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PubMed1979

Calcium oxalate dihydrate crystal growth.

Calcium oxalate dihydrate (COD), although thermodynamically unstable with respect to calcium oxalate monohydrate (COM), is more commonly seen in human urine. For the first time, a COD-seeded crystal growth assay has been developed. Seed crystals of COD were precipitated from filtered urine by the addition of ammonium oxalate and were stored dry and added to a supersaturated calcium oxalate solution to initiate an experiment. The growth rate in the COD-seeded system was 22 per cent of that for a COM-seeded system, for equivalent surface areas. Urine samples from normal subjects have similar inhibitory activity in the COD- and COM-seeded systems, as do pyrophosphate and heparin, which are known inhibitors of COM crystal growth. These results indicate that the technically simpler COM-seeded growth assay is suitable for measurement of inhibitors of calcium oxalate growth in urine.

Calcium Oxalate↗
PubMed1998

Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity.

Expression and stability of immunoglobulins in transgenic plants have been investigated and optimized by accumulation in different cellular compartments as cytosol, apoplastic space and endoplasmic reticulum (ER) as will be discussed in this review. In several cases described the highest accumulation of complete active antibodies was achieved by targeting into the apoplastic space. High-level expression of active recombinant single-chain Fv antibodies (scFv's) was obtained by retention of these proteins in the lumen of the endoplasmic reticulum. This has been shown for leaves and seeds of transgenic tobacco as well as for potato tubers. Transgenic tobacco seeds, potato tubers and tobacco leaves can facilitate stable storage of scFv's accumulated in the ER over an extended (seeds, tubers) or a short (leaves) period of time. The expression of specific scFv's in different plant species, plant organs and cellular compartments offers the possibility of blocking regulatory factors or pathogens specifically. Examples are scFv's expressed in the cytosol and the apoplastic space of transgenic plant cells modulating the infection process of plant viruses and a cytosolically expressed scFv that influenced the activity of phytochrome A protein. The immunomodulation approach has been shown to be also applicable for investigating the action of the phyto-hormone abscisic acid (ABA). High-level accumulation of specific anti-ABA scFv's in the ER of all leaf cells has been used to block the influence of ABA on the stomatal functions. Seed-specific expression of high amounts of anti-ABA-scFv's at a defined time of seed-development induced a developmental switch from seed ripening to vegetative growth. It has been demonstrated that ER retention is essential for the accumulation of sufficient scFv to bind high concentrations of ABA in the transgenic seeds.

Adjuvants, Immunologic↗
PubMed2000

Differential expression of diacylglycerol acyltransferase (DGAT) genes in olive tissues.

Fatty acids are accumulated in triacylglycerols (TAGs), in specialized organelles of seeds named oil bodies. The major site of TAG accumulation is detected in developing seed and mesocarp of certain species. We have isolated two cDNAs encoding DGAT enzymes from olives. The deduced polypeptides differ by 26 amino acids in size. However, they have high homology and almost identical hydropathy profiles. The DGAT gene is expressed in all tissues that synthesize TAGs. However, higher levels of DGAT transcripts have been detected in seed tissues of developing olive drupe. DGAT expression and mRNA accumulation in drupe tissues is developmentally regulated. Each DGAT transcript shows a distinct profile of accumulation. The existence of two different DGAT transcripts might reflect two different enzymes with discrete function and/or localization.

Acyltransferases↗
PubMed2003

Pollination-, development-, and auxin-specific regulation of gibberellin 3beta-hydroxylase gene expression in pea fruit and seeds.

To understand further how pollination, seeds, auxin (4-chloroindole-3-acetic acid [4-Cl-IAA]), and gibberellins (GAs) regulate GA biosynthesis in pea (Pisum sativum) fruit, we studied expression of the gene PsGA3ox1 that codes for the enzyme that converts GA(20) to biologically active GA(1) using real-time reverse transcription-polymerase chain reaction analysis. PsGA3ox1 mRNA levels were minimally detectable in prepollinated pericarps and ovules (-2 d after anthesis [DAA]), increased dramatically after pollination (0 DAA), then decreased by 1 DAA. Seed PsGA3ox1 mRNA levels increased at 4 DAA and again 8 to 12 DAA, when seed development was rapid. Pericarp PsGA3ox1 mRNA levels peaked coincidentally with rapid pod diameter expansion (6-10 DAA) to accommodate the growing seeds. The effects of seeds and hormones on the expression of pericarp PsGA3ox1 were investigated over a 24-h treatment period. Pericarp PsGA3ox1 mRNA levels gradually increased from 2 to 3 DAA when seeds were present; however, when the seeds were removed, the pericarp transcript levels dramatically declined. When 2-DAA deseeded pericarps were treated with 4-Cl-IAA, PsGA3ox1 mRNA levels peaked 4 h after hormone treatment (270-fold increase), then decreased. PsGA3ox1 mRNA levels in deseeded pericarps treated with indole-3-acetic acid or GA(3) were the same or lower than deseeded controls. These data show that PsGA3ox1 is expressed and developmentally regulated in pea pericarps and seeds. These data also show that pericarp PsGA3ox1 expression is hormonally regulated and suggest that the conversion of GA(20) to GA(1) occurs in the pericarp and is regulated by the presence of seeds and 4-Cl-IAA for fruit growth.

Fertility↗
PubMed2000

Separation and characterization of basic barley seed proteins.

Basic proteins in barley starchy endosperm from developing seeds were separated by two-dimensional (2-D) nonequilibrium pH gel electrophoresis. Total as well as partial extracts were analyzed. Edman degradation sequencing and immunological detection were performed after transfer of separated proteins onto membranes. Only one protein could be analyzed by N-terminal sequencing of blotted and separated proteins from the total extract. Fractionation of extracts was done using cation exchange chromatography, concanavalin A and heparin affinity chromatography. Internal sequences were determined after in-gel cleavage of proteins using trypsin or cyanogen bromide and separation of the fragments by reversed-phase chromatography or in a gel electrophoresis system for peptide separation. This resulted in a new protocol for obtaining internal sequences from proteins separated by 2-D electrophoresis. A total of 16 sequences, including nine internal sequences, were analyzed, permitting the identification of ten proteins, including five that appeared to have a blocked N-terminus. An additional protein was identified using immunological detection. Three protein sequences remained unidentified. Separated proteins were also analyzed with a glycan detection method.

Acetates↗
PubMed1982

Activity of enzymes of arginine metabolism in the cotyledons of developing and germinating pea seeds.

Ornithine carbamoyltransferase, argininosuccinate synthetase, argininosuccinate lyase, and arginase activity were measured in extracts from cotyledons of developing and germinating seeds of Pisum sativum L. The course of activity of these four urea cycle enzymes showed a similar pattern during seed development. The activity per cotyledon increased sharply initially and reached a maximum about 5 weeks after anthesis, when the relative water content of the seeds was about 60%. About 8 weeks after anthesis, the seeds were mature (air-dry) and had enzyme activities which were much lower. The activities of the enzymes differed considerably. Ornithine carbamoyltransferase showed the highest activity, followed in order of decreasing activity by arginase, argininosuccinate lyase, and finally argininosuccinate synthetase.The course of the activity of the four enzymes was different during germination. Arginase activity increased sharply 7 hours after the onset of germination and remained at a constant level during the following days. Argininosuccinate synthetase activity decreased; the other enzymes showed a small increase in activity and a subsequent decrease. Results are discussed in relation to the regulation of the arginine metabolism during pea seed development and germination.

Journal Article↗
PubMed1987

Intracellular pH of Cotton Embryos and Seed Coats during Fruit Development Determined by P Nuclear Magnetic Resonance Spectroscopy.

The pH of the phosphate-containing compartments of developing cotton seed coat and embryo tissues was determined by means of (31)P nuclear magnetic resonance spectroscopy. The pH values of these tissues varied as a function of developmental age. From 27 to approximately 38 days postanthesis, a strong pH differential existed between the two tissues; the seed coat was up to 1.4 pH units more acid than developing cotton embryos. The pattern of pH values found with this technique agrees with pH values of tissue homogenates in distilled water. The results confirm an earlier suggestion that seed coat cells are more acidic than embryo cells during key developmental stages of the seed. The pH differential between these two tissues causes abscisic acid to diffuse from seed coats to embryos against its apparent concentration gradient to prevent viviparous germination, despite a higher abscisic acid concentration in the embryo.

Journal Article↗
PubMed2003

Chlorophyll reduction in the seed of Brassica napus with a glutamate 1-semialdehyde aminotransferase antisense gene.

Chlorophyll reduction in the seed of Brassica can be achieved by downregulating its synthesis. To reduce chlorophyll synthesis, we have used a cDNA clone of Brassica napus encoding glutamate 1-semialdehyde aminotransferase (GSA-AT) to make an antisense construct for gene manipulation. Antisense glutamate 1-semialdehyde aminotransferase gene (Gsa) expression, directed by a Brassica napin promoter, was targeted specifically to the embryo of the developing seed. Transformants expressing antisense Gsa showed varying degrees of inhibition resulting in a range of chlorophyll reduction in the seeds. Seed growth and development were not affected by reduction of chlorophyll. Seeds from selfed transgenic plants germinated with high efficiency and growth of seedlings was vigorous. Seedlings from T2 transgenic lines segregated into three distinctive phenotypes: dark green, light green and yellow, indicating the dominant inheritance of Gsa antisense gene. These transgenic lines have provided useful materials for the development of a low chlorophyll seed variety of B. napus.

Amino Acid Sequence↗
PubMed2006

Genetics and biochemistry of seed flavonoids.

Flavonoids are secondary metabolites that accumulate in most plant seeds and are involved in physiological functions such as dormancy or viability. This review presents a current view of the genetic and biochemical control of flavonoid metabolism during seed development. It focuses mainly on proanthocyanidin accumulation in Arabidopsis, with comparisons to other related metabolic and regulatory pathways. These intricate networks and their fine-tuned regulation, once they are determined, should contribute to a better understanding of seed coat development and the control of PA and flavonol metabolism. In addition, flavonoids provide an interesting model to study various biological processes and metabolic and regulatory networks.

Flavonoids↗
PubMed1989

Seed dormancy and responses of caryopses, embryos, and calli to abscisic Acid in wheat.

Preharvest sprouting of wheat (Triticum aestivum L.) is associated with inadequate seed dormancy. Although abscisic acid (ABA) has often been suggested to play a central role in developing seed, its involvement in dormancy of mature seed lacks firm experimental evidence and endogenous ABA levels are not well correlated with germinability. We examined genotypic and temporal variation in wheat seed and embryo germination responses to ABA and determined whether differential sensitivity of embryos to ABA extended to growth of embryo-derived calli. Germination of Parker 76 caryopses, which have little dormancy at maturity, was only slightly inhibited by ABA, whereas germination of Clark's Cream, a highly dormant genotype, was greatly inhibited. Responsiveness of caryopses to ABA and dormancy of seeds decreased concurrently during afterripening. Germination of embryos excised from dormant and nondormant seeds was more responsive to ABA but otherwise was similar to that of caryopses, indicating that differential response to ABA occurs in the embryo. Growth of calli derived from immature embryos of two sprouting-susceptible wheat genotypes exceeded growth of calli from Clark's Cream, but no distinct differences in response to ABA among the genotypes were apparent. We concluded that the action of ABA is similar in developing and mature seeds, that genotypic and temporal variation in embryo responsiveness to endogenous ABA may be involved in dormancy, and that ABA probably acts in concert with other endogenous constituents.

Journal Article↗
PubMed2006

Jekyll encodes a novel protein involved in the sexual reproduction of barley.

Cereal seed development depends on the intimate interaction of filial and maternal tissues, ensuring nourishment of the new generation. The gene jekyll, which was identified in barley (Hordeum vulgare), is preferentially expressed in the nurse tissues. JEKYLL shares partial similarity with the scorpion Cn4 toxin and is toxic when ectopically expressed in Escherichia coli and tobacco (Nicotiana tabacum). In barley, jekyll is upregulated in cells destined for autolysis. The gene generates a gradient of expression in the nucellar projection, which mediates the maternal-filial interaction during seed filling. Downregulation of jekyll by the RNA interference technique in barley decelerates autolysis and cell differentiation within the nurse tissues. Flower development and seed filling are thereby extended, and the nucellar projection no longer functions as the main transport route for assimilates. A slowing down in the proliferation of endosperm nuclei and a severely impaired ability to accumulate starch in the endosperm leads to the formation of irregular and small-sized seeds at maturity. Overall, JEKYLL plays a decisive role in the differentiation of the nucellar projection and drives the programmed cell death necessary for its proper function. We further suggest that cell autolysis during the differentiation of the nucellar projection allows the optimal provision of basic nutrients for biosynthesis in endosperm and embryo.

Amino Acid Sequence↗
PubMed2003

The role of invertases and hexose transporters in controlling sugar ratios in maternal and filial tissues of barley caryopses during early development.

To analyse carbohydrate metabolism and its role during early seed development of barley we characterised genes encoding two cell wall-bound invertases (HvCWINV1 and HvCWINV2) and two putative hexose transporter-like genes (HvSTP1 and HvSTP2). No typical vacuolar invertase gene could be identified. Instead, a gene encoding sucrose:fructan 6-fructosyltransferase (HvSF6FT1), an enzyme with soluble acid invertase activity, was isolated and characterised. Furthermore, enzyme activities and sugar levels were measured. HvSF6FT1-mRNA levels and acid soluble invertase activity are highest in the maternal pericarp 1-2 days after flowering (DAF). HvSF6FT1 is strongly expressed in regions flanking the main vascular bundle and to a lower extent in filial endospermal transfer cells, which persist until maturity and never accumulate starch. In contrast, cell wall-bound invertase HvCWINV2 is expressed early in development mainly in the style region and later on in pericarp areas which transiently accumulate starch and undergo degradation later in development. The hexose transporter HvSTP2 shows a temporal and spatial expression pattern similar to HvCWINV2. Transcripts of HvCWINV1 have been localised within the first row of endospermal cells and in the outermost area of the nucellar projection as well as in endospermal transfer cells before starch filling; the same regions of the endosperm are labelled with a hexose transporter HvSTP1-probe. HvSTP1 is expressed at very low levels within the pericarp but much higher in the syncytial endosperm at 3 DAF and in endospermal transfer cells 7 DAF. The temporal and spatial association of HvCWINV1 and HvSTP1 expression indicates that hexoses liberated by the invertase within the endospermal cavity are taken up by the transporter to be delivered into the central uncellularised space of the endosperm to supply mitotically active endosperm cells with hexoses. The results are discussed and compared with published data on the role of soluble sugars as signal molecules in seed developmental processes.

Carbohydrate Metabolism↗
PubMed2003

Differentiation of legume cotyledons as related to metabolic gradients and assimilate transport into seeds.

Legume seed development is closely related to metabolism and nutrient transport. To analyse this relationship, a combination of biochemical, histological and transgenic approaches was used. Sugars within tissue sections have been quantitatively measured by metabolic imaging. During cotyledon differentiation glucose gradients emerge related to a particular cell type, with higher concentrations in non-differentiated premature regions. Sucrose in creases at the beginning of maturation in a layer underneath the outer epidermis expressing a sucrose transporter. Sucrose distribution is initially controlled by uptake activity and the permeability within the parenchyma and, later on, also by differences in growth and starch accumulation. Increased sucrose levels are accompanied by increased levels of sucrose synthase and ADP-Glc pyrophosphorylase mRNAs, but carbon flux into starch is initially still low. Rates increase at a stage when hexose concentrations become low, allowing increased flux through the sucrose synthase pathway. Transfer cell formation represents a regional specification of the cotyledonary epidermis for embryo nutrition characterized by increased transport-active cell surfaces and up-regulated expression of transport-related genes. The E2748 pea seed mutation blocks epidermal differentiation into transfer cells and leads to the loss of epidermal cell identity. Embryos with impaired epidermis cannot tolerate elevated levels of sucrose and respond with disorganized growth. The E2748 gene product is required for transfer cell formation in developing cotyledons with no other function during plant growth. Seed coat permeability provides a hypoxic environment for embryo development. However, at maturity, seed energy supply is not limited indicating fundamental developmental and metabolic adaptations. Results from transgenic seeds show that altered expression of single genes induces complex and unexpected changes. In AGP-antisense seeds the block in starch synthesis leads to pleiotropic effects of water and nitrogen content and induces temporal changes in seed development.

Biological Transport↗
PubMed1997

BANYULS, a novel negative regulator of flavonoid biosynthesis in the Arabidopsis seed coat.

A mutant of Arabidopsis that accumulates a high level of red pigments within the seed coat has been isolated from a population of T-DNA-transformed plants. Genetic analysis revealed that the mutation is recessive and affects maternal seed tissues only. Due to the color of the immature seeds, this mutation was named banyuls (ban). Pigments accumulated continuously from early seed development to the desiccation stage in the seed coat of the mutant. The phenotype of the double mutant banyuls/ transparent testa confirmed the flavonoid nature of the pigments and enabled assignment of the regulatory TT (Transparent Testa) genes to two groups according to their epistatic relationship to ban. The flavonoid content of germinated ban and wild-type seedlings was similar. Plants harbouring the ban mutation had a normal formation of trichomes and root hairs and were not affected in their responses to light. The seeds of ban plants exhibited reduced germination compared to wild-type which may be a direct consequence of the high level of pigments. These results suggest that BANYULS functions as a negative regulator of flavonoid biosynthesis that prevents accumulation of pigments in the seed coat during early embryogenesis in Arabidopsis.

Anthocyanins↗
PubMed2001

The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat endothelium.

Phenolic compounds that are present in the testa interfere with the physiology of seed dormancy and germination. We isolated a recessive Arabidopsis mutant with pale brown seeds, transparent testa12 (tt12), from a reduced seed dormancy screen. Microscopic analysis of tt12 developing and mature testas revealed a strong reduction of proanthocyanidin deposition in vacuoles of endothelial cells. Double mutants with tt12 and other testa pigmentation mutants were constructed, and their phenotypes confirmed that tt12 was affected at the level of the flavonoid biosynthetic pathway. The TT12 gene was cloned and found to encode a protein with similarity to prokaryotic and eukaryotic secondary transporters with 12 transmembrane segments, belonging to the MATE (multidrug and toxic compound extrusion) family. TT12 is expressed specifically in ovules and developing seeds. In situ hybridization localized its transcript in the endothelium layer, as expected from the effect of the tt12 mutation on testa flavonoid pigmentation. The phenotype of the mutant and the nature of the gene suggest that TT12 may control the vacuolar sequestration of flavonoids in the seed coat endothelium.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

An automatic seed identification technique for interstitial implants using three isocentric radiographs.

A technique for the automatic reconstruction of the spatial coordinates of seeds in an interstitial implant has been developed. Seed coordinates from three isocentric radiographs, an anterior-posterior film, and an orthogonal pair of films taken at gantry angles of +/- 45 degrees from the anterior-posterior direction are digitized and recorded in a random sequence. From this, the three-dimensional coordinates of the seeds in the implant are computed by matching the values of common coordinates along the axis of rotation. This rotate and match technique leads to an accurate and consistent identification and reconstruction of seeds with 90% of them within 2 mm of their actual location. These new computer routines have been added to an existing treatment planning system (AECL TP-11 treatment planning system, version 5A). We present our algorithm and technique along with clinical examples.

Brachytherapy↗
PubMed1986

A Comparison of Oleic Acid Metabolism in the Soybean (Glycine max [L.] Merr.) Genotypes Williams and A5, a Mutant with Decreased Linoleic Acid in the Seed.

The metabolism of oleoyl coenzyme A (CoA) was examined in developing seed from two soybean (Glycine max [L.] Merr.) genotypes: Williams, a standard cultivar and A5, a mutant containing nearly twice the oleic acid (18:1) content of Williams. The in vitro rates of esterification of oleoyl-CoA to lysophosphatides by acyl-CoA: lysophosphatidylcholine acyltransferase was similar in both genotypes and lysophosphatidyl-ethanolamine was a poor substrate. Crude extracts desaturated exogenous [1-(14)C]dioleoyl phosphatidylcholine at 14% of the rate achieved with [1-(14)C]oleoyl-CoA, and 50 micromolar lysophosphatidylcholine. The desaturase enzyme also required NADH for full activity. Extracts from Williams contained 1.5-fold more oleoyl phosphatidylcholine desaturase activity, on a fresh weight basis, than did A5 and appeared to have a similar affinity for oleoyl-CoA. There was 1.2- to 1.9-fold more linoleic acid (18:2) in phosphatidylcholine from Williams than from A5, measured at two stages of development, but both genotypes had a similar distribution of fatty acids in the one and two positions. Phosphatidylethanolamine in A5 contained relatively more linoleic acid (18:2) in the one position than did Williams. The increased oleic acid (18:1) content in A5 appeared to be a result of decreased rates of 18:1 desaturation of oleoyl-phosphatidylcholine in this genotype.

Journal Article↗