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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

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

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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

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chitooctaose, the photosynthetic parameters were improved obviously. The

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

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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

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

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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

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

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

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

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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

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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).

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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

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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

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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.

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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

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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

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