Involvement of LeMRP,an ATP‐binding cassette transporter,in shikonin transport and biosynthesis in Lithospermum erythrorhizon

• Shikonin and its derivatives are important medicinal secondary metabolites accumulating in roots of Lithospermum erythrorhizon. Although some membrane proteins have been identified as transporters of secondary metabolites, the mechanisms underlying shikonin transport and accumulation in L. erythrorhizon cells still remain largely unknown.
• In this study, we isolated a cDNA encoding LeMRP, an ATP‐binding cassette transporter from L. erythrorhizon, and further investigated its functions in the transport and biosynthesis of shikonin using the yeast transformation and transgenic hairy root methods, respectively. Real‐time PCR was applied for expression analyses of LeMRP and shikonin biosynthetic enzyme genes.
• Functional analysis of LeMRP using the heterologous yeast cell expression system showed that LeMRP could be involved in shikonin transport. Transgenic hairy roots of L. erythrorhizon demonstrated that LeMRP overexpressing hairy roots produced more shikonin than the empty vector (EV) control. Real‐time PCR results revealed that the enhanced shikonin biosynthesis in the overexpression lines was mainly caused by highly up‐regulated expression of genes coding key enzymes (LePAL, HMGR, Le4CL and LePGT) involved in shikonin biosynthesis. Conversely, LeMRP RNAi decreased the accumulation of shikonin and effectively down‐regulated expression level of the above genes. Typical inhibitors of ABC proteins, such as azide and buthionine sulphoximine, dramatically inhibited accumulation of shikonin in hairy roots.
• Our findings provide evidence for the important direct or indirect role of LeMRP in transmembrane transport and biosynthesis of shikonin.

     

A model for a bioconversion system with the promoter of the parAt gene,which confers a high level of expression of a transgene in hairy roots

The promoter of the protoplast auxin-regulated (parAt) gene of tobacco, which is expressed throughout the tissues of hairy roots, can be useful for developing a bioconversion system with hairy roots. The parAt gene is shown to be expressed in roots of seedlings and in those of mature tobacco plants. The 5-upstream region of parAt was fused to the coding sequence of the ß-d-glucuronidase (GUS) gene to generate the parAt-GUS fusion gene, which was introduced into the binary vector for Agrobacterium. Hairy roots that carried the fusion gene were obtained (parAt-GUS/hairy root) by infecting tobacco plants with A. rhizogenes carrying the fusion gene in the binary vector. Biochemical analysis with 4-methylumbelliferyl ß-d-glucuronide (MUG), a substrate for GUS, showed that the level of GUS activity was tenfold higher than that of hairy roots carrying the reporter GUS gene, which is linked to the cauliflower mosaic virus 35S RNA promoter (35S-GUS/hairy root). We also examined the rate of conversion of MUG to 4-methylumbel-liferone (MU) by hairy roots when MUG was added to the culture medium of the parAt-GUS/hairy roots. The hairy roots converted MUG to MU at more than ten times as high efficiency as the 35S-GUS/hairy roots. In addition to tobacco, the parAt-GUS gene was similarly expressed in hairy roots from Atropa and Arabidopsis. These results suggest that the promoter of the parAt gene is a useful tool for conversion of various metabolites by hairy root cultures. Correspondence to: Y. Machida     

Shikonin production and secretion by hairy root cultures of Lithospermum erythrorhizon

Summary Hairy root cultures of Lithospermum erythrorhizon were established by transformation of in vitro grown shoots with Agrobacterium rhizogenes 15834. Hairy roots cultured on Murashige and Skoog solid medium did not produce any red pigments. However, the hairy roots cultured in Root Culture solid or liquid media produced a large amount of red pigments, which were released to the medium. The addition of adsorbents to the culture medium stimulated shikonin production by ca. 3-fold. Using this method an air-lift fermenter system was established, equipped with a XAD-2 column, which continuously produced ca. 5 mg/day of shikonin during a period of more than 220 days.     

Transformation of the monocot Alstroemeria by Agrobacterium rhizogenes

An efficient procedure is described for transformation of calli of the monocotyledonous plant Alstroemeria by Agrobacterium rhizogenes. Calli were co-cultivated with A. rhizogenes strain A13 that harbored both a wild-type Ri-plasmid and the binary vector plasmid pIG121Hm, which included a gene for neomycin phosphotransferase II (NPTII) under the control of the nopaline synthase (NOS) promoter, a gene for hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a gene for -glucuronidase (GUS) with an intron fused to the CaMV 35S promoter. Inoculated calli were plated on medium that contained cefotaxime to eliminate bacteria. Four weeks later, transformed cells were selected on medium that contained 20 mg L^–1 hygromycin. A histochemical assay for GUS activity revealed that selection by hygromycin was complete after eight weeks. The integration of the T-DNA of the Ri-plasmid and pIG121Hm into the plant genome was confirmed by PCR. Plants derived from transformed calli were produced on half-strength MS medium supplemented with 0.1 mg L^–1 GA₃ after about 5 months of culture. The presence of the gusA, nptII, and rol genes in the genomic DNA of regenerated plants was detected by PCR and Southern hybridization, and the expression of these transgenes was verified by RT-PCR.     

Overexpression of phytochrome A enhances the light-induced formation of adventitious shoots on horseradish hairy roots

A full-length cDNA of the gene for phytochrome A from Arabidopsis thaliana was fused with the 35S promoter of cauliflower mosaic virus (CaMV35S-PHYA) and introduced into horseradish (Armoracia rusticana Gaert., Mey. et Scherb.) hairy roots. The phytochrome level in hairy roots transformed with CaMV35SPHYA was about three times greater than that in normal hairy roots and the rate of light-induced formation of adventitious shoots was also higher in the hairy roots transformed with CaMV35SPHYA. These results indicate that the light-induced formation of adventitious shoots on horseradish hairy roots is closely related to the phytochrome level. Received: 11 August 1998 / Revision received: 21 October 1998 / Accepted: 20 November 1998     

Isolation and characterization of a novel plant promoter that directs strong constitutive expression of transgenes in plants

A novel, constitutively expressed gene, designated MtHP, was isolated from the model legume species Medicago truncatula. Sequence analysis indicates that MtHP most likely belongs to the PR10 multi-gene family. The MtHP promoter was fused to a -glucuronidase gene to characterize its expression in different plant species. Transient assay by microprojectile bombardment and hairy root transformation by Agrobacterium rhizogenes revealed GUS expression in leaf, stem, radicle and root in M. truncatula. Detailed analysis in transgenic Arabidopsis plants demonstrated that the promoter could direct transgene expression in different tissues and organs at various developmental stages; its expression pattern was similar to that of CaMV35S promoter, and the level of expression was higher than the reporter gene driven by CaMV35S promoter. Deletion analysis revealed that even a 107 bp fragment of the promoter could still lead to a moderate level of expression. The promoter was further characterized in white clover (Trifolium repens), a widely grown forage legume species. Strong constitutive expression was observed in transgenic white clover plants. Compared with CaMV35S promoter, the level of GUS activity in transgenic white clover was higher when the transgene was driven by MtHP promoter. Thus, the promoter provides a useful alternative to the CaMV35S promoter in plant transformation for high levels of constitutive expression.     

Ri-mediated Transformation of <Emphasis Type="Italic">Glycyrrhiza uralensis</Emphasis> with a Squalene Synthase Gene (GuSQS1) for Production of Glycyrrhizin

Root of Glycyrrhiza uralensis, one of the most important medicinal plants, containing bioactive triterpene saponins (glycyrrhizin). Squalene synthase (SQS) plays a regulatory role in the biosynthesis of triterpene saponins. In the present investigation, SQS coding sequence from G. uralensis was cloned by polymerase chain reaction (PCR) and a transgenic system was developed for G. uralensis through Agrobacterium rhizogenes-mediated transformation. The SQS gene placed under a CaMV 35S promoter was transferred into G. uralensis using A. rhizogenes strain ACCC10060. The transformed hairy roots were selected on Murashige and Skoog (1962)-containing phosphinothricin (PPT) and root lines were established. The integration of SQS gene was confirmed by PCR and Southern blot. Three transgenic root lines UP1, UP24, UP31 were obtained and their growth rates were detected. The result showed that transgenic root lines but UP1 line grew faster than control hairy roots; high-performance liquid chromatography (HPLC) analysis demonstrated the highest glycyrrhizin content of transgenic roots was 2.5 mg/g dry weight and was about 2.6 times higher than control hairy roots. The nucleotide sequences GuSQS1 and GUSQS2 reported in this paper appear in the EMBL nucleotide sequence database with the accession number AM182329 and AM182330, respectively.     

Functional analysis of the Rubisco large subunit ÂN-methyltransferase promoter from tobacco and its regulation by light in soybean hairy roots

The ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) large subunit (LS) ^ɛ N-methyltransferase (Rubisco LSMT) catalyzes post-translational methylation of the ɛ-amino group of lysine-14 in the LS of Rubisco. The entire nucleotide sequence for the tobacco (Nicotiana tabacum) Rubisco LSMT (rbcMT-T) gene including the putative promoter region was recently reported, and sequence analysis of the promoter region revealed seven GT-1 motifs. The ability of several truncated rbcMT-T promoter fragments to confer light responsiveness to reporter gene expression in transgenic soybean (Glycine max) hairy roots was examined. Chimeric constructs consisting of the rbcMT-T promoter fused to a bacterial β-glucuronidase (GUS) reporter gene and transferred to soybean via Agrobacterium rhizogenes were evaluated. The rbcMT-T promoter fragments conferred expression of the reporter gene in transgenic hairy roots, callus, and cell suspension cultures based on histochemical and fluorometric GUS analyses. The results suggest a quantitative role for the number of GT-1 motifs in determining differential expression between light and dark conditions. Received: 7 January 1998 / Revision received: 23 March 1998 / Accepted: 13 April 1998     

Overexpression of the Arabidopsis thaliana squalene synthase gene in Withania coagulans hairy root cultures

Squalene synthase (SS) dimerizes two molecules of farnesyl diphosphate to synthesize squalene, a shared precursor in steroid and triterpenoid biosynthesis in plants. The SS1 gene encoding SS from Arabidopsis thaliana was introduced in Withania coagulans under the control of the CaMV35S promoter together with the T-DNA of Agrobacterium rhizogenes A4. The engineered hairy roots were studied for withanolide production and phytosterol accumulation and the results were compared with those obtained from control roots harbouring only the T-DNA from pRiA4. The increased capacity of the engineered roots for biosynthesizing phytosterols and withanolides was strongly related with the expression level of the transgene, showing the effectiveness of overexpressing 35SS1 to increase triterpenoid biosynthesis.     

Tropane alkaloids production in transgenic Hyoscyamus niger hairy root cultures over-expressing Putrescine N-methyltransferase is methyl jasmonate-dependent

The cDNA from Nicotiana tabacum encoding Putrescine N-methyltransferase (PMT), which catalyzes the first committed step in the biosynthesis of tropane alkaloids, has been introduced into the genome of a scopolamine-producing Hyoscyamus niger mediated by the disarmed Agrobacterium tumefaciens strain C58C1, which also carries Agrobacterium rhizogenes Ri plasmid pRiA4, and expressed under the control of the CaMV 35S promoter. Hairy root lines transformed with pmt presented fivefold higher PMT activity than the control, and the methylputrescine (MPUT) levels of the resulting engineered hairy roots increased four to fivefold compared to the control and wild-type roots, but there was no significant increase in tropane alkaloids. However, after methyl jasmonate (MeJA) treatment, a considerable increase of PMTase and endogenous H6Hase as well as an increase in scopolamine content was found either in the transgenic hairy roots or the control. The results indicate that hairy root lines over-expressing pmt have a high capacity to synthesize MPUT, whereas their ability to convert hyoscyamine into scopolamine is very limited. Exposure to MeJA strongly stimulated both polyamine and tropane biosynthesis pathways and elicitation led to more or less enhanced production simultaneously.     

Effects of Methyl Jasmonate on Shikonin and Dihydroechinofuran Production in Lithospermum Cell Cultures

Methyl jasmonate, when administered to Lithospermum erythrorhizoncell suspension cultures, was found to induce the productionof shikonin derivatives (the red naph-thoquinone pigments ofthe root) and dihydroechinofuran (an abnormal metabolite ofgeranylhydroquinone). Culture experiments showed that methyljasmonate caused a rapid increase in the activities of enzymesinvolved in the biosynthesis of shikonin such as p-hydroxybenzoategeran-yltransferase, which was followed by the rapid accumulationof dihydroechinofuran and the delayed production of shikonin.The induction patterns observed were similar to those elicitedby oligogalacturonides in Lithospermum cells, suggesting thatjasmonic acid or its derivative may act as a signaling moleculein the elicitation of shikonin biosynthesis. Interestingly,however, the copper ion, which is essential for inducing shikoninbiosynthesis by oligogalacturonides, was not required for shikonininduction by methyl jasmonate ¹Present address: Laboratory of Molecular & Cellular Biology,Department of Agricultural Chemistry, Kyoto University, Kitashirakawa,Kyoto, 606-01 Japan     

An improved transformation protocol for studying gene expression in hairy roots of sugar beet (Beta vulgaris L.)

A transformation protocol, based on co-inoculation with two strains of Agrobacterium, Agrobacterium tumefaciens LBA4404 and A. rhizogenes 15834 containing a binary vector with the GUS gene, was established for the induction of transgenic hairy roots from sugar beet (Beta vulgaris L.) explants. It resulted in marked improvement in the formation of hairy roots and the integration of the binary vector T-DNA into the host genome. Of 250 inoculated sugar beet hypocotyls, 84% yielded hairy roots 5–7 days after inoculation, of which 70% were co-transformed with the binary vector T-DNA. To determine stable expression of alien genes in hairy roots, the nematode resistance gene Hs1 ^pro-1 was used as a reporter gene. In addition, molecular marker analysis was applied to monitor stable incorporation of a translocation from the wild beet B. procumbens. The molecular analysis and the nematode (Heterodera schachtii) resistance test in vitro demonstrated that the genomic structure and the expression of the Hs1 ^pro-1 -mediated nematode resistance were well-maintained in all hairy root cultures even after repeated sub-culture. Received: 25 November 1997 / Revision received: 26 May 1998 / Accepted: 15 June 1998     

Influence of <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> on induction of hairy roots and benzylisoquinoline alkaloids production in Persian poppy (<Emphasis Type="Italic">Papaver bracteatum</Emphasis> Lindl.): preliminary report

Persian poppy (Papaver bracteatum Lindl.) is an important medicinal plant and source of the opium alkaloids codeine, morphine and thebaine. Transgenic root cultures of P. bracteatum Lindl. are well-defined model systems to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid biosynthesis. Agrobacterium rhizogenes was able to produce hairy roots on wounded Persian poppy seedlings. Excised shoots from 7-day-old Persian poppy were co-cultivated with the A. rhizogenes strain R15834 carrying the pBI121 binary vector. All media, except for the co-cultivation medium, included 40 mg l⁻¹ paromomycin to select for pBI121 transformants and 200 mg l⁻¹ cefotaxime to eliminate the Agrobacterium. Eight weeks after infection, paromomycin-resistant roots appeared on 45–50% of explants maintained on hormone-free medium. Isolated hairy roots were propagated in liquid medium containing 1.0 mg l⁻¹ 1-naphthaleneacetic acid to promote rapid growth. Also, callus induction and shoot regeneration of transformed Calli in vitro was achieved on B5 medium containing 1.0 mg l⁻¹ 1-naphthaleneacetic acid. Detection of the neomycin phosphotransferase gene and GUS histochemical localization confirmed the integrative transformation of root cultures. This is the first study to illustrate useful protocol to introduce foreign genes into transgenic Persian poppy hairy root cultures using A. rhizogenes strain R15834.     

Shoot and root culture of Hypericum perforatum L. transformed with Agrobacterium rhizogenes A4M70GUS

Hairy root cultures of Hypericum perforatum were obtained following inoculation of aseptically germinated seedlings with A. rhizogenes strain A4M70GUS. Effect of sucrose on the growth and biomass production of hairy root cultures was investigated. Hairy root cultures spontaneously regenerated shoots buds from which a number of shoot culture clones was established. Transformed shoot cultures exhibited good shoot multiplication, elongation and rooting on a hormone-free woody plant medium. Plants regenerated from hairy roots were similar in appearance to the normal, nontransformed plants.     

Improved metabolic action of a bacterial lysine decarboxylase gene in tobacco hairy root cultures by its fusion to arbcS transit peptide coding sequence

The gene of a bacterial lysine decarboxylase (ldc) fused to arbcS transit peptide coding sequence (tp), and under the control of the CaMV 35S promoter, was expressed in hairy root cultures ofNicotiana tabacum. The fusion of theldc to the targeting signal sequence improved the performance of the bacterial gene in the plant cells in many respects. Nearly all transgenic hairy root cultures harbouring the35S-tp-ldc gene contained distinctly higher lysine decarboxylase activity (from 1.5 to 30 pkat LDC per mg protein) than those which had been transformed with constructs in which the gene had been directly cloned behind the CaMV 35S promoter. The higher enzyme activity led to the accumulation of up to 0.7% cadaverine on a dry mass basis. In addition, part of the cadaverine pool was used for increased biosynthesis of anabasine, an alkaloid which was hardly detectable in control cultures. The best line contained anabasine levels of 0.5% dry mass, which could further be enhanced by feeding of lysine.     

Co-transformed, diploid Lolium perenne (perennial ryegrass), Lolium multiflorum (Italian ryegrass) and Lolium temulentum (darnel) plants produced by microprojectile bombardment

A total of 37 plants (30 Lolium multiflorum Lam., 6 L. perenne L., 1 L. temulentum L.) were regenerated from cell suspension colonies bombarded with plasmid DNAs encoding a hygromycin resistance gene (HYG) expressed under a CaMV35S promoter and a β-glucuronidase (GUS) gene expressed under a truncated rice actin1 promoter and first intron, or a maize ubiquitin promoter and first intron. Resistant plants were regenerated under hygromycin selection and transferred to soil. PCR analysis showed that the co-transformation frequency of the GUS gene varied from 33% to 78% of transformants, while histochemical staining of leaf tissue from soil-grown plants showed that the co-expression frequency varied from 37% to 50%. The transgenic nature of the plants was demonstrated by Southern hybridisation analysis, which also showed that the non-selected (GUS) gene was generally present at a higher copy number than the selected (HYG) gene. Received: 10 October 1997 / Revision received: 18 March 1998 / Accepted: 29 November 1998     

Hairy roots cultures from different Solanaceous species have varying capacities to produce E. coli B-subunit heat-labile toxin antigen

The gene encoding enterotoxigenic Escherichia coli B-subunit heat-labile toxin (LTB) antigen was co-transformed into hairy root cultures of Nicotiana tabacum (tobacco), Solanum lycopersicum (tomato) and Petunia parodii (petunia) under the CaMV35S promoter. Tobacco and petunia roots contained ~65–70 μg LTB g⁻¹ tissue whilst hairy roots of tomato contained ~10 μg LTB g⁻¹. Antigen at ~600 ng ml⁻¹ was detected in growth medium of tobacco and petunia. Tobacco roots with higher LTB levels showed growth retardation of ~80% whereas petunia hairy roots with similar levels of LTB showed only ~35% growth retardation, relative to vector controls. Regeneration of plants from LTB-containing tobacco hairy roots was readily achieved and re-initiated hairy roots from greenhouse-grown plants showed similar growth and LTB production characteristics as the original hairy root cultures.     

High-efficiency <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation of heat inducible sHSP18.2-GUS in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

The chimerical gene, Arabidopsis thaliana sHSP18.2 promoter fused to E. coli gusA gene, was Agrobacterium rhizogenes-mediated transformed into Nicotiana tabacum as a heat-regulatable model, and the thermo-inducible expression of GUS activity in N. tabacum transgenic hairy roots was profiled. An activation of A. rhizogenes with acetosyringone (AS) before cocultured with tobacco's leaf disc strongly promoted transgenic hairy roots formation. Transgenic hairy roots formation efficiency of A. rhizogenes precultured with 200 μM AS supplementation was 3.1-fold and 7.5-fold, respectively, compared to the formation efficiency obtained with and without AS supplementation in coculture. Transgenic hairy roots transformed with different AS concentration exhibited a similar pattern of thermo-inducibility after 10 min to 3 h heat treatments detected by GUS expression. The peak of expressed GUS specific activity, 399,530 pmol MUG per mg total protein per min, of the transgenic hairy roots was observed at 48 h after 3 h of 42°C heat treatment, and the expressed GUS specific activity was 7–26 times more than that reported in A. thaliana, tobacco BY-2 cells and Nicotiana plumbaginifolia. Interference caused by AS supplementation on the growth of transgenic hairy roots, time-course of GUS expression and its expression level were not observed.     

Hairy roots — a short cut to transgenic root nodules

To facilitate molecular studies of symbiotic nitrogen fixation a procedure for rapid production of transgenic root nodules was established on the legumeLotus corniculatus (Bird'sfoot trefoil). Regeneration of transgenic plants is not required as transgenic nodules are formed onAgrobacterium rhizogenes incited roots inoculated withRhizobium. Easy identification of transformed roots is possible using a set ofA. rhizogenes acceptor strains carrying assayable marker genes such as chloramphenicol acetyltransferase (CAT), -glucuronidase (GUS), or luciferase (LUC) under control of the cauliflower mosaic virus (CaMV) 35S promoter. Counterselection ofA. rhizogenes after infection of plants was improved using an auxotrophy marker.Abbreviations CAT chloramphenicol acetyltransferase - GUS -glucuronidase - LUC luciferase - Ri root inducing - TL left T-DNA - CaMV Cauliflower Mosaic Virus