Cca-miR398 increases copper sulfate stress sensitivity via the regulation of CSD mRNA transcription levels in transgenic Arabidopsis thaliana

MicroRNAs play crucial roles during the process of plant development under stress conditions. Copper is an essential micronutrient for most organisms and serves as an important redox-active cofactor for various functional proteins. In the present study, we investigated the effects of copper sulfate stress on hickory (Carya cathayensis) root development. We identified that hickory cca-miR398 was related to copper sulfate stress response, targeting Copper/Zinc superoxide dismutases (cytosolic (CSD1) and chloroplastic (CSD2)) and a 5b subunit of mitochondrial cytochrome C oxidase (COX5b.1) that are linked directly to stress regulatory networks. The sequence of hickory cca-miR398 is highly similar to that of Arabidopsis miR398b and miR398c, regardless of one nucleotide variation. Therefore, target genes of cca-miR398 were investigated by using 5'-Rapid-amplification of cDNA ends. An overexpression of cca-miR398 in Arabidopsis caused a reduction not only in root length and cotyledon greening, but also in the CSD1, CSD2, and CSD3 transcription levels. These reductions had greater significance in transgenic Arabidopsis than in wild-type Arabidopsis under copper sulfate stress. The level of physiological indicators also changed in transgenic Arabidopsis. In addition, the expressions of copper-responsive microRNAs, such as miR397 and miR408, were affected by the copper sulfate stress. These results showed that CSD possesses the ability to enhance copper sulfate stress response in both transgenic Arabidopsis and hickory roots by increasing the production of superoxide dismutase. Our results also demonstrated that cca-miR398 weakens hickory tolerance to copper sulfate by regulating CSD targets.