Efficient production of saikosaponins in Bupleurum falcatum root fragments combined with signal transducers

An efficient system to produce saikosaponins (saikosaponin-a and -d) in Bupleurum falcatum adventitious root fragments combined with signal transducers was developed. The roots are heterogeneous in terms of size and shape and sometimes form aggregates during cultivation. When the roots were cut to lengths of about 5 mm using a scalpel and cultivated, the root fragments did not form the aggregates, and root growth and saikosaponin production were not inhibited. After screening various signal transducers, it was clear that methyl jasmonate (MeJA) markedly promoted saikosaponin production. By comparing the effect of MeJA and related substances on saikosaponin production, we conclude that both the pentenyl and carboxylmethyl group of MeJA play an important role in the promotion of saikosaponin production. Addition of both 100 microM MeJA and 20 mM CaCl2 to the medium stimulated the content of saikosaponin in the root, with levels reaching 31.7 mg/g-dry root for 15 days of cultivation. A large amount of root fragments were prepared using a blender and cultivated (23 g-dry root/l) with 400 microM MeJA and 20 mM CaCl2, resulting in a high concentration of saikosaponins (747.3 mg/l).     

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.     

Enhanced production of saikosaponins by root culture of Bupleurum falcatum L. using two-step control of sugar concentration

 The effect of sugar concentration on the production of saikosaponins was investigated using a root culture of Bupleurum falcatum L. The formation of the lateral roots, which were induced in the presence of indolebutyric acid, was suppressed as the sugar concentration was increased. After the lateral root tips had emerged from the inoculated roots, however, high concentrations of sugar showed no inhibitory effect on the development of the lateral roots. A two-step culture, with 1% sucrose at the beginning of the culture and addition of 6% sucrose at 14 days, when lateral roots have emerged, greatly improved the productivity, affording 0.8 g/l of saikosaponin-a and -d. Received: 28 October 1999 / Revision received: 19 April 2000 / Accepted: 27 April 2000     

Up-regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra

Exogenously applied methyl jasmonate (MeJA) stimulated soyasaponin biosynthesis in cultured cells of Glycyrrhiza glabra (common licorice). mRNA level and enzyme activity of beta-amyrin synthase (bAS), an oxidosqualene cyclase (OSC) situated at the branching point for oleanane-type triterpene saponin biosynthesis, were up-regulated by MeJA, whereas those of cycloartenol synthase, an OSC involved in sterol biosynthesis, were relatively constant. Two mRNAs of squalene synthase (SQS), an enzyme common to both triterpene and sterol biosyntheses, were also up-regulated by MeJA. In addition, enzyme activity of UDP-glucuronic acid: soyasapogenol B glucuronosyltransferase, an enzyme situated at a later step of soyasaponin biosynthesis, was also up-regulated by MeJA. Accumulations of bAS and two SQS mRNAs were not transient but lasted for 7 d after exposure to MeJA, resulting in the high-level accumulation (more than 2% of dry weight cells) of soyasaponins in cultured licorice cells. In contrast, bAS and SQS mRNAs were coordinately down-regulated by yeast extract, and mRNA accumulation of polyketide reductase, an enzyme involved in 5-deoxyflavonoid biosynthesis in cultured licorice cells, was induced transiently by yeast extract and MeJA, respectively.     

A squalene epoxidase from Nigella sativa participates in saponin biosynthesis and mediates terbinafine resistance in yeast

Squalene epoxidase catalyzes the formation of 2,3-oxidosqualene from squalene and in plants is the last enzyme common to all biosynthetic pathways leading to an array of triterpene derivatives like phytosterols, brassinosteroid phytohormones or saponins. In this work, we present a squalene epoxidase gene (NSSQE1) from the triterpene saponin producing plant Nigella sativa. The gene product showed a high degree of homology to functional squalene epoxidases (SQEs) from Arabidopsis thaliana and was able to complement SQE deficient yeast that harboured a knockout mutation in the underlying erg1 gene. Moreover, the expression of the NSSQE1 gene in ERG1 wild type yeast revealed that NSSQE1 conferred resistance towards terbinafine, an inhibitor of fungal SQEs. The latter suggested that a terbinafine-dependent NSSQE1 selection marker system can be developed for yeast. The gene NSSQE1 was ubiquitously expressed in all plant tissues analysed, including roots where no triterpene saponins are produced. Therefore, we argue that NSSQE1 is a housekeeping gene for triterpene metabolism in Nigella sativa. Similar to triterpene saponins, NSSQE1 was up-regulated by methyl jasmonate in leaves and should also be functionally involved in saponin biosynthesis in Nigella sativa.     

Validation and application of reference genes for quantitative gene expression analyses in various tissues of <Emphasis Type="Italic">Bupleurum chinense</Emphasis>

It is crucial to select stable references in gene expression analyses using quantitative real-time PCR (qRT-PCR). In this work, seven frequently used reference genes, 18S, Actin, EF1α, α-tubulin, β-tubulin, Cyclophilin and Cytoplasmic ribosomal protein L2 (L2), from Bupleurum chinense DC. were evaluated as the internal control in five tissues, roots, stems, leaves, flowers and fruits, before tissue specific gene expression assays. The results showed that β-tubulin was the most stable and reliable reference gene among the seven candidate genes in the measured tissues. The expression levels of four genes involved in saikosaponins (the pharmacological active compounds of B. chinense) biosynthesis, HMGR, IPPI, FPS and β-AS, were assayed with β-tubulin as the internal control in the five tissues. All the four genes were expressed in the five tissues with different profiles and HMGR in the order of roots > flowers, stems and leaves > fruits, IPPI of stems > leaves and fruits > roots and flowers, FPS of flowers > fruits > stems and roots > leaves and β-AS of roots > flowers, stems and fruits > leaves. The genes of FPS and β-AS were expressed predominantly in flowers and roots, respectively. This study may provide a suitable internal control for quantitative gene expression assays in various tissues and give insight into the tissue expression profiles of four saikosaponins biosynthesis-involved genes of medicinal B. chinense.     

Optimization of the sucrose and ion concentrations for saikosaponin production in hairy root culture ofBupleurum falcatum

Saikosaponin productivity was examined in aBupleurum falcatum L. BFHR2 hairy root culture in response to changes in the sucrose content (2≈8%), nitrogen content (0≈250 mM NH₄NO₃), phosphate content (0≈12 mM NaH₂PO₄), and the potassium content (0≈87.2 mM KCl) of the culture media. We found that the conditions for maximal saikosaponin production differed from those for optimal root growth. Highest saikosaponin yield was achieved for 8% sucrose, 62 mM NH₄NO₃, 1.2 mM NaH₂PO₄, and 0.5 mM KCl.     

Use of the modified viral satellite DNA vector to silence mineral nutrition-related genes in plants: silencing of the tomato ferric chelate reductase gene, <Emphasis Type="Italic">FRO1</Emphasis>, as an example

Virus-induced gene silencing (VIGS) is potentially an attractive reverse-genetics tool for studies of plant gene function, but whether it is effective in silencing mineral nutritional-related genes in roots has not been demonstrated. Here we report on an efficient VIGS system that functions in tomato roots using a modified viral satellite DNA (DNAmβ) associated with Tomato yellow leaf curl China virus (TYLCCNV). A cDNA fragment of the ferric chelate reductase gene (FRO1) from tomato was inserted into the DNAmβ vector. Tomato roots agro-inoculated with DNAmβ carrying both a fragment of FRO1 and TYLCCNV used as a helper virus exhibited a significant reduction at the FRO1 mRNA level. As a consequence, ferric chelate reductase activity, as determined by visualization of the pink FeBPDS₃ complex was significantly decreased. Our results clearly demonstrated that VIGS system can be employed to investigate gene function associated with plant nutrient uptake in roots.     

<Emphasis Type="Italic">QUASIMODO1</Emphasis> is expressed in vascular tissue of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> inflorescence stems,and affects homogalacturonan and xylan biosynthesis

An insertion in the promoter of the Arabidopsis thaliana QUA1 gene (qua1-1 allele) leads to a dwarf plant phenotype and a reduction in cell adhesion, particularly between epidermal cells in seedlings and young leaves. This coincides with a reduction in the level of homogalacturonan epitopes and the amount of GalA in isolated cell walls (Bouton et al., Plant Cell 14: 2577 2002). The present study was undertaken in order to investigate further the link between QUA1 and cell wall biosynthesis. We have used rapidly elongating inflorescence stems to compare cell wall biosynthesis in wild type and qua1-1 mutant tissue. Relative to the wild type, homogalacturonan α-1-4-D-galacturonosyltransferase activity was consistently reduced in qua1-1 stems (by about 23% in microsomal and 33% in detergent-solubilized membrane preparations). Activities of β-1-4-D-xylan synthase, β-1-4-D-galactan synthase and β-glucan synthase II activities were also measured in microsomal membranes. Of these, only β-1-4-D-xylan synthase was affected, and was reduced by about 40% in qua1-1 stems relative to wild type. The mutant phenotype was apparent in inflorescence stems, and was investigated in detail using microscopy and cell wall composition analyses. Using in situ PCR techniques, QUA1 mRNA was localized to discrete cells of the vascular tissue and subepidermal layers. In mutant stems, the organization of these tissues was disrupted and there was a modest reduction in homogalacturonan (JIM5) epitopes. This study demonstrates a specific role for QUA1 in the development of vascular tissue in rapidly elongating inflorescence stems and supports a role of QUA1 in pectin and hemicellulose cell wall synthesis through affects on α-1,4-D-galacturonosyltransferase and β-1,4-D-xylan synthase activities.