Effect of Exogenous Nitro Oxide on Chilling Tolerance, Polyamine

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Effect of exogenous nitro oxide on chilling tolerance, polyamine, proline and gamma-aminobutyric acid and in bamboo shoots (Phyllostachys praecox f. prevernalis) Di Wang, Li Li, Yanqun Xu, Jarukitt Limwachiranon, Dong Li, Zhaojun Ban, and Zisheng Luo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02091 • Publication Date (Web): 24 Jun 2017 Downloaded from http://pubs.acs.org on June 26, 2017

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Journal of Agricultural and Food Chemistry

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Effect of exogenous nitro oxide on chilling tolerance,

2

polyamine, proline and gamma-aminobutyric acid and in

3

bamboo shoots (Phyllostachys praecox f. prevernalis)

4

Di Wang1, Li Li1, Yanqun Xu1, Jarukitt Limwachiranon1, Dong Li1, Zhaojun Ban2,

5

Zisheng Luo1*

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1. Zhejiang University, College of Biosystems Engineering and Food Science, Key

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Laboratory for Agro-Products Postharvest Handling of Ministry of Agriculture,

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Zhejiang Key Laboratory for Agri-Food Processing, Hangzhou, 310058, People’s

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Republic of China

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2. School of Biological and chemical Engineering / School of Light Industry, Zhejiang

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University of Science and Technology, Hangzhou 310023, China, People’s Republic

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of China

13 14

*Corresponding author

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Zisheng Luo

16

College of Biosystems Engineering and Food Science

17

Zhejiang University

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Hangzhou 310058, People’s Republic of China

19

E-mail: [email protected]

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Phone: +86-571-88982175

ACS Paragon Plus Environment


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ABSTRACT: The effects of exogenous nitro oxide (NO) on chilling resistance and

22

the metabolism of polyamine, proline and gamma-aminobutyric acid of bamboo

23

shoots were investigated. Bamboo shoots were dipped in 0.07 mM sodium

24

nitroprusside (SNP) and stored at 1 °C for 56 d. During the storage, the development

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of chilling injury of SNP treated bamboo shoots was inhibited with decreased

26

accumulation of malonaldehyde and electrical leakage. At the end of storage, the

27

chilling injury incidence of treated bamboo shoots decreased by 37.9% while their

28

malonaldehyde content and electrical leakage were 8.8% and 18.6% lower than that of

29

control, respectively. Interestingly, the endogenous NO, polyamines, γ-aminobutyric

30

acid and proline contents of treated bamboo shoot also significantly increased.

31

Consistently, the metabolisms of these nitrogenous compounds were stimulated in

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treated bamboo shoots, according to their higher (20.2%-49.8%) related enzyme

33

activities, including nitric oxide synthase, arginine decarboxylase, ornithine

34

decarboxylase,

35

△1-pyrroline-5-carboxylate synthetase. The results indicated that the SNP treatment

36

enhanced chilling tolerance of bamboo shoots, which might associate with the

37

activated metabolism of polyamines, γ-aminobutyric acid and proline. SNP treatment

38

might be an alternative technology to avoid chill injury during cold storage of bamboo

39

shoots.

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KEYWORDS: nitro oxide, bamboo shoot, chilling injury, polyamine, proline, GABA

glutamate

decarboxylase,

orn-δ-aminotransferase

41


and

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INTRODUCTION

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In recent years, bamboo shoots had received lots of consumer’s attention owing to

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their high nutrition, rich in protein and fiber with low fat and sugar content.

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Nevertheless, freshly harvested bamboo shoots stored at room temperature were

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easily lignified and infected by bacteria.1 Low temperature was reported to effectively

47

delay the lignification in bamboo shoot, resulting in prolonged shelf life.2 However,

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the chilling injury (CI), manifesting as flesh browning, tissue lignifications and

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water-soaked spots, is a major cause of quality loss in bamboo shoots stored at

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unsuitable low temperature.3 Several previous research have found that those chilling

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injury of bamboo shoots could be significantly reduced by the application of

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exogenous 1-methylcyclopropene4 or salicylic acid,5 as well as heat treatment before

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cold storage.6

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Nitro oxide (NO), as an important intracellular signal molecule, had been reported

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to involve in numerous physiological processes in plants, not only the growth and

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development, as well as the abiotic stress response.7 The application of exogenous NO

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has been reported to alleviate or retard the chilling injury in cold stored fruits and

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vegetables, such as Japanese plums,8 mango,9 cucumber10 and banana fruit.11 Besides,

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Zhu et al.12 found that the process of ripening and softening caused by intermittent

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warming to reduce the chilling injury in peach fruit was delayed by NO fumigation.

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The reduced the chilling injury in NO treated kiwifruit was attributed to the inhibited

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of ethylene biosynthesis and respiration by recent studies.8, 9 It also had been proved

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that NO treatment can protect the fruit cell membranes against oxidative stress, which



64

might associate with the NO-induced activation of antioxidant enzymes.13

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Polyamines (PAs), including putrescine (Put), spermidine (Spd) and spermine

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(Spm), play important roles in stress tolerance of plants.14 It had been reported that the

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cell osmolarity of oat, rice and maize could be regulated by PAs due to its

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polycationic nature in physiological pH values.14 Actually, studies had found that the

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increase of PAs level is always accompanied by the enhanced chilling tolerance in

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horticultural products, such as the increasing of Put content in cold stored peppers,15

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banana fruit16 and tomato fruit.17 Additionally, a cold-stress triggered accumulation of

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Put content was found both in pepper15 and red chicory.18 These bioactive PAs are

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synthesized from arginine and ornithine in plant cell, catalyzed by arginine

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decarboxylase (ADC) and ornithine decarboxylase (ODC).19 Among their metabolic

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process in plants, diamine oxidase (DAO) and polyamine oxidase (PAO) as catabolic

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enzymes accounts for oxidization of PAs.14 According to Wang et al.,16 the PAs

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induced by exogenous NO might attribute to the enhancement of ADC and ODC

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activities in cold stored banana.

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The accumulation of proline and γ-Aminobutyric acid (GABA) in plants was also

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believed to relate with the tolerance to environmental stress.20 Proline was generally

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founded to accumulate rapidly in crops in response to various abiotic stress, for

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instance, draught, high salinity, heavy metals and chilling.21 It has been proved that

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proline is involved in regulating the osmotic balance, maintaining stability of the

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structures of subcellular organelles, membrane and protain.22 A key enzyme involve in

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the biosynthesis is △1-pyrroline-5-carboxylate synthetase (P5CS), while the proline


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dehydrogenase (PDH) is important to the catabolism of proline.23 Alternatively,

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proline

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ornithine-delta-aminotransfer (OAT).24 The increase of GABA content was found to

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contribute to the reduced CI development in peach fruit and loquat fruit.25, 26 GABA is

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synthesized from α-decarboxylation of L-glutamic acid in a reaction catalyzed by

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glutamate decarboxylase (GAD) in plants.27 GABA is metabolic transformed via

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polyamine decomposing pathway, where the first step was catalyzed by DAO.28

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GABA is also converted to succinyl semialdehyde by GABA transaminase (GABA-T)

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and then converted into succinic acid, which involved in the following tricarboxylic

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acid cycle.28 According to the study of Wang et al.,16 exogenous NO could efficiently

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increase endogenous GABA and proline contents as well as result in reduced CI in

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banana.

can

also

be

synthesized

from

ornithine

via

the

catalysis

of

98

However, little information is available about the effect of exogenous NO on the

99

chilling tolerance in bamboo shoots and the involvement of polyamine, proline,

100

gamma-aminobutyric acid during the cold-stress response. Therefore, in the present

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study, the alternations of the metabolic actives of CI, PAs, GABA and proline of

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bamboo shoots with exogenous NO treatments were investigated, with an attempt to

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explore the their attribution to the crop chilling tolerance during cold storage.

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MATERIALS AND METHODS

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Bamboo shoot, treatment and storage. The bamboo shoots (Phyllostachys

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praecox f. prevernalis.) were got from a plantation in Linan, Zhejiang Province of

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China. The bamboo shoots were transferred to the laboratory in 1 h after harvested in



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fiberboard cartons package. The shoots of uniform size and free from blemishes were

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selected for the experiment.

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In preliminary experiments, the concentrations of sodium nitroprusside (SNP, the

111

dornor of NO) were 0.03, 0.05, 0.07 and 0.09 mM in which 0.07 mM SNP was

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selected for this experiment according to the chilling injury. In this experiment, the

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first group was fumigated with 0.07 mM of SNP for 30 min at 20 °C. Distilled water

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was used under the same condition as control. Every three bamboo shoots were put on

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one plastic tray and wrapped with polyethylene films. The bamboo shoots were stored

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in incubator (SANYO MIR-254, Panasonic Co., Ltd., Japan) at 1 °C with 85-90%

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relative humidity for 56 d in dark. Physiological parameters were measured every 14

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d. There were fifteen trays for each treatment, and each treatment was conducted

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independently for three biological replications.

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Chilling injury evaluation. The chilling injury symptoms representing by

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browning and water-soaked appearance on the flesh were visually checked for 30

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bamboo shoots at each time point, and the chilling injury incidence of every replicate

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were calculated by percentage.

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Determination of electrical leakage and MDA content. Electrical leakage was

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determined by the method of Li et al.29 with modifications. Twenty pieces of bamboo

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shoots slices were kept in distilled water for 30 min at 25 °C. Then, the conductance

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ratio of the extraction was determined under the condition of 25 °C (L0) and of 100 °C

128

(L1) for 30 min using conductance ratio meter. The cell membrane permeability was

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represented by the ratio of L0 to L1.


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Frozen samples (2.0 g) were weighed for MDA content assay according to the

131

method of Li et al. with modifications.29 The samples were homogenized with 8 mL

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of 0.05 M phosphate buffer (pH 7.8) and centrifuged at 4 °C for 20 min at 12,000 × g.

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The 1mL of supernatant was mixed with 3 mL of thiobarbituric acid (TBA) and then

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boiled for 15 min and centrifuged again. The absorbance at 600 nm of the supernatant

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was measured and MDA content was expressed as nanomole per gram FW.

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Determination of endo-NO content. The endo-NO content was measured

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according to the method of Zhang et al.30 with slight modifications. The samples (2.0

138

g) were homogenized with 5mL of 100 mM sodium phosphate buffer (pH 7.4) in a

139

homogenizer and centrifuged. The supernatant was collected for endo-NO assay.

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Nitric Oxide Assay Kit (Nanjing Jiancheng Biological Engineering Co., Ltd.) was

141

employed for the detection of NO content according to the manufacturer’s manual.

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The absorbance was measured at 550 nm and distilled water was used as the control.

143

The standard curve of nitrate was compared to quantify the NO content and the results

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were expressed as nanomole per gram FW.

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Determination of nitric oxide sythetase (NOS) activity. NOS activity was

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determined by the method of Zhang et al.30 with slight modifications. Two gram of

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samples were homogenized with 5mL of 100 mM sodium phosphate buffer (pH 7.4,

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containing 1mM EDTA, 10 mM EGTA, 1 mM leupeptin, 1 mM PMSF and 1% PVP)

149

and centrifuged at 4 °C for 20 min at 12,000 × g. The supernatant was collected for

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enzyme assay. Total Nitric Oxide Synthase Assay Kit (Nanjing Jiancheng Biological

151

Engineering Co., Ltd.) was used for analysis of NOS activity following the



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introduction of the manufacturer. The absorbance was measured at 530 nm. One unit

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of NOS activity was defined as the amount of 1 nmol NO formation per min. The

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results were expressed as unit per gram FW.

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Determination of PAs content. According to Zhang et al.,31 PAs content was

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detected by HPLC system (Shimadzu, Japan), equipped with a photo-diode array

157

detector (Shimadzu). Samples (2 g) were grinded in liquid nitrogen and incubated

158

with 1 mL of 5% perchloric acid (PCA) for 60 min. The extract was centrifuged for

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20 min at 12,000 × g at 4 °C and the supernatant was used for the PAs determination.

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Two milliliter extraction was incubated with 1 mL of NaOH (2 M) and 10 µL of

161

benzoyl chloride, for 30min, at 30 °C. The 2 mL of saturation NaCl was added to the

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mixture after that 2 mL of ether was added for extraction. The mix was centrifuged

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again for 5 min at 5,000 × g at 4 °C. Then 1 mL ether fraction was collected and dried

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with hot air. The residue was dissolved in 1 mL of methanol and the target compounds

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were seperated by a Kromasil HPLC C18 column (250 mm × 4.6 mm, 0.45 µm

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particle sizes, Phenomenex, Torrance, CA) using methanol and water at a flow rate of

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0.8 mL/min at 30 °C. The injection volume was 20 µL. The absorbance at 254 nm was

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measured. External standard method was used for the quantification analysis and the

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results were expressed as nanomole per gram FW.

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Determination of ADC, ODC, DAO and PAO activities. ADC and ODC

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activities were determined according to the method of Hu et al.32 with slight

172

modifications. Frozen samples (2.0 g) were homogenized with 5 mL of 100 mM

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sodium phosphate buffer (pH 8.0, containing 0.1 mM PMSF, 1 mM pyridoxal


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phosphate, 5 mM dithiothreitol (DTT), 5 mM EDTA, 25 mM ascorbic acid and 1%

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PVPP). The homogenate was centrifuged at 4 °C for 20 min at 12,000 × g and the

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supernatant was collected for enzymes analysis. The reaction solution contained 10

177

mM Tris-HCl buffer (pH 7.5, containing 5 mM EDTA, 50 µM pyridoxal phosphate, 5

178

mM DTT) and 0.3 mL enzyme extract. The reaction was initiated by adding 0.2 mL of

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25 mM L-arginine for ADC assay or L-ornithine for ODC assay. The mixture was

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blended and incubated at 37 °C for 60 min and the PCA was added. After the

181

centrifugation at 5,000 × g for 10 min ， the supernatant was collected for

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spectrophotometer determination. The absorbance was measured at 254 nm. One unit

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of ADC and ODC activity was defined as the amount of enzyme causing 0.01

184

absorbance increase per minute at the assay conditions.

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DAO and PAO activities were detected according to the method of Gao et al. 33

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Samples (2.0 g) were homogenized with 5 mL of 100 mM sodium phosphate buffer

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(pH 6.5) and centrifuged at 12,000 × g for 20 min at 4 °C. The supernatant was

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collected for the enzymes assay. The reaction mixture included 2.0 mL of 100 mM

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sodium phosphate buffer (pH 6.5), 0.2 mL colour reagent (containing 25 µL N,

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N-dimethylaniline and 10 mg/100mL 4-aminoantipyrine in 100 mM phosphate buffer

191

(pH 6.5)), 0.1 mL of 250 U/mL horseradish peroxidase solution and 0.5 mL

192

supernatant. The reaction was initiated by adding 0.2 mL of 20 mM Put for DAO or

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Spd and Spm for PAO. The changes of the absorbance at 550 nm were measured. The

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enzyme activity was defined as 0.01 increase per minute under.

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Determination of proline content. Proline content was determined by the method



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of Zhao et al. with slight modifications.34 Frozen samples (2.0 g) were homogenized

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with 5 mL of sulphosalicylic acid solution (3%, v/v) and extracted with boiling water

198

bath for 10 min. The homogenate was centrifuged at 12,000 × g for 10 min at 4 °C.

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Two milliliter supernatant was mixed with 2 mL of glacial acetic acid and 3 mL of

200

ninhydrin, and then boiled for 30 min. The mixture extracted with 4 mL of

201

methylbenzene by shaking for 30 second and the absorbance at 520 nm was measured.

202

The proline content was quantified with external standards and expressed as

203

microgram per gram FW.

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Determination of OAT, P5CS and PDH activities. The assay of OAT activity was

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performed according to Cao et al.35 with some modifications. Frozen samples (2.0 g)

206

were homogenized in 5 mL of 50 mM potassium phosphate buffer (pH 8.0, containing

207

1 mM DTT, 1% w/v PVPP, 1 mM α-ketoglutarate and 0.05 mM pyridoxal phosphate).

208

The homogenate was centrifuged at 12,000 × g for 15 min and the supernatant was

209

used for the enzymes assay. One milliliter of the supernatant was blended with 0.9 mL

210

of 50 mM potassium phosphate buffer (pH 8.0, contained 35 mM ornithine, 5 mM

211

α-ketoglutarate, 0.05 mM pyridoxine phosphate) and incubated at 25 °C for 20 min

212

and the reaction was teminated by adding 0.3 mL of 3 M of HClO4. Then 0.2 mL of 2%

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ninhydrin was added into the mixture and boiled for 20 min. The mixture was

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centrifuged as it cools and the sediment was dissolved with 1.5 mL of anhydrous

215

alcohol. The production of P5C was quantified according to the absorbance increase

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at 510 nm. One unit of OAT activity was defined as the amount of 1 µmol P5C

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formation per second.


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P5CS and PDH activities were determined by the procedure of Shang et al.36 with

219

minor changes. Frozen samples (2.0 g) were homogenized with 10mL of Tris-HCl

220

buffer (50 mM, pH 7.4), which contained MgCl2 (7 mM), KCl (0.6 mM), EDTA (3

221

mM), DTT (1 mM), and PVPP (5% v/v). The homogenate extracts were centrifuged at

222

12,000 × g for 20 min at 4 °C and the extract supernatants were used for enzymes

223

assay. P5CS activity reaction included 3 mL of 100 mM Tris-HCl buffer (pH 7.2),

224

containing 25 mM of MgCl2, 75 mM sodium glutamate, 5 mM ATP and 0.2 mL of

225

enzyme extract. The reaction was initiated by adding 0.2 mL of 0.4 mM NADPH. The

226

absorbance change at 340 nm was measured and the enzyme activity was defined as

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the amount of 1 µmol NADPH formation per second. PDH activity reaction contained

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1.6 mL of 0.15 M Na2CO3-NaHCO3 buffer (pH 10.3), 0.2 mL of 0.1 M L-proline and

229

0.2 mL of enzyme extract. The PDH activity reaction was initiated by adding 0.2 mL

230

of 10 mM NAD+. The absorbance change at 340 nm was measured and the enzyme

231

activity was defined as 0.001 decrease per minute under the assay conditions.

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Determination of GABA content. GABA content was measured based on the

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method of Deewatthanawong et al.37 with modification. Frozen samples (1.0 g) were

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homogenized with liquid nitrogen and extracted with 1mL of methanol for 10 min.

235

The extracts were blended with 1mL of 70 mM lanthanum oxide and centrifuged at

236

12,000 × g for 10 min at 4 °C. The reaction mixture contained 0.16 mL of potassium

237

phosphate buffer (0.1 M, pH of 8.6), 0.6 mM NADP+, 0.1 U GABase, 1 mM

238

α-ketoglutaric acid and 0.5 mL of the supernatant. The α-ketoglutaric acid was added

239

in mixture and incubated for 10 min at 25 °C. The reduction of NADP+ was



240

quantified according to the absorbance at 340 nm. GABA content was expressed as

241

microgram per gram FW.

242

Determination of GAD and GABA-T activities. GAD and GABA-T activities

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were analyzed through the method of Deewatthanawong et al.37 with slight

244

modifications.37 Frozen sample (2.0g) were homogenized with 5 mL of Tris-HCl

245

buffer (100 mM, pH 9.0), which contained 10% glycerin, 1 mM DTT, 5 mM EDTA,

246

0.5 mM pyridoxal phosphate and 1 mM PMSF. The mixture was centrifuged for 20

247

min at 12000 × g at 4 °C and the supernatant was prepared for enzymes analysis.

248

GAD activity reaction included 5 mL of 100 mM potassium phosphate buffer (pH 5.8),

249

40 µM pyridoxal phosphate, 3mM L-glutamic acid and 0.1 mL of supernatant. The

250

reaction was incubated at 30 °C for 60 min and stopped by adding 0.1 mL of 0.5 M

251

HClO4. The increase of absorbance at 340 nm was recorded. One unit of enzyme

252

activity was expressed as the production of 1 µmol of GABA per hour. GABA-T

253

activity reaction contained 50 mM Tris-HCl buffer (pH 8.2), 10% glycerin, 1.5 mM

254

DTT, 0.75 mM EDTA, 0.1 mM pyridoxal phosphate, 16 mM GABA, 4 mM pyruvic

255

acid and 0.2 mL enzyme extract. The mixture was blended and incubated at 30 °C for

256

60 min and the reaction was stopped by adding 0.1 mL of 100 mM sulphosalicylic

257

acid. The absorbance at 340 nm was measured and the enzyme activity was expressed

258

as the production of 1 µmol of alanine per hour.

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Statistical analysis. Data were expressed as means ± standard deviations. DPS13.5

260

was used to conduct significance test and correlation analysis of data. Least

261

significant difference (LSD) test was performed for significance test, and the


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significance level was set at P < 0.05.

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RESULTS

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Chilling injury incidence, MDA content and electrical leakage. As shown in

265

Figure 1A, exogenous NO significantly (P < 0.05) reduced the CI incidence in treated

266

bamboo shoots compared with control during the later stage of storage. At the end of

267

storage, the chilling injury incidence of SNP-treated bamboo shoots was 37.9% lower

268

than that of control groups.

269

The MDA content in both groups was increased and the increase in SNP-treated

270

bamboo shoots was significantly (P < 0.05) inhibited compared with control (Figure

271

1B). The MDA content of SNP-treated bamboo shoots was 8.8% lower than that in

272

control on day 56.

273

Electrical leakage increased gradually in all bamboo shoots during the storage time

274

(Figure 1C). However, SNP-treated bamboo shoots exhibited a significant (P < 0.05)

275

lower electrical leakage, which was 18.6% lower than control at the end of storage.

276

Endogenous NO content and NOS activity. The NO content was promoted in

277

bamboo shoots by the exogenous NO treatment (Figure 2A). SNP treated bamboo

278

shoots showed 16.4% higher NO content at day 56 compared with control. Moreover,

279

SNP treatment significantly (P < 0.05) enhanced in the activity of NOS, which was

280

20.2% higher than water-treated bamboo shoots at the end of storage.

281

Polyamine metabolism. The treatment with SNP before cold storage resulted in an

282

increased accumulation of Put and Spd contents in bamboo shoots during the whole

283

storage period (Figure 3A-B), which was to 21.6% and 18.3% than those in control



284

group at the end of storage. SNP treatment promoted the Spm content during the

285

whole storage period compared with control (Figure 3C).

286

The ADC and ODC activities of bamboo shoots in response to exogenous NO were

287

shown in Figure 4A-B. Among the cold storage, ADC activity decreased rapidly,

288

while ODC activity reached its highest value on 28 d after the SNP treatment and then

289

decreased gradually afterwards. For the bamboo shoots after same storage time, the

290

SNP treated ones exhibited significant (P < 0.05) higher level in relative to control

291

group. In contrast, DAO activity was inhibited by SNP treatment during the whole

292

storage period and the peak of the difference between two groups was found on 42 d

293

(Figure 4C). SNP treatment also significantly (P < 0.05) inhibited PAO activity, which

294

was approximately 15.9% lower in SNP treated bamboo shoots than that in control on

295

56 d (Figure 4D).

296

Proline content and P5CS, OAT and PDH activities. Proline content and OAT

297

activity of bamboo shoots were significantly (P < 0.05) elevated by exogenous NO

298

(Figure 5A-B). The activity of OAT increased at the early stage of the cold storage

299

and then declined. The highest enzyme activity of OAT was advanced in SNP treated

300

bamboo shoots, which showed up 14 d earlier than control. P5CS activity increased

301

significantly (P < 0.05) in treated bamboo shoots, showing 20.5% higher than control

302

bamboo shoots at the end of storage (Figure 5C). Whereas PDH activity in bamboo

303

shoots with the exogenous NO application was suppressed significantly (P < 0.05)

304

compared to that of control group (Figure 5D).

305

GABA content and GAD, GABA-T activities. As indicated in Figure 6A,


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significant (P < 0.05) stimulated accumulation of GABA content was found in SNP

307

treated bamboo shoots. Besides, SNP treatment promoted the GAD activity compared

308

to the control group during storage (Figure 6B). GAD activity exhibited a decrease

309

tendency as storage prolonged, but higher activity level was found in treated bamboo

310

shoots than the control at the end of storage. GABA-T activity kept increasing in

311

control during storage while this increase was significantly (P < 0.05) inhibited after

312

SNP treatment (Figure 6C). The highest value of GABA-T activity on 56 d in the

313

treated bamboo shoots was approximately 82.6% that of the control (Figure 6C).

314

DISCUSSION

315

It had been well documented that NO participated in plant response to abiotic stress,

316

such as salinity, drought, high irradiance, extreme temperature and heavy metals.38, 39,

317

40

318

reported in diverse crops, including plum,8 peach,12 mango,9 cucumber10 and

319

banana.16 The present study found that the exogenous NO treatment effectively

320

reduced the CI in bamboo shoots during cold storage. The breakage of membrane

321

integrity was the primary damage of chilling injury due to the lipid peroxidation.41

322

Besides, MDA is a common product of lipid peroxidation that reflects the extent of

323

oxidative injury.42 In our study, the lower relative electrolyte leakage and the MDA

324

content in SNP-treated bamboo shoots corresponded with lower CI. This indicated

325

that the application of SNP could effectively reduce the membrane permeability and

326

keep the cell have a better redox homeostasis in bamboo shoots under chilling stress.

327

The cold stress induced protection on the plant cell membrane and lipid oxidation

Their involvement in regulating the chilling injury tolerance in plants was also



328

were also reported in cabbage seedlings.43

329

It was well proved that several enzymes, such as NO synthase (NOS)-like enzyme,

330

nitrate reductase and nitrite reductase, contributed to the production of endogenous

331

NO in plant.44 In present study, the exogenous NO application was found to

332

significantly improve NOS activity and NO content in bamboo shoot during cold

333

storage. There are evidences suggesting that the evaluated NOS activity and NO level

334

in fruits under cold temperature might associate with an increasing cold tolerance.45

335

According to the results in present study, it was suggested that exogenous NO was

336

involved in the generation of stress-related signal molecular and activation of its

337

upsteam enzymes in plants.

338

It was well known that PAs involved in the plants response to various

339

environmental stresses, such as drought, oxidative stress, salinity, and chilling.19 PAs

340

were synthesized from both arginine and ornithine with two major biosynthesis

341

enzymes, ADC and ODC,19 and decomposed with the catalyzing of DAO and PAO in

342

plants.14 The accumulation of Put, Spd and Spm content were observed in SNP treated

343

bamboo shoots during cold storage in present study. At the same time, ODC and ADC

344

activities were induced, while DAO and PAO activities were inhibited by SNP

345

treatment, which indicated that the accumulation of polyamines in cold-stored

346

bamboo shoots might involve in the chilling tolerance. Consistent with this, Wang et

347

al.16 founded that exogenous NO induced chilling tolerance in banana fruit by

348

regulating PAs metabolism.

349

Proline accumulation had been reported as a general response of plants to various


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350

environmental stress stimuli, which adjusted cell osmotic balance and alleviated

351

cellular redox potential.19 It was well known that P5CS and OAT participated in

352

synthesizing proline, while PDH acted as a proline catabolic enzyme.46 In present

353

study, the data revealed that SNP treatment enhanced the activities of P5CS and OAT,

354

while PDH activity was inhibited in bamboo shoots during cold storage. Meanwhile, a

355

higher proline content of SNP-treated bamboo shoots was also observed under the

356

same time point. These results suggested that SNP treatment could protect membrane

357

and cold tolerance by enhancing related enzymes activity and inducing proline

358

accumulation. Besides, exogenous NO prevented the decline of OAT activity as well

359

as contributed to the synthetize of proline, which was consistent with the results

360

obtained from banana fruit and cucumber leaf.16, 10

361

GABA is regarded as a key signal molecule in plant defense to various biotic stress

362

which played essential roles in stabilizing the sub-cellular structures.47 GAD and

363

GABA-T are working separately in catalyzing glutamate to GABA and its catabolism

364

in plant organisms.48 In present study, suppressed GAD activity and evoked GABA-T

365

activity were exhibited in bamboo shoots after SNP treatment during cold storage.

366

Simultaneously, GABA content was evidently enhanced in SNP treated bamboo

367

shoots. It’s sound to hypothesize that exogenous NO has positive effect on alleviating

368

or retarding the chilling injury by increasing GABA content and the elevating activity

369

of related enzymes. The exogenous NO induced accumulation of the GABA level in

370

cherimoya fruit and inhibition of the GAD and GABA-T activities in banana fruit

371

under cold stress were also supported to our hypothesis.49, 16



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In conclusion, the present study revealed that exogenous NO induced higher

373

chilling-tolerance in postharvest bamboo shoots. This significant advantage of SNP

374

treatment was ascribed to the induction in activities of NOS, ADC, ODC, GAD, OAT

375

and P5CS as well as biosynthesis of endogenous NO, polyamine, proling Put, Spd,

376

Spm and GABA. However, further research might be required on the elucidation of

377

the mechanism in response to chilling stress at the molecular level.

378

ABBREVIATIONS USED

379

ADC, arginine decarboxylase; CI, chilling injury; DAO, diamine oxidase; DTT,

380

dithiothreitol; FW, fresh weight; GABA, γ-aminobutyric acid; GABA-T, GABA

381

transaminase; MDA, malonaldehyde; NO, nitro oxide; NOS, nitric oxide sythetase;

382

OAT,

383

△1-pyrroline-5-carboxylate synthetase; PAO, polyamine oxidase; PAs, Polyamines;

384

PCA, perchloric acid; PDH, proline dehydrogenase; Put, putrescine; SNP, sodium

385

nitroprusside; Spd, spermidine; Spm, spermine; TCA, tricarboxylic acid.

386

AUTHOR INFORMATION

387

Corresponding Author

388

E-mail: [email protected]; phone +86-571-88982175; fax +86-571-88982139.

389

ORCID

390

Zisheng Luo: 0000-0001-8232-9739

391

Funding

392

The research was financially supported by the National Natural Science Foundation of

393

China (31371856, 31571895) and the National Key Research and Development

orn-δ-aminotransferase;

ODC,

ornithine


decarboxylase;

P5CS,

Page 19 of 32


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Program of China (2017YFD0601601).

395

Notes

396

The authors declare no competing financial interest.

397

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.


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FIGURE CAPTIONS

513

Figure 1. Effect of exogenous NO on CI incidence (A), MDA content (B) and

514

electrical leakage (C) of bamboo shoots during storage at 1 °C. Values are presented

515

as means ± SD (n = 3).

516

Figure 2. Effect of exogenous NO on NOS content (A), NO activity (B) of bamboo

517

shoots during storage at 1 °C. Values are presented as means ± SD (n = 3).

518

Figure 3. Effect of exogenous NO on Put (A), Spd (B) and Spm content (C) of

519

bamboo shoots during storage at 1 °C. Values are presented as means ± SD (n = 3).

520

Figure 4. Effect of exogenous NO on ADC (A), ODC (B), DAO (C) and PAO activity

521

(D) of bamboo shoots during storage at 1 °C. Values are presented as means ± SD (n

522

= 3).

523

Figure 5. Effect of exogenous NO on proline content (A), OAT (B), P5CS (C) and

524

PDH activity (D) of bamboo shoots during storage at 1 °C. Values are presented as

525

means ± SD (n = 3).

526

Figure 6. Effect of exogenous NO on GABA content (A), GAD (B) and GABA-T

527

activity (C) of bamboo shoots during storage at 1 °C. Values are presented as means ±

528

SD (n = 3).

529



530

Figure 1

531 532


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533

Figure 2

534 535



536

Figure 3

537 538


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539

Figure 4

540 541



542

Figure 5

543 544


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Figure 6

546 547 548



549

GRAPHIC FOR TABLE OF CONTENTS

550


Page 32 of 32

Effect of Exogenous Nitro Oxide on Chilling Tolerance, Polyamine

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