Simultaneous Application of Several Exogenous dsRNAs for the Regulation of Anthocyanin Biosynthesis in Arabidopsis thaliana.

Plant surface treatment with double-stranded RNAs (dsRNAs) has gained recognition as a promising method for inducing gene silencing and combating plant pathogens. However, the regulation of endogenous plant genes by external dsRNAs has not been sufficiently investigated. Also, the effect of the simultaneous application of multiple gene-specific dsRNAs has not been analyzed. The aim of this study was to exogenously target five genes in Arabidopsis thaliana, namely, three transcription factor genes (AtCPC, AtMybL2, AtANAC032), a calmodulin-binding protein gene (AtCBP60g), and an anthocyanidin reductase gene (AtBAN), which are known as negative regulators of anthocyanin accumulation. Exogenous dsRNAs encoding these genes were applied to the leaf surface of A. thaliana either individually or in mixtures. The mRNA levels of the five targets were analyzed using qRT-PCR, and anthocyanin content was evaluated through HPLC-MS. The results demonstrated significant downregulation of all five target genes by the exogenous dsRNAs, resulting in enhanced expression of chalcone synthase (AtCHS) gene and increased anthocyanin content. The simultaneous foliar application of the five dsRNAs proved to be more efficient in activating anthocyanin accumulation compared to the application of individual dsRNAs. These findings hold considerable importance in plant biotechnology and gene function studies.

The Effect of External Treatment of Arabidopsis thaliana with Plant-Derived Stilbene Compounds on Plant Resistance to Abiotic Stresses.

Stilbenes are a group of plant phenolic secondary metabolites, with trans-resveratrol (3,5,4'-trihydroxy-trans-stilbene) being recognized as the most prominent and studied member. Stilbenes have a great potential for use in agriculture and medicine, as they have significant activities against plant pathogens and have valuable beneficial effects on human health. In this study, we analyzed the effects of direct application of stilbenes, stilbene precursor, and stilbene-rich extract solutions to the plant foliar surface for increasing the resistance of Arabidopsis thaliana to various abiotic stresses (heat, cold, drought, and soil salinity). Exogenous treatment of A. thaliana with stilbenes (trans-resveratrol, piceid, and spruce bark extract) and phenolic precursor (p-coumaric acid or CA) during germination resulted in considerable growth retardation of A. thaliana plants: a strong delay in the root and stem length of 1-week-old seedlings (in 1.3-4.5 fold) and rosette diameter of 1-month-old plants (in 1.2-1.8 fold), while the 2-month-old treated plants were not significantly different in size from the control. Plant treatments with stilbenes and CA increased the resistance of A. thaliana to heat and, to a lesser extent, to soil salinity (only t-resveratrol and spruce extract) to drought (only CA), while cold resistance was not affected. Plant treatments with stilbenes and CA resulted in a significant increase in plant resistance and survival rates under heat, with plants showing 1.5-2.3 times higher survival rates compared to untreated plants. Thus, exogenous stilbenes and a CA are able to improve plant survival under certain abiotic stresses via specific activation of the genes involved in the biosynthesis of auxins, gibberellins, abscisic acid, and some stress-related genes. The present work provides new insights into the application of stilbenes to improve plant stress tolerance.

The Endophytic Microbiome of Wild Grapevines Vitis amurensis Rupr. and Vitis coignetiae Pulliat Growing in the Russian Far East.

Many grape endophytic microorganisms exhibit high potential for suppressing the development of grape diseases and stimulating grapevine growth and fitness, as well as beneficial properties of the crop. The microbiome of wild grapevines is a promising source of biocontrol agents, which can be beneficial for domesticated grapevines. Using next-generation sequencing (NGS) and classical microbiology techniques, we performed an analysis of bacterial and fungal endophytic communities of wild grapevines Vitis amurensis Rupr. and Vitis coignetiae Pulliat growing in the Russian Far East. According to the NGS analysis, 24 and 18 bacterial taxa from the class level were present in V. amurensis and V. coignetiae grapevines, respectively. Gammaproteobacteria (35%) was the predominant class of endophytic bacteria in V. amurensis and Alphaproteobacteria (46%) in V. coignetiae. Three taxa, namely Sphingomonas, Methylobacterium, and Hymenobacter, were the most common bacterial genera for V. amurensis and V. coignetiae. Metagenomic analysis showed the presence of 23 and 22 fungi and fungus-like taxa of class level in V. amurensis and V. coignetiae, respectively. The predominant fungal classes were Dothideomycetes (61-65%) and Tremellomycetes (10-11%), while Cladosporium and Aureobasidium were the most common fungal genera in V. amurensis and V. coignetiae, respectively. A comparative analysis of the endophytic communities of V. amurensis and V. coignetiae with the previously reported endophytic communities of V. vinifera revealed that the bacterial biodiversity of V. amurensis and V. coignetiae was similar in alpha diversity to V. vinifera's bacterial biodiversity. The fungal alpha diversity of V. amurensis and V. coignetiae was statistically different from that of V. vinifera. The beta diversity analysis of bacterial and fungal endophytes showed that samples of V. vinifera formed separate clusters, while V. amurensis samples formed a separate cluster including V. coignetiae samples. The data revealed that the endophytic community of bacteria and fungi from wild V. amurensis was richer than that from V. coignetiae grapes and cultivated V. vinifera grapes. Therefore, the data obtained in this work could be of high value in the search for potentially useful microorganisms for viticulture.

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

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

Effect of VaMyb40 and VaMyb60 Overexpression on Stilbene Biosynthesis in Cell Cultures of Grapevine Vitis amurensis Rupr.

Stilbenes are plant defense compounds known to rapidly accumulate in grapevine and some other plant species in response to microbial infection and several abiotic stresses. Stilbenes have attracted considerable attention due to valuable biological effects with multi-spectrum therapeutic application. However, there is a lack of information on natural signaling pathways and transcription factors regulating stilbene biosynthesis. It has been previously shown that MYB R2R3 transcription factor genes VaMyb40 and VaMyb60 were up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to UV irradiation. In this study, the effects of VaMyb40 or VaMyb60 overexpression in cell cultures of V. amurensis on their capability to produce stilbenes were investigated. Overexpression of the VaMyb60 gene led to a considerable increase in the content of stilbenes in three independently transformed transgenic lines in 5.9-13.9 times, while overexpression of the VaMyb40 gene also increased the content of stilbenes, although to a lesser extent (in 3.4-4.0 times) in comparison with stilbene levels in the empty vector-transformed calli. Stilbene content and stilbene production in the VaMyb60-transgenic calli reached 18.8 mg/g of dry weight (DW) and 150.8 mg/L, respectively. Using HPLC analysis, we detected eight individual stilbenes: t-resveratrol diglucoside, t-piceid, t-resveratrol, ε-viniferin, δ-viniferin, cis-resveratrol, cis-piceid, t-piceatannol. T-resveratrol prevailed over other stilbenoid compounds (53.1-89.5% of all stilbenes) in the VaMyb-overexpressing cell cultures. Moreover, the VaMyb40- and VaMyb60-transformed calli were capable of producing anthocyanins up to 0.035 mg/g DW, while the control calli did not produce anthocyanins. These findings show that the VaMyb40 and VaMyb60 genes positively regulate the stilbene biosynthesis as strong positive transcription regulators and can be used in biotechnological applications for stilbene production or high-quality viticulture and winemaking.

The Specificity of Transgene Suppression in Plants by Exogenous dsRNA.

The phenomenon of RNA interference (RNAi) is widely used to develop new approaches for crop improvement and plant protection. Recent investigations show that it is possible to downregulate plant transgenes, as more prone sequences to silencing than endogenous genes, by exogenous application of double-stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs). However, there are scarce data on the specificity of exogenous RNAs. In this study, we explored whether plant transgene suppression is sequence-specific to exogenous dsRNAs and whether similar effects can be caused by exogenous DNAs that are known to be perceived by plants and induce certain epigenetic and biochemical changes. We treated transgenic plants of Arabidopsis thaliana bearing the neomycin phosphotransferase II (NPTII) transgene with specific synthetic NPTII-dsRNAs and non-specific dsRNAs, encoding enhanced green fluorescent protein (EGFP), as well as with DNA molecules mimicking the applied RNAs. None of the EGFP-dsRNA doses resulted in a significant decrease in NPTII transgene expression in the NPTII-transgenic plants, while the specific NPTII-dsRNA significantly reduced NPTII expression in a dose-dependent manner. Long DNAs mimicking dsRNAs and short DNA oligonucleotides mimicking siRNAs did not exhibit a significant effect on NPTII transgene expression. Thus, exogenous NPTII-dsRNAs induced a sequence-specific and RNA-specific transgene-suppressing effect, supporting external application of dsRNAs as a promising strategy for plant gene regulation.

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

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

External dsRNA Downregulates Anthocyanin Biosynthesis-Related Genes and Affects Anthocyanin Accumulation in Arabidopsis thaliana.

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.

Exogenous Stilbenes Improved Tolerance of Arabidopsis thaliana to a Shock of Ultraviolet B Radiation.

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.

35S promoter-driven transgenes are variably expressed in different organs of Arabidopsis thaliana and in response to abiotic stress.

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.

Physiological Conditions and dsRNA Application Approaches for Exogenously induced RNA Interference in Arabidopsis thaliana.

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.

The Grapevine Calmodulin-Like Protein Gene CML21 Is Regulated by Alternative Splicing and Involved in Abiotic Stress Response.

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-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.

Transgene suppression in plants by foliar application of in vitro-synthesized small interfering RNAs.

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.

The Effect of Abiotic Stress Conditions on Expression of Calmodulin (CaM) and Calmodulin-Like (CML) Genes in Wild-Growing Grapevine Vitis amurensis.

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.

Induction of Transgene Suppression in Plants via External Application of Synthetic dsRNA.

Recent investigations show that exogenously applied small interfering RNAs (siRNA) and long double-stranded RNA (dsRNA) precursors can be taken up and translocated in plants to induce RNA interference (RNAi) in the plant or in its fungal pathogen. The question of whether genes in the plant genome can undergo suppression as a result of exogenous RNA application on plant surface is almost unexplored. This study analyzed whether it is possible to influence transcript levels of transgenes, as more prone sequences to silencing, in Arabidopsis genome by direct exogenous application of target long dsRNAs. The data revealed that in vitro synthesized dsRNAs designed to target the gene coding regions of enhanced green fluorescent protein (EGFP) or neomycin phosphotransferase II (NPTII) suppressed their transcript levels in Arabidopsis. The fact that, simple exogenous application of polynucleotides can affect mRNA levels of plant transgenes, opens new opportunities for the development of new scientific techniques and crop improvement strategies.