Different carbon sources and their concentrations change alkaloid production and gene expression in Catharanthus roseus shoots in vitro.

To compare the effects of different carbon sources on physiological aspects, especially medicinal alkaloid biosynthesis and related gene expression in Catharantus roseus (L.) G.Don, we employed sucrose and sorbitol with two concentrations (87.64 mM, the equimolar concentration of sucrose in MS basal medium, and 150 mM) on the plant's shoots in vitro in presence of 100 µM methyl jasmonate. The production of plant alkaloids including vincristine, vinblastine, ajmalicine, vindoline and catharantine and their biosynthetic and regulatory gene expression was measured. Both treatments had incremental effects on alkaloid production, upregulated the mitogen-activated protein kinase3 (MAPK3) and a downstream responsive transcription factor, ORCA3, which resulted in elevated transcript contents of the important genes in terpenoid indol alkaloids biosynthetic pathway including peroxidase1 (PRX1), geissoschizine synthase (GS), strictosidine synthase (STR) and deacetylvindoline acetyltransferase (DAT). Defensive responses such as antioxidant enzymes (catalase, peroxidase and superoxide dismutase) activities and non-enzymatic metabolites (total phenolics, flavonoids and carotenoids) contents increased under both treatments but the effects of sorbitol were stronger. Reduced fresh weight and chlorophylls contents, increased malondialdehyde (MDA) and carotenoid contents were shown after a week under all employed treatments. It seems that replacement of sucrose with sorbitol and also, increased concentrations of both carbon sources via increasing osmotic pressure make stressful conditions for the plant especially in longer times.

Alkaloids production and antioxidant properties in Catharanthus roseus (L.) G. Don. shoots and study of alkaloid biosynthesis-related gene expression levels in response to methyl jasmonate and putrescine treatments as eco-friendly elicitors.

INTRODUCTION: This study aimed to determine the effects of methyl jasmonate (MJ) combined with putrescine as eco-friendly elicitors on secondary metabolism and gene expression of alkaloid biosynthetic pathway in Catharanthus roseus in vifro-propagated shoots. METHODS: The expression of mitogen-activated protein kinase 3 and the transcription factor, octadecanoid-responsive Catharanthus AP2-domain3, upstream of plant alkaloids' biosynthetic pathway, and of key genes in the pathway (CrPRXl, STR, DAT, and GS) are investigated as well using qRt-PCR. Antioxidant enzyme (superoxide dismutase, peroxidase, and catalase) activities and non-enzymatic antioxidants (phenolics, flavonoids, and carotenoids) contents have studied to determine the stress levels of the plant by spectrophotometer. RESULTS: Results showed increased contents of non-enzymatic antioxidants after 4-8 hr and enzymatic antioxidants activities after 24 hr. Alkaloids contents increased mostly after 1 week. The investigated signaling genes upregulated after 8 hr and biosynthetic genes after 24 hr of treatments. Combined treatments had more positive effects on gene expression levels, antioxidant responses, and secondary metabolite production than MJ individually. DISCUSSION: Increased effects of combined elicitor on genes expression may be due to cross talks between their signaling pathways. Combination of MJ and putrescine can be used as an eco-friendly elicitor for enhancing the production of economically important alkaloids in C. roseus.

Changes in medicinal alkaloids production and expression of related regulatory and biosynthetic genes in response to silver nitrate combined with methyl jasmonate in Catharanthus roseus in vitro propagated shoots.

Antihypertensive compound ajmalicine and antileukemic vincristine and vinblastine are three important terpenoid indole alkaloids produced by Catharanthus roseus (Apocynaceae). This study has been done to investigate the effects of methyl jasmonate (100 µM) and silver nitrate (50 and 100 µM) individually and simultaneously on the production of mentioned important medicinal alkaloids (vincristine, vinblastine, ajmalicine, vindoline and catharanthine) and the expression profile of related regulatory and biosynthetic genes in micropropagated shoots of C. roseus. The effects of these treatments are also investigated on non-enzymatic defensive metabolites (total phenolics, flavonoids and carotenoids) and antioxidant enzymes activities (peroxidase, EC 1.11.1.7, catalase, EC 1.11.1.6 and superoxide dismutase, EC 1.15.1.1). Changes of dry weight, quantity of lipid peroxidation, and photosynthetic pigments contents have been measured as well. The results showed increased contents of alkaloids and expression levels of investigated regulatory (Mitogen-activated protein kinase3 and Octadecanoid-responsive Catharanthus AP2-domain3) and biosynthetic (strictosidine synthase, geissoschizine synthase, deacetylvindoline acetyltransferase and peroxidase1) genes under the employed treatments. The maximum yields of these alkaloids and the highest levels of the mentioned genes expression were observed under 100 µM methyl jasmonate in combination with 100 µM of AgNO3 after seven days. The employed treatments induced increased lipid peroxidation, higher levels of enzymatic antioxidants activities and more production of non-enzymatic defensive metabolites which shows activity of plant defensive system. The results suggest that silver nitrate and methyl jasmonate signalling pathways may have cross talks and their simultaneous application make an effective combination for elicitation of medicinal alkaloids biosynthesis in C. roseus micropropagated shoots.

Assessing the tobacco-rattle-virus-based vectors system as an efficient gene silencing technique in Datura stramonium (Solanaceae).

Datura stramonium is a well-known medicinal plant, which is important for its alkaloids. There are intrinsic limitations for the natural production of alkaloids in plants; metabolic engineering methods can be effectively used to conquer these limitations. In order for this the genes involved in corresponding pathways need to be studied. Virus-Induced Gene Silencing is known as a functional genomics technique to knock-down expression of endogenous genes. In this study, we silenced phytoene desaturase as a marker gene in D. stramonium in a heterologous and homologous manner by tobacco-rattle-virus-based VIGS vectors. Recombinant TRV vector containing pds gene from D. stramonium (pTRV2-Dspds) was constructed and injected into seedlings. The plants injected with pTRV2-Dspds showed photobleaching 2 weeks after infiltration. Spectrophotometric analysis demonstrated that the amount of chlorophylls and carotenoids in leaves of the bleached plants decreased considerably compared to that of the control plants. Semi-Quantitative RT-PCR results also confirmed that the expression of pds gene in the silenced plants was significantly reduced in comparison with the control plants. The results showed that the viral vector was able to influence the levels of total alkaloid content in D. stramonium. Our results illustrated that TRV-based VIGS vectors are able to induce effective and reliable functional gene silencing in D. stramonium as an alternative tool for studying the genes of interest in this plant, such as the targeted genes in tropane alkaloid biosynthetic pathway. The present work is the first report of establishing VIGS as an efficient method for transient silencing of any gene of interest in D. stramonium.

Chromium-induced tropane alkaloid production and H6H gene expression in Atropa belladonna L. (Solanaceae) in vitro-propagated plantlets.

Hyoscyamine and scopolamine tropane alkaloids found in several solanaceous plants are anticholinergic drugs. Hyoscyamine 6ß-hydroxylase (H6H) catalyzes two consecutive oxidation reactions. The first reaction is the hydroxylation of hyoscyamine to 6ß-hydroxyhyoscyamine and the second is epoxidation of 6ß-hydroxyhyoscyamine yielding scopolamine that is the final metabolite in the tropane alkaloid biosynthetic pathway. The effects of trivalent chromium as KCr (SO4)(2) on the production of tropane alkaloids and the expression of hyoscyamine 6ß-hydroxylase gene (h6h) were studied in micro-propagated Atropa belladonna L. plantlets. The results showed that chromium treatment decreased the growth parameters (weights and lengths of the plantlets) and chlorophyll contents and increased proline contents. Moreover, semiquantitave RT-PCR analysis showed that the transcript level of H6H increased under chromium treatment. This treatment also increased hyoscyamine and scopolamine contents as shown by HPLC analysis. Changes of scopolamine contents correlate with the expression levels of h6h gene under different concentrations of chromium.

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.