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Vladimír Repka and Mária Čarná

A Global Microarray Expression Profile of Grapevine miRNAs Isolated from Cell Suspensions Pre-Treated with Apoptosis Activators

In an attempt to identify novel and apoptosis/pathogen-regulated microRNAs (miRNAs) and small interfering RNAs, we performed a robust microarray screening of small RNA population from Vitis vinifera L. cv. Limberger cell suspension exposed to apoptosis activators (e.g. methyl jasmonate) or elicitors (botrycin and cinerein) derived from necrotrophic fungus Botrytis cinerea Pers. et Fries. Using a microarray expression profiling approach, we identified 22 miRNAs. We found that a majority of these miRNAs were predicted to target stress/defense-related genes of plants. Of the 22 V. vinifera miRNAs, 11 have sequence conservation in Arabidopsis thaliana but exhibited species-specific developmental and/or stress/defense-related expression patterns. Ten of the miRNAs are highly conserved in other plant species, suggesting that even conserved miRNAs may have different regulatory roles in various species. Our results show that these grapevine miRNAs can be also induced by various apoptosis inducers. Fifty-one potential targets were predicted to the newly identified miRNAs based on sequence complementarity. In addition to miRNAs, we identified 102 other novel endogenous small RNAs in Vitis, indicating that a large number of miRNAs and other small regulatory RNAs are encoded by the Vitis vinifera genome.

Open access

Vladimír Repka and Mária Čarná

Post-Transcriptional Gene Silencing Conferred by the Ectopic Expression of the Grapevine miRNA-g1 and Inhibition of the Response by Anti-miRNA-g1 Inhibitor

Our results show that a subset (miRNA cluster 1) of grapevine (Vitis vinifera L., cv. Limberger) microRNAs (miRNAs) can be strongly induced by different apoptosis inducers including methyl jasmonate (MeJA), botrycin, cinerein and/or H2O2. We report here that the expression of endogenous miRNA-g1 and miRNA-g7 can be efficiently silenced in grapevine protoplasts using artificial miRNA (amiRNA) technology. Furthermore, we demonstrate that the ectopic expression of amiRNAs (anti-mir miRNA-g1 and pre-mir miRNA-g1) designed to target a mature miRNA-g1 directs jasmonate-induced silencing against DAD1 protein (defender against death 1). These collective results strongly support the idea that a sub-population of grapevine miRNAs induced by apoptosis may function in one of the most critical defense systems for structural and mechanical fitness.

Open access

Mária Čarná, Vladimír Repka and Ernest Šturdík

Abstract

To understand the cellular biology and biochemistry of plant cell behavior, there is not only one approach for studying proteins which are directly responsible for cellular activity. However, despite the enormous quantity of information generated by transcriptome analysis, the picture is still incomplete. The proteomic and interactomic approaches present a new point of view that so far has been missing. Comparative proteomics provide a powerful means to study products of genes and their regulation. On the other side, interactomes of different species can provide information about the evolutionary mechanisms leading to organism diversity. Then, this analysis allows scientists to better understand how complex biological processes are regulated and evolved.

Open access

Mária Čarná, Vladimír Repka and Ernest Šturdí

Proteomic Insight Into the Molecular Principles of Grapevine Habituation

Two-dimensional gel electrophoresis coupled to protein microarray analysis was used to examine, for the first time, the molecular mechanisms of grapevine (Vitis vinifera L., cv. Limberger) habituation. The examination of 2-D maps derived from control and habituated cell culture revealed the presence of 55 protein spots displaying a differential expression pattern. These facts have provide a molecular evidence suggesting that the habituated cells can be used as a model for study of cell differentiation and plant defense mechanisms. Cell death, extra-cellular alkalinization and expression of genes responsible for the formation of the defense-related proteins were analyzed in suspension cultures with hormonal autonomy (habituation). Results obtained using habituated grapevine cells compared with non-habituated cells were different and strongly depended on the concentration of elicitor applied.