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1

Effect of long-term cultivation on resveratrol accumulation in a high-producin cell culture of Vitis amurensis

100%
Dubrovina A. S., Kiselev K. V.
Acta Physiologiae Plantarum
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2012
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tom 34
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nr 3
Resveratrol is a polyphenol, present in grapes, peanuts, and other plant sources, with a wide range of valuable biological activities. We established a Vitis amurensis cell culture accumulating high levels of resveratrol by introducing the rolB gene of Agrobacterium rhizogenes in the V. amurensis genome, and studied the stability of resveratrol accumulation during 27 months of continuous subculturing. This study demonstrates a decline in the high level of resveratrol production by the rolB transgenic cell line during its long-term cultivation. Elicitation of the rolB transgenic calli with methyl jasmonate and salicylic acid, which are known to stimulate the production of plant secondary metabolites, resulted in a recovery of resveratrol accumulation in the rolB transgenic cell culture, while the empty vector-transformed culture with trace starting content of resveratrol exhibited low inducibility to the treatment.
2

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

100%
Kiselev K. V., Dubrovina A. S.
Acta Physiologiae Plantarum
|
2010
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tom 32
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nr 3
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 ammonialyase 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.
3

Influence of overexpression of the true and false alternative transcripts of calcium-dependent protein kinase CPK9 and CPK3a genes on the growth, stress tolerance, and resveratrol content in Vitis amurensis cell cultures

81%
Dubrovina A. S., Aleynova D. A., Kiselev K. V.
Acta Physiologiae Plantarum
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2016
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tom 38
|
nr 03
4

True and false alternative transcripts of calcium-dependent protein kinase CPK9 and CPK3a genes in Vitis amurensis

81%
Dubrovina A. S., Aleynova O. A., Kiselev K. V., Novikova G. V.
Acta Physiologiae Plantarum
|
2014
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tom 36
|
nr 07
Calcium-dependent protein kinases (CDPKs) are major Ca²⁺ sensors in plants playing important roles in plant development and stress responses. In the present study, RT-PCR analysis revealed that VaCPK3a and VaCPK9 cDNAs lacking extensive regions in the kinase, autoinhibitory, and Ca²⁺-binding domains were numerous in probes derived from wild grapevine Vitis amurensis. Most of the VaCPK3a and VaCPK9-modified transcripts lacked canonical splice sites and possessed short direct repeated sequences (SDRs) instead. Three VaCPK9 transcript variants were generated using canonical 5'GT and 3'AG splice sites and lacked several subdomains in the kinase domain, including ATP-binding site, which is known to be indispensable for kinase activities. These observations indicate that post-transcriptional mRNA processing might lead to production of CDPKs with abolished phosphorylation activities. Recombinant proteins VaCPK3aSF2, lacking autoinhibitory and Ca²⁺-binding domain, and VaCPK3aSF3, lacking VIII–XI kinase subdomains and autoinhibitory domain, phosphorylated exogenous substrate in a Ca²⁺- independent manner. However, reverse transcription at +65°C using heat-stable reverse transcriptase (RT) markedly lowered abundance of the unusual VaCPK3a and VaCPK9 cDNAs with SDRs, while it did not eliminate VaCPK9 cDNAs generated using canonical splice sites. The results show that VaCPK9 gene undergoes unproductive alternative splicing mediated by canonical splice sites to generate three mRNA isoforms lacking important catalytic kinase subdomains. The unusual VaCPK3a and VaCPK9 transcripts with SDRs are likely to be false alternative transcripts generated by RT template switching in vitro. The data demonstrate that using non-thermostable RTs for studying alternative splicing are not appropriate.
5

Action of ultraviolet-C radiation and p-coumaric acid on stilbene accumulation and expression of stilbene biosynthesis-related genes in the grapevine Vitis amurensis Rupr.

71%
Kiselev K. V., Ogneva Z. V., Suprun A. R., Grigorchuk V. P., Dubrovina A. S.
Acta Physiologiae Plantarum
|
2019
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tom 41
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nr 02
6

Altered somatic mutation level and DNA repair gene expression in Arabidopsis thaliana exposed to ultraviolet C, salt, and cadmium stresses

71%
Kiselev K. V., Ogneva Z. V., Dubrovina A. S., Suprun A. R., Tyunin A. P.
Acta Physiologiae Plantarum
|
2018
|
tom 40
|
nr 01
It is known that somatic mutations arising during animal growth and ageing contribute to the development of neurodegenerative and other animal diseases. For plants, several studies showed that small-scale somatic DNA mutations accumulated during Arabidopsis life cycle. However, there is a lack of data on the influence of environmental stresses on somatic DNA mutagenesis in plants. In this study, we analyzed the effects of ultraviolet C (UV-C) irradiation, high soil salinity, and cadmium (CdI₃) stresses on the level of small-scale somatic DNA mutations in Arabidopsis thaliana. The number of DNA mutations was examined in the Actin2 3′UTR (Actin-U1), ITS1-5.8rRNA-ITS2 (ITS), and ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) DNA regions. We found that somatic mutation levels considerably increased in CdI₃-treated Arabidopsis plants, while the mutation levels declined in the UV-C- and NaCl-treated A. thaliana. Cadmium is a mutagen that is known to inhibit DNA repair processes. The detected stress-induced alterations in somatic DNA mutation levels were accompanied by markedly increased expression of base excision repair genes (AtARP, AtDME, AtDML2, AtDML3, AtMBD4, AtROS, AtUNG, and AtZDP), nucleotide excision repair genes (AtDDB1a, AtRad4, and AtRad23a), mismatch repair genes (AtMSH2, AtMSH3, and AtMSH7), and photoreactivation genes (AtUVR2, AtUVR3). Thus, the results demonstrated that UV-C, high soil salinity, and cadmium stresses influence both the level of DNA mutations and expression of DNA repair genes. Salt- and UV-induced activation of DNA repair genes could contribute to the stress-induced decrease in somatic mutation level.
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