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Nectar resorption and translocation in Cucurbita pepo L. and Platanthera chlorantha Custer (Rchb.).

Nectar resorption and sugar translocation were studied in Cucurbita pepo (Cucurbitaceae) and Platanthera chlorantha (Orchidaceae) by micro-autoradiography. In both species, nectar was resorbed in pollinated and unpollinated flowers and ovules developing into seeds were found to be the main sugar sink. In C. pepo, the mobility of resorbed sugars in pollinated female flowers was higher than in unpollinated ones; male flowers showed lower mobility of resorbed sugar. In P. chlorantha, radioactivity was detected in pollinated flowers below and above labelled unpollinated ones: the nearer the flower, the stronger the accumulation of label in developing fruits.

Autoradiography↗

Quantitative analysis of anti-inflammatory and radical scavenging triterpenoid esters in evening primrose seeds.

Lipophilic triterpenoidal esters with radical scavenging and cyclooxygenase inhibitory properties were recently found in cold-pressed, nonraffinated evening primrose oil (EPO). A quantitative assay for the analysis of 3-O-trans-caffeoyl derivatives of betulinic, morolic, and oleanolic acid in evening primrose seeds was developed and validated. Extraction efficiency >99% was achieved by means of pressurized liquid extraction with two extraction cycles and 80% (v/v) ethanol at 120 degrees C. Analysis of esters was by normal-phase high-performance liquid chromatography on a Diol column and hexane/ethyl acetate (containing 0.1% formic acid) (65:35) as the eluent. The analytes were determined without further prepurification. Seeds from defined cultures of Oenothera biennis, Oenothera lamarckiana, and Oenothera ammophila, grown under identical conditions, were analyzed. The cultures originated from seeds from eight collections in the wild and from selections from five cultivars. The content of total triterpenoidal esters in seeds varied between 1.34 and 2.78 mg/g. Three types of qualitative patterns were observed for the triterpenoidal esters. The influence of different harvest times and plant treatments was studied with the cultivar Anothera. Variations between 1.5 and 2.3 mg/g were found.

Anti-Inflammatory Agents↗

Commentary: the role of cell migration in the ontogeny of the lymphoid system.

The foundations of experimental hematology were laid by histologists, and while their contributions were enormous, they were limited in their interpretation of very dynamic processes by the static nature of the methodology. The middle of the twentieth century saw the introduction of techniques for hematopoietic cell marking and development of in vitro and in vivo assays for primitive hematopoietic cells, allowing dynamic studies of hematopoiesis. Paralleling this was an understanding of cellular immunology with the discovery of the role of the thymus and the identification of T and B lymphocyte lineages. In the 1960s a series of ontogenetic studies in birds and subsequently in mice revealed that hematopoietic and lymphoid development involved migration streams of primitive cells that colonized developing primary lymphoid organs as well as spleen, marrow, and liver. The yolk sac was proposed as the ultimate origin of these lympho-hematopoietic precursors. Subsequent studies identified a region associated with the dorsal aorta as the primary site of "definitive" stem cells. These opposing views are currently achieving a compromise that recognizes that both sites contribute stem cells involved in seeding the developing tissues. The clear distinction between the local origin of the inducing microenvironment provided by the endoderm or by stroma derived from mesenchymal stem cells of mesodermal origin, and the immigrant origin of the hematopoietic stem cells and progenitors, raises intriguing questions in the current climate of stem cell plasticity, cell fusion, and discovery of stem cells in adult marrow with the capacity to generate hematopoiesis as well as other mesodermal, ectodermal, and endodermal lineages.

Animals↗

Isolation and Partial Characterization of a Seed Lectin from Tepary Bean that Delays Bruchid Beetle Development.

Four isolectin forms of a seed lectin from mature seed of tepary bean (Phaseolus acutifolius) were isolated using solubility fractionation, affinity chromatography, and high performance liquid chromatography. The subunits are polypeptides with an apparent molecular mass of 30,000 daltons. The 30 kilodalton subunits are produced starting approximately 13 days after flowering and subsequently comprise a major fraction of the proteins found in the mature seed. The amino terminus of each isolectin fraction was determined to be highly homologous with that of the subunits of common bean (Phaseolus vulgaris L.) phytohemagglutinin (PHA). The tepary isolectin cross-reacts with both erythroagglutinating and leucoagglutinating subunits of PHA antibodies, although differential cross-reactivity was noted. A seed protein fraction enriched in tepary bean lectin was found to be toxic to bean bruchid beetles (Acanthoscelides obtectus), when incorporated into their diets at incremental concentrations from (1-5% w/w) above that of PHA concentrations in mature seeds of the susceptible common bean variety "Red Kidney."

Journal Article↗

T cells developing in fetal thymus of T-cell receptor alpha-chain transgenic mice colonize gammadelta T-cell-specific epithelial niches but lack long-term reconstituting potential.

The gammadelta T cells generated during mouse fetal development are absolutely dependent on their invariant T-cell receptors (TCRs) for their function. However, there is little information on whether the epithelial homing properties of fetal T cells might also be developmentally induced by factors unrelated to TCR specificity. We have previously described TCR alpha-chain transgenic (2B4 TCR-alpha TG) mice, in which the transgenic TCR alpha-chain is expressed early, already at embryonic day 14 (E14). These mice have a large population of 'gammadelta T-cell-like' CD4- CD8- (double-negative; DN) alphabeta T cells, some of which develop during E14-E18 contemporarily to intraepithelial lymphocytes (IELs) expressing invariant TCR-gammadelta. Using the 2B4 TCR-alpha TG mouse model we have been able to more precisely study the impact of a variant TCR expression on IEL development and homing. In this study we show that TCR-alpha TG and TCR-alpha TG crossed to TCR-delta-deficient mice (TCR-alpha TG x TCR-delta-/-) carry TG TCR-alpha+ dendritic epidermal T cells (DETCs) and TCR-alpha TG+ IELs in the small intestine. The TG+ DETCs develop and seed the epidermis with similar kinetics as Vgamma5+ DETCs of normal mice, in contrast to the TCR-alphabeta+ DETCs found in TCR-delta-/- mice. However, whereas the intestinal TCR-alpha TG+ IELs persist in old mice (> 20 months), the TCR-alpha TG+ DETCs do not. The data in this study indicate that the timing of TCR expression and thereby development during ontogeny regulates the specific homing potential for fetal T cells but not their subsequent functions and properties.

Aging↗

Purification and characterization of a co(II)-sensitive alpha-mannosidase from Ginkgo biloba seeds.

An alpha-mannosidase was purified from developing Ginkgo biloba seeds to apparently homogeneity. The molecular weight of the purified alpha-mannosidase was estimated to be 120 kDa by SDS-PAGE in the presence of 2-mercaptoethanol, and 340 kDa by gel filtration, indicating that Ginkgo alpha-mannosidase may function in oligomeric structures in the plant cell. The N-terminal amino acid sequence of the purified enzyme was Ala-Phe-Met-Lys-Tyr-X-Thr-Thr-Gly-Gly-Pro-Val-Ala-Gly-Lys-Ile-Asn-Val-His-Leu-. The alpha-mannosidase activity for Man(5)GlcNAc(1) was enhanced by the addition of Co(2+), but the addition of Zn(2+), Ca(2+), or EDTA did not show any significant effect. In the presence of cobalt ions, the hydrolysis rate for pyridylaminated Man(6)GlcNAc(1) was significantly faster than that for pyridylaminated Man(6)GlcNAc(2), suggesting the possibility that this enzyme is involved in the degradation of free N-glycans occurring in developing plant cells (Kimura, Y., and Matsuo, S., J. Biochem., 127, 1013-1019 (2000)). To our knowledge, this is the first report showing that plant cells contain an alpha-mannosidase, which is activated by Co(2+) and prefers the oligomannose type free N-glycans bearing only one GlcNAc residue as substrate.

Amino Acid Sequence↗

Composition and content of glucosinolates in developing Arabidopsis thaliana.

The glucosinolate composition and content in various tissues of Arabidopsis thaliana (L.) Heynh. ecotype Columbia during development from seeds to bolting plants were determined in detail by high-performance liquid chromatography. Comparison of the glucosinolate profiles of leaves, roots and stems from mature plants with those of green siliques and mature seeds indicated that a majority of the seed glucosinolates were synthesized de novo in the silique. A comparison of the glucosinolate profile of mature seeds with that of cotyledons indicated that a major part of seed glucosinolates was retained in the cotyledons. Turnover of glucosinolates was studied by germination of seeds containing radiolabelled p-hydroxybenzylglucosinolate (p-OHBG). Approximately 70% of the content of [14C]p-OHBG in the seeds was detected in seedlings at the cotyledon stage and [14C]p-OHBG was barely detectable in young plants with rosettes of six to eight leaves. The turn-over of p-OHBG was found to coincide with the expression of the glucosinolate-degrading enzyme myrosinase, which was detectable at very low levels in seedlings at the cotyledon stage, but which dramatically increased in leaves from plants at later developmental stages. This indicates that there is a continuous turnover of glucosinolates during development and not only upon tissue disruption.

Arabidopsis↗

Fruit developmental regulation of the kiwifruit actinidin promoter is conserved in transgenic petunia plants.

We have examined the expression of actinidin, a cysteine protease found in kiwifruit, over the course of fruit development. Protease activity was first seen in fruit that had reached about half their final weight, and rose to high levels at harvest. The 5'-flanking region (nucleotides -1301 to +58) of a kiwifruit actinidin gene was fused to the beta-glucuronidase (GUS)-coding region, and the chimaeric gene was introduced into transgenic petunia plants. Induction of the GUS gene was observed during the later stages of seed pod development, closely resembling the pattern of actinidin induction in fruit tissues of kiwifruit. Some GUS expression was also detected in the vascular system of the receptacle, leaves, stems and roots. A shorter promoter fragment consisting of nucleotides -115 to +58 conferred similar spatial and temporal regulation in some of the transgenic plants.

Base Sequence↗

Transgenic overexpression of GmAPC7-CT improves seed yield and reduces susceptibility to soybean mosaic virus and Meloidogyne incognita in soybean.

Stable transgenic soybean lines overexpressing the GmAPC7-CT gene have demonstrated increased seed yield and reduced susceptibility to the soybean mosaic virus and Meloidogyne incognita. The Anaphase-Promoting Complex subunit 7 (APC7) is a core structural component of the anaphase-promoting complex or cyclosome (APC/C). The terminal region of this AtAPC7 gene has been shown in Arabidopsis thaliana to accumulate more transcripts than the full-length gene. The AtAPC7-CT gene (terminal region of the AtAPC7) encodes a protein with significant homology to a tobacco viral replication inhibitor (IVR). Its stable overexpression in transgenic A. thaliana lines resulted in notable improvements in biomass, seed yield, earliness of vegetative-reproductive transitions, and reduced susceptibility to viruses. In this study, we generated stable transgenic soybean lines overexpressing the GmAPC7-CT gene (terminal region or 3' portion of Glyma.15G096000, corresponding to the AtAPC7-CT) and evaluated seed yield and susceptibility of these lines to soybean mosaic virus and Meloidogyne incognita. The GmAPC7-CT gene is 624 nucleotides long and encodes a 207-amino acid protein with two tetratricopeptide repeat (TPR) domains. GmAPC7-CT showed 100% amino acid identity with full-length GmAPC7, 81.16% identity with AtAPC7-CT, and 87.94% identity with tobacco IVR. Stable transgenic lines demonstrated significant advancements in plant development and seed yield, with the top three lines producing up to 43% more pods, 44% more seeds, and a 16% increase in seed weight. Furthermore, these soybean lines showed up to a 70% reduction in susceptibility to soybean mosaic virus and M. incognita, reflected by decreased viral RNA load and nematode reproduction factor. Collectively, these results support a conserved role of GmAPC7-CT in soybean and AtAPC7-CT in A. thaliana, acting similarly to the tobacco IVR. Thus, our findings underscore the strong biotechnological potential of the GmAPC7-CT gene to improve key agronomic traits in soybean through genetic engineering approaches, including conventional breeding, transgenesis, and genome editing.

Glycine max↗

Inhibitors of fatty acid biosynthesis in sunflower seeds.

During de novo fatty acid synthesis in sunflower seeds, saturated fatty acid production is influenced by the competition between the enzymes of the principal pathways and the saturated acyl-ACP thioesterases. Genetic backgrounds with more efficient saturated acyl-ACP thioesterase alleles only express their phenotypic effects when the alleles for the enzymes in the main pathway are less efficient. For this reason, we studied the incorporation of [2-(14)C]acetate into the lipids of developing sunflower seeds (Helianthus annuus L.) from several mutant lines in vivo. The labelling of different triacylglycerol fatty acids in different oilseed mutants reflects the fatty acid composition of the seed and supports the channelling theory of fatty acid biosynthesis. Incubation with methyl viologen diminished the conversion of stearoyl-ACP to oleoyl-ACP in vivo through a decrease in the available reductant power. In turn, this led to the accumulation of stearoyl-ACP to the levels detected in seeds from high stearic acid mutants. The concomitant reduction of oleoyl-ACP content inside the plastid allowed us to study the activity of acyl-ACP thioesterases on saturated fatty acids. In these mutants, we verified that the accumulation of saturated fatty acids requires efficient thioesterase activity on saturated-ACPs. By studying the effects of cerulenin on the in vivo incorporation of [2-(14)C]acetate into lipids and on the in vitro activity of beta-ketoacyl-ACP synthase II, we found that elongation to very long chain fatty acids can occur both inside and outside of the plastid in sunflower seeds.

Acyl Carrier Protein↗

Control of seed mass by APETALA2.

Arabidopsis APETALA2 (AP2) encodes a member of the AP2/EREBP (ethylene responsive element binding protein) class of transcription factors and is involved in the specification of floral organ identity, establishment of floral meristem identity, suppression of floral meristem indeterminancy, and development of the ovule and seed coat. Here, we show that loss-of-function ap2 mutations cause an increase in seed mass relative to that of wild-type seeds. Analysis of an allelic series of ap2 mutations showed that increases in seed mass corresponded with the severity of defects in flower structure, indicating that AP2 activity directly influences seed mass. Experiments with male-sterile plants and deflowered wild-type plants showed that reduced fertility of ap2 mutant plants due to abnormal flower structure accounted for only part of the increase in seed mass caused by strong ap2 mutant alleles. Reciprocal cross experiments showed that AP2 acts maternally to control seed mass. The maternal effect of AP2 on seed mass involves the regulation of both embryo cell number and cell size. We show further that ap2 mutations cause changes in the ratio of hexose to sucrose during seed development, opening the possibility that AP2 may control seed mass through its effects on sugar metabolism. Together, these results identify a role for AP2 in controlling seed mass.

Arabidopsis↗

Facing herbivory as you grow up: the ontogeny of resistance in plants.

As plants develop from seeds to seedlings, juveniles and mature stages, their ontogeny can constrain the expression of resistance to herbivore damage. Nevertheless, ecological and evolutionary theories regarding interactions between plants, herbivores and their natural enemies are largely based on observations and experiments conducted at a single ontogenetic stage. Owing to resource allocation and architectural constraints in plants, and the influence of herbivore foraging behavior, resistance to herbivores is likely to change during plant development. We propose that such changes are likely to occur in a non-linear fashion and suggest that the role of ontogeny should be incorporated as an important factor in new syntheses of plant defense theory.

Journal Article↗

Differentiation of mucilage secretory cells of the Arabidopsis seed coat.

In some plant species, including Arabidopsis, fertilization induces the epidermal cells of the outer ovule integument to differentiate into a specialized seed coat cell type with a unique morphology and containing large quantities of polysaccharide mucilage (pectin). Such seed coat mucilage cells are necessary for neither viability nor germination under normal laboratory conditions. Thus, the Arabidopsis seed coat offers a unique system with which to use genetics to identify genes controlling cell morphogenesis and complex polysaccharide biosynthesis and secretion. As a first step in the application of this system, we have used microscopy to investigate the structure and differentiation of Arabidopsis seed coat mucilage cells, including cell morphogenesis and the synthesis, secretion, and extrusion of mucilage. During seed coat development in Arabidopsis, the epidermal cells of the outer ovule integument grow and differentiate into cells that produce large quantities of mucilage between the primary cell wall and plasma membrane. Concurrent with mucilage production, the cytoplasm is shaped into a column in the center of the cell. Following mucilage secretion the cytoplasmic column is surrounded by a secondary cell wall to form a structure known as the columella. Thus, differentiation of the seed coat mucilage cells involves a highly regulated series of events including growth, morphogenesis, mucilage biosynthesis and secretion, and secondary cell wall synthesis.

Adhesives↗

Spontaneous differentiation in the colonies of a nullipotent embryonal carcinoma cell line (F9).

Experiments were undertaken to reveal the spontaneous differentiation capacity of the nullipotent F9 embryonal-carcinoma (EC) cell line in colonies derived from single cells. Culture conditions which allowed the development of neuroblasts in colonies of the multipotent EC cell line (PCC3) were worked out, and comparative studies on neuroblast differentiation in PCC3 and F9 colonies were conducted. Neural-cell-specific silver impregnation, selective staining of cells having electrically excitable membranes with merocyanine 540 and the observation of nerve processes were considered as differentiation markers. The appearance of neuroblasts in F9 and PCC3 colonies could be detected from the 6th day after seeding. The development of neuroblasts was less prevalent in high-density cultures, especially in the case of F9. By the 8th day in differentiating colonies, PCC3 cells lost much of their colony-forming activity, while F9 cells preserved their original high plating efficiency, in spite of advanced differentiation. The determination of growth parameters during differentiation in colonies led to the conclusion that F9 cells had lost certain growth-control mechanisms which normally restrict the clonal growth of EC cells. It is suggested that the phenomenon of nullipotence may be analysed in terms of the coordinated regulation of proliferation and differentiation of EC cells.

Animals↗

In situ Western hybridization: a new, highly sensitive technique to detect foreign and endogenous protein distribution in rice seeds.

We have developed a highly sensitive in situ Western hybridization technique to study tissue-specific expression of foreign and endogenous genes in transgenic and non-transformed rice seed. The expression pattern of the soybean ferritin gene directed by a rice glutelin gene promoter, GluB-1, in transgenic rice seed revealed by this method was exactly the same as that revealed by immunological tissue printing but much clearer than the latter, and corresponded well to the results of GluB-1 promoter characterization studies. This method provides an alternative choice for studying the tissue-specific expression of a promoter, omitting the complicated transgenic procedure. The method can also be used to study the expression and accumulation pattern of endogenous genes, such as glutelin and prolamine genes, in non-transformed plants.

Blotting, Western↗

Genetic modification of cotton seed oil using inverted-repeat gene-silencing techniques.

Inverted-repeat-based gene constructs targeted against two key cotton seed-specific fatty acid desaturase genes, ghSAD-1, encoding stearoylacvl carrier protein delta9-desaturase and ghFAD2-1, encoding microsomal omega-6 desaturase, were transformed into cotton. The expression of ghSAD-1 and ghFAD2-1 in the inverted-repeat orientation resulted in increased levels of stearic and oleic acids, respectively. Interestingly, the content of palmitic acid in both high-stearic and high-oleic lines was substantially reduced. These materials offer the promise of developing cotton seed oil products with greatly improved nutritional appeal to consumers.

Cottonseed Oil↗

Seed-specific expression of sesame microsomal oleic acid desaturase is controlled by combinatorial properties between negative cis-regulatory elements in the SeFAD2 promoter and enhancers in the 5'-UTR intron.

The regulation of genes involved in primary lipid metabolism in plants is much less well understood than that in many other pathways in plant biology. In the investigation reported here, we have characterized transcriptional regulatory mechanisms controlling seed-specific FAD2 expression in sesame (Sesamum indicum). FAD2 codes for extra-plastidial FAD2 desaturase, which catalyzes the conversion of oleic acid to linoleic acid. Promoter analysis of the sesame FAD2 gene (SeFAD2) using the beta-glucuronidase (GUS) reporter system demonstrated that the - 660 to - 180 promoter region functions as a negative cis-element in the seed-specific expression of the SeFAD2 gene. Sesame and Arabidopsis FAD2 genes harbor one large intron within their 5'-untranslated region. These introns conferred up to 100-fold enhancement of GUS expression in transgenic Arabidopsis tissues as compared with intron-less controls. Prerequisite cis-elements for the SeFAD2 intron-mediated enhancement of gene expression and the promoter-like activity of SeFAD2 intron were identified. SeFAD2 transcripts were induced by abscisic acid (ABA) in developing sesame seeds, and the - 660 to - 548 and - 179 to - 53 regions in the SeFAD2 promoter were implicated in ABA-responsive signaling. Theses observations indicate that an intron-mediated regulatory mechanism is involved in controlling not only the seed-specific expression of the SeFAD2 gene but also the expression of plant FAD2 genes, which are essential for the synthesis of polyunsaturated fatty acids.

5' Untranslated Regions↗

Sub-cellular immunolocalization of the glucosinolate sinigrin in seedlings of Brassica juncea.

Polyclonal rat antibodies were raised to a bovine serum albumin-sinigrin conjugate and used to immunolocalize sinigrin (2-propenylglucosinolate) in imbibed seeds and developing seedlings of Brassica juncea. (L.) Czern. Sinigrin was localized to protein bodies in aleurone-like cells but shown to be absent from myrosin cells. Double labelling techniques were used to co-localize both myrosinase (beta-thioglucoside glucohydrolase, EC 3.2.3.1) and sinigrin. Myrosin grains were labelled only with the anti-myrosinase antibody, but aleurone cells were labelled with both anti-myrosinase and anti-sinigrin antibodies. High-performance liquid chromatographic analysis of conventionally fixed and dehydrated seed tissues (4 h post imbibition in water), indicated a high proportion of sinigrin was retained in fixed tissues. Over a time course of 100 h, protein bodies within aleurone-like cells degraded, fused to form the cell vacuole and lost all myrosinase labelling but retained residual sinigrin labelling. The degradation of protein bodies corresponded to a decrease in retention of sinigrin in the fixed tissues. The results describe for the first time the co-localization of a plant enzyme and its substrate, a secondary metabolite.

Antibodies↗