可溶性淀粉测定方法
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可溶性糖和可溶性淀粉：
Soluble sugar was determined by the anthrone method (Li, 2000) using sucrose as the standard. Half a gram of fresh samples was placed in a 25mL cuvette added with 10 mL distilled water, allowed to stand at 100℃ for 1 h, and filtered into 25 mL volumetric flasks. Reaction mixture of mL contained mL extracts, mL mixed reagent (1 g anthrone+50 mL ethyl acetate), 5mL H2SO4 (98%), mL distilled water. The mixture was heated at 100℃ for 1 min and absorbance read at 630nm. Soluble starch was determined by the anthrone method (Li, 2000) with sucrose as the standard. The remainder of measured soluble sugar was transferred to a 25 mL cuvette containing 10 mL distilled water and mL HClO4 mol L-1). The cuvette was placed in a boiling water bath for 30 min, cooled to 30 ℃, and filtered into 25 mL volumetric flasks. Reaction mixture of mL contained mL extracts, mL mixed reagent (1 g anthrone+50 mL ethyl acetate), 5mL H2SO4 (98%), mL distilled water. The mixture was heated at 100℃ for 1 min and absorbance read
at 630nm. Total non-structural carbohydrates (NSC) were the sum of soluble sugar and soluble starch.
Extraction and analysis of carbohydrate
Carbohydrates were analyzed essentially as described by Pharr et al. (1985) and Modore et al. (1988). Briefly, 0.5 g (FW) of cucumber seedling leaves were frozen in liquid nitrogen and grounded to fine powder, then extracted three times by 5 ml
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80% ethanol at 80℃ each. The extracts were pooled and evaporated to dryness at 50℃ in
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vacuum. The residues were dissolved in 1 ml distilled water and were filtered through lm filter. Agilent 1200HPLC system was used to analyze carbohydrate. Carbohydrate compounds were separated on a Waters Sugar Pak column 9 300 mm) at 50_C, using water as the mobile phase with the flow rate of ml min-1. Stachyose, raffinose, sucrose, glucose, galactose and fructose were identified by comparison of retention times of known standards (Sigma) and quantified by a refractive index detector (G1362A RID). Total amount of the sugars was sum of the contents of stachyose, raffinose, sucrose, glucose, galactose and fructose. Starch in the extracted tissues was digested with 10 ml 30% HClO4 (v/v) for 24 h at 25℃. The reaction was terminated by incubating at 80℃ for 10 min. After centrifugation, the supernatant was transferred to a 25 ml vitreous bottle and filled to regulated volume with distilled water. Starch was determined spectrophotometrically by reference to a glucose standard curve.
总的氨基酸测定方法：
At the end of the experiment, plants were sampled at a single day within 2 h to minimize the effect of diurnal variation. Plants were cleaned from surface salt deposits with distilled water and blotted dry. Subsequently, fresh weight of plants was determined for calculation of relative growth rates (per day). Afterwards, plants were separated into leaf laminas, roots, and rhizomes. Only living material was used, immediately frozen in liquid N2 and stored at 20℃ until analysed. Plant samples for determination of free amino acids and sugars were freeze-dried at
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20℃ in a vacuum evaporator (Christ, Germany) and pulverised (Mikro-Dismembrator; Braun Biotech, Germany). About 100 mg of powdered samples were extracted three times with 80% ethanol at room temperature, centrifuged (5000×g) and the combined supernatants were stored at
20℃ until analysed. Norleucine, as an internal standard used for recovery checks, was added to the samples during extraction. All amino acid samples were purified by ultrafiltration through Ultrafree-MC Membranes (Milipore, USA), freeze-dried at 20℃, and redried in a 2:2:1 mixture of methanol: Na-acetate:triethylamine at 4℃. Samples including standards were derivatized with phenylisothiocyanate to give phenylthiocarbamyl amino acids (Waters, Pico-Tag method), and freeze-dried at 4℃. Samples were redissolved in phosphate buffer and run on a HPLC system
(Chromasystem 500, Kontron Instruments, Germany) using a Sentry-C18 precolumn (Waters, USA) and a reverse phase Pico-Tag column ×300 mm; Waters). Separation was carried out at 46℃ with a gradient of acetate buffer and acetonitrile/ water. Standard mixtures of amino acids were used for identification and quantification of the samples. The totals of free amino acids (TotAA) were calculated as the sum of all 20 detected and quantified amino acids. Their contents are given in absolute (mmol g-1 DW) and relative (% TotAA) units. All sugar samples including standards were determined using a HPLC-system (Dionex DX-100, USA). Samples were run on PA1-Guard precolumn and PA1 column (4×250 mm; CarboPac, Dionex) with 0.15M NaOH (eluent) and detected by a Pulse Amperometric Detector with a gold electrode at 24 ± 2℃. Pulse setting was at 50, 600 and
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600mV for 360, 120 and 420 ms, respectively. Standard mixtures of sucrose, fructose and glucose were used for identification and quantification. The totals of these three sugars was designated as TotSUG. Their contents are given in absolute (mmol g-1 DW) and relative (% TotSUG) units. The osmolality in rhizomes and leaves was measured using grounded freeze-dried material. This was redissolved in distilled water, sonicated, centrifuged and supernatants were analysed. Osmolality was determined using an osmometer (Wescor Vapor Pressure Osmometer, model 5100 C, USA). The measurements were done in triplicate.
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