Cytokinin‐induced upregulation of cytokinin oxidase activity in tobacco includes changes in enzyme glycosylation and secretion

Abstract

Regulation of cytokinin oxidase (CKX) activity in relation to enzyme glycosylation and secretion was studied in wild‐type (WT) and transgenic conditionally isopentenyltransferase gene (ipt)‐expressing (IPT) tobacco (Nicotiana tabacum L. cv. Wisconsin 38) cell suspensions, calli and leaves. An increase in endogenous cytokinin content due to the tetracycline (Tc)‐induced derepression of the ipt gene transcription or surface application of N6‐benzylaminopurine (BA) resulted in significant enhancement of CKX activity in all these plant materials. As revealed by Concanavalin A‐Sepharose 4B chromatography the cytokinin‐induced enhancement of CKX activity was associated predominantly with the N‐glycoform of the enzyme (10‐ to 15‐fold increase) in calli and leaves. Application of BA to the culture media of WT and IPT cell suspensions and the derepression of the ipt by Tc substantially enhanced endogenous levels of isoprenoid cytokinins and CKX activity in both cells and the culture medium. Most CKX activity in control, BA‐ and Tc‐treated cells was associated with the non‐glycosylated form of the enzyme, whereas the majority of CKX activity in the culture media was due to the glycosylated form. The pH optimum of CKX in cells (pH 8.5) differed considerably from that in the culture medium (pH 6.0). No significant differences were found in apparent Km(iP) values of CKX between control, BA‐ and Tc‐treated IPT cells and media or between purified glycosylated and non‐glycosylated CKX. These results suggest that cytokinins induce changes in the proportions of glyco‐ and non‐glycoforms of the enzyme in multicellular calli and leaves, and influence its secretion to the cell exterior.

Cytokinins in the Bryophyte Physcomitrella patens: Analyses of Activity, Distribution, and Cytokinin Oxidase/Dehydrogenase Overexpression Reveal the Role of Extracellular Cytokinins

“…S1). The detection of considerable concentrations of extracellular cytokinin nucleotides in Physcomitrella is in accordance with the previously reported occurrence of tZRMP and iPRMP in media of tobacco cell suspension cultures (Motyka et al, 2003).…”

“…The CKX from Physcomitrella cells (plant material equivalent to approximately 1.3-1.5 g DW) was extracted and partially purified using the method of Chatfield and Armstrong (1986) as modified by Motyka et al (2003). The CKX from the media of Physcomitrella cultures (equivalent to 265 mL) was precipitated by the addition of solid ammonium sulfate directly to the medium to 80% saturation.…”

Cytokinin-Deficient Transgenic Arabidopsis Plants Show Multiple Developmental Alterations Indicating Opposite Functions of Cytokinins in the Regulation of Shoot and Root Meristem Activity

“…Our inability to detect the GFP signal in the apoplast could be explained by the weak fluorescence of secreted GFP (Batoko et al, 2000). Interestingly, an increase in the extracellular CKX fraction was triggered in tobacco cell cultures by cytokinin and was correlated with enhanced CKX glycosylation (Motyka et al, 2003). Based on the facts that AtCKX proteins contain consensus N-glycosylation sites and that protein glycosylation occurs during transport through the secretory pathway, we hypothesize that ER-located AtCKX enzymes are released to the apoplast and that the release is at least partly dependent on a triggering signal, which could be cytokinin.…”

Cytokinins - recent news and views of evolutionally old molecules

“…It is expected that all secreted CKX enzymes are post-transcriptionaly modified by glycosylation (Galuszka et al 2008). Glycosylation most probably contributes to the enzyme localisation, and also to translocation and protein stability , as the glycosylated CKXs were shown to have different pH optima and higher activity compared with the nonglycosylated forms (Kamínek and Armstrong 1990;Motyka et al 2003).…”