Expression of thehyp-1gene in early stages of development ...

Plant Cell Rep (2007) 26:211–217 DOI 10.1007/s00299-006-0240-4

GENETICS AND GENOMICS

Expression of the hyp-1 gene in early stages of development of Hypericum perforatum L. J´an Koˇsuth · Zuzana Katkovˇcinov´a · Petra Olexov´a · ˇ arov´a Eva Cell´

Received: 27 June 2006 / Revised: 24 August 2006 / Accepted: 25 August 2006 / Published online: 20 September 2006 C Springer-Verlag 2006


Abstract Level of expression of the hyp-1 gene encoding for the phenolic coupling protein which is assumed to be involved in conversion of emodin to hypericin in vitro was compared in different organs of Hypericum perforatum seedlings in early stage of development in order to find out the sites of hypericin biosynthesis. Hypericins are accumulated in multicellular dark glands distributed on the aerial parts of H. perforatum, however, the site of the final stages of their biosynthesis remains unclear. In order to verify biosynthetic capacity of the dark glands, the level of expression of the hyp-1 gene in root, stem, shoot apex, intact leaf, leaf lamina free of and leaf margins containing dark glands performed by quantitative reverse transcription real-time PCR (qRT-PCR) was compared. The results did not reveal any significant difference in the level of hyp-1 expression in the analyzed leaf tissues. Surprisingly, the highest expression level was found in roots, which contain neither any dark glands nor more than just traces of hypericin. The lowest expression level was found in the plant stem and shoot apex. The results may either indicate that the final stages of hypericin biosynthesis take place in different plant parts, mainly in roots, which are not essentially associated with the dark glands and primarily serve for hypericin accumulation or rise a question on the coding function of the respective gene in situ. Keywords Dark glands . hyp-1 gene . Hypericin biosynthesis . Hypericum perforatum . Real-time RT-PCR Communicated by K. Toriyama ˇ arov´a (
) J. Koˇsuth · Z. Katkovˇcinov´a · P. Olexov´a · E. Cell´ ˇ arik Institute of Biology and Ecology, Faculty of Science, P. J. Saf´ University in Koˇsice, M´anesova 23, 041 54 Koˇsice, Slovakia e-mail: [email protected]

Abbreviations EF-1α: Translation elongation factor 1α . qRT-PCR: Quantitative reverse transcription real-time PCR . TLC: Thin layer chromatography

Introduction Secondary metabolites in medicinal plant species have, along with multiple physiological functions throughout the plant’s life cycle, an importance as a source of active pharmaceuticals. The major constraint in genetic manipulation of secondary metabolite pathways has been the pure characterisation of biosynthetic reactions at the level of intermediates and, consequently, very few genes available from plant secondary metabolism (Verpoorte and Memelink 2002). The genus Hypericum, comprising more than 400 species, belongs from the pharmacological point of view to the most important genera. Interest is concentrated especially at the study of naphthodianthrones, hypericin and pseudohypericin, with anti-viral and anti-cancer effects (Bombardelli and Marazzoni 1995), but also on acylphloroglucinols, which contribute to the anti-depressive activity of the plant extract (M¨uller et al. 2001). It was found that hypericin and its derivates are accumulated and probably also synthesized in specialized multicellular structures with high metabolic activity. The anatomy and histology of these structures were earlier described by Curtis and Lersten (1990). Recently, the black nodule ultrastructure of H. perforatum leaves in progressive developing stages was published by Onelli et al. (2002). These black nodules appear on the aerial parts of the plant, primarily on the leaf margins and flower petals already in early stages of development. The occurrence of these structures is unique and characteristic just for some species of the genus Hypericum. Up-to-date knowledge on ultrastructure of the dark glands does not provide sufficient Springer

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evidence on the site(s) of hypericin biosynthesis in the plant. There is only one sequenced gene which is supposed to be involved in hypericin biosynthesis. According to Bais et al. (2003) the hyp-1 gene encodes for phenolic coupling protein which catalyzes in vitro direct and specific conversion of emodin to hypericin which, however, has not been proved by other authors so far. The aim of this work was to search for the possible site(s) of hypericin biosynthesis based on the expression level of the hyp-1 gene in early stages of ontogeny of H. perforatum L. seedlings and to compare the level of hyp-1 expression in different plant parts with regard to presence/absence of the black nodules.

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slight modification. Total RNA concentration was quantified at 260 nm wavelength (Secomam UviLight XTD2 spectrophotometer) and after staining of RNA with RiboGreen RNA quantification reagent (Molecular Probes). Fluorescent quantification was performed at 480/520 nm (excitation/emission, with fluorescence reader FLUOstar BMG). For quantification, 1:1000 dilution of the RiboGreen dye was used. Reverse transcription (RT) was performed at 37◦ C in 20 µl volume using 5 µg of the total RNA, 10 mM anchored oligoT primer and 200 U M-MLV reverse transcriptase (Invitrogene) according to the manufacturer’s instruction. RT-PCR and qRT-PCR

Material and methods Plant material Stems, leaves, roots and shoot apices were isolated from plants derived from seeds of tetraploid (2n = 4x = 32) H. perforatum donor plants cultured under in vitro and ex vitro conditions. The seedlings cultured in vitro were kept and clonally multiplied on hormone-free RM basal culture medium (Linsmaier and Skoog 1965), modified, under artificial illumination of 33 µmol m−2 s−1 , 16/8 h photoperiod, 70% relative humidity and 23◦ C temperature. The ex vitro plants were grown under laboratory conditions. The seedlings cultured 3 months after the last subculture and 12-month-old ex vitro plants were used for isolation of RNA and hypericin assay. The leaves of ex vitro cultured plants were dissected under stereomicroscope into leaf margins containing dark glands and the interior parts of the leaves free of glands. Verification of ploidy The ploidy level was determined based on the DNA content of the nuclei isolated from approximately 20 mg of H. perforatum leaves chopped in 1 ml Otto I buffer (0.1 M citric acid monohydrate; 0.5% Tween 20). The chopped plant leaves were filtered through 42 µm nylon mesh, the flowthrough was discarded and the pellet of the isolated nuclei was dissolved in 100 µl Otto I buffer. Immediately before the analysis 1 ml of Otto II buffer (0.4 M Na2 HPO4 ·12H2 O) was added and the sample was treated with RNase (Sigma) and stained with propidium iodide. The DNA content of the stained nuclei was measured by FlowCytometer FACSCalibur (Becton-Dickinson) at the flow rate 20–30 particles per second. RNA isolation and reverse transcription Total RNA was isolated from the selected parts of H. perforatum plants according to Jaakola et al. (2001) with a Springer

PCR primers for the reverse transcription PCR (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR) were designed using the GenTool Lite 1.0 software. The primers for amplification of the hyp-1 were based on the published H. perforatum sequence (AY148090). Degenerative primers were constructed for the reference gene, translation elongation factor 1α (EF-1α) according to the ClustalX nucleotide sequence alignments of the gene from Arabidopsis thaliana, Manihot esculentum and Euphorbia esula. The PCR prodr SV Gel and PCR Clean-Up ucts were purified by Wizard
System (Promega) and directly sequenced. qRT-PCRs were performed in duplicates by iCycler iQ Real-Time PCR Detection System (BioRad) in 30 µl reaction volume containing: 1 × iQTM SYBR Green Supermix (0.2 mM dNTP, 3 mM MgCl2 ), 0.5 µM forward and reverse primer and 50 ng of reverse transcribed RNA/cDNA. The reaction conditions were as follows: 95◦ C 3 min, 40 cycles (94◦ C 30 s, 52◦ C (hyp-1) or 56◦ C (EF1α) 30 s, 72◦ C 30 s), 74◦ C 4 min followed by melting curve analysis to confirm amplification of the desired single and specific product. Evaluation of the gene expression level The relative expression levels of the hyp-1 and EF1α genes were evaluated by the method of standard curve. Standard curves for hyp-1 and EF1α were obtained by amplification of a serially diluted mixture of cDNA samples (four-fold dilutions), with four to five dilution points, each in duplicate. The calculated resulting relative expression of the hyp-1 gene was normalized either to relative EF1α expression (hyp-1/EF1α) or to the total RNA load (hyp-1/RNA). Finally, the level of normalized hyp-1 gene expression in the analyzed samples was evaluated in relation to expression of normalized hyp-1 expression in the leaf of the same plant to obtain proportion between hyp-1 expressions in different parts of the same plant.

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Hypericin assay The presence of hypericins, including hypericin and pseudohypericin, in the plant samples was detected and quantified in extracts by spectrophotometry as follows: 50 mg of dried and homogenised parts of the plants were extracted twice with 15 ml chloroform in a sonicator and filtered (the samples were kept in chloroform for 2 days during second chloroform extraction to remove the overlapping pigments). The dried powder was extracted with 15 ml methanol and evaporated in a water bath. This was followed by addition of chloroform, the supernatant was discarded and the solid phase containing hypericin and its derivatives was dissolved in methanol and filtered. Absorbance of the extracts was measured at hypericin absorbance maximum of 590 nm wavelength by spectrophotometer Secomam UviLight XTD2. The total content of hypericins was quantified after extrapolation from calibration curve of the hypericin standard (Roth). All samples were analyzed in two or three replicates. The presence of hypericin in the samples was proved by thin layer chromatography on silica gel TLC plates which were developed in toluene/ethyl acetate/formic acid (50/40/10, v/v/v) (Briskin et al. 2000). Hypericin standard was run with the samples.

margin); intact leaves of H. perforatum plants. Using the real-time RT-PCR, relative expression of the hyp-1 gene in different organs (root, stem, leaves and young leaves comprising shoot apex) of in vitro and ex vitro grown plants was also compared. The results were evaluated either in relation to the level of expression of internal reference gene, translation elongation factor-1 alpha (EF1α) or concerning to total amounts of RNA/cDNA given to each qRT-PCR reaction. Since there is no sequence information about the EF1α gene in H. perforatum or any close relatives, degenerative primers for amplification of the EF1α gene fragment were designed. The sequence homology of the proposed EF1α gene fragment was verified by sequence analysis. More than 90% of the nucleotide sequence homology within the known plant EF1α genes was found, proving that the desired product was amplified. Using melt curve analysis, amplification of just one product during real-time RT-PCR was confirmed for the EF1α and the hyp-1 genes, respectively. The EF1α was used to normalize the expression levels of the hyp-1 gene in different parts of the leaf. On the other hand, the expression of the hyp-1 gene in different organs was evaluated concerning to same amount of total RNA/cDNA given to the each amplification reaction as the EF1α expression level in root, stem and leaf varied significantly.

Results

Hyp-1 expression in leaves and dark glands

Dark glands are well known to accumulate hypericin in some species of the genus Hypericum. It has also been proposed that these metabolically active structures might be the site ˇ arov´a et al. 1994; Onelli et al. of hypericin biosynthesis (Cell´ 2002). To verify the hypothesis, we evaluated the content of hypericins and expression level of hyp-1 gene, the major gene involved in hypericin biosynthesis (Bais et al. 2003) in different parts of tetraploid H. perforatum plants. The ploidy level was verified in all analyzed plants by flow cytometry. For quantification of the steady-state mRNA level of hyp-1 gene in the samples, the fluorescence-based quantitative realtime reverse transcription (qRT-PCR) with double-stranded DNA-specific binding dye SYBR Green I was performed. qRT-PCR was employed to study the hyp-1 gene expression in different parts of the leaf: internal part of the leaves with dissected marginal parts containing dark glands (leaf interior); marginal dark gland-rich part of the leaves (leaf

For the hyp-1 expression analysis in the marginal and interior parts of the leaves the ex vitro grown plants was used only because of impossibility to dissect the leaf margin from the vitrified leaf tissue of in vitro grown plants. Comparison of the EF1α-normalized hyp-1 gene expression in intact leaves and in the dissected parts of the leaves showed that the expression level in all analyzed samples is very similar (Table 1). No significant differences were detected between the samples of leaf margin, intact leaves or leaf interior. The expression level in leaf interior was slightly lower than that in the intact leaf (approx. 0.778-fold) or in the dark gland-containing leaf margin (approx. 0.631-fold). On the other hand, the hyp-1 expression level in the leaf margin containing dark glands was slightly higher than the expression in intact leaf (approx. 1.233-fold). Based on these findings, the hyp-1 expression level in intact leaf represents an average value.

Table 1

EF1α and hyp-1 expression level in different parts of ex vitro grown H. perforatum leaf

Identifier

Mean hyp-1/RNA ± SD

Mean EF1α/RNA ± SD

Mean hyp-1/EFα1 ± SD

Mean hyp-1/EF1α/leaf ± SD

Intact leaf Leaf interior Leaf margin

175.00 ± 1.95 133.00 ± 17.10 112.00 ± 23.60

60.90 ± 2.17 59.50 ± 6.94 31.60 ± 0.35

2.874 ± 0.107 2.235 ± 0.388 3.544 ± 0.748

1.000 ± 0.037 0.778 ± 0.135 1.233 ± 0.260

SD: standard deviation. Springer

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Trace 107.65 595.06 n.t Trace 99.037 375.384 n.t Trace 95.106 428.28 n.t 1.324 ± 0.299 0.043 ± 0.007 1.000 ± 0.138 0.036 ± 0.002 3.622 ± 0.336 0.643 ± 0.089 1.000 ± 0.210 0.141 ± 0.027 5.845 ± 0.698 0.614 ± 0.050 1.000 ± 0.072 0.318 ± 0.063 1.609 ± 0.261 0.048 ± 0.08 1.128 ± 0.156 0.041 ± 0.002 2.108 ± 0.196 0.374 ± 0.052 0.582 ± 0.122 0.082 ± 0.016 3.112 ± 0.372 0.327 ± 0.026 0.532 ± 0.038 0.169 ± 0.034 0.333 ± 0.035 1.910 ± 0.267 1.000 ± 0.144 1.699 ± 0.356 0.740 ± 0.158 1.460 ± 0.342 1.000 ± 0.294 2.030 ± 0.468 3.257 ± 0.313 2.378 ± 0.189 1.000 ± 0.022 1.757 ± 0.170 n.t.: not tested; d.w.: dry weight; SD: standard deviation.

0.441 ± 0.107 0.081 ± 0.011 1.000 ± 0.141 0.062 ± 0.006 2.680 ± 0.228 0.938 ± 0.085 1.000 ± 0.040 0.287 ± 0.047 19.036 ± 1.191 1.459 ± 0.108 1.000 ± 0.099 0.558 ± 0.105

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e.v. Root e.v. Stem e.v. Leaf e.v. Sh.Apex i.v. Root1 i.v. Stem1 i.v. Leaf1 i.v. Sh.Apex1 i.v. Root2 i.v. Stem2 i.v. Leaf2 i.v. Sh.Apex2

3.54 ± 0.79 0.65 ± 0.06 8.03 ± 0.80 0.49 ± 0.02 15.60 ± 1.25 5.46 ± 0.47 5.82 ± 0.16 1.67 ± 0.27 75.00 ± 5.42 5.75 ± 0.13 3.94 ± 0.28 2.20 ± 0.39

2.37 ± 0.10 13.60 ± 1.79 7.12 ± 0.68 12.10 ± 0.33 7.40 ± 0.35 14.60 ± 1.58 10.00 ± 2.08 20.30 ± 2.02 24.10 ± 2.29 17.60 ± 1.37 7.40 ± 0.11 13.00 ± 1.24

Mean hyp1/ EF1α ± SD Mean EF1α/ RNA/leaf ± SD Mean hyp-1/ RNA/leaf ± SD

Mean EF1α/ RNA ± SD

Expression of hyp-1 in stem, root and shoot apex and production of hypericins

Mean hyp-1/ RNA ± SD Identifier

Table 2

The content of hypericin and expression of hyp-1 in different parts of the in vitro (i.v.) and ex vitro (e.v.) grown plants of H. perforatum

Mean hyp-1/EF1α/leaf ± SD

Hypericin content (µg/g d.w.)

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The differences between the hyp-1 expression levels in marginal, dark gland-rich part of the leaf and intact leaf were relatively small; therefore, further experiments were aimed at the study of the hyp-1 expression level in different organs of the plant. The evaluation of the hyp-1 expression was performed with regard to the total RNA/cDNA added to the qRT-PCR reaction as the expression level of the internal reference gene, EF1α, varied among the analyzed plant organs isolated from both, in vitro and ex vitro grown plants. The expression level of the EF1α in root was 0.333- and 0.740-fold lower than in the leaf but in the stem and the shoot apex it was 1.699- and 2.378-fold higher. On the contrary, the EF1α expression in the root of one in vitro grown plant was 3.275 times higher than in the leaf tissue (Table 2). Therefore, the EF1α could not be used as a proper internal reference gene for this purpose and the expression of the surveyed hyp-1 gene was evaluated in relation to the same total RNA given to each analyzed sample. As shown in Table 2, the highest level of the hyp-1 expression in ex vitro cultivated H. perforatum plants was detected in the leaves but very weak in the stem and shoot apex (0.062fold and 0.081-fold of hyp-1 expression in leaf, respectively). The hyp-1 expression level in roots reached less than half of the expression in leaf. Contrariwise in vitro grown plants expressed significantly higher levels of the hyp-1 gene in roots (2.680-fold or 19.036fold higher than in the leaves). The hyp-1 gene expression in the shoot apex was lower than in the leaf but the difference as compared with the ex vitro grown plants was not as high (0.287- and 0.558-fold of leaf expression). Unlike in ex vitro plants the expression of hyp-1 in stem of in vitro grown plants was comparable with the expression in leaf (0.938-fold and 1.459-fold the expression in leaf). Preliminary TLC analyses proved the presence of hypericin in leaves and stems but not in roots. Subsequently, the content of hypericin in the samples of root, stem and intact leaf was determined spectrophotometricaly. Because of small amounts of plant tissue from individual plants available for analysis, the content of hypericin was not measured in the shoot apices or interior and inferior parts of the leaf. As expected, the hypericin content was higher in the leaves (375–595 µg/g d.w. than in the stems (95–107 µg/g d.w.) but in the roots only traces could be detected (Table 2).

Discussion The genus Hypericum belongs from the pharmacological point of view to the most important genera. Interest is concentrated especially at the study of naphthodianthrones,

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hypericin and pseudohypericin, with anti-viral and anticancer effects (Bombardelli and Marazzoni 1995). It was proposed that the physiological role of hypericins in plants is in the chemical defence of H. perforatum against some phytophagous insects (Fields et al. 1990; Guillet et al. 2000). The localization of hypericin in the plant is well documented. It was found that these compounds are localized (Briskin and Gawienowski 2001) and probably also synthesized in the ˇ arov´a et al. 1994; Onelli et al. 2002) which dark glands (Cell´ are dispersed over the all above-ground parts of the plant (flowers, capsules, leaves, stems) but not in the roots (H¨olzl and Petersen 2003). The stems usually contain less dark glands on the surface and also lower amounts of hypericin. Dark glands are metabolically active multicellular structures which do not degenerate during the course of ontogeny of the gland (Ciccacarelli et al. 2001; Curtis and Lersten 1990; Onelli et al. 2002). They are present on the leaves already in early ontogenetic stages. During the course of ontogeny the dimensions of the glands increase, probably due to hypericin accumulation. In the present study, we aimed at the verification of the hypothesis that the black nodules fulfil, besides the role of hypericin accumulation, also a role of biosynthetic capacity in H. perforatum. The prerequisite for biosynthesis of any metabolite in an organism is the presence of enzyme(s) involved in its biosynthetic pathway. Although the effectors are proteins, mRNA is a blueprint. We studied the expression of mRNA encoding for Hyp-1 protein, the only known enzyme involved in the biosynthetic pathway of hypericin (Bais et al. 2003). The differences in the expression pattern of the hyp-1 gene in different plant parts/organs were studied quantitatively. If the dark glands represent not only the site of hypericin accumulation but also its synthesis, the expression level of the gene(s) involved in this pathway should be elevated. Surprisingly, we found that there is not significantly elevated level of the hyp-1 gene in dark glands-rich part of the leaf in comparison with the intact leaves or internal parts of the leaf free of dark glands. The hyp-1 gene expression in the interior part of the leaves is lower but the difference in the expression pattern between the leaf margin and the leaf interior is relatively small, approximately 1.6-fold difference. The expression of hyp-1 in the interior parts without any dark glands is not significantly lower implying that the expression of hyp-1 gene and hypericin biosynthesis would not be directly associated with dark glands containing tissue. Cell cultures represent very ingenious system for study and production of plant secondary metabolites. Many authors propose that certain degree of differentiation and production of dark gland-like structures is necessary for hypericin production in the cell cultures (Pasqua et al. 2003). On the other hand, there are few data indicating the presence of traces of hypericin in undifferentiated cell cultures (Gadzovska et al.

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2005; Kartnig et al. 1996; Kirakosyan et al. 2000b). However, it is assumed that hypericin production and accumulation in cell culture is associated with some kind of differentiation at the cell level (Kirakosyan et al. 2000a, 2004). The dark glands are considered as the limiting factor for production of the active compounds (Pasqua et al. 2003) probably due to necessity of dark glands for deposition of the synthesized hypericin. As it is known that fully mature dark glands with secretory activity are present on leaves already in early ontogenic stage (Onelli et al. 2002), we tested if the expression the hyp-1 gene is not accomplished mainly in early developmental stages of the leaves and afterwards the expression slows down. Therefore, we tested the gene expression also in plant leaves in early developmental stage forming shoot apex of the plant. The acquired results show that the expression of the hyp-1 gene in this early stage of leaf ontogeny is relatively low, especially in older, ex vitro grown plants. On the contrary, the expression of the EF1α in this early ontogenic stage is very high. Similarly, high expression level of this gene in plant meristem was found out by Ursin et al. (1991). This corroborate with the assumption that hypericin is synthesised during the course of ontogeny and that the glands are not the only site of hypericin biosynthesis. It is interesting that there are significant differences in the hyp-1 expression between the in vitro and ex vitro grown plants. We suppose that this could be due to different age of the in vitro and ex vitro grown plants and differences between normal, ex vitro, and vitrified, in vitro, grown plants. Consistent with the known variability in hypericin production between different genotypes (Koperd´akov´a et al. 2006; Koˇsuth et al. 2003), the variability in the level of expression was also found between different in vitro grown plants, whereas the hyp-1/EF1α ratio in the studied organs remained more or less stable. This applies especially for the leaf and stem tissue (ratio: 0.582 and 0.532 in leaf, or 0.374 and 0.327 in stem samples) where the analyzed samples are expected to be more homogenous. In the sample of stem of ex vitro grown plants very low level of the hyp-1 expression was detected. This seems to be consistent with the function and structure of the stem with prevailing vascular tissues. The hyp-1 expression in stems of in vitro grown plants was comparable with the leaves probably due to structural differences of stem between in vitro and ex vitro grown plants. The most interesting results were obtained from the study of the gene expression in roots, the tissue/organ where the presence of hypericin, in amounts detectable by TLC and spectrophotometry, respectively, was not proved. In addition, the roots do not contain morphological structures serving as a site for its accumulation. There are several possible reasons: (i) the sites of hypericin biosynthesis and accumulation might be different; (ii) Springer

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the level of steady state mRNA does not prove presence of the encoding polypeptide; (iii) the proposed in vitro function of the respective gene has not been proved in vivo; (iv) it is still not clear whether the hyp-1 gene is really involved in hypericin biosynthesis. In the genus Hypericum, the roots have never been used as a source of hypericin. However, the ability of the roots to accumulate xanthones, which partially share the pathway with hypericin was documented by Pasqua et al. (2003). Naphtodianthrones were mentioned to be present just in traces or not detected at all (H¨olzl and Petersen 2003). If assumed that hypericins fulfil a role as a defensive allelochemicals against phytophagous insects in aerial parts of the plant (Fields et al. 1990), there is no need to accumulate the compounds in root, although due to the dark condition, hypericin would not activate by light there and it would not be toxic for the plant. From this point of view, there is another important function of the dark nodules – to maintain this secreted material (hypericin) in the dark to avoid killing actively secreting cells (Onelli et al. 2002). Regarding the role of light in hypericin production in vivo, it was also found that increasing the light intensity during growth period increased the number of dark glands associated with the leaves of H. perforatum. It also resulted in increased level of hypericin (Briskin and Gawienowski 2001). The authors suggested several reasons. The observation that higher light intensity led to rise of the content of hypericins in leaf was interpreted in terms of increased amounts of carbon available for the biosynthesis of hypericins. Also, the increased number of dark glands could suggest an additional photomorphogenic effect of light on dark gland development. Alternatively, increased light intensity might serve to increase the number of glands serving as final deposition sites for hypericin following their biosynthesis at another site of the plant. Similarly, nicotine was proved to be synthesized in roots of tobacco plants and afterwards transported to the leaf and other aerial tissues of the plant (Katoh et al. 2005). Our results of gene expression analysis indicate that hypericin could be synthesized in roots and the dark glands serve just for hypericin accumulation and for protection of hypericin from light activation. On the other side, if assumed that the dark glands are not only the site of accumulation but also synthesis of hypericin, the respective gene may not be related to hypericin biosynthesis and its function in vivo should be further proved by gene inactivation or other techniques. Since the results are based on the expression of mRNA, the blueprint, and the existence of the blueprint does not prove the presence of a compound for which it codes (Bartlett 2002), it would be therefore essential to supplement and link the mRNA analyses with analyses of protein expression and function.

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Plant Cell Rep (2007) 26:211–217 Acknowledgement This work was supported by grants APVV-20003704 and VEGA 1/2326/05.

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