Impact of VaCCaMK gene overexpression and its splicing isoforms on cell growth and stilbene accumulation in Vitis amurensis Rupr

Impact of VaCCaMK gene overexpression and its splicing isoforms on cell growth and stilbene accumulation in Vitis amurensis Rupr | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Impact of VaCCaMK gene overexpression and its splicing isoforms on cell growth and stilbene accumulation in Vitis amurensis Rupr Konstantin V. Kiselev, Alina A. Beresh, Alexey A. Ananev, Olga A. Aleynova, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9503080/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Calcium (Ca²⁺) functions as an essential intracellular second messenger in plants, mediating processes such as pathogen defence, stress adaptation and enzyme activation. Plants possess multiple families of calcium‑binding proteins, and among these, calcium/calmodulin‑dependent protein kinases (CCaMKs) remain among the least characterized. Also, their dual capacity to bind both Ca²⁺ and calmodulin makes them a subject of significant research interest. Usually, there is one CCaMK gene per plant genome, but when we cloned the full-length VaCCaMK cDNA sequence, we found several transcripts with missing exons, so it is possible that alternative splicing increases the overall diversity of CCaMK sequences. This study investigated the role of CCaMKs in Vitis amurensis Rupr. under abiotic stress conditions using grapevine cell cultures overexpressing the VaCCaMK1 gene and its alternatives VaCCaMK1- s1, -s2. Results demonstrated that VaCCaMK1 ‑overexpressing cultures did not exhibit increased tolerance to salt, osmotic, cold, or heat stress. Additionally, the content of secondary metabolites, as represented by stilbenes mainly produced by used grapevine cells, remained largely unchanged. However, a significant increase in both fresh and dry cell mass was observed compared with the control group: fresh biomass increased 1.1–1.7‑fold, and dry biomass 1.1–1.8‑fold. These findings indicate that VaCCaMK genes do not affect plant cell susceptibility to the tested abiotic stresses. VaCCaMK1-s1 or - s2 the overexpression had a similar but weaker effect on grape cells. Nevertheless, VaCCaMK appear to act as a positive regulator of cell growth and development in V. amurensis , suggesting their potential role in enhancing biomass accumulation. abiotic stress calcium gene expression secondary metabolites tolerance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Calcium (Ca 2+ ) is a universal intracellular second messenger in plants, playing a pivotal role in transducing extracellular signals into coordinated cellular responses (McCormack et al. 2005 ; Kudla et al. 2010 ). It regulates a wide array of physiological processes, including pathogen defence, stress adaptation, enzyme activation, stomatal movement, photomorphogenesis, and growth regulation (Reddy et al. 2011 ; Kiselev, Dubrovina, 2025 ). The specificity and versatility of Ca 2+ signalling are achieved through a complex network of calcium‑binding proteins that decode the spatiotemporal patterns of Ca 2+ concentration changes, often referred to as “calcium signatures”, into specific downstream responses (Harper and Harmon 2005 ). The majority of Ca 2+ sensor proteins contain multiple EF‑hand motifs: conserved helix‑loop‑helix structures in which Ca 2+ ions are coordinated within the acidic Ca 2+ ‑coordinating loop (Halling et al. 2016 ). The major plant EF‑hand‑containing Ca 2+ ‑binding proteins include calmodulins (CaMs), calmodulin‑like proteins (CMLs), Ca 2+ ‑dependent protein kinases (CDPKs), calcineurin B‑like proteins (CBLs), and calcium/calmodulin‑dependent protein kinases (CCaMKs) (Hashimoto and Kudla 2011 ; Mohanta et al. 2019 ). Among the diverse families of calcium‑binding proteins in plants CCaMKs occupy a unique position due to their dual capacity to bind both Ca 2+ ions and calmodulin. This dual regulatory mechanism allows CCaMKs to integrate calcium signals and transduce them into phosphorylation events, thereby modulating the activity of target proteins (Takezawa et al. 1996 ). CCaMKs are best known for their critical roles in plant–microbe interactions, particularly in establishing symbioses with nitrogen‑fixing rhizobia and arbuscular mycorrhizal fungi. In these processes, CCaMK acts as a central component of the common symbiosis signaling pathway, where it decodes Ca 2+ oscillations and activates downstream transcriptional reprogramming (Hayashi et al. 2010 ). Despite their importance in biotic interactions, the functions of CCaMKs in abiotic stress responses remain poorly understood. Abiotic stresses such as salinity, drought, osmotic imbalance, and extreme temperatures represent major constraints on plant growth and productivity worldwide (Kumar, 2020 ). These stressors often trigger rapid increases in cytosolic Ca 2+ concentrations, suggesting that calcium signalling pathways, including CCaMKs, may play a role in stress perception and adaptation (Xu et al. 2022 ). However, direct evidence linking CCaMK activity to abiotic stress tolerance is limited, and the molecular mechanisms involved are largely unexplored (Contreras Delgado et al. 2026 ). Grape Vitis amurensis is an excellent model for studying stress tolerance mechanisms, as it exhibits remarkable resilience to cold, pathogens, and other environmental challenges (Liu, Li, 2013 ; Xin et al. 2013 ). Grapevine cell cultures provide a controlled system to investigate molecular and physiological responses without the complexity of whole‑plant architecture. The present study aimed to elucidate the role of VaCCaMK genes in abiotic stress responses and cell growth regulation in V. amurensis . Specifically, we investigated the effects of overexpressing the full‑length grape VaCCaMK gene and its alternatively spliced variants ( VaCCaMK-s1 , VaCCaMK-s2 ) in grapevine cell cultures under salt, osmotic, cold, and heat stress conditions. We also assessed changes in stilbene production, key secondary metabolites with antioxidant and antimicrobial properties, as well as biomass accumulation. 2. Results and Discussion 2.1. Cloning and sequencing of VaCCaMK mRNA transcripts Previously, the grapevine Vitis vinifera L. VvCCaMK sequence was determined during whole-genome sequencing in BioProject PRJNA1013121 (Jaillon et al. 2007 ; Shi et al. 2023 ). The protein-coding sequence of VvCCaMK consists of 1563 bp and 7 exons. Next, using primers for the beginning and end of the protein-coding sequence of the known VvCCaMK gene (GenBank XM_002273306) on cDNA obtained from a wild-growing V . amurensis grape leaf, we obtained the full-length protein-coding sequence of the VaCCaMK gene. The resulting VaCCaMK sequence was highly homologous to the VvCCaMK gene in terms of nucleotide composition (Identities 99.2%) and deduced amino acid sequence (Identities 99.6%) (Fig. 1 ). In the available grape V. amurensis genome (Wang et al. 2024 ), we found only one copy of the VaCCaMK gene. Table 1 CCaMK genes in plant species with known functions. Species Monocots or eudicots Gene functions Accession no. Reference Lily Lilium longiforum , LlCCaMK Monocots Preferentially expressed in developing anthers Q43531 Patil et al. 1995 Wheat, Triticum aestivum , TaCCaMK Monocots Overexpressing TaCCaMK in Arabidopsis plants reduced their sensitivity to abscisic acid (ABA) treatment during seed germination and root elongation. Increased root length HM595635 Yang et al. 2011 Maize, Zea mays , ZmCCaMK Monocots It plays a key role in the antioxidant protection of plants induced by brassinosteroids and in increasing their resistance to drought ABD67491 Liu et al. 2020 Lotus japonicus , LjCCaMK Eudicots Single amino-acid replacement in a CCaMK is sufficient to turn fully differentiated root cortical cells into meristematic founder cells of root nodule primordia CAJ76700 Tirichine et al. 2006 Medicago truncatula , MtCCaMK Eudicots It ensures the formation of nodules when the plant interacts with nitrogen-fixing bacteria Q6RET7 Routray et al. 2013 Tobacco, Nicotiana tabacum , NtCCaMK Eudicots It has been shown that their expression is strictly timed to the stage of anther development, when the buds are 0.5–1.0 cm in size, which coincides with meiosis AAD28791 Liu et al. 1998 It is important to note that while we were trying to obtain a full-length VaCCaMK sequence, we cloned various short VaCCaMK variants, even though we used a special heat-stable reverse transcriptase and high-fidelity DNA polymerase. Among these short VaCCaMK variants, there were two main sequences that were repeated several times in two independent transformations: VaCCaMK-s1 and VaCCaMK-s2 . We did not find such short variants in the V. amurensis genome. (Wang et al. 2024 ). It was only after the third transformation attempt and the analysis of more than 20 clones that we were able to obtain the standard sequence VaCCaMK . We showed that the E1 and E4 exon sequences of VaCCaMK-s1 had 8-bp short direct repeats (SDR): TGTTTCAC, resulting that the partial sequence of E1 and E4 and the complete sequence of E2 and E3 are missing from the VaCCaMK-s1 transcript sequence and at the same time, the protein reading frame does not get messed up (Fig. 2 a). This results in a 906 bp cDNA sequence that can be used to produce a shortened protein missing part of the kinase domain (Fig. 2 b). The production of this VaCCaMK-s1 transcript can be attributed to mechanisms of genomic instability and DNA recombination (Ogihara et al. 1988 ), but due to its high abundance during cloning, we decided to include it in our further research. A feature of VaCCaMK-s2 transcript was that the entire 6-th exon was missing (Fig. 2 a), what is a typical example of alternative splicing (Dubrovina et al. 2013 ). This results in a 1503 bp cDNA sequence that can be used to produce a shortened protein missing second EF-hand, protein with two EF-hands (Fig. 2 b). Also, due to its high abundance during cloning, we decided to include it in our further research. Next, we performed a promoter analysis (2000 bp upstream of the start codon) of the VaCCaMK gene using PlantCARE. We identified over a hundred different cis -acting elements (supplementary Table S1 ); however, only those with well‑established functions are presented in the (Fig. 3 ). Thus, the VaCCaMK gene promoter contains the highest number of light‑responsive elements (6 elements). It also includes elements associated with phytohormone regulation: one for auxin, one for abscisic acid, and one for gibberellins. Additionally, elements responsive to stress hormones were detected: one for salicylic acid and one for methyl jasmonate. Elements associated with abiotic stresses were also present, albeit in smaller numbers (2 for drought and 1 for wounding). 2.2. Expression of VaCCaMK mRNA transcripts in grapevine V. amurensis leaves under abiotic stress conditions In this section, we analyzed the expression of the VaCCaMK gene in grape leaves under various common abiotic stresses: water deficit, salt stress, osmotic stress, high temperatures, low temperatures, and ultraviolet С irradiation. It was shown that VaCCaMK expression increased in most stresses, but this increase was small and often within the error limits. Only salt stress significantly increased VaCCaMK expression by 1.4–1.9 times at all time points compared to untreated control cells (Fig. 4 ). Therefore, it was important to see how overexpression of the VaCCaMK gene would affect resistance to the described stresses, primarily salt stress. 2.3. VaCCaMK1 gene overexpression in the grapevine V. amurensis cell cultures Using agrobacterial transformation we independently obtained three cell lines for each used gene construction: F1, F2, and F3 for VaCCaMK gene, S1-1, S1-2, and S1-3 for VaCCaMK-s1 gene, or S2-1, S2-2, and S2-3 for VaCCaMK-s2 gene. Next, we showed that the resulting transgenic lines actively expressed the corresponding transgenes (Fig. 5 a), resulting in a significant increase in the overall expression of VaCCaMK in all transgenic cell lines, which was 3.1–8.4 times higher than in the control KA0 cell culture (Fig. 5 b). At the same time, overexpression of the VaCCaMK transgenes was found to reduce expression of the endogenous VaCCaMK gene by 1.1–2.7‑fold, although this reduction did not reach statistical significance. It was further shown that overexpression of the VaCCaMK gene and its alternative transcripts VaCCaMK-s1 and VaCCaMK-s2 increases the accumulation of fresh and dry biomass by the resulting transgenic grape cells. The accumulation of biomass by transgenic cells was greatest with overexpression of the VaCCaMK gene: the accumulation of fresh weight significantly increased by 1.6–1.7 times and dry mass by 2.2–2.5 times (Fig. 6 ). Overexpression of the VaCCaMK-s2 transcript increased the accumulation of fresh biomass by 1.5–1.7 times and dry by 1.9–2.4 times (Fig. 6 ). Overexpression of the VaCCaMK-s1 transcript also activated an increase in fresh and dry weight, but this increase was to a lesser extent (1.0-1.5 and 1.8–2.4 times) and was not significant for all three cell lines (Fig. 6 ). It is well established that plant calcium sensors play key roles in stress resistance. We therefore investigated the effect of VaCCaMK transcript overexpression on the tolerance of transgenic grape cells to temperature extremes (high and low), salt stress, and osmotic stress (Fig. 7 ). The data show that the applied conditions significantly reduced fresh biomass accumulation in the control KA0 cell culture (Fig. 7 ), confirming the efficacy of the selected temperatures and substance concentrations. However, no significant differences in growth were observed between VaCCaMK -transgenic cells and KA0 control cells under these stress conditions. All cell lines overexpressing the VaCCaMK gene and its short variants exhibited reduced growth at 16°C compared to the control; however, these differences relative to KA0 cell growth were not statistically significant (Fig. 7 ). Additionally, several VaCCaMK -expressing cell lines showed marginally enhanced growth at elevated temperatures (33°C), though the observed increase fell within the range of measurement error (Fig. 7 a,b). We also analyzed the content of valuable secondary metabolites capable of being synthesized by grape cells – specifically, stilbenes. In grape cell cultures, stilbenes occur in eight forms: resveratrol diglucoside, trans-piceid, cis-piceid, trans-piceatannol, trans-resveratrol, cis-resveratrol, ε‑viniferin, and δ‑viniferin (Aleynova et al. 2023 ). Following transformation with the VaCCaMK gene and its short variants, the stilbene content was in the range of 0.26–0.50 mg/g dry weight (DW), whereas the stilbene content in the control cell culture KA0 was 0.38 ± 0.05 mg/g DW (supplementary Table S2). Thus, transformation with the VaCCaMK gene did not lead to significant changes in stilbene accumulation. 3. Conclusions In this study, we successfully cloned and sequenced the full‑length VaCCaMK gene from V. amurensis , revealing a high degree of homology (99.6% at the amino acid level) with the VvCCaMK gene of V. vinifera . During cloning, two short transcript variants, VaCCaMK‑s1 and VaCCaMK‑s2 , were also identified. VaCCaMK‑s1 arises due to genomic instability and DNA recombination, featuring 8‑bp short direct repeats and a truncated cDNA sequence (906 bp). VaCCaMK‑s2 is a product of alternative splicing, lacking the 6-th exon and resulting in a 1503 bp cDNA sequence. Typically, plant genomes contain a single CCaMK gene, implying strong evolutionary conservation of this kinase function (Wang et al. 2015 ). However, recent findings suggest that post‑transcriptional regulation, particularly alternative splicing, may expand the functional diversity of CCaMK proteins. During cloning of the full‑length VaCCaMK cDNA from grape V. amurens is, a wild grapevine species notable for its high cold and disease resistance (Liu, Li, 2013 ), we identified multiple transcript variants with missing exons. This observation raises the possibility that alternative splicing generates functionally distinct isoforms of CCaMK proteins, potentially enabling fine‑tuned regulation of calcium signaling under different conditions. This is an interesting example, and it is possible that other low‑copy plant genes undergo similar modifications; however, this requires further research. Transgenic cell lines overexpressing VaCCaMK and its variants exhibited increased biomass accumulation, most notably with the full‑length gene (fresh weight: 1.6–1.7 times; dry mass: 2.2–2.5 times). However, no significant improvement in tolerance to temperature extremes, salt, or osmotic stress and no increase in the secondary metabolites was observed in transgenic cells compared to controls. Previous studies have demonstrated that overexpression of the TaCCaMK gene enhances wheat root growth (Yang et al. 2011 ). Similarly, CCaMK genes are frequently involved in nodule formation and development during bacterial‑plant interactions (Tirichine et al. 2006 ; Routray et al. 2013 ). Therefore, existing publications also indicate the growth‑promoting properties of these genes in other plant species, which is consistent with our findings. Overall, our findings characterize VaCCaMK and its splice variants in V. amurensis and suggest their role in biomass regulation rather than in abiotic stress tolerance or stilbene production, this is also important for plant biotechnology in the regulation of the biosynthetic properties of plant cells. 4. Materials and Methods 4.1. Plant material and drought, salt, cold and heat treatments of V. amurensis leaves Wild-type 10-12-year-old plants of gape V. amurensis were sampled from a non-protected natural population near Vladivostok (Akademgorodok, Russia). For VaCCaMK expression analysis, the V. amurensis vines were collected in September 2025 and divided into cuttings (excised young stems, approximately 8 cm long, with one healthy leaf). The cuttings were then placed in cups filled with filtered sterile water. Salt treatment was performed by adding 50 and 100 mM of NaCl to the water in cups. Cold and heat treatments were performed by 16°C and 33°C in a growth chamber (TSO-1/80, Smolenskoe SKTB SPU, Smolensk, Russia). Mannitol treatment (osmotic stress) was applied by adding 0.2 and 0.3 M of D-mannitol (Serva, New York, USA) to the water in cups (Aleynova et al. 2024 ). 4.2. Overexpression of VaCCaMK transgenes in cell cultures of V. amurensis The VaCCaMK transgene was used in three forms, including VaCCaMK (full-length), short VaCCaMK-s1 and VaCCaMK-s2 . To generate the construction for plant cell transformation, the sequences of the VaCCaMK gene transcript were amplified from cDNA of grapevine V. amurensis leaves by PCR using the primers presented in the supplementary Table S3. We used a heat-stable RNAscribe RT (Biolabmix, Novosibirsk, Russia) to obtain cDNA and then in PCR we used a high-fidelity Tersus polymerase (Evrogen, Moscow, Russia) to obtain VaCCaMK PCR products for cloning to plasmids. We have previously shown that this completely eliminates mutations or deletions (Kiselev et al. 2011 ; Dubrovina et al. 2014 ). The obtained VaCCaMK PCR products were subcloned into a pJET1.2 using the CloneJET PCR Cloning Kit (ThermoFisher Scientific, Waltham, MA, USA) and sequenced using an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manuphacturer’s instructions. Next, we performed PCR with the forward primer containing a Bgl II restriction site and the reverse primer containing a Sal I restriction site (supplementary Table S3). The VaCCaMK transcripts were cloned into the pSAT1 vector (Tzfira et al., 2015) by the Bgl II and Sal I sites. Then, the expression cassette from pSAT1 with the VaCCaMK genes was cloned into the pZP-RCS2-nptII vector (Tzfira et al., 2015) using the PalAI (AscI) sites. All transgenes in the used vectors were under the control of the double cauliflower mosaic virus promoter. The VaCCaMK overexpression constructs of (pZP-RCS2-VaCCaMK-nptII, pZP-RCS2- VaCCaMK-s1-nptII, or pZP-RCS2-VaCCaMK-s2-nptII) or empty vector (pZP-RCS2-nptII) were introduced into the Agrobacterium tumefaciens strain (GV3101::pMP90), which was used for the transformation of the suspension V7 culture of V. amurensis . 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 ) and are maintained in our laboratory. All VaCCaMK transgenic cell lines and KA0 cell line were obtained again: three lines for each genetic construction used, except for the one vector KA0 cell line, which overexpressed only the nptII gene and used as control. Grapevine cell lines were grown in the dark at 24–25°C for 35 days on Murashige and Skoog modified medium (Dubrovina et al., 2010 ) supplemented with 0.5 mg/L BAP, 2 mg/L NAA, and 8 g/L agar in the dark. Inoculum biomass was 0.15–0.17 g. 4.3. Nucleic acid purification and quantitative real-time PCR (qPCR) Cetyltrimethylammonium bromide (CTAB)-based extraction was used for total RNA isolation as described (Kiselev et al., 2013 ). cDNAs for qPCR were prepared using the RNAscribe RT kit (Biolabmix, Novosibirsk, Russia) with oligo(dT)15 at 55°C for 50 min as described in the manufacturer's protocol. The mRNA transcript levels of the transgenes were determined by the 2 −ΔΔCT method (Livak, Schmittgen, 2001 ) with two internal controls, including VaGAPDH (XM_002263109) and VaActin1 (DQ517935) for grape V. amurensis as described (Aleynova et al., 2022 ). Primers designed for qPCRs are shown in the supplementary Table S3. 4.4. Analysis of promoter cis-acting elements The PlantCARE database ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html , accessed on 15 Apryl 2026) was used to predict cis-regulatory elements in the 2000 bp promoter regions upstream of the transcription start sites of the VaCCaMK gene. The results were visualized using gggenes (version 0.6.0) (Wilkins, 2025 ) and tidyverse (version 2.0.0) (Wickham et al. 2019 ) R packages. 4.5. HPLC and mass spectrometry stilbene analysis Stilbenes levels were analyzed by HPLC with diode array detection as described (Dubrovina et al., 2010 ). The extracts were separated on Shim-pack GIST C18 column the on HPLC LC-20AD XR analytical system (Shimadzu, Japan), equipped with an SPD-M20A photodiode array detector. 4.5. Statistical analysis Three independent experiments with ten technical replicates in each experiment were performed for biomass accumulation and three independent experiments with three technical replicates in each experiment for the stilbene analysis in the callus cell lines. For the analysis of the VaCCaMK total, transgene, and endogenous expression, we performed three independent experiments with ten technical replicates (five qPCR reactions normalized to one internal control gene and five qPCR reactions – to the second internal gene in each independent experiment). Data are presented as mean ± standard error (SE) and were evaluated by Student’s t test performed in Microsoft Excel Standard 2019 (Microsoft Office, Microsoft, Redmond, Washington, USA). Declarations Author Contributions: KVK and ASD performed research design, interpretation and paper preparation. AAB, AAA, EVT, and OAA performed experiments with agrobacterial transformation, experiments on the cell lines, RNA isolations, RT-qPCRs, and data analysis. NNN carried out the bioinformatics and statistical analyses. ARS conducted the HPLS analysis. Funding: The research was carried out within the state assignment of Ministry of Science and Higher Education of the Russian Federation (theme No. 124012200181-4). Data Availability Statement: The data presented in this study are available within the article and Supplementary Materials. Compliance with Ethical Standards: We declare that we have no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References Aleynova OA, Suprun AR, Ananev AA, Nityagovsky NN, Ogneva ZV, Dubrovina AS, Kiselev KV (2022) Effect of calmodulin–like gene (CML) overexpression on stilbene biosynthesis in cell cultures of Vitis amurensis Rupr. Plants (Basel) 11:171 Aleynova OA, Kiselev KV, Suprun AR, Ananev AA, Dubrovina AS (2023) Involvement of the calmodulin–like protein gene VaCML92 in grapevine abiotic stress response and stilbene production. 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J Open Source Softw 4:1686 Wilkins D (2025) gggenes: Draw gene arrow maps in ggplot2. R package version 0.6.0. Available online: (accessed on 15 April 2026) Xin H, Zhu W, Wang L, Xiang Y, Fang L, Li J, Sun X, Wang N, Londo JP, Li S (2013) Genome–wide transcriptional profile analysis of Vitis amurensis and Vitis vinifera in response to cold stress. PLoS ONE, 8(3), e58740 Xu T, Niu J, Jiang Z (2022) Sensing mechanisms: calcium signaling mediated abiotic stress in plants. Front Plant Sci 13:925863 Yang C, Li A, Zhao Y et al (2011) Overexpression of a wheat CCaMK gene reduces ABA sensitivity of Arabidopsis thaliana during seed germination and seedling growth. Plant Mol Biol Rep 29:681–692 Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterialsinfo.docx supplementary.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 05 May, 2026 Reviewers agreed at journal 04 May, 2026 Reviewers invited by journal 04 May, 2026 Editor assigned by journal 28 Apr, 2026 Submission checks completed at journal 28 Apr, 2026 First submitted to journal 23 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9503080","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":634515761,"identity":"f6a21cd5-52d4-4933-a0a9-832fb3438f0f","order_by":0,"name":"Konstantin V. Kiselev","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACCWYg8YCBQc4AxGAwsCBSSwIDgzFUiwQRWhggWhI3IPHxA8l29ocPEioOp29nZz784UOBBIP5jAT8WqSZeYwNEs4czt3ZzJYmOQPoMJkbBLTIMfOwSSS2peVuOMxjxswD1CIhQVAL+/Mfif/S0g0O8xh/JkqLNDODGUNig00CUIuBNFFaJJt5jCUSjtkYwvzCI8HzAL8WifPHH374UCMhb85/GBhif2zkJNgJ2IIBeEhUPwpGwSgYBaMAGwAAJqw1pDOvq9IAAAAASUVORK5CYII=","orcid":"","institution":"Federal Scientific Center of the East Asia Terrestrial Biodiversity, FEB RAS","correspondingAuthor":true,"prefix":"","firstName":"Konstantin","middleName":"V.","lastName":"Kiselev","suffix":""},{"id":634515762,"identity":"9611897a-6e7a-4431-af06-eb464f82e6ac","order_by":1,"name":"Alina A. Beresh","email":"","orcid":"","institution":"Federal Scientific Center of the East Asia Terrestrial Biodiversity, FEB RAS","correspondingAuthor":false,"prefix":"","firstName":"Alina","middleName":"A.","lastName":"Beresh","suffix":""},{"id":634515763,"identity":"d2d69186-0501-441c-bcca-eb5df447579c","order_by":2,"name":"Alexey A. Ananev","email":"","orcid":"","institution":"Federal Scientific Center of the East Asia Terrestrial Biodiversity, FEB RAS","correspondingAuthor":false,"prefix":"","firstName":"Alexey","middleName":"A.","lastName":"Ananev","suffix":""},{"id":634515764,"identity":"1ea7a32f-f877-4644-a7db-b695f509da97","order_by":3,"name":"Olga A. Aleynova","email":"","orcid":"","institution":"Federal Scientific Center of the East Asia Terrestrial Biodiversity, FEB RAS","correspondingAuthor":false,"prefix":"","firstName":"Olga","middleName":"A.","lastName":"Aleynova","suffix":""},{"id":634515765,"identity":"0cd7f10c-a32f-4603-b765-0e4f2cb339b3","order_by":4,"name":"Nikolay N. Nityagovsky","email":"","orcid":"","institution":"Federal Scientific Center of the East Asia Terrestrial Biodiversity, FEB RAS","correspondingAuthor":false,"prefix":"","firstName":"Nikolay","middleName":"N.","lastName":"Nityagovsky","suffix":""},{"id":634515766,"identity":"452d096c-5634-447b-b199-dcd56930a5ab","order_by":5,"name":"Evgeniya V. Trubetskaya","email":"","orcid":"","institution":"Federal Scientific Center of the East Asia Terrestrial Biodiversity, FEB RAS","correspondingAuthor":false,"prefix":"","firstName":"Evgeniya","middleName":"V.","lastName":"Trubetskaya","suffix":""},{"id":634515767,"identity":"48b10322-b1bb-4ee1-bd56-907026cc70c6","order_by":6,"name":"Andrey R. Suprun","email":"","orcid":"","institution":"Federal Scientific Center of the East Asia Terrestrial Biodiversity, FEB RAS","correspondingAuthor":false,"prefix":"","firstName":"Andrey","middleName":"R.","lastName":"Suprun","suffix":""},{"id":634515768,"identity":"a51b40f1-780a-4259-9c86-82445172ae75","order_by":7,"name":"Alexandra S. Dubrovina","email":"","orcid":"","institution":"Federal Scientific Center of the East Asia Terrestrial Biodiversity, FEB RAS","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"S.","lastName":"Dubrovina","suffix":""}],"badges":[],"createdAt":"2026-04-23 06:53:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9503080/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9503080/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109067786,"identity":"c0c7898c-68dd-4a50-9c05-5fbe2bd7fe53","added_by":"auto","created_at":"2026-05-12 10:00:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":570261,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment of the conserved domains of several plant CCaMKs. K – kinase domain (red font), CaM – calmodulin binding domain (green font), EF – three EF-hands or the visinin-like domain (blue font). The DDBJ/GenBank/EMBL accession numbers of the above four CCaMKs were listed in Table 1.\u003c/p\u003e","description":"","filename":"Fig.1domains.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9503080/v1/55c253bb90af94cdf2d1997e.jpg"},{"id":109014145,"identity":"df5a0808-a5f2-49dc-b6ac-a23011c326cb","added_by":"auto","created_at":"2026-05-11 17:13:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":106202,"visible":true,"origin":"","legend":"\u003cp\u003ea) The structure representation of \u003cem\u003eVaCCaMK\u003c/em\u003e mRNA splice variants in grapevine \u003cem\u003eVitis amurensis \u003c/em\u003e(not to scale). Exons (E) and introns (I) are shown using boxes and lines, respectively, with white dashed boxes representing untranslated regions (UTRs). In blue font arrows and F and R – primes used for \u003cem\u003eVaCCaMK\u003c/em\u003e expression levels in the leaves of grapevine \u003cem\u003eV. amurensis\u003c/em\u003e cuttings. b) CCaMK functional domains: N – N-terminal domain, K – kinase domain (red font), CaM – calmodulin binding domain (green font), EF – three EF-hands or the visinin-like domain (blue font), C - C-terminal domain.\u003c/p\u003e","description":"","filename":"Fig.3promotor.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9503080/v1/a0adf5e7f2725a4c836b3d5b.jpg"},{"id":109068057,"identity":"af4393a9-f091-4583-9e21-8c67acf205d4","added_by":"auto","created_at":"2026-05-12 10:03:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":219975,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of \u003cem\u003ecis\u003c/em\u003e-acting elements in the promotor region of the grape \u003cem\u003eVaCCaMK\u003c/em\u003e gene. \u003cem\u003eCis\u003c/em\u003e-regulatory element analysis of \u003cem\u003eVaCCaMK\u003c/em\u003e gene. Sequences 2000 bp upstream of the start codon were extracted and cis-acting elements were predicted by PlantCARE and visualized using gggenes (version 0.6.0) (Wilkins, 2025) and tidyverse (version 2.0.0) (Wickham et al. 2019) R packages.\u003c/p\u003e","description":"","filename":"Fig.2shema.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9503080/v1/43bb199a6a2f47b9b804e06d.jpg"},{"id":109204420,"identity":"7c75713d-d836-46d3-9499-773d2aad13dd","added_by":"auto","created_at":"2026-05-13 14:59:30","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":99483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eVaCCaMK\u003c/em\u003e expression in grape \u003cem\u003eVitis amurensis\u003c/em\u003e leaves under different stress conditions. The \u003cem\u003eVaCCaMK\u003c/em\u003e expression levels were determined by quantitative RT-PCR and analyzed before stress conditions (0 h) and after 6 h, 12 h, and 24 h of treatments. C – non-stress conditions (filtered water, 25\u003csup\u003eo\u003c/sup\u003eC); WD – water-deficit stress (cuttings constantly laid on a paper towel, 25\u003csup\u003eo\u003c/sup\u003eC); NaCl – salt stress (constantly 0.4 M NaCl, 25\u003csup\u003eo\u003c/sup\u003eC); Man – mannitol (constantly 0.4 M mannitol, 25\u003csup\u003eo\u003c/sup\u003eC); 37 – heat stress (constantly filtered water, 37\u003csup\u003eo\u003c/sup\u003eC); 10 or 4 – cold stress (constantly filtered water, 10\u003csup\u003eo\u003c/sup\u003eC or and 4\u003csup\u003eo\u003c/sup\u003eC), UV-C – ultraviolet C irradiation (for 20 min at a wavelength of 254 nm, 230 μW cm\u003csup\u003e−2\u003c/sup\u003e, 15 cm from the lamp VL-215.MC, provided by Vilber Lourmat company (Marne-la-Vallée, France). The data are presented as mean ± standard error; r.u. – related units. Means followed by the same letter were not different using Student’s t test, where \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 was considered to be statistically significant.\u003c/p\u003e","description":"","filename":"Fig.4express.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9503080/v1/8360e66895559bec7d627568.jpg"},{"id":109067949,"identity":"ed8fc1a7-ca28-4fe3-8c40-835f861c0731","added_by":"auto","created_at":"2026-05-12 10:02:51","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":121166,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification the transgene (a), total (b), and endogenous (c) \u003cem\u003eVaCCaMK\u003c/em\u003eexpression in the transgenic cell cultures of \u003cem\u003eV. amurensis\u003c/em\u003e performed by quantitative real-time RT-PCR. RNA was extracted from the vector control (KA0), \u003cem\u003eVaCCaMK\u003c/em\u003e-transformed (F1, F2, F3), \u003cem\u003eVaCCaMK-s1\u003c/em\u003e-transformed (s1-1, s1-2, s1-3), and \u003cem\u003eVaCCaMK-s2\u003c/em\u003e-transformed (s2-1, s2-2, s2-3) cell lines of \u003cem\u003eV. amurensis\u003c/em\u003e. The data are presented as mean ± standard error; r.u. – related units. *p \u0026lt; 0.05; **p \u0026lt; 0.01 versus values of fluorescence in the KA0 cell culture transformed with the empty vector (Student’s \u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"Fig.5overexpress.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9503080/v1/992fa96effb82b10f4126b63.jpg"},{"id":109067966,"identity":"dd7dc151-5c77-4a60-88e2-d6962f74ef06","added_by":"auto","created_at":"2026-05-12 10:02:54","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":121488,"visible":true,"origin":"","legend":"\u003cp\u003eFresh and dry biomass accumulation in the cell lines KA0 (vector control), F1, F2, F3 (overexpressed \u003cem\u003eVaCCaMK\u003c/em\u003e gene), S1-1, S1-2, S1-3 (overexpressed \u003cem\u003eVaCCaMK-s1\u003c/em\u003etranscript), S2-1, S2-2, S2-3 (overexpressed \u003cem\u003eVaCCaMK-s2\u003c/em\u003e transcript) cell lines. The callus tissue samples were harvested from the 35 days old cultures. The data are presented as mean ± standard error; r.u. – related units. Means followed by the same letter were not different using Student’s \u003cem\u003et\u003c/em\u003e test. p \u0026lt; 0.05 was considered to be statistically significant.\u003c/p\u003e","description":"","filename":"Fig.6weight.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9503080/v1/02a2cfbfc4828d9e653d4992.jpg"},{"id":109203760,"identity":"7fa34c56-833d-4ec6-9e7b-6bc93ce4a3f7","added_by":"auto","created_at":"2026-05-13 14:45:42","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":437033,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of cold (16, 16\u003csup\u003eo\u003c/sup\u003eС), heat (35, 35\u003csup\u003eo\u003c/sup\u003eС), salt (NaCl, 1: 50 mM and 2: 100\u0026nbsp;mM), and mannitol (Man, 1: 200 and 2: 300 mM) induced osmotic stresses on the growth of 35-day-old transgenic callus cell cultures of \u003cem\u003eVitis amurensis\u003c/em\u003e. KA0 (vector control), F1, F2, F3 (a, overexpressed \u003cem\u003eVaCCaMK\u003c/em\u003e gene), S1-1, S1-2, S1-3 (b, overexpressed \u003cem\u003eVaCCaMK-s1\u003c/em\u003e transcript), S2-1, S2-2, S2-3 (c, overexpressed \u003cem\u003eVaCCaMK-s2\u003c/em\u003e transcript) cell lines. The data are presented as mean ± standard error; for 100% - growth in the control conditions for each cell line. Statistical significance was determined by Student’s t‑test: *p\u0026nbsp;\u0026lt;\u0026nbsp;0.05 and **p \u0026lt; 0.01 versus growth values of the respective cell line cultured under stress‑free control conditions (100%, marked in green).\u003c/p\u003e","description":"","filename":"Fig.7stress.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9503080/v1/efb1a3d89028c2728a8b6a53.jpg"},{"id":109208241,"identity":"08bbd760-fddf-4659-addd-b84c326bfae5","added_by":"auto","created_at":"2026-05-13 15:24:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1937600,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9503080/v1/8a289b5a-b27c-4660-beec-ff531a297589.pdf"},{"id":109014141,"identity":"dda4966b-7f78-4f9f-90d7-282a49da129c","added_by":"auto","created_at":"2026-05-11 17:13:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13942,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialsinfo.docx","url":"https://assets-eu.researchsquare.com/files/rs-9503080/v1/80e4444c38587fd7e9e63d84.docx"},{"id":109014143,"identity":"f213f1a2-becb-4064-972d-697cd6bd9111","added_by":"auto","created_at":"2026-05-11 17:13:37","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23368,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9503080/v1/a3f874798ffce62e2b7fb766.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impact of VaCCaMK gene overexpression and its splicing isoforms on cell growth and stilbene accumulation in Vitis amurensis Rupr","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCalcium (Ca\u003csup\u003e2+\u003c/sup\u003e) is a universal intracellular second messenger in plants, playing a pivotal role in transducing extracellular signals into coordinated cellular responses (McCormack et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Kudla et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). It regulates a wide array of physiological processes, including pathogen defence, stress adaptation, enzyme activation, stomatal movement, photomorphogenesis, and growth regulation (Reddy et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kiselev, Dubrovina, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The specificity and versatility of Ca\u003csup\u003e2+\u003c/sup\u003e signalling are achieved through a complex network of calcium‑binding proteins that decode the spatiotemporal patterns of Ca\u003csup\u003e2+\u003c/sup\u003e concentration changes, often referred to as \u0026ldquo;calcium signatures\u0026rdquo;, into specific downstream responses (Harper and Harmon \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe majority of Ca\u003csup\u003e2+\u003c/sup\u003e sensor proteins contain multiple EF‑hand motifs: conserved helix‑loop‑helix structures in which Ca\u003csup\u003e2+\u003c/sup\u003e ions are coordinated within the acidic Ca\u003csup\u003e2+\u003c/sup\u003e‑coordinating loop (Halling et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The major plant EF‑hand‑containing Ca\u003csup\u003e2+\u003c/sup\u003e‑binding proteins include calmodulins (CaMs), calmodulin‑like proteins (CMLs), Ca\u003csup\u003e2+\u003c/sup\u003e‑dependent protein kinases (CDPKs), calcineurin B‑like proteins (CBLs), and calcium/calmodulin‑dependent protein kinases (CCaMKs) (Hashimoto and Kudla \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Mohanta et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the diverse families of calcium‑binding proteins in plants CCaMKs occupy a unique position due to their dual capacity to bind both Ca\u003csup\u003e2+\u003c/sup\u003e ions and calmodulin. This dual regulatory mechanism allows CCaMKs to integrate calcium signals and transduce them into phosphorylation events, thereby modulating the activity of target proteins (Takezawa et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). CCaMKs are best known for their critical roles in plant\u0026ndash;microbe interactions, particularly in establishing symbioses with nitrogen‑fixing rhizobia and arbuscular mycorrhizal fungi. In these processes, CCaMK acts as a central component of the common symbiosis signaling pathway, where it decodes Ca\u003csup\u003e2+\u003c/sup\u003e oscillations and activates downstream transcriptional reprogramming (Hayashi et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite their importance in biotic interactions, the functions of CCaMKs in abiotic stress responses remain poorly understood. Abiotic stresses such as salinity, drought, osmotic imbalance, and extreme temperatures represent major constraints on plant growth and productivity worldwide (Kumar, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These stressors often trigger rapid increases in cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e concentrations, suggesting that calcium signalling pathways, including CCaMKs, may play a role in stress perception and adaptation (Xu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, direct evidence linking CCaMK activity to abiotic stress tolerance is limited, and the molecular mechanisms involved are largely unexplored (Contreras Delgado et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2026\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGrape \u003cem\u003eVitis amurensis\u003c/em\u003e is an excellent model for studying stress tolerance mechanisms, as it exhibits remarkable resilience to cold, pathogens, and other environmental challenges (Liu, Li, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Xin et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Grapevine cell cultures provide a controlled system to investigate molecular and physiological responses without the complexity of whole‑plant architecture. The present study aimed to elucidate the role of \u003cem\u003eVaCCaMK\u003c/em\u003e genes in abiotic stress responses and cell growth regulation in \u003cem\u003eV. amurensis\u003c/em\u003e. Specifically, we investigated the effects of overexpressing the full‑length grape \u003cem\u003eVaCCaMK\u003c/em\u003e gene and its alternatively spliced variants (\u003cem\u003eVaCCaMK-s1\u003c/em\u003e, \u003cem\u003eVaCCaMK-s2\u003c/em\u003e) in grapevine cell cultures under salt, osmotic, cold, and heat stress conditions. We also assessed changes in stilbene production, key secondary metabolites with antioxidant and antimicrobial properties, as well as biomass accumulation.\u003c/p\u003e"},{"header":"2. Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cloning and sequencing of VaCCaMK mRNA transcripts\u003c/h2\u003e \u003cp\u003ePreviously, the grapevine \u003cem\u003eVitis vinifera\u003c/em\u003e L. \u003cem\u003eVvCCaMK\u003c/em\u003e sequence was determined during whole-genome sequencing in BioProject PRJNA1013121 (Jaillon et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Shi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The protein-coding sequence of \u003cem\u003eVvCCaMK\u003c/em\u003e consists of 1563 bp and 7 exons. Next, using primers for the beginning and end of the protein-coding sequence of the known \u003cem\u003eVvCCaMK\u003c/em\u003e gene (GenBank XM_002273306) on cDNA obtained from a wild-growing \u003cem\u003eV\u003c/em\u003e. \u003cem\u003eamurensis\u003c/em\u003e grape leaf, we obtained the full-length protein-coding sequence of the \u003cem\u003eVaCCaMK\u003c/em\u003e gene. The resulting \u003cem\u003eVaCCaMK\u003c/em\u003e sequence was highly homologous to the \u003cem\u003eVvCCaMK\u003c/em\u003e gene in terms of nucleotide composition (Identities 99.2%) and deduced amino acid sequence (Identities 99.6%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the available grape \u003cem\u003eV. amurensis\u003c/em\u003e genome (Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), we found only one copy of the \u003cem\u003eVaCCaMK\u003c/em\u003e gene.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eCCaMK\u003c/em\u003e genes in plant species with known functions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003cp\u003eor eudicots\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGene functions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAccession no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLily \u003cem\u003eLilium longiforum\u003c/em\u003e, LlCCaMK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePreferentially expressed in developing anthers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQ43531\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePatil et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1995\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWheat, \u003cem\u003eTriticum aestivum\u003c/em\u003e, TaCCaMK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOverexpressing \u003cem\u003eTaCCaMK\u003c/em\u003e in Arabidopsis plants reduced their sensitivity to abscisic acid (ABA) treatment during seed germination and root elongation. Increased root length\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHM595635\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaize, \u003cem\u003eZea mays\u003c/em\u003e, ZmCCaMK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIt plays a key role in the antioxidant protection of plants induced by brassinosteroids and in increasing their resistance to drought\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eABD67491\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLiu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLotus japonicus\u003c/em\u003e, LjCCaMK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSingle amino-acid replacement in a CCaMK is sufficient to turn fully differentiated root cortical cells into meristematic founder cells of root nodule primordia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCAJ76700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTirichine et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMedicago truncatula\u003c/em\u003e, MtCCaMK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIt ensures the formation of nodules when the plant interacts with nitrogen-fixing bacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQ6RET7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRoutray et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTobacco, \u003cem\u003eNicotiana tabacum\u003c/em\u003e, NtCCaMK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIt has been shown that their expression is strictly timed to the stage of anther development, when the buds are 0.5\u0026ndash;1.0 cm in size, which coincides with meiosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAAD28791\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLiu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1998\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIt is important to note that while we were trying to obtain a full-length \u003cem\u003eVaCCaMK\u003c/em\u003e sequence, we cloned various short \u003cem\u003eVaCCaMK\u003c/em\u003e variants, even though we used a special heat-stable reverse transcriptase and high-fidelity DNA polymerase. Among these short \u003cem\u003eVaCCaMK\u003c/em\u003e variants, there were two main sequences that were repeated several times in two independent transformations: \u003cem\u003eVaCCaMK-s1\u003c/em\u003e and \u003cem\u003eVaCCaMK-s2\u003c/em\u003e. We did not find such short variants in the \u003cem\u003eV. amurensis\u003c/em\u003e genome. (Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). It was only after the third transformation attempt and the analysis of more than 20 clones that we were able to obtain the standard sequence \u003cem\u003eVaCCaMK\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eWe showed that the E1 and E4 exon sequences of \u003cem\u003eVaCCaMK-s1\u003c/em\u003e had 8-bp short direct repeats (SDR): TGTTTCAC, resulting that the partial sequence of E1 and E4 and the complete sequence of E2 and E3 are missing from the \u003cem\u003eVaCCaMK-s1\u003c/em\u003e transcript sequence and at the same time, the protein reading frame does not get messed up (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). This results in a 906 bp cDNA sequence that can be used to produce a shortened protein missing part of the kinase domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The production of this \u003cem\u003eVaCCaMK-s1\u003c/em\u003e transcript can be attributed to mechanisms of genomic instability and DNA recombination (Ogihara et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1988\u003c/span\u003e), but due to its high abundance during cloning, we decided to include it in our further research.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA feature of \u003cem\u003eVaCCaMK-s2\u003c/em\u003e transcript was that the entire 6-th exon was missing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), what is a typical example of alternative splicing (Dubrovina et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This results in a 1503 bp cDNA sequence that can be used to produce a shortened protein missing second EF-hand, protein with two EF-hands (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Also, due to its high abundance during cloning, we decided to include it in our further research.\u003c/p\u003e \u003cp\u003eNext, we performed a promoter analysis (2000 bp upstream of the start codon) of the \u003cem\u003eVaCCaMK\u003c/em\u003e gene using PlantCARE. We identified over a hundred different \u003cem\u003ecis\u003c/em\u003e-acting elements (supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e); however, only those with well‑established functions are presented in the (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Thus, the \u003cem\u003eVaCCaMK\u003c/em\u003e gene promoter contains the highest number of light‑responsive elements (6 elements). It also includes elements associated with phytohormone regulation: one for auxin, one for abscisic acid, and one for gibberellins. Additionally, elements responsive to stress hormones were detected: one for salicylic acid and one for methyl jasmonate. Elements associated with abiotic stresses were also present, albeit in smaller numbers (2 for drought and 1 for wounding).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Expression of VaCCaMK mRNA transcripts in grapevine V. amurensis leaves under abiotic stress conditions\u003c/h2\u003e \u003cp\u003eIn this section, we analyzed the expression of the \u003cem\u003eVaCCaMK\u003c/em\u003e gene in grape leaves under various common abiotic stresses: water deficit, salt stress, osmotic stress, high temperatures, low temperatures, and ultraviolet С irradiation. It was shown that \u003cem\u003eVaCCaMK\u003c/em\u003e expression increased in most stresses, but this increase was small and often within the error limits. Only salt stress significantly increased \u003cem\u003eVaCCaMK\u003c/em\u003e expression by 1.4\u0026ndash;1.9 times at all time points compared to untreated control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Therefore, it was important to see how overexpression of the \u003cem\u003eVaCCaMK\u003c/em\u003e gene would affect resistance to the described stresses, primarily salt stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. VaCCaMK1 gene overexpression in the grapevine V. amurensis cell cultures\u003c/h2\u003e \u003cp\u003eUsing agrobacterial transformation we independently obtained three cell lines for each used gene construction: F1, F2, and F3 for \u003cem\u003eVaCCaMK\u003c/em\u003e gene, S1-1, S1-2, and S1-3 for \u003cem\u003eVaCCaMK-s1\u003c/em\u003e gene, or S2-1, S2-2, and S2-3 for \u003cem\u003eVaCCaMK-s2\u003c/em\u003e gene. Next, we showed that the resulting transgenic lines actively expressed the corresponding transgenes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), resulting in a significant increase in the overall expression of VaCCaMK in all transgenic cell lines, which was 3.1\u0026ndash;8.4 times higher than in the control KA0 cell culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). At the same time, overexpression of the \u003cem\u003eVaCCaMK\u003c/em\u003e transgenes was found to reduce expression of the endogenous \u003cem\u003eVaCCaMK\u003c/em\u003e gene by 1.1\u0026ndash;2.7‑fold, although this reduction did not reach statistical significance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt was further shown that overexpression of the \u003cem\u003eVaCCaMK\u003c/em\u003e gene and its alternative transcripts \u003cem\u003eVaCCaMK-s1\u003c/em\u003e and \u003cem\u003eVaCCaMK-s2\u003c/em\u003e increases the accumulation of fresh and dry biomass by the resulting transgenic grape cells. The accumulation of biomass by transgenic cells was greatest with overexpression of the \u003cem\u003eVaCCaMK\u003c/em\u003e gene: the accumulation of fresh weight significantly increased by 1.6\u0026ndash;1.7 times and dry mass by 2.2\u0026ndash;2.5 times (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Overexpression of the \u003cem\u003eVaCCaMK-s2\u003c/em\u003e transcript increased the accumulation of fresh biomass by 1.5\u0026ndash;1.7 times and dry by 1.9\u0026ndash;2.4 times (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Overexpression of the \u003cem\u003eVaCCaMK-s1\u003c/em\u003e transcript also activated an increase in fresh and dry weight, but this increase was to a lesser extent (1.0-1.5 and 1.8\u0026ndash;2.4 times) and was not significant for all three cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt is well established that plant calcium sensors play key roles in stress resistance. We therefore investigated the effect of \u003cem\u003eVaCCaMK\u003c/em\u003e transcript overexpression on the tolerance of transgenic grape cells to temperature extremes (high and low), salt stress, and osmotic stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The data show that the applied conditions significantly reduced fresh biomass accumulation in the control KA0 cell culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), confirming the efficacy of the selected temperatures and substance concentrations. However, no significant differences in growth were observed between \u003cem\u003eVaCCaMK\u003c/em\u003e-transgenic cells and KA0 control cells under these stress conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll cell lines overexpressing the \u003cem\u003eVaCCaMK\u003c/em\u003e gene and its short variants exhibited reduced growth at 16\u0026deg;C compared to the control; however, these differences relative to KA0 cell growth were not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Additionally, several \u003cem\u003eVaCCaMK\u003c/em\u003e-expressing cell lines showed marginally enhanced growth at elevated temperatures (33\u0026deg;C), though the observed increase fell within the range of measurement error (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea,b).\u003c/p\u003e \u003cp\u003eWe also analyzed the content of valuable secondary metabolites capable of being synthesized by grape cells \u0026ndash; specifically, stilbenes. In grape cell cultures, stilbenes occur in eight forms: resveratrol diglucoside, trans-piceid, cis-piceid, trans-piceatannol, trans-resveratrol, cis-resveratrol, ε‑viniferin, and δ‑viniferin (Aleynova et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Following transformation with the \u003cem\u003eVaCCaMK\u003c/em\u003e gene and its short variants, the stilbene content was in the range of 0.26\u0026ndash;0.50 mg/g dry weight (DW), whereas the stilbene content in the control cell culture KA0 was 0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mg/g DW (supplementary Table S2). Thus, transformation with the \u003cem\u003eVaCCaMK\u003c/em\u003e gene did not lead to significant changes in stilbene accumulation.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Conclusions","content":"\u003cp\u003eIn this study, we successfully cloned and sequenced the full‑length \u003cem\u003eVaCCaMK\u003c/em\u003e gene from \u003cem\u003eV. amurensis\u003c/em\u003e, revealing a high degree of homology (99.6% at the amino acid level) with the \u003cem\u003eVvCCaMK\u003c/em\u003e gene of \u003cem\u003eV. vinifera\u003c/em\u003e. During cloning, two short transcript variants, \u003cem\u003eVaCCaMK‑s1\u003c/em\u003e and \u003cem\u003eVaCCaMK‑s2\u003c/em\u003e, were also identified. \u003cem\u003eVaCCaMK‑s1\u003c/em\u003e arises due to genomic instability and DNA recombination, featuring 8‑bp short direct repeats and a truncated cDNA sequence (906 bp). \u003cem\u003eVaCCaMK‑s2\u003c/em\u003e is a product of alternative splicing, lacking the 6-th exon and resulting in a 1503 bp cDNA sequence.\u003c/p\u003e \u003cp\u003eTypically, plant genomes contain a single \u003cem\u003eCCaMK\u003c/em\u003e gene, implying strong evolutionary conservation of this kinase function (Wang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, recent findings suggest that post‑transcriptional regulation, particularly alternative splicing, may expand the functional diversity of CCaMK proteins. During cloning of the full‑length \u003cem\u003eVaCCaMK\u003c/em\u003e cDNA from grape \u003cem\u003eV. amurens\u003c/em\u003eis, a wild grapevine species notable for its high cold and disease resistance (Liu, Li, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), we identified multiple transcript variants with missing exons. This observation raises the possibility that alternative splicing generates functionally distinct isoforms of CCaMK proteins, potentially enabling fine‑tuned regulation of calcium signaling under different conditions. This is an interesting example, and it is possible that other low‑copy plant genes undergo similar modifications; however, this requires further research.\u003c/p\u003e \u003cp\u003eTransgenic cell lines overexpressing \u003cem\u003eVaCCaMK\u003c/em\u003e and its variants exhibited increased biomass accumulation, most notably with the full‑length gene (fresh weight: 1.6\u0026ndash;1.7 times; dry mass: 2.2\u0026ndash;2.5 times). However, no significant improvement in tolerance to temperature extremes, salt, or osmotic stress and no increase in the secondary metabolites was observed in transgenic cells compared to controls. Previous studies have demonstrated that overexpression of the \u003cem\u003eTaCCaMK\u003c/em\u003e gene enhances wheat root growth (Yang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Similarly, CCaMK genes are frequently involved in nodule formation and development during bacterial‑plant interactions (Tirichine et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Routray et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Therefore, existing publications also indicate the growth‑promoting properties of these genes in other plant species, which is consistent with our findings.\u003c/p\u003e \u003cp\u003eOverall, our findings characterize \u003cem\u003eVaCCaMK\u003c/em\u003e and its splice variants in \u003cem\u003eV. amurensis\u003c/em\u003e and suggest their role in biomass regulation rather than in abiotic stress tolerance or stilbene production, this is also important for plant biotechnology in the regulation of the biosynthetic properties of plant cells.\u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Plant material and drought, salt, cold and heat treatments of V. amurensis leaves\u003c/h2\u003e \u003cp\u003eWild-type 10-12-year-old plants of gape \u003cem\u003eV. amurensis\u003c/em\u003e were sampled from a non-protected natural population near Vladivostok (Akademgorodok, Russia). For \u003cem\u003eVaCCaMK\u003c/em\u003e expression analysis, the \u003cem\u003eV. amurensis\u003c/em\u003e vines were collected in September 2025 and divided into cuttings (excised young stems, approximately 8 cm long, with one healthy leaf). The cuttings were then placed in cups filled with filtered sterile water. Salt treatment was performed by adding 50 and 100 mM of NaCl to the water in cups. Cold and heat treatments were performed by 16\u0026deg;C and 33\u0026deg;C in a growth chamber (TSO-1/80, Smolenskoe SKTB SPU, Smolensk, Russia). Mannitol treatment (osmotic stress) was applied by adding 0.2 and 0.3 M of D-mannitol (Serva, New York, USA) to the water in cups (Aleynova et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Overexpression of VaCCaMK transgenes in cell cultures of V. amurensis\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eVaCCaMK\u003c/em\u003e transgene was used in three forms, including \u003cem\u003eVaCCaMK\u003c/em\u003e (full-length), short \u003cem\u003eVaCCaMK-s1\u003c/em\u003e and \u003cem\u003eVaCCaMK-s2\u003c/em\u003e. To generate the construction for plant cell transformation, the sequences of the \u003cem\u003eVaCCaMK\u003c/em\u003e gene transcript were amplified from cDNA of grapevine \u003cem\u003eV. amurensis\u003c/em\u003e leaves by PCR using the primers presented in the supplementary Table S3.\u003c/p\u003e \u003cp\u003eWe used a heat-stable RNAscribe RT (Biolabmix, Novosibirsk, Russia) to obtain cDNA and then in PCR we used a high-fidelity Tersus polymerase (Evrogen, Moscow, Russia) to obtain \u003cem\u003eVaCCaMK\u003c/em\u003e PCR products for cloning to plasmids. We have previously shown that this completely eliminates mutations or deletions (Kiselev et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Dubrovina et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The obtained VaCCaMK PCR products were subcloned into a pJET1.2 using the CloneJET PCR Cloning Kit (ThermoFisher Scientific, Waltham, MA, USA) and sequenced using an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manuphacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eNext, we performed PCR with the forward primer containing a Bgl II restriction site and the reverse primer containing a Sal I restriction site (supplementary Table S3). The \u003cem\u003eVaCCaMK\u003c/em\u003e transcripts were cloned into the pSAT1 vector (Tzfira et al., 2015) by the Bgl II and Sal I sites. Then, the expression cassette from pSAT1 with the \u003cem\u003eVaCCaMK\u003c/em\u003e genes was cloned into the pZP-RCS2-nptII vector (Tzfira et al., 2015) using the PalAI (AscI) sites. All transgenes in the used vectors were under the control of the double cauliflower mosaic virus promoter.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eVaCCaMK\u003c/em\u003e overexpression constructs of (pZP-RCS2-VaCCaMK-nptII, pZP-RCS2- VaCCaMK-s1-nptII, or pZP-RCS2-VaCCaMK-s2-nptII) or empty vector (pZP-RCS2-nptII) were introduced into the \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain (GV3101::pMP90), which was used for the transformation of the suspension V7 culture of \u003cem\u003eV. amurensis\u003c/em\u003e. The V7 callus cultures were established in 2017 from young stems of the wild-growing mature \u003cem\u003eV. amurensis\u003c/em\u003e vines near Vladivostok as described in (Tyunin et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and are maintained in our laboratory.\u003c/p\u003e \u003cp\u003eAll \u003cem\u003eVaCCaMK\u003c/em\u003e transgenic cell lines and KA0 cell line were obtained again: three lines for each genetic construction used, except for the one vector KA0 cell line, which overexpressed only the \u003cem\u003enptII\u003c/em\u003e gene and used as control. Grapevine cell lines were grown in the dark at 24\u0026ndash;25\u0026deg;C for 35 days on Murashige and Skoog modified medium (Dubrovina et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) supplemented with 0.5 mg/L BAP, 2 mg/L NAA, and 8 g/L agar in the dark. Inoculum biomass was 0.15\u0026ndash;0.17 g.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Nucleic acid purification and quantitative real-time PCR (qPCR)\u003c/h2\u003e \u003cp\u003eCetyltrimethylammonium bromide (CTAB)-based extraction was used for total RNA isolation as described (Kiselev et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). cDNAs for qPCR were prepared using the RNAscribe RT kit (Biolabmix, Novosibirsk, Russia) with oligo(dT)15 at 55\u0026deg;C for 50 min as described in the manufacturer's protocol. The mRNA transcript levels of the transgenes were determined by the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method (Livak, Schmittgen, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) with two internal controls, including \u003cem\u003eVaGAPDH\u003c/em\u003e (XM_002263109) and \u003cem\u003eVaActin1\u003c/em\u003e (DQ517935) for grape \u003cem\u003eV. amurensis\u003c/em\u003e as described (Aleynova et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Primers designed for qPCRs are shown in the supplementary Table S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Analysis of promoter cis-acting elements\u003c/h2\u003e \u003cp\u003eThe PlantCARE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on 15 Apryl 2026) was used to predict cis-regulatory elements in the 2000 bp promoter regions upstream of the transcription start sites of the \u003cem\u003eVaCCaMK\u003c/em\u003e gene. The results were visualized using gggenes (version 0.6.0) (Wilkins, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and tidyverse (version 2.0.0) (Wickham et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) R packages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.5. HPLC and mass spectrometry stilbene analysis\u003c/h2\u003e \u003cp\u003eStilbenes levels were analyzed by HPLC with diode array detection as described (Dubrovina et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The extracts were separated on Shim-pack GIST C18 column the on HPLC LC-20AD XR analytical system (Shimadzu, Japan), equipped with an SPD-M20A photodiode array detector.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.5. Statistical analysis\u003c/h2\u003e \u003cp\u003eThree independent experiments with ten technical replicates in each experiment were performed for biomass accumulation and three independent experiments with three technical replicates in each experiment for the stilbene analysis in the callus cell lines. For the analysis of the \u003cem\u003eVaCCaMK\u003c/em\u003e total, transgene, and endogenous expression, we performed three independent experiments with ten technical replicates (five qPCR reactions normalized to one internal control gene and five qPCR reactions \u0026ndash; to the second internal gene in each independent experiment). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE) and were evaluated by Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test performed in Microsoft Excel Standard 2019 (Microsoft Office, Microsoft, Redmond, Washington, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKVK and ASD performed research design, interpretation and paper preparation.\u0026nbsp;AAB, AAA, EVT,\u0026nbsp;and OAA performed\u0026nbsp;experiments with agrobacterial transformation, experiments on the cell lines, RNA isolations, RT-qPCRs, and data analysis.\u0026nbsp;NNN carried out the bioinformatics and statistical analyses. ARS conducted the HPLS analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The research was carried out within the state assignment of Ministry of Science and Higher Education of the Russian Federation (theme No. 124012200181-4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe data presented in this study are available within the article and Supplementary Materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards:\u003c/strong\u003e We declare that we have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAleynova OA, Suprun AR, Ananev AA, Nityagovsky NN, Ogneva ZV, Dubrovina AS, Kiselev KV (2022) Effect of calmodulin\u0026ndash;like gene (CML) overexpression on stilbene biosynthesis in cell cultures of Vitis amurensis Rupr. 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Available online: (accessed on 15 April 2026)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXin H, Zhu W, Wang L, Xiang Y, Fang L, Li J, Sun X, Wang N, Londo JP, Li S (2013) Genome\u0026ndash;wide transcriptional profile analysis of Vitis amurensis and Vitis vinifera in response to cold stress. PLoS ONE, 8(3), e58740\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu T, Niu J, Jiang Z (2022) Sensing mechanisms: calcium signaling mediated abiotic stress in plants. Front Plant Sci 13:925863\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang C, Li A, Zhao Y et al (2011) Overexpression of a wheat CCaMK gene reduces ABA sensitivity of Arabidopsis thaliana during seed germination and seedling growth. Plant Mol Biol Rep 29:681\u0026ndash;692\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"abiotic stress, calcium, gene expression, secondary metabolites, tolerance","lastPublishedDoi":"10.21203/rs.3.rs-9503080/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9503080/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCalcium (Ca\u0026sup2;⁺) functions as an essential intracellular second messenger in plants, mediating processes such as pathogen defence, stress adaptation and enzyme activation. Plants possess multiple families of calcium‑binding proteins, and among these, calcium/calmodulin‑dependent protein kinases (CCaMKs) remain among the least characterized. Also, their dual capacity to bind both Ca\u0026sup2;⁺ and calmodulin makes them a subject of significant research interest. Usually, there is one \u003cem\u003eCCaMK\u003c/em\u003e gene per plant genome, but when we cloned the full-length \u003cem\u003eVaCCaMK\u003c/em\u003e cDNA sequence, we found several transcripts with missing exons, so it is possible that alternative splicing increases the overall diversity of \u003cem\u003eCCaMK\u003c/em\u003e sequences. This study investigated the role of CCaMKs in \u003cem\u003eVitis amurensis\u003c/em\u003e Rupr. under abiotic stress conditions using grapevine cell cultures overexpressing the \u003cem\u003eVaCCaMK1\u003c/em\u003e gene and its alternatives \u003cem\u003eVaCCaMK1-\u003c/em\u003es1, -s2. Results demonstrated that \u003cem\u003eVaCCaMK1\u003c/em\u003e‑overexpressing cultures did not exhibit increased tolerance to salt, osmotic, cold, or heat stress. Additionally, the content of secondary metabolites, as represented by stilbenes mainly produced by used grapevine cells, remained largely unchanged. However, a significant increase in both fresh and dry cell mass was observed compared with the control group: fresh biomass increased 1.1\u0026ndash;1.7‑fold, and dry biomass 1.1\u0026ndash;1.8‑fold. These findings indicate that \u003cem\u003eVaCCaMK\u003c/em\u003e genes do not affect plant cell susceptibility to the tested abiotic stresses. \u003cem\u003eVaCCaMK1-s1\u003c/em\u003e or -\u003cem\u003es2\u003c/em\u003e the overexpression had a similar but weaker effect on grape cells. Nevertheless, VaCCaMK appear to act as a positive regulator of cell growth and development in \u003cem\u003eV. amurensis\u003c/em\u003e, suggesting their potential role in enhancing biomass accumulation.\u003c/p\u003e","manuscriptTitle":"Impact of VaCCaMK gene overexpression and its splicing isoforms on cell growth and stilbene accumulation in Vitis amurensis Rupr","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 17:13:24","doi":"10.21203/rs.3.rs-9503080/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-05T05:04:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"203724580989805328148925200680472586405","date":"2026-05-04T08:51:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-04T08:34:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-29T03:06:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-29T03:06:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2026-04-23T06:36:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c02fd36f-e534-4591-8c50-1dd50b4b293c","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-05T05:04:29+00:00","index":14,"fulltext":""},{"type":"reviewerAgreed","content":"203724580989805328148925200680472586405","date":"2026-05-04T08:51:16+00:00","index":12,"fulltext":""},{"type":"reviewersInvited","content":"7","date":"2026-05-04T08:34:12+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T17:13:25+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 17:13:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9503080","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9503080","identity":"rs-9503080","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}