Subcellular localization and biochemical comparison of cytosolic and secreted cytokinin dehydrogenase enzymes from maize

Abstract

Cytokinin dehydrogenase (CKX; EC 1.5.99.12) degrades cytokinin hormones in plants. There are several differently targeted isoforms of CKX in plant cells. While most CKX enzymes appear to be localized in the apoplast or vacuoles, there is generally only one CKX per plant genome that lacks a translocation signal and presumably functions in the cytosol. The only extensively characterized maize CKX is the apoplastic ZmCKX1; a maize gene encoding a non-secreted CKX has not previously been cloned or characterized. Thus, the aim of this work was to characterize the maize non-secreted CKX gene (ZmCKX10), elucidate the subcellular localization of ZmCKX10, and compare its biochemical properties with those of ZmCKX1. Expression profiling of ZmCKX1 and ZmCKX10 was performed in maize tissues to determine their transcript abundance and organ-specific expression. For determination of the subcellular localization, the CKX genes were fused with green fluorescent protein (GFP) and overexpressed in tomato hairy roots. Using confocal microscopy, the ZmCKX1-GFP signal was confirmed to be present in the apoplast, whereas ZmCKX10-GFP was detected in the cytosol. No interactions of ZmCKX1 with the plasma membrane were observed. While roots overexpressing ZmCKX1-GFP formed significantly more mass in comparison with the control, non-secreted CKX overexpression resulted in a small reduction in root mass accumulation. Biochemical characterization of ZmCKX10 was performed using recombinant protein produced in Pichia pastoris. In contrast to the preference for 2,6-dichlorophenolindophenol (DCPIP) as an electron acceptor and trans-zeatin, N(6)-(Delta(2)-isopentenyl)adenine (iP) and N(6)-(Delta(2)-isopentenyl)adenosine (iPR) as substrates for ZmCKX1, the non-secreted ZmCKX10 had a range of suitable electron acceptors, and the enzyme had a higher preference for cis-zeatin and cytokinin N-glucosides as substrates.

CYTOKININ OXIDASE/DEHYDROGENASE4 Integrates Cytokinin and Auxin Signaling to Control Rice Crown Root Formation

“…For example, in Arabidopsis, the only nontargeted CKX, AtCKX7, is restricted to the cytosol (Galuszka et al, 2007;Köllmer et al, 2014). Similarly, in maize, ZmCKX10 lacked a translocation signal and also localized in the cytosol (Smehilová et al, 2009). Our results demonstrated that OsCKX4 is also a cytosolic cytokinin degradation enzyme.…”

“…to be localized in the apoplast or vacuoles, there is generally one CKX per plant genome that lacks a translocation signal and, thus, presumably functions in the cytosol (Smehilová et al, 2009). For example, in Arabidopsis, the only nontargeted CKX, AtCKX7, is restricted to the cytosol (Galuszka et al, 2007;Köllmer et al, 2014).…”

Cytokinins - recent news and views of evolutionally old molecules

“…The substrate binds above the isoalloxazine plane of the FAD cofactor and its amino group forms a hydrogen bond with an aspartic acid-glutamic acid (Asp-Glu) pair that facilitates the oxidation (Malito et al 2004). Studies on the substrate specificity of Arabidopsis thaliana and maize CKX isoforms revealed that not only free bases and ribosides of isoprenoid CKs, including cis-zeatin (Šmehilová et al 2009;Pertry et al 2010;Gajdošová et al 2011), but also CK 9-glucosides and nucleotides are effectively cleaved by individual isoforms (Galuszka et al 2007;Kowalska et al 2010). It was also presented that aromatic CKs are degraded by CKX as well, although with lower reaction rates (Frébortová et al 2004;Galuszka et al 2007).…”

Antioxidant enzymatic protection during tobacco leaf ageing is affected by cytokinin depletion

“…cytokinin N-glucosides and cis-zeatins, with the highest decrease found in old leaves and the lowest decline in young ones. As both N-glucosides and cis-zeatins were reported to serve as substrates for the CKX (Armstrong 1994, Galuszka et al 2007, Š mehilová et al 2009) the decrease in concentrations of these derivatives in AtCKX2 plants was obviously a direct consequence of their higher degradation by the CKX activities. The levels of active cytokinin bases and ribosides were rather low even in control leaves and were not considerably altered in transformed plants.…”