A. S. Dubrovina

Phenylalanine ammonia-lyase and stilbene synthase gene expression in rolB transgenic cell cultures of Vitis amurensis

Transformation of Vitis amurensis callus culture by the plant oncogene rolB of Agrobacterium rhizogenes results in high (up to 3.15% dry wt.) levels of resveratrol in the transformed culture. The present study deals with the effect of rolB on phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) gene expression in two rolB transgenic V. amurensis callus cultures with different levels of rolB expression and resveratrol production. The total expression of PALs and STSs in rolB transgenic cultures increased 1.3–3.8 times compared with the control culture. In the rolB transgenic cultures expression of VaPAL1, VaPAL2, and six STS genes was increased, while expression of VaPAL3 and VaSTS6 was not significantly changed. These results suggest that rolB increases resveratrol production via selective enhancement of expression of individual genes from PAL and STS gene families. We propose that increase of VaPAL3, VaSTS1, and VaSTS6 transcript levels is not strongly required for high resveratrol production by rolB transgenic cell cultures.

Expression of calcium-dependent protein kinase (CDPK) genes under abiotic stress conditions in wild-growing grapevine Vitis amurensis.

Calcium-dependent protein kinases (CDPKs), which are important sensors of Ca(2+) flux in plants, are known to play essential roles in plant development and adaptation to abiotic stresses. In the present work, we studied expression of CDPK genes under osmotic and temperature stress treatments in wild-growing grapevine Vitis amurensis Rupr., which is known to possess high adaptive potential and a high level of resistance against adverse environmental conditions. In this study, using RT-PCR with degenerate primers, DNA sequencing and frequency analysis of RT-PCR products, we identified 13 CDPK genes that are actively expressed in healthy V. amurensis cuttings under high salt, high mannitol, desiccation, and temperature stress conditions. 12 CDPKs, namely VaCPK1, VaCPK2, VaCPK3, VaCPK9, VaCPK13, VaCPK16, VaCPK20, VaCPK21, VaCPK25, VaCPK26, VaCPK29 and VaCPK30, were novel for Vitaceae, and their full cDNAs were obtained and described. Quantitative real-time RT-PCR demonstrated that mRNA levels of 10 VaCPK genes were differentially up-regulated under the osmotic and temperature stress treatments, while the abundance of 3 VaCPK transcript variants, VaCPK3a, VaCPK25, and VaCPK30, was not markedly changed. Expression profiling of the VaCPK genes in leaves, leaf petioles, stems, inflorescences, berries, and seeds of V. amurensis revealed that the genes exhibit different organ-specific expression patterns. The stimulatory effect of abiotic stress on the expression of the VaCPK1, 2, 3, 9, 13, 16, 20, 21, 26, and VaCPK29 genes is suggestive of their implication in the grapevine response to osmotic and temperature stresses, while the variability in their organ-specific expression patterns indicates that the enzymes perform distinct biological functions.

Resveratrol content and expression of phenylalanine ammonia-lyase and stilbene synthase genes in rolC transgenic cell cultures of Vitis amurensis

Resveratrol, a naturally occurring polyphenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In the present investigation, we show that transformation of Vitis amurensis Rupr. with the oncogene rolC of Agrobacterium rhizogenes increased resveratrol production in the two transformed callus cultures 3.7 and 11.9 times. The rolC-transformed calli were capable of producing 0.099% and 0.144% dry weight of resveratrol. We characterized phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) gene expression in the two rolC transgenic callus cultures of V. amurensis. In the rolC transgenic culture with higher resveratrol content, expression of VaPAL3, VaSTS3, VaSTS4, VaSTS5, VaSTS6, VaSTS8, VaSTS9, and VaSTS10 was increased; while in the rolC culture with lower resveratrol content, expression of VaPAL3 and VaSTS9 was increased. We suggest that transformation of V. amurensis calli with the rolС gene induced resveratrol accumulation via selective enhancement of expression of individual PAL and STS genes involved in resveratrol biosynthesis. We compared the data on PAL and STS gene expression in rolC transgenic calli with the previously obtained results for rolB transgenic calli of V. amurensis. We propose that the transformation of V. amurensis with the rolC and rolB genes of A. rhizogenes increased resveratrol accumulation through different regulatory pathways.

Enhanced resveratrol accumulation in rolB transgenic cultures of Vitis amurensis correlates with unusual changes in CDPK gene expression.

It has been established that transformation of Vitis amurensis callus culture with the plant oncogene rolB of Agrobacterium rhizogenes results in a high level of resveratrol production in the transformed culture. In the present report, we investigated two rolB transgenic V. amurensis cell cultures with different levels of rolB expression and resveratrol production. We examined whether the calcium ion flux and later steps of the calcium-mediated signal transduction pathway play a role in resveratrol biosynthesis in the rolB transgenic cultures. It has been shown that the calcium channel blockers, LaCl(3), verapamil, and niflumic acid, significantly reduced the accumulation of resveratrol in the rolB transgenic cultures. The number of the calcium-dependent protein kinase (CDPK) transcript variants and abundance of some of the transcripts were considerably altered in the rolB transgenic cell cultures, as revealed by frequency analysis of RT-PCR products and real-time PCR. Some unusual CDPK transcripts with deletions and insertions in the kinase domain were isolated from cDNA probes of rolB-transformed cells. These results suggest that active resveratrol biosynthesis in rolB transgenic cultures of V. amurensis is Ca2+ dependent. We propose that the rolB gene has an important role in regulation of calcium-dependent transduction pathways in transformed cells.

Structure and expression profiling of a novel calcium-dependent protein kinase gene, CDPK3a, in leaves, stems, grapes, and cell cultures of wild-growing grapevine Vitis amurensis Rupr.

Key messageVaCDPK3ais actively expressed in leaves, stems, inflorescences, and berries ofVitis amurensisand may act as a positive growth regulator, but is not involved in the regulation of resveratrol biosynthesis.AbstractCalcium-dependent protein kinases (CDPKs) are known to play important roles in plant development and defense against biotic and abiotic stresses. It has previously been shown that CDPK3a is the predominant CDPK transcript in cell cultures of wild-growing grapevine Vitis amurensis Rupr., which is known to possess high resistance against environmental stresses and to produce resveratrol, a polyphenol with valuable pharmacological effects. In this study, we aimed to define the full cDNA sequence of VaCDPK3a and analyze its organ-specific expression, responses to plant hormones, temperature stress and exogenous NaCl, and the effects of VaCDPK3a overexpression on biomass accumulation and resveratrol content in V. amurensis calli. VaCDPK3a was actively expressed in all analyzed V. amurensis organs and tissues and was not transcriptionally regulated by salt and temperature stresses. The highest VaCDPK3a expression was detected in young leaves and the lowest in stems. A reduction in the VaCDPK3a expression correlated with a lower rate of biomass accumulation and higher resveratrol content in calli of V. amurensis under different growth conditions. Overexpression of the VaCDPK3a gene in the V. amurensis calli significantly increased cell growth for a short period of time but did not have an effect on resveratrol production. Further subculturing of the transformed calli resulted in cell death and a decrease in expression of the endogenous VaCDPK3a. The data suggest that while VaCDPK3a acts as a positive regulator of V. amurensis cell growth, it is not involved in the signaling pathway regulating resveratrol biosynthesis and resistance to salt and temperature stresses.

A new method for analyzing gene expression based on frequency analysis of RT-PCR products obtained with degenerate primers

This paper presents a new method of gene expression analysis: frequency analysis of RT-PCR products obtained with degenerate primers (FAPP). The main advantage of the new approach compared to the present methods of gene expression analysis is that it is applicable to non-model plant objects whose gene sequences are unknown. The advantages and disadvantages of FAPP are described in detail using data on calcium-dependent protein kinase, stilbene synthase, and phenylalanine ammonia-lyase gene expression in two cell cultures of Vitis amurensis. We compared the expression profiles obtained by FAPP to those obtained by real-time PCR and expressed sequence tags.

The effect of salicylic acid on phenylalanine ammonia-lyase and stilbene synthase gene expression in Vitis amurensis Cell Culture

Resveratrol is a phytoalexin with antibacterial, antiviral and cancer-preventing effects. The objective of the study was to identify PAL and STS genes of Vitis amurensis Rupr. encoding the phenylalanine ammonia-lyases (PAL) and stilbene synthases (STS), the key enzymes involved in the resveratrol biosynthesis. A V. amurensis Rupr. cell culture characterized by low resveratrol level was chosen as a model object. Salicylic acid (SA), a known secondary metabolism inducing agent, was used for enhancement of resveratrol production in this culture. PAL and STS gene expression was analyzed using the reverse transcription PCR and real-time PCR techniques. SA was originally found to specifically enhance the expression of VaPAL3, VaSTS2, VaSTS3, VaSTS4, VaSTS5, VaSTS6, and VaSTS8 of multigene families VaPAL and VaSTS. The results obtained were compared with the earlier published data on PAL and STS gene expression in the rolB transformed V. amurensis cell cultures characterized by high levels of resveratrol. The effects of SA treatment and the rolB transformation on VaPAL and VaSTS gene expression were found to be considerably different.

Regulation of stilbene biosynthesis in plants

AbstractMain conclusionThis review analyzes the advances in understanding the natural signaling pathways and environmental factors regulating stilbene biosynthesis. We also discuss the studies reporting on stilbene content and repertoire in plants. Stilbenes, including the most-studied stilbene resveratrol, are a family of phenolic plant secondary metabolites that have been the subject of intensive research due to their valuable pharmaceutical effects and contribution to plant disease resistance. Understanding the natural mechanisms regulating stilbene biosynthesis in plants could be useful for both the development of new plant protection strategies and for commercial stilbene production. In this review, we focus on the environmental factors and cell signaling pathways regulating stilbene biosynthesis in plants and make a comparison with the regulation of flavonoid biosynthesis. This review also analyzes the recent data on stilbene biosynthetic genes and summarizes the available studies reporting on both stilbene content and stilbene composition in different plant families.

The calcium-dependent protein kinase gene VaCPK29 is involved in grapevine responses to heat and osmotic stresses

Ca2+-dependent protein kinases (CDPKs or CPKs) are essential primary sensors of Ca2+ in plants and are known to play important roles in plant abiotic and biotic stress responses. Vitis amurensis is a wild grapevine species with a high level of cold and disease resistance. It has previously been shown that transcription of 10 CDPK genes of V. amurensis was elevated under salt, desiccation, high mannitol, cold, and heat stress conditions. Expression of VaCPK29 was induced under high and low temperatures, water deficit, and high mannitol stress in plant cuttings of V. amurensis. The present study revealed that the callus cell cultures of V. amurensis and soil-grown plants of Arabidopsis thaliana overexpressing VaCPK29 exhibited higher tolerance to heat and high mannitol stress in comparison with the control transformed with the empty vector. Cold, salt, and drought stress tolerance of the transgenic V. amurensis calli and A. thaliana plants was comparable to that of the controls. The stress-responsive genes AtDREB1A, AtDREB2A, AtRD29A, AtRD29B, and AtABF3 were up-regulated in the VaCPK29-overexpressing A. thaliana plants under heat stress. Taken together, the data indicate that the VaCPK29 gene may act as a positive regulator in the grapevine response to heat and osmotic stresses.

The methylation status of plant genomic DNA influences PCR efficiency

During the polymerase chain reaction (PCR), which is a versatile and widely used method, certain DNA sequences are rapidly amplified through thermocycling. Although there are numerous protocols of PCR optimization for different applications, little is known about the effect of DNA modifications, such as DNA methylation, on PCR efficiency. Recent studies show that cytosine methylation alters DNA mechanical properties and suggest that DNA methylation may directly or indirectly influence the effectiveness of DNA amplification during PCR. In the present study, using plant DNA, we found that highly methylated plant DNA genomic regions were amplified with lower efficiencies compared to that for the regions methylated at a lower level. The correlation was observed when amplifying stilbene synthase (STS1, STS10) genes of Vitis amurensis, the Actin2 gene of Arabidopsis thaliana, the internal transcribed spacer (AtITS), and tRNAPro of A. thaliana. The level of DNA methylation within the analyzed DNA regions has been analyzed with bisulfite sequencing. The obtained data show that efficient PCRs of highly methylated plant DNA regions can be hampered. Proteinase K treatment of the plant DNA prior to PCR and using HotTaq DNA polymerase improved amplification of the highly methylated plant DNA regions. We suggest that increased DNA denaturation temperatures of the highly methylated DNA and contamination with DNA-binding proteins contribute to the hampered PCR amplification of highly methylated DNA. The data show that it is necessary to use current DNA purification protocols and commercial kits with caution to ensure appropriate PCR product yield and prevent bias toward unmethylated DNA amplification in PCRs.