SRA

Nanomaterials (NMs) are on the nanoscale level range ca. 1-100 nm and due to the peculiar physico-chemical properties are widely used in different areas. The present study aimed to characterize the responses of zucchini (Cucurbita pepo L.) treated with copper oxide (CuO) NPs from seed germination to the flowering stage. CuO NPs treatment did not negatively impact plant morphology and growth as well as the pollen formation and viability. Physiological analyses were followed by a complete RNAseq-based transcriptomic analysis in the different plant tissues. Mitochondrial and chloroplast functions emerged as a critical component in plant response. Evidence of CuO NPs internalization and biotransformation was a driving force to measure the impacts at both physiological and molecular levels. Overall design: Cucurbita pepo L. (cv. Costata Romanesco) seeds were pre-germinated in vermiculite for 10 days prior to transplanting to soil. Zucchini seeds were purchased from Johnny's Selected Seeds (Albion, ME). The experimental soil was collected from the Connecticut Agricultural Experiment Station (CAES) in New Haven, CT, USA. Individual solutions of CuO NPs and CuO bulk material in water (30% water capacity of soil/vermiculite mixture) were probe sonicated to maximize dispersion. CuO bulk material and CuSO4·5H2O were purchased from Sigma Aldrich (St. Louis, MO, US). CuO NPs, CuO (bulk) or (copper sulfate) CuSO4 stock dispersions were slowly added to 500g of soil. The final concentration of CuO NPs and bulk in pots was 100 mg kg-1 while for CuSO4·5H2O (copper sulfate pentahydrate) was 320 mg kg-1, representing a total concentration of Cu about 80 mg kg-1 for all the treatments. Zucchini seedlings were planted (one each pot) and grown indoor under supplemental fluorescent lighting (60 µE m2 sec) under a photoperiod of 16h light, at approximately 22-28 °C until complete flowering. The plants were top watered for 60d growth period. Total RNA was extracted from 0.1 g of fresh plant material using a Sigma-Aldrich Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO). Three biological replicates per treatment were extracted. After quality control samples were sent to IGA Technologies Srl (Udine, IT) for RNA sequencing service. TruSeq Stranded mRNA kit (Illumina, San Diego, CA) has been used for library preparation following the manufacturer's instructions. RNA samples were quantified and quality tested by Agilent 2100 Bioanalyzer RNA assay (Agilent Technologies, Santa Clara, CA). Final libraries were checked Agilent Bioanalyzer DNA assay (Agilent Technologies, Santa Clara, CA). Libraries were prepared for sequencing and sequenced on single-end 75 bp mode on NextSeq 500 (Illumina, San Diego, CA). Alignment of reads to the reference transcriptome available on Cucurbitgenomics database (http://cucurbitgenomics.org/) has been performed using STAR software with default parameters. The resulting raw data have been normalized and the differentially expressed genes were identified using a 2.3 threshold of FPKM data (in log2).