Cytokinin activity of N6-benzyladenine derivatives assayed by interaction with the receptors in planta, in vitro, and in silico

Abstract

Biological effects of hormones in both plants and animals are based on high-affinity interaction with cognate receptors resulting in their activation. The signal of cytokinins, classical plant hormones, is perceived in Arabidopsis by three homologous membrane receptors: AHK2, AHK3, and CRE1/AHK4. To study the cytokinin-receptor interaction, we used 25 derivatives of potent cytokinin N-benzyladenine (BA) with substituents in the purine heterocycle and/or in the side chain. The study was focused primarily on individual cytokinin receptors from Arabidopsis. The main in planta assay system was based on Arabidopsis double mutants retaining only one isoform of cytokinin receptors and harboring cytokinin-sensitive reporter gene. Classical cytokinin biotest with Amaranthus seedlings was used as an additional biotest. In parallel, the binding of ligands to individual cytokinin receptors was assessed in the in vitro test system. Quantitative comparison of results of different assays confirmed the partial similarity of ligand-binding properties of receptor isoforms. Substituents at positions 8 and 9 of adenine moiety, elongated linker up to 4 methylene units, and replacement of N by sulfur or oxygen have resulted in the suppression of cytokinin activity of the derivative toward all receptors. Introduction of a halogen into position 2 of adenine moiety, on the contrary, often increased the ligand activity, especially toward AHK3. Features both common and distinctive of cytokinin receptors in Arabidopsis and Amaranthus were revealed, highlighting species specificity of the cytokinin perception apparatus. Correlations between the extent to which a compound binds to a receptor in vitro and its ability to activate the same receptor in planta were evaluated for each AHK protein. Interaction patterns between individual receptors and ligands were rationalized by structure analysis and molecular docking in sensory modules of AHK receptors. The best correlation between docking scores and specific binding was observed for AHK3. In addition, receptor-specific ligands have been discovered with unique properties to predominantly activate or block distinct cytokinin receptors. These ligands are promising for practical application and as molecular tools in the study of the cytokinin perception by plant cells.

Cytokinin perception in potato: new features of canonical players

“…In addition to natural CKs, there are many synthetic ones. Receptors exhibit different affinities for these compounds ( Lomin et al , 2015 ; Savelieva et al , 2018 ). We studied the ligand specificity of putative receptors in competitive experiments where binding of labeled CK was carried out in the presence of various concentrations of certain unlabeled ligands.…”

Regulation of vacuolar H+-ATPase activity of BY-2 tobacco suspension culture cells during growth by elongation

“…V. 483. pp. 341-343. doi: 10.1134 Lomin SN et al (2018) Studies of cytokinin receptor-phosphotransmitter interaction provide evidences for the initiation of cytokinin signalling in the endoplasmic Возьмите на заметку: Знание рН-зависимости активности рецептора фитогормона может способствовать появлению новых представлений о субклеточной локализации и функции рецептора, а также об особенностях гомеостаза, транспорта и сигналинга гормона. Том 1, № 4, С.…”

Distinct Peculiarities of In Planta Synthesis of Isoprenoid and Aromatic Cytokinins

“…Since the discovery of kinetin in 1955 by Miller, Skoog et al [3], a large number of various CKs as well as corresponding nucleoside and nucleotide derivatives were synthesized or isolated from different plant sources [4,5]. Among these numerous molecular forms, only CKs as nucleobases possess hormonal activity, but not their nucleoside or nucleotide derivatives [6,7]. The structural diversity of CKs and CK derivatives in vivo is obviously due to complex pathways of their biosynthesis.…”

“…In the present study, a new approach was proposed for the comparative analysis of the biosynthesis pathway of aromatic and isoprenoid CKs. We synthesized a series of ribo-, 2 -deoxyribo-and 5 -deoxyribonucleoside derivatives of natural iP, BA, KIN, and also synthetic cytokinin analog N 6 -phenylethyladenine, which possesses a high hormonal activity [7]. To investigate the possibility of biochemical conversion in vivo into active CKs in comparison with corresponding ribonucleosides, a series of 2 -deoxyribonucleosides was obtained.…”