title: A fluorometric assay for trehalose in the picomole range.
abstract: BACKGROUND
  Trehalose is a non-reducing disaccharide that is used as an osmolyte, transport sugar, carbon reserve and stress protectant in a wide range of organisms. In plants, trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is thought to be a signal of sucrose status. Trehalose itself may play a role in pathogenic and symbiotic plant-microbe interactions, in responses to abiotic stress and in developmental signalling, but its precise functions are unknown. A major obstacle to investigating its function is the technical difficulty of measuring the very low levels of trehalose usually found in plant tissues, as most of the established trehalose assays lack sufficient specificity and/or sensitivity.
  
  
  RESULTS
  A kinetic assay for trehalose was established using recombinant Escherichia coli cytoplasmic trehalase (treF), which was shown to be highly specific for trehalose. Hydrolysis of trehalose to glucose is monitored fluorometrically and the trehalose content of the tissue extract is determined from an internal calibration curve. The assay is linear for 0.2-40 pmol trehalose, and recoveries of trehalose were ≥88%. A. thaliana Col-0 rosettes contain about 20-30 nmol g-1FW of trehalose, increasing to about 50-60 nmol g-1FW in plants grown at 8°C. Trehalose is not correlated with sucrose content, whereas a strong correlation between Tre6P and sucrose was confirmed. The trehalose contents of ear inflorescence primordia from the maize ramosa3 mutant and wild type plants were 6.6±2.6 nmol g-1FW and 19.0±12.7 nmol g-1FW, respectively. The trehalose:Tre6P ratios in the ramosa3 and wild-type primordia were 2.43±0.85 and 6.16±3.45, respectively.
  
  
  CONCLUSIONS
  The fluorometric assay is highly specific for trehalose and sensitive enough to measure the trehalose content of very small amounts of plant tissue. Chilling induced a 2-fold accumulation of trehalose in A. thaliana rosettes, but the levels were too low to make a substantial quantitative contribution to osmoregulation. Trehalose is unlikely to function as a signal of sucrose status. The abnormal inflorescence branching phenotype of the maize ramosa3 mutant might be linked to a decrease in trehalose levels in the inflorescence primordia or a downward shift in the trehalose:Tre6P ratio.
authors.affiliation: Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche, Seconda Università degli Studi di Napoli, Via Vivaldi 43, I-81100, Caserta, Italy.
authors.forename: Petronia
authors.initials: P
authors.lastname: Carillo
authors.affiliation: Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, Potsdam-Golm, 14476, Germany.
authors.forename: Regina
authors.initials: R
authors.lastname: Feil
authors.affiliation: Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, Potsdam-Golm, 14476, Germany.|INRA Bordeaux, University of Bordeaux, UMR1332 Fruit Biology and Pathology, F-33883, Villenave d'Ornon, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Gibon
authors.affiliation: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.|Laboratory of Plant Genetics and Breeding, Department of Biological Production, Faculty of Bioresource Sciences, Kaidobata-nishi 241-438, Shimo-Shinjyo Nakano, Akita City 010-0195, Japan.
authors.forename: Namiko
authors.initials: N
authors.lastname: Satoh-Nagasawa
authors.affiliation: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.
authors.forename: David
authors.initials: D
authors.lastname: Jackson
authors.affiliation: Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, Potsdam-Golm, 14476, Germany.|Metanomics GmbH, Tegeler Weg 33, Berlin, 10589, Germany.
authors.forename: Oliver E
authors.initials: OE
authors.lastname: Bläsing
authors.affiliation: Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, Potsdam-Golm, 14476, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Stitt
authors.affiliation: Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, Potsdam-Golm, 14476, Germany.
authors.forename: John Edward
authors.initials: JE
authors.lastname: Lunn