Inhibitor trials – roots only

Young E. grandis seedlings were grown as above – germinating for one month on 1% agar and then growing for an additional six weeks on MS media covered with a sterile cellophane membrane. Their roots were cut and allowed to commence regrowing to ensure an active growth phase. At T₀, the starting length and position of the roots were recorded. Roots were treated with either AdOx (5 nM-5 μM), AMI-1 (50 nM-50 μM), 2,3-dimethoxynitrostyrene (30 nM-30 μM in 1% DMSO), 1% DMSO control or water control and root growth was measured 24 h later. The first inhibitor, AdOx, is a general methylation inhibitor and indirectly blocks the reaction providing the methyl group that is added by methyltransferases to their respective substrates including arginine. Thus all methylation processes, including lysine methylation, are inhibited by AdOx. The second inhibitor, AMI-1, more specifically inhibits protein arginine methylation by specifically blocking PRMTs but not PKMTs [58]. The more recently described DMNS only inhibits the activity of PRMT1 (and PRMT8, not present in E. grandis; [59]). Alignment of PRMT protein sequences from human and E. grandis genomes shows that EgPRMT1 has the appropriate cysteine residue to be inhibited by DMNS, while the other E. grandis PRMTs do not (Additional file 5: Figure S1). One millilitre of solution was administered directly to each root system daily over the test period of two weeks. Between 17 and 30 replicates were performed for each treatment type. Lateral rooting was analyzed after two weeks of treatment. Root tip samples were taken and observed with a Zeiss Stemi 2000-C stereomicroscope (Germany). All plants were alive and healthy at the time of harvest, indicating that the dosage of inhibitors was sub-lethal.