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Sample Set Information

ID SE137
Title Metabolite Signature during Short-Day Induced Growth Cessation in Populus.
Description The photoperiod is an important environmental signal for plants, and influences a wide range of physiological processes. For woody species in northern latitudes, cessation of growth is induced by short photoperiods. In many plant species, short photoperiods stop elongational growth after a few weeks. It is known that plant daylength detection is mediated by Phytochrome A (PHYA) in the woody hybrid aspen species. However, the mechanism of dormancy involving primary metabolism remains unclear. We studied changes in metabolite profiles in hybrid aspen leaves (young, middle, and mature leaves) during short-day-induced growth cessation, using a combination of gas chromatography–time-of-flight mass spectrometry, and multivariate projection methods. Our results indicate that the metabolite profiles in mature source leaves rapidly change when the photoperiod changes. In contrast, the differences in young sink leaves grown under long and short-day conditions are less distinct. We found short daylength induced growth cessation in aspen was associated with rapid changes in the distribution and levels of diverse primary metabolites. In addition, we conducted metabolite profiling of leaves of PHYA overexpressor (PHYAOX) and those of the control to find the discriminative metabolites between PHYAOX and the control under the short-day conditions. The metabolite changes observed in PHYAOX leaves, together with those in the source leaves, identified possible candidates for the metabolite signature (e.g., 2-oxo-glutarate, spermidine, putrescine, 4-amino-butyrate, and tryptophan) during short-day-induced growth cessation in aspen leaves.
Authors Kusano M, Jonsson P, Fukushima A, Gullberg J, Sjöström M, Trygg J, Moritz T.
Reference Front Plant Sci. 2011 Jul 12;2:29. doi: 10.3389/fpls.2011.00029. eCollection 2011.

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Sample Information

Title hybrid aspen (P. tremula × P. tremuloides)
Organism - Scientific Name Populus tremula × Populus tremuloides
Organism - ID NCBI taxonomy 47664
Compound - ID
Compound - Source
Preparation Twenty-eight hybrid aspen trees were grown under long photoperiods (long-day conditions, LDs) of 18 h/6 h day/night cycle using photosynthetic active radiation (PAR) light for 12 h at 400 μmol m−2s−1, and extended for another 6 h at 30 μmol m−2s−1. After 3 months, 20 consecutive leaves (the length of the first leaf below the apex was approximately 1 cm) and the apex (defined as apical tissue from which all major leaf primordia had been removed) were sampled from seven plants (LD0-samples). Two days later another seven plants were sampled (LD2-samples), and the daylength was changed to short winter photoperiods (short-day conditions, SDs): 12/12 h day/night). After 2 and 6 days under short photoperiods (SD2 and SD6), seven plants were sampled.

To examine the effect of 35S: : oat PHYA-overexpression, we sampled 10 leaves from 22-PHYAOX and 21-control plants. We used two different photoperiods [LD0 and short photoperiod at day 7 (SD7)] to obtain PHYOX and the control plants (wild-type, WT). After removal from the plant samples were dipped in liquid nitrogen and stored at −80°C until required.

Sample Preparation Details ID

Analytical Method Information

Method Details ID MS1
Sample Amount 1 μL

Analytical Method Details Information

Instrument GC:Agilent 6890
Instrument Type
Ionization EI
Ion Mode Positive
Description Leaf samples were crushed, extracted, and their metabolite profiles were analyzed according to (Gullberg et al., 2004). Stable isotope reference compounds (15 ng μl−1 each of [13C3]-myristic acid, [13C4]-hexadecanoic acid, [2H4]-succinic acid, [13C5, 15N]-glutamic acid, [2H7]-cholesterol, [13C5]-proline, [13C4]-disodium α-ketoglutarate, [13C12]-sucrose, [2H4]-putrescine, [2H6]-salicylic acid, and [13C6]-glucose) were added to an extraction mixture of chloroform:MeOH:H2O (3:1:1). The samples (10 mg fresh weight each) were then extracted in 1 ml of the extraction mixture using a MM 301 Vibration Mill (Retsch GmbH & Co. KG, Haan, Germany) at a frequency of 30 Hz s−1 for 3 min using a 3-mm of tungsten carbide bead (Retsch GmbH & Co. KG, Haan, Germany) per tube to increase the extraction efficiency. After extraction samples were placed in an Eppendorf centrifuge (Model 5417C) for 10 min at 14,000 rpm. Following this, 200 μl of the supernatant was transferred to a GC-vial and evaporated to dryness. The samples were then derivatized by shaking them with 30 μl of methoxyamine hydrochloride (15 mg ml−1) in pyridine for 10 min at 5°C. Samples were then incubated overnight at room temperature. The samples were then trimethylsilylated by adding 30 μl of MSTFA with 1% TMCS and incubating for 1 h at room temperature. After silylation, 30 μl of heptane was added.

The samples were analyzed according to Gullberg et al. (2004) using gas chromatography–time-of-flight mass spectrometry (GC–MS). We used blank control samples and a series of n-alkanes (C12–C40) to allow us to calculate retention indices (Schauer et al., 2005). One microliter of each derivatized sample was injected using a split/splitless injector in splitless mode of an Agilent 7683 autosampler (Agilent, Atlanta, GA, USA) into an Agilent 6890 gas chromatograph equipped with a 10-m × 0.18-mm i.d. fused silica capillary column with a chemically bonded 0.18 μm DB 5-MS stationary phase (J&W Scientific, Folsom, CA, USA). The injector temperature was 270°C, the septum purge flow rate was 20 ml min−1 and the purge was turned on after 60 s. The gas flow rate through the column was 1 ml min−1, the column temperature was held at 70°C for 2 min, then increased by 40°C min−1 to 320°C, and held for 2 min. The column effluent was introduced into the ion source of a Pegasus III time-of-flight mass spectrometer, GC–MS (LECO Corp., St Joseph, MI, USA). The transfer line and the ion source temperatures were 250 and 200°C, respectively. Ions were generated by a 70-eV electron beam at an ionization current of 2.0 mA, and 30 spectra s−1 were recorded in the mass range 50–800 m/z. The acceleration voltage was turned on after a solvent delay.


Data Analysis Information

Title Data analysis
Data Analysis Details ID DS1
Recommended decimal places of m/z

Data Analysis Details Information

Title Statistical Data Analysis
Description Multivariate statistical investigations were performed using SIMCA-P + 12 software (Umetrics, Umeå, Sweden). All variables were log10-transformed, centered, and scaled to unit variance for the analysis. To connect the information of two-block variables (X and Y) to each other, we used an orthogonal projection to latent structures (OPLS). OPLS is one of the supervised methods which is commonly applied in metabolomics. An OPLS regression model (Trygg and Wold, 2002) was calculated to investigate potential relationships between the metabolic compositions (X) of the aspen leaves and their positions (Y) on the stem. Peak areas under the resolved GC–MS peaks were used as descriptors (X) and the leaf positions as the response (Y) in the OPLS model. R2X is the cumulative modeled variation in X, R2Y is the cumulative modeled variation in Y, and Q2Y is the cumulative predicted variation in Y, according to cross-validation. The range of these parameters is 0–1, where 1 indicates a perfect fit.
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