The interaction of soil micromycetes with "hot" particles in a model system]

A model system which permits observing for a long time and fixing interaction of fungi with a radiation source has been created on the basis of an isolated "hot" particle, deficient mineral medium (saccharose content 60 mg/l) and suspension of fungal conidia. Five species (six strains) of micromycetes isolated from radionuclide-contaminated soils and fifteen "hot" particles have been tested. It has been found out for the first time that Cladosporium cladosporioides and Penicillium roseo-purpureum are able actively overgrow "hot" particles whose radioactivity did not exceed 3.1-1.0(-7) Ci by gamma-spectrum and to destroy them 50-150 days later. Certain changes in morphology of fungi-destructors of "hot" particles are revealed. A problem on ecological significance of the found phenomenon is discussed.     

Cell-type specific,coordinate expression of two ADP-glucose pyrophosphorylase genes in relation to starch biosynthesis during seed development of Vicia faba L.

Several cDNA clones encoding two different ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27) polypeptides denoted VfAGPC and VfAGPP were isolated from a cotyledonary library of Vicia faba L. Both sequences are closely related to AGPase small-subunit sequences from other plants. Whereas mRNA levels of VfAGPP were equally high in developing cotyledons and leaves, the mRNA of VfAGPC was present in considerable amounts only in cotyledons. During development of cotyledons, both mRNAs accumulated until the beginning of the desiccation phase and disappeared afterwards. The increase of AGPase activity in cotyledons during the phase of storage-product synthesis was closely followed by the accumulation of starch. The AGPase activity in crude extracts of cotyledons was insensitive to 3-phosphoglycerate whereas the activity from leaves could be activated more than five-fold. Inorganic phosphate inhibited the enzyme from both tissues but was slightly more effective on the leaf enzyme. There was a correlation at the cellular level between the distribution of VfAGPP and VfAGPC mRNAs and the accumulation of starch, as studied by in-situ hybridisation and by histochemical staining in parallel tissue sections of developing seeds, respectively. During the early phase of seed development (12–15 days after fertilization) VfAGPase mRNA and accumulation of starch were detected transiently in the hypodermal, chlorenchymal and outer parenchymal cell layers of the seed coat but not in the embryo. At 25 days after fertilization both synthesis of VfAGPase mRNA and biosynthesis of starch had started in parenchyma cells of the inner adaxial zone of the cotyledons. During later stages, the expression of VfAGPase and synthesis of starch extended over most of the cotyledons but were absent from peripheral cells of the abaxial zone, provascular and procalyptral cells.Abbreviations AGPase ADP-glucose pyrophosphorylase - DAF days after fertilization - Glc1P glucose-1-phosphate - 3-PGA 3-phosphoglycerate - VfAGPC AGPase subunit of Vicia faba mainly expressed in cotyledons - VfAGPP AGPase subunit of Vicia faba mainly expressed in leaves and cotyledons - pVfAGPC, pVfAGPP plasmids containing VfAGPC and VfAGPP, respectively This work was supported by the Bundesministerium für Forschung und Technologie BCT 0389, Molekular- und Zellbiologie von höheren Pflanzen und Pilzen. U.W acknowledges additional support by the Fonds der chemischen Industrie. We thank Elsa Fessel for excellent technical assistance.     

Seed Architecture Shapes Embryo Metabolism in Oilseed Rape

Constrained to develop within the seed, the plant embryo must adapt its shape and size to fit the space available. Here, we demonstrate how this adjustment shapes metabolism of photosynthetic embryo. Noninvasive NMR-based imaging of the developing oilseed rape (Brassica napus) seed illustrates that, following embryo bending, gradients in lipid concentration became established. These were correlated with the local photosynthetic electron transport rate and the accumulation of storage products. Experimentally induced changes in embryo morphology and/or light supply altered these gradients and were accompanied by alterations in both proteome and metabolome. Tissue-specific metabolic models predicted that the outer cotyledon and hypocotyl/radicle generate the bulk of plastidic reductant/ATP via photosynthesis, while the inner cotyledon, being enclosed by the outer cotyledon, is forced to grow essentially heterotrophically. Under field-relevant high-light conditions, major contribution of the ribulose-1,5-bisphosphate carboxylase/oxygenase–bypass to seed storage metabolism is predicted for the outer cotyledon and the hypocotyl/radicle only. Differences between in vitro– versus in planta–grown embryos suggest that metabolic heterogeneity of embryo is not observable by in vitro approaches. We conclude that in vivo metabolic fluxes are locally regulated and connected to seed architecture, driving the embryo toward an efficient use of available light and space.     

High-resolution histographical mapping of glucose concentrations in developing cotyledons of Vicia faba in relation to mitotic activity and storage processes: glucose as a possible developmental trigger

Previous studies provided evidence that the carbohydrate status triggers developmental processes in the growing cotyledons of Vicia faba . We describe here the high-resolution mapping of glucose concentrations in tissue sections of developing faba bean cotyledons by quantitative bioluminescence and single-photon imaging. Patterns of local glucose distributions are compared with tissue cell type, mitotic index and the distribution pattern of starch. During cotyledon differentiation, gradients in the glucose concentration emerge which are related to the particular cell type. Higher concentrations are found in non-differentiated premature regions of the cotyledon whereas mature starch-accumulating regions contain particularly low concentrations of glucose. In addition, glucose concentration is correlated to mitotic activity. The glucose distribution pattern is therefore related to the developmental gradient. Our data provide for the first time evidence for steep glucose gradients across developing plant embryos and favour the idea that sugar gradients may have morphogenic functions in developing cotyledons.     

Temperature-dependent endogenous oxygen concentration regulates microsomal oleate desaturase in developing sunflower seeds

Oleoyl-phosphatidylcholine desaturase (FAD2) is a key enzyme involved in fatty acid desaturation in oilseeds, which is affected by environmental temperature. The results of this study show that FAD2 is regulated in vivo via temperature-dependent endogenous oxygen concentrations in developing sunflower (Helianthus annuus L.) seeds. By combining in vivo oxygen profiling, in situ hybridization of FAD2 genes, an assay of energy status, fatty acid analysis, and an in vitro FAD2 enzyme activity assay, it is shown that: (i) the oil-storing embryo is characterized by a very low oxygen level that is developmentally regulated. Oxygen supply is mainly limited by the thin seed coat. (ii) Elevations of external oxygen supply raised the energy status of seed and produced a dramatic increase of the FAD2 enzyme activity as well as the linoleic acid content. (iii) A clear negative correlation exists between temperature and internal oxygen concentration. The changes occurred almost instantly and the effect was fully reversible. The results indicate that the internal oxygen level acts as a key regulator for the activity of the FAD2 enzyme. It is concluded that a major mechanism by which temperature modifies the unsaturation degree of the sunflower oil is through its effect on dissolved oxygen levels in the developing seed.     

Antisense inhibition of the plastidial glucose-6-phosphate/phosphate translocator in Vicia seeds shifts cellular differentiation and promotes protein storage

The glucose-6-phosphate/phosphate translocator (GPT) acts as an importer of carbon into the plastid. Despite the potential importance of GPT for storage in crop seeds, its regulatory role in biosynthetic pathways that are active during seed development is poorly understood. We have isolated GPT1 from Vicia narbonensis and studied its role in seed development using a transgenic approach based on the seed-specific legumin promoter LeB4. GPT1 is highly expressed in vegetative sink tissues, flowers and young seeds. In the embryo, localized upregulation of GPT1 at the onset of storage coincides with the onset of starch accumulation. Embryos of transgenic plants expressing antisense GPT1 showed a significant reduction (up to 55%) in the specific transport rate of glucose-6-phosphate as determined using proteoliposomes prepared from embryos. Furthermore, amyloplasts developed later and were smaller in size, while the expression of genes encoding plastid-specific translocators and proteins involved in starch biosynthesis was decreased. Metabolite analysis and stable isotope labelling demonstrated that starch biosynthesis was also reduced, although storage protein biosynthesis increased. This metabolic shift was characterized by upregulation of genes related to nitrogen uptake and protein storage, morphological variation of the protein-storing vacuoles, and a crude protein content of mature seeds of transgenics that was up to 30% higher than in wild-type. These findings provide evidence that (1) the prevailing level of GPT1 abundance/activity is rate-limiting for the synthesis of starch in developing seeds, (2) GPT1 exerts a controlling function on assimilate partitioning into storage protein, and (3) GPT1 is essential for the differentiation of embryonic plastids and seed maturation.     

Ectopic expression of an amino acid transporter (VfAAP1) in seeds of Vicia narbonensis and pea increases storage proteins

Storage protein synthesis is dependent on available nitrogen in the seed, which may be controlled by amino acid import via specific transporters. To analyze their rate-limiting role for seed protein synthesis, a Vicia faba amino acid permease, VfAAP1, has been ectopically expressed in pea (Pisum sativum) and Vicia narbonensis seeds under the control of the legumin B4 promoter. In mature seeds, starch is unchanged but total nitrogen is 10% to 25% higher, which affects mainly globulin, vicilin, and legumin, rather than albumin synthesis. Transgenic seeds in vitro take up more [14C]-glutamine, indicating increased sink strength for amino acids. In addition, more [14C] is partitioned into proteins. Levels of total free amino acids in growing seeds are unchanged but with a shift toward higher relative abundance of asparagine, aspartate, glutamine, and glutamate. Hexoses are decreased, whereas metabolites of glycolysis and the tricarboxylic acid cycle are unchanged or slightly lower. Phosphoenolpyruvate carboxylase activity and the phosphoenolpyruvate carboxylase-to-pyruvate kinase ratios are higher in seeds of one and three lines, indicating increased anaplerotic fluxes. Increases of individual seed size by 20% to 30% and of vegetative biomass indicate growth responses probably due to improved nitrogen status. However, seed yield per plant was not altered. Root application of [15N] ammonia results in significantly higher label in transgenic seeds, as well as in stems and pods, and indicates stimulation of nitrogen root uptake. In summary, VfAAP1 expression increases seed sink strength for nitrogen, improves plant nitrogen status, and leads to higher seed protein. We conclude that seed protein synthesis is nitrogen limited and that seed uptake activity for nitrogen is rate limiting for storage protein synthesis.