Novel Cytokinin Derivatives Do Not Show Negative Effects on Root Growth and Proliferation in Submicromolar Range

Abstract

BackgroundWhen applied to a nutrition solution or agar media, the non-substituted aromatic cytokinins caused thickening and shortening of the primary root, had an inhibitory effect on lateral root branching, and even showed some negative effects on development of the aerial part at as low as a 10 nanomolar concentration. Novel analogues of aromatic cytokinins ranking among topolins substituted on N9-atom of adenine by tetrahydropyranyl or 4-chlorobutyl group have been prepared and tested in standardized cytokinin bioassays [1]. Those showing comparable activities with N6-benzylaminopurine were further tested in planta.Methodology/Principal FindingsThe main aim of the study was to explain molecular mechanism of function of novel cytokinin derivatives on plant development. Precise quantification of cytokinin content and profiling of genes involved in cytokinin metabolism and perception in treated plants revealed several aspects of different action of m-methoxytopolin base and its substituted derivative on plant development. In contrast to standard cytokinins, N9- tetrahydropyranyl derivative of m-topolin and its methoxy-counterpart showed the negative effects on root development only at three orders of magnitude higher concentrations. Moreover, the methoxy-derivative demonstrates a positive effect on lateral root branching and leaf emerging in a nanomolar range of concentrations, in comparison with untreated plants.Conclusions/SignificanceTetrahydropyranyl substitution at N9-position of cytokinin purine ring significantly enhances acropetal transport of a given cytokinins. Together with the methoxy-substitution, impedes accumulation of non-active cytokinin glucoside forms in roots, allows gradual release of the active base, and has a significant effect on the distribution and amount of endogenous isoprenoid cytokinins in different plant tissues. The utilization of novel aromatic cytokinin derivatives can distinctively improve expected hormonal effects in plant propagation techniques in the future.

N9-substituted aromatic cytokinins with negligible side effects on root development are an emerging tool for in vitro culturing

“…2). Similarly to our previous results, 14 the application of 50 nM 3MeOBA9THPP had positive effect on primary root elongation when compared with untreated controls (cp. primary root lengths: 11.4 ± 1.2 cm vs. 9.9 ± 0.6 cm).…”

“…14 We found that the THP group can be cleaved off from its parent CK both in the root and the leaf. Subsequently, the free base can be glycosylated at the N9 position to provide the glucoside or riboside.…”

“…Indeed, we found that acropetal transport of 3MeOBA9THPP was significantly strengthened when compared with free base transport. 14 Moreover, when roots were treated with 3MeOBA9THPP, the total 3MeOBAP CK pool in the xylem sap was about 10 times higher than that in control plants treated with the free base. This unexpected finding suggested that the concentration of active CKs in the aerial part of the plants would probably be higher in case of 3MeOBA9THPP treatment, explaining the observed positive effect on the maize shoot.…”

“…1) and 4-chlorobutyl (yielding 3MeOBA9ClButP) as substituents and tested their activity in standardized CK activity assays. 10,14 Although the substitution at the N9 position very Moreover, 3MeOBA9ClButP even showed a distinct positive effect on lateral root formation. In contrast, for maize 3MeOBA9ClButP and 3MeOBAP9G had a slightly negative effect on root proliferation in the submicromolar concentration range (8-40 nM).…”

“…Hence, the positive effect of 3MeOBA9THPP treatment on plant development is correlated with the endogenous CK levels, strongly suggesting that the observed phenotypic effects are proportional to the concentration of the active CK forms, which result from the metabolic conversions of exogenously applied CK and from successful homeostasis of endogenous isoprenoid CKs. 14 To address the question of biological activity of a given CK form, we can employ two types of biological assays, both of which are based on heterologous expression of the plant CK histidine kinase (HK) receptor in E. coli strain KMI001. The receptor activation test reflects the ability of the ligand to initiate signal transduction from the tested HK receptor and makes use of the reporter gene lacZ under the control of cps promoter.…”