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.

We detected and quantified six stilbenes ( cis -piceid, t -piceid, t -ε-viniferin, cis -ε-viniferin, t -resveratrol, and t -δ-viniferin) in the leaves, petioles, berry skins, and seeds of wild-growing Vitis amurensis . The highest content of stilbenes and expression of stilbene biosynthesis genes were in the probes collected in the autumn and after ultraviolet elicitation. Stilbenes, including the best-studied stilbene resveratrol, are known to display valuable bioactivities and protect plants against various pathogens. There is a lack of studies on stilbene quantities and spectrum combined with an analysis of the stilbene biosynthesis pathway gene expression in Vitaceae species, despite grapevine is an important source of stilbenes. This study presents an analysis of stilbene spectrum, stilbene content, and expression of stilbene biosynthesis genes both in natural conditions and after ultraviolet (UV-C) elicitation in the leaves, petioles, berry skins, and seeds of wild-growing Vitis amurensis, a highly stress-tolerant plant species. Using HPLC analysis, we detected six main stilbenes: cis-piceid (up to 0.257 mg/g of dry weight (DW) of plant material), t-piceid (up to 0.055 mg/g DW), t-ε-viniferin (up to 0.122 mg/g DW), cis-ε-viniferin (up to 0.031 mg/g DW), t-resveratrol (from 0.004 to 0.121 mg/g DW), and t-δ-viniferin (up to 0.019 mg/g DW). The stilbenes were actively synthesized in the leaves (total stilbenes 0.39 mg/g DW) and berry skins (total stilbenes 0.249 mg/g DW) of V. amurensis collected in the autumn. qRT-PCR revealed that the stilbene synthase (STS), resveratrol O-glucosyltransferase (Glu1), and polyphenol oxidase (PPO1) genes were actively expressed in the analyzed tissues. The resveratrol methyltransferase (Romt1) gene, which is known to catalyze biosynthesis of pterostilbene, was also expressed, but no pterostilbene has been detected in V. amurensis. The content of all detected stilbenes and expression of stilbene biosynthesis genes increased after UV-C treatment, except for Romt1. The data are important for understanding the stilbene biosynthesis in grapevine.

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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.

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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.

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