可溶性淀粉测定方法

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 7.5 mL contained 0.5 mL extracts, 0.5 mL mixed reagent (1 g anthrone+50 mL ethyl acetate), 5mL H2SO4 (98%), 1.5 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 1.0 mL HClO4 (9.2 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 7.5 mL contained 0.5 mL extracts, 0.5 mL mixed reagent (1 g anthrone+50 mL ethyl acetate), 5mL H2SO4 (98%), 1.5 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 80% ethanol at 80℃each. The extracts were pooled and evaporated to dryness at 50℃ in vacuum. The residues were dissolved in 1 ml distilled water and were filtered through 0.22 lm filter. Agilent 1200HPLC system was used to analyze carbohydrate. Carbohydrate compounds were separated on a Waters Sugar Pak column (6.5 9 300 mm) at 50_C, using water as the mobile phase with the flow rate of 0.5 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℃ for10 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,