The occurrence of cytokinins and their biosynthesis pathways in epithelioma papulosum cyprini cells

Cytokinins are small signaling molecules present in all kingdoms of life; however, their biological relevance outside of plants is poorly characterized. Cytokinin detection across kingdoms has been shown to be inconsistent and is often organism or cell line dependent. To ensure optimal detection in epithelioma papulosum cyprini (EPC), a cold-blooded vertebrate cell line, a range of biomasses were evaluated. Cytokinins were extracted by solid-phase extraction and then detected using high performance liquid chromatography and tandem mass spectrometry. The objectives were to establish endogenous CK profiles and characterize their biosynthesis in EPC cells. Among the fourteen CKs that were scanned for, only four forms were detected overall within the samples analyzed, including, N6-(Δ2-isopentenyl) adenine-9-riboside (iPR), N6-(Δ2-isopentenyl) adenine-9-riboside-5’ (mono/di/tri)phosphate (iPNT), and cis-zeatin-9-riboside-5’ (mono/di/tri)phosphate (cZNT) and 2‐methylthio‐zeatin (2MeSZ). The total cytokinin concentrations ranged from 0.03 pmol/g to 681.025 pmol/gfwt. The results from this study provided evidence that EPC cells can produce and process cytokinins.