Showing papers on "Endosperm published in 1991"

Early embryogenesis in Arabidopsis thaliana. II. The developing embryo

Abstract: Embryo differentiation in Arabidopsis thaliana follows the classical Capsella variation of the Onagrad type. Fertilization occurs approximately 3 h after flowering, whereupon vacuolar organization in the zygote changes and the cell elongates rapidly to approximately three times its original length. Cytoplasmic polarization is maintained. During the first two division steps there is very little increase in total cell volume, and during subsequent divisions vacuole number increases, with a concomitant decrease in size. Plastids remain undifferentiated up to the late globular stage, after which grana begin to develop. Ribosomal concentration increases significantly after fertilization. Differences between embryo proper cells become evident by the heart stage; vacuole, plastid, and mitochondrial abundance, size, and complexity vary within the embryo. There are no plasmodesmatal connections with the endosperm or integuments. Suspensor development is complete by the early globular stage, when it consists of sev...

The Physiology of Starch and Protein Deposition in the Endosperm of Wheat

Abstract: Yield and protein percentage are key issues in the production and marketing of wheat. Yield is a measure of the activity of processes contributing to deposition of starch in the grain, and protein percentage, while not independent of yield, reflects processes in nitrogen metabolism. This paper considers starch and protein deposition in the endosperm of wheat from a physiological point of view and, in particular, explores the extent to which deposition of starch or protein can be manipulated and increased independently of the other product. Rate and duration of both starch and protein deposition in the endosperm of wheat are all independent events, controlled by separate mechanisms. Consideration of this independence can contribute to promoting specific responses within the plant that culminate in starch and protein deposition, whether attempts at improvement be genetic or agronomic in approach. The capacity, or potential, of the grain to accumulate dry matter is established during the grain enlargement phase, that is within the first 15-20 days after anthesis. Genetic, morphological and physiological factors influence development of this capacity, but a major determinant is a substrate effect on mitotic activity in the endosperm. Grain filling commences 10-15 days after anthesis and occupies the last 20-30 days until the grain ripens. Grain filling is the deposition of polymeric product in cells and organelles formed during the grain enlargement phase. Undoubtedly, stress curtails assimilate supply during grain filling but, under adequate growing conditions, both the rate and duration of starch deposition during grain filling are determined mainly by factors that operate within or close to the grain itself. On the other hand, the rate and duration of protein deposition are determined mainly by factors of supply external to the grain. This contrast can be considered in its simplest form as starch deposition lying on an asymptotic region of a rate-versus-supply relationship, while protein deposition lies on a linear region. Strategies for improving starch and protein deposition in wheat are discussed. Starch yield and protein yield should be selected as independent traits in cultivar improvement, and crop management should reflect differences in source-sink relations for starch deposition and protein deposition during the grain filling stage.

Genomic imprinting in endosperm its effect on seed development in crosses between species and between different ploidies of the same species and its implications for the evolution of apomixis

Abstract: If a mother sometimes has offspring by more than one father and if genes in the offspring are active in acquiring resources from maternal tissues, theory predicts that alleles at some loci in the offspring will evolve different patterns of gene expression depending on the gene’s parent of origin (genomic imprinting). The criteria for the evolution of imprinting are satisfied in many seed plants, and imprinting has been reported from the endosperm of angiosperm seeds. This paper’s purpose is to show that imprinting phenomena in endosperm can provide a coherent explanation of some failures of experimental crosses, and of the prevalence of pseudogamy among apomictic angiosperms. As a consequence of imprinting, seed development comes to depend on a particular ratio of maternal and paternal genomes in endosperm. This ratio is normally two maternal genomes to each paternal genome. Imprinting probably accounts for the failure of crosses between diploids and their autotetraploids, because the 2m: 1p ratio is disturbed in such crosses. Imprinting may also account for the breakdown of endosperm in crosses between related species, if the expression of maternal and paternal genomes in endosperm is out of balance. When a cross fails because of such an imbalance, the reciprocal cross will have the opposite imbalance and a complementary phenotype would be expected. The embryological evidence is consistent with this prediction. For example, many incompatible crosses show delayed wall formation in one direction of the cross, but precocious wall formation in the other direction. Typically, seed development can be classified as showing ‘paternal excess’ or ‘maternal excess’. Paternal excess is associated with unusually vigorous early growth of the endosperm, and maternal excess with the opposite. This pattern is consistent with natural selection on paternal gene expression favouring larger seeds. Genetic evidence from maize confirms an association between paternal gene expression and larger kernel size, and maternal gene expression and smaller kernel size. Genomic imprinting creates a requirement for both maternal and paternal genomes in imprinted tissues. In mammals, imprinting is expressed in derivatives of the zygote. The requirement for a paternal genome has constituted a block to the evolution of parthenogenesis, because all the genes in a parthenogenetic embryo are maternal. In angiosperms, imprinting is primarily expressed in the endosperm rather than the embryo. If the effects of imprinting in the embryo are small, an asexually produced embryo can develop, provided that it is associated with a viable endosperm. Many a Pom^ ts are pseudogamous. That is, the endosperm is fertilized and contains maternal and paternal genes embryo is asexual and contains maternal genes only. Thus, the division of labour between the embryo the endosperm during development of the seed can be seen as a preadaptation for apomixis. Some apomicts are autonomous. That is, the embryo and the endosperm both develop without fertilization. Genomic imprinting in endosperm would seem to constitute a barrier to the evolution of autonomous apomixis. Thus, there is a problem, not previously appreciated, in understanding how autonomous apomixis is possible

Regulation of synthesis and transport of secreted proteins in cereal aleurone.

Abstract: Publisher Summary This chapter discusses the regulation of synthesis and transport of secreted proteins in cereal aleurone. It is one of the few digestive tissues found in plants, and the synthesis and secretion of hydrolytic enzymes by this tissue are controlled primarily by the plant hormones gibberellic acid (GA) and abscisic acid (ABA), and by the calcium ion. The aleurone cells from various cereals have the same basic structure and function and play the same role in germination-related processes. The most extensively studied aleurone enzyme is α-amylase. α-Amylase plays a role in the endosperm during germination; it is made in very large amounts in aleurone; it is easily assayed, purified, and detected on electrophoretic gels; it is very stable; and it consists of a single polypeptide chain. The mechanisms that regulate the intracellular transport and secretion of hydrolytic enzymes from the barley aleurone cell as well as the mechanisms involved in the targeting of proteins to subcellular compartments and the identity of these compartments are reviewed in the chapter.

Characterization of an immunoglobulin binding protein homolog in the maize floury-2 endosperm mutant.

Abstract: The maize b-70 protein is an endoplasmic reticulum protein overproduced in the floury-2 (fl2) endosperm mutant. The increase in b-70 levels in fl2 plants occurs during seed maturation and is endosperm specific. We have used amino acid sequence homology to identify b-70 as a homolog of mammalian immunoglobulin binding protein (BiP). Purified b-70 fractions contain two 75-kilodalton polypeptides with pl values of 5.3 and 5.4. Both 75-kilodalton polypeptides share several properties with BiP, including the ability to bind ATP and localization within the lumen of the endoplasmic reticulum. In addition, both b-70 polypeptides can be induced in maize cell cultures with tunicamycin treatment. Like BiP, the pl 5.3 form of b-70 is post-translationally modified by phosphorylation and ADP-ribosylation. However, modification of the pl 5.4 species was not detected in vitro or in vivo. Although the b-70 gene is unlinked to fl2, b-70 overproduction is positively correlated with the fl2 gene and is regulated at the mRNA level. In contrast, the fl2 allele negatively affects the accumulation of the major endosperm storage proteins. The physical similarity of b-70 to BiP and its association with abnormal protein accumulation in fl2 endoplasmic reticulum may reflect a biological function to mediate protein folding and assembly in maize endosperm.

opaque-2 modifiers increase gamma-zein synthesis and alter its spatial distribution in maize endosperm.

Abstract: Through the action of opaque-2 modifier genes, the soft, floury endosperm of opaque-2 mutants is converted to a vitreous phenotype. This change in endosperm texture is associated with a twofold to threefold increase in gamma-zein content. To investigate the effect of opaque-2 modifiers on the expression of gamma-zein genes, we analyzed the synthesis and distribution of gamma-zein protein and the level of gamma-zein mRNAs in developing endosperms of the inbreds W64A and W64Ao2, a modified opaque-2 mutant Pool 34 QPM, and their reciprocal F1 hybrids. We also characterized the number and organization of gamma-zein genes in these and related maize genotypes. Our studies show that opaque-2 modifiers are semidominant genes, resulting in a twofold to threefold increase in gamma-zein gene expression in both opaque-2 and normal genetic backgrounds. The increase in gene expression appears to be a consequence of enhanced mRNA transcription or stability rather than gene amplification because gamma-zein genes occur in one or two copies in modified as well as nonmodified genetic backgrounds. Ultrastructural studies showed that gamma-zein occurs in high concentrations in the first few subaleurone cells of nonmodified endosperms, but high concentrations of gamma-zein occur in the subaleurone and central endosperm cells of modified opaque-2 mutants. The increased concentration and distribution of gamma-zein in modified endosperms are highly correlated with the activity of opaque-2 modifier genes.

Influence of water deficit on maize endosperm development : enzyme activities and RNA transcripts of starch and zein synthesis, abscisic Acid, and cell division.

Abstract: In maize (Zea mays L.), drought during the post-pollination stage decreases kernel growth and often leads to grain yield losses. Kernels in the apical region of the ear are more severely affected than basally positioned kernels. We hypothesized that water deficit during early endosperm development might inhibit kernel growth by decreasing endosperm cell division, and that this response might be mediated by changes in endosperm abscisic acid (ABA) levels. Greenhouse-grown maize, cultivar Pioneer 3925, was subjected to water limitation from 1 to 15 days after pollination (DAP), spanning the period of endosperm cell division and induction of storage product accumulation. Water deficit decreased the number of endosperm nuclei during the treatment period; the most substantial effect was in the apical region of ears. Correspondingly, endosperm fresh weight, starch accumulation and dry mass at maturity were decreased by water limitation. Abscisic acid concentrations in endosperm were quantified by enzyme-linked immunosorbent assay. Water deficit increased ABA concentration in apical-region endosperm by four-fold compared to controls. ABA concentrations were also increased in middle and basal regions of the ear, but to a lesser extent. Two key enzymes in the starch synthesis pathway, sucrose synthase and granule-bound ADP-glucose starch synthase, and zein, the major storage protein in maize endosperm, were studied as markers of storage product synthesis. Water deficit did not affect sucrose synthase enzyme activity or RNA transcript abundance relative to total RNA. However, ADP-glucose starch synthase activity and RNA transcript abundance decreased slightly in apical-region endosperm of water-limited plants by 15 DAP, compared with well-watered controls. In contrast to starch, there was no treatment effect on the accumulation of zein, evaluated at either the polypeptide or RNA level. We conclude that under the conditions tested, the establishment of starch and zein synthetic potential in endosperm was only slightly affected by plant water deficit during the early phase of kernel growth, and that capacity for growth and starch accumulation was affected by the extent to which cell division was inhibited. Based on correlative changes in ABA concentration and cell division we suggest that ABA may play a role in inhibiting endosperm cell division during water limitation.

A novel seed protein gene from Vicia faba is developmentally regulated in transgenic tobacco and Arabidopsis plants.

Abstract: We have isolated a novel gene, denoted USP, from Vicia faba var. minor, which corresponds to the most abundant mRNA present in cotyledons during early seed development; however, the corresponding protein does not accumulate in cotyledons. The characterized USP gene with its two introns is 1 of about 15 members of a gene family. A fragment comprising 637 bp of 5′ flanking sequence and the total 5′ untranslated region was shown to be sufficient to drive the mainly seed-specific expression of two reporter genes, coding for neomycin phosphotransferase 11 and β-glucuronidase, in transgenic Arabidopsis thaliana and Nicotiana tabacum plants. We showed that the USP promoter becomes active in transgenic tobacco seeds in both the embryo and the endosperm, whereas its activity in Arabidopsis is detectable only in the embryo. Moreover, we demonstrated a transient activity pattern of the USP promoter in root tips of both transgenic host species.

Increased expression of the maize immunoglobulin binding protein homolog b-70 in three zein regulatory mutants.

Abstract: Plants carrying floury-2, Defective endosperm-B30, or Mucronate mutations overproduce b-70, a maize homolog of the mammalian immunoglobulin binding protein. During endosperm development in these mutants, levels of both b-70 protein and RNA increase dramatically between 14 days and 20 days after pollination. At later stages, b-70 RNA levels decline while protein levels remain high. The increase in b-70 RNA levels is endosperm specific and dependent on gene dosage in the floury-2 mutant. In all three mutants, the increases in b-70 RNA and protein levels are inversely proportional to changes in zein synthesis. Although b-70 polypeptides can be extracted from purified protein bodies, they carry a carboxy-terminal endoplasmic reticulum retention signal, HDEL. We propose that induction of b-70 in these mutants is a cellular response to abnormally folded or improperly assembled storage proteins and probably reflects its role as a polypeptide chain binding protein.

The microtubular cytoskeleton during development of the zygote, proembryo and free-nuclear endosperm in Arabidopsis thaliana (L.) heynh

Abstract: The microtubular cytoskeleton has been studied during development of the zygote, proembryo and free-nuclear endosperm in A. thaliana using immunofluorescence localization of tubulin in enzymatically isolated material. Abundant micro tubules (MTs) are found throughout proembryogenesis. Microtubules in the coenocytic endosperm are mainly internal. By contrast, there is a re-orientation of MTs to a transverse cortical distribution during zygote development, predominantly in a subapical band which accompanies a phase of apical extension. The presence of these cortical arrays coincides with the elongation of the zygote. Cortical arrays also accompany elongation of the cylindrical suspensor. Extensive networks of MTs ramify throughout the cytoplasm of cells in the proembryo proper. Perinuclear arrays are detected in a number of cell types and MTs contribute to typical mitotic configurations during nuclear divisions. Preprophase bands of MTs are absent throughout megasporogenesis and embryo-sac development and do not occur in endosperm cell divisions. We have observed MTs throughout the first division cycle of the zygote. By placing the observed stages in a most probable sequence, we have identified this cell cycle as the point during embryogenesis at which a preprophase band is reinstated as a regular feature of cell division. Preprophase bands were observed to predict planes of cytokinesis in cell divisions up to the octant stage.

Gamma-Zein Content is Ralated to Endosperm Modification in Quality Protein Maize

Abstract: Opaque-2 (o2) is a mutation in one of the regulatory loci that control storage protein gene transcription in maize (Zea mays L.) kernels. It results in a significant reduction in the prolamine content of the endosperm, which becomes chalky and soft. Quality Protein Maize (QPM) genotypes are modified, hard-endosterm, o2 mutants that carry genetic factors responsible for increased synthesis of the γ-zein protein (mol. wt. 27 kD). The genes encoding this storage protein are not under regulatory control of the 07 locus

Biochemical changes and in-vitro protein digestibility of the endosperm of germinating dolichos lablab

Abstract: A gradual decrease was noted in total protein, soluble protein and protein fractions such as albumin, globulin, prolamine andglutelin in the endosperm of Dolichos lablab L germinated for 8 days. Prolamine showed a maximum decrease of 85% whereas only a 25% decrease was noted in the globulin fraction. Both acid and alkaline protease activities increased during germination, and the acid protease activity was consistently higher than the alkaline protease activity throughout. Electrophoretic studies showed that high molecular weight polypeptide bands disappeared with the appearance of new low molecular weight polypeptide bands in the endosperm proteins of the germinating seeds. Total phenolics and tannin contents and trypsin inhibitory activity also increased during the progressive germination. Germination increased the in-vitro protein digestibility of the endosperm of Dolichos lablab in the presence of proteolytic enzymes like pepsin, trypsin and pancreatin. It showed a negative but non-significant correlation with trypsin inhibitory activity, phenolics and tannin contents. Amylase activity also increased gradually up to the sixth day of germination which coincided with the increase in the reducing sugars.

Primary structure of a novel barley gene differentially expressed in immature aleurone layers.

Abstract: As a direct approach to elucidate the molecular biology of barley aleurone cell development, we differentially screened an aleurone cDNA library made from poly(A)+ RNA of immature grains for clones representing transcripts present in the aleurone but not in the starchy endosperm. For one of these clones, B22E, which hybridies to a 0.7 kb transcript, Northern and in situ hybridization revealed that expression is under complex spatial, temporal and hormonal control in barley grains. cDNAs corresponding to B22E transcripts were isolated from aleurone/pericarp and embryo of developing grains, and from germinating scutella. Among these were the nearly full-length aleurone/pericarp clone p1322E.a16 (541 bp). cDNAs matching the sequence of this clone (type 1 transcript) were found for all tissues investigated. In addition, cDNAs with an extra 12 bp insertion (type 2 transcript) were obtained from germinating scutella. The two different transcripts can encode novel barley proteins of 115 and 119 amino acids, respectively. A gene designated B22EL8 was isolated and sequenced; it encodes the type 1 B22E transcript and contains two introns of 145 and 125 bp. Particle bombardment of barley, aleurone with a B22EL8 promoter-GUS (β-glucuronidase) construct demonstrates that the promoter (3 kb) is active in developing barley grains. The promoter is not, however, active in the seeds of tobacco plants transgenic for the B22EL8 gene, indicating the existence of sequences specific for monocots. A comparison of 1.4 kb of upstream sequence of B22E with the maize c1 promoter reveals a number of short, identical sequences which may be responsible for aleurone cell-specific gene transcription.

Monocot regulatory protein Opaque-2 is localized in the nucleus of maize endosperm and transformed tobacco plants.

Abstract: Protein targeting to the nucleus has been studied extensively in animal and yeast systems; however, nothing is known about nuclear targeting in plants. The Opaque-2 (O2) gene produces a regulatory protein that is responsible for inducing transcription of the alpha-zein class of storage proteins in maize kernels. The cloned O2 gene encodes a protein that contains a leucine zipper DNA binding domain that can interact with zein gene promoters. We have used immunolocalization to show that the O2 protein is present in nuclei in the maize endosperm tissues known to produce alpha-zeins. In addition, neither embryo tissue from wild-type kernels nor endosperm from kernels harboring a null o2 allele contain the O2 protein. Analysis of a transposable, element-induced o2 allele, o2-m20, revealed that sectors of endosperm cells contained the nuclear-localized O2 protein, indicating excision of the transposable element. To study further the nuclear transport of the O2 protein, we have transformed this gene, under the control of a constitutive promoter, into tobacco. Plants were shown to have detectable levels of steady-state O2 mRNA and O2 protein. Immunolocalization of O2 protein in transformed tobacco plants indicated that the O2 protein was transported into tobacco nuclei. Therefore, we have developed a system to study nuclear targeting in plants and have established that the nuclear transport machinery is similar in monocots and dicots.

Normal and lysine-containing zeins are unstable in transgenic tobacco seeds.

Abstract: Chimeric genes composed of the β-phaseolin promoter, an α-zein coding sequence and its modified versions containing lysine codons, and a β-zein polyadenylation signal were inserted into the genome of tobacco by Agrobacterium-mediated transformation. α-Zein mRNA levels in the transgenic tobacco seeds 20 days after self-pollination varied between 1.0% and 2.5% of the total mRNA population. At 25 days after pollination the 19 kDa α-zein was immunologically detected with a polyclonal antiserum in protein extracts from the seeds of transgenic plants. The transgenic plant with the highest level of zein gene expression had an α-zein content that was approximately 0.003% of the total seed protein. The amount of α-zein in other transgenic plants varied between 1 × 10−4% and 1 × 10−5% of the total seed protein. The differences in the amounts of mRNA and protein did not correlate with the lysine substitutions introduced into the α-zein protein. Polysomes translating α-zein mRNA isolated from tobacco seeds contained fewer ribosomes than those from maize endosperm, but this did not appear to be the cause of the inefficient protein synthesis. In vivo labelling and immunoprecipitation indicated that newly synthesized α-zein was degraded in tobacco seeds with a half-life of less than 1 hour.

Improved Germination and Modified Imbibition of shrunken-2 Sweet Corn by Seed Disinfection and Solid Matrix Priming

Abstract: Generally, sweet corn cultivars (Zea mays L.) carrying the shrunken-2 (sh2) gene have lower germination and seedling vigor than normal or sugary (su) cultivars. Seeds of sh2 'How Sweet It Is' (HSII) and 'Crisp N'Sweet 711' (CNS-711) were imbibed for 6 hours. Rapid water uptake, higher seed leakage, and fungal infection were found in HSII, the lower germinating cultivar. Imbibition rate and leakage conductivity were reduced in both cultivars during the first 5 hours at 5C as compared with 25C. Sodium hypochlorite was an effective seed disinfectant. When the seeds were primed with sodium hypochlorite via solid matrix priming (SMP), germination under stressful con- ditions (soilcold test) was significantly improved in both cultivars. Primed seeds had significantly lower imbibitional rates and leakage conductivity than nonprimed seeds. The superior germination measured in primed and disinfected seeds was possibly due to the lower imbibitional rate and reduced seed fungal infection. The incorporation of the mutant sh2 gene in sweet corn has greatly improved sweet corn eating quality (Garwood et al., 1976). The sh2 supersweet hybrids have high levels of sugar in the endosperm and excellent postharvest sugar retention (Laugh- nan, 1953). However, germination and seedling vigor are often poor, especially under stress conditions (Styer et al., 1980). Small endosperm (Warm, 1980), susceptibility to seed- and soil- borne diseases (Pieczarka et al., 1978), and dysfunction in re- serve mobilization (Styer and Cantliffe, 1983) have been re- ported as causes of reduced seed viability and seedling vigor. Imbibition, or seed hydration, is the first step in germination. Rapid water uptake can negatively affect germination (Powell and Mathews, 1978). If water absorption is reduced during the early stages of imbibition, tissues develop in an organized man- ner, allowing sufficient time for membrane rearrangement and thus possibly reducing imbibition injury (Woodstock and Tao, 1981). Throughout imbibition, seeds lose a wide variety of car- bohydrates, mineral nutrients, proteins, and organic acids (Nor- din, 1984). Leakage can provide essential nutritive substances for fungi to develop on and around the seed (Schroth and Cook, 1964). Berger and Wolf (1974) reported that poor stand in sh2 sweet corn was associated with seed rot and damping off. Fu- sarium moniliforme (Sheldon) penetrated sh2 corn kernels via small cracks in the pericarp, where the pathogen localized be- tween the pericarp and aleurone layer, and eventually moved into the endosperm and embryo (Styer and Cantliffe, 1983). Sodium hypochlorite has been used as a seed disinfectant in pepper (Fieldhouse and Sasser, 1975) and corn (Schoen and Kulik, 1977). Similarly, seedlings of 'Iochief' and 'Earlivee' sweet corn were less infected with F. moniliforme after sodium hypochlorite seed disinfection (Anderegg and Guthrie, 1981). Seed priming has been a successful presowing seed treatment to improve the rate and uniformity of seed emergence under stress conditions. SMP controls seed hydration by the physical and osmotic characteristic of a solid matrix carrier (Kubik et al., 1989). The biochemical mechanisms involved in osmotic priming treatments are not entirely understood (Bradford, 1986).

Genetic analysis of salt tolerance during germination in Lycopersicon.

Abstract: The salt-tolerant cultivated tomato (Lycopersicon esculentum) accession, ‘PI174263’, and a sensitive cv, ‘UCT5’, were crossed to develop reciprocal F1, F2 and BC1 populations for genetic analysis of salt tolerance in tomatoes during seed germination. Variation was partitioned into embryo, endosperm and maternal (testa and cytoplasmic) components. Generation means analysis indicated that there were no significant embryo (additive, dominance or epistatic) effects on germination performance under salt stress. Highly significant endosperm additive and testa dominance effects were detected. The proportion of the total variance explained by the model containing these two components was R2=98.2%. Variance component analysis indicated a large genetic variance with additive gene action as the predominant component. Furhter inspection indicated that this variance was attributable to endosperm additive effects on germinability under salt stress. Narrow-sense heritability was estimated as moderately high. Implications for breeding procedures are discussed.

Three high-lysine mutations control the level of ATP-binding HSP70-like proteins in the maize endosperm.

Abstract: The synthesis and deposition of seed storage proteins in maize are affected by several dominant and recessive mutants. The effect of three independent mutations, floury-2 (fl2), Defective endosperm-B30 (De-B30), and Mucronate (Mc), that reduce zein level in the endosperm were investigated. These mutations also control the level of b-70, a polypeptide bound to protein bodies, which is separable into the two isoforms b-70I and b-70II by two-dimensional gel electrophoresis. Both isoforms are overexpressed 10-fold in fl2; however, only b-70I is present in De-B30 and Mc, which contain an amount of total b-70 isoforms fivefold higher than in the wild type. Both b-70I and b-70II resemble heat shock protein (HSP70) in that they bind ATP, cross-react with anti-HSP antibodies, and have N-terminal sequence homology to HSP70. All maize protein body-located b-70 characteristics are typical of those of chaperone-like HSPs. A third protein, b-70III, similar in size to but slightly more acidic than b-70I and b-70II, also binds ATP and reacts with the same antibody, providing evidence for the presence in endosperm extracts of a cytosolic chaperone-like protein. The level of b-70III was not altered by the mutations studied. The results suggested that the repression effect of the three mutations on zein accumulation may be mediated by the alteration of a zein transport or zein assembly process involving b-70I and b-70II.

Seeds of tomato (Lycopersicon esculentum Mill.) which develop in a fully hydrated environment in the fruit switch from a developmental to a germinative mode without a requirement for desiccation

Abstract: Seed water content is high during early development of tomato seeds (10–30 d after pollination (DAP)), declines at 35 DAP, then increases slightly during fruit ripening (following 50 DAP). The seed does not undergo maturation drying. Protein content during seed development peaks at 35 DAP in the embryo, while in the endosperm it exhibits a triphasic accumulation pattern. Peaks in endosperm protein deposition correspond to changes in endosperm morphology (i.e. formation of the hard endosperm) and are largely the consequence of increases in storage proteins. Storage-protein deposition commences at 20 DAP in the embryo and endosperm; both tissues accumulate identical proteins. Embryo maturation is complete by 40 DAP, when maximum embryo protein content, size and seed dry weight are attained. Seeds are tolerant of premature drying (fast and slow drying) from 40 DAP.

Replication of a geminivirus derived shuttle vector in maize endosperm cells.

Abstract: A maize (Zea mays L.) endosperm cell culture has been shown to efficiently replicate DNA sequences derived from wheat dwarf virus (WDV), a monopartite monocot geminivirus. To analyze sequences necessary for viral replication and to verify their application for a plant gene expression vector, we have developed a 3.7 kilobase pairs Escherichia coli--plant cell shuttle vector, pWI-11. The p15A origin of replication, functional in E. coli, was introduced into the viral sequences. We have replaced the coding region of the coat protein gene by that of bacterial neomycin phosphotransferase II (NPT II) gene. The resulting NPT II gene fusion can serve as a selectable marker in both plant and E. coli systems. Into a unique cloning site in this pWI-11 vector, we introduced a gene fusion carrying the bacterial beta-glucuronidase (GUS) coding region under control of the cauliflower mosaic virus 35S (CaMV35S) gene promoter and terminator. By transferring these viral sequences into protoplasts derived from maize endosperm cell cultures, we have demonstrated that the plasmid pWI-11 can replicate in maize endosperm cells, that the GUS reporter gene introduced into pWI-11 can be expressed at high level in the transformed cells, and that the replicating viral DNA can be rescued from endosperm cells by transforming E. coli in the presence of kanamycin. The level of GUS gene expression increased progressively in transformed endosperm cells during a prolonged culture period, coinciding with replication of the viral sequences in these cells.

Transformation of the developing barley endosperm by particle bombardment.

Abstract: Delivery of DNA into intact cells of the developing barley (Hordeum vulgare L.) endosperm was performed with the BIOLISTIC particle gun. It is shown that the proximal 532 base pairs (bp) of the upstream region of a B1-hordein gene drive the expression of the β-glucuronidase (GUS) gene (uidA) in sub-aleurone and starchy-endosperm cells but not in cells devoid of starch, i.e. developing aleurone cells. The 35S promoter from cauliflower mosaic virus was active in all three cell types. This cell-specific activity of the hordein promoter was verified by a detailed histological study of the regions of the extruded endosperms expressing the uidA gene. The analysis included a histological study of the developing endosperm as a base for classifying the different cell types in the developing endosperm.

Avena fatua L. seed and seedling nutrient dynamics as influenced by mycorrhizal infection of the maternal generation

Abstract: . The objective of this study was to determine how mycorrhizal infection of one generation of plants influences the nutrient dynamics of seeds and seedlings comprising the subsequent generation. We showed that, for Avena fatua L., seeds produced by mycorrhizal (M) plants consistently contained significantly more phosphorus (particularly the phytate P and residual P fractions) than seeds produced by non-mycorrhizal (NM) plants. We also followed the development of spikelets produced by M and NM plants. The rates of increase in spikelet dry weight and nitrogen content were largely unaffected by mycorrhizal infection. However, the rate of P accumulation into spikelets was significantly increased by mycorrhizal infection. Greater endosperm P reserves in seeds produced by M plants were associated with greater rates of P accumulation in resultant seedlings. Moreover, offspring plants (all NM) produced by M mother plants had significantly higher root and rhizosphere phosphatase, ATPase and phytase activities than offspring plants produced by NM mother plants. This persistent maternal effect has never before been described. Our results suggest that mycorrhizal infection of one generation of plants may have substantial positive effects on the offspring generation, and thus, may influence plant population dynamics.

Tissue-Specific Expression and Anaerobically Induced Posttranscriptional Modulation of Sucrose Synthase Genes in Sorghum bicolor M.

Abstract: We have used antibodies directed against the two sucrose synthase (SS) isozymes, and the cDNA clones corresponding to the two nonallelic genes in maize to describe sorghum (Sorghum bicolor) SS genes and their expressions at protein and RNA levels. Western blot analyses have shown evidence of two SS isozymes, SS1 and SS2, in sorghum; these were similar, but not identical, to maize isozymes in size, charge, subunit composition, and epitope specificities against both monoclonal and polyclonal antibodies. Tissue-specific distributions of isozymes and genomic Southern hybridization data are consistent with a hypothesis that the SS1 and SS2 isozymes are encoded by two nonallelic genes, designated here as Sus1 and Sus2, respectively. Northern blot hybridizations on root RNAs showed gene-specific transcript patterns and, as in maize, the SS2-specific transcripts were slightly larger than the SS1-specific transcripts. Interestingly, no difference in the size of the SS1 and SS2 polypeptides was detected. Anaerobic induction led to significant elevations in steady-state levels of both SS1 and SS2 transcripts, but there was no detectable increase in the levels of the SS proteins. Thus, both the SS genes in sorghum were significantly regulated at the posttranscriptional level; whereas in maize, only one of the two SS genes was affected in this fashion. Another difference between maize and sorghum SS isozymes was in endosperm-specific polymerization among the SS subunits. Unlike maize endosperm where only the two SS homotetramers are seen, sorghum endosperm showed five SS isozymes attributable to a random copolymerization of SS1 and SS2 subunits, presumably due to a simultaneous expression of both genes in the endosperm cells. Physiological and molecular bases of these differences between these two crop plant species remains to be elucidated.

A novel approach to differentiated embryos in the absence of endosperm

Abstract: In all of the Poaceae tested (Bromus, Festuca, Hordeum, Lolium, Poa, Triticum) the formation of grains without endosperm was induced from unpollinated ovules by treatment with the following synthetic auxins: DIG; 2,4-D; 2,4,5-T; or CPAA. Cytokinins (BAP, ZTN) as well as adenine or gibberellic acid (GA3) alone were ineffective. In parthenogenetic lines auxin treatment resulted in grains with mature embryos without endosperm. Differences in embryo differentiation were found, which were dependent on the synthetic auxins used, their concentrations, and the developmental stages of the treated spikes or panicles. Thus, the regulation of embryogenesis by the endosperm can be replaced by exogenous auxin application. The developing proembryos of grasses did not need nutritive support from the endosperm. Auxin treatment to give mature embryos without endosperm enables the screening of apomictic species for sexual plants and sexual species for parthenogenetic individuals. It opens ways for inducing haploid parthenogenesis and improves methods for overcoming interspecific crossing barriers.

cDNA cloning, characterization and expression of an endosperm-specific barley peroxidase.

Abstract: A barley peroxidase (BP 1) of pI ca. 8.5 and Mr 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from a cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C-terminal amino acid residues of mature BP 1. The clone pcR7 encodes an additional C-terminal sequence of 22 residues, which apparently are removed during processing. BP 1 is less than 50% identical to other sequenced plant peroxidases. Analyses of RNA and protein from aleurone, endosperm and embryo tissue showed maximal expression 15 days after flowering, and high levels were found only in the endosperm. BP 1 was not expressed in the leaves.