Effect of Sulfated Chitooligosaccharides on Wheat Seedlings (Triticum

Subscriber access provided by GAZI UNIV

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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.

Page 1 of 27

Journal of Agricultural and Food Chemistry

1

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

2

under Salt Stress

3

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

4

Pengcheng Lia*

5

a

6

b

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

7 8 9 10 11 12 13 14

*Corresponding author:

15

Pengcheng Li

16

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

17

E-mail addresses: [email protected].

18

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

19 20

Song Liu

21

E-mail addresses: [email protected].

22

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

ACS Paragon Plus Environment


23

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

24

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

25

stress. The antioxidant enzyme activities, chlorophyll contents and fluorescence

26

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

27

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

28

increase the chlorophyll contents and modulate fluorescence characters in wheat

29

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

30

antioxidant enzymes containing superoxide dismutase, catalase, peroxidase, ascorbate

31

peroxidase, glutathione reductase and dehydroascorbate reductase. Similarly, the

32

mRNA expression levels of several antioxidant enzymes were efficiently modulated

33

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

34

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

35

photochemical efficiency of wheat seedlings was associated with its enhanced

36

capacity for antioxidant enzymes which prevented structure degradation of the

37

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

38

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

39

sulfate group.

40

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

41

activities; photochemical efficiency; gene expression

42 43 44

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


Page 2 of 27

Page 3 of 27


45

Several physiological processes especially photosynthesis in plants are affected by

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

of plant.

57

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

58

antioxidant activity of polysaccharides depends on several structural parameters, such

59

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

60

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

61

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

62

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

63

oligosaccharides5, aminoethyl chitosan10, and low molecular weight chitosans11

64

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

65

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

66

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



67

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

68

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

69

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

70

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

71

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

72 73 74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

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

82

turbidity method.

83

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

84

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

85

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

86

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

87

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

88

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


Page 4 of 27

Page 5 of 27


89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

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

97

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

98

different group were determined.

99

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

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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

108

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

109

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

110

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



111

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

112

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

113

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

114

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

115

the enzyme activities were determined immediately.

116

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

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

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

126

read at 470 nm.

127

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

128

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

129

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

130

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

131

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

132

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


Page 6 of 27

Page 7 of 27


133

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

134

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

135

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

136

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

137

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

138

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

139

expression levels of genes were analysed using comparative threshold cycle method

140

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

141

Table.S1.

142

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

143

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

144

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

145

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

146

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

147

adaptation for 30 min.

148

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

149

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

150

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

151

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

152 153 154

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



155

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

156

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

157

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

158

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

159

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

160

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

161

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

162

treatment (P

Effect of Sulfated Chitooligosaccharides on Wheat Seedlings (Triticum

Recommend Documents