An alternative cytokinin biosynthesis pathway

Abstract

Studies of de novo cytokinin biosynthesis in isopentenyltransferase (ipt)-transformed Arabidopsis thaliana, involving in vivo deuterium labeling and mass spectrometry, showed that the biosynthetic rate of zeatinriboside-5'-monophosphate was around 66-fold higher than that of isopentenyladenosine-5'-monophosphate (iPMP), the proposed primary product of the Agrobacterium ipt. Double tracer analysis, using [(2)H(6)] isopentenyladenosine and deuterium oxide, provided evidence for an alternative, iPMP-independent, biosynthetic pathway for zeatin-type cytokinins, present in both ipt-expressing and wild-type Arabidopsis thaliana. Reduction of the biosynthetic flux in the alternative pathway by use of mevastatin, an inhibitor for 3-hydroxy-3-methylglutaryl CoA reductase, indicated a terpenoid origin for the side-chain precursor of the iPMP independent pathway.

Figure 1

The established model of de novo cytokinin biosynthesis in plants with the formation of iPMP as the first cytokinin. iP, Isopentenyladenine; Z, zeatin.

Figure 2

Cytokinin levels in wild-type (wt) and ipt transgenic A. thaliana plants in the 12 h after induction of ipt expression by spraying with 30 μM dexamethasone (Dex). IP, isopentenyladenine; Z, zeatin.

Figure 3

In vivo deuterium labeling of ZMP (■) and iPMP (○) in transgenic A. thaliana plants, incubated on deuterium-enriched media containing dexamethasone to induce ipt expression. Enrichment is expressed as the ratio of deuterium labeled substance (tracer)/unlabeled substance (tracee), after correction for natural isotope distribution. Initiation of cytokinin overproduction is marked by an arrow. Standard deviation is indicated by error bars. The slope of the ZMP 6- to 12-h regression line was significantly higher than the slope of the 0- to 3-h data (P < 0.05, dummy variable analysis).

Figure 4

(a) Typical mass isotopomer profiles for iPMP and ZMP purified from ipt-expressing A. thaliana plants after feeding with ²H₂O/[²H₆]iPA double tracers for 6 or 12 h. (b) 12-h double tracer experiment repeated in the presence of metyrapone. Empty bars represent unlabeled ZMP including the natural isotope distribution. Tracers indicated: [²H₆]iPMP, red; [²H_n]ZMP species with label originating from ²H₂O, blue.

Figure 5

(a) ZMP mass isotopomer profiles from A. thaliana wild-type plants incubated in a liquid medium containing 1 μM [²H₆]iPA and 30% ²H₂O for 24 h. (b) In the presence of metyrapone. Empty bars represent unlabeled ZMP including the natural isotope distribution. Tracers indicated: [²H₅]ZMP formed from [²H₆]iPMP, red; [²H_n]ZMP species with label originating from ²H₂O, blue. Standard deviation is indicated by error bars. Significance level of labeling: P < 0.05 (Student's t test).

Figure 6

Effect of mevastatin on the in vivo deuterium incorporation into ZMP via the iPMP-independent pathway. Metyrapone added in both experiments to block iPMP-ZMP conversion. Standard deviation is indicated by error bars. P < 0.05 (Student's t test).

Figure 7

Biosynthetic scheme describing the iPMP-dependent pathway (black arrows), with iPMP as the primary intermediate and the iPMP-independent pathway (blue arrows) producing ZMP as the first cytokinin. The main flow of the tracers used in this work is indicated: [²H₆]iPA, red and ²H₂O, blue.