Intracellular protein-differential expression in Brassica napus hairy roots in response to the presence of the azo dye Naphthol Blue Black

BONILLA JOSE; MAGALLANES NOGUERA, CYNTHIA; PAEZ DANIELA; KURINA-SANZ MARCELA; CALLEGARI EDUARDO

Simposio; 7th Annual Eastern South Dakota Research Symposium; 2023

The dichotomy between progress and the damage to the ecosystem caused by the anthropogenic activities is notable. To this, the scientific community must respond with applicable knowledge to reverse the imbalance caused and prevent future contamination. In this sense, phytoremediation appears as a low-cost and easy-to-manage sustainable technology. In previous works, Brassica napus hairy roots showed the ability to remove 100 % of 180 µg mL-1 Naphthol Blue Black in 10 days, using both growing and resting cells culture methods, at pH 5.8 and 8.0. In this work, we aimed to analyze the molecular response of B. napus hairy roots by analyzing the differential expression of intracellular proteins in the presence of Naphthol Blue Black, in order to understand the homeostatic mechanisms involved in the tolerance and removal of the azo dye.Intracellular proteins were obtained at 48 h of culture from 1 g of fresh weight of B. napus hairy roots, grown in the absence and in the presence of 180 µg mL-1 Naphthol Blue-Black, through phenol extraction supplemented with SDS and sonication treatments1. Samples were separated into two aliquots: the first one was used for in solution proteomic analysis and the other to be analyzed by GelLC-MS/MS2. In both cases, reduction and alkylation of proteins were carried out using DTT and Iodoacetamide, and digestion was carried out with Trypsin. Tryptic peptides were then concentrated, separated, and analyzed by nanoscale Ultra Performance LC (EASY-nLC™ 1200, Thermo Fisher Scientific), coupled to MS/MS with a nanoscale Electrospray ionization source (nanoESI-MS/MS; Orbitrap Exploris™ 240, Thermo Fisher Scientific). The generic peak and mass list were obtained with Proteome Discoverer v2.5 (Thermo Fisher Scientific) coupled with Mascot v2.8.1 (local license, www.matrixscience). We used the database corresponding to B. napus v20220629 (139,334 sequences; 47,116,931 residues, www.uniprot.org). In solution and in gel data were combined to obtain the global protein profiles. The comparative study was carried out with ProteoIQ v2.8 (www.premierbiosoft.com). Proteins found exclusively in dye-exposed hairy roots were classified based on their biological function with Gene Ontology (http://geneontology.org/). We worked with biological triplicates and analytical duplicates.