Tungstate, a molybdate analog inactivating nitrate reductase, deregulates the expression of the nitrate reductase structural gene

Nitrate reductase (NR, EC 1.6.6.1) from higher plants is a homodimeric enzyme carrying a molybdenum cofactor at the catalytic site. Tungsten can be substituted for molybdenum in the cofactor structure, resulting in an inactive enzyme. When nitratefed Nicotiana tabacum plants were grown on a nutrient solution in which tungstate was substituted for molybdate, NR activity in the leaves decreased to a very low level within 24 hours while NR protein accumulated progressively to a level severalfold higher than the control after 6 days. NR mRNA level in molybdate-grown plants exhibited a considerable day-night fluctuation. However, when plants were treated with tungstate, NR mRNA level remained very high. NR activity and protein increased over a 24-hour period when nitrate was added back to N-starved molybdate-grown plants. NR mRNA level increased markedly during the first 2 hours and then decreased. In the presence of tungstate, however, the induction of NR activity by nitrate was totally abolished while high levels of NR protein and mRNA were both induced, and the high level of NR mRNA was maintained over a 10-hour period. These results suggest that the substitution of tungsten for molybdenum in NR complex leads to an overexpression of the NR structural gene. Possible mechanisms involved in this deregulation are discussed.     

In Vivo Inhibition of Seed Development and Reserve Protein Accumulation in Recombinants of Abscisic Acid Biosynthesis and Responsiveness Mutants in Arabidopsis thaliana

In Arabidopsis thaliana, seed development in recombinants of the ABA-deficient aba mutant with the ABA response mutants abi1 or abi3 is compared to wild type and the monogenic parents. Aberrant seed development occurred in the aba,abi3 recombinant and was normal in aba,abi1, abi3 and aba,abi1 seeds. Embryos of the recombinant aba,abi3 seeds maintained the green color until maturity, the seeds kept a high water content, did not form the late abundant 2S and 12S storage proteins, were desiccation intolerant, and often showed viviparous germination. Application of ABA, and particularly of an ABA analog, to the roots of plants during seed development partially alleviated the aberrant phenotype. Seeds of aba,abi3 were normal when they developed on a mother plant heterozygous for Aba. In contrast to seed development, the induction of dormancy was blocked in all monogenic mutants and recombinants. Dormancy was only induced by embryonic ABA; it could not be increased by maternal ABA or ABA applied to the mother plant. It is concluded that endogenous ABA has at least two different effects in developing seeds. The nature of these responses and of the ABA response system is discussed.     

Transformation of Brassica napus and Brassica oleracea Using Agrobacterium tumefaciens and the Expression of the bar and neo Genes in the Transgenic Plants

An efficient and largely genotype-independent transformation method for Brassica napus and Brassica oleracea was established based on neo or bar as selectable marker genes. Hypocotyl explants of Brassica napus and Brassica oleracea cultivars were infected with Agrobacterium strains containing chimeric neo and bar genes. The use of AgNO₃ was a prerequisite for efficient shoot regeneration under selective conditions. Vitrification was avoided by decreasing the water potential of the medium, by decreasing the relative humidity in the tissue culture vessel, and by lowering the cytokinin concentration. In this way, rooted transformed shoots were obtained with a 30% efficiency in 9 to 12 weeks. Southern blottings and genetic analysis of S1-progeny showed that the transformants contained on average between one and three copies of the chimeric genes. A wide range of expression levels of the chimeric genes was observed among independent transformants. Up to 25% of the transformants showed no detectable phosphinotricin acetyltransferase or neomycin phosphotransferase II enzyme activities although Southern blottings demonstrated that these plants were indeed transformed.     

Red light-induced accumulation of ubiquitin-phytochrome conjugates in both monocots and dicots

Phytochrome is rapidly degraded in vivo after photoconversion from the stable red-absorbing (Pr) form to the far red-absorbing (Pfr) form. Previously, we have shown in etiolated oat seedlings that ubiquitin-phytochrome conjugates (Ub-P) appear after Pfr formation suggesting that oat phytochrome is rapidly degraded by a ubiquitin-dependent proteolytic pathway. Here, we extend this observation to etiolated tissue from other monocotyledonous (corn [Zea mays. (L.)] and rye [Secale cereale (L.)] and dicotyledonous species (pea [Pisum sativum (L,)] and zucchini squash [Cucurbita pepo (L.)]). Following Pfr formation by red light, all four species synthesized a heterogeneous series of Ub-P that appeared and disappeared concomitant with the degradation of the chromoprotein. When Pfr was photoconverted back to Pr by a far-red light pulse, degradation of phytochrome ceased and the levels of Ub-P concomitantly dropped. In pea and zucchini squash, loss of Ub-P after photoconversion of Pfr back to Pr was rapid, occurring with a half-life of approximately 5 to 10 minutes. These data indicate that the accumulation of Ub-P after Pfr formation is a general phenomenon in etiolated seedlings of higher plants and further support the hypothesis that plants degrade Pfr via Ub-P intermediates.     

Biosynthesis of the Cyanogenic Glucoside Dhurrin in Seedlings of Sorghum bicolor (L.) Moench and Partial Purification of the Enzyme System Involved

The cyanogenic glucoside dhurrin is rapidly synthesized in etiolated seedlings of Sorghum bicolor (L.) Moench. The dhurrin content of the seedlings increases sigmoidally with the germination time. Shoots of 10 centimeters height contain 850 nanomoles of dhurrin per shoot corresponding to 6% of the dry weight. The biosynthetic activity sharply rises upon germination and reaches a maximum level of 10 nanomoles dhurrin/(hour × shoot) after 48 hours when the shoots are 3 centimeters high. This maximum level is followed by a sharp decline in activity when germination time exceeds 65 hours. Dhurrin and the dhurrin-synthesizing enzyme system are primarily located in the upper part of the etiolated shoot where both are evenly distributed between the coleoptile, the primary leaves and the upper 0.5 centimeter of the first internode including the shoot apex. Dhurrin constitutes 30% of the dry weight of the upper 1.2 centimeter of 10 centimeter high shoots. The seed and root contain neither dhurrin nor the dhurrin-synthesizing enzyme system. The codistribution of dhurrin and the enzyme system throughout the seedling indicates that production and storage sites are located within the same cell. Purification of the dhurrin-synthesizing enzyme by gel filtration or by sucrose gradient centrifugations results in a tenfold increase in specific activity. Further purification is accompained by a decline in specific activity due to loss of essential components as demonstrated by reconstitution experiments.     

Biochemical and Histochemical Localization of Monoterpene Biosynthesis in the Glandular Trichomes of Spearmint (Mentha spicata)

The primary monoterpene accumulated in the glandular trichomes of spearmint (Mentha spicata) is the ketone (−)-carvone which is formed by cyclization of the C₁₀ isoprenoid intermediate geranyl pyrophosphate to the olefin (−)-limonene, hydroxylation to (−)-trans-carveol and subsequent dehydrogenation. Selective extraction of the contents of the glandular trichomes indicated that essentially all of the cyclase and hydroxylase activities resided in these structures, whereas only about 30% of the carveol dehydrogenase was located here with the remainder located in the rest of the leaf. This distribution of carveol dehydrogenase activity was confirmed by histochemical methods. Electrophoretic analysis of the partially purified carveol dehydrogenase from extracts of both the glands and the leaves following gland removal indicated the presence of a unique carveol dehydrogenase species in the glandular trichomes, suggesting that the other dehydrogenase found throughout the leaf probably utilizes carveol only as an adventitious substrate. These results demonstrate that carvone biosynthesis takes place exclusively in the glandular trichomes in which this natural product accumulates.     

The development of epidermal feet in preparation for metamorphosis in an insect

Insect epidermal cell surfaces can be seen by scanning electron microscopy after removal of the basal lamina. This let us study surface changes in the 5th larval stage of Calpodes ethlius (Lepidoptera, Hesperiidae) in preparation for metamorphosis at the end of the stadium, in particular changes in the basal cell processes or feet, intercellular lymph spaces, filopodia and hemidesmosomes. The feet develop in three phases, initiation, elongation and contraction. Initial growth begins immediately after ecdysis and continues until commitment to pupation 66 hr later. During this phase the feet are randomly oriented. Elongation and orientation begin after commitment to pupation. Orientation is probably achieved by selective survival and growth of those feet that are axially oriented rather than by reorientation. As the larva shortens to the pupal form late in the stadium, contraction of the feet occurs and the cells become columnar. The feet finally disappear as the cells rearrange themselves into new positions in the pupal epidermis. The lateral margins of the feet are united by adhesions even when their interdigitations are most complex. The adhesions separate an intercellular lymph space from the haemolymph. The lymph space remains small through most of the stadium, but enlarges with the loss of lateral junctions as the feet contract and eventually extends along most of the length of the columnar cells. Filopodia then form and span the gaps between the cells as though they have been induced by the separation and loss of lateral cell to cell contact. Scanning electron microscopy also shows that hemidesmosomes reflect the axial alignment of the cells even before the orientation of the feet. The hemidesmosome plaques are linear structures having a constant width of 0.15 - 0.2 mum and variable length. They arise in short sections and lengthen by the linear addition of more sections with the same width. Late in the stadium they lose their axial alignment and may become branched.     

Ultrastructure of the embryonic dorsal organ of Orchestia cavimana (crustacea, amphipoda); with a note on localization of chloride and on the change in calcium-deposition before the embryonic moult

The transitory dorsal organ of Orchestia cavimana appears simultaneously with the development of the germ layer and is gradually reduced during the last 2-3 days of embryonic development. It represents the only direct connection of the embryo with the chorion or-after the embryonic moult-with the embryonic envelope. The shape is hemispherical and it consists of about 50 bottle-shaped cells, arranged radially around a centre. This centre is filled with different kinds of extracellular material which forms a central plug apically and a central cone below it. The bottle-shaped cells taper apically. The neck region of these cells is characterized by numerous microvilli which project into the intercellular space. This space is filled with an electron dense substance and is in contact with the central cone. In the basal neck region numerous mitochondria are associated with the microvilli. The high density of mitochondria is characteristic for the nuclear region. The cytoplasm of the basal region below the nucleus contains numerous calcium granules. Evidence for the concentration of chloride in the apical dorsal organ is shown. Before the embryonic moult and during the duplication of the egg-volume the number of calcium granules in the dorsal organ and the integument is reduced. Simultaneously calcium granules appear in the now visible periembryonic space. This suggests that part of the calcium is shifted into this space. The function of the dorsal organ is discussed. Besides the probable main function-transport activity of ions-its role before and during embryonic moult and its part in the utilisation of yolk are discussed.     

Production of bovine identical twins via transfer of demi-embryos without zonae pellucidae

Embryos were collected nonsurgically from n?turally-cycling or superovulated donors 7 d after estrus. Forty-four morulae, early blastocysts and blastocysts classified as good to excellent were bisected using a fine glass needle to produce forty-four identical demi-embryos. The bisected demi-embryos, without zonae pellucidae, were nonsurgically transferred, either by twin or single transfer. An additional forty-eight embryos were collected from the same donors and transferred as a control. Among the twin transfers, 8 of the 13 recipients became pregnant (61.5%). Seven of them conceived twin fetuses (87%) and one a single fetus. However, only two sets of normal identical twin calves were obtained. Among the single transfers, 72.6% (45/62) of bisected embryos without zonae pellucidae resulted in pregnancy, of which 48.4% (30/62) were identical twins, and 24.2% (15/62) were singletons. Another 27.4% (17/62) of the recipients did not became pregnant. The pregnancy rate for whole embryos with zonae pellucidae was 72.9% (35/48). These data show that there was no significant difference between the pregnancy rates of bisected embryos without zonae pellucidae and whole embryos with zonae pellucidae transferred 7 d after estrus. Bisection of bovine embryos was simplified and even morula stage embryos were transferred without zonae pellucidae.     

Beyond abortion: refusal of caesarean section

Social pressures and legal restrictions are proliferating against pregnant women. A dramatic infringement on women's rights is the court ordered cesarean section, as illustrated by the case of Angela C., a terminally ill cancer patient, 26 weeks pregnant, whose refusal of a cesarean section was overridden by a District of Columbia court. The premature infant and the mother died within two days. This case epitomizes a developing judicial pattern whose ethical reasoning the author criticizes. Within the context of the right to privacy and the concept of viability, which could legally override that right, Mahowald analyzes different situations where cesarean delivery is refused. After arguing that court ordered cesarean sections are inconsistent with court refusals to force persons to undergo less invasive procedures (e.g., bone marrow donation for the benefit of family members), she proposes alternatives to the present inconsistent practice.