Enhanced h6h transcript level,antioxidant activity and tropane alkaloid production in Hyoscyamus reticulatus L. hairy roots elicited by acetylsalicylic acid

Hyoscyamine (Hyos) and scopolamine (SCP) are drugs widely used as antimuscarinic to treat diseases such as Parkinson’s or to calm schizoid patients. In this study, with the aim of enhancing tropane alkaloid production in H. reticulatus hairy root cultures, the effects of the signalling molecule acetylsalicylic acid (ASA) were investigated at different concentrations (0, 0.01, 0.1 and 1 mM) and inoculation times (24 and 48 h). As well as reducing biomass production, ASA treatment significantly enhanced the activity of catalase, guaiacol peroxidase and ascorbate peroxidase (p < 0.01), which was highest at 48 h of exposure to 1 mM of ASA. The highest accumulation of Hyos and SCP (1.6- and 3.5-fold more than in the control, respectively) was obtained at 24 h of exposure to 0.1 mM ASA. Additionally, semi-quantitative RT-PCR analysis showed an increased expression of the hyoscyamine-6-beta-hydroxylase (h6h) gene, involved in the last Hyos and SCP biosynthetic step, which correlated with the enhanced level of Hyos and SCP production under ASA elicitation. Our findings suggest that ASA, by stimulating the expression of key biosynthetic genes and enzymes, can be applied to increase commercial tropane alkaloid production in a H. reticulatus hairy root system.     

Characteristics of growth and tropane alkaloid production in Hyoscyamus albus hairy roots transformed with Agrobacterium rhizogenes A4

Summary Hairy root culture of Hyoscyamus albus was established by transformation with Agrobacterium rhizogenes strain A4. The growth and production of five tropane alkaloids were investigated under various culture conditions. Among the four basal culture media tested, Woody Plant medium was the best for growth of the hairy roots, but a high amount of tropane alkaloids was obtained with Gamborg's B5 medium. Sucrose concentration in B5 medium had little effect on the growth, while 3% sucrose was suitable for the alkaloid production. Addition of KNO₃ to Woody Plant medium affected the growth, whereas the alkaloid content was not markedly improved. Supplement of some metal ions to B5 medium stimulated the alkaloid production. In particular, Cu²⁺ remarkably enhanced both the growth and the alkaloid yield. The hairy roots cultured under 16 h/day light survived for more than 32 days compared with those cultured in the dark.Abbreviations EDTA ethylenediaminetetraacetic acid - HPLC high performance liquid chromatography - MeOH methanol - MS medium Murashige and Skoog medium - WP medium McCown's Woody Plant medium - B5 medium Gamborg B5 medium - wt weight     

Optimization of induction and culture conditions and tropane alkaloid production in hairy roots of <Emphasis Type="Italic">Anisodus acutangulus</Emphasis>

In this study, an efficient transformation system for the medicinal plant Anisodus acutangulus was successfully developed and optimized using Agrobacterium rhizogenes. Three bacterial strains, A4, R1601, and modified C58C1 and three explant types, leaf blade, petiole, and stem, were examined. The highest transformation efficiency of 94.44% was achieved using strain C58C1 with stem explants. Over 20 independent hairy root lines were successfully established with strain C58C1 using stem explants, all of which contained the ro/B and ro/C genes as confirmed by polymerase chain reaction (PCR). Out of four media compositions, the liquid 1/2 MS medium was found the most suitable for hairy root growth. The maximum biomass of one hairy root line increased up to 80 times in liquid 1/2 MS medium after a 30 day culture period. Different hairy root lines displayed a varied capacity for tropane alkaloid production and the best hairy root line (T4) from the C58C1-stem combination produced up to 10.21 mg/g (dw) of hyoscyamine, which was about 1.5-fold higher than in the wild type plants. To our knowledge, this is the first report to demonstrate the production of tropane alkaloids in hairy roots of A. acutangulus.     

Influence of exogenous hormones on growth and secondary metabolite production in hairy root cultures of Cichorium intybus L. CV. Lucknow local

Summary The effect of exogenously fed hormones on hairy root cultures of Cichorium intybus L. ev. Lucknow Local was studied. It was seen that auxin in the presence of low levels of kinetin induces rapid disorganization in hairy root cultures of C. intybus, ultimately to form suspension cultures, and this process was associated with the decrease in coumarin content in the cells. Of various treatments, it was observed that with an increase in the auxin: cytokinin ratio, the biomass decreased with the increase in disorganization index during the culture period of 28 d. The disorganization index was less when the inoculum size was enhanced to 10-fold. The total endogenous indole-3-acetic acid titers and indole-3-acetic acid oxidase activity also decreased with an increase in disorganization index, and was independent of initial inoculum size, with only a magnitude difference. The total coumarin content strictly correlated with growth in all the treatments. In contrast, exogenously supplied gibberellic acid at the 0.5 mg l⁻¹ level enhanced growth, coumarin content, and branching patterns over the control and other treatments on day 28. The exogenously fed growth regulators had an effect on growth, auxin and coumarin biosyntheses, wherein transformed roots treated with increasing concentration of auxin to cytokinin ratios lost their ability for coumarin biosynthesis. The behavior of hairy roots from an Indian cultivar of chicory upon growth regulator treatment is discussed in terms of growth, coumarin and auxin biosyntheses.     

Intermittent lead‐induced stress on antioxidant enzyme activity and subcellular distribution of Pb in Pogonatherum crinitum seedlings

• Pogonatherum crinitum is a promising lead (Pb) hyperaccumulator due to its high Pb tolerance and accumulation ability. However, the mechanisms that support Pb accumulation and tolerance in P. crinitum are not yet clearly understood.
• An indoor hydroponic experiment was conducted by cultivating P. crinitum seedlings exposed to intermittent Pb stress for 60 days, divided into four stages (T1, T2, T3 and T4), with a 15‐day duration per stage. The following concentrations of Pb were used: 0, 500, 0, 500 mg·l^?1 and 0, 1000, 0, 1000 mg·l^?1). Antioxidant enzyme activity, Pb concentration and subcellular distribution of Pb were measured at each of the above stages.
• The results showed that superoxide dismutase (SOD) activity in shoots, and SOD, peroxidase (POD) and malondialdehyde (MDA) activity in shoots and roots significantly increased from T1 (no Pb stress) to T2 (Pb stress) in both 500 mg·l^?1 and 1000 mg·l^?1 treatments; however, no significant difference was noted between stages T3 (no Pb stress) and T4 (Pb stress). There was no obvious effect of Pb stress on catalase (CAT) activity in shoots and roots among different stages. The Pb concentration in shoots was up to 5090.90 mg·kg^?1 and 7573.57 mg·kg^?1, and the bioconcentration factor (BFC) was 10.18 and 7.57 for the 500 mg·l^?1 and 1000 mg·l^?1 treatments, respectively, which confirmed the Pb hyperaccumulator characteristics of P. crinitum. For plants under Pb stress, most of the Pb was fixed in the cell walls, with a smaller amount in leaves and root vacuoles.
• Both SOD and POD scavenging of reactive oxygen radicals and fixing and compartmentalisation of Pb in the cell wall might play important roles in detoxification of P. crinitum seedlings in response to Pb stress. There was no phased response of P. crinitum to intermittent Pb stress and the physiological response to Pb stress may be contiguous.

     

Changes in antioxidant enzyme activity in the fine roots of black poplar (Populus nigra L.) and cottonwood (Populus deltoides Bartr. ex Marsch) in a heavy-metal-polluted environment

The effect of heavy metal deposition onto soil from a copper smelter on lipid peroxidation and antioxidant enzyme activity in the fine roots of two poplars (Populus nigra L. and Populus deltoides Bartr. ex Marsch) was analyzed. The subjects were mature trees growing in real environments. In both analyzed species, heavy metals stimulated the overproduction of free radicals in fine roots (measured as malondialdehyde level), which was directly proportional to advancing senescence. In young fine roots, heavy metals caused a decrease in guaiacol peroxidase activity and presumably disturbed the lignification process. Catalase was highly sensitive to the presence of heavy metals in the soil. In contrast, ascorbate peroxidase and glutathione reductase activities were unaffected by heavy metals. In the case of superoxide dismutase, a clear increase in enzyme activity was observed only in P. nigra under drought conditions, whereas it was inhibited in polluted stands.     

Interactive effects of salicylic acid and silicon on oxidative damage and antioxidant activity in spinach (<Emphasis Type="Italic">Spinacia oleracea</Emphasis> L. cv. Matador) grown under boron toxicity and salinity

We investigated individual and combined effects of salinity, soil boron (B), silicon (Si) and salicylic acid (SA) on the activities of major antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT and ascorbate peroxidase, APX) and non-enzymatic antioxidants (AA), proline, chlorophyll, anthocyanin, H₂O₂ concentration, stomatal resistance (SR), lipid peroxidation (MDA), membrane permeability (MP), and the uptake of sodium (Na), chloride (Cl), boron and Si of spinach plants. In general, salinity significantly increased H₂O₂ and proline concentrations, antioxidant activity, membrane permeability, lipid peroxidation and SR of the spinach plants, indicating that they were stressed, whereas application of B only increased proline concentration. However, plant fresh weights did not decline with either treatment. The application of Si decreased H₂O₂ and increased the activity of SOD and CAT. The application of SA increased SOD activity. Neither SA nor Si had any effect on the proline concentration, or MP. However, application of Si increased chlorophyll concentration and decreased lipid peroxidation (MDA concentration). Si treatment had no effect on SR. The concentration of B in the tissues, which was strongly increased by B treatment, was decreased by NaCl. As a result of salinity, concentrations of Na⁺ and Cl⁻ ions were increased in the plant tissues, and application of Si slightly increased these concentrations. These results indicate that exogenous Si application increases stress tolerance of spinach, a plant that is naturally reasonably resistant to combined salinity and B toxicity, by the enhancement of antioxidant mechanisms that reduce membrane damage. Exogenous SA has a less obvious effect, although the levels of salinity and boron stress applied were not sufficient in this experiment to reduce plant fresh weight.     

Synergistic effect of auxins and polyamines in hairy roots of <Emphasis Type="Italic">Cichorium intybus</Emphasis> L. during growth,coumarin production and morphogenesis

Hairy roots of Cichorium intybus obtained by infecting with different Agrobacterium rhizogenes strains (LMG-150 and A20/83) were studied for total endogenous indole-3-acetic acid (IAA) levels and indole-3-acetic acid oxidase (IAAO) activity. The roots initiated by LMG- 150 showed higher endogenous IAA levels as well as IAAO activity as compared to the roots from A20/83. Coumarin production in roots obtained by both of these strains strictly correlated with growth, with higher content in the roots obtained by LMG- 150. Moreover roots from LMG-150 showed increased growth index, length of primary roots and number of secondary and tertiary roots. The roots derived from LMG-150 were studied for total endogenous IAA and IAAO activity under the exogenous administration of polyamines and fungal elicitors. The treatment with putrescine (Put) at 1.5 mM level showed maximum endogenous IAA levels and IAAO activity as compared to the control and other polyamine administration, it also supported faster growth in terms of biomass accumulation, and total coumarin production. Of the various treatments, mycelial extract (ME) and culture media filtrate (CMF) of Pythium aphanidermatum and Phytopthora parasitica var. nicotiana, the treatment with 1 % CMF of P. parasitica var. nicotiana, resulted in maximum IAA levels and IAAO activity, which was supported by maximum biomass, coumarin production as compared to the control and other elicitor treatments. Two different regenerants of chicory obtained through A. rhizogenes LMG-150 designated as T-I and T-II, were studied for total endogenous IAA levels and IAAO activity. T-II showed higher titers of IAA with higher activity of IAAO as compared to T-I. Endogenous titer of IAA and IAAO activity was found to be maximum in transformed roots as compared to T-I, T-II, normal roots and normal plants. Our work showed a variation in endogenous auxin levels in these transformed plants. There exists a synergistic effect of endogenous IAA titers and polyamines in regulating root morphogenesis. Fungal elicitors influenced growth and coumarin production and an elicitor preparation of 1 % CMF of P. parasitica var. nicotiana gave spontaneous regeneration of shoots. The implications of these results are discussed.     

The effects of copper, iron and zinc on digestive enzyme activity in the hybrid tilapia Oreochromis niloticus (L.) ×Oreochromis aureus (Steindachner)

The present experiment was conducted to study effects of Cu, Fe and Zn on activities of digestive enzymes of the hybrid tilapia Oreochromis niloticus×Oreochromis aureus. The acidic protease activities increased 65·5 and 55·1% by addition of homogenates of digesta‐containing stomach with copper (75 mg l⁻¹) and zinc (50 mg l⁻¹) respectively. Addition of Cu and Zn increased the activities of protease in the hepatopancreas homogenates by 132·7 and 38·1% respectively, and reduced the activity of protease in the digesta‐containing intestine homogenates by 11·0 and 13·8% respectively. Addition of Fe (50 mg l⁻¹) increased the acidic protease activity by 96·7% but did not alter the activities of protease in the intestine and hepatopancreas. Addition of Cu markedly inhibited activities of amylase in intestine and hepatopancreas homogenates, while Zn addition showed no effects. Addition of Fe reduced activities of amylase in the intestine homogenates by 47·9% but had no effect on amylase activities in the hepatopancreas. When Cu (75 mg kg⁻¹), Fe (50 mg kg⁻¹) and Zn (50 mg kg⁻¹) were supplemented to basal diet for 3 weeks, the activities of amylase in hepatopancreas homogenates increased 125·3, 215·6 and 70·0%, respectively, the activities of amylase in intestine increased 79·8, 74·6 and 48·5%, respectively, and the activities of lipase in intestine increased 90·5, 149·8 and 84·0%, respectively. Supplementation of Cu, Fe or Zn into diet had no effects on activity of protease in all digestive organs. Therefore, the results suggest that effects of Cu, Fe and Zn on activity of digestive enzymes in vitro were different from those seen in vivo, and that the positive effects of Cu, Fe and Zn supplemented to fish diet would be valuable information for formulating fish feed.     

镉及其与钙组合对褐脉少花龙葵毛状根生长、抗氧化酶活性和吸收镉的影响

为了探讨利用褐脉少花龙葵毛状根来修复重金属镉(Cd)污染的可能性,采用溶液培养法研究了Cd单独及其与钙(Ca)组合对褐脉少花龙葵毛状根生长、抗氧化酶超氧化物歧化酶(SOD)和过氧化物酶(POD)活性及对Cd吸收的影响。结果表明,Cd≤50μmol/L时能促进毛状根生长,而高于100μmol/LCd则抑制毛状根生长,使其侧根根尖变褐和变短,数目减少。与对照相比,不同浓度Cd培养的毛状根可溶性蛋白含量和SOD活性先升高后逐渐下降;其丙二醛(MDA)含量显著提高;100μmol/LCd使毛状根POD活性逐渐升高,但300μmol/LCd则使毛状根POD活性逐渐降低。与对照(仅添加100μmol/L或300μmol/LCd的毛状根)相比,Cd和10～30mmol/LCaCl2组合培养使毛状根可溶性蛋白含量和MDA含量降低;但提高其SOD活性;而100μmol/LCd和10～30mmol/LCaCl2结合培养的毛状根POD活性均比对照低;而300μmol/LCd和10～30mmol/LCaCl2结合培养的毛状根POD活性则均比对照提高。原子吸收分光光度法测定结果表明,毛状根吸收和吸附的重金属Cd含量随着培养基中Cd浓度的升高而增加。但外源加入10～30mmol/LCaCl2能减少毛状根对Cd的吸收,并调节其抗氧化酶SOD和POD活性,降低其膜脂过氧化水平而解除重金属Cd对毛状根生长的抑制或毒害。