Deuteriumin vivo labelling of cytokinins inArabidopsis thaliana analysed by capillary liquid chromatography/frit-fast atom bombardment mass spectrometry

Åstot, Crister; Doležal, Karel; Moritz, Thomas; Sandberg, Göran

doi:10.1002/(sici)1096-9888(200001)35:1<13::aid-jms901>3.0.co;2-i

Cited by 20 publications

(8 citation statements)

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“…The in vivo deuterium-labeling experiments were performed according to ref. 27. The biosynthetic rate is presented as the tracer͞tracee ratio, which is the incorporation of deuterium into the different cytokinin metabolites corrected for the natural isotope abundance obtained from unlabeled standard of the respective cytokinin metabolite.…”

Section: Methodsmentioning

confidence: 99%

“…The apical part of Arabidopsis plants grown on soft agar was separated from roots at the hypocotyls junction, and the different plant tissues were incubated separately in one-half of MS medium containing 1.5% sucrose and 30% 2 H 2 O for 12 h. The rate of cytokinin biosynthesis was measured according to ref. 27. In a second experiment, leaves of 6-wk-old tobacco plants were analyzed for pool size and synthesis rate of ZMP and ZR.…”

Section: Methodsmentioning

confidence: 99%

“…The aim of the present study was to investigate the true nature of the interaction on the homeostatic level between these two hormones. For this purpose, we used in vivo deuterium-labeling technology to measure the rate of synthesis of both IAA and cytokinins (21,26,27). The advantage of the approach of probing the synthesis rate is that 2 H 2 O penetrates all plant organs and a labeling process starts in essentially every biosynthetic pathway of the plant cell.…”

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Auxin regulation of cytokinin biosynthesis in Arabidopsis thaliana : A factor of potential importance for auxin–cytokinin-regulated development

Nordström

Tarkowski

Tarkowská

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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One of the most long-lived models in plant science is the belief that the long-distance transport and ratio of two plant hormones, auxin and cytokinin, at the site of action control major developmental events such as apical dominance. We have used in vivo deuterium labeling and mass spectrometry to investigate the dynamics of homeostatic cross talk between the two plant hormones. Interestingly, auxin mediates a very rapid negative control of the cytokinin pool by mainly suppressing the biosynthesis via the isopentenyladenosine-5 -monophosphate-independent pathway. In contrast, the effect of cytokinin overproduction on the entire auxin pool in the plant was slower, indicating that this most likely is mediated through altered development. In addition, we were able to confirm that the lateral root meristems are likely to be the main sites of isopentenyladenosine-5 -monophosphate-dependent cytokinin synthesis, and that the aerial tissue of the plant surprisingly also was a significant source of cytokinin biosynthesis. Our demonstration of shoot-localized synthesis, together with data demonstrating that auxin imposes a very rapid regulation of cytokinin biosynthesis, illustrates that the two hormones can interact also on the metabolic level in controlling plant development, and that the aerial part of the plant has the capacity to synthesize its own cytokinin independent of long-range transport from the root system.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Auxin regulation of cytokinin biosynthesis in Arabidopsis thaliana : A factor of potential importance for auxin–cytokinin-regulated development

Nordström

Tarkowski

Tarkowská

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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509

397

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“…We also used a recently developed method where cytokinin biosynthesis was monitored after deuterium incorporation into cytokinins (25). When plants are incubated on a liquid growth medium enriched with deuterium oxide, general labeling occurs throughout essentially all biosynthetic pathways of the plant cell.…”

Section: Table 1 Effect Of Metyrapone On Cytokinin Nucleotide Contenmentioning

confidence: 99%

“…However, our investigation is based solely on samples taken in the initial induction phase during which no effect from the long-term growth alteration is expected. We have previously developed powerful tools for analyzing cytokinins by mass spectrometry (24) and for quantitative analysis of cytokinin biosynthesis by in vivo deuterium labeling (25). In this paper we present evidence, acquired by using these techniques, for an alternative, iPMP-independent, biosynthetic pathway of zeatin-type cytokinins, operational in both wild-type and ipt-transgenic plants.…”

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An alternative cytokinin biosynthesis pathway

Åstot

Doležal

Nordström

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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125

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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 H6] isopentenyladenosine and deuterium oxide, provided evidence for an alternative, iPMPindependent, 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. C ytokinins are an important class of plant growth regulators, defined by their ability to promote cell division in tissue culture in the presence of auxins (1, 2). Virtually all naturally occurring cytokinins identified to date are adenine species substituted at N 6 with an isoprenoid or aromatic side chain. In this text, cytokinins will refer solely to the isoprenoid cytokinin bases and their sugar conjugates. Cytokinins affect many plant developmental processes including cell division, cell differentiation, chlorophyll senescence, and apical dominance (3).Efforts to elucidate the biosynthetic origin of cytokinins in plants have been inconclusive. Early suggestions that tRNA degradation could be the major source of free, active cytokinins (4) were disproved when calculations of tRNA turnover rates showed that a tRNA-independent de novo biosynthetic pathway also must be present in plants (5). A major breakthrough was the discovery of a cytokinin biosynthetic enzyme in the slime mold, Dictyostelium discoideum (6). Cell-free extracts from this organism can convert AMP and dimethylallyl-pyrophosphate (DMAPP) to the free cytokinins isopentenyladenosine-5Ј-monophosphate (iPMP) and the corresponding nucleoside (isopentenyladenosine, iPA). This finding, and studies on the metabolism of isopentenyl-type cytokinins (7,8), led to the proposal that iPMP is also the primary cytokinin intermediate in plants, and zeatin cytokinins are formed by hydroxylation of iPMP and its derivatives (9, 10) ( Fig. 1).Later, the product of the T-DNA gene 4 (ipt) of the crown gall-forming bacterium, Agrobacterium tumefaciens, also was described as a DMAPP:AMP isopentenyltransferase (ipt) (11,12). AMP was found to be the preferred adenylic substrate for the Agrobacterium ipt enzyme but searches for alternative side-chain donors have been limited. However, when de novo biosynthesis in crown gall tissue of Vinca rosea was traced with 14 C-adenine, in vivo production of iPMP was undetectable, whereas zeatinriboside-5Ј-monophospate (ZMP) production was strong (13). After studies of 14 C-isopententyladenine metabolism in this system, it was proposed that the very low levels of iPMP were caused by rapi...

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