Calcium and methyl jasmonate cross-talk in the secondary metabolism of grape cells

In grape cell cultures cv. Gamay Fréaux var. Teinturier, Ca was shown to decrease cell pigmentation through the inhibition of anthocyanin biosynthesis, while stimulating stilbenoids accumulation. Because methyl jasmonate (MeJA) is a well-known inducer of secondary metabolism in grape cells, and Ca antagonizes its stimulatory effect over several enzymes of core metabolic branches, in the present study we hypothesized that Ca and MeJA signaling pathways interact to regulate specific secondary metabolism routes. Grape cultured cells were elicited with MeJA or with MeJA + Ca and an UPLC-MS-based targeted metabolomic method was implemented to characterize their polyphenolic profiles. Results were compared with the profile of cells elicited with Ca only, previously reported. Data was complemented with gene expression analysis, allowing the assembly of a metabolic map that unraveled routes specifically regulated by both elicitors. MeJA + Ca specifically boosted E-resveratrol and E--viniferin levels by 180% and 140%, respectively, in comparison to cells treated with MeJA only, while the stimulatory effect of MeJA over flavonoid synthesis was inhibited by Ca. In parallel, Ca downregulated most flavonoid pathway genes, including LAR1, ANS, BAN and ANR. Ca was able to mimic or potentiate the effect of MeJA on the expression of JA signaling genes, including JAR1, PIN and PR10. Transcript/metabolite correlation networks exposed the central influence of FLS1, STS, CDPK17 and COI1 in polyphenolic biosynthetic routes. This study highlights the potential of the MeJA-Ca combination for diverting polyphenolic metabolism towards the production of specific metabolites of interest, highly relevant in a biotechnological perspective.This work was supported by the “Contrato-Programa” UIDB/04050/2020 funded by portuguese national funds through the FCT I.P. The work was also supported by FCT, CCDR-N (Norte Portugal Regional Coordination and Development Commission) and European Funds (FEDER/POCI/COMPETE2020) through the project AgriFood XXI (NORTE-01-0145-FEDER-000041) and the research projects BerryPlas tid (PTDC/BIA-FBT/28165/2017 and POCI-01-0145-FEDER-028165) and MitiVineDrought (PTDC/BIA-FBT/30341/2017 and POCI-01-0145- FEDER-030341). A.T. was supported by a postdoctoral researcher con tract/position within the project “BerryPlastid”. This article/publication is based upon work from COST Action CA 17111 INTEGRAPE, supported by COST (European Cooperation in Science and Technology) – in the context of a STSM to V.M. The R´egion-Centre Val de Loire (France) supported this work under the grant agreement to Projects CEPATLAS and VINODRONE to A.L.info:eu-repo/semantics/publishedVersio

Silybum marianum cell cultures stably transformed with Vitis vinifera stilbene synthase accumulate t-resveratrol in the extracellular medium after elicitation with methyl jasmonate or methylated β-cyclodextrins

The growing demand for t-resveratrol for industrial uses has generated considerable interest in its production. Heterologous resveratrol production in plant cell suspensions, apart from requiring the introduction of only one or two genes, has the advantage of high biomass yield and a short cultivation time, and thus could be an option for large-scale production. Silybum marianum is the source of the flavonolignan silymarin. Phenylpropanoid synthesis in cultures of this species can be activated by elicitation with methyl jasmonate and methylated β-cyclodextrins, with products of the pathway (coniferyl alcohol and some isomers of the silymarin complex) being released into the medium. Given that stilbene synthase shares the same key precursors involved in flavonoid and /or monolignol biosynthesis, we explored the potential of metabolically engineered S. marianum cultures for t-resveratrol production. Cell suspensions were stably transformed with Vitis vinifera stilbene synthase 3 and the expression of the transgene led to extracellular t-resveratrol accumulation at the level of milligrams per litre under elicitation. Resveratrol synthesis occurred at the expense of coniferyl alcohol. Production of silymarin was less affected in the transgenic cultures, since the flavonoid pathway is limiting for its synthesis, due to the preferred supply of precursors for the monolignol branch. The fact that the expressed STS gene took excessively produced precursors of non-bioactive compounds (coniferyl alcohol), while keeping the metabolic flow for target secondary compounds (i.e. silymarin) unaltered, opens a way to extend the applications of plant cell cultures for the simultaneous production of both constitutive and foreign valuable metabolites.This work has been supported by a grant from the Spanish Ministry of Science and Innovation (BIO2014-51861-R). Diego Hidalgo is a predoctoral fellow of Mexican CONACYT

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