Untitled Document

Leaf, root and seed developmental stage tissues were collected from Soybean line Williams 82 plants grown in the controlled growth chamber conditions

Drought stress at the vegetative stage: One-month old soybean plants were subjected to gradual stress by withholding water and the samples were collected in three biological replicates. We have monitored relative water content (RWC), leaf water potential, and turface-soil mixture water potential and moisture content to assess the stress level. Leaf RWC 95%, Leaf water potential -0.3 MPa, Soil water content 20% (v/v) for well watered samples and Leaf RWC 65%, Leaf water potential -1.6 MPa, and Soil water content is 9.6% (v/v) for the water stressed samples. Soybean root and leaf samples were collected in triplicates and immediately frozen under liquid nitrogen.

GC-MS profiling of soybean metabolites:

I. Extraction of Polar and Nonpolar Metabolites from soybean tissues

Soybean tissues were ground to fine powder and 20 mg was transferred to 15 mL glass tube. Chloroform (5 mL) was added, and the mixture was shaken in a shaking water bath at 50 °C for 30 min with 50 rpm. Internal standard (IS) for nonpolar metabolites, docosanol-chloroform 10µL of 200µg/mL was added during this step. Followed by 5 mL of deionized water and IS for the polar metabolites, aqueous ribitol 10 μL of 200 ug/mL was added sequentially, and after each addition, the mixture was shaken in a water bath at 50 °C for another 2 hours at 80rpm. After cooling down to room temperature, separate the upper (polar) and lower (nonpolar) fractions by centrifugation and transfer the polar and non-polar phase into two 4 mL sample vials. Both polar and non-polar samples were dried slowly under argon and the samples were stored at -20 °C pending further processing.

II. Derivatization of Polar Extracts

An aliquot of the polar fraction was evaporated to dryness and oximated with 120µL methoxylamine hydrochloride (20 mg mL-1) in anhydrous pyridine at 100 °C for 60 min. After cooling down the samples were then silylated at 65 °C for 60 min with 80 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). A subsample (40 μL) was taken and added to an autosampler vial containing a mixture of n-alkanes (undecane, tridecane, hexadecane, eicosane, tetracosane, triacontane, tetratriacontane, and octatriacontane) to serve as retention index (RI) markers. The sample was diluted with pyridine (1:1) and analyzed by GC-MS.

III. Derivatization of Nonpolar Extracts.

The entire nonpolar fraction was evaporated to dryness and transesterified at 50 °C overnight with 1.25 M (v/v) methanolic hydrochloric acid and followed by the samples were evaporated to dryness under argon. The extract was solubilized and silylated with MSTFA (80 μL) at 65 °C for 60 min. A subsample (40 μL) was prepared for analysis by GC-MS as described for the polar fraction.

IV. Metabolite profiling by GC-MS

The polar and nonpolar samples were analyzed similarly using a Thermo Finnigan DSQ Ultra GC--MS system (Thermo Fischer, USA). Samples were analyzed in sequences each containing blank controls and the respective reference samples. Samples were injected in a splitless mode for 0.2 min with the split flow 30 mL/min. Chromatography was effected on a DB5-MS column (15 m × 0.25 mm × 0.25 μm; J&W, Folsom, CA) using helium at 1.0 mL/min constant flow. The GC temperatures were 80 °C for 2.0 min, increased at 5 °C min-1 to 300 °C, and then keep for 6 min. The GC-MS interface temperature was 280 °C. MS acquisition conditions were electron impact (EI) ionization at 70 eV, source temperature of 250 °C, and detector gain 3 x 105. The mass spec data were acquired using the Xcalibur software package V. 1.2.