Effect of Sulfated Chitooligosaccharides on Wheat Seedlings (Triticum

Feb 29, 2016 - Relationship between the Degree of Polymerization of Chitooligomers and Their Activity Affecting the Growth of Wheat Seedlings under Sa...
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Effect of Sulfated Chitooligosaccharides on Wheat Seedlings (Triticum aestivum L.) under Salt Stress ping zou, Kecheng Li, Song Liu, Xiaofei He, Ronge Xing, Xiaoqian Zhang, and Pengcheng Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05624 • Publication Date (Web): 29 Feb 2016 Downloaded from http://pubs.acs.org on March 3, 2016

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Effect of Sulfated Chitooligosaccharides on Wheat Seedlings (Triticumaestivum L.)

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under Salt Stress

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Ping Zouab, Kecheng Lia, Song Liua*, XiaofeiHea, XiaoqianZhanga, Ronge Xinga,

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Pengcheng Lia*

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a

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b

Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China Institute of tobacco research of CAAS, Qingdao266101, China

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*Corresponding author:

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Pengcheng Li

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Tel.: +86 532 82898707; fax: +86 532 82968951.

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E-mail addresses: [email protected].

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Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China

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Song Liu

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E-mail addresses: [email protected].

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Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China

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Abstract: In this study, sulfated chitooligosaccharide (SCOS) was applied to wheat

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seedlings in order to investigate its effect on the plants’ defence response under salt

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stress. The antioxidant enzyme activities, chlorophyll contents and fluorescence

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characters of wheat seedlings were determined at a certain time. The results showed

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that treatment with exogenous SCOS could decrease the content of malondialdehyde,

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increase the chlorophyll contents and modulate fluorescence characters in wheat

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seedlings under salt stress. In addition, SCOS was able to regulate the activities of

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antioxidant enzymes containing superoxide dismutase, catalase, peroxidase, ascorbate

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peroxidase, glutathione reductase and dehydroascorbate reductase. Similarly, the

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mRNA expression levels of several antioxidant enzymes were efficiently modulated

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by SCOS. The results indicated that SCOS could alleviate the damage of salt stress by

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adjusting the antioxidant enzyme activities of plant. And the effect of SCOS on

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photochemical efficiency of wheat seedlings was associated with its enhanced

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capacity for antioxidant enzymes which prevented structure degradation of the

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photosynthetic apparatus under NaCl stress. Furthermore, the effective activities of

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alleviating salt stress indicated the activities of SCOS were closely related with the

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sulfate group.

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Key words: wheat; sulfated chitooligosaccharides; salt stress; antioxidant enzyme

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activities; photochemical efficiency; gene expression

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Introduction Salt stress is an important factor that limits crop growth, productivity and yield.

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Several physiological processes especially photosynthesis in plants are affected by

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salt stress. The influences of salt stress on photosynthesis are either direct (such as the

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limitation of the stomata) or indirect, for instance, the oxidative stress1. The damage

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of oxidative stress to plant is related to the excess production of reactive oxygen

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species (ROS)2. ROS can exert a series of physiological responses including changes

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in cellular structure and degradation of proteins3. The antioxidant enzymes strictly

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regulate the ROS production by ROS scavenging pathways. The major antioxidant

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enzymes of plants include superoxide dismutase (SOD), catalase (CAT), peroxidase

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(POD), glutathione reductase (GR), ascorbate peroxidase (APX) and

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dehydroascorbate reductase (DHAR) etc.4,5. All of them play critical roles in ROS

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removing which is directly with the relevant of defence against various abiotic stress

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of plant.

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Polysaccharides have been demonstrated to scavenge free radicals in vitro6-9. The

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antioxidant activity of polysaccharides depends on several structural parameters, such

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as the molecular weight6, type and position of substitute groups, such as acetyl7,

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sulfate and phosphate8,9. Chitosan and its derivatives exhibit pronounced antioxidant

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activity. Like other polysaccharides, the antioxidant activity of chitosan appears to be

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dependent on its molecular weight, substitution groups and so on. N-carboxymethyl

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oligosaccharides5, aminoethyl chitosan10, and low molecular weight chitosans11

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showed high ROS scavenging effects. Xing12et al. found that sulfated chitosan also

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had strong scavenging ability on free radicals. Thus, sulfated chitosan could be a

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potential type of antioxidants for reducing salt stress on plants. Our previous study on

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different molecular weight of chitooligosaccharide (COS), showed enhancing salt

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tolerance of wheat seedlings under 0.01% 1300 Da of COS. Therefore, the purpose of

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present study to investigate the effect of sulfated chitooligosaccharide (SCOS) and

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COS on wheat seedlings under salt stress. Furthermore, we evaluated the expression

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of some of salt-associated genes in wheat treated with SCOS.

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Materials and methods Preparation of SCOS. SCOS was obtained according to the method given by Xing

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et al.13. The weight average molecular weight (Mw) was measured using a high

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performance liquid chromatography (HPLC, Agilent Technologies, USA). A TSK

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G3000-PWXL column was utilized for chromatography. A 0.2 M CH3COOH/0.1 M

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CH3COONa aqueous solution was used as mobile phases at a flow velocity of 0.8

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ml/min. Fourier transform infrared (FT-IR) spectra of samples were detected in the

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range of 4000 to 400 cm−1 regions using a FT-IR spectrometer (Thermo Scientific

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Nicolet iS10, USA) in KBr discs. The sulfate content was measured by BaCl2-gelatin

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turbidity method.

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Plant material and treatments. The wheat (Triticumaestivum L.Jimai 22) seeds

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were surface sterilized with a 1% sodium hypochlorite solution. 10 min later they

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were thoroughly washed with deionized water. Then seeds were soaked in deionized

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water for 5 h and then transferred into a petri dish with moist gauze for germination at

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25°C for 24 h in the dark. Germinated seeds were sowed in petri dishes with nylon

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mesh and were grown in Hoagland solution in incubator. The day/night cycle was 14

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h/10 h, at 25°C/20°C, respectively. The relative humidity was 60% and the strength of

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illumination was 800 µmolm−2s−1. 10 d after sowing, wheat seedlings were transferred

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to Hoagland solution with 100 mMNaCl. Meanwhile, the wheat seedlings were

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divided into five groups, containing a control check (CK, neither SCOS nor NaCl)

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group, NaCl stressed as a negative control, salicylic acid (0.01% SA) treated as a

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positive control, a SCOS-NaCl stressed (treated with 0.01% SCOS) group and a

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COS-NaCl stressed (treated with 0.01% COS) group. The nutrient solution was

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renewed every other day. After 2 h, 5 d and 10 d of treatment, the growth situation,

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physiological indices and genes expression of salt tolerance of wheat seedlings in

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different group were determined.

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Growth parameters. After 10 d of NaCl treatment, wheat seedlings of each group

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were harvested for determination of shoot length, root length and wet weight; after

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which samples were dried at 105°C for 2 h to obtain dry weight.

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Lipid peroxidation degrees. Malondialdehyde (MDA) content indicates the degree

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of lipid peroxidation in plants. It was detected using a thiobarbituric acid (TBA)

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reaction14. After 2 h, 5 d and 10 d of NaCl treatment respectively, 0.5 g leaf samples

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were homogenized in 10% (w/v) trichloroacetic acid (TCA). Then the homogenates

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were centrifuged at 4000 g for 10 min. Afterwards, 2 ml of 0.6% TBA was added into

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2 ml supernatant, and the solution was heated in boiling water for 15 min and cooled

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immediately afterwards. Next, the solution was centrifuged at 10,000 g for 15 min.

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Then the absorbance was read at 450 nm, 532 nm and 600 nm, separately. The MDA

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content was calculated as µg MDA g-1 fresh weight (FW).

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Antioxidant enzyme activities. After 2 h, 5 d and10 d of NaCl treatment

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respectively, the second fully expanded leaves samples (0.5 g) were homogenized in

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liquid nitrogen and brought up to a volume of 5 ml by cold sodium phosphate buffer

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(pH 7.8). Then the solution was centrifuged at 12000 g at 4°C for 15 min, after which

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the enzyme activities were determined immediately.

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The total soluble protein was detected using the means of Bradford15. A total of 100

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µl supernatant and 5 ml of Coomassie brilliant blue G250 staining were mixed, and

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then the absorbance was reported at 595 nm. SOD activity was determined by the

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inhibition of the photoreduction of nitroblue tetrazolium (NBT)16. One unit of SOD

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was defined as the amount of enzyme corresponding to 50% inhibition of the NBT

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reduction. The decrease in the absorbance at 240 nm was used for definition of CAT

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activity. The CAT activity was calculated as H2O2 reduced mg-1 protein min-117. The

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means reported by Seckinet al.14 was used to determine POD activity. The POD

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activity was calculated on account of the rate of formation of guaiacol

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dehydrogenation and defined as µmol GDHP mg-1 protein min-1. The absorbance was

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read at 470 nm.

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APX was measured by the procedure of Nguyen18. The APX activity was

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calculated from the decline in absorbance at 290 nm. DHAR activity was estimated

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according to Mishra19 by assaying the decline of DHA at 265 nm. DHAR activity is

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defined as µmol DHA reduced mg-1 protein min-1. The activity of GR was determined

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by the means of Mandhania20. The decrease in absorbance was read at 340 nm. GR

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activity was defined as 0.1 µmol oxidized NADPH mg-1 protein min-1.

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Analysis of genes expression. Total RNA was extracted from wheat leaves (0.2 g)

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using PureLink® RNA Mini Kit (Life Technologies, USA). Total RNA was quantified

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by UV spectrophotometer. RevertAidTM First Strand cDNA Synthesis Kit (Takara,

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Dalian, China) was used for the synthesis of first-strand cDNA. qRT-PCR was

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performed in an Eppendorf Master cycler (Eppendorf, Hamburg, Germany) using the

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SYBR ExScript qRT-PCR Kit (Takara, Dalian, China) as reported by Li et al.21. The

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expression levels of genes were analysed using comparative threshold cycle method

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(2-∆∆Ct) with β-actin as the control. Specific primers for each gene were designed in

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Table.S1.

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Chlorophyll contents and fluorescence characters. After 2 h, 5 d and 10 d of

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NaCl treatment, chlorophyll a (Chl (a)), chlorophyll b (Chl (b)) and total chlorophyll

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(Chl (a+b)) content were measured with 95% ethanol22. Chlorophyll fluorescence

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parameters were detected utilizing a chlorophyll fluorescence system (PAM-2100,

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Walz, Germany). The chlorophyll fluorescence parameters were assayed after dark

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adaptation for 30 min.

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Statistical analysis. Each test was performed in triplicate, and the results were

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averaged. Data were subjected to ANOVA analysis by SPSS (version 19.0) and

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Duncan's test (P<0.05) to compare the mean value of different treatments. Each of

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the data points were expressed as the average ± SD of three independent replicates.

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Results and discussion Preparation of SCOS. The Mw of sulfated COS measured by HPLC is

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approximately 1600 Da (Fig.S1) and the sulfate content of SCOS is 48.5%. Fig.S2

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depicts the FT-IR spectrum of SCOS, due to the sulfo groups, characteristic

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absorptions at 1204 and 794 cm-1 were assigned to S=O and C–O–S bond stretching

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respectively. Because of the pyranose units in the polysaccharide, the peak at 934

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cm-1proved the cyclic pyranosyl rings were not destroyed by microwave radiation23.

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Plant growth and biomass accumulation. As shown in Table 1, shoot length, root

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length, wet weight and dry weight were significantly inhibited under 100 mM NaCl

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treatment (P