Degradation of the plant hormone cytokinin is controlled by cytokinin oxidase/dehydrogenase (CKX) enzymes. The molecular and cellular behavior of these proteins is still largely unknown. In this study, we show that CKX1 is a type II single-pass membrane protein that localizes predominantly to the endoplasmic reticulum (ER) in Arabidopsis (Arabidopsis thaliana). This indicates that this CKX isoform is a bona fide ER protein directly controlling the cytokinin, which triggers the signaling from the ER. By using various approaches, we demonstrate that CKX1 forms homodimers and homooligomers in vivo. The amino-terminal part of CKX1 was necessary and sufficient for the protein oligomerization as well as for targeting and retention in the ER. Moreover, we show that protein-protein interaction is largely facilitated by transmembrane helices and depends on a functional GxxxG-like interaction motif. Importantly, mutations rendering CKX1 monomeric interfere with its steady-state localization in the ER and cause a loss of the CKX1 biological activity by increasing its ER-associated degradation. Therefore, our study provides evidence that oligomerization is a crucial parameter regulating CKX1 biological activity and the cytokinin concentration in the ER. The work also lends strong support for the cytokinin signaling from the ER and for the functional relevance of the cytokinin pool in this compartment.

• MeSH
• Arabidopsis metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• membránové proteiny chemie   metabolismus   MeSH
• multimerizace proteinu * MeSH
• oxidoreduktasy chemie   metabolismus   MeSH
• proteinové domény MeSH
• proteiny - lokalizační signály MeSH
• proteiny huseníčku chemie   metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• sekvence aminokyselin MeSH
• stabilita proteinů MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• cytokinin oxidase MeSH Prohlížeč
• membránové proteiny MeSH
• oxidoreduktasy MeSH
• proteiny - lokalizační signály MeSH
• proteiny huseníčku MeSH
• rekombinantní fúzní proteiny MeSH
• zelené fluorescenční proteiny MeSH

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.

• MeSH
• biosenzitivní techniky metody   MeSH
• cytokininy analýza   metabolismus   MeSH
• enzymy imobilizované chemie   metabolismus   MeSH
• isopentenyladenosin analogy a deriváty   analýza   metabolismus   MeSH
• kukuřice setá enzymologie   MeSH
• limita detekce MeSH
• oxidoreduktasy chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokinin oxidase MeSH Prohlížeč
• cytokininy MeSH
• enzymy imobilizované MeSH
• isopentenyladenosin MeSH
• N(6)-(delta(2)-isopentenyl)adenine MeSH Prohlížeč
• oxidoreduktasy MeSH
• zeatin riboside MeSH Prohlížeč