Relationship between the Degree of Polymerization of Chitooligomers

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The relationship between the degrees of polymerization of chitooligomers and its activity of affecting the growth of wheat seedlings under salt stress Xiaoqian Zhang, Kecheng Li, Song Liu, Ping Zou, Ronge Xing, Huahua Yu, Xiaolin Chen, Yukun Qin, and Pengcheng Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03665 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 25, 2016

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Journal of Agricultural and Food Chemistry

1

The relationship between the degrees of polymerization of

2

chitooligomers and its activity of affecting the growth of wheat seedlings

3

under salt stress

4

Xiaoqian Zhang†,‡, Kecheng Li*,†,§, Song Liu†, Ping Zouǁ, Ronge Xing†, Huahua

5

Yu†, Xiaolin Chen†, Yukun Qin†, Pengcheng Li*,†

6

†

7

Chinese Academy of Sciences, Qingdao 266071, China

8

‡

University of Chinese Academy of Sciences, Beijing 100049, China

9

§

Nantong Marine Science and Technology R&D center, IOCAS, Jiangsu

Key Laborotory Experimental Marine Biology, Institute of Oceanology,

10

226006, China

11

ǁ

Institute of Tobacco Research of CAAS, Qingdao 266101, China

12 13 14 15 16

*Corresponding author.

17

Pengcheng Li

18

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

19

E-mail addresses: [email protected].

20

Kecheng Li

21

Tel.: +86 532 82898641; fax: +86 532 82968780.

22

E-mail addresses: [email protected]. 1

ACS Paragon Plus Environment


23 24

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ABSTRACT Seven chitooligomers (COSs) with determined degrees of polymerization

25

(DPs)

(chitotetraose

to

chitooctaose,

DP

8-10,

26

heterogeneous COS with various DPs were firstly applied to explore the

27

relationship between the DP of COSs and its effect on growth of wheat

28

seedlings under salt stress. The results showed that COS could promote the

29

growth of wheat seedlings under salt stress. Moreover, chitohexaose,

30

chitoheptaose and chitooctaose exhibited stronger activity compared with

31

other COS samples, which suggested that its activity had a closely relationship

32

with its DP. After 10 days of treatment with chitohexaose, chitoheptaose and

33

chitooctaose, the photosynthetic parameters were improved obviously. The

34

soluble sugar and proline contents were improved by 26.7%-53.3% and

35

43.6.0%-70.2%, respectively, while the concentration of malondialdehyde

36

(MDA) was reduced by 36.8% - 49.6%. In addition, the antioxidant enzymes

37

activities were clearly activated. At molecular level, the results revealed that

38

they could obviously induce the expression of Na+/H+ antiporter genes.

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KEYWORDS ： Chitooligomers; Degrees of polymerization; Salt stress;

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Photosynthesis; Antioxidant enzyme activities; Gene expression

41

2


DP

10-12)

and

a

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INTRODUCTION

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Salt stress is one of the most serious abiotic stresses and it can lead to

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the reduction of agricultural productivity.1 High salt concentration makes it

45

more difficult for roots to absorb water and disturbs the homeostasis of cellular

46

ions resulting in osmotic stress, ion toxicity and generation of reactive oxygen

47

species (ROS). Plants had developed protective system to avert the adverse

48

effects resulted from salt stress. At the metabolism level, plant could sythesis

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compatible solutes to maintain cell turgor. Additionally, plant could also activate

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redox system to clear excessive ROS and keep the cellular redox balance. At

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the molecular level, salt stress could induce the gene expression of antioxidant

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enzymes, regulatory proteins and ion transporters to reduce the adverse

53

effects induced by salinity.2

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However, compared with the normal physiological conditions, salt stress

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could inhibit the photosynthesis, break metabolic balance and damage cellular

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structures, and ultimately results in the reduction of crop yield.3 Therefore it is

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vital to develop practical methods for improving the salt tolerance of plants.

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Except the soil improvement and conventional breeding, exogenous

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application of biostimulators is also an effective method to enhance the salt

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tolerance of plants and it has great practical perspectives on alleviating the salt

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tolerance of plants.

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Chitooligomers (COS) is partially depolymerized products of chitosan,

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which is consisted of D-glucosamine and N-acetyl-D-glucosamine.1 It has

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shown various functional properties (e.g. antitumor, antioxidant, antimicrobial, 3



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wound healing and immune-enhancing effects) that made it possible to apply

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to many fields including biomedicine and food.4, 5 In agriculture, COS has the

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ability to promote plants growth and induce plant innate immunity.6-8 In addition,

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it was reported that COS exhibited positive effects on salt stress alleviation

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both in safflower and sunflower.9 Recently, Zou and colleagues also reported

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that exogenous COS and its derivatives could enhance the plant growth under

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salt stress.10 The bioactivity of COS closely related to its DP. However, prior

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studies about the effect of COS on plant growth under salt stress were mostly

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performed using heterogeneous COS with various DPs and the ability of each

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individual COS remains unknown.

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In recent years, many researchers focused on the development of

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separation techniques of COS with single DP. A series of COSs with

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well-defined DP could be obtained and has drawn considerable attention. In

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order to determine the active ingredient and better understand biological action

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of COS, some studies were recently carried out with COS with single or narrow

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DP, such as antitumor activity,11 antibacterial activity5 and elicitor activity of

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plant defence.12 These studies showed that a DP of at least 4 is essential for

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COSs to induce biological responses. Our prior study also suggested that

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those COSs with DP > 4 exhibited a better bioactivity in promoting the growth

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of plants.13

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In order to clarify the function–structure relationship between the DP of

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COSs and its activity on affecting the growth of wheat seedlings under salt 4


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stress, this work was conducted using eight COSs with DP > 4, including

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seven COSs with determined DP (chitotetraose to chitooctaose, DP 8–10, DP

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10–12) and a heterogeneous COS with various DPs (mix). The DP effects of

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COSs on photosynthetic parameters, lipid peroxidation degrees, compatible

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solutes, antioxidant enzyme activities and gene expression of Na+/H+

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antiporters were investigated and the optimal DP was identified under salt

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stress. These results were fundamental to the study of action mechanism of

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COS on promoting plant growth under salt stress and the preparation of plant

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growth regulator.

96 97

MATERIALS AND METHODS

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Preparation of COS with different DPs. Seven COS samples with

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determined DP were prepared following the method described by Li et al.14, 15

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including five single COSs, chitotetraose (≥98%), chitopentaose (≥98%),

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chitohexaose (≥98%), chitoheptaose (≥93%), chitooctaose (≥90%), DP 8–10

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(12.0%, 53.1%, 28.0%) and DP 10–12 (18.4%, 49.4%, 22.3%), respectively.

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The COS mixture (DP2-12) was prepared by the method reported by

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Trombotto et al.16

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Plant material and treatments. Wheat (Triticum aestivum L. Jimai 22)

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seeds were used in our study. After germinated at 25℃ for 24 h in the dark,

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seeds were transplanted into Petri dishes (11.5 cm in diameter) with Hoagland

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solution in a light growth chamber with 25℃/20℃ and 14-h/10-h light/dark 5



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cycle settings. After the second leaf was fully developed, the wheat seedlings

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were divided into ten experimental groups randomly with three replicates each,

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including a control group (CK, without NaCl and sprayed with distilled water), a

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NaCl (100 mM) stressed group (sprayed with distilled water) as a negative

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control and eight NaCl (100 mM) stressed groups (sprayed with 50 mg/L

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chitotetraose, chitopentaose, chitohexaose, chitoheptaose, chitooctaose, DP

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8–10, DP 10–12 and mix, separately). The volume of COS solutions or

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deionized water sprayed on each sample was 45 mL. After 10 days treatment,

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the leaves of wheat seedlings were used to measure the physiological

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parameters and the relative expression level of Na+/H+ antiporter genes.

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Determination

of

chlorophyll

contents

and

photosynthetic

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characteristics. Chlorophyll contents were assayed with spectrophotometer

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at 665 nm and 649 nm according to the description by Zou et al.17 The second

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functional leaves were used to measure the photosynthetic characteristics.

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Intercellular CO2 concentration (Ci), transpiration rate (Tr),

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conductance (Gs) and photosynthetic rate (Pn) were measured by portable

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photosynthesis system (L.MAN-LCPro-SD, BioScientific Ltd., UK). Gas flow

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rate was set at 200 µmol s–1, photosynthetic photon flux density and CO2

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concentration were maintained at 800 µmol m−2s−1 and 395 ± 5 µmolmol−1,

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separately.

stomatal

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Lipid peroxidation degrees. Malondialdehyde (MDA) content was

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measured with thiobarbituric acid (TBA) reaction according to the method 6


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described by Heath and Packer.18 Samples (0.2g) was homogenized in 10%

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trichloroacetic acid (TCA) and centrifuged for 10 min at 4000 × g. And then 2

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mL aliquot of extract was mixed with the same volume of 0.6% TBA and then

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bathed in boiling water for 30 min. Next, the cooled reaction liquid was

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centrifuged and then used to determine the MDA content.

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Soluble sugar and proline contents. The anthrone method was used to

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measure the content of soluble sugar.19 3 mL reaction mixture includng 2.1 mM

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anthrone, 1.09 mM thiourea and 1.08 M H2SO4 was mixed with leaf extract and

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then bathed in boiling water for 10 min. Content of soluble sugar was

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quantitatively estimated at an absorbance of 620 nm. Free proline content was

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measured with the ninhydrin acid reagent method according to Bates20.

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L-proline was used as a standard to make a calibration curve.

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Antioxidant enzyme activities. The second functional leaves (0.2g)

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were ground in liquid nitrogen and used to extract crude enzyme. Superoxide

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dismutase (SOD) activity was assayed according to the method described by

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Beauchamp et al with some appropriate modifications.21 After 30 µL of enzyme

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extract was added, the reaction mixture was exposed to the light (4000 lx) for

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15 min to start the reaction. SOD activity was measured in the absorbance of

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560 nm. Catalase (CAT) activity in leaf samples was measured by the method

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reported by Lu et al.22 The CAT activity was measured at 240 nm with a UV

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spectrophotometer. The determination of peroxidase (POD) activity was

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followed by the method used by Seckin et al.23 The absorbance values at 470 7



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nm were read to determine the POD activities.

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Quantitative real-time PCR (qRT-PCR) analysis. Total RNA was

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extracted using RNAprep Pure Plant Kit (TIANGEN, China). After that the total

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RNA was used as template to synthesis complementlary DNA (cDNA) by

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PrimeScriptTM RT reagent Kit (Takara, Dalian, China). Then, RT-PCR was

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performed with SYBR® Premix Ex TaqTM (Tli RNaseH Plus) (Takara, Dalian,

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China) according to the manufacturer’s instructions and each test was

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performed in triplicate. 2-△△CT method was used to quantification. β-actin was

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selected as an internal control in each experiment. Table 1 listed the

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sequences of all gene-specific primers.

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Statistical analysis. Each experimental value represent the average ±

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standard deviations (SD) of three biological samples. Statistical analyses of

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the data of physiological characteristics were performed using ANOVA

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analysis and Duncan’s multiple range tests (P< 0.05) by SPSS (version 19.0).

167 168

RESULTS AND DISCUSSION

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DP effects of COS on chlorophyll contents and photosynthetic

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parameters. As the material basis of photosynthesis, chlorophyll played a

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leading role in photosynthesis24. As shown in Table 2, compared with the

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control group (CK), salt stress lead to a decrease (P < 0.05) of chlorophyll

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contents. However, the chlorophyll contents were increased at different

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degrees in the plants treated with COSs with different DPs (chitotetraose to 8


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chitooctaose, DP 8–10, DP 10–12 and mix) under salt stress. Our study

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showed that chitohexaose, chitoheptaose, chitooctaose could improve the

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Chl-a contents obviously. In addition, Chl-b contents of COS treatments

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increased in the order of chitohexaose, chitoheptaose, chitooctaose > DP 8-10,

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mix > chitotetraose, chitopentaose, DP 10-12. Consequently, chitohexaose,

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chitoheptaose and chitooctaose increased the total chlorophyll contents

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by14.9%-19.2%, which was more effective than other single or narrow COSs

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(chitotetraose, chitopentaose, DP 8–10, DP 10–12).

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Photosynthesis is one of the most important metabolic pathway in plants

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and it had a closely relationship with the plant growth under salt stress.25 The

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effects of eight COSs on photosynthetic parameters of wheat seedlings were

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showed in Table 3. The value of Gs was dramatically decreased responsed to

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salt stress and it’s undoubtedly resulted from the osmotic effect of the Na+.3

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Present results suggested that the imposition of salt stress reduced the value

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of Pn, Tr, Gs and Ci of wheat seedlings apparently (P4.5 In addition, the study performed by Yamada et al. revealed that

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N-acetyl COSs larger than hexaose were more effective than biose and triose

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in the activity of inducing the formation of phytoalexins in suspension-cultured

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rice cells.46 Most of these studies suggested that chitohexaose, chitoheptaose

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and chitooctaose seemed to possess more biological activity, which were in

357

agreement with our findings about the function-structure relationship between

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DP of COS and its effect on plant growth under salt stress. The application of

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COS with different DPs in wheat seedlings under salt stress could increase the

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contents of chlorophyll and proline, decrease the MDA concentration, enhance

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the photosynthesis, activate antioxidant enzymes activities, and induce the

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expression of salt-related genes. Furthermore, it could also be found that a

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significantly difference of elicitor activity existed in COS samples (chitotetraose,

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to chitooctaose, DP 8–10, DP 10–12 and mix) at metabolism and gene

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expression level. Chitohexaose, chitoheptaose and chitooctaose generally had

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better activity in reducing the adverse effect of salt stress and further enhance

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the plant growth. In addition, the mix exhibited an intermediate effect among all

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COS samples, which may result from its heterogeneous components that

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contained both high-activity and low-activity single COS.

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The observed activity difference among eight COSs tested in this study

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may be explained by the specific plasma membrane receptors. It had been

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reported that the recognition of chitin oligosaccharide elicitor were mediated by 17



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a plasma membrane receptor (CEBiP) in the rice cells.47 In Arabidopsis, as a

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CEBiP homolog, chitin elicitor receptor kinase 1 (AtCERK1) had been

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established as a chitin receptor. Chitin binding induced phosphorylation of the

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intracellular kinase domain of AtCERK1 and started a series of defense

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reaction, including Cl- and K+ efflux, cytoplasmic acidification, synthesis of

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jasmonic acid, burst out of ROS and expression of some specific responsive

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genes.48,49 Moreover, COSs with different DPs had a different ability in binding

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the

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N-acetyl-D-glucosamine (NAG) could act as an optimal ligand to initiate the

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cross-linking of AtCERK1-ECD, and then deliver the signaling induced by

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chitin. Moreover, (NAG)7 and (NAG)8 were more effective than other chitin

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oligomers tested.50 Thus chitohexaose, chitoheptaose and chitooctaose may

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have a more suitable size for recognizing and activating the plasma membrane

386

receptor, and further trigger the physiological and biochemical reaction

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downstream and relieve the salt stress of plant as is found in our results.

388

However, the exact mechanisms still need to be further studied.

receptors.

Liu

et

al.

further

found

that

the

heptamer

of

389 390

AUTHOR INFORMATION

391

*Corresponding author.

392

*(P. L.) Phone: +86 532 82898707. Fax: +86 532 82968951. Email:

393

[email protected].

394

*(K. L.) Phone: +86 532 82898641. Fax: +86 532 82968780. Email: 18


Page 19 of 36


395

[email protected].

396

Funding

397

The study was supported by the National Natural Science Foundation of

398

China

(No.

41406086),

the

Commonweal Item

of

State

399

Oceanic Administration of China (No. 201305016-2, 201405038-2) and

400

Nantong Applied Basic Research Projects (MS12015124).

401

Notes

402

The authors declare no competing financial interest.

403 404

References

405

(1) Ruiz-Lozano, J. M.; Porcel, R.; Azcon, C.; Aroca, R. Regulation by

406

arbuscular mycorrhizae of the integrated physiological response to salinity in

407

plants: new challenges in physiological and molecular studies. J. Exp. Bot.

408

2012, 63, 4033-4044.

409

(2) Krasensky, J.; Jonak, C. Drought, salt, and temperature stress-induced

410

metabolic rearrangements and regulatory networks. J. Exp. Bot. 2012, 63,

411

1593-1608.

412

(3) Munns, R.; Tester, M. Mechanisms of salinity tolerance. Annu. Rev. Plant

413

Biol. 2008, 59, 651-681.

414

(4) Kim, S.; Rajapakse, N. Enzymatic production and biological activities of

415

chitosan oligosaccharides (COS): A review. Carbohyd. Polym. 2005, 62,

416

357-368. 19



417

(5) Li, K.; Xing, R.; Liu, S.; Qin, Y.; Yu, H.; Li, P. Size and pH effects of

418

chitooligomers on antibacterial activity against Staphylococcus aureus. Int. J.

419

Biol. Macromol. 2014, 64, 302-305.

420

(6) Nguyen, A. D.; Vo, T. P. K.; Tran, T. D. Research on impact of chitosan

421

oligomers on biophysical characteristics, growth, development and drought

422

resistance of coffee. Carbohyd. Polym. 2011, 84, 751-755.

423

(7) Nge, K. L.; Nwe, N.; Chandrkrachang, S.; Stevens, W. F. Chitosan as a

424

growth stimulator in orchid tissue culture. Plant Sci. 2006, 170, 1185-1190.

425

(8) Chatelain, P. G.; Pintado, M. E.; Vasconcelos, M. W. Evaluation of

426

chitooligosaccharide application on mineral accumulation and plant growth in

427

Phaseolus vulgari. Plant Sci. 2014, 215, 134–140.

428

(9) Jabeen, N.; Ahmad, R. The activity of antioxidant enzymes in response to

429

salt stress in safflower (Carthamus tinctorius L.) and sunflower (Helianthus

430

annuus L.) seedlings raised from seed treated with chitosan. J. Science Food

431

Agr. 2013, 93, 1699-1705.

432

(10) Zou, P.; Li, K. C.; Liu, S.; He, X. F.; Zhang, X. Q.; Xing, R. E.; Li, P. C.

433

Effect of Sulfated Chitooligosaccharides on Wheat Seedlings (Triticum

434

aestivum L.) under Salt Stress. J. Agr. Food Chem. 2016, 64, 2815-2821.

435

(11) Xiong, C. N.; Wu, H. G.; Wei, P.; Pan, M.; Tuo, Y. Q.; Kusakabe, I.; Du, Y.

436

G. Potent angiogenic inhibition effects of deacetylated chitohexaose separated

437

from chitooligosaccharides and its mechanism of action in vitro. Carbohyd.

438

Res. 2009, 344, 1975-1983. 20


Page 20 of 36

Page 21 of 36


439

(12) Yamada, A.; Shibuya, N.; Kodama, O.; Akatsuka, T. Induction of

440

Phytoalexin

441

N-Acetyl-chitooligosaccharides. Biosci. Biotech. Bioch. 2014, 57, 405-409.

442

(13) Zhang, X. Q.; Li, K. C.; Liu, S.; Xing, R. G.; Yu, H. H.; Chen, X. L.; Li, P. C.

443

Size effects of chitooligomers on the growth and photosynthetic characteristics

444

of wheat seedlings. Carbohyd. Polym. 2016, 138, 27-33.

445

(14) Li, K. C.; Xing, R. E.; Liu, S.; Qin, Y. K.; Li, B.; Wang, X. Q.; Li, P. C.

446

Separation and scavenging superoxide radical activity of chitooligomers with

447

degree of polymerization 6-16. Int. J. Biol. Macromol. 2012, 51, 826-830.

448

(15) Li, K. C.; Liu, S.; Xing, R. G.; Yu, H. H.; Qin, Y. K.; Li, R. F.; Li, P. C.

449

High-resolution separation of homogeneous chitooligomers series from 2-mers

450

to 7-mers by ion-exchange chromatography. J. Sep. Sci. 2013, 36, 1275-1282.

451

(16) Trombotto, S.; Ladaviere, C.; Delolme, F.; Domard, A. Chemical

452

preparation and structural characterization of a homogeneous series of

453

chitin/chitosan oligomers. Biomacromolecules 2008, 9, 1731-1738.

454

(17) Zou, P.; Li, K. C.; Liu, S.; Xing, R. G.; Qin, Y. K.; Yu, H. H.; Zhou, M. M.; Li,

455

P. C. Effect of chitooligosaccharides with different degrees of acetylation on

456

wheat seedlings under salt stress. Carbohyd. Polym. 2015, 126, 62-69.

457

(18) Heath, R. L.; Packer, L. Photoperoxidation in isolated chloroplasts: I.

458

Kinetics and stoichiometry of fatty acid peroxidation. Arch. biochem.

459

biophys. 1968, 125,189-198.

460

(19) Sanchez, F. J.; Manzanares, M.; de Andres, E. F.; Tenorio, J. L.; Ayerbe, L.

Formation

in

Suspension-cultured

21


Rice

Cells

by


461

Turgor maintenance, osmotic adjustment and soluble sugar and proline

462

accumulation in 49 pea cultivars in response to water stress. Field Crops Res.

463

1998, 59, 225-235.

464

(20) Bates, l. S. Rapid determination of free proline content for water-stress

465

studies. Plant Soil 1973, 39, 205–207.

466

(21) Beauchamp, C.; Fridovich, I. Superoxide dismutase: improved assays and

467

an assay applicable to acrylamide gels. Anal. biochem. 1971, 44, 276-287.

468

(22) Lu, Z. Q.; Liu, D. L.; Liu, S. K. Two rice cytosolic ascorbate peroxidases

469

differentially improve salt tolerance in transgenic Arabidopsis. Plant Cell Rep.

470

2007, 26, 1909-1917.

471

(23) Seckin, B.; Sekmen, A. H.; Turkan, I. An Enhancing Effect of Exogenous

472

Mannitol on the Antioxidant Enzyme Activities in Roots of Wheat Under Salt

473

Stress. J. Plant Growth Reg. 2009, 28, 12-20.

474

(24) Zeng, D.; Luo, X. Physiological effects of chitosan coating on wheat

475

growth and activities of protective enzyme with drought tolerance. Open J. Soil

476

Sci. 2012, 2, 282-288.

477

(25) Arfan, M.; Athar, H. R.; Ashraf, M. Does exogenous application of salicylic

478

acid through the rooting medium modulate growth and photosynthetic capacity

479

in two differently adapted spring wheat cultivars under salt stress? J. Plant

480

Physiol. 2007, 164, 685-694.

481

(26) Powles, S. B. Photoinhibition of photosynthesis induced by visible light.

482

Annu. Rev. Plant Physiol. 1984, 35, 15-44. 22


Page 22 of 36

Page 23 of 36


483

(27) Munns, R. Comparative physiology of salt and water stress. Plant cell

484

environ. 2002, 25, 239-250.

485

(28) Gunes, A.; Inal, A.; Alpaslan, M.; Eraslan, F.; Bagci, E. G.; Cicek, N.

486

Salicylic

487

symptomatic for oxidative stress and mineral nutrition in maize (Zea mays L.)

488

grown under salinity. J. Plant Physiol. 2007, 164, 728-736.

489

(29) Meloni, D. A.; Oliva, M. A.; Martinez, C. A.; Cambraia, J. Photosynthesis

490

and activity of superoxide dismutase, peroxidase and glutathione reductase in

491

cotton under salt stress. Environ. Exp. Bot. 2003, 49, 69-76.

492

(30) Bor, M.; Özdemir, F.; Türkan, I. The effect of salt stress on lipid

493

peroxidation and antioxidants in leaves of sugar beet Beta vulgaris L. and wild

494

beet Beta maritima L. Plant Sci. 2003, 164, 77-84.

495

(31) Fridovich, I. Biological effects of the superoxide radical. Arch. Biochem.

496

Biophys. 1986, 247, 1-11.

497

(32) Misra, N.; Saxena, P. Effect of salicylic acid on proline metabolism in lentil

498

grown under salinity stress. Plant Sci. 2009, 177, 181-189.

499

(33) Szabados, L.; Savoure, A. Proline: a multifunctional amino acid. Trends

500

plant sci. 2010, 15, 89-97.

501

(34) Khatkar, D.; Kuhad, M. S. Short-term salinity induced changes in two

502

wheat cultivars at different growth stages. Bio. Plantarum 2000, 43, 629-632.

503

(35) Ma, L. J.; Li, Y. Y.; Yu, C. M.; Wang, Y.; Li, X. M.; Li, N.; Chen, Q.; Bu, N.

504

Alleviation of exogenous oligochitosan on wheat seedlings growth under salt

acid

induced

changes

on

some

physiological

23


parameters


505

stress. Protoplasma 2012, 249, 393-399.

506

(36) Foyer, C. H.; Noctor, G. Oxidant and antioxidant signalling in plants: a

507

re-evaluation of the concept of oxidative stress in a physiological context. Plant

508

Cell Environ. 2005, 28, 1056-1071.

509

(37) Sudhakar, C.; Lakshmi, A.; Giridarakumar, S. Changes in the antioxidant

510

enzyme efficacy in two high yielding genotypes of mulberry (Morus alba L.)

511

under NaCl salinity. Plant Sci. 2001, 161, 613-619.

512

(38) Tomida, H.; Fujii, T.; Furutani, N.; Michihara, A.; Yasufuku, T.; Akasaki, K.;

513

Maruyama, T.; Otagiri, M.; Gebicki, J. M.; Anraku, M. Antioxidant properties of

514

some different molecular weight chitosans. Carbohyd. Res. 2009, 344,

515

1690-1696.

516

(39) Apse, M. P.; Aharon, G. S. Snedden, W. A.; Blumwald, E. Salt tolerance

517

conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis.

518

Science 1999, 285, 1256-1258.

519

(40)

520

Regulation of SOS1, a plasma membrane Na+/H+ exchanger in Arabidopsis

521

thaliana, by SOS2 and SOS3. Proc. Natl. Acad. Sci. U. S. A. 2002, 99,

522

8436-8441.

523

(41) Fukuda, A.; Nakamura, A.; Hara, N.; Toki, S.; Tanaka, Y. Molecular and

524

functional analyses of rice NHX-type Na+/H+ antiporter genes. Planta 2011,

525

233, 175-188.

526

(42) Shi, H.; Ishitani, M.; Kim, C.; Zhu, J. K. The Arabidopsis thaliana salt

Qiu, Q. S.; Guo, Y.; Dietrich, M. A.; Schumaker, K. S.; Zhu, J. K.

24


Page 24 of 36

Page 25 of 36


527

tolerance gene SOS1 encodes a putative Na+/H+ antiporter. Proc. Natl. Acad.

528

Sci. USA 2000, 97, 6896-6901.

529

(43) Yokoi, S.; Quintero, F. J.; Cubero, B.; Ruiz, M. T.; Bressan, R. A.;

530

Hasegawa, P.M.; Pardo, J. M. Differential expression and function of

531

Arabidopsis thaliana NHX Na+/H+ antiporters in the salt stressresponse. Plant J.

532

2002, 30, 529-539.

533

(44) Zhu, J. K. Regulation of ion homeostasis under salt stress. Curr. Opin.

534

Plant Biol. 2003, 6, 441-445.

535

(45) Wei, X. L.; Wang, Y. F.; Zhu, Q.; Xiao, J. B.; Xia, W. S. Effects of chitosan

536

pentamer and chitosan hexamer in vivo and in vitro on gene expression and

537

secretion of cytokines. Food Agr. Immunol. 2009, 20, 269-280.

538

(46) Yamada, A.; Shibuya, N.; Kodama, O.; Akatsuka, T. Induction of

539

Phytoalexin

540

N-Acetyl-chitooligosaccharides. Biosci. Biotech. Biochem. 1993, 57, 405-409.

541

(47) Kaku, H.; Nishizawa, Y.; Ishii-Minami, N.; Akimoto-Tomiyama, C.; Dohmae,

542

N.; Takio, K.; Minami, E.; Shibuya, N. Plant cells recognize chitin fragments for

543

defense signaling through a plasma membrane receptor. Proc. Natl. Acad. Sci.

544

USA 2006, 103, 11086-11091.

545

(48) Petutschnig, E. K.; Jones, A. M. E.; Serazetdinova, L.; Lipka, U.; Lipka, V.

546

The Lysin Motif Receptor-like Kinase (LysM-RLK) CERK1 is a major

547

chitin-binding protein in Arabidopsis thaliana and subject to chitin-induced

548

phosphorylation. J. Biol. Chem. 2010, 285, 28902-28911.

Formation

in

Suspension-cultured

25


Rice

Cells

by


549

(49) Shibuya, N.; Minami, E. Oligosaccharide signalling for defence responses

550

in plant. Physiol. Mol.Plant P. 2001, 59, 223-233.

551

(50) Liu, T. T.; Liu, Z. X.; Song, C. J.; Hu, Y. F.; Han, Z. F.; She, J.; Fan, F. F.;

552

Wang, J. W.; Jin, C. W.; Chang, J. B.; Zhou, J. M.; Chai, J. J. Chitin-induced

553

dimerization activates a plant immune receptor. Science 2012, 336,

554

1160-1164.

555 556

26


Page 26 of 36

Page 27 of 36


557

Figure 1. DP effects of COS on MDA content in leaves of Jimai-22 under salt

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stress. Values are the mean ± SD of three replicates. Different letters indicate

559

significant differences at P < 0.05.

560

Figure 2. DP effects of COS on soluble sugar contents in leaves of Jimai-22

561

under salt stress. Values are the mean ± SD of three replicates. Different

562

letters indicate significant differences at P < 0.05.

563

Figure 3. DP effects of COS on proline contents in leaves of Jimai-22 under

564

salt stress. Values are the mean ± SD of three replicates. Different letters

565

indicate significant differences at P < 0.05.

566

Figure 4. DP effects of COS on SOD (a), POD (b) and CAT (c) activities in

567

leaves of Jimai-22 under salt stress. Values are the mean ± SD of three

568

replicates. Different letters indicate significant differences at P < 0.05.

569

Figure 5. DP effects of COS on the relative transcript level of SOS1 (a) and

570

NHX2 (b) gene in leaves of Jimai-22 under salt stress. Values are the mean ±

571

SD of three replicates. Different letters indicate significant differences at P

Relationship between the Degree of Polymerization of Chitooligomers

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