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

Interaction of Plants and Endophytic Microorganisms: Molecular Aspects, Biological Functions, Community Composition, and Practical Applications

Endophytes are microorganisms that live asymptomatically inside plant tissues [...

Biodiversity of endophytic bacteria and fungi of wild grapes

The diversity of endophytic bacteria and fungi of V. amurensis grape plants growing in the suburbs of Vladivostok in the summer and autumn periods of 2018-2020 was analyzed. About 600 strains of bacteria and 160 strains of fungi were inoculated from peripherally sterilized leaves and stems of V. amurensis. Isolated bacteria were representatives of 36 genera: Actinobacterium, Acinetobacter, Agrobacterium, Arthrobacter, Bacillus, Buttiauxella, Curtobacterium, Duganella, Erwinia, Enterobacter, Frigoribacterium, Frondihabitans, Klebsiella, Leclercia, Lelliottia, Methylobacterium, Microbacterium, Mucilaginibacter, Luteibacter, Lysinimonas, Pantoea, Paenibacillus, Parageobacillus, Pedobacter, Phyllobacterium, Plantibacter, Pseudomonas, Pullulanibacillus, Raoultella, Rhizobium, Sphingomonas, Staphylococcus, Stenotrophomonas, Streptomyces, Serratia, Xanthomonas. The largest number of strains were representatives of the genera Erwinia, Pantoae and Pseudomonas. Endophytic grape fungi were represented by 25 genera: Alternaria, Annulohypoxylon, Aureobasidium, Biscogniauxia, Cladosporium, Colletotrichum, Coniochaeta, Coprinellus, Davidiellaceae, Didymella, Discosia, Epicoccum, Fusarium, Hypoxylon, Neosetophoma, Nemania, Neurospora, Nigrospora, Paraphoma, Penicillium, Pestalotiopsis, Pestalosphaeria, Phoma, Trichoderma, Xylaria. The largest number of representatives were of the genus Didymella, Cladosporium and Colletotrichum

The Bark of the Spruce Picea jezoensis Is a Rich Source of Stilbenes

Stilbenes are plant phenolic secondary metabolites that show beneficial effects on human health and possess high antifungal activity against plant pathogens. Currently, a search for plant sources with high stilbene levels is relevant, since stilbene content in various plant species can vary substantially and is often at a low level. In this paper, the bark and wood of Picea jezoensis were analyzed for the content and composition of stilbenes and compared with other known stilbene sources. The HPLC-MS analysis of P. jezoensis bark and wood extracted with different solvents and at different temperatures revealed the presence of 11 and 5 stilbenes, respectively. The highest number of stilbenes of 171 and 229 mg per g of the dry weight (mg/g DW) was extracted from the bark of P. jezoensis using methanol or ethanol at 60 °C for 2 h. Trans-astringin, trans-piceid, and trans-isorhapontin prevailed over other stilbenoids (99% of all detected stilbenes). The most abundant stilbene was trans-isorhapontin, reaching 217 mg/g DW or 87% of all stilbenes. An increase in the extraction time from 2 to 6 h did not considerably increase the detected level of stilbenes, while lower extraction temperatures (20 and 40 °C) significantly lowered stilbene yield. The content of stilbenes in the P. jezoensis bark considerably exceeded stilbene levels in other stilbene-producing plant species. The present data revealed that the bark of P. jezoensis is a rich source of stilbenes (primarily trans-isorhapontin) and provided effective stilbene extraction procedures

The Biodiversity of Grapevine Bacterial Endophytes of <i>Vitis amurensis</i> Rupr.

In this paper, the composition profiles of bacterial endophytes in wild-growing Amur grape Vitis amurensis Rupr. grown in the south of the Russian Far East were analyzed using both a cultivation-dependent (sowing bacteria) and a cultivation-independent (next generation sequencing, NGS) approach. Both methods revealed the prevalent endophytes in V. amurensis were represented by Gammaproteobacteria—40.3–75.8%, Alphaproteobacteria—8.6–18.7%, Actinobacteria—9.2–15.4%, and Bacilli—6.1–6.6%. NGS also showed a large proportion of Bacteroidia (12.2%) and a small proportion of other classes (less than 5.7%). In general, NGS revealed a greater variety of classes and genera in the endophytic bacterial community due to a high number of reads (574,207) in comparison with the number of colonies (933) obtained after the cultivation-dependent method. A comparative analysis performed in this study showed that both wild grape V. amurensis from Russia and domesticated cultivars of V. vinifera from Germany and California (USA) exhibit the same basic composition of endophytic bacteria, while the percentages of major taxa and minor taxa showed some differences depending on the plant organ, grape individuals, environmental conditions, and sampling time. Furthermore, the obtained data revealed that lower temperatures and increased precipitation favored the number and diversity of endophytic bacteria in the wild Amur grape. Thus, this study firstly described and analyzed the biodiversity of endophytic bacteria in wild grapevine V. amurensis

Simultaneous Application of Several Exogenous dsRNAs for the Regulation of Anthocyanin Biosynthesis in <i>Arabidopsis thaliana</i>

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

Physiological Conditions and dsRNA Application Approaches for Exogenously induced RNA Interference in <i>Arabidopsis thaliana</i>

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 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

The Diversity of Fungal Endophytes from Wild Grape Vitis amurensis Rupr

Grapevine endophytic fungi have great potential for application in agriculture and represent an important source of various compounds with valuable biological activities. Wild grapevine is known to host a great number of rare and unidentified endophytes and may represent a rich repository of potential vineyard biocontrol agents. This investigation aimed to study the fungal endophytic community of wild grape Vitis amurensis Rupr. using a cultivation-dependent (fungi sowing) and a cultivation-independent (next-generation sequencing, NGS) approach. A comprehensive analysis of the endophytic fungal community in different organs of V. amurensis and under different environmental conditions has been performed. According to the NGS analysis, 12 taxa of class level were presented in different grapevine organs (stem, leaf, berry, seed). Among the 12 taxa, sequences of two fungal classes were the most represented: Dothideomycetes&mdash;60% and Tremellomycetes&mdash;33%. The top five taxa included Vishniacozyma, Aureobasidiaceae, Cladosporium, Septoria and Papiliotrema. The highest number of fungal isolates and sequences were detected in the grape leaves. The present data also revealed that lower temperatures and increased precipitation favored the number and diversity of endophytic fungi in the wild Amur grape. The number of fungi recovered from grape tissues in autumn was two times higher than in summer. Thus, this study is the first to describe and analyze the biodiversity of the endophytic fungal community in wild grapevine V. amurensis

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