ANALYSIS OF GENE EXPRESSION IN ARABIDOPSIS THALIANA SUSPENSION CULTURE CELLS WITH REDUCED NDB2 GENE EXPRESSION
Abstract
Background. The alternative respiratory pathway of plant mitochondria is not linked to ATP synthesis and, therefore, is not directly controlled by the energy status of the cell. This alternative pathway includes rotenone insensitive NAD(P)H type II dehydrogenases located on both the outer and inner surfaces of the inner mitochondrial membrane, ubiquinone, and alternative oxidase (AOX). It is supposed that alternative NAD(P)H dehydrogenases have functions similar to those of AOX, including thermogenesis, prevention of ROS formation, oxidation of overage reducing agents for the metabolic pathways maintenance, etc. Publications on the successful NDB2 suppression expression are still insufficient. For example, Arabidopsis plants lacking both AOX1a and NDB2 were shown to be more sensitive to combined drought and high light treatment, while plants overexpressing these genes showed increased tolerance and ability to post-stress recovery.
Purpose. The aim of this work was to study how the suppression of NDB2 expression in the heterotrophic cells of Arabidopsis suspension culture will affect the expression of other alternative respiratory pathway genes, uncoupling proteins, as well as genes of heat shock proteins under non-stress conditions.
Materials and methods. For RNA isolation, 5 ml of suspension culture cells were collected. RNA was isolated using reagents from the GeneJET Plant RNA Purification Mini Kit (Thermo Scientific, Lithuania) according to the manufacturer’s instructions.
The first strand of cDNA was synthesized using the Thermo Scientific reagent kit (Lithuania), according to the manufacturer’s recommendations. RT-PCR was carried out on the CFX96™ Real-Time PCR Detection System (Bio-Rad, the USA), using a qPCR mix-HS SYBR reagent kit (Evrogen, Russia) according to the manufacturer’s instructions. The analysis of RT-PCR data was performed using the SFX Manager software (Bio-Rad, USA). All experiments were carried out in two analytical and three biological replicates. A gene encoding glyceraldehyde-3-phosphate dehydrogenase, GAPD, was used as a reference gene.
Results. The amount of NDB2 mRNA in the AS5 suspension culture cells was reduced by 8.2 times compared to the control, Col-0. Suppression of the NDB2 expression in AS5 Arabidopsis suspension culture cells resulted in an increase of transcripts amount not only NDB4 gene, but also NDB1, as well as NDA2 and NDC1; at the same time, the NDB3 and NDA1 expression genes decreased in comparison with Col-0 cells. We did not find any changes in the AOX1a, AOX1b, and AOX1d expression levels genes in AS5 cells compared to the control, but the quantity of AOX1c gene transcripts increased slightly. During analyzing the expression levels of the genes encoding the uncoupling proteins, UCP1 gene expression was increased in AS5 cells. In the cells of the AS5 line suspension culture, the expression levels of all the HSPs genes studied by us, except for HSP17.7, increased.
Conclusion. Therefore, the results obtained suggest that the suppression of NDB2 gene expression in Arabidopsis heterotrophic cells in the absence of stress alters redox status of cells, which in turn leads to changes in the level of accumulation of other NAD(P)H dehydrogenases genes transcripts and the increase of HSPs gene expression, while the key AOX genes expression does not change.
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