The Effect of Stress Hormones, Ultraviolet C, and Stilbene Precursors on Expression of Calcineurin B-like Protein (CBL) and CBL-Interacting Protein Kinase (CIPK) Genes in Cell Cultures and Leaves of Vitis amurensis Rupr

Calcium serves as a crucial messenger in plant stress adaptation and developmental processes. Plants encode several multigene families of calcium sensor proteins with diverse functions in plant growth and stress responses. Several studies indicated that some calcium sensors may be involved in the regulation of secondary metabolite production in plant cells. The present study aimed to investigate expression of calcineurin B-like proteins (CBL) and CBL-interacting protein kinase (CIPK) in response to conditions inducting biosynthesis of stilbenes in grapevine. We investigated CBL and CIPK gene expression in wild-growing grapevine Vitis amurensis Rupr., known as a rich stilbene source, in response to the application of stilbene biosynthesis-inducing conditions, including application of stress hormones (salicylic acid or SA, methyl jasmonate or MeJA), phenolic precursors (p-coumaric acids or CA), and ultraviolet irradiation (UV-C). The influence of these effectors on the levels of 13 VaCBL and 27 VaCIPK mRNA transcripts as well as on stilbene production was analyzed by quantitative real-time RT-PCR in the leaves and cell cultures of V. amurensis. The data revealed that VaCBL4-1 expression considerably increased after UV-C treatment in both grapevine cell cultures and leaves. The expression of VaCIPK31, 41-1, and 41-2 also increased, but this increase was mostly detected in cell cultures of V. amurensis. At the same time, expression of most VaCBL and VaCIPK genes was markedly down-regulated both in leaves and cell cultures of V. amurensis, which may indicate that the CBLs and CIPKs are involved in negative regulation of stilbene accumulation (VaCBL8, 10a-2, 10a-4, 11, 12, VaCIPK3, 9-1, 9-2, 12, 21-1, 21-2, 33, 34, 35, 36, 37, 39, 40, 41-3, 41-4). The results obtained provide new information of CBL and CIPK implication in the regulation of plant secondary metabolism in response to stress hormones, metabolite precursors, and UV-C irradiation.

The plant calcium sensor proteins include calmodulins (CaMs), calmodulin-like proteins (CML), calcium-dependent protein kinases (CDPKs), calcineurin B-like proteins (CBLs), calcineurin B-like proteins (CBL), and CBL-interacting protein kinases (CIPK) [4][5][6]. There are scarce data on the functions and regulation of the CBL-CIPK calcium signaling Plants 2023, 12, 1562 2 of 11 network. A novel type of calcium sensor, termed as CBLs, was identified in plant cells 20 years ago [7]. CBLs specifically target a family of plant-specific CIPK. To decode a calcium signal, CBL binds Ca 2+ and interacts with CIPK, leading to activation of the kinase. Then, the CBL-CIPK complex phosphorylates downstream target proteins and changes their biological activities [8]. Data from the current literature report on the functions of the plant CBL-CIPK network in mineral uptake, stomatal movement, pH regulation, maintenance of Na+/K+ homeostasis, etc. [8].
Ishitani et al. [9] demonstrated that the CBL4-CIPK24 complex of Arabidopsis thaliana migrated to the plasma membrane where it activated the Na + /H + antiporter (SOS1) located on the plasma membrane and the vacuolar H + -ATPase, resulting in enhanced plant salt tolerance. Other CBL-CIPK complexes have been studied in Arabidopsis, such as CBL1/CBL9-CIPK23 that was observed to localize to the plasma membrane and regulate K + in roots and stomatal guard cells [10]. Overexpression of OsCBL8 and OsCIPK15 in rice enhanced plant salt tolerance [11]. OsCIPK31 mutants exhibited hypersensitive phenotypes to abscisic acid (ABA), salt, mannitol, and glucose. Compared with wild-type rice plants, OsCIPK31 mutants exhibited retarded germination and slow seedling growth [12]. Moreover, TaCIPK23 has been shown to participate in wheat cell signaling in response to ABA and drought by mediating the crosstalk between ABA-induced signaling and other pathways associated with adaptation to drought [13]. In potatoes, StCIPK10 has been shown to enhance both cellular scavenging of reactive oxygen species and cellular modulation of corresponding osmoregulatory substances to strengthen plant drought and osmotic stress tolerance [14].
A number of articles have shown that plant calcium sensor proteins, such as CDPKs and CMLs, are involved in the plant pathogen response [15,16] and plant defense tools such as secondary metabolite production [17]. It is proposed that CDPKs are involved in stress hormone-and light-mediated activation of plant defense reactions and secondary metabolite production in response to biotic and abiotic cues [16]. Considering CBL and CIPK, there is scarce information on their involvement in plant defense reactions. It is known that overexpression of StCBL4 and StCIPK2 individually and synergistically increased the tolerance of potato plants to soilborne plant pathogen fungi Rhizoctonia solani in Nicotiana benthamiana [18].
Knockdown of the TaCIPK14 gene in wheat significantly increased wheat resistance to the pathogenic fungus Puccinia striiformis (Pst), whereas overexpression of TaCIPK14 resulted in enhanced wheat susceptibility to Pst by decreasing different aspects of the defense response, including accumulation of reactive oxygen species (ROS) and expression of pathogenesis-relative genes [19]. Currently, there is no information about CBL-CIPK participation in the regulation of biosynthesis of plant secondary metabolites.
In this study, we aimed for a comprehensive analysis of CBL and CIPK gene expression in the wild-growing grapevine Vitis amurensis Rupr. under the treatments inducing active production of stilbenes, which are important defense compounds in grapevines and some other plant families [20,21]. For this, we induced stilbene biosynthesis using stress hormones, ultraviolet C irradiation, and stilbene precursors to measure CBL and CIPK mRNA levels by quantitative real-time RT-PCR (qRT-PCR) in the leaves and callus cell culture of V. amurensis.

Stilbene Content in V. Amurensis after SA, MeJA, CA, and UV-C Treatments
To test the involvement of VaCBL and VaCIPK genes in the regulation of stilbene synthesis in grapevine, we exposed vine cuttings and calli V. amurensis to different doses of SA, MeJA, stilbene precursors, and UV-C. We used experimental design and chemical concentrations for stress hormones (salicylic acid or SA, methyl jasmonate or MeJA), CA, and UV-C as described [22][23][24][25]. Previously, we found that the doses of the treatments (50-200 µM for SA and MeJA, 100-500 µM for CA, and 20 min at a wavelength of 254 nm, 230 µW cm −2 , 15 cm from the UV-C lamp) exhibited the highest stimulating effect on Plants 2023, 12, 1562 3 of 11 stilbene biosynthesis. The highest stilbene content in the leaves of V. amurensis vines was observed 24 h after MeJA or UV-C treatments [26,27]. The highest stilbene content in callus cell cultures of V. amurensis was detected at 35 day of cultivation [22,28]. Therefore, we used the same time periods for stilbene analysis.
First, we analyzed the composition of stilbenes in the leaves and V7 callus cell culture of V. amurensis. The V7 callus cultures were established in 2017 from young stems of mature V. amurensis vines [29]. The HPLC analysis revealed the presence of eight major stilbenoids in the tissues of V. amurensis at a concentration of 0.001 mg/g DW or higher ( Figure 1, Table S1). It is possible that other stilbenes were present in the analyzed tissues of V. amurensis, but in trace amounts (less than 0.001 µg/g DW). The main focus of the present paper was to analyze the repertoire and content of the major stilbenes, and, therefore, we analyzed the content of the above-mentioned compounds. Table S1). It is possible that other stilbenes were present in the analyzed tissues of V. amurensis, but in trace amounts (less than 0.001 µg/g DW). The main focus of the present paper was to analyze the repertoire and content of the major stilbenes, and, therefore, we analyzed the content of the above-mentioned compounds.
All treatments considerably increased total stilbene content in both the cell culture and leaves of V. amurensis ( Figure 2, Table S1). In the calli of V. amurensis, the content of stilbenes increased most significantly after application of MeJA (200 µM) and UV-C (24 h post-treatment), up to 2.3-3.2 mg/g DW, which is 10.2-and 14.1-fold higher than in the untreated control calli, respectively ( Figure 1a). For the leaves of V. amurensis, the highest stimulating effect on stilbene biosynthesis was provided by the application of SA (200 µM), CA (200 µM), and UV-C (24 h) with the increase in stilbene levels by 4.4, 5.4, and 5.5 times in comparison with the untreated control leaves, respectively (Figure 1b). We found that the UV-C treatment had the highest positive effect on stilbene biosynthesis compared with the effects of MeJA, SA, and CA, potentially due to the involvement of stilbenes in plant UV protection [20]. and leaves of V. amurensis ( Figure 2, Table S1). In the calli of V. amurensis, the content of stilbenes increased most significantly after application of MeJA (200 µM) and UV-C (24 h post-treatment), up to 2.3-3.2 mg/g DW, which is 10.2-and 14.1-fold higher than in the untreated control calli, respectively (Figure 1a). For the leaves of V. amurensis, the highest stimulating effect on stilbene biosynthesis was provided by the application of SA (200 µM), CA (200 µM), and UV-C (24 h) with the increase in stilbene levels by 4.4, 5.4, and 5.5 times in comparison with the untreated control leaves, respectively (Figure 1b). We found that the UV-C treatment had the highest positive effect on stilbene biosynthesis compared with the effects of MeJA, SA, and CA, potentially due to the involvement of stilbenes in plant UV protection [20].
A detailed analysis of the individual stilbenes in the V7 probes (Table S1) revealed that the significant increase in the total stilbene content after all treatments was mainly due to an increase in the content of viniferins (ε-and δ-viniferin), t-resveratrol, and tresveratrol diglucoside (Table S1), while in the leaves, the content of viniferins (ε-and δviniferin), t-resveratrol, and t-piceid was mostly enhanced after all used treatments (Table S1). A detailed analysis of the individual stilbenes in the V7 probes (Table S1) revealed that the significant increase in the total stilbene content after all treatments was mainly due to an increase in the content of viniferins (ε-and δ-viniferin), t-resveratrol, and t-resveratrol diglucoside (Table S1), while in the leaves, the content of viniferins (ε-and δ-viniferin), t-resveratrol, and t-piceid was mostly enhanced after all used treatments (Table S1).

CBL and CIPK Expression after SA, MeJA, CA, and UV-C Treatments
Previously, eight CBL genes were described based on the well-known genome of cultivated grapevine V. vinifera [30]. However, in the NCBI GeneBank, two transcripts are associated with the CBL4 gene of V. vinifera, four transcripts with the CBL10a gene, and two transcripts with the CBL13 gene. These transcripts differed in the 3 -end of the CBL coding region and included modifications of the last exon ( Figure S1), which resembled splicing-related variations. The specific primers were designed for all CBL transcripts found in the NCBI database (Table S2). Thus, we analyzed the levels of 13 VaCBL transcript variants (Figure 3). The qRT-PCR analysis revealed that the level of VaCBL4-1 transcript considerably increased at all time points after UV-C treatment both in the V7 cell culture and grapevine leaves (Figure 3a,b). The levels of VaCBL5, VaCBL10a-3, and VaCBL13-1 transcripts also considerably increased at both doses of SA (VaCBL10a-3) and MeJA (VaCBL5, VaCBL10a-3, VaCBL13-1), but this increase was detected only in cell cultures (Figure 3a). The levels of other VaCBL transcript variants were not significantly changed after the SA, MeJA, CA, and UV-C treatments of the grapevine V. amurensis cell cultures (Figure 3a). In contrast to the cell cultures, the treatments of V. amurensis vine cuttings led to a significant decrease in the levels of the most VaCBL transcripts (Figure 3b). Furthermore, expression of VaCBL10a-1, 10b, and 13-3 transcripts increased in some individual probes (Figure 3). It is possible that VaCBL4-1 is implicated in stilbene biosynthesis in grapevines as a positive regulator in response to UV-C irradiation. Transcripts VaCBL10a-1, 10b, and 13-3, VaCBL5, VaCBL10a-3, and VaCBL13-1 may also have a slight positive effect on the biosynthesis of stilbenes, because their expression increased with only one dose of the used inductors ( Figure 3). The data obtained revealed that expression levels of five VaCBLs considerably decreased at both doses of the used stilbene biosynthesis inductors in the leaves of V. amurensis, including VaCBL8 (UV-C), VaCBL10a-2 (MeJA, CA), VaCBL10a-4 (MeJA), VaCBL11 (UV-C), and VaCBL12 (MeJA) transcripts (Figure 3b). Furthermore, down-regulation of CBL transcript levels was detected at individual doses for the VaCBL4-2, VaCBL5, and VaCBL10a-4 ( Figure 3).
Then, we analyzed transcript levels of VaCIPK genes in response to SA, MeJA, CA, and UV-C (Figure 4). Previously, 20 CIPK genes were described for grapevines based on the wellknown genome of cultivated grapevine V. vinifera [30]. However, in the NCBI GeneBank, two transcripts are associated with the CIPK9 gene of V. vinifera, two transcripts-with the CIPK21 gene, and six transcripts-with the CIPK41 gene. These transcripts differ in the 3 -end of the CBL coding region and include modifications of the last exon ( Figure S1). The specific primers were designed for all CBL transcripts found in the NCBI database (Table S2). Thus, we analyzed the levels of 27 VaCIPK transcript variants (Figure 3). and UV-C (Figure 4). Previously, 20 CIPK genes were described for grapevines based on the well-known genome of cultivated grapevine V. vinifera [30]. However, in the NCBI GeneBank, two transcripts are associated with the CIPK9 gene of V. vinifera, two transcripts-with the CIPK21 gene, and six transcripts-with the CIPK41 gene. These transcripts differ in the 3′-end of the CBL coding region and include modifications of the last exon ( Figure S1). The specific primers were designed for all CBL transcripts found in the NCBI database (Table S2). Thus, we analyzed the levels of 27 VaCIPK transcript variants (Figure 3).

Discussion
Previous data revealed the involvement of some CDPK and CML genes in the regulation of stilbene production in V. amurensis [29,31,32]. Five families of calcium sensor genes are known in plants; these are CDPK, CaM, CML, CBL, and CIPK [4][5][6]. Expression of CDPK, CaM, and CML was studied in grapevine tissues under stilbene biosynthesisinducing conditions, including stress hormones (SA and MeJA), phenolic precursors (CA), UV-C, transformation by agrobacterium rol genes, leading to a strong increase in stilbene accumulation [29,[31][32][33][34][35][36][37]. Thus, together with this study, the expression of main plant calcium sensors has been studied in grapevine cell cultures and leaves under conditions inducing stilbene biosynthesis.
It has been shown that the plant transformation with the rol genes of Agrobacterium rhizogenes was accompanied by a strong increase in t-resveratrol content and activation of the expression of VaCDPK1d, VaCDPK1e, VaCDPK1-L, and VaCDPK2a genes [34]. A number of CML genes, such as VaCML52, VaCML65, VaCML93, and VaCML95 [29], were highly up-regulated in the leaves and cell cultures of wild grapevine V. amurensis in the stilbene-modulating conditions. At the same time, CaM gene expression did not increase significantly [29].
A number of studies revealed that overexpression of the AtCPK1 gene from A. thaliana contributes to a significant increase in the content of isoflavonoids in soybean [38]. Overexpression of AeCDPK6 promoted hyperoside (flavonol, 3-O-galactoside of quercetin) accumulation in an AeMYB30-dependent manner in the flowers of okra plants [39]. The grapevine V. amurensis calli overexpressing the VaCPK1, VaCPK26, and VaCPK20 genes were capable of producing up to 0.6, 0.4, and 4.2 mg/g DW of t-resveratrol, respectively [31,36]. However, overexpression of other CDPK genes in grapevine callus cultures did not significantly affect the biosynthesis of stilbenes [37]. For CMLs, it has been shown that overexpression of the VaCML65 gene led to a considerable and consistent increase in the content of stilbenes of 3.8-23.7 times (up to 19 mg/g DW of total stilbenes or up to 18 mg/g DW of t-resveratrol) in all transformed lines [32].
In this work, the expression of VaCBLs and VaCIPKs has been analyzed after treatment with SA, MeJA, CA, and UV-C. Taken together, the data obtained in this study indicated that several VaCBLs (VaCBL4-1, VaCBL10a-3, VaCBL13-1) and VaCIPKs (VaCIPK31, VaCIPK41-1, VaCIPK41-2) can be considered as possible positive activators of stilbene biosynthesis, because their expression was considerably increased in after stilbene biosynthesis induction. The VaCBL4-1 transcript is the most interesting candidate in the positive regulation of stilbene biosynthesis, because the VaCBL4-1 transcript level markedly increased at all time points both in cell culture and grapevine leaves after UV-C treatment, which is the strongest inductor of stilbene biosynthesis (Figure 3). At the same time, the number of CBLs probably involved in negative regulation of stilbene accumulation is greater than the number of CBLs involved in the stimulation of stilbene accumulation. The results revealed that a high number of VaCBL (VaCBL8, 10a-2, 10a-4, 11, 12) and VaCIPK (VaCIPK3, 9-1, 9-2, 12, 21-1, 21-2, 33, 34, 35, 36, 37, 39, 40, 41-3, 41-4) family members may act as possible blockers of stilbene biosynthesis, since expression of these genes markedly decreased in the probes with high stilbene content. These genes are perhaps involved in grapevine primary metabolism and are responsible for growth and development regulation, since expression of these genes is suspended under the imposed stress caused by the stress hormones, stilbene precursors, and UV-C. In general, these findings need to be verified in future studies, potentially by establishing transgenic plants or plant cell cultures overexpressing the CBLs or CIPKs genes.
In conclusion, this study indicates the role of some CBL and CIPK family members in the regulation of secondary metabolite production in plant cells and provides new data on stilbene biosynthesis regulation in grapevines. According to this study, several CBL and CIPK genes can be used in biotechnology as stimulators for stilbene biosynthesis in plant cells.

V. amurensis Plant Material
We used young vines of wild-growing grapevine V. amurensis Rupr. (Vitaceae) sampled from a non-protected natural population near Vladivostok, Russia [40]. The V. amurensis vines collected in June 2022 were divided into cuttings (excised young stems 7-8 cm long with one healthy leaf) that were placed in individual 100 mL beakers with 50 mL filtered water and used for the treatments. The cuttings of V. amurensis were grown under a photoperiod of 11/13 h light/dark at a light intensity of 70 µmol m −2 s −1 at 15 • C and darkness at 10 • C, humidity 60-70%.
The V7 callus cultures were established in 2017 from young stems of mature V. amurensis vines as described [29]. For stilbene analysis, the V7 calli were cultivated with 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 solid Murashige and Skoog (MS) modified W B/A medium [41,42] supplemented with 0.5 mg/L 6-benzylaminopurine, 2 mg/L α-naphthaleneacetic acid, and 8 g/L agar.

Plant Treatments and Chemicals
Sterile solutions of SA diluted in ethyl alcohol, MeJA and CA diluted in dimethyl sulfoxide (DMSO) were added aseptically to desired concentrations (50 and 200 µM of SA and MeJA, 100 and 500 µM of CA) to the autoclaved cultivated media for cell cultures or to the filtered water for the vine cuttings of V. amurensis. DMSO (200 µL/L) or ethyl alcohol (200 µL/L) were added for the control treatments (V7 or L). The UV-C treatments were conducted for 20 min at a wavelength of 254 nm (230 µW cm −2 , 15 cm from the lamp) in the dark using UV lamp VL-215.MC provided by Vilber Lourmat company (Marne-la-Vallée, France) as described [24]. Control V. amurensis cuttings were placed in the dark in individual 100 mL beakers with 50 mL filtered water. After the UV-C treatments, the treated and control cuttings were grown under a photoperiod of 11/13-h light (70 µmol m −2 s −1 , 15 • C)/dark (10 • C) for 1 h and 24 h.

Stilbene Analysis by High Performance Liquid Chromatography (HPLC) and Mass Spectrometry
Total stilbene content was measured by HPLC as described [43,44]. Briefly, the extracts were separated on Zorbax C18 column (150 mm, 2.1-mm i.d., 3.5-lm part size, Agilent Technologies, USA). 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 for 60 min. Mass spectrometry was performed using a 1260 Infinity analytical system (Agilent Technologies, Santa Clara, CA, USA) for quantification of all components as described [43]. HPLC 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 for quantification of all components as described [44].

RNA Isolation, Reverse Transcription, and Expression Analysis of VaCBLs and VaCIPKs
Total RNA extraction was performed using the cetyltrimethylammonium bromide (CTAB)-based extraction as described [33]. Complementary DNAs were synthesized using 1.5 µg of RNA by the MMLV Reverse Transcription PCR Kit with oligo(dT)15 (RT-PCR, Evrogen, Moscow, Russia). The RT-PCR reactions were performed in 50 µL aliquots of the reaction mixture, which contained 1 × RT buffer, 1 mM each of the 4 dNTPs, 2 µM of dithiothreitol (DTT), 1 µM of oligo(dT)15 primer, 250 U of MMLV-polymerase at 37 • C for 1.5 h. The 1 µL samples of reverse transcription products were then amplified by qPCR.