VaCPK20 gene overexpression significantly ...

Appl Microbiol Biotechnol (2014) 98:5541–5549 DOI 10.1007/s00253-014-5625-7

APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY

VaCPK20 gene overexpression significantly increased resveratrol content and expression of stilbene synthase genes in cell cultures of Vitis amurensis Rupr O. A. Aleynova-Shumakova & A. S. Dubrovina & A. Y. Manyakhin & Y. A. Karetin & K. V. Kiselev

Received: 30 November 2013 / Revised: 15 February 2014 / Accepted: 17 February 2014 / Published online: 4 March 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Resveratrol, a naturally occurring plant phenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In the present investigation, we show that transformation of a Vitis amurensis Rupr. cell suspension with the gene VaCPK20 for a calcium-dependent protein kinase (CDPK) under the control of double CaMV 35S promoter increased resveratrol production in five independently transformed cell lines in 9-68 times compared with control cells. The VaCPK20-transformed calli were capable of producing 0.04-0.42 % dry wt. of resveratrol, while the control calli produced up to 0.008 % dry wt. of resveratrol Also, we characterized expression of stilbene synthase (STS) genes in the five VaCPK20-transgenic cell lines of V. amurensis. In all VaCPK20-transgenic cell lines, expression of VaSTS7 increased; while expression of VaSTS1 decreased. We suggest that transformation of V. amurensis calli with the VaCPK20 gene induced resveratrol accumulation via enhancement of expression of the VaSTS7 gene involved in resveratrol O. A. Aleynova-Shumakova : A. S. Dubrovina : K. V. Kiselev (*) Laboratory of Biotechnology, Institute of Biology and Soil Science, Far East Branch of Russian Academy of Sciences, Vladivostok 690022, Russia e-mail: [email protected] A. Y. Manyakhin Mountain-Taiga station, Far East Branch of Russian Academy of Sciences, Posyolok Gornotaezhnoe, Primorsky krai, Ussuriisky region 692533, Russia Y. A. Karetin : K. V. Kiselev Department of Cell Biology and Genetics, The School of Natural Sciences, Far Eastern Federal University, Vladivostok 690090, Russia Y. A. Karetin Laboratory of Embryology, A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, Palchevsky St. 17, Vladivostok 690059, Russia

biosynthesis. The obtained data first demonstrate that overexpression of a CDPK gene resulted in increased accumulation of a stilbenoid phytoalexine in transgenic plant cells. We propose that the VaCPK20 gene could play an important role in the regulation of resveratrol biosynthesis in grape cells. Keywords Calcium . Callus culture . CDPK . Grape . Resveratrol . Plant phenol . STS . Vitis amurensis

Introduction Trans-resveratrol (3,4',5-trihydroxy-trans-stilbene) is a plant phenol derived from grapes, mulberries, peanuts, and other plant sources. Resveratrol displays a wide range of biological activities, including anti-inflammatory, antioxidant, and platelet antiaggregatory properties, and modulation of lipoprotein metabolism (Shankar et al. 2007; Kiselev 2011). Resveratrol has been shown to possess chemopreventive properties against certain cancers and cardiovascular diseases, and to have positive effects on age longevity (Aggarwal et al. 2004; Shankar et al. 2007). The influence of trans-resveratrol on plant physiology is also interesting, especially for agriculture. Trans-resveratrol shows antifungal activity (Jeandet et al. 2002; Adrian and Jeandet 2006); in leaves and berries, it acts as a phytoalexin that is produced in response to stresses, such as wounding or pathogen attack (Langcake and Pryce 1976). Therefore, the study of regulation of resveratrol biosynthesis in plant cells is of a great interest for plant molecular biology and biotechnology. Due to the capability for active continuous growth rate, grape cell cultures are a convenient model for studying regulation of resveratrol production in plant cells. A variety of resveratrol-producing plant species contain low resveratrol levels (up to 0.03 % dry wt.), and the treatment of the cultures with UV irradiation, elicitors, or other agents did not result in a

5542

considerable increase in resveratrol production (Ku et al. 2005; Wang et al. 2010; Kiselev 2011). Also, it has been shown that transformation of Vitis amurensis with the rolB and rolC genes of Agrobacterium rhizogenes greatly enhances resveratrol production in the transformed calli (Kiselev et al. 2007, 2009a; Dubrovina et al. 2010). High level of rolB expression resulted in more than a 100-fold increase in resveratrol production (up to 3.15 % dry wt.) in the callus culture transformed with rolB gene compared to the control cell culture transformed with the empty vector. It has been shown that active resveratrol biosynthesis in the rolB transgenic cultures of V. amurensis is Ca2+dependent (Dubrovina et al. 2009; Kiselev et al. 2012). Furthermore, application of an antagonist of calcium-dependent protein kinases (CDPK) in plant cells N-(6-aminohexyl)-5-chloro-1naphthalenesulfonamide (W7) significantly decreased resveratrol production and expression of STS genes in grape cells producing higher levels of resveratrol after treatments with coumaric acid, salicylic acids, and phenylalanine (Kiselev et al. 2013a). In plants, CDPKs are key calcium sensor proteins in calcium-mediated signaling (Dixit and Chelliah 2013) and exist as a multigene family (Cheng et al. 2002; Asano et al. 2005; Ray et al. 2007). We showed that expression of some CDPK genes was increased in cell cultures of V. amurensis with high levels of resveratrol content (Dubrovina et al. 2009; Kiselev et al. 2013a). Taken together, the data indicate that Ca2+ ion flux and later steps of the Ca2+mediated signal transduction pathway, including CDPKs, could be involved in the regulation of the resveratrol biosynthesis in the grapevine. Previously, 13 full cDNAs of CDPKs from V. amurensis, namely VaCDPK1, VaCDPK2, VaCDPK3, VaCDPK9, VaCDPK13, VaCDPK16, VaCDPK20, VaCDPK21, VaCDPK25, VaCDPK26, VaCDPK29, VaCDPK30, and VaCDPK3a were obtained and described (Kiselev et al. 2013b; Dubrovina et al. 2013). Comparison of the deduced amino acid sequence of VaCDPK20 with Arabidopsis CPKs revealed the highest homology with AtCPK1 (81 % positives, AK1 isoform, GenBank acc. no. NM_120569, L14771), AtCPK2 (83 % positives, GenBank NM_111902), and AtCPK20 (84 % identity, GenBank NM_129449). The phenylalanine ammonia lyase (PAL, EC 4.3.1.5) enzyme was identified as one of the possible substrates for phosphorylation of AtCPK1 (Cheng et al. 2001). PAL is the first enzyme in the phenylpropanoid pathway for biosynthesis of plant secondary metabolites. The enzyme catalyzes monooxidative deamination of phenylalanine to produce cynnamate. PAL is an important enzyme that links primary metabolism to secondary metabolism (Wang et al. 2013). Thus, information about PAL phosphorylation by CDPK provides a link between CDPKs and secondary metabolism in plants (Cheng et al. 2001). VaCDPK20 is a close homologue to AtCPK1 and, therefore, the purpose of this study was to examine the effects of

Appl Microbiol Biotechnol (2014) 98:5541–5549

VaCPK20 overexpression on resveratrol production and expression of stilbene synthase (STS) genes in cell cultures of V. amurensis.

Materials and methods Plant materials and growth conditions The V2 callus culture of wild-growing grapes V. amurensis Rupr. (Vitaceae) was established in 2002 as described previously (Kiselev et al. 2009a). The V2 cell culture is cultivated in the Laboratory of Biotechnology at the Institute of Biology and Soil Science, Far East Branch of the Russian Academy of Sciences (Vladivostok, Russia) and is publicly available. The KA-0 empty vector-transformed cell line was obtained in 2012 by co-cultivation of the V2 cell suspension with Agrobacterium tumefaciens GV3101:pMP90 strain containing pZP-RCS2-nptII (Tzfira et al. 2005), which contained only the kanamycin (Km) resistance gene, nptII, as described previously (Kiselev et al. 2009a, 2013b). The VaCPK20 transgenic V. amurensis independently transformed callus cell lines (designated KA-09-I, KA-09-II, KA-09-III, KA-09-IV, and KA-09-V) were obtained in 2013 by transformation of the V2 cell suspension with A. tumefaciens strain GV3101:pMP90 containing pZP-RCS2-VaCPK20-nptII as described previously (Kiselev et al. 2009a). The grape cell cultures were cultivated at 35-day subculture intervals in the dark at 24-25 °C in test tubes with 15 ml of the solid Murashige and Skoog modified WB/A medium supplemented with 0.5 mg/l 6-benzylaminopurine (BA) and 2.0 mg/l α-naphthaleneacetic acid (NAA) (Kiselev et al. 2009a).

Isolation and sequencing of VaCPK20 Full length cDNA coding sequence of VaCPK20 (VaCPK3c) gene was amplified based on the known sequence of VvCPK20 gene (GenBank accession number XM_002284714) from Vitis vinifera (Jaillon et al. 2007; Velasco et al. 2007) using primers 5'ATG GGG AAC ACA TGT GTA GGA, 5'TTA GGT TAT TTT GTA TCC CTT CTT and RNA isolated from inflorescences of wild-growing V. amurensis as reported (Dubrovina et al. 2013). The target amplicons were isolated from the gel using Cleanup Mini (Evrogen, Moscow, Russia). The cDNA of VaCPK20 (1,707 bp) was subcloned into a pTZ57R/T plasmid (Fermentas, Vilnius, Lithuania) and sequenced. Multiple sequence alignments and a phylogenetic tree based on pairwise alignment were done with ClustalX program (Altschul et al. 1990).


Overexpression of VaCPK20 in V. amurensis cell cultures To generate the construction for plant cell transformation, the full-length cDNA of VaCPK20 (GenBank accession number KC488322) were amplified by PCR using the forward primer 5'GCT CCT CGA GAT GGG GAA CAC ATG TGT AGG A and reverse primer 5'TCG AGG ATC CTT AGG TTA TTT TGT ATC CCT TCT T from pTZ57-VaCPK20. The forward primer contained a XhoI restriction site and the reverse primer contained a BamHI restriction site, which are underlined. The full-length cDNA of VaCPK20 was cloned into the pSAT1 vector (Tzfira et al. 2005) by the XhoI and BamHI sites under the control of the double cauliflower mosaic virus (CaMV 35S) promoter. Then, the expression cassette from pSAT1 with the VaCPK20 gene was cloned into the pZP-RCS2-nptII vector (Tzfira et al. 2005) using the PalAI (AscI) sites. The pZPRCS2-nptII construction also carried the nptII gene under the control of the double CaMV 35S promoter. The used restriction enzymes were obtained from SibEnzyme (Novosibirsk, Russia). Plasmid DNA samples (pSAT1 and pZP-RCS2nptII) were kindly provided by Professor Alexander Krichevsky (State University of New York, Stony Brook, USA). The overexpression construct of VaCPK20 (pZP-RCS2VaCPK20-nptII) or empty vector (pZP-RCS2-nptII) was introduced into the A. tumefaciens strain GV3101:pMP90 and transformed into the V. amurensis suspension culture V2 by co-cultivation with the bacterial cells as described (Kiselev et al. 2013b). Briefly, calli of the V2 culture (1 g) were transferred to 40 ml of liquid WB/A medium in 250 ml Erlenmeyer flasks and cultivated at 24-25 °C in the dark on a rotary shaker (100 rpm). An overnight suspension of A. tumefaciens GV3101 cells (200-900 μl with 150,000-300,000 colonyforming units per milliliter) was added to a 3-5-day-old V2 suspension culture of V. amurensis. After 2 days, cefotaxime (Cf) was added to a final concentration of 250 mg/l. Then, after 1-3 days, the transformed cells of V. amurensis were transferred to a fresh WB/A solid medium supplemented with 250 mg/l cefotaxim and 10-20 mg/l kanamycin sulfate (Km). After transformation, the calli were cultivated for a 3-month period in the presence of 10-15 mg/l of Km to select transgenic cells and for a 4-month period in the presence of 250 mg/l of Cf to suppress the bacteria. Expression of the nptII genes was verified using primes and PCR conditions described earlier (Kiselev et al. 2007). The absence of A. tumefaciens was confirmed by PCR of the VirB2 gene using primers 5'ATG CGA TGC TTT GAA AGA TAC CG and 5'TTA GCC ACC TCC AGT CAG CG as described (Kiselev et al. 2013b). For analyzing the total VaCPK20 expression, we used primers S1 5'TCG AGA AGG AGG ATC ATT TGT and A1 5'TTA GGT TAT TTT GTA TCC CTT CTT; S1 and A1 designed to the 3' end of the

5543

protein coding region of VaCPK20 mRNA. For VaCPK20 transgenic expression, we used primers S1 and A2 5'GAG AGA CTG GTG ATT TTT GCG; A2 designed to the CaMV 35S terminator in the pSAT1 vector. For VaCPK20 endogenous expression, we used primers S1 and A3 5'TTG TGG CAT TCG AGG ATC AAG; A3 designed to the 3' UTR of the of VaCPK20 mRNA. Total RNA isolation and quantitative RT-PCR To t a l R N A i s o l a t i o n w a s p e r f o r m e d u s i n g t h e cetyltrimethylammonium bromide-based extraction developed by Bekesiova et al. (1999) with slight modifications (Kiselev et al. 2013b). Complementary DNAs were synthesized, as described previously (Kiselev et al. 2009b; Kiselev and Dubrovina 2010). The cloned RT-PCR products were sequenced using an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, USA), as described (Tyunin et al. 2012). cDNAs of STS1-STS10 were amplified using TaqMan Real-time PCR (Syntol, Moscow, Russia). The amplification conditions, gene-specific primer pairs, and TaqMan-probes for the STS1-STS10, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and Actin1 genes of V. amurensis are presented in Shumakova et al. (2011). cDNAs of VaCPK20, GAPDH, and Actin1 were amplified using EvaGreen Real-time PCR (Biotium, Hayward, USA). The qRT-PCRs were performed using a Real-time PCR Kit (Syntol) in an thermocycler supplied with multicolor Real-Time PCR detection system (DNA Technology, Moscow, Russia). Expression was calculated by the 2–ΔΔCT method (Giulietti et al. 2001). Scaling option is highest (the highest expressing sample accrued the value 1 in the relative mRNA calculation). VaActin1 (GenBank acс. no. DQ517935) and VaGAPDH (GenBank acс. no. GU585870) genes were used as endogenous controls to normalize variance in the quality and the amount of cDNA used in each real-time RT-PCR experiment. qRT-PCR data shown were obtained from at least two independent experiments and are averages of eight technical replicates for each independent experiment (four qPCR reactions normalized to VaActin1 and four qPCR reactions normalized to VaGAPDH expression). High-performance liquid chromatography The dried and powdered callus culture samples (100 mg) were extracted with 96 % EtOH (3 ml) for 2 h at 60 °С. An ethanolic solution of resveratrol (3,4′,5-trihydroxy-trans-stilbene approx. 99 % GC; Sigma-Aldrich; St. Louis, USA) was used as the standard to calculate the resveratrol content in V. amurensis extracts. The analytical HPLC was carried out using a Shimadzu 10 series HPLC system equipped with UVVIS detector (Tokyo, Japan). Extracts and fractions were analyzed using a 5 μm, 250 mm×4.6 mm Phenomenex Luna C18 column (Phenomenex, Torrance, CA) thermostated at

5544

30 °C as described (Dubrovina et al. 2010; Kiselev et al. 2013c). Statistical analysis The statistical analysis was carried out using the Statistica 10.0 program (StatSoft Inc, Boston, USA). The data are presented as mean ± standard error (SEM) and were tested by paired Student's t test. The 0.05 level was selected as the point of minimal statistical significance in all analyses.

Results Genetic transformation of V. amurensis with the VaCPK20 gene and selection of the transgenic cell lines The V2 suspension culture was incubated with Agrobacterium strains, bearing constructs pZP-RCS2-nptII (empty vector, KA-0 cell line) or pZP-RCS2-VaCPK20-nptII (the VaCPK20 gene under the control of double CaMV 35S promoter, five KA-09 cell lines). The KA-09 strain of Agrobacterium was inoculated in multiple separate flasks with cell suspensions of V. amurensis to establish independently transformed KA-09 cell lines. We selected transgenic cell aggregates in the presence of 10-20 mg/L Km for 3 months and established several kanamycin-resistant lines. Previously, Km sensitivity of the parent V2 culture was tested, and it was shown that these calli completely ceased to grow during the second month of cultivation at such low Km concentration as 10 mg/L (Dubrovina et al. 2010). Therefore, cell selection in the presence of 1020 mg/L Km for 3 months was adequate to establish the transgenic cell lines. During the first month after transformation, we selected the fast-growing calli from certain primary small aggregates, which appeared in the presence of Km and established several Km-resistant independent clonal lines KA0, KA09-I, KA09-II, KA09-III, KA09-IV, and KA09-V. The vector culture KA-0 reproduced morphological, growth and biosynthetic characteristics of the parent V2 culture, indicating that transformation by the empty vector did not cause significant perturbations in transformed cells. Previously, the average resveratrol content in the parent V2 cell culture was 0.026±0.01 (% dry wt.), and the culture accumulated 186.8±20.0 fresh biomass (g/L) at the end of the 35-day subculture interval (Kiselev et al. 2009a). But during subsequent cultivation of the V2 cell culture, we detected some decreasing of resveratrol production; in the present investigation period (2012-2013) the average resveratrol content in the parent V2 cell culture was 0.017±0.011 %, and the culture accumulated 191.7±20.7 g/L fresh biomass. The average resveratrol content in the KA-0 cell line transformed by empty vector was 0.013±0.011 % and KA-0 cell line accumulated 189.9±19.1 g/L fresh biomass. The KA-0 calli reproduced


morphological, growth, and biosynthetic characteristics of the parent V2 culture; this indicating that transformation by the empty vector did not cause significant perturbations in transformed cells. The KA-0 cell line was used as a control in all further experiments, including the present investigation. The fast-growing KA-09-I, KA-09-II, KA-09-III, KA-09IV, and KA-09-V cell lines were used for further investigation. The formation of stable callus phenotype of the used cell lines was observed 2 months after removal of Km from the nutrient medium. These calli represented friable vigorously growing homogenous tissues, which did not appear to have undergone differentiation, only the КА09-I cells compared with other KA-09 lines produced denser calli with brownish or whitish downy calli aggregates. We did not observe the formation of any differentiated structures, such as roots or shoot-like structures, in the KA-09-transformed cell lines as on the WB/A medium with standard hormone composition (BA 0.5 and NAA 2 mg/L) and on the medium with decreased rates of BA and/or NAA in the dark (data not shown). The semiquantitative RT-PCR has shown that the nptII gene was transcribed in all obtained transgenic cell lines (Fig. 1a). The absence of A. tumefaciens was confirmed using PCR to control the presence or absence of the VirB2 gene (Fig. 1b). The identity of the nptII and VirB2 RT-PCR products was confirmed by DNA sequencing. Using different primers sets, we analyzed total, transgenic, and endogenous VaCPK20 expression. The five independently transformed transgenic KA-09-I, KA-09-II, KA-09-III, KA-09-IV, and KA-09-V cell lines actively expressed the exogenous VaCPK20, and its expression was approximately at the same level in the five KA09 lines (Fig. 1b). The highest total expression of exogenous and endogenous VaCPK20 was detected in the KA-09-I and KA-09-IV calli. The total VaCDPK20 expression in the KA-09-II, KA-09-III, and KA09-V was in 1.2-1.8 times lower compared with the expression in the KA-09-I and KA-09-IV calli (Fig. 1b). The expression of endogenous VaCPK20 gene was at the approximately same level in the five transgenic KA-09 cell lines. Expression of endogenous VaCPK20 did not considerably differ from that in the control KA-0 cell culture (Fig. 1b). Production of resveratrol by the VaCDPK20-transformed cell lines of V. amurensis and their growth parameters In the Table 1, we presented fresh and dry biomass accumulation, resveratrol content, and resveratrol production by the empty vector-transformed cell culture KA-0 and VaCPK20overexpressing cell lines KA-09-I, KA-09-II, KA-09-III, KA09-IV, and KA-09-V. VaCPK20 overexpression in four from five VaCPK20-transgenic cell lines increased fresh biomass accumulation in 1.1-1.6 times compared with KA-0 cells cultivated on W-B/A medium, but this enhancement was statistically significant only for KA-09-III and KA-09-IV cell


5545

We analyzed stilbene production in V. amurensis cells by means of HPLC which revealed that trans-resveratrol was exclusively produced by these calli. According to the HPLC determinations, the KA-0 calli on W-B/A medium accumulated 0.008 % dry wt. of resveratrol, while all VaCPK20-transgenic cells contained significantly higher resveratrol quantity: 0.043-0.418 % dry wt. (Table 1). Thus, transformation of the V2 cell suspension of V. amurensis with the VaCPK20 gene of V. amurensis increased resveratrol production in the transformed cell lines 9-68 times compared to the control KA-0 (Table 1). The addition of CDPK antagonist W7 to KA-09-I VaCPK20-overexpressing cell line reversed this effect: the resveratrol content and production considerably decreased in the W7-treated KA-09-I cells and, as a result, did not significantly differ from that in the control KA-0 cells (Table 1). We tested the effect of decreased hormone (BA and NAA) concentrations on the level of resveratrol accumulation in the empty vector-transformed KA0 and VaCPK20-overexpressing KA09 calli. Resveratrol measurements revealed that the highest resveratrol content in the VaCPK20-transformed cell lines was under BA 0.5 and NAA 2 mg/L and BA 0.5 mg/L treatments (Table 1). Therefore, the standard medium composition (BA 0.5 and NAA 2 mg/L) was used to analyze stylbene synthase (STS) gene expression. Expression of STS genes

Fig. 1 Expression of the nptII, Virb2, VaActin, and VaCDPK20 genes in grape cell cultures. (a) Separation of RT-PCR products of nptII, Virb2, and VaActin genes by gel electrophoresis. RNA was extracted from KA-0, KA-09-I, KA-09-II, KA-09-III, KA-09-IV, and KA-09-V cell lines during the second month of Km-free cultivation. (b) Quantification of the total, transgene, and endogenous VaCDPK20 mRNAs performed by real-time PCR. Pc positive controls (pSAT1-VaCDPK20 for the VaCDPK20 gene, A. tumefaciens strain GV3101:pMP90 for Virb2 gene, and DNA of V. amurensis calli for the actin gene); Nc negative control (PCR mixture without plant DNA), 1, 2—RNAwas extracted from KA-0 and KA-04-III 40-day callus cultures, respectively. qRT-PCR data were obtained from at least two independent experiments and are averages of eight technical replicates for each independent experiment (four qPCR reactions normalized to VaActin1 and four qPCR reactions normalized to VaGAPDH expression) and presented as mean ± SE. * p