Distinct metabolism of N‐glucosides of isopentenyladenine and trans‐zeatin determines cytokinin metabolic spectrum in Arabidopsis

Abstract

Summary The diversity of cytokinin (CK) metabolites suggests their interconversions are the predominant regulatory mechanism of CK action. Nevertheless, little is known about their directionality and kinetics in planta. CK metabolite levels were measured in 2‐wk‐old Arabidopsis thaliana plants at several time points up to 100 min following exogenous application of selected CKs. The data were then evaluated qualitatively and by mathematical modeling. Apart from elevated levels of trans‐zeatin (tZ) metabolites upon application of N6‐(Δ2‐isopentenyl)adenine (iP), we observed no conversions between the individual CK‐types – iP, tZ, dihydrozeatin (DHZ) and cis‐zeatin (cZ). In particular, there was no sign of isomerization between tZ and cZ families. Also, no increase of DHZ‐type CKs was observed after application of tZ, suggesting low baseline activity of zeatin reductase. Among N‐glucosides, those of iP were not converted back to iP while tZ N‐glucosides were cleaved to tZ bases, thus affecting the whole metabolic spectrum. We present the first large‐scale study of short‐term CK metabolism kinetics and show that tZ N7‐ and N9‐glucosides are metabolized in vivo. We thus refute the generally accepted hypothesis that N‐glucosylation irreversibly inactivates CKs. The subsequently constructed mathematical model provides estimates of the metabolic conversion rates.

trans-Zeatin-N-glucosides have biological activity in Arabidopsis thaliana

“…These bioassay results are strong evidence that tZNGs are not simply converted to tZ as recently reported [27]; one would expect nearly identical results in all bioassays if this were the case. Instead, these data suggest some type of context-dependent cytokinin activity for tZNGs, though the exact nature of this is not yet understood.…”

“…This possible tZ increase does not appear to be the result of hydrolysis of tZNGs to tZ base, as this would likely result in an effect similar to that of exogenous tZ application, which is not observed (Fig 7). This result also appears to provide genetic evidence countering that described in a recent metabolic study showing conversion of tZNGs to tZ [27]. It must be noted, however, that this assay only included cotyledon tissue; while it does not appear that tZNGs are hydrolyzed to tZ in cotyledons, this data cannot be generalized other organs.…”

“…It was recently reported that within minutes of exogenous application, tZNGs were hydrolyzed to tZ base [27]. While the physiological, transcriptomic, and proteomic data above appear to refute this claim, or at least suggest tZNGs are not fully converted to tZ, we decided to also use a genetic approach to test this hypothesis.…”

“…Recently, a metabolic study suggested that tZNGs are converted to active tZ in Arabidopsis within minutes of exogenous application [27] but it is important to note the fact that Hošek et al used Arabidopsis cell lines for some of their work. CKNGs are largely localized to the extracellular space [28], so using a liquid-grown cell line may have significant impacts on CK homeostasis.…”

Steady-State Levels of Cytokinins and Their Derivatives May Serve as a Unique Classifier of Arabidopsis Ecotypes

“…The pool of CK-glucosides that can potentially contribute to levels of active CK appears to be larger than previously expected. The generally accepted hypothesis that N-glucosylation irreversibly inactivates CKs was recently put into question [55]. Thus, release of active CKs from CK glucosides including the N-glucosides, may substantially affect the whole metabolic pattern.…”