Isolation and characterization of a copalyl diphosphate synthase gene promoter from Salvia miltiorrhiza

The promoter, 5' UTR, and 34-nt 5' fragments of protein encoding region of the Salvia miltiorrhiza copalyl diphosphate synthase gene were cloned and characterized. No tandem repeats, miRNA binding sites, or CpNpG islands were observed in the promoter, 5' UTR, or protein encoding fragments. The entire isolated promoter and 5' UTR is 2235 bp long and contains repetitions of many cis-active elements, recognized by homologous transcription factors, found in Arabidopsis thaliana and other plant species. A pyrimidine-rich fragment with only 6 non-pyrimidine bases was localized in the 33-nt stretch from nt 2185 to 2217 in the 5' UTR. The observed cis-active sequences are potential binding sites for trans-factors that could regulate spatio-temporal CPS gene expression in response to biotic and abiotic stress conditions. Obtained results are initially verified by in silico and co-expression studies based on A. thaliana microarray data. The quantitative RT-PCR analysis confirmed that the entire 2269-bp copalyl diphosphate synthase gene fragment has the promoter activity. Quantitative RT-PCR analysis was used to study changes in CPS promoter activity occurring in response to the application of four selected biotic and abiotic regulatory factors; auxin, gibberellin, salicylic acid, and high-salt concentration.


Introduction
Tanshinones are abietane-type norditerpenoid quinones found in the Chinese medicinal herb Salvia miltiorrhiza Bunge [1,2].They are synthesized from a common precursor geranylgeranyl diphosphate (GGPS) in a sequential pair of cyclization reactions [1].The first reaction, catalyzed by copalyl diphosphate synthase (EC 5.5.1.12),is based on carbon-carbon double-bond protonation that leads to the production of copalyl diphosphate (CPS).Subsequent cyclization and rearrangement reactions, catalyzed by kaurene-like synthase (KLS), result in the formation of a miltiradiene moiety, a key intermediate in tanshinone biosynthesis [3,4].Both CPS and KSL enzymes were previously cloned in S. miltiorrhiza.Their cDNA, together with that of GGPS synthase and farnesyl synthase, was then incorporated in modular miltiradiene biosynthesis pathway in yeast (Saccharomyces cerevisiae).This approach allowed miltiradiene concentrations 365 mg/L to be reached in a 15-L bioreactor [5].CPS is also the first committed enzyme in plant hormone gibberellin biosynthesis [1].
Metabolic engineering approaches to increase the concentration of particular metabolite are generally based on the overexpression of crucial, rate-limiting enzymes in homologic or heterologic hosts [5].Instead of using strong, constitutive promoters to achieve overexpression of crucial enzyme genes, the transcriptional regulation of particular pathway in native plant condition could be achieved by controlling the overexpression of particular trans-factors, which play a significant role in regulation of rate-limiting pathway enzymes [6].
Therefore, data related to promoter structure and distribution of cis-active elements are important and could be used to produce modified or synthetic promoters, that are active under particular environmental conditions or respond positively to the action of particular trans-factor.Such promoters could be used to regulate the expression of genes encoding rate-limiting enzymes of a particular secondary metabolite pathway in a concerted way.As a result, greater concentrations of a particular secondary metabolite, often of significant medical importance can be achieved [7].
However, the detailed promoter structure of CPS gene is as yet unknown.Available results of deletion mutagenesis of the CPS gene from Arabidopsis thaliana indicate that several promoter regions together with intron 1 and 2, are needed for efficient expression of the gus reporter gene.The CPS gene region localized at −997 to −726, containing the putative ACGT (−934 to −931) cis-active motif is responsible for seed specific gene expression [8,9].
In the present report, the isolation and in silico characterization of CPS promoter and 5'-untranslated region (5' UTR) from S. miltiorrhiza (GenBank accession number KF718290.2) is described.Moreover, both the activity of isolated fragment and its relative strength as compared to the CAMV35S promoter were revealed in the RT-PCR analysis of the transformed S. miltiorrhiza plants.The influence of auxin, gibberellin, salicylic acid, and high-salt concentration on promoter activity was assessed: gus gene expression being measured 0, 12, 24, 48, 72, and 96 hours after hormonal treatment, or during a 96-hour exposure to 100 mM NaCl.

Plant material
Salvia miltiorrhiza seeds were provided by the Medicinal Garden of the Department of Pharmacognosy at the Faculty of Pharmacy, Medical University of Łódź (Poland).Young plants up to 8 weeks old were cultivated at 26°C ±2°C and natural light in pots of 0.5 L (diameter 12 cm) containing composite soil.

Isolation of genomic DNA
The genomic DNA was isolated from fresh plant material according to Khan et al. [10].The purity and concentration of DNA was assessed on the basis of A 260 , A 260/280 , and A 260/230 parameters using a P300 Nanophotometer (Implen, Germany).

Promoter isolation and in silico analysis
The putative promoter region of the S. miltiorrhiza CPS gene was isolated using Genome Walker™ Universal Kit (Clontech, USA) according to the manufacturer's instructions.A 5'-terminal fragment of cDNA sequence deposited in GenBank at accession number EU003997.1 was used as a target for two gene specific primers GSP1 and GSP2 presented in the Fig. 1.Amplified DNA fragments were TOPO-TA cloned into a pCR®II-TOPO® vector (Life Technologies, USA) and sequenced.The obtained sequence was searched for cis-active elements, tandem repeats, CpG/CpNpG islands and microRNA binding sites using PlantPAN and PlantPAN 2.0 [11,12].TSSP software and RegSite Plant DB, (Softberry Inc., USA) were employed to localize TATA-box and transcription initiation site (TIS).
The reliability of PlantPAN2.0was ascertained as described by Chow et al. [12].The A. thaliana transcription factor (TF) binding sites used in PlantPAN2.0were experimentally verified by high-throughput, in vitro protein binding microarray technology.In total, 12 829 TFs and 615 position weight matrices were integrated into PlantPAN2.0.The IUPAC consensus motifs of plant TFs were extracted manually from the Uniprot database.These matrices were supplemented with 548 up-to-date matrices from JASPAR, TRANSFAC (public release 7.0), and PLACE.Finally, 19 960 TFs and 1143 matrices of TF binding sites from 76 plant species were introduced into Plant-PAN2.0[12].Match™ software was used in PlantPAN2.0 to scan putative cis-acting sequences against its library of positional weight matrices.The cut-off profiles of TF-matrices and matrices without TF, curated in PlantPAN2.0and TRANSFACR, were trained using the following essential parameters: minimize false negative matches (minFN), minimize false positive matches (minFP), and minimize the sum of both error rates (minSUM).To calculate the minFN, random sequences (100 000 sequences, 50 bp per sequence) were generated and scanned with TFBS weight matrices using Match™ without any cut-off threshold.To find the minFN cut-off, the core and matrix similar score were estimated by selecting a score recognizing at least 90% of the oligonucleotides.The minFN cut-off was used to scan the 30 000 reliable promoters of A. thaliana, Oryza sativa, and Glycine max.As a result, the minFP value was estimated as a score that recognized the best 1% of hits.As the trained minFP is set as the cut-off profile in PlantPAN2.0,strict scanning results are obtained [12].

Preparing the CPS-pKGWFS7 plasmid
The 2.3-kb SalI/EcoRI fragment, encompassing the CPS promoter was cloned into the donor pENTR™ 4 Dual Selection plasmid.Finally, the CPS promoter was transferred into the acceptor Gateway™ vector pKGWFS7 through GATEWAY™ LR homology recombination among attL1/attL2 and attR1/attR2 sites, catalyzed by the Gateway® LR Clonase® II enzyme mix (Thermo Fisher Scientific, USA).

Plant transformation
The Agrobacterium tumefaciens GV2260 competent cells were transformed using heat shock protocol [13] by CPS-pKGWFS7 or pXK2FS7 plasmids.The A. tumefaciens GV2260 line was a kind gift from Prof. Dirk Inzé (Gent University, Belgium).The leaves of S. miltiorrhiza plants were used as explants and transformed according to Yan and Wang [14].

PCR analysis
Total genomic DNA was isolated from young leaves of putative transformants and untransformed (control) plants using the Isolate II Plant DNA kit (Bioline, Singapore).The purity and concentration of DNA was assessed on the basis of A 260 , A 260/280 , and A 260/230 parameters using a P300 Nanophotometer (Implen, Germany).The GUS gene fragment (162 bp) was amplified using GUS F 5'-TCAGC-GCGAAGTCTTTATAC-3' and GUS R 5'-ATAACATACGGCGTGACATC-3' primers.
The PCR reaction was performed in a volume of 25 µL.A 50-ng sample of freshlyprepared genomic DNA was used as a template.The primer concentration was 0.4 µM.The PCR reaction mixture contained also 2.5 mM Mg 2+ , nucleotide triphosphates (0.25 mM each), 1 U of TaqNova DNA polymerase (DNA Gdańsk, Poland) and an appropriate, 1×-concentrated PCR buffer.The PCR reaction parameters were as follows: initial denaturation (95°C, 3 min), denaturation (95°C, 1 min), primer annealing (54°C for gus and 70°C for egfp, 1 min), extension (72°C, 1 min), and final extension (72°C, 3 min).In total, 40 PCR cycles were performed.The salt adjusted (50 mM Na + ) temperatures of primer melting (T M ) were computed by the OligoCalc on-line calculator [15].The temperature of primer annealing was set 2-4°C below the calculated salt adjusted T M .The PCR reaction was realized in the MyCycler™ Thermal Cycler (Biorad, USA).

Fluorescence analysis
The detection of EGFP fluorescence in leaf cross sections of transformed or control plants was carried out using a model Axio Scope A1, Carl Zeiss, Germany.An Ax-ioCam MRm Rev. 3 FireWire camera was used to capture the fluorescence image.Observations and image captures were carried out at 150× magnification using EC Plan-Neofluar objectives.Blue light was provided by an LED 470 nm module.The 520-nm emission filter was used for observations under blue light.
The emission spectrum of chlorophyll a, produced by photosystems I and II is approximately in the range of 640-800 nm.As the excitation spectrum for EGFP is localized between 470 and 600 nm (emission max. at 509 nm), the 520-nm emission filter efficiently removes the chlorophyll a autofluorescence, allowing only the EGFP signal to pass [16].

Confirmation of plant transformation
The transformation of plant material was confirmed by PCR analysis of genomic DNA.The results of the ethidium-bromide agarose gel electrophoresis of PCR products specific for transformed plants are presented in Fig. S1.Plant transformation was confirmed through microscope fluorescence analysis (Fig. S2a-c).
The leaves of transformed plants leaves revealed strong EGFP-mediated fluorescence (Fig. S2a,b), while only unspecific fluorescence localized on vascular bundle elements was observed in control plants (Fig. S2c).This phenomenon is mediated by lignified secondary cell wall fluorescence in the green range with emission spectra of 440-540 nm, which overlaps with EGFP's emission spectra of 507 nm [17].
High-salt conditions were obtained by adding 100 mM NaCl (5.84 g/L) to MS solid medium containing 1.5% of cell culture grade agar (Sigma Aldrich, Poznań, Poland).Transgenic S. miltiorrhiza plants, transformed by CPS-pKGWFS7 plasmid, growing on solid MS medium without 100 mM NaCl were used as controls.

RNA isolation and cDNA synthesis
Total RNA isolation was performed in accordance with the protocol given in the Isolate Plant II RNA kit (Bioline, Singapore).Plant leaves were cut off and frozen instantly in liquid nitrogen.Approximately 80-100 mg of plant material was processed.The digestion of putative genomic DNA impurities, by RNase-free DNaseI (4 U/sample) was one of the steps used in the Isolate Plant II RNA kit.To check the efficiency of DNaseI digestion, RNA samples without the subsequent reverse transcription reaction were used as negative controls in quantitative, real-time PCR reaction.The RNA concentration was determined spectrophotometrically using a p300 Nanophotometer (Implen, Germany).The isolated RNA had an A 260/A280 ratio of 1.6-1.8.The isolated RNA samples were stored at −80°C until analysis.
Each type of RNA samples was obtained in triplicate.The RT-PCR reaction was carried out using an Enhanced Avian HS RT-PCR Kit (Sigma-Aldrich, Poland), according to the manufacturer's protocol.The final concentration of RNA in the reaction mixture was 0.01 µg/µL.The reaction mixture consisted of : 1 µL of dNTPs (1 mM final), 1 µL of anchored oligo (dT) 23 (3.5 µM final), 2 µL of 10× buffer, 1 µL of RNase inhibitor (20 U), 1 µL of Enhanced Avian Reverse Transcriptase (RT; 20 U), volume of RNA sample (required to achieve final concentration of 0.01 µg/µL), and distilled water up to a final volume 20 µL.The reaction was performed at 42°C for 1 hour.The enzyme was inactivated at 80°C for 5 minutes.Synthesized cDNA was stored at −20°C until analysis.As a reference gene the ubiquitin was used.Controls without RT were performed to ensure that the DNase I digestion was complete and samples were not contaminated with genomic DNA.

Real-time PCR
The amount of GUS and ubiquitin transcripts was analyzed by means of real-time PCR.Experiments were performed in duplicate to ensure reproducibility of the technique.Amplification reactions were performed using Rotor-Gene 6000 (Corbet) and SYBR Green Jump Start Tag ReadyMix™ (Sigma-Aldrich, Poland) according to the manufacturer's instructions.
Ethidium-bromide gel electrophoresis and the alignment of primers and Populus trichocarpa cDNA sequence (FJ438462.1),confirmed the size of the PCR reaction product to be 192 bp.The same analysis performed for gus gene primers and E. coli gus gene cDNA sequence (AAA68923.1)validated the size of PCR product to be 162 bp [18,19].
The reaction mixture for both genes consisted of 7.5 µL SYBR-Green ReadyMix, 0.7 µL of each primer, 1 µL of cDNA and distilled water to a final volume of 16 µL.The reactions for GUS and ubiquitin were carried out in separate tubes.Samples were tested in triplicate, and the means of the obtained C T values for both GUS and ubiquitin were calculated.In each experiment, a negative control, also tested in triplicate, was included.
The 2 −ΔΔC T method by Livak and Schmittgen [20,21] was used to calculate relative changes in gene expression determined from real-time quantitative PCR experiments.Results are shown as the mean ±SD of three determinations in three technical repeats.The Rotor-Gene 6000 Series Software 1.7 (Corbett Life Science, Qiagen) was used to analyze the qPCR results.
Significant differences between plants transformed by CPS-pKGWFS7 or pXK2FS7 as well as material treated by biotic or abiotic factors were assessed by one-way ANOVA.A p value <0.05 was considered to be significant [21].
The normalized GUS expression in plants transformed by pXK2FS7 plasmid was 1 (0.52-1.82), while the same parameter in plants transformed by CPS-pKGWFS7 was 0.18 (0.12-0.28;Fig. 1a,b).Therefore, the relative strength of the CPS promoter represents 0.18 (0.12-0.28) of that of a strong, constitutively active CAMV35S.

Microarray co-expression studies
Microarray data for A. thaliana publicly available through the web interface of the database at http://bbc.botany.utoronto.cawere used for co-expression studies.Transfactors and other protein co-expressed with A. thaliana CPS gene (At4g02780) were compared with trans-factors revealed by the in silico searches of S. miltiorrhiza CPS promoter region.Expression Angler software (University of Toronto, Canada; http:// bbc.botany.utoronto.ca)was used to identify A. thaliana genes that responds similarly in terms of their gene expression levels relative to the control.All samples in the database are subject to such control.The Pearson correlation coefficient (r) was used to identify the co-regulated genes.The cut-of value for r was set up in the range of 0.65-1.00.Results are formatted after median centering and normalization [22].
Excel was used to calculate the p value, based on the Pearson correlation coefficient (r), using the following formula [23]: , where n is a sample number (224).

Results
The structure of CPS promoter and 5'UTR of CPS gene The positions of both, the TIS and translation initiation sites allowed the 5' UTR to be qualified (Fig. 2).The TATA-box was localized −33 nt from the TIS.Furthermore, the sequence around the TIS at adenyl nucleotide 2168 (AGACAA, nt 2164-2169) was closely related to the consensus sequence WnT/aC/t A/cw (where W or w = A or T; n = any nt), found in 217 dicot promoters (Fig. 2) [24].
The in silico analysis of the CPS promoter region from S. miltiorrhiza indicated a lack of tandem repeats, CpG/CpNpG islands, and microRNA (miRNA) target sequences.
However, the in silico analysis revealed the presence of a pyrimidine-rich segment (PRS) located in the 33-nt fragment 2185-2217 in the 5' UTR, wherein only six nucleotides were not pyrimidines (Fig. 2).
A hypothetical leaf specific cis-active motif and corresponding trans-factor ASF-2 were identified (Tab. 1, Tab. 2) [25].The potential influence of CPS on leaf development supported the presence of cis-active motif for Athb1 trans-factor (Tab. 1, Tab. 2) [26,27].Presence of cis-active motif for trans-factors DPBF1 and 2 supported the view that CPS gene in S. miltiorrhiza could participate in embryo development [28].
Our results suggest, that the transcription rate of S. miltiorrhiza CPS may potentially depend on the availability of ethylene, auxin, salicylic acid, dark and light (Tab.1, Tab. 2).The results imply the presence of cis-active motifs, which could potentially be recognized by putative S. miltiorrhiza homologs of the previously characterized Ethylene Response Factor 1 [29,30].The cis-active motifs associated with response to light and dark were found, together with the respective trans-factor GT1 which recognize them (Tab.1, Tab. 2) [31][32][33].However, the other potential cis-active elements associated with the response to light and auxin as well as the regulation of the circadian clock were also found, without the corresponding trans-factors [34][35][36][37][38][39].
The outcome of the in silico analysis given in Tab. 1 indicated that S. miltiorrhiza CPS promoter activity could be controlled through the numerous stress factors occurring in the internal and external environment of the plant as anaerobic conditions, pathogen infection, dehydration, and protein unfolding [40][41][42][43].

Analysis of trans-factors co-expressed with Arabidopsis thaliana CPS gene
Large datasets of microarray studies performed on A. thaliana allowed to calculate Pearson correlation coefficients (r) among expression level of particular genes.Expression Angler software was used to find out transcription factors and other proteins that are co-expressed with A. thaliana CPS gene (At4g02780) and compare them to trans-factors putatively interacting with A. miltiorrhiza CPS promoter region, predicted by the in silico searches [22,23].
The following microarray dataset compendiums were used: AtGenExpress hormone and chemical, AtGenExpress abiotic stress, AtGenExpress pathogen, and AtGenExpress Plus -extended tissue.The AtGenExpress pathogen compendium contained four genes co-expressed with A. thaliana CPS gene.None of them was a trans-factor.Many more (224) co-regulated genes, including trans-factors were found in the AtGenExpress Plus -extended tissue compendium [22,23].It was found that p < 0.00001 for all obtained r values.
Tab. 1 The cis-active motifs in S. miltiorrhiza CPS promoter region and corresponding homologous trans-factors from A. thaliana and other species, active in hormone, light, and stress-conditions response.

Cis-active motif Cis-active motif site Trans-factor and references Species
Plant embryogenesis 2 5'-GATA-3' Element conserved among promoters of light-responsive genes, particularly chlorophyll a/bbinding proteins (Cab).The sequence is recognized by the ASF-2 factor.However, the DNA binding by ASF-2 is not related to light-sensitive but rather leaf-specific expression [25].

5'-CAATTAAATC-3'
A sequence motif conserved in promoter regions of tomato Lhc (Light-harvesting complex) genes.The Lhc complex participates in transmitting circadian rhytmicity.Identical or very similar (consensus CAANNNNATC) motifs were found in promoter regions of circadian controlled Arabidopsis thaliana Lhc b1 1, Lhc b1 2, Lhc a3, and Lhc a4 genes.The sequence is absent in the promoter of non-circadian expressed Lhc b gene of Pinus contorta [34,35].
The sequence is the core region of sequence over represented in light induced promoters (SOR-LIP 1) of light-inducible genes regulated by a phytochrome A system.Among other SORLIPs, the SORLIP1 is the most over-represented and most statistically significant element.The localization of SORLIP 1 appears to be strain independent, because the strongest hits are observed when both strands are considered.The core 5'-GCCAC-3' sequence may be flanked by A at the 5' and G or A at the 3' end [36,37].7 5'-GAAAAA-3' GT-1 motif found in the promoter of Glycine max calmodulin isoform SCaM-4 which plays a role in pathogen-and salt-induced SCaM-4 gene expression [31].

5'-AAACAAA-3'
The sequence motif found in majority (9) of 13 analyzed promoters of genes belonging to the ethanol fermentation pathway, that are known to be positively regulated in anaerobic conditions.The group of analyzed genes belongs to the seven different plant species [40].

5'-TTGAC-3'
The sequence localized in the promoter region of A. thaliana NPR1 gene.The expression of NPR1 is induced by pathogen infection or treatment with defense-inducing compounds such as salicylic acid.As a result of NPR1 overexpression, transgenic plants are more resistant to broad spectrum of microbial pathogens.The activation of NPR1 promoter depends on recognition of 5'-TTGAC-3' sequence by A. thaliana WRKY18 protein.Mutations of the 5'-TTGAC-3' sequence abolished their recognition by A. thaliana WRKY18, resulting in decreased NPR1 expression and inhibition of pathogen resistance development [41].
5'-TAACTG-3' Gel mobility experiment indicated that the consensus sequence 5'-TAACTG-3' is recognized specifically by A. thaliana MYB2 trans-factor.The expression of MYB2 gene is increased by dehydration and inhibited by rehydration.A beta-glucuronidase reporter gene driven by the Atmyb2 promoter was induced by dehydration and salt stress in transgenic Arabidopsis plants [42].
The analysis of obtained data indicated that numerous trans-factors participate in such processes as plant morphogenesis, plant defense, response to light, hormones, and osmotic stress as well as regulation of apoptosis (Tab.3) [44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60].Comparison of the obtained data with trans-factors identified by the in silico searches of S. miltiorrhiza CPS promoter showed no common protein (Tab. 1, Tab. 3).Therefore, the potential link between the regulation of A. thaliana and S. miltiorrhiza CPS gene expression may not be evaluated at the level of particular transcription factor.The common elements in the regulation of both genes are related only to reaction to particular biotic and abiotic factors.According to the above information, the co-expression microarray data indicated positive correlation of A. thaliana CPS expression with factors participating in response to red light (AtMYB18/LAF1), circadian clock regulation (CCT motif family protein) and auxin metabolism (AtIDD16; Tab. 3) [45,52,53].
Tab. 3 Transcription factors co-expressed with CPS in A. thaliana.Pearson correlation coefficient (r) characterizes the co-regulation rate (p < 0.00001).

Trans-factor r Function
Plant morphogenesis These results resembled data obtained by in silico searches in relation to light and hormone response (Tab.1).Moreover, microarray data implied that CPS gene in A. thaliana is co-regulated with cysteine/histidine-rich C1 domain family proteins, playing a role in plant defense processes (Tab.3) [54].Previous in silico searches of CPS promoter from S. miltiorrhiza also implied such a function, played here by WRKY (Tab. 1) [41].
Other examples of closely related functions played by both genes was regulation of apoptosis.The apoptosis was regulated in A. thaliana by the putative ubiquitin-ligase and F-box/RNI-like/FBD-like domains-containing protein, both co-expressed with the CPS gene (Tab.3) [56,57].Also in the S. miltiorrhiza, CPS promoter was observed a cis-active motif participating in the response to protein unfolding (Tab. 1) [43].The occurrence of a functional link between protein unfolding and apoptosis was supported by the activation of apoptosis pathway in reaction to the presence of unfolded proteins [43,56].Also, the reaction to plant dehydration could be achieved in the S. miltiorrhiza CPS promoter by MYB2 trans-factor (Tab. 1) [42].Microarray data revealed that the A. thaliana CPS is co-regulated with AtMYB41, playing a related role in response to osmotic stress (Tab.3) [55].
Proteins co-expressed with CPS in A. thaliana include an enzyme of gibberellin 2-oxidase activity (GA2OX3; Tab. 3) [60].Although GA2OX3 is not a trans-factor, it plays an important role in gibberellin biosynthetic and catabolic processes, implying a regulatory link between gibberellin precursor biosynthesis catalyzed by CPS and next stages of biosynthesis and catabolism of the plant hormone [2,60].
Contrary to previous results, the analysis of obtained microarray data in relation to plant morphogenesis indicated rather different functions as compared to in silico searches.The microarray data implied a role of A. thaliana CPS in development of such elements as pollen, sieve, seed, ovule, and gametophyte (Tab.3) [44,[46][47][48][49][50][51].According to the in silico searches, the S. miltiorrhiza CPS promoter should participate rather in leaf development (Tab. 1) [26,27].This point of view was supported by identification in A. thaliana trans-factor participating in leaf morphogenesis (AtIDD16) [45].
Another example of processes that were revealed only by microarray data and not confirmed by the in silico searches were response to nitrogen and anthocyanin biosynthetic pathway regulation (Tab.3) [51,58,59].

Calibration of RT-PCR reaction
Calibration curves for gus and ubiquitin genes were y = −3.25x− 7.3968 (R 2 = 1) and y = −3.23x− 6.8942 (R 2 = 1), respectively.RNA samples containing the potential contamination of genomic DNA, that were not treated by reverse transcriptase, indicated no amplification.

Response to biotic and abiotic factors
RT-PCR analysis was used to experimentally confirm the activity of the CPS promoter in response to high-salt concentration, salicylic acid, auxin, and gibberellic acid as described in "Material and methods" (Fig. 3a-d).Factors that potentially affect CPS promoter activity were selected on the basis of in silico searches (salicylic acid, highsalt, and auxin) and microarray co-expression studies (gibberellic acid).Treatment by auxin did not significantly affect the gus gene expression (Fig. 3a).
The response to salicylic acid was observed relatively early, with a 1.75-fold (1.41-2.17)increase in gus gene expression observed after 12 hours (Fig. 3c).At later stages, salicylic acid was observed to have an inhibitory effect, with expression being only 0.37-times (0.35-0.39) of control values after 24 hours.Following this, gus gene activity increased strongly: 18.57-fold (18.00-19.30)after 72 hours and 13.52-fold (10.41-17.66)after 96 hours.
More time was needed for a positive response to gibberellic acid to be observed than for salicylic acid in our experimental model (Fig. 3b).No response to gibberellin was observed before 24 h, when a 1.49-fold (1.46-1.52)increase in gus gene expression was seen.Dynamic changes in the rate of gus transcription occurred later, since the expression increased strongly after 72 h to 3.90-fold (3.27-4.63),before finally falling to 0.24 (0.20-0.29) after 96 h.
Generally speaking, a putative biphasic or positive feedback loop response was observed for the two analyzed plant hormones [61].

Discussion
The isolated CPS gene fragment indicates moderate activity based on the GUS reporter gene expression in transformed Salvia miltiorrhiza.The relative strength of isolated fragment represents 0.18 (0.12-0.28) of that of the CAMV35S activity.
The in silico analysis of the S. miltiorrhiza CPS promoter revealed a lack of tandem repeats, CpG/CpNpG islands, and miRNA target sites.Therefore, an increased mutation frequency, methylation-dependent control and targeting by miRNAs are unlikely to happen [62][63][64].
Searching the 5' UTR for potential transcription regulatory elements, revealed the presence of a 33-nt long PRS, running from nucleotide 2185 to nucleotide 2217 (Fig. 2), that may participate in the proper organization of spliceosomal complexes [65].The in silico analysis reveals that the CPS promoter could play a role in such processes as response to ethylene, dark, light, auxin, and salicylic acid, but these results have not yet been validated experimentally in S. miltiorrhiza [29][30][31][33][34][35][36][37][38][39].Also, the potential influence of such stress factors as anaerobic conditions, dehydration, pathogen infection, and protein unfolding on CPS expression should be verified and validated experimentally [40][41][42][43].Comparison of results obtained by the in silico search to the outcome of microarray data, indicated no shared, co-regulated proteins.Only the common biological processes that may be regulated by CPS genes in A. thaliana and S. miltiorrhiza were observed.Therefore, the careful examination of microarray data confirmed and further increased the number of potential processes that may be regulated by CPS gene as compared to the in silico search results.Among them are red light response, circadian clock regulation, auxin metabolism, plant defense, regulation of apoptosis, and osmotic stress adaptation [52][53][54][55][56][57].The results of in silico searches and microarray co-expression studies differ with regard to plant morphogenesis.While the in silico studies suggest that CPS promoter has a potential role in plant embryogenesis and leaf development, the microarray co-expression tests indicate that the CPS promoter has potential participation in a broader range of morphogenesis processes as pollen, sieve, seed, ovule, and gametophyte development [25,28,[44][45][46][47][48][49][50][51].
However, all the observed biological processes revealed by the in silico searches and microarray data should be experimentally validated in S. miltiorrhiza.Also, novel functions related to nitrogen metabolism and anthocyanin biosynthetic pathway regulation that are revealed by microarray data and are not confirmed by the in silico results require experimental verification by the RT-PCR studies on S. miltiorrhiza.
Results of RT-PCR experiments indicated that the CPS promoter is not regulated by auxin.Therefore, the cis-active element, putatively responding to auxin treatment 5'-TGTCCCAT-3' (Tab. 1) is rather not active in our experimental model.
Obtained RT-PCR analysis experimentally confirmed that the CPS promoter was positively regulated by gibberellin and salicylic acid.The outcome of high-salt treatment suggests the occurrence of negative regulation.The initial increase of gus gene expression controlled by CPS promoter was seen to fall and then rise again over a longer observation period.This biphasic response found in the case of gibberellin and salicylic acid could suggest that the initial increase of CPS activity could produce more gibberellin precursors and finally more endogenous gibberellin, which could stimulate promoter activity at later stages of observation.This hypothesis should be verified by the analysis of endogenous changes in gibberellin occurring in the course of a 96-h period of CPS gene stimulation by gibberellin and salicylic acid.Earlier works suggest that increased activity of CPS and ent-kaurene synthase (KS) are able to increase the concentration of kaurenoic acid.However, the concentration of GA did not significantly change, suggesting that CPS and KS do not affect the efficiency of later stages of gibberellin biosynthesis [66].
The experimentally validated information regarding trans-factors recognizing the cis-active elements, commonly found in the promoters of crucial, regulatory enzymes, could be used to improve the content of particular secondary metabolites in plant tissues.Similar approaches, based around the concerted regulation of crucial pathway enzymes through overexpression of AtPAP1 trans-factor, have facilitated the greater anthocyanin production [67].
An interesting avenue of future research could be the cloning, isolation, and functional characterization of the homologous, or as yet unknown trans-factors of S. miltiorrhiza.This could be done by using yeast one-hybrid screen technology to search cDNA libraries against known cis-active motif sequences from the promoter region [68].

Fig. 1
Fig. 1 Quantitative RT-PCR analysis of GUS expression, performed on S. miltiorrhiza plants transformed by pXK2FS7 (a) or CPS-pKGWFS7 (b) plasmids.Results presented as normalized gus expression.

Fig. S2
Fig. S2Fluorescence microscope pictures obtained from leaves fragments prepared from plants transformed by CPS-pKGWFS7 and pXK2FS7 plasmids as well as control, untransformed plants.
[28]cular properties of cis-active elements found in the S. miltiorrhiza CPS promoter by in silico searches.Sequence found in the proximal region of carrot Dc3 gene promoter (−117 to −35).Binding of the sequence (consensus ACACNNG) by basic leucine zip trans-factors DPBF-1 and 2 regulates plant embryogenesis.The sequence was used to clone cDNA of DPBF-1 and 2 in carrot by yeast one-hybrid system[28].