A highly selective biosensor with nanomolar sensitivity based on cytokinin dehydrogenase

We have developed a N6-dimethylallyladenine (cytokinin) dehydrogenase-based microbiosensor for real-time determination of the family of hormones known as cytokinins. Cytokinin dehydrogenase from Zea mays (ZmCKX1) was immobilised concurrently with electrodeposition of a silica gel film on the surface of a Pt microelectrode, which was further functionalized by free electron mediator 2,6-dichlorophenolindophenol (DCPIP) in supporting electrolyte to give a bioactive film capable of selective oxidative cleavage of the N6- side chain of cytokinins. The rapid electron shuffling between freely diffusible DCPIP and the FAD redox group in ZmCKX1 endowed the microbiosensor with a fast response time of less than 10 s. The immobilised ZmCKX1 retained a high affinity for its preferred substrate N6-(Δ2-isopentenyl) adenine (iP), and gave the miniaturized biosensor a large linear dynamic range from 10 nM to 10 µM, a detection limit of 3.9 nM and a high sensitivity to iP of 603.3 µAmM−1cm−2 (n = 4, R2 = 0.9999). Excellent selectivity was displayed for several other aliphatic cytokinins and their ribosides, including N6-(Δ2-isopentenyl) adenine, N6-(Δ2-isopentenyl) adenosine, cis-zeatin, trans-zeatin and trans-zeatin riboside. Aromatic cytokinins and metabolites such as cytokinin glucosides were generally poor substrates. The microbiosensors exhibited excellent stability in terms of pH and long-term storage and have been used successfully to determine low nanomolar cytokinin concentrations in tomato xylem sap exudates

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

Selectivity of <i>Zm</i>CKX1-based cytokinin microbiosensor.

<p>Amperometric responses of cytokinins and analogues at 10 µM normalised to that of 10 µM iP. Operating potential, +350 mV. Key: <i>N</i>⁶-(Δ²-isopentenyl) adenine (iP), kinetin (K), <i>N</i>⁶-(Δ²-isopentenyl) adenosine (iPR), <i>N</i>⁶-(Δ²-isopentenyl) adenosine-5′-monophosphate (iPMP), <i>trans</i>-zeatin-9-glucoside (Z9G), <i>N</i>⁶-(Δ²-isopentenyl) adenosine-7-<i>beta</i>-D-glucoside (iP7G), <i>N</i>⁶-(Δ²-isopentenyl) adenosine-9-<i>beta</i>-D-glucoside (iP9G), (<i>RS</i>)-dihydrozeatin (DZ), <i>cis</i>-zeatin (cZ), <i>trans</i>-zeatin (Z), <i>trans</i>-zeatin riboside (ZR) and <i>cis</i>-zeatin riboside (cZR), Thidiazuron (TDZ), gibberellic acid (GA), <i>N</i>⁶-benzyladenine (<i>N</i>⁶-BAP), abscisic acid (ABA) and adenosine (Ado).</p

Performance of the cytokinin microbiosensor.

<p>(A) Amperometric response to iP at concentrations of 0.1, 0.2, 0.5, 1, 2 and 5 µM. Operating potential, +350 mV; (B) Amperometric response to iP at concentrations of 20, 50, 100, 200 and 500 nM. Operating potential, +350 mV; (C) Calibration plot of the microbiosensor at low iP concentrations ranging 10 to 200 nM, inset shows corresponding calibration over a larger concentration range; (D) Linear fit on a Lineweaver – Burk plot for a <i>Zm</i>CKX1-based cytokinin microbiosensor.</p

Optimum conditions of cytokinin microbiosensor.

<p>(A) Dependence of microbiosensor response on the concentration of DCPIP. Operating potential: +300 mV; (B) Response to 5 µM iP at different operating potentials in phosphate buffer (pH 6.5) containing 10 µM DCPIP as supporting electrolyte.</p

Phylogenetic tree for the IPT proteins shows diverse pattern for the different subtypes.

<p>Maximum likelihood phylogeny derived by the full-protein 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 with a red arrow. The tRNA IPTs clades were named as suggested before [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138468#pone.0138468.ref009" target="_blank">9</a>].</p

Amino acid sequence alignment of ZmCKX1 and <i>NoCKX1</i>.

<p>Protein sequences were aligned using the ClustalW interface in BioEdit 7.0.5.3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138468#pone.0138468.ref055" target="_blank">55</a>]. Major differences in conserved regions are indicated by boxes. Amino acid residues which were mutated to match consensus sequence are marked by asterisk. Histidine in the motif ¹⁰⁴GHS is involved in FAD covalent linkage, ³⁹⁴PHPWLN and ⁵⁰⁴HFG are conserved C-terminal domains and the residues ¹⁶⁹D, ³⁷⁸V and ³⁸¹E are involved in substrate biding. FAD binding domain (pfam01565) and cytokinin binding domain (pfam09265) of <i>NoCKX1</i> identified by NCBI´s conserved domain search [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138468#pone.0138468.ref066" target="_blank">66</a>] are shown in red and underlined letters, respectively. Numbering refers to the sequence of ZmCKX1.</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

Concentration of cytokinins in <i>Nostoc</i> cells.

<p>^aAbbreviations: iP, <i>N</i>^<i>6</i>- (Δ²-isopentenyl) adenine; iPR, <i>N</i>^<i>6</i>- (Δ²-isopentenyl) adenine 9-riboside; iPNT, <i>N</i>^<i>6</i>- (Δ²-isopentenyl)adenine nucleotides; tZ, <i>trans</i>-zeatin; tZR, <i>trans</i>-zeatin 9-riboside; tZNT, <i>tran</i>s-zeatin nucleotides; tZOG, <i>trans</i>-zeatin-O-glucoside; tZROG, <i>trans</i>-zeatin 9-riboside-O-glucoside; cZ, <i>cis</i>-zeatin; cZR, <i>cis</i>-zeatin 9-riboside; cZNT, <i>ci</i>s-zeatin nucleotides; cZOG, <i>cis</i>-zeatin-O-glucoside; cZROG, <i>cis</i>-zeatin 9-riboside-O-glucoside; DHZ, dihydrozeatin; DHZR, dihydrozeatin 9-riboside; DHZNT, dihydrozeatin nucleotides; DHZOG, dihydrozeatin-O-glucoside; DHZROG, dihydrozeatin 9-riboside-O-glucoside.</p><p>Concentration of cytokinins in <i>Nostoc</i> cells.</p

List of vectors and <i>E</i>. <i>coli</i> expression strains used in <i>NoCKX1</i> study.

<p>^apCIOX vector is a modified version of the commercial pET SUMO vector of Invitrogen. It was a kind gift from prof. Andrea Mattevi.</p><p>List of vectors and <i>E</i>. <i>coli</i> expression strains used in <i>NoCKX1</i> study.</p