Seasonal cardenolide production and Dop5betar gene expression in natural populations of Digitalis obscura

Productivity variations and seasonal fluctuations of cardenolides have been studied in 10 natural populations of Digitalis obscura distributed in three bioclimatic belts. Main cardenolides in D. obscura plants are those of the series A and such predominance (ca. 80-85%) over the series B metabolites is independent of the population studied or the degree of maturity of the leaves. Primary glycosides represent ca. 50-60% of total cardenolides; this percentage did not vary among populations or with the leaf age but increased in summer and decreased in winter. A correlation analysis between plant biomass and cardenolide content showed a positive relationship of these parameters, which, according to the bioclimatic distribution of the populations, suggests that certain environmental conditions may cause marked decreases in plant biomass together with a reduction in productivity. Cardenolide contents changed in the timecourse of the four seasons as a multiple response to distinct plant and/or environmental factors. The lowest production was recorded in May, followed by a fast cardenolide accumulation in summer, a decreasing phase in autumn, and a stationary phase in winter. We also analysed the seasonal expression of the gene encoding the progesterone 5beta-reductase, enzyme producing the required 5beta-configured intermediaries of cardenolides. A fragment of the isolated partial genomic sequence was used as a probe for Northern analysis to study the seasonal gene expression in selected populations. The expression pattern showed increasing levels from February to July and a further reduction in autumn, although harmful climatic conditions seems to induce overexpression of this gene.     

Photosynthetic acclimation of higher plants to growth in fluctuating light environments

This paper describes a study into the potential of plants to acclimate to light environments that fluctuate over time periods between 15 min and 3 h. Plants of Arabidopsis thaliana (L.) Heynh., Digitalis purpurea L. and Silene dioica (L.) Clairv. were grown at an irradiance 100 mol m^-2 s^-1. After 4–6 weeks, they were transferred to light regimes that fluctuated between 100 and either 475 or 810 mol m^-2 s^-1, in a regular cycle, for 7 days. Plants were shown, in most cases, to be able to undergo photosynthetic acclimation under such conditions, increasing maximum photosynthetic rate. The extent of acclimation varied between species. A more detailed study with S. dioica showed that this acclimation involved changes in both Rubisco protein and cytochrome f content, with only marginal changes in pigment content and composition. Acclimation to fluctuating light, at the protein level, did not fully reflect the acclimation to continuous high light - Rubisco protein increased more than would be expected from the mean irradiance, but less than expected from the high irradiance; cytochrome f increased when neither the mean nor the high irradiance would be expected to induce an increase.This revised version was published online in October 2005 with corrections to the Cover Date.     

Gene Control in Somatic Embryos of Digitalis lanata: Expression of the β-Glucuronidase Gene Fused to a Plastocyanin Promoter

Digitalis lanata was transformed by agrobacteria-mediated gene transfer with a chimeric reporter gene encoding for β-glucuronidase (CUS) from Escherichia coll under the control of the plastocyanin 3 (Pc3) promoter from Spinada oleracea (Pc3::uidA fusion gene). Transformed cell lines were regenerated to plants via somatic embryos. CUS activity was determined fluorometrically and histochemically. The Pc3::uidA fusion gene was expressed in the late globular and bipolar stages of somatic embryos. Expression started in globular embryos (stage-1-globules) in that part of the parenchymatic tissue which later on formed the cotyledons. No GUS activity was detectable in the parenchymatic tissue forming the root pole, in cells of the developing procambium or in epidermal cells. These tissues were free of GUS activity also in bipolar embryos. The parenchymatic cells of the cotyledons and the primary cortex of the hypocotyl of germinating embryos showed GUS activity, in contrast to the epidermal cells and the cells of the central cylinder.     

The digitalis-like steroid hormones: new mechanisms of action and biological significance

Digitalis-like compounds (DLC) are a family of steroid hormones synthesized in and released from the adrenal gland. DLC, the structure of which resembles that of plant cardiac glycosides, bind to and inhibit the activity of the ubiquitous cell surface enzyme Na(+), K(+)-ATPase. However, there is a large body of evidence suggesting that the regulation of ion transport by Na(+), K(+)-ATPase is not the only physiological role of DLC. The binding of DLC to Na(+), K(+)-ATPase induces the activation of various signal transduction cascades that activate changes in intracellular Ca(++) homeostasis, and in specific gene expression. These, in turn, stimulate endocytosis and affect cell growth and proliferation. At the systemic level, DLC were shown to be involved in the regulation of major physiological parameters including water and salt homeostasis, cardiac contractility and rhythm, systemic blood pressure and behavior. Furthermore, the DLC system has been implicated in several pathological conditions, including cardiac arrhythmias, hypertension, cancer and depressive disorders. This review evaluates the evidence for the different aspects of DLC action and delineates open questions in the field.     

Expression of recombinant Digitalis lanata EHRH. cardenolide 16′-O-glucohydrolase in Cucumis sativus L. hairy roots

The coding sequence for the Digitalis lanata EHRH. cardenolide 16′-O-glucohydrolase was inserted downstream of the 35S promoter in the binary vector pBI121 resulting in plant expression vector pBI121cgh. Cotyledon explants excised from 10-day-old seedlings of Cucumis sativus L. were transformed using Agrobacterium rhizogenes 15834 harbouring pBI121cgh. Hairy roots were obtained from infected cotyledon explants in vitro 10 days after inoculation. PCR amplification of coding sequences for cgh I, rolB and rolC from Ri plasmid showed that the aimed sequences were inserted into the genome of transformed cucumber hairy roots. Glycolytic activity of the transgenic CGH I was measured by HPLC using Lanatoside glycosides as substrate. Therefore, the cgh I transformed cucumber hairy roots may provide a valuable model for biotransformation of natural compounds by recombinant enzymes.This report is dedicated to Prof. W. Roos on the occasion of his 60th birthday.     

Dual activity of the H1-H2 domain of the (Na(+)+K+)-ATPase

(Na⁺+K⁺)-ATPase is a target receptor of digitalis (cardiac glycoside) drugs. It has been demonstrated that the H1-H2 domain of the α-subunit of the (Na⁺+K⁺)-ATPase is one of the digitalis drug interaction sites of the enzyme. Despite the extensive studies of the inhibitory effect of digitalis on the (Na⁺+K⁺)-ATPase, the functional property of the H1-H2 domain of the enzyme and its role in regulating enzyme activity is not completely understood. Here we report a surprise finding: instead of inhibiting the enzyme, binding of a specific monoclonal antibody SSA78 to the H1-H2 domain of the (Na⁺+K⁺)-ATPase elevates the catalytic activity of the enzyme. In the presence of low concentration of ouabain, monoclonal antibody SSA78 significantly protects enzyme function against ouabain-induced inhibition. However, higher concentration of ouabain completely inactivates the (Na⁺+K⁺)-ATPase even in the presence of SSA78. These results suggest that the H1-H2 domain of the (Na⁺+K⁺)-ATPase is capable of regulating enzyme function in two distinct ways for both ouabain-sensitive and -resistant forms of the enzyme: it increases the activity of the (Na⁺+K⁺)-ATPase during its interaction with an activator; it also participates in the mechanism of digitalis or ouabain-induced inhibition of the enzyme. Understanding the dual activity of the H1-H2 domain will help better understand the structure-function relationships of the (Na⁺+K⁺)-ATPase and the biological processes mediated by the enzyme.     

Seed bank spatial pattern in a temperate secondary forest

Abstract. Seed bank spatial pattern was studied in a secondary forest dominated by Fagus sylvatica and Betula celtiberica in the Urkiola Natural Park (N Spain). Soil samples were taken every 2 m in a regular grid (196 points) and divided into two fractions (0–3 cm and 3–10 cm deep). The viable seed bank was studied by monitoring seedling emergence for ten months. The effect of different factors on seed bank composition and patterning was analysed using constrained ordination as a hypothesis testing tool. Furthermore, the existence of spatial autocorrelation was evaluated by geostatistical analysis. Seed density was high, 7057 seed.m^?2, with a few species dominating. Species composition in the various layers were significantly correlated. The seed bank showed significant spatial structure, which was partially explainable by the spatial structure of the canopy and understorey vegetation. Spatial clumping from 0–8 m was observed in seed bank density and composition, mainly due to the pattern of two abundant taxa Juncus effusus and Ericaceae. The Ericaceae seed bank was related to the spatial distribution of dead stumps of Erica arborea. J. effusus was not present in the above‐ground vegetation, which indicates that its seed bank was formed in the past. As expected, the seed bank of this forest reflects its history, which is characterized by complex man‐induced perturbations. The seed bank appears to be structured as a consequence of contrasting driving forces such as canopy structure, understorey composition and structural and microhabitat features.     

Effect of calcium restriction on cardenolide accumulation in two cell lines of Digitalis thapsi grown under different light regimes

The effect of calcium-deprivation on growth and the production of cardenolides in two undifferentiated cell lines of Digitalis thapsi maintained under three different light regimes (16 h photoperiod, darkness, or continuous light) was investigated. Growth was stimulated by continuous light in both cell lines cultured in complete medium. The light regime did not affect cardenolide accumulation in the cells of the hypocotyl-derived line; by contrast, continuous light or darkness increased the production in the leaf-derived line. The elimination of calcium favoured cardenolide production independently of the origin of the suspensions and the light regime, this beneficial effect being predominantly manifested under continuous light.     

An assessment of genetic relationships within the genus Digitalis based on PCR-generated RAPD markers

RAPD markers were used to study inter-specific variation among six species of the genus Digitalis: D. obscura, D. lanata, D. grandiflora, D. purpurea, D. thapsi and D. dubia, and the hybrid D. excelsior (D. purpurea×D. grandiflora). A total of 91 highly reproducible bands amplified with four arbitrarily chosen decamer primers were obtained. Homology of the co-emigrating RAPD markers was tested by blot hybridisation and sequencing of selected bands. The application of a range of statistical approaches for RAPD data analysis, including distance and parsimony methods, family clustering and the analysis of molecular variance (AMOVA), indicated that these molecular markers were taxonomically informative in Digitalis. The species relationships revealed were fully consistent with those previously obtained using morphological affinities. The hybrid D. excelsior seems to have stronger affinity to the section Digitalis than to Grandiflorae. This is the first known report of the application of RAPD markers for the study of genetic relationships among species of the genus Digitalis. Received: 20 October 1999 / Accepted: 9 November 1999     

Factors affecting shoot proliferation and vitrification inDigitalis obscura cultures

Summary Variations of composition and consistency of the culture medium and time of exposure to growth regulators were assayed to optimize normal caulogenic response ofDigitalis obscura hypocotyls cultured in vitro. The effects of the culture conditions on physiologic changes related to vitrification of the regenerated plants were also investigated. Liquid medium increased the bud-forming capacity of the explants but induced buds failed to develop into shoots and showed symptoms of vitrification. On agar-solidified media, maximum multiplication rates were achieved with 0.7% agar. Increasing agar concentration reduced vitrification but lowered the propagation rate. Changes in the strength of the macronutrients of Murashige and Skoog did not significantly affect the bud-forming capacity of the explants. In contrast, a drastic inhibitory effect on both bud formation and shoot elongation was produced when NH₄NO₃ was omitted. Reduction of NH₄NO₃ to one-half or one-fourth of the level of the original formulation not only increased the bud-forming capacity ofD. obscura hypocotyls but also resulted in less vitrification. Modifications of time and method of exposure to growth regulators neither improved the multiplication rates nor overcame vitrification. Cardenolide content was lower in vitrified than in normal cultures and coincided with an overall reduction of photosynthetic pigments, lignin, and dry matter.