Accumulation of mannitol in the cytoplasm and vacuole during the expansion of sepal cells associated with flower opening in Delphinium × belladonna cv. Bellamosum.

MAIN CONCLUSION: The role of mannitol differs from that of glucose, fructose and sucrose in sepal cell expansion associated with flower opening in Delphinium × belladonna. Sepals of Delphinium × belladonna are colored and much larger than the petals. To determine whether the role of mannitol in sepal growth associated with flower opening differs from those of ubiquitous metabolic sugars including glucose, fructose and sucrose, we investigated changes in cell number, subcellular concentrations of soluble carbohydrates, and osmotic potential in sepals during flower opening in Delphinium × belladonna cv. Bellamosum. The number of epidermal cells in the sepals did not increase from the stage when sepal pigmentation started, whereas the cell area increased during flower opening, indicating that petal growth during flower opening depends on cell expansion. Mannitol concentrations in the vacuole at three different stages were approximately 100 mM, which were much higher than the other carbohydrate concentrations, but they decreased slightly at open stage. In contrast, mannitol concentration in the cytoplasm was 56 mM at bud stage, but it increased to 104 mM at open stage. Glucose and fructose concentrations in the vacuole at open stage increased to 45 and 56 mM, respectively. Total osmotic potential in apoplast and symplast, which was partially due to soluble carbohydrates, was almost constant during flower opening. Therefore, mannitol may be acting constitutively as the main osmoticum in the vacuole where it may contribute to the maintenance of the osmotic balance between the cytoplasm and vacuole in open flowers. The role of mannitol differs from those of glucose, fructose, and sucrose in sepal cell expansion in Delphinium × belladonna.

[Pharmacognostical study of Atropa belladonna].

Based on the research of plant taxonomy and botanical investigation, microscopic characteristics of the root, stem, leaf transverse section and powder of Atropa belladonna were studied for identification of the herb. The research detailed and made clear to the description identification and microscopic characteristics of officinal parts of the herbs. The work provided reference for the identification of A. belladonna herbs and pieces of work in the future, as well as a theoretical basis for the further research, development, medicinal use and the upgrading of quality standards.

Differential production of tropane alkaloids in hairy roots and in vitro cultured two accessions of Atropa belladonna L. under nitrate treatments.

Plants are a potential source of a large number of valuable secondary metabolites. In vitro cultures are being considered as an alternative to agricultural processes for studying valuable secondary metabolites. In this way, nutritive factors are important parameters influencing the production of these compounds in plants. Effects of nitrate concentrations (KNO3) on the production of two tropane alkaloids, hyoscyamine and scopolamine, and the growth of aerial parts and roots of two in vitro propagated accessions of Atropa belladonna and hairy roots were investigated. As hairy roots cultures are able to keep a stable production of alkaloids over long periods of subculturing, they are considered as an interesting option for the study of alkaloid biosynthesis. A hairy roots culture of Atropa belladonna was established by transformation with Agrobacterium rhizogenes strain AR15834. The results of our study showed that a rise in KNO3 concentration caused a decline in hairy roots growth, and had a remarkable effect on the alkaloid content. The alkaloid concentrations obtained in the hairy roots were 3-20 times higher than that in the plants at 35 mM of KNO3. Increasing the nitrate concentration in the medium of hairy roots also improved the hyoscyamine/scopolamine ratio, while it increased the scopolamine/hyoscyamine ratio in the studied plants.

Eukaryotic genome evolution: rearrangement and coevolution of compartmentalized genetic information.

The plant cell operates with an integrated, compartmentalized genome consisting of nucleus/cytosol, plastids and mitochondria that, in its entirety, is regulated in time, quantitatively, in multicellular organisms and also in space. This genome, as do genomes of eukaryotes in general, originated in endosymbiotic events, with at least three cells, and was shaped phylogenetically by a massive and highly complex restructuring and intermixing of the genetic potentials of the symbiotic partners and by lateral gene transfer. This was accompanied by fundamental changes in expression signals in the entire system at almost all regulatory levels. The gross genome rearrangements contrast with a highly specific compartmental interplay, which becomes apparent in interspecific nuclear-plastid cybrids or hybrids. Organelle exchanges, even between closely related species, can greatly disturb the intracellular genetic balance ("hybrid bleaching"), which is indicative of compartmental coevolution and is of relevance for speciation processes. The photosynthetic machinery of plastids, which is embedded in that genetic machinery, is an appealing model to probe into genomic and organismic evolution and to develop functional molecular genomics. We have studied the reciprocal Atropa belladonna-Nicotiana tabacum cybrids, which differ markedly in their phenotypes, and found that transcriptional and post-transcriptional processes can contribute to genome/plastome incompatibility. Allopolyploidy can influence this phenomenon by providing an increased, cryptic RNA editing potential and the capacity to maintain the integrity of organelles of different taxonomic origins.

Expression of functional mammalian P450 2E1 in hairy root cultures.

P450 2E1 is an important mammalian liver enzyme known to metabolize a wide range of compounds including several common environmental pollutants. The medicinal plant, Atropa belladonna, was transformed with Agrobacterium rhizogenes containing a binary vector with rabbit P450 2E1 in either the sense or antisense orientation. The resulting "hairy roots" were isolated and grown in liquid medium. Production of P450 2E1 protein was verified in the roots containing the 2E1 gene in the sense orientation. Transgenic and control root cultures were dosed with the environmental pollutant, trichloroethylene (TCE), and were analyzed for the TCE metabolites, chloral and trichloroethanol. The root cultures expressing the mammalian P450 2E1 had increased levels of the metabolites compared to the levels in the control roots. This method represents a quick way to screen transformants for expression of foreign genes before regeneration of whole plants, and also as a possible source of foreign protein for purification.

Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only.

Atropa belladonna leaf disks were infected by a wild strain Agrobacterium rhizogenes 15834 harboring the Ri-TL-DNA and by a disarmed Agrobacterium tumefaciens strain harboring a construction with only rol ABC and npt II genes. Thirteen root lines were established and examined for their growth rate and alkaloid productivity to evaluate the possible role of rol genes in morphological differentiation and in tropane alkaloid formation. A great diversity has been observed in the growth rate of these 13 root lines. The root biomass increased up to 75 times. The total alkaloid contents were similar in the root lines obtained by infection with A. rhizogenes 15834 and A. tumefaciens rol ABC. The last ones accumulated between 4 (1.1 mg g(-1) DW) and 27 (8 mg g(-1) DW) times more alkaloids than the intact roots (0.3 mg g(-1) DW). This work has shown that the rol ABC genes were sufficient to increase tropane alkaloid production in A. belladonna hairy root cultures.

Hairy root culture in a liquid-dispersed bioreactor: characterization of spatial heterogeneity.

A liquid-dispersed reactor equipped with a vertical mesh cylinder for inoculum support was developed for culture of Atropa belladonna hairy roots. The working volume of the culture vessel was 4.4 L with an aspect ratio of 1.7. Medium was dispersed as a spray onto the top of the root bed, and the roots grew radially outward from the central mesh cylinder to the vessel wall. Significant benefits in terms of liquid drainage and reduced interstitial liquid holdup were obtained using a vertical rather than horizontal support structure for the biomass and by operating the reactor with cocurrent air and liquid flow. With root growth, a pattern of spatial heterogeneity developed in the vessel. Higher local biomass densities, lower volumes of interstitial liquid, lower sugar concentrations, and higher root atropine contents were found in the upper sections of the root bed compared with the lower sections, suggesting a greater level of metabolic activity toward the top of the reactor. Although gas-liquid oxygen transfer to the spray droplets was very rapid, there was evidence of significant oxygen limitations in the reactor. Substantial volumes of non-free-draining interstitial liquid accumulated in the root bed. Roots near the bottom of the vessel trapped up to 3-4 times their own weight in liquid, thus eliminating the advantages of improved contact with the gas phase offered by liquid-dispersed culture systems. Local nutrient and product concentrations in the non-free-draining liquid were significantly different from those in the bulk medium, indicating poor liquid mixing within the root bed. Oxygen enrichment of the gas phase improved neither growth nor atropine production, highlighting the greater importance of liquid-solid compared with gas-liquid oxygen transfer resistance. The absence of mechanical or pneumatic agitation and the tendency of the root bed to accumulate liquid and impede drainage were identified as the major limitations to reactor performance. Improved reactor operating strategies and selection or development of root lines offering minimal resistance to liquid flow and low liquid retention characteristics are possible solutions to these problems.