Biochemical and Structural Aspects of Cytokinin Biosynthesis and Degradation in Bacteria

It has been known for quite some time that cytokinins, hormones typical of plants, are also produced and metabolized in bacteria. Most bacteria can only form the tRNA-bound cytokinins, but there are examples of plant-associated bacteria, both pathogenic and beneficial, that actively synthesize cytokinins to interact with their host. Similar to plants, bacteria produce diverse cytokinin metabolites, employing corresponding metabolic pathways. The identification of genes encoding the enzymes involved in cytokinin biosynthesis and metabolism facilitated their detailed characterization based on both classical enzyme assays and structural approaches. This review summarizes the present knowledge on key enzymes involved in cytokinin biosynthesis, modifications, and degradation in bacteria, and discusses their catalytic properties in relation to the presence of specific amino acid residues and protein structure

Prussian Blue acts as a mediator in a reagentless cytokinin biosensor

An electrochemical biosensor for detection of the plant hormone cytokinin is introduced. Cytokinin homeostasis in tissues of many lower and higher plants is controlled largely by the activity of cytokinin dehydrogenase (CKX, EC 1.5.99.12) that catalyzes an irreversible cleavage of N(6)-side chain of cytokinins. Expression of Arabidopsis thaliana CKX2 from Pichia pastoris was used to prepare purified AtCKX2 as the basis of the cytokinin biosensor. Prussian Blue (PrB) was electrodeposited on Pt microelectrodes prior to deposition of the enzyme in a sol-gel matrix. The biosensor gave amperometric responses to several cytokinins. These responses depended on the presence of both the enzyme and the Prussian Blue. Thus Prussian Blue must act as an electron mediator between the FAD centre in CKX2 and the Pt surface

Figural artwork of Egon Schiele and his artistic techniques

Department of Theory of Art and ArtworksKatedra teorie umění a tvorbyFaculty of HumanitiesFakulta humanitních studi

Biochemical characterization of putative adenylate dimethylallyltransferase and cytokinin dehydrogenase from Nostoc sp. PCC 7120

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.</p

SDS-PAGE analysis of purification of <i>NoCKX1</i> expressed from different vectors.

<p>A/ Purification of <i>NoCKX1</i> expressed from pMAL-c4X vector. Lane 1: molecular mass standard; lane 2: fusion protein (<i>NoCKX1</i> with maltose binding protein) after purification on amylose column; lane 3: same protein after cleavage by factor Xa protease. B/ Purification of <i>NoCKX1</i> expressed from pCIOX vector. Lane 1: molecular mass standard; lane 2: fusion protein (<i>NoCKX1</i> with SUMO and histidine tag) after purification on Ni-NTA column. Proteins were separated in 10% SDS-polyacrylamide gel.</p

Phylogenetic tree for the CKX proteins shows a clear distinction between plants and bacterial sequences.

<p>Maximum likelihood phylogeny derived by the CKX domain sequences identified in both plants (green) and bacteria (orange). The robustness of the phylogenetic tree was assessed using 500 bootstrap repetitions. The sequence identifiers and species corresponding to the abbreviations are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138468#pone.0138468.s004" target="_blank">S2 Table</a>. The investigated sequence is marked by a red arrow.</p