loss of individual fitness) by this supernumerary fertilization product was dependent upon compensatory gains in the inclusive fitness of related embryos. The result of the loss of individual fitness by one of the two original products of double fertilization was the establishment of endosperm, a highly modified embryo/organism that reproduces cryptically through behavior that enhances the fitness of its associated embryo within a seed. Finally, although triploid endosperm remains a synapomorphy of angiosperms, inclusive fitness analysis demonstrates that the embryo-nourishing properties of endosperm initially evolved in a diploid condition.

Gibberellins and pea seed development

Abstract: The gibberellin (GA)-biosynthesis mutations, lh i , ls and Ie 5839 have been used to investigate the role(s) of the GAs in seed development of the garden pea (Pisum sativum L.). Seeds homozygous for lh i possess reduced GA levels, are more likely to abort during development, and weigh less at harvest, compared with wild-type seeds due to expression of the lh i mutation in the embryo and/ or endosperm. Compared with wild-type seeds, the lh i mutation reduces endogenous GA1 and gibberellic acid (GA3) levels in the embryo/endosperm a few days after anthesis and fertilizing lh i plants with wild-type pollen dramatically increases GA1 and GA3 levels in the embryo/ endosperm and restores normal seed development. By contrast, the ls and le 5839 mutations do not appear to reduce GA levels in the embryo/endosperm of seeds a few days after anthesis, and do not affect embryo or endosperm development. However, both the ls and lh i mutations substantially reduce endogenous GA levels in embryos at contact point (the first day the liquid endosperm disappears). Levels of GAs in seeds from crosses involving the ls and lh i mutations suggest that GAs are synthesised in both the embryo/endosperm and testa and that the expression of ls depends on the tissue and developmental stage examined. These results suggest that GAs (possibly GA1 and/or GA3) play an important role early in pea seed development by regulating the development of the embryo and/or endosperm. By contrast, the high GA levels found in wild-type seeds at contact point (and beyond) do not appear to have a physiological role in seed development.

opaque-15, a maize mutation with properties of a defective opaque-2 modifier.

Abstract: An opaque mutation was identified that reduces gamma-zein synthesis in maize endosperm The mutation, opaque-15, causes a 2- to 3-fold reduction in gamma-zein mRNA and protein synthesis and reduces the proportion of the 27-kDa gamma-zein A gene transcript Although the protein bodies in opaque-15 are similar in size and morphology compared to wild type, there are fewer of them in developing endosperm cells The opaque-15 mutation maps near the telomere of chromosome 7L, coincident with an opaque-2 modifier locus Based on its phenotype, opaque-15 appears to be a mutation of an opaque-2 modifier gene

Endosperm development in Zea mays; implication of gametic imprinting and paternal excess in regulation of transfer layer development

Abstract: Fertilisation in maize (Zea mays), in common with most angiosperms, involves two fusion events: one of the two sperm nuclei unites with the egg cell nucleus, while the other sperm nucleus fuses with the two central cell nuclei giving rise to the triploid endosperm. Since deviation from this nuclear ratio (2:1 maternal/paternal) in the endosperm can result in abortion, it has been suggested that the genomes of the sperm and/or central cell are differentially imprinted during sexual development. By crossing a normal diploid maize line as female with its autotetraploid counterpart, an unbalanced genomic ratio (2:2 maternal/paternal) is created in the endosperm which often results in the eventual abortion of the tissue. Detailed structural comparison of these aberrant endosperms with normal endosperms reveals that the formation of the transfer cell layer, a tissue formed some 8 days after pollination and responsible for the transport of nutrients into the endosperm, is almost completely suppressed under conditions of paternal genomic excess. The first structural analysis of the development of this tissue in normal and aberrant endosperms is reported, and the implications of regulating the formation of such a tissue by gametically imprinted genes are discussed in the light of current theories on the consequences of genomic imbalance on early embryonic development.

The cellular pathway of photosynthate transfer in the developing wheat grain. II. A structural analysis and histochemical studies of the pathway from the crease phloem to the endosperm cavity

Abstract: In the developing wheat grain, photosynthate is transferred longitudinally along the crease phloem and then laterally into the endosperm cavity through the crease vascular parenchyma, pigment strand and nucellar projection. In order to clarify this cellular pathway of photosynthate unloading, and hence the controlling mechanism of grain filling, the potential for symplastic and apoplastic transfer was examined through structural and histochemical studies on these tissue types. It was found that cells in the crease region from the phloem to the nucellar projection are interconnected by numerous plasmodesmata and have dense cytoplasm with abundant mitochondria. Histochemical studies confirmed that, at the stage of grain development studied, an apoplastic barrier exists in the cell walls of the pigment strand. This barrier is composed of lignin, phenolics and suberin. The potential capacity for symplastic transfer, determined by measuring plasmodesmatal frequencies and computing potential sucrose fluxes through these plasmodesmata, indicated that there is sufficient plasmodesmatal cross-sectional area to support symplastic unloading of photosynthate at the rate required for normal grain growth. The potential capacity for membrane transport of sucrose to the apoplast was assessed by measuring plasma membrane surface areas of the various cell types and computing potential plasma membrane fluxes of sucrose. These fluxes indicated that the combined plasma membrane surface areas of the sieve element–companion cell (se–cc) complexes, vascular parenchyma and pigment strand are not sufficient to allow sucrose transfer to the apoplast at the observed rates. In contrast, the wall ingrowths of the transfer cells in the nucellar projection amplify the membrane surface area up to 22-fold, supporting the observed rates of sucrose transfer into the endosperm cavity. We conclude that photosynthate moves via the symplast from the se–cc complexes to the nucellar projection transfer cells, from where it is transferred across the plasma membrane into the endosperm cavity. The apoplastic barrier in the pigment strand is considered to restrict solute movement to the symplast and block apoplastic solute exchange between maternal and embryonic tissues. The implications of this cellular pathway in relation to the control of photosynthate transfer in the developing grain are discussed.

Adenosine diphosphate glucose pyrophosphorylase genes in wheat: differential expression and gene mapping

Abstract: A full-length cDNA clone representing the large (shrunken-2) subunit of ADP-glucose pyrophosphorylase (AGP; EC 2.7.7.27) has been isolated from a cDNA library prepared from developing grain of hexaploid wheat (Triticum aestivum L., cv. Chinese Spring). The 2084-bp cDNA insert contains an open reading frame of 1566 nucleotides and primer-extension analysis indicated that the 5′ end is 10 nucleotides shorter than the mRNA. The deduced protein contains 522 amino acids (57.8 kDa) and includes a putative transit peptide of 62 amino acids (6.5 kDa). The similarity of the deduced protein to the small subunit of AGP and to other AGP genes from plants and microorganisms is discussed. Northern hybridisation shows that the Agp1 genes (encoding the small subunit in the wheat endosperm) and the Agp2 genes (encoding the large subunit in the wheat endosperm) are differentially expressed in the wheat grain. Transcripts from both gene sets accumulate to high levels in the endosperm during grain development with the majority of the expression in the endopsperm rather than the embryo and pericarp layers. Although enzyme activity is detected in developing grains prior to 10 d post anthesis, only the Agp1 genes are active at this time (the Agp2 genes are not expressed until 10 d post anthesis). The possibility that the enzyme expressed during early grain development is a homotetramer of small subunits is discussed. The Agp1 and Agp2 genes are arranged as triplicate sets of single-copy homoeoloci in wheat. The Agp2 genes are located on the long arms of chromosomes 1A, 1B and 1D, about 80 cM from the centromere. The Agp1 genes have been mapped to a position just distal to the centromere on the long arms of chromosomes 7A, 7B and 7D.