Effect of Calmodulin-like Gene (CML) Overexpression on Stilbene Biosynthesis in Cell Cultures of Vitis amurensis Rupr.

Stilbenes are plant phenolics known to rapidly accumulate in grapevine and other plants in response to injury or pathogen attack and to exhibit a great variety of healing beneficial effects. It has previously been shown that several calmodulin-like protein (CML) genes were highly up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to stilbene-modulating conditions, such as stress hormones, UV-C, and stilbene precursors. Both CML functions and stilbene biosynthesis regulation are still poorly understood. In this study, we investigated the effect of overexpression of five VaCML genes on stilbene and biomass accumulation in the transformed cell cultures of V. amurensis. We obtained 16 transgenic cell lines transformed with the VaCML52, VaCML65, VaCML86, VaCML93, and VaCML95 genes (3-4 independent lines per gene) under the control of the double CaMV 35S promoter. HPLC-MS analysis showed that overexpression of the VaCML65 led to a considerable and consistent increase in the content of stilbenes of 3.8-23.7 times in all transformed lines in comparison with the control calli, while biomass accumulation was not affected. Transformation of the V. amurensis cells with other analyzed VaCML genes did not lead to a consistent and considerable effect on stilbene biosynthesis in the cell lines. The results indicate that the VaCML65 gene is implicated in the signaling pathway regulating stilbene biosynthesis as a strong positive regulator and can be useful in viticulture and winemaking for obtaining grape cultivars with a high content of stilbenes and stress resistance.

1. Introduction

Stilbenes are part of a vast group of plant polyphenolic compounds with antimicrobial activities synthesized via the phenylpropanoid pathway by a broad range of unrelated plant families [1,2,3]. Plant stilbenes are well-known to confer a variety of health benefits and possess promising pharmacological applications [4,5,6]. Stilbenes are also classified as phytoalexins that rapidly accumulate in plants in response to injury or microbial pathogens and contribute to plant environmental stress resistance [3,7]. Trans-resveratrol (t-resveratrol) is the most prominent stilbene with a variety of valuable biologically active properties and was widely used in the food and medicine industries [8]. Stilbenes are known to be involved in plant constitutive and inducible defense reactions against plant fungal and bacterial pathogens, nematodes, and herbivores [7,9]. Biosynthesis of natural stilbenoids is activated in response to a number of other environmental stresses, such as ultraviolet (UV) irradiation, wounding, drought, or unfavorable temperatures [3,9].

Although there is a lack of information about the signaling pathways and biochemical events regulating stilbene biosynthesis in plant tissues, it is known that (1) hormone signaling (salicylic acid (SA), methyl jasmonate (MeJA), ethylene, jasmonic acid, and abscisic acid) [10,11,12,13,14,15]; (2) production of reactive oxygens species (ROS) [10,12,15]; (3) stress-induced increase in calcium cytoplasmic concentration [10,11,16,17], and (4) activation of calcium-dependent protein kinases or CDPKs [18,19,20] are implicated in the activation of stilbene biosynthesis in plant tissues. These signaling events eventually lead to the activation of specific transcription factors responsible for the induction of stilbene synthases (STSs) and other stilbene biosynthetic genes [13,21,22]. Furthermore, our recent results demonstrated high responsiveness of plant calmodulin-like protein (CML) genes to the agents stimulating the biosynthesis of stilbenes, including stilbene precursors, SA and MeJa, and UV-C exposure [23], which suggested plant CML genes as promising candidates for the activation of plant defense reactions and stilbene production. Thus, it is known that calcium signaling is implicated in the regulation of stilbene biosynthesis in grapevine [3].

Plants encode several classes of calcium sensor proteins, which perceive and decode the alterations in calcium cytoplasmic concentration induced in response to stress and developmental cues [24,25]. The plant calcium sensor proteins include calmodulins (CaMs), calmodulin-like proteins (CMLs), calcium-dependent protein kinases (CDPKs), calcineurin B-like proteins (CBLs), and CBL-interacting protein kinases (CIPKs) [26,27]. Most of the calcium sensor protein subfamilies are encoded by multigene families where the most numerous are CMLs, including 50 genes in Arabidopsis [28] and at least 54 CML genes in grapevine [29]. While the high number and diversity of plant CMLs suggest their importance in plant survival and productivity, the biological functions of this class of plant calcium-binding proteins remain poorly studied.

CML proteins were classified as “sensor relays” lacking “sensor responder” domains and catalytic activity [26,30]. After calcium binding, CMLs undergo conformational changes and interact with their target proteins whose activity is then altered. CMLs transmit the information from the environmental and developmental cues to various downstream biochemical events leading to a proper physiological response. The available research findings show that plant CMLs are involved in the regulation of various developmental processes, such as trichome branching or seed development, and plant abiotic and biotic stress responses [31,32,33,34]. It was shown that the expression of particular CML genes was activated in response to pathogen attack [35] or to stress hormones, such as methyl jasmonate or MeJA (AtCML39) and salicylic acid or SA (AtCML43) [36,37,38]. A number of studies show that overexpression of CML genes in plants improved plant abiotic stress resistance or contributed to plant–pathogen protection [39,40,41,42,43]. For example, CaCML13 acts positively in pepper immunity against Ralstonia solanacearum infection forming a feedback loop with CabZIP63 transcriptional factor [35].

At present, the relationship of plant CMLs and other calcium sensor proteins with plant defense reactions and production of plant secondary metabolites remains almost unexplored. Recently, it was shown that a number of CML genes, such as VaCML52, 65, 93, and 95, were highly up-regulated in the leaves and cell cultures of wild grape Vitis amurensis Rupr. in response to stilbene precursors, stress hormones (MeJA, SA) and UV-irradiation [23], which are known as potent inductors of stilbene biosynthesis. The Amur grape, Vitis amurensis Rupr., displays a high level of stress resistance and stilbene content compared to other Vitaceae species and is now commercially cultivated [3,44,45,46]. Vines of V. amurensis are used as a resistant rootstock to breed materials for novel cultivars. Elucidating the molecular mechanisms and pathways underlying the stilbene biosynthesis in V. amurensis is of high interest.

In the present study, we aimed to investigate and verify the function of the VaCML52, 65, 93, and 95 genes as potential positive regulators of stilbene biosynthesis in the grapevine by overexpressing the CML genes in cell cultures of V. amurensis. We also transformed V. amurensis with the VaCML86 gene, whose expression was not essentially changed in response to the stilbene-modulating conditions, with the purpose to use it as an additional control. Results of the present investigation demonstrated that the VaCML65 gene is a strong positive regulator of stilbene biosynthesis, while the VaCML52, 86, 93, and 95 genes are not actively involved in this process. The VaCML65 gene can be useful in viticulture and winemaking for obtaining grape cultivars with a high content of stilbenes and resistance to environmental stresses.

2. Results and Discussion

2.1. Genetic Transformation and Selection of the VaCML-Transgenic Cell Lines

To establish Vitis amurensis Rupr. cell cultures overexpressing the full-length VaCML52, VaCML65, VaCML86, VaCML93, and VaCML95 genes and the control cell line, the V7 suspension culture of V. amurensis was incubated with A. tumefaciens strains to bear the pZP-RCS2-VaCML-nptII construct for VaCML-transgenic cells or the pZP-RCS2-nptII for the control KA0 cell line. Then, we selected transgenic callus cell aggregates in the presence of 10–15 mg/L of kanamycin (Km) for four months and established several Km-resistant lines as described [19]. The semiquantitative RT-PCR has shown that the nptII gene was transcribed in all obtained transgenic cell lines, and the absence of A. tumefaciens was confirmed using RT-PCR to control the presence or absence of the VirB2 gene (Supplementary Figure S1). The selected transformed calli represented friable vigorously growing homogenous tissues, which did not undergo differentiation on the WB/A medium supplemented with 6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA) in the dark. For further analysis, we used 16 transgenic cell lines transformed with the VaCML genes: three VaCML52-transformed cell lines (52-1, 52-2, 52-3), four VaCML65-transformed cell lines (65-1, 65-2, 65-3, 65-4), three VaCML86-transformed cell lines (86-1, 86-2, 86-3), three VaCML93-transformed cell lines (93-1, 93-2, 93-3), and four VaCML95-transformed cell lines (95-1, 95-2, 95-3, 95-4) (Table 1). The control KA0 transformed calli reproduced morphological, growth, and biosynthetic characteristics of the parent V7 culture.

Table 1

Biomass accumulation and total stilbene production in the cell lines of Vitis amurensis overexpressing the VaCML52, VaCML65, VaCML86, VaCML93, or VaCML95 genes.

Cell Line	Overexpressed CML Gene	Fresh Weight, g/L	Dry Weight, g/L	Total Stilbene Production, mg/L
KA0	-	163.7 ± 13.3 b	6.65 ± 1.02 a	4.69 ± 1.54 d
52-1	VaCML52	155.0 ± 12.8 b	8.07 ± 1.17 a	13.82 ± 5.37 c
52-2		165.1 ± 14.4 b	6.77 ± 0.82 a	14.35 ± 4.88 c
52-3		165.2 ± 10.1 b	7.83 ± 0.97 a	8.93 ± 3.66 cd
65-1	VaCML65	131.3 ± 8.5 b	7.11 ± 0.88 a	135.66 ± 34.20 a
65-2		195.1 ± 15.5 ab	6.75 ± 0.91 a	56.67 ± 18.62 b
65-3		137.3 ± 14.9 b	6.91 ± 0.93 a	27.84 ± 13.25 b
65-4		201.1 ± 16.6 ab	8.81 ± 1.32 a	26.94 ± 10.19 bc
86-1	VaCML86	158.7 ± 11.2 b	6.69 ± 0.75 a	6.42 ± 1.88 d
86-2		158.9 ± 10.4 b	6.54 ± 0.98 a	3.22 ± 0.91 d
86-3		149.1 ± 8.9 b	5.71 ± 0.74 a	5.05 ± 1.74 d
93-1	VaCML93	197.1 ± 13.7 ab	8.75 ± 1.43 a	26.46 ± 11.47 bc
93-2		153.8 ± 12.2 b	6.21 ± 0.88 a	4.31 ± 1.12 d
93-3		218.5 ± 13.4 a	8.77 ± 1.50 a	11.07 ± 5.15 c
95-1	VaCML95	198.8 ± 10.8 ab	7.81 ± 0.99 a	3.78 ± 0.74 d
95-2		154.7 ± 11.1 b	6.75 ± 0.79 a	9.28 ± 4.13 cd
95-3		225.2 ± 15.6 a	8.64 ± 1.17 a	2.81 ± 0.65 d

The callus tissue samples were harvested from the 35-day-old cultures (three independent experiments with ten technical replicates for weight calculations and three independent experiments with two technical replicates for total stilbene measurement). Means followed by the same letter in one column were not different using one-way analysis of variance (ANOVA), followed by the Tukey HSD multiple comparison test.

The VaCML-transgenic cell lines were confirmed for expression of the transgene and endogenous VaCML52, 65, 86, 93, and 95 expressions by qRT-PCR (Figure 1). Most of the VaCML-transformed cell lines actively expressed the transgenes (Figure 1a–e). Additionally, the total expression of VaCML52, 65, 86, 93, and 95 genes exceeded that in the KA0 control in most of the VaCML-transformed calli, except for VaCML95 (Figure 1k–o). The analysis of the endogenous VaCML expression revealed that expression of endogenous VaCML86 and VaCML93 was not affected in all cell lines, while expression of VaCML52, 65, and 95 was altered in a part of the cell lines obtained in comparison with that in the control KA0 (Figure 1f–j). On one side, it is known that plant transgenes and/or plant homologous endogenous genes can be silenced as a result of induction of RNA interference (RNAi) and generation of transgene-derived small interfering RNA (siRNAs) [47,48]. In the course of RNAi, double-stranded RNAs (dsRNAs) are processed by a ribonuclease into siRNAs, which are incorporated in the RNA-induced silencing complex that provides cleavage, destabilization, or hindering translation of any homologous mRNAs [49,50]. On the other side, other studies show that in contrast to a conventional transgene, an endogene-resembling transgene was more stably expressed and poorly processed into small RNAs [51,52]. Therefore, there is a need to verify whether either transgene and/or endogene was silenced and to what degree after plant genetic transformation and establishment of plant transgenic cell cultures. The data obtained in the present work show that expression of the endogenous VaCML52 and VaCML95 genes was down-regulated after transformation in the 52-2, 52-3, and 95-3 lines, which suggested that the decrease in endogenous expression, in this case, might be caused by the activation of the RNAi process.

Figure 1

Quantification of the transgene (a–e), total (f–j), and endogenous (k–o) VaCML gene expression in the transgenic cells of Vitis amurensis performed by quantitative RT-PCR. RNA was extracted from control V. amurensis cell line transformed with the empty vector harboring only nptII selective marker (KA0) and V. amurensis cell lines transformed with the VaCML52 (a,f,k, VaCML52-1, -2, and -3), VaCML65 (b,g,l, VaCML65-1, -2, -3, and -4), VaCML86 (c,h,m, VaCML86-1, -2, and -3), VaCML93 (d,i,n, VaCML93-1, -2, and -3), and VaCML95 (e,j,o, VaCML95-1, -2, -3, and -4) genes. The data are presented as mean ± SE (two independent experiments with eight technical replicates). Means on each figure followed by the same letter were not different using one-way analysis of variance (ANOVA), followed by the Tukey HSD multiple comparison test.

2.2. Stilbene Content and Biomass Accumulation in the Grapevine VaCML-Transgenic Cell Lines

It is known that the highest content of stilbenes in callus cell cultures of V. amurensis was typical for the 35th day of cultivation [53]. Therefore, cell culture samples were collected from the 35-day-old calli for stilbene extraction and biomass analysis. In Table 1, we presented fresh and dry biomass accumulation in the VaCML-transformed cell lines of V. amurensis. Transformation of the V. amurensis cells with the analyzed VaCML genes did not substantially affect the fresh and dry growth parameters in most obtained cell lines, except for the fresh weight of the 93-3 and 95-2 cell lines (Table 1).

Using HPLC, we determined the content and composition of stilbenes in the obtained VaCML-transgenic cell lines. Overexpression of the VaCML65 gene led to a considerable increase in the content of stilbenes in all obtained transgenic lines in 3.8–23.7 times (Figure 2). Stilbene content in the VaCML65-transgenic cell lines reached 19.1 mg per g of the dry cells weight (DW) and stilbene production reached 136 mg per l (Figure 2, Table 1). This is one of the greatest values for cell cultures among the known data ([3], Figure 2, Table 1). This value was approximately 27 times higher than stilbene production in control cell culture KA0 (Table 1) and 4 times higher than stilbene production in VaCPK20 transgenic cell lines (up to 35 mg/L, [19]), but 1.2 times lower than that in the rolB transgenic cell culture of V. amurensis (152 mg/L [16]).

Figure 2

Total stilbene content in the cell lines of Vitis amurensis transformed with the VaCML52, VaCML65, VaCML86, VaCML93, or VaCML95 genes in mg per g of the dry weight (DW). KA0—control cell line transformed with the empty vector harboring only nptII selective marker; 52-1, 2, 3—cell lines transformed with the VaCML52 gene; 65-1, 2, 3, 4—cell lines transformed with the VaCML65 gene; 86-1, 2, 3—cell lines transformed with the VaCML86 gene; 93-1, 2, 3—cell lines transformed with the VaCML93 gene; 95-1, 2, 3—cell lines transformed with the VaCML95 gene. Means followed by the same letter were not different using one-way analysis of variance (ANOVA), followed by the Tukey HSD multiple comparison test (three independent experiments with two technical replicates). p < 0.05 was considered statistically significant.

Considering other VaCML genes, we observed an increase in the content and production of stilbenes in the 52-2 and 93-1 cell lines transformed with the VaCML52 and VaCML93 genes (Figure 2; Table 1). We consider this effect rather as a non-specific transformation effect since only one cell line out of three for VaCML52 or VaCML93 showed significant changes in the production of stilbenes. All other cell lines transformed with VaCML52, VaCML86, VaCML93, and VaCML95 showed no significant changes in the content and production of stilbenes (Figure 2; Table 1).

Overexpression of the VaCML65 gene did not change the spectrum of detected individual stilbenes (Table 2). The increase in the total content of stilbenes in the four VaCML65-transgenic cell lines was due to a strong increase in the content of t-resveratrol (Table 2). For example, in the VaCML65-transgenic lines, the content of t-resveratrol increased up to 18.1 mg/g DW, i.e., 4.6–43 times in comparison with resveratrol levels detected in the control KA0 cell line (Table 2). The content of other stilbenoid compounds in the VaCML65-transformed cells increased to a lesser extent or even decreased (Table 2). The data indicate that overexpression of the VaCML65 gene led to the increase in the content of stilbenes via activation of t-resveratrol biosynthesis (Table 2).

Table 2

The content of individual stilbenes (mg per g of the dry weight (DW)) in the transgenic Vitis amurensis cell lines transformed with VaCML52, VaCML65, VaCML86, VaCML93, or VaCML95 genes.

Cell Lines	Overexpressed CML Gene	t-Resveratrol Diglucoside	t-Piceid	t-Resveratrol	ε-Viniferin	δ-Viniferin	cis-Resveratrol	cis-Piceid	Piceatannol
KA0	-	0.12 ± 0.07 c	0.09 ± 0.03 b	0.42 ± 0.11 f	0.05 ± 0.02 b	0.12 ± 0.03 b	0 a	0 a	0.01 ± 0.01 a
52-1	VaCML52	0.18 ± 0.05 c	0.08 ± 0.03 b	1.06 ± 0.35 ef	0.08 ± 0.04 ab	0.28 ± 0.09 ab	0.01 ± 0.01 a	0.01 ± 0.01 a	0.01 ± 0.01 a
52-2		0.17 ± 0.06 c	0.07 ± 0.03 b	1.62 ± 0.70 de	0.06 ± 0.02 b	0.17 ± 0.05 ab	0 a	0.01 ± 0.01 a	0.01 ± 0.01 a
52-3		0.23 ± 0.09 c	0.07 ± 0.02 b	0.58 ± 0.21 f	0.09 ± 0.03 ab	0.18 ± 0.06 ab	0 a	0 a	0 a
65-1	VaCML65	0.26 ± 0.09 c	0.14 ± 0.04 b	18.06 ± 7.09 a	0.12 ± 0.06 ab	0.46 ± 0.16 a	0.01 ± 0.01 a	0.01 ± 0.01 a	0.02 ± 0.01 a
65-2		0.25 ± 0.08 c	0.12 ± 0.03 b	7.79 ± 3.31 ab	0.06 ± 0.03 b	0.17 ± 0.05 ab	0 a	0.01 ± 0.01 a	0.01 ± 0.01 a
65-3		0.72 ± 0.17 a	0.40 ± 0.13 a	2.31 ± 0.47 cd	0.12 ± 0.06 ab	0.25 ± 0.11 ab	0 a	0.01 ± 0.01 a	0.01 ± 0.01 a
65-4		0.68 ± 0.18 ab	0.25 ± 0.08 ab	1.95 ± 0.32 cd	0.08 ± 0.02 ab	0.35 ± 0.14 ab	0 a	0.01 ± 0.01 a	0.01 ± 0.01 a
86-1	VaCML86	0.22 ± 0.11 c	0.08 ± 0.03 b	0.33 ± 0.12 fg	0.07 ± 0.02 b	0.25 ± 0.09 ab	0.01 ± 0.01 a	0 a	0.01 ± 0.01 a
86-2		0.13 ± 0.07 c	0.05 ± 0.03 b	0.15 ± 0.04 g	0.08 ± 0.05 ab	0.19 ± 0.11 ab	0 a	0 a	0 a
86-3		0.25 ± 0.11 c	0.08 ± 0.03 b	0.29 ± 0.10 fg	0.05 ± 0.02 b	0.21 ± 0.08 ab	0.01 ± 0.01 a	0.01 ± 0.01 a	0.01 ± 0.01 a
93-1	VaCML93	0.14 ± 0.05 c	0.04 ± 0.02 b	2.32 ± 1.03 cd	0.15 ± 0.05 a	0.37 ± 0.11 ab	0.01 ± 0.01 a	0.01 ± 0.01 a	0.01 ± 0.01 a
93-2		0.31 ± 0.12 bc	0.07 ± 0.03 b	0.21 ± 0.09 g	0.02 ± 0.01 b	0.09 ± 0.04 b	0 a	0.02 ± 0.01 a	0a
93-3		0.32 ± 0.16 bc	0.11 ± 0.06 b	0.63 ± 0.24 f	0.05 ± 0.03 b	0.17 ± 0.10 ab	0 a	0 a	0 a
95-1	VaCML95	0.16 ± 0.06 c	0.06 ± 0.02 b	0.15 ± 0.06 g	0.03 ± 0.01 b	0.10 ± 0.03 b	0.01 ± 0.01 a	0.01 ± 0.01 a	0.01 ± 0.01 a
95-2		0.23 ± 0.07 c	0.05 ± 0.02 b	1.01 ± 0.37 ef	0.03 ± 0.02 b	0.09 ± 0.03 b	0 a	0.01 ± 0.01 a	0.01 ± 0.01 a
95-3		0.11 ± 0.03 c	0.06 ± 0.02 b	0.14 ± 0.07 g	0.02 ± 0.01 b	0.08 ± 0.03 b	0 a	0.01 ± 0.01 a	0 a

KA0—control cell line of V. amurensis transformed with the “empty” vector harboring only nptII selective marker; 52-1, 2, 3—V. amurensis cell lines transformed with the VaCML52 gene; 65-1, 2, 3, 4—V. amurensis cell lines transformed with the VaCML65 gene; 86-1, 2, 3—V. amurensis cell lines transformed with the VaCML86 gene; 93-1, 2, 3—V. amurensis cell lines transformed with the VaCML93 gene; 95-1, 2, 3—V. amurensis cell lines transformed with the VaCML95 gene. The callus tissue samples were harvested from the 35-day-old cell cultures. Means followed by the same letter in one row were not different using one-way analysis of variance (ANOVA), followed by the Tukey HSD multiple comparison test (three independent experiments with two technical replicates). p < 0.05 was considered statistically significant.

Then, it was important to verify whether the enhanced production of stilbenes in cell cultures overexpressing the VaCML65 gene was due to the activation of biosynthesis or to a reduction in the degradation of these compounds. For this purpose, we analyzed the expression of several important stilbene biosynthesis genes (Supplementary Figure S2), including five phenylalanine ammonia-lyase (PAL), ten stilbene synthase (STS), and five genes of VaMyb14, VaMyb15, VaMyb40, VaMyb60, and VaMyb107 transcription factors (Figure 3), which are known as possible positive regulators of stilbene biosynthesis [54]. Expression of these VaMyb genes was considerably increased in grapevine cells with a high stilbene content [54].

Figure 3

Quantification the VaPAL1-5 (a) and VaSTS1-5 (b), VaSTS6-10 (c), and VaMyb14, 15, 40, 60, and 107 (d) gene expression in the VaCML65-transgenic cell lines of Vitis amurensis performed by quantitative PCR (qRT-PCR). RNA was extracted from the vector control (KA0), VaCML65-1, -2, -3, and -4-transformed cell lines of V. amurensis. Means on each figure followed by the same letter were not different using one-way analysis of variance (ANOVA), followed by the Tukey HSD multiple comparison test (two independent experiments with eight technical replicates). p < 0.05 was considered to be statistically significant. n.d.—not detected.

We showed that overexpression of the VaCML65 gene led to considerably increased mRNA transcript levels of the VaPAL1, 2, 5 (Figure 3a) and VaSTS2, 3, 4, 5, 6, 8, and 10 genes (Figure 3b,c) in the four VaCML65-transgenic cell lines. The results show that the enhanced content of stilbenes in the obtained VaCML65-transgenic grape cells was associated with activation of stilbene biosynthesis via a considerable increase in the expression of certain PAL and STS genes (Figure 3). Moreover, some Myb transcription factors may be involved in this process, for example, VaMyb60, because expression levels of VaMyb60 increased in all lines after transformation with VaCML65 gene (Figure 3d).

3. Conclusions

In this paper, we investigated the effect of overexpression of five grapevine CML genes, which were highly up-regulated in wild-growing grapevine V. amurensis in response to stilbene-inducing conditions (stress hormones, phenolic precursors, and UV irradiation), and were suggested as promising candidates playing important roles in stilbene biosynthesis [43]. Previously, it was shown that up-regulation of the VaCML52, VaCML65, VaCML93, and VaCML95 genes was induced after cultivation in the presence of SA, coumaric acid, MeJA, or UV treatment and associated with an increase in the content of stilbenes in both callus cell cultures and leaves of V. amurensis [43]. However, only VaCML65 overexpression considerably induced stilbene levels in all independent VaCML65-transgenic cell lines. The data indicate that the VaCML65 gene is involved in the signaling pathway regulating stilbene biosynthesis and acts as a positive regulator of t-resveratrol production. The proposed model of the signaling pathway leading to stilbene biosynthesis induction with the involvement of VaCML65 in this process was presented in Figure S2. Briefly, after signal perception, stimulated receptors are proposed to induce calcium influx, which then leads to the activation of calcium sensor proteins, including VaCML65. It is possible that VaCML65 together with mitogen-activated protein kinases (MAPK) cascade and hormone signaling could activate expression of PAL, STS, and other genes responsible for stilbene biosynthesis, probably through activation of some transcription factors (e.g. VaMyb60).

The absence of a stable and considerable stilbene-modulating effect in the cell lines of V. amurensis overexpressing the VaCML52, VaCML86, VaCML93, and VaCML95 genes indicates that these VaCML genes are not directly involved in the regulation of stilbene biosynthesis in grapevine. It is also possible that additional proteins, which are not present in the cell cultures, are necessary for the work of the VaCML52, VaCML86, VaCML93, and VaCML95 for their implication as positive regulators for stilbene production. In conclusion, the results are important for understanding the signaling pathways and mechanisms regulating the biosynthesis of plant secondary metabolites and might be in demand in plant biotechnology and agriculture.

4. Materials and Methods

4.1. Plant Material and Cell Cultures

The V7 callus cultures were established in 2017 from young stems of the wild-growing mature V. amurensis vines near Vladivostok as described in Tyunin et al. 2019 [55]. All transgenic cell lines were obtained by Agrobacterium-mediated transformation as described in Aleynova-Shumakova et al., 2014 [19]. Briefly, the control KA0 cell culture transformed with the empty vector was obtained in 2020 by co-cultivation of the V7 cell suspension with Agrobacterium tumefaciens GV3101:pMP90 strain containing pZP-RCS2-nptII [56], which contained only the Km resistance gene, nptII.

To generate the construction for plant cell transformation, the full-length cDNA of VaCML52, VaCML65, VaCML86, VaCML93, and VaCML95 genes (GenBank accession number MN540595, MN540606, MN540576, MN540582, MN540584) were amplified by PCR using the primers presented in the Supplementary Table S1. The forward primer contained a BglII (for CML52), Psp124B I (or SacI, for CML65, 93, 95) or HindIII (for CML86) restriction site and the reverse primer contained a BamHI (for all CML genes) restriction site, which are underlined. The full-length cDNA of CMLs was cloned into the pSAT1 vector [56] by the BglII/Psp124B I/HindIII and BamHI sites under the control of the double cauliflower mosaic virus (CaMV 35S) promoter. Then, the expression cassette from pSAT1 with the CML genes was cloned into the pZP-RCS2-nptII vector [56] using the PalAI (AscI) sites. The pZP-RCS2-nptII construction also carried the nptII gene under the control of the double CaMV 35S promoter (Supplementary Figure S3). The used restriction enzymes were obtained from SibEnzyme (Novosibirsk, Russia).

The independently transformed VaCML-transgenic callus cell lines of V. amurensis, designated 52-1, 52-2, 52-3 (VaCML52 gene); 65-1, 65-2, 65-3, 65-4 (VaCML65 gene); 86-1, 86-2, 86-3 (VaCML86 gene); 93-1, 93-2, 93-3 (VaCML93 gene); 95-1, 95-2, 95-3, 95-4 (VaCML95 gene), were obtained in 2020 by transformation of the V7 cell suspension with A. tumefaciens strain GV3101::pMP90 containing pZP-RCS2-VaCML-nptII as described previously [19,57]. The VaCML genes and the selective marker nptII gene were driven under the double CaMV 35S promoter. Transcript level of the nptII gene was verified using semiquantitative RT-PCR with the primers and PCR conditions described earlier [46]. The absence of A. tumefaciens was confirmed by RT-PCR of the VirB2 gene using primers listed in Table S1. All transgenic cell lines were cultivated on the solid Murashige and Skoog modified WB/A medium [58] supplemented with 0.5 mg/L BAP, 2 mg/L NAA, and 8 g/L agar in the dark in 100 mL flasks with 20 mL of the medium. For stilbene analysis, the V. amurensis calli were cultivated at 35-day subculture intervals in the dark at 24–25 °C in test tubes (height 150 mm, internal diameter 146 mm) with 7–8 mL of the WB/A medium.

4.2. Stilbene Analysis by High-Performance Liquid Chromatography (HPLC) and Mass Spectrometry

The content of stilbenes was measured by HPLC as described [43,59]. Briefly, the extracts were separated on Zorbax C18 column (column temperature 40 °C, 150 mm, 2.1-mm i.d., 3.5-lm part size, Agilent Technologies, Santa Clara, CA, USA) the on HPLC LC-20AD XR analytical system (Shimadzu, Kyoto, Japan). The mobile phase consisted of a gradient elution of 0.1% aqueous acetic acid (A) and acetonitrile (B). The gradient profile with a flow rate of 0.2 mL/min was: 0 min 0% B; 35 min 40% B; 40 min 50% B; 50 min 100% B and then eluent B until 60 min. The injected volume was 5 µL. Liquid chromatography-high-resolution mass spectrometry for quantification of all components was performed using a 1260 Infinity analytical system (Agilent Technologies, USA) as described [20,59]. HPLC for quantification of all components was performed using LC-20 analytical HPLC system (Shimadzu, Japan) equipped with an SPD-M20A photodiode array detector, LC-20ADXR pump, Shim-pack XR-ODS II column and SIL-20ACXR auto sampler as described [46].

4.3. RNA Isolation, Reverse Transcription and Quantitative Real-Time PCR (qRT-PCR)

Total RNA extraction was performed using the cetyltrimethylammonium bromide-based extraction as described [47]. Complementary DNAs were synthesized using the MMLV Reverse transcription PCR Kit with oligo(dT)15 (RT-PCR, Evrogen, Moscow, Russia) as described [60]. qRT-PCRs were performed using the real-time PCR kit (Evrogen) and EvaGreen Real-time PCR dye (Biotium, Hayward, CA, USA) using total cDNAs as described [28,46]. The expression was calculated by the 2−ΔΔCT method with two internal controls, VaGAPDH and VaActin1, as described [61]. The primers used for qRT-PCRs are listed in Table S1. We used different primer sets for analyzing expression of the exogenous (transgene) and endogenous VaCML genes. To analyze transcript levels of the VaCML transgenes, the primers were designed according to the promoter and terminator sequences of the transgenes (Figure S4; Table S1). To analyze expression of the endogenous VaCML genes, the primers were designed to the untranslated (UTR) and specific coding gene regions of the VaCMLs (Figure S4; Table S1). To analyze transcript levels of both VaCML transgenes and VaCML endogenes (total expression), the primers were designed to protein-coding regions of VaCML genes (Figure S4; Table S1).

4.4. Statistical Analysis

For quantification of the VaCML, VaPAL, VaSTS, and VaMyb gene expression we used two independent experiments with eight technical replicates (four qPCR reactions normalized to VaGAPDH and four qPCr reactions—to VaActin gene in each independent experiment). For callus tissue weight calculations, we used three independent experiments with ten technical replicates and three independent experiments with two technical replicates for total stilbene measurement. 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. A value of p < 0.05 was considered significant.