Isolation of the 3ß-HSD promoter from Digitalis ferruginea subsp. ferruginea and its functional characterization in Arabidopsis thaliana.

BACKGROUND: Although members of the SDR gene family (short chain dehydrogenase) are distributed in kingdom of life, they have diverse roles in stress tolerance mechanism or secondary metabolite biosynthesis. Nevertheless, their precise roles in gene expression or regulation under stress are yet to be understood. METHODS: As a case study, we isolated, sequenced and functionally characterized the 3ß-HSD promoter from Digitalis ferruginea subsp. ferruginea in Arabidopsis thaliana. RESULTS: The promoter fragment contained light and stress response elements such as Box-4, G-Box, TCT-motif, LAMP element, ABRE, ARE, WUN-motif, MYB, MYC, W box, STRE and Box S. The functional analysis of the 3ß-HSD promoter in transgenic Arabidopsis seedlings showed that the promoter was expressed in cotyledon and root elongation zone in 2 days' seedlings. However, this expression was extended to hypocotyl and complete root in 6 days' seedlings. In 20 days-old seedlings, promoter expression was distributed to the whole seedling including hydathodes aperture, vascular bundle, shoot apical meristem, trichomes, midrib, leaf primordia, hypocotyl and xylem tissues. Further, expression of the promoter was enhanced or remained stable under the different abiotic stress conditions like osmotic, heat, cold, cadmium or low pH. In addition, the promoter also showed response to methyl jasmonate (MeJA) application. The expression could not be induced in wounded cotyledon most likely due to lack of interacting elements in the promoter fragment. CONCLUSIONS: Taken together, the 3ß-HSD promoter could be a candidate for the development of transgenic plants especially under changing environmental conditions.

Plastidial Expression of 3ß-Hydroxysteroid Dehydrogenase and Progesterone 5ß-Reductase Genes Confer Enhanced Salt Tolerance in Tobacco.

The short-chain dehydrogenase/reductase (SDR) gene family is widely distributed in all kingdoms of life. The SDR genes, 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and progesterone 5-ß-reductases (P5ßR1, P5ßR2) play a crucial role in cardenolide biosynthesis pathway in the Digitalis species. However, their role in plant stress, especially in salinity stress management, remains unexplored. In the present study, transplastomic tobacco plants were developed by inserting the 3ß-HSD, P5ßR1 and P5ßR2 genes. The integration of transgenes in plastomes, copy number and transgene expression at transcript and protein level in transplastomic plants were confirmed by PCR, end-to-end PCR, qRT-PCR and Western blot analysis, respectively. Subcellular localization analysis showed that 3ß-HSD and P5ßR1 are cytoplasmic, and P5ßR2 is tonoplast-localized. Transplastomic lines showed enhanced growth in terms of biomass and chlorophyll content compared to wild type (WT) under 300 mM salt stress. Under salt stress, transplastomic lines remained greener without negative impact on shoot or root growth compared to the WT. The salt-tolerant transplastomic lines exhibited enhanced levels of a series of metabolites (sucrose, glutamate, glutamine and proline) under control and NaCl stress. Furthermore, a lower Na+/K+ ratio in transplastomic lines was also observed. The salt tolerance, mediated by plastidial expression of the 3ß-HSD, P5ßR1 and P5ßR2 genes, could be due to the involvement in the upregulation of nitrogen assimilation, osmolytes as well as lower Na+/K+ ratio. Taken together, the plastid-based expression of the SDR genes leading to enhanced salt tolerance, which opens a window for developing saline-tolerant plants via plastid genetic engineering.

Effects of cytokinins, gibberellic acid 3, and gibberellic acid 4/7 on in vitro growth, morphological traits, and content of steviol glycosides in Stevia rebaudiana.

Steviol glycosides (SGs) and gibberellic acids share a part of their biosynthesis pathways. Despite the widespread studies on the effect of gibberellic acid 3 (GA3), the effect of gibberellic acid 4 and 7 (GA4/7) on Stevia rebaudiana has never been studied. This study aimed at a comparative evaluation of different hormone effects, i.e., 1 mg L-1 GA4/7, 1 mg L-1 GA3, or 0.5 mg L-1 kinetin and 0.5 mg L-1 6-benzylaminopurine (BAP) (KB 0.5), on in vitro propagation, growth, morphological properties, and content of SGs in leaf samples of stevia. In comparison with the control group (hormone-free), the treatments of KB 0.5 or GA3 produced the highest biomasses and largest leaf areas. The three hormonal treatments produced a similar number of leaves, the ratio of fresh to dry weight, and leaf length. GA4/7-treated explants produced the highest ratio of leaf area to leaf length. The effect of GA4/7 on shoot elongation was greater than that of the control or even GA3. While the effect of GA3 on rebaudioside-A (Reb-A) production was similar to that of the control (16.2 and 18.04 mg g-1, respectively), GA4/7 resulted in a lower amount of it (13.31 mg g-1). Except for GA4/7, which induced more stevioside accumulation, the treatments' effects were comparable to that of the control. The ratio of stevioside to Reb-A was the highest for GA4/7 (2.62), followed by GA3 (1.93), and then the two others. Sum of Reb-A and stevioside content was not changed by the use of any of the treatments.

An Overview of Cardenolides in Digitalis - More Than a Cardiotonic Compound.

The genus Digitalis L. containing species, commonly known as the "foxglove", is the main source of cardenolides, which have various pharmacological properties effective against certain pathological conditions including myocardial infarction, arterial hypertension, cardiac dysfunction, angina, and hypertrophy. Togehter with a prime effect of controlling the heart rhythm, many workers demonstrated that lanatoside C and some other cardiac glycosides are effective in several cancer treatments such as prostate and breast cancers. Due to digoxigenin derivatives of cardenolides, which are mainly used for medicinal purposes, such as digoxigenin, D. lanata as a main source is of great interest for commercial scale production of cardenolides in Europe. Phytochemical studies on cardenolides, naturally occurring plant secondary metabolites, have mainly focused on the species of the genus Digitalis L., as the members of this family have a high level and diverse content of cardenolides. During the last few decades, plant tissue culture techniques have been optimised for many plant species including Digitalis, however, the production capacity of cardenolides somehow failed to reach a commercially desired extent. In this review paper, the genus Digitalis is evaluated in terms of its main botanical and physiological features, traditional uses, molecular genetics and metabolomics, cellular mechanism of action, medicinal uses, clinical pharmacology, drug interactions, therapy in the management of cardiovascular disorders, potential utility of therapy in extracardiac conditions, and toxicity.

Effect of zinc oxide (ZnO) nanoparticles on physiology and steviol glycosides production in micropropagated shoots of Stevia rebaudiana Bertoni.

This study aims to address the effects of different concentrations (0, 0.1, 1.0, 10, 100 or 1000 mg L-1) of engineered zinc oxide (ZnO) nanoparticles (34 nm in size) on growth parameters, steviol glycosides (rebaudioside A and stevioside) production and antioxidant activities in the tissue culture grown shoots of Stevia rebaudiana Bertoni. The highest percentage of shoot formation (89.6%) at 1 mg L-1 of ZnO nanoparticles concentration suggests a positive influence of ZnO nanoparticles on S. rebaudiana growth as compared to other treatments with or without ZnO nanoparticles. Additionally, HPLC results illustrate a significant enhancement of steviol glycosides (almost doubled as compared to the control) in micropropagated shoots grown under an oxidative stress of 1 mg L-1 of ZnO nanoparticles. This finding is further affirmed by an increased 2,2-diphenyl-1-picryl hydrazyl (DPPH) scavenging activity, total anti-oxidant capacity, total reducing power, total flavonoid content and total phenolic content, with an ascending oxidative pressure and generation of reactive oxygen species (ROS). However, the antioxidant activities, formation of secondary metabolites and the physiological parameters showed a sudden decline after crossing a threshold of 1 mg L-1 concentration of ZnO nanoparticles and falls to a minimum at 1000 mg L-1, elucidating maximum phytotoxic effect of ZnO nanoparticles at this concentration. This is the first study evaluating both the favorable and adverse effects of ZnO nanoparticles employed to a highly valuable medicinal plant, S. rebaudiana.

Evaluation of biochemical markers during somatic embryogenesis in Silybum marianum L.

In present report effects of explants type, basal media and plant growth regulators (PGRs) were tested for induction of indirect somatic embryogenesis in medicinally important plant Silybum marianum L. Leaf, petiole and root explants were exploited in vitro on B5 (Gamborg), SH (Schenk and Hildebrandt) and MS (Murashige and Skoog) media for induction of embryogenic callus followed by somatic embryogenesis. Highest callus induction frequency (76 ± 4.8 %) was recorded when petiole explants of in vitro derived plantlets were cultured on B5 medium supplemented with 1.5 mg l-1 2,4-dichlorophenoxyacetic acid (2,4_D) in combination with 1.5 mg l-1 Thidiazuron (TDZ). Induction and multiplication of somatic embryos were observed, when the embryogenic calluses were sub-cultured on to B5 medium containing 0.5 mg l-1 2,4-D plus 1.5 mg l-1 TDZ. At this PGRs treatment, 77 % of the cultures responded with 39.1 somatic embryos per callus. Furthermore, MS0 medium was indicated more reponsive for growth and maturation of somatic embryos. Analysis of biochemical markers during various growth phases in somatic embryogenesis revealed that somatic embryos exhibited highest level of total carbohydrate, starch, ascorbic acid and total free amino acids. However, higher protein levels were detected in non-embryogenic callus. Nevertheless, considerable amount of silymarin (4.1 mg g-1 DW) was detected in somatic embryos than other growth phases. Thus, the present study concluded that biochemical and physiological changes during embryogenesis are influenced by interplay of explants type, basal media and PGRs.