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Stilbene synthases (STS) are plant enzymes that are responsible for the biosynthesis of stilbenes, which are plant phenolic compounds with valuable biological properties. Stilbenes also play important roles in plant tolerance to biotic and abiotic stresses. Therefore, plants that overexpress STS genes can be more resistant to various stresses. This paper investigated the effects of STS gene overexpression in Arabidopsis thaliana (L.) Heynh. Columbia-0 plants on stilbene content and tolerance to the following abiotic stresses: low and high temperatures, salinity, drought, and ultraviolet irradiation (UV-B and UV-C). We used VaSTS1 and VaSTS7 genes from grapevine (Vitis amurensis Rupr.) expressed under the double cauliflower mosaic virus 35S (CaMV35S) promoter. This study firstly demonstrated that overexpression of the VaSTS1 and VaSTS7 genes in A. thaliana plants considerably increased plant tolerance to UV-B and UV-C, while the tolerance to the low and high temperatures, salinity, and drought was not affected. We showed that the highest trans-piceid and trans-resveratrol total content was in ST1 A. thaliana plants that overexpressed the VaSTS1 gene in the range 8.28 - 22.66 µg g-1(f.m.). ST7 plants that overexpressed the VaSTS7 gene showed only trans-resveratrol at 0.02 - 0.08 µg g-1(f.m). Stilbene content and UV tolerance in transgenic A. thaliana plants correlated with STS transgene expression. STS expression, UV tolerance, and stilbene content was higher in VaSTS1 transgenic plants compared with that in VaSTS7 transgenic plants.

The cauliflower mosaic virus (CaMV) 35S promoter is known as the most frequently used promoter in plant biotechnology. Although it is widely considered to be a strong constitutive promoter exhibiting high transcriptional activity, the transcriptional stability of CaMV 35S has not been extensively studied. Using the model plant species Arabidopsis thaliana, this study aimed for a comprehensive expression analysis of two widely used plant transgenes, neomycin phosphotransferase II (NPTII) and enhanced green fluorescent protein (EGFP), regulated by a double CaMV 35S promoter depending on the organ type, time of day, plant age, and in response to abiotic stress conditions. Quantitative real-time PCR (qRT-PCR) analysis revealed that the NPTII and EGFP transcript levels were markedly higher in the cotyledons, young leaves, and roots than in the inflorescences, stems, and adult leaves of three independent transgenic A. thaliana lines. The expression of NPTII and EGFP varied during the day and was elevated with the plant age. Drought and cold stress considerably affected the expression of the transgenes, while heat, high salinity, and wounding had no significant effect. This study shows that transgenes driven by a common constitutive promoter can exhibit marked variations in transcriptional activity depending on plant organ, physiological conditions, and in response to abiotic stress. Therefore, to ensure high and stable transgene activity, considerable attention should be given to the transgenic plant material and incubation conditions before harvesting the plant material.

Stilbenes are natural compounds protecting plants against microbial pathogens and known to possess valuable biologically active properties. In the present study, we established transgenic grapevine callus cell cultures overexpressing three stilbene synthase (STS) genes of spruce Picea jezoensis PjSTS1a, PjSTS2, and PjSTS3. Transformation of Vitis amurensis calli with the PjSTS1a, PjSTS2, and PjSTS3 genes significantly increased total content of stilbenes in 3.6-6, 2.5-2.9, and 4.1-16.1 times, respectively, in comparison with the control calli. The most pronounced positive effect on the accumulation of stilbenes was observed for the PjSTS3-overexpressing calli where the total content of stilbenes was increased up to 3.1 mg/g DW, and the stilbene production reached 25.4 mg/L. These values were higher than those achieved for the grapevine callus cell cultures overexpressing three STS genes from V. amurensis. Thus, transformation of grapevine cell cultures with spruce STS genes with a relatively low degree of homology to the endogenous VaSTSs is a more effective strategy for induction of plant secondary metabolite biosynthesis than using the grapevine genes for the overexpression experiments.

Excessive ultraviolet B (UV-B) irradiation is one of the most serious threats leading to severe crop production losses. It is known that secondary metabolite biosynthesis plays an important role in plant defense and forms a protective shield against excessive UV-B irradiation. The contents of stilbenes and other plant phenolics are known to sharply increase after UV-B irradiation, but there is little direct evidence for the involvement of stilbenes and other plant phenolics in plant UV-B protection. This study showed that foliar application of trans-resveratrol (1 and 5 mM) and trans-piceid (5 mM) considerably increased tolerance to a shock of UV-B (10 min at 1800 µW cm−2 of irradiation intensity) of four-week-old Arabidopsis thaliana plants that are naturally incapable of stilbene production. Application of trans-resveratrol and trans-piceid increased the leaf survival rates by 1–2%. This stilbene-induced improvement in UV-B tolerance was higher than after foliar application of the stilbene precursors, p-coumaric and trans-cinnamic acids (only 1–3%), but less than that after treatment with octocrylene (19–24%), a widely used UV-B absorber. Plant treatment with trans-resveratrol increased expression of antioxidant and stress-inducible genes in A.thaliana plants and decreased expression of DNA repair genes. This study directly demonstrates an important positive role of stilbenes in plant tolerance to excessive UV-B irradiation, and offers a new approach for plant UV-B protection.

Exogenous application of double-stranded RNAs (dsRNAs) and small-interfering RNAs (siRNAs) to plant surfaces has emerged as a promising method for regulation of essential genes in plant pathogens and for plant disease protection. Yet, regulation of plant endogenous genes via external RNA treatments has not been sufficiently investigated. In this study, we targeted the genes of chalcone synthase (CHS), the key enzyme in the flavonoid/anthocyanin biosynthesis pathway, and two transcriptional factors, MYBL2 and ANAC032, negatively regulating anthocyanin biosynthesis in Arabidopsis. Direct foliar application of AtCHS-specific dsRNAs and siRNAs resulted in an efficient downregulation of the AtCHS gene and suppressed anthocyanin accumulation in A. thaliana under anthocyanin biosynthesis-modulating conditions. Targeting the AtMYBL2 and AtANAC032 genes by foliar dsRNA treatments markedly reduced their mRNA levels and led to a pronounced upregulation of the AtCHS gene. The content of anthocyanins was increased after treatment with AtMYBL2-dsRNA. Laser scanning microscopy showed a passage of Cy3-labeled AtCHS-dsRNA into the A. thaliana leaf vessels, leaf parenchyma cells, and stomata, indicating the dsRNA uptake and spreading into leaf tissues and plant individual cells. Together, these data show that exogenous dsRNAs were capable of downregulating Arabidopsis genes and induced relevant biochemical changes, which may have applications in plant biotechnology and gene functional studies.

Recent studies have revealed that foliar application of double-stranded RNAs (dsRNAs) or small-interfering RNAs (siRNAs) encoding specific genes of plant pathogens triggered RNA interference (RNAi)-mediated silencing of the gene targets. However, a limited number of reports documented silencing of plant endogenes or transgenes after direct foliar RNA application. This study analyzed the importance of physiological conditions (plant age, time of day, soil moisture, high salinity, heat, and cold stresses) and different dsRNA application means (brush spreading, spraying, infiltration, inoculation, needle injection, and pipetting) for suppression of neomycin phosphotransferase II (NPTII) transgene in Arabidopsis thaliana, as transgenes are more prone to silencing. We observed a higher NPTII suppression when dsRNA was applied at late day period, being most efficient at night, which revealed a diurnal variation in dsRNA treatment efficacy. Exogenous NPTII-dsRNA considerably reduced NPTII expression in 4-week-old plants and only limited it in 2- and 6-week-old plants. In addition, a more discernible NPTII downregulation was detected under low soil moisture conditions. Treatment of adaxial and abaxial leaf surfaces by brushes, spraying, and pipetting showed a higher NPTII suppression, while infiltration and inoculation were less efficient. Thus, appropriate plant age, late time of day, low soil moisture, and optimal dsRNA application modes are important for exogenously induced gene silencing.

Recent research has shown that plants can uptake long dsRNAs and dsRNA-derived siRNAs that target important genes of infecting fungi or viruses when applied on the surface of plant leaves. The external RNAs were capable of local and systemic movement inducing plant resistance against the pathogens. Few studies have been made for plant gene regulation by foliar application of RNAs. In this study, several types of ssRNA and siRNA duplexes targeting the neomycin phosphotransferase II (NPTII) transgene were in vitro-synthesized and externally applied to the leaf surface of 4-week-old transgenic Arabidopsis thaliana plants. External application of the synthetic NPTII-encoding siRNAs down-regulated NPTII transcript levels in transgenic A. thaliana 1 and 7 days post-treatment with a higher and more consistent effect being observed for siRNAs methylated at 3′ ends. We also analyzed the effects of external NPTII-encoding dsRNA precursors and a dsRNA-derived heterogenous siRNA mix. Digestion of the NPTII-dsRNA to the heterogeneous siRNAs did not improve efficiency of the transgene suppression effect. Key Points• Foliar application of siRNAs down-regulated a commonly used transgene in Arabidopsis. • A more consistent effect was observed for methylated siRNAs. • The findings are important for development of plant gene regulation approaches.

Plant calmodulins (CaMs) and calmodulin-like proteins (CMLs) are important plant Ca2+-binding proteins that sense and decode changes in the intracellular Ca2+ concentration arising in response to environmental stimuli. Protein Ca2+ sensors are presented by complex gene families in plants and perform diverse biological functions. In this study, we cloned, sequenced, and characterized three CaM and 54 CML mRNA transcripts of Vitis amurensis Rupr., a wild-growing grapevine with a remarkable stress tolerance. Using real-time quantitative RT-PCR, we analyzed transcript abundance of the identified VaCaMs and VaCMLs in response to water deficit, high salinity, high mannitol, cold and heat stresses. Expression of VaCaMs and 32 VaCMLs actively responded to the abiotic stresses and exhibited both positive and negative regulation patterns. Other VaCML members showed slight transcriptional regulation, remained essentially unresponsive or responded only after one time interval of the treatments. The substantial alterations in the VaCaM and VaCML transcript levels revealed their involvement in the adaptation of wild-growing grapevine to environmental stresses.

An inhibitor of DNA methylation 5-azacytidine (5A) is a chemical analog of the nucleoside cytidine. This study investigated the influence of 5A-induced DNA hypomethylation on the accumulation of somatic DNA mutations (nucleotide substitutions, indels) in the Actin2 3′ untranslated region, nuclear internal transcribed spacer ITS1-5.8rRNA-ITS2, and the ribulose-1,5-bisphosphate carboxylase/oxygenase gene of Arabidopsis thaliana and analyzed concurrent changes in the expression of DNA methyltransferase and DNA repair genes. The 5A treatment (20 mg per 100 g of soil) decreased DNA methylation, and the detected 5A-induced demethylation was associated with the up-regulation of the DNA methyltransferase genes: chromomethylase AtCMT3, methyltransferase AtMETI, and domains rearranged methyltransferases AtDRM1 and AtDRM2. Cultivation of plants in the presence of 5A led to a considerable increase in the number of single nucleotide substitutions in the analyzed DNA regions of 5A-treated A. thaliana. The 5A treatment significantly increased the transcriptions of 7 DNA repair genes (endonuclease AtARP, DNA demethylases AtDME and AtDML2, DNA glycosylase AtMBD4, DNA damage-binding protein AtDDB1, and photolyases AtUVR2 and AtUVR3) out of the 17 analyzed genes from the base excision repair, nucleotide excision repair, and photoreactivation pathways. However, 5A decreased the transcription of DNA 3′-phosphatase AtZDP, DNA repair protein AtRad23a, mismatch repair proteins AtMsh2 and AtMsh3. It is possible that the changes in the transcription of the DNA repair genes contributed to the detected increase in the number of single nucleotide substitutions that accumulated in the 5A-treated A. thaliana. Taken together, the data indicate that there is an interaction between the processes of DNA methylation and mutation accumulation.

This paper investigated the effects of p-coumaric acid (CA) on stilbene biosynthesis in healthy cuttings of grapevine Vitis amurensis Rupr. under control conditions and after ultraviolet (UV-C) irradiation. It has been shown that the content of the detected six stilbenes (cis-piceid, t-piceid, t-e-viniferin, cis-e-viniferin, t-resveratrol, and t-δ-viniferin) increased after feeding with CA up to 0.5 mg g−1 of the dry weight (DW), which is 2.4 times higher than that under control conditions. Feeding with CA and UV-C irradiation exerted a combined positive effect on the content of stilbenes increasing it to 0.7 mg g−1 DW. In addition, the CA treatment improved viability of the cuttings after the UV-C treatment. The elevation in stilbene content induced by CA and UV-C correlated with induction of stilbene biosynthetic genes encoding stilbene synthases, resveratrol O-glucosyltransferase, polyphenol oxidase, and cationic peroxidase. The data indicate that feeding with the precursors of phenolic compounds could be an effective strategy for activation of stilbene production.

Little is known about the contributions of DNA methylation/demethylation to plant aging and senescence. We used Arabidopsis thaliana to study how increasing age of an annual plant species influences DNA methylation. Based on methylation-sensitive DNA fragmentation assay, it could be concluded that aging A. thaliana was accompanied by DNA demethylation. Bisulfite sequencing reveals that cytosine methylation within the Actin2 3’ untranslated region and internal transcribed spacer with 5.8S rRNA (ITS1-5.8SrRNA-ITS2) DNA regions decreased with A. thaliana growth and aging. We show that transcription of methyltransferase genes, chromomethyltransferase AtCMT3 and methyltransferse AtMETI, significantly decreased during development and aging of the A. thaliana plants, whereas expression of demethylase genes - repressor of silencing AtROS1, demeter AtDME, and demeter-like AtDML2 and AtDML3 - increased at least at some stages of plant development. The data obtained in the present study suggest that plant DNA regions may undergo demethylation during plant aging via reduction of DNA methylation processes and activation of active DNA demethylation.

Stilbenes are valuable plant phytoalexins, the biosynthesis of which is characteristic of different groups of phylogenetically unrelated plants. It is believed that all the stilbenes are the derivatives of resveratrol (3,5,4'-trihydroxy-trans-stilbene) or compounds close to it (pinosylvin or piceatannol). The last stage of the resveratrol biosynthesis takes place with the involvement of stilbene synthase or resveratrol synthase (STS). The family Pinaceae is characterized by the presence of the derivatives of pinosylvin (genus Pinus) and piceatannol (genus Picea), the biosynthetic pathways of which are scarcely examined. Previously, in different species of the genus Picea, only two stilbene synthase genes were described. On the basis of RNA isolated from the needles of spruce Picea jezoensis, the full-length cDNAs of the four stilbene synthase genes, PjSTS1a, PjSTS1b, PjSTS2, and PjSTS3, were obtained. Then, using the clone frequency analysis and real-time PCR, expression of the PjSTS1a, PjSTS1b, PjSTS2, and PjSTS3 genes was examined in the needles of P. jezoensis accessions of different age and sampled in different seasons (spring, summer, autumn, winter). Among the analyzed transcripts, the PjSTS1a and PjSTS1b genes were the most frequent, indicating their higher level of expression compared to other STS genes. The highest level of PjSTS1a and PjSTS1b expression was observed in autumn, while the level of PjSTS2 and PjSTS3 expression was the highest in spring and winter. Moreover, the highest PjSTS expression was detected in the young tissues of P. jezoensis in autumn, which may indicate a higher level of stilbene biosynthesis in these tissues.

Calmodulin-like proteins (CMLs) represent a large family of plant calcium sensor proteins involved in the regulation of plant responses to environmental cues and developmental processes. In the present work, we identified four alternatively spliced mRNA forms of the grapevine CML21 gene that encoded proteins with distinct N-terminal regions. We studied the transcript abundance of CML21v1, CML21v2, CML21v3, and CML21v4 in wild-growing grapevine Vitis amurensis Rupr. in response to desiccation, heat, cold, high salinity, and high mannitol stress using quantitative real-time RT-PCR. The levels of all four splice variants of VaCML21 were highly induced in response to cold stress. In addition, VaCML21v1 and VaCML21v2 forms were highly modulated by all other abiotic stress treatments. Constitutive expression of VaCML21v2 and VaCML21v4 improved biomass accumulation of V. amurensis callus cell cultures under prolonged low temperature stress. Heterologous expression of the grapevine CML21v2 and VaCML21v4 splice variants in Arabidopsis improved survival rates of the transgenic plants after freezing. The VaCML21v2 overexpression enhanced activation of the cold stress-responsive marker genes AtDREB1A and AtDREB2A, while VaCML21v4 overexpression&mdash, AtCOR47, AtRD29A, AtRD29B, and AtKIN1 genes after freezing stress in the transgenic Arabidopsis. The results indicate that the grapevine CML21 gene acts as a positive regulator in the plant response to cold stress. The detected variety of CML21 transcripts and their distinct transcriptional responses suggested that this expansion of mRNA variants could contribute to the diversity of grapevine adaptive reactions.

It is known that somatic mutations arising during animal growth and ageing contribute to the development of neurodegenerative and other animal diseases. For plants, several studies showed that small-scale somatic DNA mutations accumulated during Arabidopsis life cycle. However, there is a lack of data on the influence of environmental stresses on somatic DNA mutagenesis in plants. In this study, we analyzed the effects of ultraviolet C (UV-C) irradiation, high soil salinity, and cadmium (CdI3) stresses on the level of small-scale somatic DNA mutations in Arabidopsis thaliana. The number of DNA mutations was examined in the Actin2 3′UTR (Actin-U1), ITS1-5.8rRNA-ITS2 (ITS), and ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) DNA regions. We found that somatic mutation levels considerably increased in CdI3-treated Arabidopsis plants, while the mutation levels declined in the UV-C- and NaCl-treated A. thaliana. Cadmium is a mutagen that is known to inhibit DNA repair processes. The detected stress-induced alterations in somatic DNA mutation levels were accompanied by markedly increased expression of base excision repair genes (AtARP, AtDME, AtDML2, AtDML3, AtMBD4, AtROS, AtUNG, and AtZDP), nucleotide excision repair genes (AtDDB1a, AtRad4, and AtRad23a), mismatch repair genes (AtMSH2, AtMSH3, and AtMSH7), and photoreactivation genes (AtUVR2, AtUVR3). Thus, the results demonstrated that UV-C, high soil salinity, and cadmium stresses influence both the level of DNA mutations and expression of DNA repair genes. Salt- and UV-induced activation of DNA repair genes could contribute to the stress-induced decrease in somatic mutation level.