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.

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.

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.

The most serious problems for cultivated grapes are pathogenic microorganisms, which reduce the yield and quality of fruit. One of the most widespread disease of grapes is “gray mold”, caused by the fungus Botrytis cinerea. Some strains of Bacillus, such as Bacillus halotolerans, Bacillus amyloliquefaciens, and Bacillus velezensis, are known to be active against major post-harvest plant rots. In this study, we showed that the endophytic bacteria B. velezensis strain AMR25 isolated from the leaves of wild grapes Vitis amurensis Rupr. exhibited antimicrobial activity against grape pathogens, including B. cinerea. The genome of B. velezensis AMR25 has one circular chromosome with a length of 3,909,646 bp. with 3689 open reading frames. Genomic analysis identified ten gene clusters involved in the nonribosomal synthesis of polyketides (macrolactin, bacillene, and difficidin), lipopeptides (surfactin, fengycin, and bacillizin), and bacteriocins (difficidin). Also, the genome under study contains a number of genes involved in root colonization, biofilm formation, and biosynthesis of phytohormones. Thus, the endophytic bacteria B. velezensis strain AMR25 shows great promise in developing innovative biological products for enhancing plant resistance against various pathogens.

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.

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.