Interaction of methyl jasmonate, wounding and fungal elicitation during sesquiterpene induction in Hyoscyamus muticus in root cultures

The ability of methyl jasmonate (MeJa) to induce sesquiterpene production in root cultures of Hyoscyamus muticus has been studied. Although MeJa alone could not induce sesquiterpene in unwounded culture, MeJa added in the presence of wounding displayed a dose-dependent response, saturating at 50 μM. The ability to respond to MeJa declined with an increase in time between MeJa contact and wounding; however, responsiveness could be recovered by re-wounding of tissue prior to MeJa contact, suggesting that additional signaling related to wounding is required for sesquiterpene pathway induction. The saturation level of sesquiterpene induction with fungal elicitor was four times higher than the saturation level achieved by MeJa, with clear differences in sesquiterpene composition. Fungal elicitation results in a higher level of lubimin and a lower level of solavetivone production; whereas, methyl jasmonate induces predominantly solavetivone and little or no lubimin production. This suggests that fungal elicitation induces enzymes further down the sesquiterpene pathway which are not affected by MeJa. The induction of roots in contact with subsaturated levels of elicitor can be enhanced to saturation production levels by the addition of small amounts of MeJa (5–10 μmoles/l). In these studies, MeJa was consistently found to favor the earlier metabolite (solavetivone), while fungal elicitation promoted conversion to subsequent metabolites in the pathway (lubimin). The interactive role of MeJa in signal transduction for secondary metabolic production is discussed. Received: 8 June 1997 / Revision received: 13 August 1997 / Accepted: 13 September 1997     

Enhanced recovery of solavetivone from Agrobacterium transformed root cultures of Hyoscyamus muticus using integrated product extraction

The integrated recovery of solavetivone from fungus elicited "hairy root" cultures of Hyoscyamus muticus is examined using volatile organic solvents and solid-phase adsorbents in an external loop extraction configuration. Hexane and pentane are shown to be toxic when added directly to the culture; however, growth of roots is not inhibited when cultures are exposed to media saturated with these hydrocarbons. Solid-phase neutral adsorbents, XAD-7 and XAD-16, display higher capacity and better solavetivone partitioning capability than the hydrocarbons; however, their selectivity for the sesquiterpene solavetivone is poor in comparison with hexane. In both cases, the integration of product recovery through extraction resulted in a doubling of product formation by alleviating feedback repression. Implications of these results to the recovery of secondary metabolites from plant root cultures are discussed. (c) 1993 John Wiley & Sons, Inc.     

Jasmonates induce over-accumulation of methylputrescine and conjugated polyamines in Hyoscyamus muticus L. root cultures

 Jasmonic acid (JA) and its methyl ester (MeJA) at concentrations ranging from 0.001 to 10 μM provoked large increases in methylputrescine levels in normal and hairy roots of Hyoscyamus muticus L.; generally, levels of free putrescine and perchloric acid-soluble conjugated putrescine, spermidine and spermine also increased dramatically. More ¹⁴C-putrescine was formed when hairy roots were incubated with labelled ornithine than with arginine; conjugated ¹⁴C-putrescine was also rapidly formed. In accord with these results, ornithine decarboxylase (EC 4.1.1.17) activity was higher than that of arginine decarboxylase (EC 4.1.1.19), and MeJA enhanced these activities about two- and fourfold, respectively. Although treatment of root cultures with jasmonates enhanced precursor (putrescine, methylputrescine) levels and accumulation of secondary metabolites such as acid-soluble conjugated di-/polyamines, it provoked only modest increases in tropane alkaloid tissue levels. Received: 24 March 1999 / Revision received: 5 October 1999 / Accepted: 26 October 1999     

Effect of elicitation on growth, respiration, and nutrient uptake of root and cell suspension cultures of Hyoscyamus muticus

The elicitation of Hyoscyamus muticus root and cell suspension cultures by fungal elicitor from Rhizoctonia solani causes dramatic changes in respiration, nutrient yields, and growth. Cells and mature root tissues have similar specific oxygen uptake rates (SOUR) before and after the onset of the elicitation process. Cell suspension SOUR were 11 and 18 micromol O2/g FW x h for non-elicited control and elicited cultures, respectively. Mature root SOUR were 11 and 24 micromol O2/g FW x h for control and elicited tissue, respectively. Tissue growth is significantly reduced upon the addition of elicitor to these cultures. Inorganic yield remains fairly constant, whereas yield on sugar is reduced from 0.532 to 0.352 g dry biomass per g sugar for roots and 0.614 to 0.440 g dry biomass per g sugar for cells. This reduction in yield results from increased energy requirements for the defense response. Growth reduction is reflected in a reduction in root meristem (tip) SOUR, which decreased from 189 to 70 micromol O2/g FW x h upon elicitation. Therefore, despite the increase in total respiration, the maximum local oxygen fluxes are reduced as a result of the reduction in metabolic activity at the meristem. This distribution of oxygen uptake throughout the mature tissue could reduce mass transfer requirements during elicited production. However, this was not found to be the case for sesquiterpene elicitation, where production of lubimin and solavetivone were found to increase linearly up to oxygen partial pressures of 40% O2 in air. SOUR is shown to similarly increase in both bubble column and tubular reactors despite severe mass transfer limitations, suggesting the possibility of metabolically induced increases in tissue convective transport during elicitation.     

Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering

 In order to test the possibility of enhancing the production of pharmaceutically valuable scopolamine in transgenic cultures, the 35S-h6h transgene that codes for the enzyme hyoscyamine-6β-hydroxylase (EC 1.14.11.11) was introduced into Hyoscyamus muticus L. strain Cairo (Egyptian henbane). This plant was chosen for its capability to produce very high amounts of tropane alkaloids (up to 6% of the dry weight in the leaves of mature plant). To our knowledge, this is the first time such a large population of transgenic cultures has been studied at the morphological, chemical and genetic levels. A great variation was observed in the tropane alkaloid production among the 43 positive transformants. The best clone (KB7) produced 17 mg/l scopolamine, which is over 100 times more than the control clones. However, conversion of hyoscyamine to scopolamine was still incomplete. The expression of h6h was found to be proportional to the scopolamine production, and was the main reason behind the variation in the scopolamine/hyoscyamine ratio in the hairy-root clones. These results indicate that H. muticus strain Cairo has a potential for even more enhanced scopolamine production with more efficient gene-expression systems. Received: 24 December 1998 / Accepted: 13 January 1999     

An unusual root tip formation in hairy root culture of Hyoscyamus muticus

Summary Hairy root cultures of Hyoscyamus muticus were established using Agrobacterium rhizogenes ATCC 15834. In one out of 8 clones established, an unusual root tip formation was observed after transfer of cultures from half-strength Murashige and Skoog (1962) to White's medium (1939). This phenomenon was associated with the production of a fine brownish cell suspension culture. Hairy root development resumed after transfer of the root tips from White to half-strength Murashige and Skoog medium. After plating the isolated brownish cells on hormone-free half-strength Murashige and Skoog or White solid medium, callus proliferation was observed, and then redifferentiation of hairy roots occurred. The polymerase chain reaction analysis of the H. muticus hairy root (clone Z2) revealed that only the tl region of the T-DNA was integrated. The growth and the production of five tropane alkaloids by this clone were examined.Abbreviations PCR Polymerase Chain Reaction - MS medium Murashige and Skoog Medium - 1/2 MS medium half-strength MS medium - WP medium Woody Plant medium - RC medium Root Culture medium - WH medium White medium - HPLC High Performance Liquid Chromatography - wt. weight     

Effect of nitrogen and sucrose on the primary and secondary metabolism of transformed root cultures of Hyoscyamus muticus

Abatract The effect of carbon and nitrogen sources on two well-established hairy root clones, LBA1S and C58A, of Hyoscyamus muticus strain Cairo, were investigated. Both clones exhibited completely different patterns with regards to their growth rate, hyoscyamine accumulation, and fatty acid contents. Clone C58A grew faster and yielded more biomass (17.4 g l^-1, in 21 days), but produced less hyoscyamine. The maximum hyoscyamine content (120 mg l^-1) in clone LBA1S was reached in 28 days. Neither of the clones could use lactose or fructose as the sole carbon source, nor ammonium as the sole nitrogen source. The growth in the medium containing glucose was significantly reduced compared to that containing sucrose. Clone LBA1S was sensitive to the changes in sucrose concentration and an increase in ammonium in the culture medium, whereas C58A tolerated these changes better but was more sensitive to the increase in total nitrogen. Lipid synthesis was active in the exponential growth phase, and the total fatty acid content varied from 5 to 34 mg g^-1 of dry root material. The major fatty acids were linoleic, palmitic and linolenic. There were considerable differences in the total amount of lipids and in their relative ratios when different nutrients were applied.Abbreviations DW dry weight - FA fatty acids - FFA free fatty acids - FW fresh weight     

Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus

In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.     

Embryogenesis and plant regeneration from callus cultures derived from unpollinated ovaries of Hyoscyamus muticus L.

Unpollinated ovaries of Hyoscyamus muticus L. (commonly known as Egyptian henbane) were cultured on Murashige and Skoog and Bourgin and Nitsch media supplemented with various growth hormones to study the organogenesis, embryogenesis and regeneration of plantlets. Embryogenesis was reported for callus grown on both media containing 0.05 mg/l α-naphthaleneacetic acid and 0.5 mg/l 6-benzylaminopurine. Differentiation of roots and shoots from the calli also occurred in these media. Albinism or chlorophyll deficiency and variation in ploidy level were observed among the ovary-derived plantlets. Received: 7 April 1997 / Revised received: 2 August 1997 / Accepted: 2 September 1997     

Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.)

In this study, the effects of ploidy level and culture medium were studied on the production of tropane alkaloids. We have successfully produced stable tetraploid hairy root lines of Hyoscyamus muticus and their ploidy stability was confirmed 30?months after transformation. Tetraploidy affected the growth rate and alkaloid accumulation in plants and transformed root cultures of Egyptian henbane. Although tetraploid plants could produce 200% higher scopolamine than their diploid counterparts, this result was not observed for corresponding induced hairy root cultures. Culture conditions did not only play an important role for biomass production, but also significantly affected tropane alkaloid accumulation in hairy root cultures. In spite of its lower biomass production, tetraploid clone could produce more scopolamine than the diploid counterpart under similar growth conditions. The highest yields of scopolamine (13.87?mg?l^?1) and hyoscyamine (107.7?mg 1^?1) were obtained when diploid clones were grown on medium consisting of either Murashige and Skoog with 60?g/l sucrose or Gamborg??s B5 with 40?g/l sucrose, respectively. Although the hyoscyamine is the main alkaloid in the H. muticus plants, manipulation of ploidy level and culture conditions successfully changed the scopolamine/hyoscyamine ratio towards scopolamine. The fact that hyoscyamine is converted to scopolamine is very important due to the higher market value of scopolamine.     

Stimulation of solavetivone synthesis in free and immobilized cells of Hyoscyamus muticus by Rhizoctonia solani fungal components

Summary Three elicitor fractions including spent culture medium, hydrolyzed mycelial pellet, and hydrolyzed mycelial biomass supernatant were tested for their ability to induce sesquiterpene formation in freely suspended and immobilized cells. Immobilization enhanced the effectiveness of the hydrolysate elicitors, but not that derived from spent culture medium. When effectiveness of the elicitors is compared based on product yield, spent culture medium is shown to be far more effective than hydrolysates as an elicitor.     

Somatic Embryogenesis and Plant Regeneration from Tissue Cultures of Hyoscyamus muticus L.

Somatic embryogenesis and regeneration of plantlets was achieved In callus cultures derived from cotyledonary leaf pieces of Hyoscyamus muticus L on MS medium enriched with 2 mg/l 2,4–0 and 0.5 mg/l BAP. For embryogenesis and organogenesis varying concentrations of NAA with or without BAP were added In the medium. Organogenesis was also achieved when callus was transferred to the hormone free medium.     

Tropane Alkaloids from Callus Cultures, Differentiated and Shoots of Hyoscyamus muticus L.

Alkaloid production has been observed in cotyledonary leaf derived callus tissues, and also in in vitro differentiated shoots, and roots of Hyoscyamus muticus. The callus tissue was developed form cotyledonary leaf explants on Murashige and Skoog medium enriched with 2 mg 1^-1 2, 4-D and 0.5 mg 1^-1 BAP. Cotyledonary leaf derived callus was proliferated in the same medium for 2 passages (1 passage 28-30 days). Green and compact callus was used for alkaloid extraction. Shoots and roots formed on MS medium containing 0.05 mg 1^-1 NAA and 0.5 mg 1^-1 BAP, and also compact, nodular and embryogenic calli from which these shoots and roots differentiated, were used for alkaloid extraction. Chromatographic studies performed with TLC showed the presence of hyoscyamine as the major alkaloid present in the callus tissues, differentiated shoots and roots. However, alkaloid content varied in different tissues. Differentiated roots were found to contain maximum amount of hyoscyamine.     

The effect of inoculum size on the growth of cell and root cultures ofHyoscyamus muticus: Implications for reactor inoculation

Cell suspensions inoculated at low cell concentrations displayed a typical growth reduction, whereas root cultures displayed an improvement in growth. Specific growth rate ofHyoscyamus muticus cell suspensions decreased from 0.25 to 0.12 d⁻¹ as inoculum concentration was reduced from 4.0 to 0.02 g fresh weight per liter. In contrast, roots show an increase in growth rate from 0.24 to 0.43 d⁻¹. These contrasting growth patterns can be explained as the result of: a) the high specific surface area of cells as compared to roots and, b) the differentiated structure of roots. The dispersed nature of cell suspensions makes them more prone to leakage of key growth factors/cellular contents to medium. The results of this work indicate that cell cultures require substantially higher inoculum concentrations. In contrast, roots can be inoculated at very low concentrations. These facts imply that whereas seed vessels must be employed by cell suspensions, their use for root cultures is a compromise between an easier handling of an entwined root mass and the reduction of the contamination risk of large medium volumes.     

Simultaneous determination of scopolamine, hyoscyamine and littorine in plants and different hairy root clones of Hyoscyamus muticus by micellar electrokinetic chromatography

Hyoscyamus muticus hairy root clones were established following infection with Agrobacterium rhizogenes strains A4, LBA-9402 and 15834 and with A. tumefaciens strain C58C1pRTGus104. The accumulation of tropane alkaloids hyoscyamine, littorine and scopolamine was evaluated by micellar electrokinetic capillary electrophoresis. Littorine was reported for the first time in these clones as well as in the roots of the intact plant and confirmed by collision induced dissociation-mass spectrometry. Tropane alkaloid content in hairy roots was compared with leaves and roots of normal plants at two vegetative stages. Significant differences appeared between the alkaloid contents of the different clones. In particular, all the hairy root clones and the roots of the intact plant produced 1.5-3 and 4.5-9 times more littorine than scopolamine, respectively. The only exception was clone KB7, carrying the h6h gene, which overproduced scopolamine. The aerial parts of H. muticus plants did not contain any littorine, thus indicating different transportation or translocation mechanisms of the various tropane alkaloids.     

Effect of Temperature and Kinetin on the Germination and Endogenous Hormones of Hyoscyamus muticus Seeds

Germination of Hyoscyamus muticus L. seeds was investigated under different temperature, light and kinetin treatments. Diurnal temperature fluctuation of 25°C for 14 h and 5°C for 10 h, strongly promoted germination. Kinetin (60 mg/1) substituted for the periodic temperature changes. Both kinetin and diurnal temperature fluctuation increased the IAA level in the seeds prior to germination. A natural inhibitor (R_f 0.6–0.8) did not disappear in response to either treatment.     

Selection for Hyoscyamine and Cinnamoyl Putrescine Overproduction in Cell and Root Cultures of Hyoscyamus muticus

Hairy root cultures of Hyoscyamus muticus have been shown to produce stable levels of tropane alkaloids comparable to those found in whole plants. In contrast, cell cultures of this and other solanaceous species produce only trace amounts of alkaloids but can be used for selection of metabolic variants. We have taken advantage of both systems and the ability to convert between them in vitro in an effort to select for increased production of the tropane alkaloid hyoscyamine. Hairy roots were converted into cell suspensions by addition of 1 mg/L 2,4-dichlorophenoxyacetic acid to Murashige-Skoog medium (T. Murashige and F. Skoog [1962] Physiol Plant 15: 473-497) and screened for resistance to the amino acid analog p-fluorophenylalanine (PFP). Cells that could grow in media containing 400 [mu]M PFP were selected and cloned from single cells. The resistant cells accumulated high levels of cinnamoyl putrescines, which share the same biosynthetic precursors as hyoscyamine. Hairy root cultures were regenerated from both PFP-sensitive and PFP-resistant cells by removing 2,4-dichlorophenoxyacetic acid from the medium. Resistance to PFP continued to be expressed in regenerated roots. Higher levels of hyoscyamine were found in hairy roots regenerated from PFP-resistant cells than were found in controls. We suggest that the precursors overproduced by the PFP-resistant cells can be diverted into the hyoscyamine pathway upon the regeneration of root cultures.     

Purification and characterization of hyoscyamine 6 beta-hydroxylase from root cultures of Hyoscyamus niger L. Hydroxylase and epoxidase activities in the enzyme preparation

Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of l-hyoscyamine to 6 beta-hydroxyhyoscyamine in the biosynthetic pathway leading to scopolamine [Hashimoto, T. & Yamada, Y. (1986) Plant Physiol. 81, 619-625] was purified 310-fold from root cultures of Hyoscyamus niger L. The enzyme has an average Mr of 41,000 as determined by gel filtration on Superose 12 and exhibited maximum activity at pH 7.8 l-Hyoscyamine and 2-oxoglutarate are required for the enzyme activity, with respective Km values of 35 microM and 43 microM. Fe2+, catalase and a reductant such as ascorbate significantly activated the enzyme. 2-Oxoglutarate was not replaced by any of ten other oxo acids tested, nor was Fe2+ by nine other divalent cations tested. The enzyme was inhibited moderately by EDTA, Tiron and various oxo acids and aliphatic dicarboxylic acids, and strongly by nitroblue tetrazolium and divalent cations Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+. Several pyridine dicarboxylates and o-dihydroxyphenyl derivatives inhibited the hydroxylase. Pyridine 2,4-dicarboxylate and 3,4-dihydroxybenzoate are competitive inhibitors with respect to 2-oxoglutarate with the respective Ki values of 9 microM and 90 microM. Several alkaloids with structures similar to l-hyoscyamine were hydroxylated by the enzyme at the C-6 position of the tropane moiety. The enzyme preparation also epoxidized 6,7-dehydrohyoscyamine, a hypothetical precursor of scopolamine, to scopolamine (Km 10 microM). This epoxidation reaction required the same co-factors as the hydroxylation reaction and the epoxidase activities were found in the same fractions with the hydroxylase activities during purification. Two possible pathways for scopolamine biosynthesis are discussed in the light of the hydroxylase and epoxidase activities found in the partially purified preparation of hyoscyamine 6 beta-hydroxylase.     

Landrace of japonica rice,Akamai exhibits enhanced root growth and efficient leaf phosphorus remobilization in response to limited phosphorus availability

Aims

Phosphorus (P) acquisition through extensive root growth and P allocation to different plant organs through efficient remobilization are important for acclimation of crop plants to P-limited environments. This study elucidated changes in rice root growth and leaf P-remobilization and their influence on grain yield under P deficiency.

Methods

Two pot experiments were conducted with (P100) and without (P0) inorganic P supply using two Japanese rice cultivars: Akamai (Yamagata) and Koshihikari. Multiple harvests were made until the panicle initiation stage. Root and shoot growth response, P acquisition, and temporal leaf P-remobilization efficiency were measured. A separate experiment ascertained the final yield and grain P status.

Results

The Akamai rice cultivar showed enhanced root growth and more acquired soil P. The Akamai root dry weight was 66% greater than that of Koshihikari under P0. Confronting P deficiency, Akamai remobilized some P from its lower mature leaves to upper younger leaves starting from early growth. The remobilized P fraction increased to 72% at panicle initiation under P0. Under P0, Akamai exhibited two-fold higher leaf P-remobilization efficiency than under P100.

Conclusions

Enhanced root growth that facilitates acquisition of more soil P through better soil exploration coupled with efficient leaf P remobilization from the early growth stage improves adaptation of Akamai rice cultivar to P-limited environments. Nevertheless, P-starvation responses did not facilitate higher grain yields in P-limited conditions.