Role of the non-mevalonate pathway in indole alkaloid production by Catharanthus roseus hairy roots

The 1-deoxy-D-xylulose-5-phosphate (DXP) pathway (non-mevalonate pathway) leading to terpenoids via isopentenyl diphosphate (IPP) has been shown to occur in most bacteria and in all higher plants. Treatment with the antibiotic fosmidomycin, a specific inhibitor of DXP reductoisomerase, considerably inhibited the accumulation of the alkaloids ajmalicine, tabersonine, and lochnericine by Catharanthus roseus hairy root cultures in the exponential growth phase. However, fosmidomycin did not significantly affect alkaloid levels in stationary phase hairy root cultures. Feeding with 1-deoxy-D-xylulose, 10-hydroxygeraniol, or loganin resulted in significant increases in alkaloid production by exponential phase hairy root cultures. These results suggest that the DXP pathway is a major provider of carbon for the monoterpenoid pathway leading to the formation of indole alkaloids in C. roseus hairy roots in the exponential phase.     

Effect of the engineered indole pathway on accumulation of phenolic compounds in Catharanthus roseus hairy roots

Catharanthus roseus has been well-known to contain indole alkaloids effective for treatment of diverse cancers. We examined the intracellular accumulation profiles of phenolic compounds in response to ectopic overexpression of tryptophan feedback-resistant anthranilate synthase holoenzyme (ASalphabeta) in C. roseus hairy roots. Among 13 phenolic compounds measured, 6 phenolic compounds were detected in late exponential phase ASalphabeta hairy roots. Uninduced and induced ASalphabeta hairy roots accumulated up to 1.2 and 4.5 mg/g DW over a 72-h period, respectively. Upon induction, in parallel with a rapid increase in tryptophan in the first 48 h, accumulation of phenolic compounds tended to increase to a maximum level (4.5 mg/g DW) at 48 h, after which phenolic levels decreased back to the uninduced level by 72 h. Naringin was a predominant form that comprised about 72% and 36% of the total content of phenolic compounds in the uninduced and induced lines, respectively. Upon induction, accumulation of catechin drastically increased with the highest level (3.6 mg/g) occurring at 48 h, whereas that of all others except for salicylic acid showed no statistical difference. Catechin is a final product of the flavonoid pathway, and thus metabolic flux into this pathway is transiently increased by overexpression of AS. Like catechin, salicylic acid is very sensitive to induction as it began to increase to 5-fold within 4 h of induction, but unlike catechin, no significant accumulation of salicylic acid was noted after 4 h of induction. The results suggest differential regulation of this particular biosynthesis branch within the phenolic pathway.     

Influence of fungal elicitors on production of ajmalicine by cell cultures of Catharanthus roseus

Suspension cultures of Catharanthus roseus (C. roseus) were elicited with fungal cell wall fragments of Aspergillus niger (A. niger), Fusarium moniliforme (F. moniliforme), and Trichoderma viride (T. viride). The effects of elicitor dosage, exposures time, and age of subculture on ajmalicine accumulation were studied. A higher concentration of elicitor extract responded positively to C. roseus suspension cultures. Ajmalicine accumulation increased by about 3-fold when cells were treated with A. niger, F.moniliforme, and T. viride. The maximum ajmalicine production (75 microg g(-1) dry weight (DW)) was observed in cells treated with T. viride. Cell cultures were elicited with 5% preparation of A. niger, F. moniliforme, and T. viride and exposed for 24, 48, 72, and 96 h. for elicitation. Suspension cultures elicited with T. viride for 48 h showed a 3-fold increase (87 microg g(-1) DW) in ajmalicine contents, whereas A. niger and F. moniliforme synthesized a 2-fold increase in alkaloid and yielded 52 and 56 microg g(-1) DW ajmalicine, respectively. C. roseus cells of different age (5,10, 15, 20, and 25 days old) were treated with a 5% elicitor of A. niger, F. moniliforme, and T. viride and investigated elicitors activity at different age of cell cultures. Maximum yield 166 microg g(-1) DW of ajmalicine was synthesized in 20 day old suspension cultures treated with T. viride. A longer period of incubation of cell cultures with elicitors adversely affected the ajmalicine synthesis.     

Economic assessment of plant cell culture for the production of ajmalicine

Cost estimates have been prepared for commercial-scale production of ajmalicine-rich Catnaranthus roseus biomass using plant cell culture. At the current state of the technology the cost would be approximately $7.30/lb dry biomass ($3215/kg ajmalicine). Naturally-grown C. roseus roots have a 50% lower ajmalicine concentration but would cost only ca. $0.70/lb ($619/kg ajmalicine). The principal reason for the high cost of the plant cell culture route is not the slow specific growth rate (0.35 day(-1)), but rather the slow specific product accumulation rate (0.26 mg/g day). This rate will have to be increased by a factor of 40 to make the process competitive.     

Differential Secretion and Accumulation of Terpene Indole Alkaloids in Hairy Roots of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> Treated with Methyl Jasmonate

The induction of several secondary metabolites in plants is one of the most commonly observed effects after the external addition of methyl jasmonate (MeJA). After the elicitation of Catharanthus roseus hairy roots with different concentrations of MeJA, changes in the accumulation of alkaloids such as ajmalicine, serpentine, ajmaline and catharanthine were observed. In addition to the increased accumulation of alkaloids in the tissues, the root exudation of phytochemicals increased compared to that of the non-treated control hairy roots. Moreover, MeJA induced differential secretion of several C. roseus hairy root metabolites.     

Expression of the Arabidopsis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy roots

In plants, the indole pathway provides precursors for a variety of secondary metabolites. In Catharanthus roseus, a decarboxylated derivative of tryptophan, tryptamine, is a building block for the biosynthesis of terpenoid indole alkaloids. Previously, we manipulated the indole pathway by introducing an Arabidopsis feedback-insensitive anthranilate synthase (AS) alpha subunit (trp5) cDNA and C. roseus tryptophan decarboxylase gene (TDC) under the control of a glucocorticoid-inducible promoter into C. roseus hairy roots [Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004a. Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels. Biotechnol. Bioeng. 86, 718-727; Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004b. Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine. Metabol. Eng. 6, 268-276]. Inducible expression of either or both transgenes did not lead to significant increases in overall alkaloid levels despite the considerable accumulation of tryptophan and tryptamine. In an attempt to more successfully engineer the indole pathway, a wild type Arabidopsis ASbeta subunit (ASB1) cDNA was constitutively expressed along with the inducible expression of trp5 and TDC in C. roseus hairy roots. Transgenic hairy roots expressing both trp5 and ASB1 show a significantly greater resistance to feedback inhibition of AS activity by tryptophan than plants expressing only trp5. In fact, a 4.5-fold higher concentration of tryptophan is required to achieve 50% inhibition of AS activity in plants overexpressing both genes than in plants expressing only trp5. In addition, upon a 3 day induction during the exponential phase, a trp5:ASB1 hairy root line produced 1.8 times more tryptophan (specific yield ca. 3.0 mg g(-1) dry weight) than the trp5 hairy root line. Concurrently, tryptamine levels increase up to 9-fold in the induced trp5:ASB1 line (specific yield ca. 1.9 mg g(-1) dry weight) as compared with only a 4-fold tryptamine increase in the induced trp5 line (specific yield ca. 0.3 mg g(-1) dry weight). However, endogenous TDC activities of both trp5:ASB1 and trp5 lines remain unchanged irrespective of induction. When TDC is ectopically expressed together with trp5 and ASB1, the induced trp5:ASB1:TDC hairy root line accumulates tryptamine up to 14-fold higher than the uninduced line. In parallel with the remarkable accumulation of tryptamine upon induction, alkaloid accumulation levels were significantly changed depending on the duration and dosage of induction.     

Overexpression of a Catharanthus tryptophan decarboxylase (tdc) gene leads to enhanced terpenoid indole alkaloid (TIA) production in transgenic hairy root lines of Rauwolfia serpentina

To enhance the production of terpenoid indole alkaloids in Rauwolfia serpentina, Catharanthus tryptophan decarboxylase (Crtdc) gene was over-expressed in transgenic hairy root cultures using Agrobacterium rhizogenes-mediated transformation. Among six transgenic hairy root lines, line RT4 accumulated the highest alkaloid content, with 0.1202 % dry weight (DW) reserpine and 0.0064 % DW ajmalicine, after 10 weeks of culture. Whereas, wild-type roots accumulated 0.0596 ± 0.003 % DW reserpine and 0.0011 ± 0.001 % DW ajmalicine. Transgenic hairy root line RT7 produced the lowest alkaloid content (reserpine: 0.0896 ± 0.002 % DW; ajmalicine: 0.002 ± 0.0 % DW). On the basis of alkaloid content the six hairy root lines were grouped as RT4/RT2 > RT3/RT5 > RT7/RT8. Analysis of gene expression profile indicated that Crtdc was expressed at a higher level in transgenic lines, which could be correlated with enhanced metabolite accumulation in roots. This study confirms that over-expression of Crtdc is a superlative method to improve the biosynthetic potential of Rauwolfia hairy root cultures. Enhanced reserpine and ajmalicine production can serve as an alternative choice to provide resources for relative pharmaceutical industries.     

Transient studies of light-adapted cultures of hairy roots of Catharanthus roseus: growth and indole alkaloid accumulation

Cultures of C. roseus transgenic ("hairy") root clones LBE-6-1 and LBE-4-2 were adapted with periodic daily illumination to investigate the effect of light on growth and nutrient utilization, and the accumulation of the indole alkaloids. Light-adapted roots appeared green and had radially thickened morphology compared with dark-grown controls. Their growth rates were higher than dark-grown controls, with 45% lower doubling times: LBE-6-1, 3.6 days; LBE-4-2, 2.8 days. Relative to dark-grown controls, light-adapted growth increased the biomass (DW) of LBE-6-1 by 25%, but had no effect on the DW of LBE-4-2. The macronutrients NH4+, NO3-, Pi, and sugars, were depleted completely by light-adapted root cultures in that order. The specific and total levels of the indole alkaloid serpentine was enhanced and of tabersonine was lowered in both root clones, while the overall trends of growth and non-growth association of tabersonine and serpentine, respectively, remained unaltered by light adaptation. Ajmalicine accumulation was enhanced in LBE-6-1, but lowered in LBE-4-2; its accumulation was growth-associated in dark-grown LBE-6-1, but appeared non-growth associated in light-adapted cultures. The accumulation of tabersonine-related compounds, lochnericine, and h?rhammericine exhibited growth-associated trends, and were either negatively affected or unaffected by light adaptation of LBE-6-1. Neither vindoline nor its precursor, deacetylvindoline, was detected.     

建立金线莲原球茎悬浮体系生产次生代谢产物

研究植物激素浓度和培养周期对金线莲原球茎悬浮培养生长及其代谢产物积累的影响,以增加金线莲悬浮培养的生长量,提高次生代谢产物的生产。结果表明,MS培养基添加S-3307 1.0mg/L,6-BA0.5mg/L和3%的蔗糖适合总生物量的生长(214.45g/L,FW和18.23g/L DW)。而MS培养基添加S-3307 1.0mg/L,6-BA 3.0mg/L和5%的蔗糖,总黄酮,总酚和多糖的干重(5.43mg/g,2.87mg/g和243.23mg/g)达到最大化。研究原球茎悬浮培养过程,发现经过7个星期培养就能获得最大的生物质总量(225.98 g/L的FW和18.53 g/L的DW)、总黄酮干重(5.09mg/g)和总酚干重(2.04mg/g),而多糖生产达到其峰值(229.36mg/g干重)是在培养后5个星期。     

蔗糖和光对三裂叶野葛毛状根生长及次生物质产生的影响

研究了蔗糖浓度和光对固体培养的三裂叶野葛毛状根生长及其总异黄酮和葛根素产生的影响。结果表明：在供试的分别添加1%、3%、5%、7%和9%蔗糖的MS固体培养基中,3%蔗糖能促进三裂叶野葛毛状根的生长及其异黄酮类化合物和葛根素的积累;培养20d后,其生物量达到0.48g(DW,干重)/瓶,总异黄酮和葛根素含量分别为25.44mg/g(DW)和11.64mg/g(DW)。与添加3%蔗糖的MS培养基培养的三裂叶野葛毛状根相比,含5%蔗糖的培养基培养的毛状根干重增殖倍数提高了7.0％,而含1％、7％和9％蔗糖的培养基培养的毛状根干重增殖倍数分别下降62.4％、42.8％和65.3％;其总异黄酮含量分别降低574％、13％和33.4％,葛根素含量分别下降47.9％、15.8％和35.1％,但其毛状根培养物的可溶性糖含量则分别增加了0.52、1.45和1.54倍。暗培养30d的毛状根的生物量达到0.83g(DW)/瓶,分别比蓝光和白光培养的毛状根提高37.1%和23.3%。在蓝光和白光下培养的部分毛状根的表面呈淡绿色;但白光处理的毛状根中总异黄酮含量比蓝光和暗培养处理的分别提高了14.7%和19.2%;蓝光抑制毛状根中葛根素含量的积累,白光和暗培养的毛状根培养物中的葛根素含量分别是蓝光处理的1.61倍和1.52倍。     

Nitric oxide potentiates oligosaccharide-induced artemisinin production in Artemisia annua hairy roots

The purpose of the present study was to characterize the generation of nitric oxide （NO） in Artemisia annua roots induced by an oligosaccharide elicitor （OE） from Fusarium oxysporum mycelium and the potentiation role of NO in the elicitation of artemisinin accumulation. The OE （0.3 mg total sugar/mL） induced a rapid production of NO in cultures, which exhibited a biphasic time course, reaching the first plateau within 1.5 h and the second within 8 h of OE treatment. Artemisinin content in 20-day-old hairy roots was increased from 0.7mg/g dry wt to 1.3 mg/g dry wt by using the OE treatment for 4d. In the absence of OE, the NO donor sodium nitroprusside （SNP） at 10, 50 ~1 and 100 ~1 enhanced the growth of hairy roots, but had no effect on artemisinin synthesis, The combination of SNP with OE increased artemisinin content from 1.2 mg/g drywt to 2.2 mg/g dry wt, whereas the maximum production of artemisinin in cultures was 28.5 mg/L, a twofold increase over the OE treatment alone. The effects of SNP on the OE-induced artemisinin were suppressed strongly by the NO scavenger 2-（4- carboxyphenyl）-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide （cPTIO）. The results suggest that NO can strongly potentiate elicitor-induced artemisinin synthesis in A. annua hairy roots.     

Effect of plant growth regulators and elicitors on rhinacanthin accumulation in hairy root cultures of Rhinacanthus nasutus (L.) Kurz

Several biologically active secondary metabolites like anthraquinones, sterols, triterpenes, flavonoids and naphthoquinones are present in Rhinacanthus nasutus. Naphthoquinones are important group of compounds generally known as rhinacanthin (RC) consists of 15 derivatives named RC A–D and G–Q of which RC-C, RC-D and RC-N have various medicinal properties. The individual role of two auxins i.e. indole-3-butyric acid (IBA) and α naphthalene acetic acid (NAA) and two elicitors i.e. methyl jasmonate (MJ) and salicylic acid (SA) in Murashige and Skoog medium on hairy root growth and RC (RC-C, RC-D and RC-N) accumulation was investigated in the present study. Time course study revealed that IBA and NAA at 2.5 μM showed maximum fresh weight (FW) and dry weight (DW) 4 weeks after culture. However, RC production was maximum after 6 weeks of culture on both media. A concentration-dependent response was observed when various concentrations of MJ (2.0, 5.0, 10 and 15 μM) and SA (10, 50, 100 and 150 μM) were supplemented in the medium. On MJ and SA media the FW and DW decreased as the concentration of elicitors increased. However, this decrease was more severe in MJ treated cultures. All the MJ and SA treated cultures showed significantly higher amount of RC-C, RC-D and RC-N in hairy roots harvested 7 days after elicitation as compared to control. Of the two elicitors, MJ was more efficient in inducing RC accumulation than SA. The highest RC content (6.3 mg/g DW RC-C; 1.1 mg/g DW RC-D and 0.61 mg/g DW RC-N) was observed after treatment with 10 μM MJ which was about 1.7-, 2.5- and 3.5-fold higher RC-C, RC-D and RC-N respectively than the control.     

Effects of Agrobacterium rhizogenes strains and other parameters on production of isoflavonoids in hairy roots of Pueraria candollei Grah. ex Benth. var. candollei

Using several explants of Pueraria candollei Grah. ex Benth. var. candollei and two strains of Agrobacterium rhizogenes (ATCC 15834 and 43057), hairy root cultures were established. Including 100???M acetosyringone in the culture medium enhanced frequency of hairy root induction by up to 58?%. Subsequently, effects of inoculum size (IS) and temperature on growth and production of isoflavonoids in hairy roots were determined. Conditions of 1?%?IS and 32?°C promoted the highest accumulation of total isoflavonoid content, up to 31.0?±?22.6?mg/g dry weight (DW), in hairy roots. Moreover, culture of hairy roots at 32?°C decreased browning of hairy roots. Furthermore, this temperature promoted accumulation of the secondary metabolite daidzein; whereas, hairy root cultures at the stationary phase accumulated higher amounts of the isoflavonoid puerarin rather than daidzein.     

Effects of stress factors, bioregulators, and synthetic precursors on indole alkaloid production in compact callus clusters cultures of Catharanthus roseus

Compact callus cluster (CCC) cultures established from Catharanthus roseus consist of cohesive callus aggregates displaying certain levels of cellular or tissue differentiation. CCC cultures synthesize about two-fold more indole alkaloids than normal dispersed-cell cultures. Our studies here show that additions of KCl, mannitol, and a variety of synthetic precursors and bioregulators to the CCC cultures markedly improved indole alkaloid production and release of these alkaloids into the medium. Treatment with 250 mM mannitol and 4 g/l KCl yielded 42.3 mg l(-1) and 33.6 mg l(-1)of ajmalicine, respectively; these amounts were about four-fold higher than the control. Succinic acid, tryptamine, and tryptophan feedings also significantly increased ajmalicine (41.5 mg l(-1), 36.9 mg l(-1), and 31.8 mg l(-1), respectively) and catharanthine (21.1 mg l(-1), 17.2 mg l(-1), and 18 mg l(-1), respectively) production by the CCC cultures, while geraniol feeding inhibited biomass and alkaloid accumulation. We also found that tetramethyl ammonium bromide could significantly improve ajmalicine production (49.3 mg l(-1)) and catharanthine production (18.3 mg l(-1)) in C. roseus CCC cultures. The mechanisms responsible for these treatment effects are discussed herein.     

Flavonoid content and antioxidant activity of Artemisia vulgaris L. “hairy” roots

We investigated the effect of Agrobacterium rhizogenes-mediated transformation on antioxidant activity of Artemisia vulgaris “hairy” roots. It appeared that transformation may increase flavonoid content as well as DPPH-scavenging activity and ability to reduce Fe³⁺ as compared to the non-transformed plants. Some “hairy” roots accumulated flavonoids up to 73.1?±?10.6?mg RE/g DW (while the amount of flavonoids in the leaves of non-transformed plants was up to 49.4?±?5.0?mg RE/g DW). DPPH-scavenging activity of some “hairy” root lines was 3–3.8 times higher than such one of the roots of the control plants. The Fe³⁺-reducing power of most transgenic root extracts exceeded such power of the extracts of the roots of the control plants. The decrease in SOD activity was found in the most “hairy” root lines compared to the control roots. The increase of flavonoid content correlated with the increase of ability of extracts to scavenge DPPH*- radical and Fe³⁺ - reducing power. No correlation between SOD activity of extracts and concentration of flavonoids was found (p?≥?0.2).Thus, transformation has led to the alteration in flavonoid accumulation and antioxidant activity in A. vulgaris “hairy” roots. Transgenic roots with high-antioxidant properties can be selected after A. rhizogenes-mediated transformation.     

不同理化因子对雪莲毛状根生长和总黄酮生物合成的影响

在 1 2MS液体培养基上研究了不同理化因子对水母雪莲毛状根生长和总黄酮生物合成的影响。实验结果表明 :氮源总浓度 (包括NH+4和NO-3)为 30mmol L ;NH⁺₄ NO^-₃比例为 5∶2 5 ;2 %蔗糖和 3%葡萄糖组合 ;0.5mg LGA₃和 0.5mg LIBA ;pH5.8;18h d的光照 (光强为 35.0.0lx) ;2.4℃ ;摇床转速为 100rmin有利于毛状根生长及总黄酮的生物合成。在此培养条件下 ,经过21d的培养毛状根生长量达到 12.8g L(DW) ,总黄酮合成量为 192.2mg L ,即总黄酮含量占毛状根干重的 15 % ,约为干重野生水母雪莲植株总黄酮含量的 2.5倍。     

Enhancement of ajmalicine production in Catharanthus roseuscell cultures with methyl jasmonate is dependent on timing and dosage of elicitation

The optimum growth stage for enhancing ajmalicine production in Catharanthus roseus cultures with methyl jasmonate (MJ) was after 6 d growth. MJ added at 10 or 100 microm on day 6 gave a maximum ajmalicine production of 10.2 mg l(-1), a 300% increase over that of non-elicited cultures.     

Effects of buffered media upon growth and alkaloid production of Catharanthus roseus hairy roots

The influence of buffered media upon the growth and alkaloid productivity of Catharanthus roseus hairy root culture was examined. As expected, the buffers minimized shifts in the pH of the media and had slightly negative effects upon growth. The growth of the hairy roots remained optimal in unbuffered media. The specific yield of lochnericine was significantly lower in response to the addition of buffers, while tabersonine was significantly higher. In contrast, the specific yields of ajmalicine, serpentine, and horhammericine remained unchanged.