Expression of Genes Associated with Nickel Resistance in Red Oak (Quercus rubra).

The main objectives of the present study were to 1) Determine the level of nickel (Ni) toxicity in red oak (Quercus rubra) and 2) assess gene expression dynamics in response to nickel stress. Ni treatments included three doses (150 mg / kg, 800 mg / kg, and 1,600 mg/kg) of nickel nitrate salt. Water and potassium nitrate were used as controls. The experimental design was a completely randomized block with 16 replications. Total RNA was extracted from roots and leaves. Several genes associated with Ni resistance in model and non-model plants were targeted in this study. They include genes for Serine acetyltransferase, Glutathione reductase, Nicotianamine synthase, Metal transporter Nramp3, 1-aminocyclopropane-1-carboxylic acid, and deaminase (ACC). PCR primers were designed by matching gene sequences to the Q. rubra genome. When possible, primers were designed to span the exon-exon border of the gene. They were checked for hairpins, self, and hetero-dimers using the OligoAnalyzer 3.1. The cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit by Life Technologies. RT-qPCR was performed using the Dynamo HS SYBR Green qPCR Kit by Life Technologies and conducted according to the manufacturer’s protocol. Each sample was amplified with the MJ Research PTC-200 Thermal Cycler in triplicates. The data were analyzed using the MJ Opticon Monitor 3.1 by BioRad and C(t) values were determined. C(t) values were only normalized to the housekeeping genes. The screening assays revealed that all the Q. rubra genotypes tested were highly resistant to nickel toxicity as no symptom or damages were observed even in seedling treated with the highest dose of 1,600 mg of Ni per Kg of dry soil. Expression of targeted genes in these Q. rubra genotypes treated with different Ni doses will be discussed.