Final full-text version of the paper available at: http://www.biochemj.org/
Tre6P (trehalose 6-phosphate) is implicated in sugar-signalling pathways in plants, but its exact functions in vivo are uncertain. One of the main obstacles to discovering these functions is the difficulty of measuring the amount of Tre6P in plant tissues. We have developed a highly specific assay, using liquid chromatography coupled to MS-Q3 (triple quadrupole MS), to measure Tre6P in the femto-picomole range. The Tre6P content of sucrose-starved Arabidopsis thaliana seedlings in axenic culture increased from 18 to 482 pmol·g−1FW (fresh weight) after adding sucrose. Leaves from soil-grown plants contained 67 pmol·g−1FW at the end of the night, which rose to 108 pmol·g−1FW after 4 h of illumination. Even greater changes in Tre6P content were seen after a 6 h extension of the dark period, and in the starchless mutant, pgm. The intracellular concentration of Tre6P in wild-type leaves was estimated to range from 1 to 15 μM. It has recently been reported that the addition of Tre6P to isolated chloroplasts leads to redox activation of AGPase (ADPglucose pyrophosphorylase) [Kolbe, Tiessen, Schluepmann, Paul, Ulrich and Geigenberger (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 11118–11123]. Using the new assay for Tre6P, we found that rising sugar levels in plants are accompanied by increases in the level of Tre6P, redox activation of AGPase and the stimulation of starch synthesis in vivo. These results indicate that Tre6P acts as a signalling metabolite of sugar status in plants, and support the proposal that Tre6P mediates sucrose-induced changes in the rate of starch synthesis.
This research was supported by theMax Planck Society and by the BMBF (Federal Ministry
of Education and Research)-funded project GABI (Genomanalyse im biologischen System
Pflanze) Verbund Arabidopsis III ‘Gauntlets, Carbon and Nutrient Signaling: Test Systems,
and Metabolite and Transcript Profiles’ (0312277A). R.M. was supported by a Ramón y
Cajal research contract. We thank Melanie Höhne for excellent technical assistance. We
also thank Dr Michael Dahl (University of Konstanz, Germany) and Dr Carlos Gancedo
(Unidad de Bioquimica y Genetica de Levaduras, Instituto de Investigaciones Biomedicas
Alberto Sols, C. S. I. C.- Universidad Autonoma de Madrid, Madrid, Spain) for the gifts of
the pSG2 plasmid and Yarrowia lipolytica HXK1 over-expressing yeast strain respectively.
Peer reviewed