Bikinin-like inhibitors targeting GSK3/Shaggy-like kinases: characterisation of novel compounds and elucidation of their catabolism in planta

Abstract

BackgroundPlant GSK-3/Shaggy-like kinases are key players in brassinosteroid (BR) signalling which impact on plant development and participate in response to wounding, pathogens and salt stress. Bikinin was previously identified in a chemical genetics screen as an inhibitor targeting these kinases. To dissect the structural elements crucial for inhibition of GSK-3/Shaggy-like kinases by bikinin and to isolate more potent compounds we synthesised a number of related substances and tested their inhibitory activity in vitro and in vivo using Arabidopsis thaliana.ResultsA pyridine ring with an amido succinic acid residue in position 2 and a halogen in position 5 were crucial for inhibitory activity. The compound with an iodine substituent in position 5, denoted iodobikinin, was most active in inhibiting BIN2 activity in vitro and efficiently induced brassinosteroid-like responses in vivo. Its methyl ester, methyliodobikinin, showed improved cell permeability, making it highly potent in vivo although it had lower activity in vitro. HPLC analysis revealed that the methyl residue was rapidly cleaved off in planta liberating active iodobikinin. In addition, we provide evidence that iodobikinin and bikinin are inactivated in planta by conjugation with glutamic acid or malic acid and that the latter process is catalysed by the malate transferase SNG1.ConclusionBrassinosteroids participate in regulation of many aspects of plant development and in responses to environmental cues. Thus compounds modulating their action are valuable tools to study such processes and may be an interesting opportunity to modify plant growth and performance in horticulture and agronomy. Here we report the development of bikinin derivatives with increased potency that can activate BR signalling and mimic BR action. Methyliodobikinin was 3.4 times more active in vivo than bikinin. The main reason for the superior activity of methyliodobikinin, the most potent compound, is its enhanced plant tissue permeability. Inactivation of bikinin and its derivatives in planta involves SNG1, which constitutes a novel pathway for modification of xenobiotic compounds.

The Roles of Phosphorylation and SHAGGY-Like Protein Kinases in Geminivirus C4 Protein Induced Hyperplasia

“…Previously, bikinin (30 μM) was shown to induce significant developmental changes by 3 days post treatment [ 33 ] and to inhibit meristem development in 8-day-old seedlings [ 55 ], a change also seen in the presence of the C4 protein [ 9 ]. A recent study of bikinin-like inhibitors showed that bikinin was inactivated by conjugation with glutamic acid or malic acid [ 56 ], suggesting that concentrations of bikinin at ≥30 μM might be needed to see long term effects. This is supported by an IC 50 of 23.3 μM for bikinin in a hypocotyl elongation assay (56).…”

Two homolog wheat Glycogen Synthase Kinase 3/SHAGGY - like kinases are involved in brassinosteroid signaling

“…Foliar spraying cannot be directly compared to uptake over several days via the seedling roots or from the medium by tiny embryos. Differences in tissue permeability as well as inactivation of the compound in planta [ 42 ] may require higher concentrations if the uptake occurs via the leaves over a limited time. PCZ was used as alternative to BRZ that had little effect on wheat seedling growth.…”

Brassinosteroids Are Master Regulators of Gibberellin Biosynthesis in Arabidopsis

“…(B) Genes of Arabidopsis involved in GO GA biosynthetic process (GO:0009686) that contain the non-E-Box motif within 750 bp upstream of the transcriptional start site. (Rozhon et al, 2010(Rozhon et al, , 2014. BES1 was enriched on the identified binding site in all three promoters assessed ( Figure 7D).…”

“…For ChIP, 35Sp:BES1-CFP plants (Rozhon et al, 2014) were grown for 21 d in standard growth conditions. The plants where sprayed at noon with 10 mM epi-BL and DMSO as control.…”