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Article

Influence of the 135 bp Intron on Stilbene Synthase VaSTS11 Transgene Expression in Cell Cultures of Grapevine and Different Plant Generations of Arabidopsis thaliana

by
Konstantin V. Kiselev
*,
Zlata V. Ogneva
,
Olga A. Aleynova
,
Andrey R. Suprun
,
Alexey A. Ananev
,
Nikolay N. Nityagovsky
and
Alexandra S. Dubrovina
Laboratory of Biotechnology, Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch of the Russian Academy of Sciences, 690022 Vladivostok, Russia
*
Author to whom correspondence should be addressed.
Horticulturae 2023, 9(4), 513; https://doi.org/10.3390/horticulturae9040513
Submission received: 19 March 2023 / Revised: 10 April 2023 / Accepted: 18 April 2023 / Published: 20 April 2023
(This article belongs to the Special Issue Plant Tissue and Organ Cultures for Crop Improvement in Omics Era)
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Abstract

:
Modern plant biotechnology often faces the problem of obtaining a stable and powerful vector for gene overexpression. It is known that introns carry different regulatory elements whose effects on transgene expression have been poorly studied. To study the effect of an intron on transgene expression, the stilbene synthase 11 (VaSTS11) gene of grapevine Vitis amurensis Rupr. was selected and overexpressed in grapevine callus cell cultures and several plant generations of Arabidopsis thaliana as two forms, intronless VaSTS11c and intron-containing VaSTS11d. The STS genes play an important role in the biosynthesis of stilbenes, valuable plant secondary metabolites. VaSTS11d contained two exons and one intron, while VaSTS11c contained only two exons, which corresponded to the mature transcript. It has been shown that the intron-containing VaSTS11d was better expressed in several generations of transgenic A. thaliana than VaSTS11c and also exhibited a lower level of cytosine methylation. As a result, the content of stilbenes in the VaSTS11d-transgenic plants was much higher than in the VaSTS11c-transgenic plants. Similarly, the best efficiency in increasing the content of stilbenes was also observed in grapevine cell cultures overexpressing the intron-containing VaSTS11d transcript. Thus, the results indicate that an intron sequence with regulatory elements can have a strong positive effect on both transgene expression level and its biological functions in plants and plant cell cultures.

1. Introduction

Modern plant biotechnology and genetic engineering often face the problem of obtaining a stable and powerful expression vector [1]. Plant organisms perceive the introduced transgene as foreign DNA and, with the help of epigenetic mechanisms, reduce the level of transgene expression [2]. Thus, an important challenge in the field of biotechnology is the vector design for the stable and strong expression of plant transgenes in crop plant tissue improvements and ornamental plant modifications.
Commonly, plant transgenes contain no introns, a feature shared with transposons, which are also prime targets for gene silencing [2]. Given that introns are very common in plant genes but are often lacking in introduced transgenes and plant transposons, it has been hypothesized that introns may contribute to gene silencing suppression [3]. It has been found that small RNA libraries of Arabidopsis thaliana were strongly enriched for exon sequences derived from intronless genes [3]. According to the study, compared to intronless transcripts, spliced transcripts possess a reduced ability to act as a substrate for an RNA-dependent RNA polymerase (RDR) and are less effective substrates for gene silencing. Therefore, it is possible that a mechanism for the intron-mediated suppression of gene silencing exists in plants and other organisms [3].
According to recent findings, aberrant RNAs derived from intronless transgenes and endogenes are eliminated via the RNA-directed RNA polymerase 6 (RDR6)/DICER-LIKE 4 (DCLs) pathway [4]. However, it has been assumed that aberrant RNAs derived from intron-containing transgene and endogene sequences are preferentially targeted to exonucleolytic RNA decay pathways [3,4,5].
While gene intronic regions do not encode proteins, they may encode for non-protein-coding RNA (ncRNAs), which perform gene regulation functions [6]. Using nematode Caenorhabditis elegans, it has been shown that such intron-derived ncRNAs may play an important role in the regulation of gene expression [6]. It has been shown that the products of the lin-4 gene of C. elegans are small ncRNAs derived from introns. They regulate the expression of the lin-14 gene via an antisense RNA-RNA interaction, which participates in the regulation of the postembryonic development of these animals [6]. Moreover, introns may contain regulatory elements acting as binding sites for transcription factors that can regulate transgene expression [7].
In this study, using several generations of Arabidopsis and grapevine callus cell cultures, we analyzed the expression of the grapevine stilbene synthase VaSTS11 gene, as a representative of the multigenic STS family with the shortest intron in Vitis amurensis Rupr. Stilbenes are a group of plant phenolic compounds with antimicrobial activities and a wide range of health-beneficial effects [8,9,10,11]. Stilbenes are synthesized via the phenylpropanoid pathway by a broad range of unrelated plant families [11,12,13]. Stilbene synthase (STS; EC 2.3.1.95) is known as a key enzyme in stilbene biosynthesis, catalyzing the formation of simple monomeric stilbenes (e.g., resveratrol) from coenzyme A-esters of cinnamic acid derivatives and three malonyl-CoA units in a single reaction [14]. Then, t-resveratrol and other monomeric stilbenes may be metabolized to form other stilbenes, such as pterostilbene via the methylation of resveratrol by resveratrol O-methyltransferase or Romt [15]; piceid via resveratrol glycosylation by glucosyltransferases [16]; or viniferins via oxidation by polyphenol oxidase or PPO [17].
In this study, we analyzed the difference in the expression and cytosine methylation of the intronless transgene VaSTS11c and the intron-containing transgene VaSTS11d that was transgenic homozygous for the transgene A. thaliana in the fourth (T4) and seventh (T7) plant generations. The level of expression of the two VaSTS11 transgene forms and the stilbene production level were also analyzed in callus cell cultures of V. amurensis.

2. Results and Discussion

2.1. VaSTS11 Transgene Expression in the VaSTS11-Transgenic A. thaliana

For quantitative RT-PCR (RT-qPCR), the same primers were used for the amplification of the VaSTS11d and VaSTS11c transcripts based on their identical exon sequences. Therefore, we can compare the expression levels of VaSTS11d and VaSTS11c in different plant generations of the transgenic Arabidopsis using the two independent plant lines for VaSTS11d and VaSTS11c (Figure 1) obtained by the floral dip method [18]. The highest transgene expression level was detected for the intron-containing VaSTS11d transgene in the T4 plants of both ST11d-1 and ST11d-2 plant lines (Figure 1). The intron-containing VaSTS11d expression was considerably higher than the expression of the intronless VaSTS11c in the T4 generation in both transgenic plant lines. In the T7 generation, the VaSTS11d expression level in the ST11d1 and ST11d-2 plant lines considerably decreased (by 3.6–4.1-fold) compared to T4 and reached the values of the intronless VaSTS11c in one of the transgenic lines (Figure 1). A decrease in the transgene expression level was also detected for VaSTS11c in the T7 VaSTS11c-transgenic plant generation as compared to the T4 VaSTS11c-transgenic plants. The VaSTS11c expression level decreased by 1.2–2.8 times, so the VaSTS11c mRNA level in the T7 plants did not significantly differ from the unspecific amplification in WT and KA0 control plants (Figure 1).

2.2. Stilbene Content in the VaSTS11-Transgenic A. thaliana

Then, the stilbene levels in the obtained VaSTS11d- and VaSTS11c-transgenic plant lines were analyzed using HPLC. Recent investigations show that trans-resveratrol and trans-piceid were the prevalent stilbenes in transgenic A. thaliana overexpressing different STS genes [19]. However, in the VaSTS11-transgenic plant lines (ST11d-1, ST11d-2, ST11c-1, and ST11c-2), we consistently detected only trans-piceid (Figure 2a) and did not detect peaks of other stilbenes. The highest content of trans-piceid (17.9 µg/g of dry weight, Figure 2b) was detected in the T4 generation of the intron-containing ST11d-1 line previously demonstrating the highest expression of the VaSTS11 transgene (Figure 1). In the T7 generation of the ST11d-1 line, the content of trans-piceid was considerably lower than its content in the T4 generation—by 1.4 times, reaching 12.4 µg/g of dry weight (Figure 2b). For the ST11d-2 line, we also observed a decrease in stilbene content in the T7 generation in comparison to T4 plants, but the difference was not statistically considerable. The lowest content of stilbenes was detected in the intronless ST11c-2 line in the T7 generation, only 0.3 µg/g of dry weight (Figure 2b). In all VaSTS11-transgenic lines, the total stilbene content positively correlated (r = 0.95) with the level of VaSTS11 transgene expression and significantly decreased in the T7 generation compared with that in the T4 generation.
There were two studies where the sorghum SbSTS1 gene was overexpressed in Arabidopsis tt4 mutants, which could not make flavonoids, resulting in the content of stilbenes reaching 600 μg/g FW (cis-piceid, resveratrol diglucoside, and t- and cis-resveratrol acetylhexosides) in Arabidopsis leaves [20,21]. This stilbene content is one of the highest levels in transgenic Arabidopsis plants. It is possible that this level may have been reached due to an excess in stilbene precursors in these plants, since the tt4 mutant line could not produce flavonoids that require the same precursors as stilbenoid compounds. The biosynthesis pathways of the flavonoids and stilbenes use the same precursors [22,23].
In other papers, transformations of wild-type A. thaliana plants with the grapevine VaSTS1 and VaSTS7 genes led to stilbene production in A. thaliana with stilbene content reaching 22.7 μg/g FW for VaSTS1 and 0.1 μg/g FW for VaSTS7 [24]. Thus, stilbene levels in the VaSTS11-transgenic A. thaliana were closer to the average stilbene levels in STS-transgenic A. thaliana.

2.3. VaSTS11 Cytosine Methylation in Transgenic A. thaliana

Using bisulfite sequencing, we analyzed the cytosine DNA methylation level of the 394 bp fragment of the 3′ end of the STS coding region in the VaSTS11d and VaSTS11c transgenes. The total level of cytosine DNA methylation in the VaSTS11c transgene of the T4 plant generation was 1.2–1.4 times higher than the total level of cytosine VaSTS11d methylation in the T4 generation (Figure 3). In all transgenic lines, the transgene cytosine methylation increased in plants of the T7 generation in comparison with the T4 plants. Moreover, it reached very high values—up to 91.1–94.2% in the intronless ST11c-1 and ST11c-2 lines (Figure 3). The data indicate that the VaSTS11c transgene in these plants in the T4 and T7 generations is high and can be indicated as hypermethylated [25].

2.4. Search for Consensus Patterns of Plant Regulatory Sequences in the VaSTS11 Intron Using NSITE-PL

Using the NSITE-PL plant regulatory element recognition database [7], we identified four regulatory elements in the region of 135 nucleotides of the VaSTS11 intron: Gap box 2, EIN3 BS2, E-box, and ANAC089 BS 2 (Figure 4 and Figure S1, Table 1).
Gap box 2 (TTTTCAT)—a regulatory element located in the regulatory region of the chloroplast localized glyceraldehyde-3-phosphate dehydrogenase (GapB) gene. Gap box binding factor (GAPF) interacts with this element [26]. A dehydrogenase is an enzyme of the oxidoreductase group that oxidizes a substrate by reducing an electron acceptor. Dehydrogenases are important enzymes in plant primary metabolism [27].
EIN3 BS2 (ATGTATAC)—a regulatory element located in the regulatory region of the ethylene-insensitive 3 (EIN3)-binding F box protein 2 (EBF2) gene of A. thaliana, which is a part of the Skp, Cullin, F-box containing complex (SCF complex). SCF is a multi-protein E3 ubiquitin ligase complex that catalyzes the ubiquitination of proteins destined for 26S proteasomal degradation. It is located in the nucleus and is involved in the ethylene-response pathway [28]. EIN3, or ethylene-insensitive 3, is the binding factor for EIN3 BS2, and is a nuclear transcription factor that initiates downstream transcriptional cascades for ethylene responses.
E-box (CACTTG)—a regulatory element located in the promoter region of the flowering factor gene CRY2-interacting bHLH 1, cryptochrome-interacting main helix-loop-helix (CIB1). The CIB1 protein recognizes E-box and activates the transcription of the flowering factor of A. thaliana FT (Flowering locus T) by binding to cryptochrome CRY2 in the presence of blue light [29]. However, representatives of the CIB1-CIB5 proteins do not need the presence of blue light and CRY2 to enhance transcription of the target gene, only the presence of a recognizable regulatory E-box element is sufficient [29].
ANAC089 BS 2 (CATCCT)—a regulatory element located in the regulatory region of the chloroplastic stromal ascorbate peroxidase sAPX gene. sAPX scavenges hydrogen peroxide in plant cells [30]. Protein ANAC089 interacts with this regulatory element. NAC—NAM, ATAF, and CUC domain proteins comprise one of the largest plant-specific transcription factor families, represented by ~105 genes in Arabidopsis. ANAC089—one of the proteins of the NAC family transcription factor that negatively regulates floral initiation.
Probably, the presence of described regulatory elements within the VaSTS11 intron signals to the GAPF, EIN3, CIB, bHLH122, and ANAC089 A. thaliana proteins about the need to activate transcription of the VaSTS11d transgene. The VaSTS11c transgene does not bear such regulatory elements and therefore exhibits a lower expression level in general. This assumption can explain the differences in the level of VaSTS11 expression, VaSTS11 cytosine methylation, and stilbene contents in the two analyzed generations of A. thaliana.
We also noticed that the flower number was 2–3 times higher in VaSTS11d-overexpressing A. thaliana than in VaSTS11c-overexpressing ones. The analysis revealed that there are regulatory elements both stimulating (E-box) and inhibiting (ANAC089 BS 2) flowering. Interestingly, the number of known E-box-interacting proteins is higher than the number of ANAC089-interacting proteins. Perhaps E-box serves as a signal to accelerate flowering in the intron-containing VaSTS11d-transgenic A. thaliana.
GapB—chloroplast localized glyceraldehyde-3-phosphate dehydrogenase; GeneID: 840895 (GAPB), TAIR: AT1G42970. GAPF—Gap box binding factor. EBF2—ethylene-insensitive 3 (EIN3)-binding F box protein 2 of Arabidopsis thaliana, part of the Skp, Cullin, F-box-containing complex (SCF complex) is a multi-protein E3 ubiquitin ligase complex that catalyzes the ubiquitination of proteins destined for 26S proteasomal degradation, it is located in the nucleus and is involved in the ethylene-response pathway; GeneID:832606, TAIR:AT5G25340. EIN3—ethylene-insensitive 3, a nuclear transcription factor that initiates downstream transcriptional cascades for ethylene responses; GeneID:821625, TAIR:AT3G20770. FT—flowering locus T, promotes flowering and FT is expressed in leaves and is induced by long-day treatment; GeneID:842859, TAIR:AT1G65480. CIB1—transcription factor, cryptochrome-interacting basic-helix-loop-helix 1. CIB1 interacts with CRY2 (cryptochrome 2) in a blue-light-specific manner in yeast and Arabidopsis cells, and it acts together with additional CIB1-related proteins to promote CRY2-dependent floral initiation. CIB1 positively regulates FT expression; GeneID:829605, TAIR:AT4G34540. CIB2—cryptochrome-interacting basic-helix-loop-helix 2; GeneID:834912, TAIR:AT5G48560. CIB3—cryptochrome-interacting basic-helix-loop-helix 3; GeneID:819922, TAIR:AT3G07340. CIB4—cryptochrome-interacting basic-helix-loop-helix 4; GeneID:837549, TAIR:AT1G10120. CIB5—cryptochrome-interacting basic-helix-loop-helix 5; GeneID:839167, TAIR:AT1G26260. At3g14205—phosphoinositide phosphatase family protein, suppressor of actin 2 (SAC2); GeneID:820638, TAIR:AT3G14205. ERF6 (At4g17490)—ethylene responsive element binding factor 6, encodes a member of the ERF (ethylene response factor) subfamily B-3 of ERF/AP2 transcription factor family (ATERF-6). It is involved in the response to reactive oxygen species and light stress; GeneID:827463, TAIR:AT4G17490. bHLH122—encodes a basic helix-loop-helix-type (bHLH) transcription factor involved in photoperiodism flowering; GeneID:841537, TAIR:AT1G51140. sAPX—a chloroplastic stromal ascorbate peroxidase that scavenges hydrogen peroxide in plant cells; GeneID:826396, TAIR:AT4G08390. ANAC089—NAM (no apical meristem), ATAF (Arabidopsis transcription activation factor), and CUC (cup-shaped cotyledon) or NAC domain containing protein 89, a membrane-tethered transcription factor that negatively regulates floral initiation; GeneID:832289, TAIR:AT5G22290. * http://www.softberry.com/berry.phtml?topic=nsitep&group=programs&subgroup=promoter, accessed on 1 April 2023 [7].
Thus, we analyzed the VaSTS11 gene, as a representative of the multigenic STS family with the shortest intron in V. amurensis, introns in other genes are much larger (Figure 5), so they carry even more regulatory elements that may have a different effect on transgene expression.

2.5. Stilbene and Biomass Accumulation in the Grapevine VaSTS11-Transgenic Cell Lines

In order to verify the higher effect of plant transformation with the intron-containing VaSTS11d on the transgene transcript level and stilbene production in comparison with VaSTS11c, we applied the vector constructions with the VaSTS11d and VaSTS11c to another model system, i.e., agrobacterium-mediated transformation of grapevine cell cultures with the intron-containing VaSTS11d and intronless VaSTS11c transgenes.
To establish VaSTS11-transgenic cell cultures of V. amurensis, the V7 suspension culture of V. amurensis was incubated with the A. tumefaciens strains. After two days in suspension, cefotaxime (250 mg/L) was added to remove agrobacteria, and on the fifth day of cultivation the suspension cells were transferred onto the solid supplemented with the selective antibiotic Km (10–20 mg/L). Then, we selected transgenic callus cell aggregates in the presence of Km for four months and established several Km-resistant independently obtained callus cell lines as described [31]. These calli did not undergo differentiation on the WB/A in the dark. For further analysis, we used the control VC transgenic cell line and six transgenic cell lines independently transformed with the VaSTS11c and VaSTS11d genes: three VaSTS11c-transformed cell lines (11c-1, 11c-2, and 11c-3) and three VaSTS11d-transformed cell lines (11d-1, 11d-2, and 11d-3) of V. amurensis (Figure 6).
The VaSTS11c- and VaSTS11d-transgenic cell lines of V. amurensis were proved by RT-qPCR for expression of the VaSTS11 transgenes. All of the VaSTS11-transformed cell lines actively expressed the transgenes, except for the 11c-1 line (Figure 6a). The highest expression of the VaSTS11 transgene was detected in the 11d-3 cell line and the lowest in the 11c-1 line (Figure 6a). In general, the expression of the VaSTS11 gene was 1.2–1.9 times higher in the 11d lines than in the 11c lines (Figure 6a).
Higher transgene expression in VaSTS11d-transgenic lines correlated (r = 0.77) with a higher stilbene content in comparison with the transgene expression and stilbene content in VaSTS11c lines (Figure 6a,b). The highest total stilbene content was detected in the 11d-3 and 11d-2 cell lines reaching 7.3 and 8.5 mg/g DW, which were 5.9 and 6.8 times higher than in the control VC cell line (Figure 6b; Table 2). In general, the total stilbene content and production was 1.6–2.6 times higher in VaSTS11d-transgenic lines than in VaSTS11c-transgenic lines (Figure 6b; Table 2). The enhancement in the total content of stilbenes in transgenic grapevine cell lines was primarily due to an increase in the content of t-resveratrol (Table S1).
The callus tissue samples were harvested from the 35-day-old cultures. The data are presented as mean ± standard error (SE) and were evaluated by one-way analysis of variance (ANOVA), followed by the Tukey HSD multiple comparison test performed in Excel using the XLSTAT software, Version 2023, where p < 0.05 was considered to be statistically significant.
The highest stilbene production was observed in the 11d-3 and 11d-2 VaSTS11-transgenic cell lines and reached 77.3 mg/L and 91.8 mg/L, respectively (Table 2). A comparison of the data with the previously published papers revealed that these are the highest values of stilbene levels produced by plant cell cultures overexpressing STS genes [19,33,34,35,36]. Previously, the highest stilbene production level reached 25.4 mg/L in a cell culture of V. amurensis overexpressing the PjSTS3 gene from spruce Picea jezoensis [36], which is 3.6 times less than the content of stilbenes in STS11d-2 and 1.3 times less than the content of stilbenes in the STS11c-2 cell line (Table 2). Thus, grapevine cell cultures also showed better properties of the intron-containing VaSTS11d transgene in the activation of stilbene biosynthesis in comparison with VaSTS11c.

3. Conclusions

Using intron-containing and intronless transgene forms, we found that the intron-containing VaSTS11d was better expressed than the intronless transgene sequence (VaSTS11c) in T4 and T7 plant generations of transgenic Arabidopsis and in one grapevine cell culture. The data revealed that the function of the plant transgene with an intron was more pronounced since the content and production of stilbenes was higher in the VaSTS11d-transgenic Arabidopsis plants and grape cells compared with VaSTS11c-transgenic plants. The same results were shown on transgenic Arabidopsis plants, where introducing an intron into a transgene reduced silencing more than four-fold [3].
The active expression of the VaSTS11d transgene with the intron was associated with a lower level of transgene cytosine methylation. A reduction in DNA methylation assumed that pre-mRNA splicing involves interactions between the cap-binding complex and components of the spliceosome. These interactions reduce the ability of the spliced transcript to act as a substrate for an RNA-dependent RNA polymerase [3]. Probably, the intron could exert an effect on STS expression via an ncRNA which performs gene regulatory functions [6]. Intron-containing genes are suggested to be preferentially channeled to exonucleolytic RNA decay pathways [3,5].
It is possible that the regulatory elements in the intron of VaSTS11 had a side effect on plant development, since in our case the plants began to flower faster, which can be explained by the presence of the flowering regulatory elements, but this suggestion requires further investigation.
Modern agricultural biotechnology is heavily dependent on using Agrobacterium to create transgenic plants, and it is difficult to think of an area of plant science research that has not benefited from this technology [37]. However, the data of the present study indicate that the transgene lost its properties after the seventh plant generation. Thus, there was no long-term fixation of this gene in the Arabidopsis genome, and this requires separate investigation.
In summary, the data obtained revealed that transgenes with introns have a great potential for use in plant biotechnology, and the results are important for understanding transgene heritage and obtaining vector constructions for effective and stable transgene expression in several plant generations.

4. Materials and Methods

4.1. Plant Material and Cell Cultures

Plants of Arabidopsis thaliana (L.) Heynh. ecotype Columbia-0 (stored by our lab) were grown in pots filled with commercially available rich soil (“Universalniy”, Fasko, Moscow, Russia) in an environmental control chamber (Sanyo MLR-352, Panasonic, Tokyo, Japan) kept on a 16/8 h d/night cycle at +22 °C and a light intensity of 120 μmol m−2 s−1.
For experiments, A. thaliana seeds were sterilized for 40–50 min in glass with chlorine vapors, which were released when 3 mL concentrated HCl was added to the 100 mL of bleach (Sayanskhimplast, 7%, Sayansk, Russia). Then, sterile seeds were germinated in Petri dishes in an environmental chamber (+22 °C and 120 μmol m−2 s−1) on 1/2 Murashige and Skoog medium (MS), pH 5.6 solidified with 0.8% agar. Then, the seedlings grown on the MS medium in Petri dishes for 7–8 days were transferred to commercially available soil.
The V7 callus cultures were established in 2017 from young stems of wild-growing mature V. amurensis vines near Vladivostok as described in [38]. The V7 cells were grown for 32–35 days in dark on MS-modified WB/A medium [39] supplemented with 0.5 mg/L BAP, 2 mg/L NAA, and 8 g/L agar in the dark.

4.2. Overexpression of VaSTS11c and VaSTS11d in Arabidopsis Plants and Cell Cultures of V. amurensis

The VaSTS11 transgene was used in two forms, including intronless VaSTS11c and intron-containing VaSTS11d (Figure 7a). The VaSTS11d transgene contained two exons and one intron: exon 1 (E1, 180 bp), exon 2 (E2, 1000 bp), and intron (I, 135 bp). The VaSTS11c transgene contained only two exons: E1 and E2 (Figure 1a and Figure S1).
To generate the construction for plant cell transformation, the full-length sequences of VaSTS11d gene and VaSTS11c transcript were amplified by PCR using the primers presented in the Supplementary Figure S1 and Table S2. For amplification of VaSTS11c transcript, we used cDNA obtained from a leaf of V. amurensis, and for VaSTS11d we used DNA from a leaf of V. amurensis. We designed primers to the 5′ and 3′ ends of the VaSTS11d and VaSTS11c cDNA coding sequences based on the known VaSTS11d and VaSTS11c sequences in V. amurensis, respectively (GenBank accession number OQ645979, OQ658380).
The obtained PCR products, VaSTS11d and VaSTS11c, were subcloned into a pJET1.2 using CloneJET PCR Cloninig Kit (ThermoFisher Scientific, Waltham, MA, USA) and sequenced using an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Then, we performed PCR with the forward primer containing a BglII restriction site and the reverse primer containing a Sal I restriction site (Supplementary Table S2).
The VaSTS11d and VaSTS11c were cloned into the pSAT1 vector [40] by the BglII and Sal I sites. Next, the expression cassette from pSAT1 with the STS genes was cloned into the pZP-RCS2-nptII vector [40,41] using the PalAI (AscI) sites. The pZP-RCS2-nptII construction also carried the nptII gene. All transgenes in the used vectors were under the control of the double cauliflower mosaic virus (CaMV 35S) promoter. The overexpression constructs of VaSTS11 (pZP-RCS2-VaSTS11d-nptII or pZP-RCS2-VaSTS11c-nptII) or empty vector (pZP-RCS2-nptII) were introduced into the of Agrobacterium tumefaciens strain (GV3101::pMP90), which was used for the floral dip transformation of A. thaliana [18] or for the transformation of the suspension V7 culture of V. amurensis [31,38,42].
Two independent fertile T3 homozygous lines of A. thaliana transformed with the pZP-RCS2-VaSTS11d-nptII (ST11d-1, ST11d-2) or pZP-RCS2-VaSTS11c-nptII (ST11c-1, ST11c-2) were chosen for detailed analyses. The transgenic lines used in this study were homozygous plants with a single-copy insertion. We determined the transgene copy number in accordance with the previously published work [43]. We identified the Arabidopsis homozygous lines by germination of all seeds (green seedling) of T3 transgenic Arabidopsis plants on Petri dishes on 1/2 MS (pH 5.6, solidified with 0.8% agar) with the addition of a selective antibiotic Km (50 mg/L).
Additionally, we obtained the control VC transgenic V. amurensis cell line and six transgenic cell lines independently transformed with the VaSTS11c and VaSTS11d genes: three VaSTS11c-transformed cell lines (11c-1, 11c-2, and 11c-3) and three VaSTS11d-transformed cell lines (11d-1, 11d-2, and 11d-3) of V. amurensis.
The T1-T4 generations of A. thaliana were selected in the presence of a selective antibiotic kanamycin (Km) at a concentration of 50 mg/L, but then we had to reduce the Km dose to 25 mg/L, since the T5 and T6 generations exhibited slower growth and did not produce seeds at 50 mg/L of Km. Then, the Km concentration was reduced to 12.5 mg/L for the T7 and T8 generations. Notably, the T8 generation grew slowly and did not produce seeds even at this low Km concentration. Km concentration was not further reduced, since non-transgenic plants have been capable of growing and producing seeds at a Km concentration less than 10 mg/L, i.e., transgene selection would not be achieved.
All further experiments were carried out on the T4 and T7 generations of transgenic A. thaliana plants (Figure 7b). We used the T4 transgenic plant generation, since it was the first generation with a lot of seeds and plant biomass, which is necessary for efficient nucleic acid isolation and HPLC analysis. The T7 generation was used because this was the last generation of the transgenic A. thaliana, which could grow and produce seeds on Km (12.5 mg/L). The T8 generation of the transgenic A. thaliana plants presented small plants (rosettes less than 1 cm), which quickly formed stems with flowers, but these flowers did not produce seeds.
To confirm the elimination of A. tumefaciens, we used RT-qPCR of the VirB2 gene using primers presented in Table S2. The transgenic calli were incubated in 100 mL flasks with 50 mL of the solid MS-modified medium [39] supplemented with 0.5 mg/L BAP, 2 mg/L NAA, and 8 g/L agar in the dark. For biomass accumulation and stilbene analysis, the grapevine cell cultures were incubated at 35-day subculture intervals in the dark at 24–25 °C in test tubes (height 150 mm, internal diameter 14 mm) with 7–8 mL of the medium.

4.3. HPLC and Mass Spectrometry Stilbene Analysis

Stilbene levels were analyzed by HPLC with diode array detection (HPLC-DAD) as described [39,44]. The extracts were separated on Shim-pack GIST C18 column (150 mm, 2.1 nm i.d., 3 nm part size; Shimadzu, Japan) on the HPLC LC-20AD XR analytical system (Shimadzu, Japan), equipped with an SPD-M20A photodiode array detector. The mobile phase consisted of a gradient elution of 0.1 % aqueous formic acid (A) and acetonitrile (B). An amount of 1 µL of the sample extract was injected with a constant column temperature maintained at 40 °C.

4.4. Nucleic Acid Purification and RT-qPCR

The cetyltrimethylammonium bromide (CTAB)-based extraction was used for total DNA isolation as described [45]. The CTAB-based extraction was used for total RNA isolation as described [46]. cDNAs were produced using the MMLV Reverse transcription PCR Kit with oligo(dT)15 (RT-PCR, Evrogen, Moscow, Russia) as described [47].
The mRNA transcript levels of the transgenes were determined by the 2−ΔΔCT method [32] with two internal controls, incuding AtGAPDH (NM_111283.4) and AtEF (XM_002864638) for Arabidopsis, and VaGAPDH (XM_002263109) and VaActin1 (DQ517935) for grape V. amurensis as described [48]. The primers designed for RT-qPCRs are shown in Table S2.
RT-qPCR reactions were performed in volumes of 20 µL using the real-time PCR kit (Evrogen) as described [46,47], containing 1 x Taq buffer, 2.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 µM of each oligonucleotide primer, 1x SybrGreen I Real-time PCR dye, 1 µL cDNAs, and 1 unit of Taq DNA polymerase (Evrogen). Analysis was performed in DTprime 4M1 Thermal Cycler (DNA-technology, Moscow, Russia) programmed for an initial denaturation step of 2 min at 95 °C followed by 50 cycles of 10 s at 95 °C and 25 s at 62 °C.

4.5. Statistical Analysis

For the analysis of the VaSTS transgene expression, we performed two independent experiments with ten technical replicates (five RT-qPCR reactions normalized to one internal control gene and five RT-qPCR reactions normalized to the second internal gene in each independent experiment). Three independent experiments with ten technical replicates in each experiment were performed for callus tissue weight analysis and three independent experiments with two technical replicates in each experiment for the stilbene analysis. The data are shown as mean ± standard error (SE) and were evaluated by Student’s t test or by one-way analysis of variance (ANOVA), followed by the Tukey HSD multiple comparison test performed in Excel using the XLSTAT software, Version 2023, where p < 0.05 was considered to be statistically significant.

Supplementary Materials

https://www.mdpi.com/article/10.3390/horticulturae9040513/s1, Figure S1: Nucleotide sequence of the VaSTS11d transgene, 135-nt intron is highlighted with an underscore and a thick font. Table S1: The content of individual stilbenes (mg per g of the dry weight (DW)) in the transgenic cell lines of Vitis amurensis transformed with VaSTS11d or VaSTS11c gene transcripts. Table S2: Primers used for amplification of Arabidopsis thaliana and Vitis amurensis cDNAs in PCR.

Author Contributions

A.S.D. and K.V.K. performed research design, interpretation, and paper preparation. A.A.A. and O.A.A. performed experiments with cell cultures, RNA and DNA isolations, and data analysis. A.R.S. performed HPLC analysis. A.A.A., Z.V.O. and N.N.N. performed RT-qPCRs and participated in data analysis. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the grant of the Russian Science Foundation # 22-16-00078, https://rscf.ru/en/project/22-16-00078/ (accessed on 9 April 2023) (Agrobacterial transformation, HPLC analysis, bisulfite sequencing, real-time qPCR). The collection and maintenance of plant material was carried out within the state assignment of Ministry of Science and Higher Education of the Russian Federation (theme No. 121031000144-5).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Quantification of the VaSTS11d and VaSTS11c transgene expression levels in transgenic Arabidopsis thaliana of T4 and T7 generation performed by quantitative RT-PCR. Wt—wild-type A. thaliana plants; KA0—the control KA0 A. thaliana plants transformed with the vector harboring only the nptII selective marker; ST11d-1, ST11d-2—two transgenic lines of A. thaliana plants transformed with the intron-containing VaSTS11d; ST11c-1, 2—A. thaliana plants transformed with the intronless VaSTS11c; r.u.—related units; T4 and T7—VaSTS11d- and VaSTS11c-transgenic A. thaliana in the fourth (T4) and seventh (T7) generations. The data are presented as mean ± SE (two independent experiments with eight technical replicates). Means followed by the same letter were not different using Student’s t test, where p < 0.05 was considered to be statistically significant.
Figure 1. Quantification of the VaSTS11d and VaSTS11c transgene expression levels in transgenic Arabidopsis thaliana of T4 and T7 generation performed by quantitative RT-PCR. Wt—wild-type A. thaliana plants; KA0—the control KA0 A. thaliana plants transformed with the vector harboring only the nptII selective marker; ST11d-1, ST11d-2—two transgenic lines of A. thaliana plants transformed with the intron-containing VaSTS11d; ST11c-1, 2—A. thaliana plants transformed with the intronless VaSTS11c; r.u.—related units; T4 and T7—VaSTS11d- and VaSTS11c-transgenic A. thaliana in the fourth (T4) and seventh (T7) generations. The data are presented as mean ± SE (two independent experiments with eight technical replicates). Means followed by the same letter were not different using Student’s t test, where p < 0.05 was considered to be statistically significant.
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Figure 2. HPLC detection (a,b) and quantification (c) of the trans-piceid content in the VaSTS11d and VaSTS11c transgenic Arabidopsis thaliana. Wt—wild-type A. thaliana plants; KA0—the control KA0 A. thaliana plants transformed with the vector harboring only the nptII selective marker; ST11d-1, ST11d-2—two A. thaliana plant lines transformed with the VaSTS11d gene; ST11c-1, ST11c-2—two A. thaliana plant lines transformed with the VaSTS11c gene; T4 and T7—VaSTS11d and VaSTS11c expression in transgenic A. thaliana of the T4 and T7 generations. The data are presented as mean ± SE (two independent experiments with eight technical replicates). Means followed by the same letter were not different using Student’s t test, where p < 0.05 was considered to be statistically significant.
Figure 2. HPLC detection (a,b) and quantification (c) of the trans-piceid content in the VaSTS11d and VaSTS11c transgenic Arabidopsis thaliana. Wt—wild-type A. thaliana plants; KA0—the control KA0 A. thaliana plants transformed with the vector harboring only the nptII selective marker; ST11d-1, ST11d-2—two A. thaliana plant lines transformed with the VaSTS11d gene; ST11c-1, ST11c-2—two A. thaliana plant lines transformed with the VaSTS11c gene; T4 and T7—VaSTS11d and VaSTS11c expression in transgenic A. thaliana of the T4 and T7 generations. The data are presented as mean ± SE (two independent experiments with eight technical replicates). Means followed by the same letter were not different using Student’s t test, where p < 0.05 was considered to be statistically significant.
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Figure 3. Cytosine methylation level (%) within the selected part of the protein coding regions of the VaSTS11d and VaSTS11c genes. Wt—wild-type A. thaliana plants; KA0—the control KA0 A. thaliana plants transformed with the vector harboring only the nptII selective marker; ST11d-1, ST11d-2—A. thaliana plants transformed with the VaSTS11d gene; ST11c-1, ST11c-2—A. thaliana plants transformed with the VaSTS11c gene; T4 and T7—VaSTS11d and VaSTS11c expression in transgenic A. thaliana of the T4 and T7 generations; n.m.—not measured. The data are presented as mean ± SE (two independent experiments with eight technical replicates). Means followed by the same letter were not different using Student’s t test, where p < 0.05 was considered to be statistically significant.
Figure 3. Cytosine methylation level (%) within the selected part of the protein coding regions of the VaSTS11d and VaSTS11c genes. Wt—wild-type A. thaliana plants; KA0—the control KA0 A. thaliana plants transformed with the vector harboring only the nptII selective marker; ST11d-1, ST11d-2—A. thaliana plants transformed with the VaSTS11d gene; ST11c-1, ST11c-2—A. thaliana plants transformed with the VaSTS11c gene; T4 and T7—VaSTS11d and VaSTS11c expression in transgenic A. thaliana of the T4 and T7 generations; n.m.—not measured. The data are presented as mean ± SE (two independent experiments with eight technical replicates). Means followed by the same letter were not different using Student’s t test, where p < 0.05 was considered to be statistically significant.
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Figure 4. Regulatory elements found in the single 135 bp intron of the VaSTS11 gene using NSITE-PL database [7].
Figure 4. Regulatory elements found in the single 135 bp intron of the VaSTS11 gene using NSITE-PL database [7].
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Figure 5. Schematic representation of the introns in the 38 functional Vitis vinifera VvSTSs genes (except VvSTS12 and VvSTS25 due to the lack of intron 1 sequence) and the Vitis amurensis VaSTS11 gene, a close homolog of the VvSTS16 and VvSTS22 genes.
Figure 5. Schematic representation of the introns in the 38 functional Vitis vinifera VvSTSs genes (except VvSTS12 and VvSTS25 due to the lack of intron 1 sequence) and the Vitis amurensis VaSTS11 gene, a close homolog of the VvSTS16 and VvSTS22 genes.
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Figure 6. Quantification the VaSTS11 transgene mRNAs performed by quantitative RT-PCR (a) and quantification of total stilbene content performed by HPLC (b) in the transgenic cell cultures of Vitis amurensis. VC—the control V. amurensis cell line transformed with the vector harboring only the nptII selective marker; 11d-1, 11d-2, and 11d-3—V. amurensis cell lines independently transformed with VaSTS11d; 11c-1, 11c-2, and 11c-3—V. amurensis cell lines independently transformed with VaSTS11c; r.u.—related units calculated as described in [32]; fluorescence in VC is indicated as “1”. The data are presented as mean ± SE (two independent experiments with eight technical replicates for quantitative RT-PCR and three independent experiments with two technical replicates). Means followed by the same letter were not different using Student’s t test, where p < 0.05 was considered to be statistically significant.
Figure 6. Quantification the VaSTS11 transgene mRNAs performed by quantitative RT-PCR (a) and quantification of total stilbene content performed by HPLC (b) in the transgenic cell cultures of Vitis amurensis. VC—the control V. amurensis cell line transformed with the vector harboring only the nptII selective marker; 11d-1, 11d-2, and 11d-3—V. amurensis cell lines independently transformed with VaSTS11d; 11c-1, 11c-2, and 11c-3—V. amurensis cell lines independently transformed with VaSTS11c; r.u.—related units calculated as described in [32]; fluorescence in VC is indicated as “1”. The data are presented as mean ± SE (two independent experiments with eight technical replicates for quantitative RT-PCR and three independent experiments with two technical replicates). Means followed by the same letter were not different using Student’s t test, where p < 0.05 was considered to be statistically significant.
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Figure 7. (a) Schematic representation of the VaSTS11d and VaSTS11c transgene; E1—first exon; I—intron; E2—second exon. (b) Schematic representation of the selection procedure for transgenic Arabidopsis thaliana; T0—transformed A. thaliana plants; T1–T8—different generations of A. thaliana transgenic plants; Km—kanamycin.
Figure 7. (a) Schematic representation of the VaSTS11d and VaSTS11c transgene; E1—first exon; I—intron; E2—second exon. (b) Schematic representation of the selection procedure for transgenic Arabidopsis thaliana; T0—transformed A. thaliana plants; T1–T8—different generations of A. thaliana transgenic plants; Km—kanamycin.
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Table
Table 1. Information on the regulatory element (RE) found in the singular 135 bp VaSTS11 intron sequences in the NSITE-PL database.
Table 1. Information on the regulatory element (RE) found in the singular 135 bp VaSTS11 intron sequences in the NSITE-PL database.
RE NameRE Registration Number in the NSITE-PL Database GeneRE Binding Factors
Gap box 2 1027, RSP01020GapB GAPF
EIN3 BS22013, RSP01979EBF2 EIN3
E-box2473, RSP02439FT CIB1; CIB2, CIB3, CIB4, CIB5
E-box2475, RSP02441FT CIB2; CIB4; CIB5
E-box2645, RSP02611At3g14205 bHLH122
E-box2647, RSP02613ERF6 (At4g17490) bHLH122
E-box2649, RSP02615ERF6 (At4g17490) bHLH122
ANAC089 BS 22664, RSP02630sAPX ANAC089
Table
Table 2. Biomass and stilbene accumulation in the cell lines of Vitis amurensis overexpressing the VaSTS11c or VaSTS11d gene transcripts. Means followed by the same letter were not different using Student’s t test, where p < 0.05 was considered to be statistically significant.
Table 2. Biomass and stilbene accumulation in the cell lines of Vitis amurensis overexpressing the VaSTS11c or VaSTS11d gene transcripts. Means followed by the same letter were not different using Student’s t test, where p < 0.05 was considered to be statistically significant.
Cell Line Overexpressed STS GeneFresh Weight, g/LDry Weight, g/LTotal Stilbene Content, mg/g DWTotal Stilbene Production, mg/L
KA0-222.1 ± 12.2 b9.77 ± 0.84 a1.24 ± 0.17 c12.1 ± 2.6 d
11d-1VaSTS11d260.1 ± 14.7 a10.98 ± 0.77 a3.71 ± 1.07 b40.7 ± 7.5 b
11d-2262.2 ± 15.1 a10.81 ± 0.92 a8.49 ± 1.25 a91.8 ± 8.9 a
11d-3232.4 ± 13.3 ab10.64 ± 0.65 a7.26 ± 1.40 ab77.3 ± 7.7 a
11c-1VaSTS11c253.6 ± 15.2 ab9.81 ± 0.74 a2.28 ± 1.02 bc22.4 ± 5.0 cd
11c-2247.4 ± 15.5 ab9.88 ± 0.98 a3.21 ± 0.38 b31.7 ± 5.3 bc
11c-3238.7 ± 16.6 ab9.84 ± 0.75 a2.66 ± 0.37 b26.2 ± 6.1 bc
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Kiselev, K.V.; Ogneva, Z.V.; Aleynova, O.A.; Suprun, A.R.; Ananev, A.A.; Nityagovsky, N.N.; Dubrovina, A.S. Influence of the 135 bp Intron on Stilbene Synthase VaSTS11 Transgene Expression in Cell Cultures of Grapevine and Different Plant Generations of Arabidopsis thaliana. Horticulturae 2023, 9, 513. https://doi.org/10.3390/horticulturae9040513

AMA Style

Kiselev KV, Ogneva ZV, Aleynova OA, Suprun AR, Ananev AA, Nityagovsky NN, Dubrovina AS. Influence of the 135 bp Intron on Stilbene Synthase VaSTS11 Transgene Expression in Cell Cultures of Grapevine and Different Plant Generations of Arabidopsis thaliana. Horticulturae. 2023; 9(4):513. https://doi.org/10.3390/horticulturae9040513

Chicago/Turabian Style

Kiselev, Konstantin V., Zlata V. Ogneva, Olga A. Aleynova, Andrey R. Suprun, Alexey A. Ananev, Nikolay N. Nityagovsky, and Alexandra S. Dubrovina. 2023. "Influence of the 135 bp Intron on Stilbene Synthase VaSTS11 Transgene Expression in Cell Cultures of Grapevine and Different Plant Generations of Arabidopsis thaliana" Horticulturae 9, no. 4: 513. https://doi.org/10.3390/horticulturae9040513

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