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PubMed1971

The pentose phosphate pathway in relation to fat synthesis in the developing castor oil seed.

Slices of 25- to 28-day-old developing castor bean endosperm were incubated with various (14)C- and (3)H-labeled substrates to determine the amount of glucose dissimilated in the pentose phosphate pathway and to determine the use of the reduced nucleotides so produced in fatty acid synthesis. Ten to 12% of the metabolized glucose traversed the pentose phosphate pathway, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) production would be sufficient to supply 51 to 68% of the reducing equivalents required for fat synthesis. However, using (3)H-NADPH produced from 3-(3)H-glucose as a tracer, it was found that only 40% of the NADPH produced in the pentose phosphate pathway was used in fat synthesis. Thus the actual contribution of the reducing equivalents generated from the pentose phosphate pathway to fat synthesis was 20 to 27% of that required. Because of the methods and assumptions, this value represents a minimal estimate of NADPH used in fat synthesis, and the actual contribution may be somewhat higher. However, tritium from (3)H-NADH generated from 1-(3)H-ethanol was incorporated into fatty acids, and it is contended that NADH may supply a large proportion of the reducing equivalents necessary for fat synthesis in this tissue.

Journal Article↗
PubMed1994

Changes in Soybean Fruit Ca2+ (Sr2+) and K+ (Rb+) Transport Ability during Development.

Mineral uptake by soybean (Glycine max [L.] Merrill) seeds during development can significantly affect seed quality and value. Little is known about seed mineral transport mechanisms and control processes, although it is clear that each mineral displays a characteristic accumulation pattern. Ion-specific accumulation patterns could result from changes in source availability, in transport kinetics through the seed pod and seed coat, or in the mineral uptake capability of the embryo. Ca2+ and K+ have negligible and high phloem mobilities, respectively. Ca2+ accumulation lags behind dry matter (C and N) and K+ accumulation in soybean embryos. To eliminate source availability influences, the Ca2+ and K+ uptake ability of isolated embryos and of seeds in pod culture was examined during seed development. Sr2+ and Rb+ were used as transport analogs of Ca2+ and K+, respectively. Sr2+ and Rb+ uptake rates by isolated embryos increased with seed fresh weight, indicating that the embryo was not limiting Ca2+ accumulation. However, the pod-cultured embryo Sr2+ and Rb+ uptake rate trends differed: Rb+ uptake increased with seed fresh weight, whereas Sr2+ uptake rates remained constant or decreased slightly. Ovule Sr2+ influx data suggest that the pod and seed coat impose a transport barrier that could account for the relative decline in embryo Ca2+ content during development.

Journal Article↗
PubMed1999

Purification of a multicatalytic protease complex from developing winged bean seeds by indirect immunoaffinity chromatography.

Many protease inhibitors have been characterized from leguminous seeds but very little is known about seed proteases which are supposedly regulated by these inhibitors. We have developed an indirect immunoaffinity chromatography system for the purification of cognate proteases from the same source, based on preferential high salt elution of the enzyme from a ternary complex of the protease, the inhibitor, and the anti-inhibitor IgG. Using anti-winged bean chymotrypsin inhibitor (WbCI) IgG as an affinity ligand, a multicatalytic protease complex has been purified from developing winged bean (Psophocarpus tetragonolobus) seeds. The purified preparation resolves into two large proteolytically active components when subjected to gel permeation chromatography under nondenaturing conditions, while SDS/PAGE analysis shows the presence of approximately 15 polypeptide chains in the 20- to 115-kDa range. The preparation cleaves known synthetic peptide substrates of trypsin, chymotrypsin, and V8 protease and it is only partially inhibited by a number of class-specific protease inhibitors. Western blot analysis shows the presence of WbCI in the purified preparation even after its extensive removal by the IgG-Sepharose column. The versatility of the indirect immunoaffinity chromatography system is attested by its extension to the soybean seeds.

Adenosine Triphosphatases↗
PubMed1982

Carbamoyl phosphate synthetase activity from the cotyledons of developing and germinating pea seeds.

Carbamoyl phosphate synthetase activity was measured in partially purified extracts from cotyledons of developing and germinating seeds of Pisum sativum L. Some properties of the enzyme were established. During cotyledon development, the activity initially increased sharply but decreased during further development. The activity from germinating seeds was only one-tenth of the maximum activity at an early developmental phase. The results are discussed in relation to pea seed development and germination.

Journal Article↗
PubMed2001

Development and functions of seed transfer cells.

In secretion or absorption processes, solutes are transported across the plasmalemma between the symplastic and apoplastic compartments. For this purpose, certain plant cells have developed a specialised transfer cell morphology characterised by wall ingrowths, which amplify the associated plasmalemma surface area up to 20-fold. Detailed studies on the function and development of transfer cells in the context of seed filling have been carried out mainly in cereal endosperm, and for the cotyledon and seed coat cells of legumes. The major solutes transferred are amino acids, sucrose and monosaccharides. The contributions of recently identified symporter proteins to solute transfer are reviewed here, as is the role of apoplastic invertases in promoting solute assimilation. Expression of invertase and monosaccharide transporters early in both cereal and legume seed development orchestrates the distribution of free sugars which play an important role in regulating transfer cell function and determining final endosperm or embryo cell number. Transfer cell differentiation is subject to developmental control, and may also be modulated by sugar levels. The most abundant genes specifically expressed in the transfer layer of maize endosperm encode small antipathogenic proteins, pointing to a role for these cells in protecting the developing endosperm against pathogen ingress. The functional characterisation of the corresponding transfer layer-specific promoters has provided a tool for dissecting transfer cell functions. Transfer cells are highly polar in their organisation, the characteristic cell wall ingrowths developing on one face only. The presence of cytoskeletal components bordering wall ingrowths is documented, but their role in establishing transfer cell morphology remains to be established.

Journal Article↗
PubMed1987

Comparison of in situ and in vitro regulation of soybean seed growth and development.

The growth characteristics of soybean (Glycine max [L.] Merr.) embryos in culture and seeds in situ were found to be similar, but developmental differences were observed. Embryos placed in culture when very small (<2 milligrams dry weight) failed to attain the maximal growth rates attained by embryos which were more mature when placed in culture. When nutrient levels were maintained in the culture medium, embryos continued to grow indefinitely, reaching dry weights far in excess of seeds matured in situ. Apparently, maternal factors were important in early and late development during the determination of maximum growth rate and the cessation of growth. Embryo growth rate was not affected by substituting glucose plus fructose for sucrose in the medium, nor by hormone treatments, including abscisic acid. Glutamine was found to give substantially better growth than glutamate, however. Contrary to prior reports, the response of soybean embryo growth rate to irradiance was found to be primarily an artifact of the effect of irradiance on media temperature. Across seven genotypes the correlation coefficient between seed growth rate in situ and embryo growth rate in vitro was 0.94, indicating essentially all of the variability of in situ seed growth rate between cultivars could be attributed to inherent growth rate differences associated with the embryos. The response to temperature was very similar for both embryos in culture and seeds in situ at temperatures below 30 degrees C. Beyond that temperature, embryo growth rate continued to increase, while seed growth rate did not. The implication is that in situ seed growth rate is determined by the inherent growth potential of the embryo at low to moderate temperatures; however, at higher temperatures, the maternal plant is unable to support the rapid growth rates that the embryo is capable of attaining under conditions of unlimited assimilate supply.

Journal Article↗
PubMed1984

Synthesis of the crystalloid protein complex in vivo in the endosperm of developing castor bean seeds.

The synthesis of subunit polypeptides of the crystalloid protein complex has been examined in endosperm from developing castor bean (Ricinus communis L. cv Hale) seeds. Pulse-label and -chase studies in vivo have shown that synthesis initially involves the formation of high molecular weight precursors (50 to 60 kilodaltons) comprising peptide-linked acidic and basic polypeptides. Precursor processing involves the posttranslational cleavage of the peptide bond to yield authentic and polypeptides. This processing has a half-life of 35 to 40 minutes and is preceded by a 45- to 60-minute lag period. Both precursor and subunit polypeptides are shown to exhibit similar molecular weight and pI heterogeneity, and this is suggested to be due to the expression of a multigene family.

Journal Article↗
PubMed2006

Development of a seeded scaffold in the great omentum: feasibility of an in vivo bioreactor for bladder tissue engineering.

OBJECTIVES: Tissue engineering is very promising in bladder reconstruction. However, one of the main problems is to limit the development of ischaemic fibrosis during tissue maturation. We describe a model using the omentum as an in vivo bioreactor for a previously seeded scaffold. METHODS: Bladder biopsies were taken from five female pigs, from which both urothelial and smooth muscle cells cultures were made. These cultured cells were used to seed a sphere-shaped small intestinal submucosa (SIS) matrix, which was transferred into the omentum after 3 wk of cell growth. The grafts were harvested 3 wk later and histologic, immunohistochemical, and functional studies were performed. RESULTS: We obtained a highly vascularized tissue-engineered construct that contracted in response to acetylcholine stimulation. The wall thickness was 4mm, on average. Histologic and immunostaining analysis of the construct confirmed the presence of a multilayer urothelium on the luminal aspect and deeper fascicles organised tissue composed of differentiated smooth muscle cells and mature fibroblasts without evidence of inflammation or necrosis. Large- and small-diameter vessels were clearly identified histologically in the tissue obtained. CONCLUSION: The omentum permitted in vivo maturation of seeded scaffolds with the development of a dense vascularisation that is anticipated to prevent fibrosis and loss of contractility. This in vivo maturation into the omentum could be the first step before in situ implantation of the construct.

Animals↗
PubMed1982

Isozymes of the glycolytic enzymes in endosperm from developing castor oil seeds.

Ion filtration chromatography on diethylaminoethyl-Sephadex A-25 has been used to separate two isozymes each of triose phosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, glycerate 3-phosphate kinase, enolase, and phosphoglycerate mutase from homogenates of developing castor oil (Ricinus communis L. cv. Baker 296) seeds. Crude plastid fractions, prepared by differential centrifugation, were enriched in one of the isozymes, whereas the cytosolic fractions were enriched in the other. These data (and data published previously) indicate that plastids from developing castor oil seeds have a complete glycolytic pathway and are capable of conversion of hexose phosphate to pyruvate for fatty acid synthesis. The enzymes of this pathway in the plastids are isozymes of the corresponding enzymes located in the cytosol.

Journal Article↗
PubMed2002

Proteomic analysis of the endoplasmic reticulum from developing and germinating seed of castor (Ricinus communis).

Endoplasmic reticulum (ER) has been prepared and analysed from germinating and developing castor bean endosperm. A combination of one- and two-dimensional (1-D and 2-D) gel electrophoresis was used to study the complexity of sample and protein differences between the two stages. The ER of the developing oilseed is central to the synthesis, sorting and storage of protein and lipid reserves while the germinating seed is concerned with their degradation. Sample complexity has been reduced by separation of ER proteins into lumenal, peripheral membrane and integral membrane subfractions. Membrane proteins pose specific problems in aggregation and binding to passive surfaces. We have overcome this by collection of membranes at density gradient interfaces and by silanization of plastic ware. Several major components have been identified from 1-D gels by N-terminal sequencing and matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprints. These include protein disulphide isomerase (PDI), calreticulin and developing-ER-specific oleate-12-hydroxylase involved in the biosynthesis of ricinoleic acid. In excess of 300 spots are detectable in each developmental fraction by high sensitivity 2-D gels. This is the first 2-D electrophoretic analysis of plant ER. These gels reveal significant differences between germinating and developing ER. Preparative loading 2-D gels of germinating ER have been run and 14 selected spots characterized by quadrupole time of flight tandem mass spectrometry (Q-TOF MS/MS). Ten of these proteins were assigned function on the basis of identity with existing castor database entries, or by homology with other species. Two proteins, aspartate proteinase precursor and N-carbamyl-L-aminohydrolase-like protein, appear to be absent from developing profiles. Most of the proteins identified are concerned with roles in protein processing and storage, and lipid metabolism which occur in the ER. Data from three of the assigned spots included unidentified peptides indicating the presence of more than one protein in these spots following 2-D electrophoresis. More extensive analysis will have to await developments in genomics but the basic separation technologies to simplify sample identity for a plant ER preparation have been established.

Amino Acid Sequence↗
PubMed1998

Changes in chemical composition, minerals and amino acids during seed growth and development of four safflower cultivars.

Investigation of four safflower (Carthamus tinctorius L.) cultivars (S208, S400, S541 and S303) showed that when the seeds were harvested at different stages of growth and development (10, 20, 30, and 40 days) after flowering, moisture content significantly decreased with time. Oil, protein, ash and crude fiber were increased up to day 30. Thereafter, these parameters started to decline gradually with time. The cultivars differed in their final values; oil content of the seeds varied from 10.90 to 45.40%, moisture varied from 4.20 to 8.10% and from 8.50 to 11.10%, protein from 12.10 to 20.30% and from 13.40 to 29.60%, ash from 2.30 to 5.40% and from 2.80 to 6.50%, for the seeds and defatted meal, respectively. Crude fiber for the defatted meal was found to vary from 29.50 to 38.60%. Carbohydrate for all cultivars decreased rapidly up to day 40 with final values varying from 28.10 to 63.30% and from 56.70 to 70.30% for the seeds and defatted meal, respectively. Mineral content (Cu, Zn, Fe, Mg, Mn) fluctuated while phosphorus content significantly increased with time for all cultivars. Amino acid content of the defatted meal increased with time up to day 30 after which it started to decline gradually for all cultivars.

Amino Acids↗
PubMed1976

[Formation of the respiratory enzyme apparatus in growing cells. IV. Composition of mitochondrial proteins synthesized in developing and germinating corn seeds].

The mitochondriogenesis in the developing maize embryo involves the accumulation of organelles with the relatively reduced matrix antigen content and relatively abundant several detergent-soluble membrane components, as compared with the mitochondria from differentiated root cells. Several new antigens are incorporated into the mitochondria of embryonic axes and scutella of imbibing seeds. While the qualitative protein patterns are constant, the ratio of individual antigens changes during the subsequent differentiation of root and scutellum cells. At the same time similar spectra consisting of 18--20 groups of mitochondrial polypeptides with the molecular weight from less than 12,000 to 115,000 are synthesized but the ratio of individual polypeptide groups changes differentially.

Antigens↗
PubMed2000

Fast activation of a time-dependent outward current in protoplasts derived from coats of developing Phaseolus vulgaris seeds.

An outward current that appeared to activate instantaneously in response to depolarising voltage pulses at low sampling frequencies predominated in the plasma membrane of ground-parenchyma protoplasts derived from coats of developing Phaseolus vulgaris L. (cv. Redland Pioneer) seeds. However, the outward current showed time-dependent activation when higher sampling frequencies were used to measure the current. Activation of the current was best described as a double-exponential time course with the fast and slow time constants being 1 and 20 ms, respectively. The current also exhibited a rapid deactivation that followed a double-exponential time course with time constants of approximately 2 and 30 ms, respectively. "Tail-current" analysis allowed us to show that this current exhibited a low selectivity between K- and Cl- (PK:Cl = 1.8). Such a fast-activating current may account for some of the reports of time-independent, instantaneous currents that have been observed in plasma membranes of plant cells digitised at low sampling frequencies. Therefore, when "instantaneous" currents appear it is advisable to characterise these currents using higher sampling frequencies with correspondingly higher filtering frequency cut-offs.

Fabaceae↗
PubMed2005

Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: dynamic cell seeding and construct development.

Human mesenchymal stem cells (hMSCs) have great potential for therapeutic applications. A bioreactor system that supports long-term hMSCs growth and three-dimensional (3-D) tissue formation is an important technology for hMSC tissue engineering. A 3-D perfusion bioreactor system was designed using non-woven poly (ethylene terepthalate) (PET) fibrous matrices as scaffolds. The main features of the perfusion bioreactor system are its modular design and integrated seeding operation. Modular design of the bioreactor system allows the growth of multiple engineered tissue constructs and provides flexibility in harvesting the constructs at different time points. In this study, four chambers with three matrices in each were utilized for hMSC construct development. The dynamic depth filtration seeding operation is incorporated in the system by perfusing cell suspensions perpendicularly through the PET matrices, achieving a maximum seeding efficiency of 68%, and the operation effectively reduced the complexity of operation and the risk of contamination. Statistical analyses suggest that the cells are uniformly distributed in the matrices. After seeding, long-term construct cultivation was conducted by perfusing the media around the constructs from both sides of the matrices. Compared to the static cultures, a significantly higher cell density of 4.22 x 10(7) cell/mL was reached over a 40-day culture period. Cellular constructs at different positions in the flow chamber have statistically identical cell densities over the culture period. After expansion, the cells in the construct maintained the potential to differentiate into osteoblastic and adipogenic lineages at high cell density. The perfusion bioreactor system is amenable to multiple tissue engineered construct production, uniform tissue development, and yet is simple to operate and can be scaled up for potential clinical use. The results also demonstrate that the multi-lineage differentiation potential of hMSCs are preserved even after extensive expansion, thus indicating the potential of hMSCs for functional tissue construct development. The system has important applications in stem cell tissue engineering.

Adipocytes↗
PubMed2006

Succession of bacterial communities during early plant development: transition from seed to root and effect of compost amendment.

Compost amendments to soils and potting mixes are routinely applied to improve soil fertility and plant growth and health. These amendments, which contain high levels of organic matter and microbial cells, can influence microbial communities associated with plants grown in such soils. The purpose of this study was to follow the bacterial community compositions of seed and subsequent root surfaces in the presence and absence of compost in the potting mix. The bacterial community compositions of potting mixes, seed, and root surfaces sampled at three stages of plant growth were analyzed via general and newly developed Bacteroidetes-specific, PCR-denaturing gradient gel electrophoresis methodologies. These analyses revealed that seed surfaces were colonized primarily by populations detected in the initial potting mixes, many of which were not detected in subsequent root analyses. The most persistent bacterial populations detected in this study belonged to the genus Chryseobacterium (Bacteroidetes) and the family Oxalobacteraceae (Betaproteobacteria). The patterns of colonization by populations within these taxa differed significantly and may reflect differences in the physiology of these organisms. Overall, analyses of bacterial community composition revealed a surprising prevalence and diversity of Bacteroidetes in all treatments.

Bacteria↗
PubMed2000

The forms and sources of cytokinins in developing white lupine seeds and fruits.

A comprehensive range of cytokinins (CK) was identified and quantified by gas chromatography-mass spectrometry in tissues of and in xylem and phloem serving developing white lupine (Lupinus albus) fruits. Analyses were initiated at anthesis and included stages of podset, embryogenesis, and seed filling up to physiological maturation 77 d post anthesis (DPA). In the first 10 DPA, fertilized ovaries destined to set pods accumulated CK. The proportion of cis-CK:trans-CK isomers was initially 10:1 but declined to less than 1:1. In ovaries destined to abort, the ratio of cis-isomers to trans-isomers remained high. During early podset, accumulation of CK (30-40 pmol ovary(-1)) was accounted for by xylem and phloem translocation, both containing more than 90% cis-isomers. During embryogenesis and early seed filling (40-46 DPA), translocation accounted for 1% to 14% of the increases of CK in endosperm (20 nmol fruit(-1)) and seed coat (15 nmol fruit(-1)), indicating synthesis in situ. High CK concentrations in seeds (0.6 micromol g(-1) fresh weight) were transient, declining rapidly to less than 1% of maximum levels by physiological maturity. These data pose new questions about the localization and timing of CK synthesis, the significance of translocation, and the role(s) of CK forms in reproductive development.

Cytokinins↗
PubMed2004

Storage behavior of Chionanthus retusus seed and asynchronous development of the radicle and shoot apex during germination in relation to germination inhibitors, including abscisic acid and four phenolic glucosides.

Studies on seed storage of Chionanthus retusus Lindl. & Paxt. revealed an orthodox behavior, one which showed both desiccation and freezing tolerance. An epicotyl after-ripening dormancy was expressed in C. retusus seeds by slow growth of the shoot apex relative to more rapid growth of the radicle when seeds were germinated at 30/20 degrees C. Although these seeds exhibit radicle protrusion, they must be after-ripened for another 8-10 weeks at 30/20 degrees C in order to obtain normal shoot growth. Removal of the endosperm, however, quickly stimulated cotyledon and shoot emergence without the additional after-ripening. Water-soluble glucoside phenolics, GL-3, Nuzhenide, ligustroside and oleoside dimethyl ester are present at relatively high levels in endosperm of freshly harvested seeds. These glucoside phenolics are excreted from the endosperm during subsequent after-ripening. Embryo and endosperm tissue from seed germinating at 30/20 degrees C (germination being defined by protrusion of the radicle) had a 10 times lower abscisic acid (ABA) content than similar tissues from freshly harvested mature seed. However, no shoot growth occurred even with the 10-fold reduction in ABA and a concomitant increase in endogenous gibberellins A1, A4 and A20. Thus, epicotyl dormancy during the first 8 weeks of after-ripening at 30/20 degrees C may be controlled by factors other than high ABA, i.e., the slow development of the shoot apex following radicle protrusion may be controlled more by high levels of glucoside phenolics than by diminished ABA and elevated GA levels.

Abscisic Acid↗