MicroRNA and regulation of gene expression of Cu/Zn-SOD in Thellungiella salsuginea plants under effect of the different concentrations of copper

One of the important element of posttranscriptional regulation of gene activity is amechanism for silencing genes, due to RNA interference, which is associated with expressionof small RNAs. In Arabidopsis thaliana L. plants found two major classes ofsmall RNAs in size 21-23 base pair involved in the suppression of gene expression:siRNA (small interfering) and miRNA (microRNA). There is increasing evidence that understress conditions changes as the expression of miRNA and expression of they targetsgenes and the activity of miRNA-protein complexes.The purpose of present study was to examine the role of microRNAs in posttranscriptionalregulation of genes Cu/Zn-SOD and CCS gene in a plant Thellungiella salsugineaunder the influence of different concentrations of copper, given that copper is a trace elementessential for plant nutrition and is a member of the cofactors of plastocyanin, Cu/Zn-SOD, cytochrome c and laccase. Object of research is plants Thellungiella salsuginea,Pallas were grown in water culture in a medium of Johnson. At the age of 6 weeks theplants were divided into groups and subjected to treatment CuSO4 (0,1 and 100 μM). Experimentswere performed in three biological and three analytical replications.Isolation of total RNA was performed TRIzol® reagent. Level of expression ofmiRNAs was assessed by Northern blot hybridization. Evaluation of protein contentCSD1 and CCS1 performed using Western blot analysis by standard methods Laemmli(1970). The level of expression of genes other studied was performed by RT-PCR withgene-specific primers.Several studies have shown that in the conditions of a copper lack decrease contentof the protein Cu/Zn-SOD and its enzymatic activity. It was also shown that inA. thaliana and other higher plants, various concentrations of copper regulate expressionof mRNA CCS1, encoding a chaperone copper, delivering Cu to apoprotein differentisoenzymes of Cu/Zn-SOD.As have shown Nozern-blot hybridization of total RNA Th. salsuginea, at entering100 μM CuSO4 in a nutrient medium occurred almost full inhibition of an expressionmiR398. In experiments with absence of copper in a nutrient medium observed increaseexpression of miR398.The study of CSD1 expression showed that after 24 hours absence of copper in thegrowth medium caused a decrease expression of this gene. When the copperconcentration was 100 μM in all parts of the plant Th. salsuginea observed increase inmRNA CSD1. However, western blot analysis with specific forms for the cytosolic Cu/Zn-SOD antibody revealed that this protein was determined only in the leaves of plants at allstudied concentrations of copper in the medium. This give evidence that the synthesis ofthe protein may be exercised only in the leaves. At high concentrations of copper(100 μM) in the leaves was shown enhance of CSD1 expression, however, increase of theprotein content CSD1 didn`t happened. On the other hand, the study of gene expressionCCS1 copper chaperone for SOD in leaves and roots showed that the amount of mRNAof this gene increased in the leaves at a concentration of CuSO4 1 and 100 μM, in theabsence of copper as expression of this gene significantly decreased in the leaves androots. CCS1 protein found only in the leaves in the control plants (0,25 μM CuSO4), aswell as 1 μM CuSO4 in the nutrient medium. In terms of different concentrations ofcopper in the growth medium of plants observed reciprocal relationship between theexpression of miR398 and genes expression CSD1 and CCS1. We can assume that underconditions of different concentrations of copper in the growth medium, there is amiR398-mediated regulation of genes CSD1 and CCS1. It is likely that the increase dueto miR398 regulation of mRNA CCS1 in the absence of copper in the growth mediumreduces the number of copper delivered to the protein CSD1 and, consequently, reducedits contents in the leaves. Probably, in the absence of copper in the nutrient medium maybe coming redistribution of copper ions between Cu/Zn-SOD and copper-containingproteins other important, for example, such as plastocyanin.

Keywords

Authors

References