Functionalized carbon nanotube toxicity in Arabidopsis leaves

Polyethilenimine (PEI) functionalized single walled carbon nanotubes (SWNTs) and carbon-based nanomaterials enable delivery of DNA and RNA in plants. Given the broad-scale use of PEI-functionalized nanomaterials in plants, we sought to investigate the reaction of plant tissues to treatment with PEI-SWNTs and pristine SWNTs. To this end, we infiltrated Arabidopsis thaliana leaves with pristine single walled carbon nanotubes used in RNA silencing applications (SWNTs) and polyethyleneimine-functionalized SWNTs used for plasmid DNA delivery (PEI-SWNTs). We used Arabidopsis as it is a well characterized model plant, for which genomic and detailed gene function information is readily available. To minimize the effects caused by the introduction of exogenous nucleic acids, in SWNT preparations we used single stranded RNA targeting Green Fluorescent Protein (GFP) with no target sequence in the Arabidopsis genome, and a plasmid that expresses GFP in PEI-SWNT preparations. For our experiments herein, we used ~25-50 fold higher concentrations of SWNTs and PEI-SWNTs compared to standard concentrations used in biomolecule delivery assays. Water-infiltrated plant leaves served as a negative control to distinguish between the SWNT-specific response and the response to the infiltration process itself. We performed RNA sequencing (RNA-seq) with RNA extracted from leaves two days after infiltration to identify changes in the leaf transcriptomic profile in response to the three treatments, compared to non-infiltrated leaves. Overall design: Young leaves of 6-week old wild-type Col-0 Arabidopsis thaliana plants in vegetative stage were selected to be fully infiltrated (approx. vol 40 µL) with a 1 mL needleless syringe (BD, cat. no. 14-823-434) loaded with water, SWNTs+ssRNA (50 ng/µL) or pDNA+PEI-SWNTs (50 ng/µL) prepared as described previously. Two days after infiltration, five biological replicates containing four leaves of each treatment from different plants and from non-treated plants were collected in a 2 mL Eppendorf tube with two 3.2 mm chrome steel beads (RPI, cat. no. 9840) and flash frozen in liquid N2 immediately. Frozen samples were ground in a Mini-beadbeater (Biospec Products, cat. no. 3110Bx) tissue homogenizer for 5 seconds at 25 Hz frequency, twice. RNA was extracted using RNeasy Plant Mini Kit (Qiagen cat. 74904) using RNase-Free DNase (Qiagen cat. 79254) following manufacturer instructions. Total RNA concentration was measured using the Qubit™ RNA BR Assay Kit (Thermo Fisher). RNA quality was checked using the 2100 Bioanalyzer with RNA 6000 Nano Kit (Agilent). RNA integrity number (RIN) scores were confirmed to be >8. Libraries were prepared using Kapa Biosystems library preparation kit with mRNA selection with poly-A magnetic beads. Libraries were pooled and sequenced on an Illumina NovaSeq S4 flow cell in a NovaSeq 6000 Platform with 150 paired end reads.