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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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Effect of exogenous nitro oxide on chilling tolerance,
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polyamine, proline and gamma-aminobutyric acid and in
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bamboo shoots (Phyllostachys praecox f. prevernalis)
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Di Wang1, Li Li1, Yanqun Xu1, Jarukitt Limwachiranon1, Dong Li1, Zhaojun Ban2,
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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
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Zhejiang University
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Hangzhou 310058, People’s Republic of China
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E-mail:
[email protected] 20
Phone: +86-571-88982175
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ABSTRACT: The effects of exogenous nitro oxide (NO) on chilling resistance and
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the metabolism of polyamine, proline and gamma-aminobutyric acid of bamboo
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shoots were investigated. Bamboo shoots were dipped in 0.07 mM sodium
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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
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accumulation of malonaldehyde and electrical leakage. At the end of storage, the
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chilling injury incidence of treated bamboo shoots decreased by 37.9% while their
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malonaldehyde content and electrical leakage were 8.8% and 18.6% lower than that of
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control, respectively. Interestingly, the endogenous NO, polyamines, γ-aminobutyric
30
acid and proline contents of treated bamboo shoot also significantly increased.
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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
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activities, including nitric oxide synthase, arginine decarboxylase, ornithine
34
decarboxylase,
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△1-pyrroline-5-carboxylate synthetase. The results indicated that the SNP treatment
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enhanced chilling tolerance of bamboo shoots, which might associate with the
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activated metabolism of polyamines, γ-aminobutyric acid and proline. SNP treatment
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might be an alternative technology to avoid chill injury during cold storage of bamboo
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shoots.
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KEYWORDS: nitro oxide, bamboo shoot, chilling injury, polyamine, proline, GABA
glutamate
decarboxylase,
orn-δ-aminotransferase
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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
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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
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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
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However, little information is available about the effect of exogenous NO on the
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chilling tolerance in bamboo shoots and the involvement of polyamine, proline,
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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
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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
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(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
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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
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g) were homogenized with 5mL of 100 mM sodium phosphate buffer (pH 7.4) in a
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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
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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.
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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)
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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
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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
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detector (Shimadzu). Samples (2 g) were grinded in liquid nitrogen and incubated
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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
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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
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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
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mM Tris-HCl buffer (pH 7.5, containing 5 mM EDTA, 50 µM pyridoxal phosphate, 5
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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
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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
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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
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(pH 6.5)), 0.1 mL of 250 U/mL horseradish peroxidase solution and 0.5 mL
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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
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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
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ninhydrin, and then boiled for 30 min. The mixture extracted with 4 mL of
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methylbenzene by shaking for 30 second and the absorbance at 520 nm was measured.
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The proline content was quantified with external standards and expressed as
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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)
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were homogenized in 5 mL of 50 mM potassium phosphate buffer (pH 8.0, containing
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1 mM DTT, 1% w/v PVPP, 1 mM α-ketoglutarate and 0.05 mM pyridoxal phosphate).
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The homogenate was centrifuged at 12,000 × g for 15 min and the supernatant was
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used for the enzymes assay. One milliliter of the supernatant was blended with 0.9 mL
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of 50 mM potassium phosphate buffer (pH 8.0, contained 35 mM ornithine, 5 mM
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α-ketoglutarate, 0.05 mM pyridoxine phosphate) and incubated at 25 °C for 20 min
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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
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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
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minor changes. Frozen samples (2.0 g) were homogenized with 10mL of Tris-HCl
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buffer (50 mM, pH 7.4), which contained MgCl2 (7 mM), KCl (0.6 mM), EDTA (3
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mM), DTT (1 mM), and PVPP (5% v/v). The homogenate extracts were centrifuged at
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12,000 × g for 20 min at 4 °C and the extract supernatants were used for enzymes
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assay. P5CS activity reaction included 3 mL of 100 mM Tris-HCl buffer (pH 7.2),
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containing 25 mM of MgCl2, 75 mM sodium glutamate, 5 mM ATP and 0.2 mL of
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enzyme extract. The reaction was initiated by adding 0.2 mL of 0.4 mM NADPH. The
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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
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0.2 mL of enzyme extract. The PDH activity reaction was initiated by adding 0.2 mL
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of 10 mM NAD+. The absorbance change at 340 nm was measured and the enzyme
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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.
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The extracts were blended with 1mL of 70 mM lanthanum oxide and centrifuged at
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12,000 × g for 10 min at 4 °C. The reaction mixture contained 0.16 mL of potassium
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phosphate buffer (0.1 M, pH of 8.6), 0.6 mM NADP+, 0.1 U GABase, 1 mM
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α-ketoglutaric acid and 0.5 mL of the supernatant. The α-ketoglutaric acid was added
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in mixture and incubated for 10 min at 25 °C. The reduction of NADP+ was
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quantified according to the absorbance at 340 nm. GABA content was expressed as
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microgram per gram FW.
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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
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modifications.37 Frozen sample (2.0g) were homogenized with 5 mL of Tris-HCl
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buffer (100 mM, pH 9.0), which contained 10% glycerin, 1 mM DTT, 5 mM EDTA,
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0.5 mM pyridoxal phosphate and 1 mM PMSF. The mixture was centrifuged for 20
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min at 12000 × g at 4 °C and the supernatant was prepared for enzymes analysis.
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GAD activity reaction included 5 mL of 100 mM potassium phosphate buffer (pH 5.8),
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40 µM pyridoxal phosphate, 3mM L-glutamic acid and 0.1 mL of supernatant. The
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reaction was incubated at 30 °C for 60 min and stopped by adding 0.1 mL of 0.5 M
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HClO4. The increase of absorbance at 340 nm was recorded. One unit of enzyme
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activity was expressed as the production of 1 µmol of GABA per hour. GABA-T
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activity reaction contained 50 mM Tris-HCl buffer (pH 8.2), 10% glycerin, 1.5 mM
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DTT, 0.75 mM EDTA, 0.1 mM pyridoxal phosphate, 16 mM GABA, 4 mM pyruvic
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acid and 0.2 mL enzyme extract. The mixture was blended and incubated at 30 °C for
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60 min and the reaction was stopped by adding 0.1 mL of 100 mM sulphosalicylic
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acid. The absorbance at 340 nm was measured and the enzyme activity was expressed
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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
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was used to conduct significance test and correlation analysis of data. Least
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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
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Figure 1A, exogenous NO significantly (P < 0.05) reduced the CI incidence in treated
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bamboo shoots compared with control during the later stage of storage. At the end of
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storage, the chilling injury incidence of SNP-treated bamboo shoots was 37.9% lower
268
than that of control groups.
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The MDA content in both groups was increased and the increase in SNP-treated
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bamboo shoots was significantly (P < 0.05) inhibited compared with control (Figure
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1B). The MDA content of SNP-treated bamboo shoots was 8.8% lower than that in
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control on day 56.
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Electrical leakage increased gradually in all bamboo shoots during the storage time
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(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.
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Endogenous NO content and NOS activity. The NO content was promoted in
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bamboo shoots by the exogenous NO treatment (Figure 2A). SNP treated bamboo
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shoots showed 16.4% higher NO content at day 56 compared with control. Moreover,
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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.
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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
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storage period (Figure 3A-B), which was to 21.6% and 18.3% than those in control
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group at the end of storage. SNP treatment promoted the Spm content during the
285
whole storage period compared with control (Figure 3C).
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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
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SNP treated ones exhibited significant (P < 0.05) higher level in relative to control
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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
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(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
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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
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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).
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DISCUSSION
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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
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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
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Besides, MDA is a common product of lipid peroxidation that reflects the extent of
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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
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keep the cell have a better redox homeostasis in bamboo shoots under chilling stress.
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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
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were also reported in cabbage seedlings.43
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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
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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
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in fruits under cold temperature might associate with an increasing cold tolerance.45
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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.
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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
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enzymes, ADC and ODC,19 and decomposed with the catalyzing of DAO and PAO in
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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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environmental stress stimuli, which adjusted cell osmotic balance and alleviated
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cellular redox potential.19 It was well known that P5CS and OAT participated in
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synthesizing proline, while PDH acted as a proline catabolic enzyme.46 In present
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study, the data revealed that SNP treatment enhanced the activities of P5CS and OAT,
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while PDH activity was inhibited in bamboo shoots during cold storage. Meanwhile, a
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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
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and cold tolerance by enhancing related enzymes activity and inducing proline
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accumulation. Besides, exogenous NO prevented the decline of OAT activity as well
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as contributed to the synthetize of proline, which was consistent with the results
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obtained from banana fruit and cucumber leaf.16, 10
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GABA is regarded as a key signal molecule in plant defense to various biotic stress
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which played essential roles in stabilizing the sub-cellular structures.47 GAD and
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GABA-T are working separately in catalyzing glutamate to GABA and its catabolism
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in plant organisms.48 In present study, suppressed GAD activity and evoked GABA-T
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activity were exhibited in bamboo shoots after SNP treatment during cold storage.
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Simultaneously, GABA content was evidently enhanced in SNP treated bamboo
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shoots. It’s sound to hypothesize that exogenous NO has positive effect on alleviating
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or retarding the chilling injury by increasing GABA content and the elevating activity
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of related enzymes. The exogenous NO induced accumulation of the GABA level in
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cherimoya fruit and inhibition of the GAD and GABA-T activities in banana fruit
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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
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chilling-tolerance in postharvest bamboo shoots. This significant advantage of SNP
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treatment was ascribed to the induction in activities of NOS, ADC, ODC, GAD, OAT
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and P5CS as well as biosynthesis of endogenous NO, polyamine, proling Put, Spd,
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Spm and GABA. However, further research might be required on the elucidation of
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the mechanism in response to chilling stress at the molecular level.
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ABBREVIATIONS USED
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ADC, arginine decarboxylase; CI, chilling injury; DAO, diamine oxidase; DTT,
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dithiothreitol; FW, fresh weight; GABA, γ-aminobutyric acid; GABA-T, GABA
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transaminase; MDA, malonaldehyde; NO, nitro oxide; NOS, nitric oxide sythetase;
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OAT,
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△1-pyrroline-5-carboxylate synthetase; PAO, polyamine oxidase; PAs, Polyamines;
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PCA, perchloric acid; PDH, proline dehydrogenase; Put, putrescine; SNP, sodium
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nitroprusside; Spd, spermidine; Spm, spermine; TCA, tricarboxylic acid.
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AUTHOR INFORMATION
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Corresponding Author
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E-mail:
[email protected]; phone +86-571-88982175; fax +86-571-88982139.
389
ORCID
390
Zisheng Luo: 0000-0001-8232-9739
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Funding
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The research was financially supported by the National Natural Science Foundation of
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China (31371856, 31571895) and the National Key Research and Development
orn-δ-aminotransferase;
ODC,
ornithine
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decarboxylase;
P5CS,
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Program of China (2017YFD0601601).
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Notes
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The authors declare no competing financial interest.
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REFERENCES
398
(1) Luo, Z.; Xu, X.; Cai, Z. Yan, B. Effect of ethylene and 1-Methylcyclopropene on lignification of postharvest
399
bamboo shoot. Food Chem. 2007, 105, 521-527.
400
(2) Kleinhenz, V.; Gosbee, M.; Elsmore, S. Storage methods for extending shelf life of fresh, edible bamboo
401
shoots [Bambusa oldhamii (Munro)]. Postharvest Biol. Tec. 2000, 19, 253–264.
402
(3) Luo, Z.; Xu, X.; Yan, B. Accumulation of lignin and involvement of enzymes in bamboo shoot during storage.
403
Eur. Food Res. Technol. 2008a, 226, 635-640.
404
(4) Luo, Z.; Xu, X.; Yan, B. Use of 1-methylcyclopropene for alleviating chilling injury and lignification of
405
bamboo shoot (Phyllostachys praecox f. prevernalis) during cold storage. J. Sci. Food Agric. 2008b, 88, 151-157.
406
(5) Luo, Z.; Wu, X.; Xie, Y. Alleviation of chilling injury and browning of postharvest bamboo shoot by salicylic
407
acid treatment. Food Chem. 2012, 131, 456-461.
408
(6) Luo, Z.; Feng, S.; Pang, J.; Mao, L.; Shou, H.; Xie, J. Effect of heat treatment on lignification of postharvest
409
bamboo shoots ( Phyllostachys praecox f. prevernalis.). Food Chem. 2012, 135, 2182–2187.
410
(7) Caro, A.; Puntarulo, S. Nitric oxide generation by soybean embryonic axes. Possible effect on mitochondrial
411
function. Free Radical Res. 1999, 31, 205-212.
412
(8) Singh, S. P.; Singh, Z.; Swinny, E. E. Postharvest nitric oxide fumigation delays fruit ripening and alleviates
413
chilling injury during cold storage of Japanese plums (Prunus salicina Lindell.). Postharvest Biol. Tec. 2009, 53,
414
101-108.
415
(9) Zaharah, S. S.; Singh, Z. Postharvest nitric oxide fumigation alleviates chilling injury, delays fruit ripening and
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
416
maintains quality in cold-stored ‘Kensington Pride’ mango. Postharvest Biol.Tec. 2011, 60, 202-210.
417
(10) Yang, H.; Wu, F.; Cheng, J. Reduced chilling injury in cucumber by nitric oxide and the antioxidant response.
418
Food Chem. 2011, 127, 1237-1242.
419
(11) Wang, Y.; Luo, Z.; Khan, Z. U. Effect of nitric oxide on energy metabolism in postharvest banana fruit in
420
response to chilling stress. Postharvest Biol. Tec. 2015, 108, 21-27.
421
(12) Zhu, L. Q.; Jie, Z.; Zhu, S. H. Effect of a combination of nitric oxide treatment and intermittent warming on
422
prevention of chilling injury of ‘Feicheng’ peach fruit during storage. Food Chem. 2010, 121, 165-170.
423
(13) Zhu, S.; Sun, L.; Liu, M. Effect of nitric oxide on reactive oxygen species and antioxidant enzymes in
424
kiwifruit during storage. J. Sci. Food Agric. 2008, 88, 2324-2331.
425
(14) Gupta, K.; Dey, A.; Gupta, B. Plant polyamines in abiotic stress responses. Acta. Physiol. Plant. 2013, 35,
426
2015-2036.
427
(15) Serrano, M.; Martinez Madrid, M. C.; Martinez, G. Role of polyamines in chilling injury of fruit and
428
vegetables. Food Sci. Technol. Int. 1996, 2, 195-199.
429
(16) Wang, Y. S.; Luo, Z. S.; Mao, L. C.; Ying, T. J. Contribution of polyamines metabolism and GABA shunt to
430
chilling tolerance induced by nitric oxide in cold-stored banana fruit. Food Chem. 2016, 197, 333-339.
431
(17) Goyal, R. K.; Fatima, T.; Topuz, M. Pathogenesis-related protein 1b1 (PR1b1) is a major tomato fruit protein
432
responsive to chilling temperature and upregulated in high polyamine transgenic genotypes. Front. Plant Sci. 2016,
433
21, 901-915.
434
(18) Botondi, R.; Massantini, R.; Anelli, G. Postharvest physiology of red chicory stored at 0 and 6 ℃. Ital. J. Food
435
Sci. 1996, 8, 273-282.
436
(19) Alcázar, R.; Altabella, T.; Marco, F. Polyamines: molecules with regulatory functions in plant abiotic stress
437
tolerance. Planta. 2010, 231, 1237-1249.
ACS Paragon Plus Environment
Page 20 of 32
Page 21 of 32
Journal of Agricultural and Food Chemistry
438
(20) Zhang, Y.; Jin, P.; Huang, Y. Effect of hot water combined with glycine betaine alleviates chilling injury in
439
cold-stored loquat fruit. Postharvest Biol. Tec. 2016, 118, 141-147.
440
(21) Radić, S.; Babić, M.; Škobić, D. Ecotoxicological effects of aluminum and zinc on growth and antioxidants in
441
Lemna minor, L. Ecotox. Environ. Safe. 2010, 3, 336-342.
442
(22) Hamilton, R. D.; Heckathorn, S. A. Mitochondrial adaptations to NaCl. Complex I is protected by
443
anti-oxidants and small heat shock proteins, whereas complex II is protected by proline and betaine. Plant Physiol.
444
2001, 3, 1266-1274.
445
(23) Planchet, E.; Verdu, I.; Delahaie, J. Abscisic acid-induced nitric oxide and proline accumulation in
446
independent pathways under water-deficit stress during seedling establishment in Medicago truncatula. J. Exp. Bot.
447
2014, 8, 2161-2170.
448
(24) Iqbal, N.; Umar, S.; Khan, N, A. A new perspective of phytohormones in salinity tolerance: Regulation of
449
proline metabolism. Environ. Exp. Bot. 2014, 2, 34-42.
450
(25) Shan, T.; Jin, P.; Zhang, Y. Exogenous glycine betaine treatment enhances chilling tolerance of peach fruit
451
during cold storage. Postharvest Biol. Tec. 2016, 1, 104-110.
452
(26) Zhang, Y.; Jin, P.; Huang, Y. Effect of hot water combined with glycine betaine alleviates chilling injury in
453
cold-stored loquat fruit. Postharvest Biol. Tec. 2016, 118, 141-147.
454
(27) Shimajiri, Y.; Oonishi, T.; Ozaki, K. Genetic manipulation of the γ-aminobutyric acid (GABA) shunt in rice:
455
overexpression of truncated glutamate decarboxylase (GAD) and knockdown of γ-aminobutyric acid transaminase
456
(GABA-T) lead to sustained and high levels of GABA accumulation in rice ker. Plant Biotechnol. J. 2013, 11,
457
594-604.
458
(28) Akihiro, T.; Koike, S.; Tani, R. Biochemical Mechanism on GABA accumulation during fruit development in
459
tomato. Plant Cell Physiol. 2008, 49, 1378-1389.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
460
(29) Li, D.; Limwachiranon, J.; Li, L. Involvement of energy metabolism to chilling tolerance induced by
461
hydrogen sulfide in cold-stored banana fruit. Food Chem. 2016, 208, 272-278.
462
(30) Zhang, X. H.; Shen, L.; Li, F. J.; Meng, D. M.; Sheng, J. P. Methyl salicylate-induced arginine catabolism is
463
associated with up-regulation of polyamine and nitric oxide levels and improves chilling tolerance in cherry
464
tomato fruit. J. Agric. Food Chem. 2011, 59, 9351-9357.
465
(31) Zhang, X. H.; Shen, L.; Li, F. J.; Meng, D. M.; Sheng, J. P. Hot air treatment-induced arginine catabolism is
466
associated with elevated polyamines and proline levels and alleviates chilling injury in postharvest tomato fruit. J.
467
Sci. Food Agric. 2013, 93, 3245-3251.
468
(32) Hu, X. H.; Zhang, Y.; Shi, Y.; Zhang, Z.; Zou, Z. R.; Zhang, H.; Zhao, J. Z. Effect of exogenous spermidine
469
on polyamine content and metabolism in tomato exposed to salinity-alkalinity mixed stress. Plant Physiol. Bioch.
470
2012, 57, 200-209.
471
(33) Gao, H. B.; Jia, Y. X.; Guo, S. R.; Lv, G. Y.; Wang, T.; Juan, L. Exogenous calcium affects nitrogen
472
metabolism in root-zone hypoxia-stressed muskmelon roots and enhances short-term hypoxia tolerance. J. Plant
473
Physiol. 2011, 168, 1217-1225.
474
(34) Zhao, D. Y.; Shen, L.; Fan, B.; Liu, K. L.; Yu, M. M.; Zheng, Y.; Ding, Y.; Sheng, J. P. Physiological and
475
genetic properties of tomato fruits from 2 cultivars differing in chilling tolerance at cold storage. J. Food Sci. 2009,
476
74, C348-C352.
477
(35) Cao, S. F.; Cai, Y. T.; Yang, Z. F.; Zheng, Y. H. MeJA induces chilling tolerance in loquat fruit by regulating
478
proline and gamma-aminobutyric acid contents. Food Chem. 2012, 133, 1466–1470.
479
(36) Shang, H. T.; Cao, S. F.; Yang, Z. F.; Cai, Y. T.; Zheng, Y. H. Effect of exogenous gamma-aminobutyric acid
480
treatment on proline accumulation and chilling injury in peach fruit after long-term cold storage. J. Agric. Food
481
Chem. 2011, 59, 1264-1268.
ACS Paragon Plus Environment
Page 22 of 32
Page 23 of 32
Journal of Agricultural and Food Chemistry
482
(37) Deewatthanawong, R.; Rowell, P.; Watkins, C. B. Gamma-Aminobutyric acid (GABA) metabolism in CO2
483
treated tomatoes. Postharvest Biol.Tec. 2010, 57, 97-105.
484
(38) Arasimowicz-Jelonek, M.; Floryszak-Wieczorek, J.; Kubiś, J. Interaction between polyamine and nitric oxide
485
signaling in adaptive responses to drought in cucumber. J. Plant Growth Regul. 2009, 2, 177-186.
486
(39) Santa-Cruz, D. M.; Pacienza, N. A.; Polizio, A. H. Nitric oxide synthase-like dependent NO production
487
enhances heme oxygenase up-regulation in ultraviolet-B-irradiated soybean plants. Phytochemistry. 2010, 15,
488
1700-1707.
489
(40) Khan, M. N.; Siddiqui, M. H.; Mohammad, F. Interactive role of nitric oxide and calcium chloride in
490
enhancing tolerance to salt stress. Nitric Oxide-Biol Ch. 2012, 4, 210-218.
491
(41) Hodges, D. M.; DeLong, J. M.; Forney, C. F. Improving the thiobarbituric acid-reactive-substances assay for
492
estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta.
493
1999, 207, 604-611.
494
(42) Bao, G.; Bi, Y.; Li, Y. Overproduction of reactive oxygen species involved in the pathogenicity of Fusarium,
495
in potato tubers. Physiol. Mol. Plant P. 2014, 86, 35-42.
496
(43) Sung, C. H.; Hong, J. K. Sodium nitroprusside mediates seedling development and attenuation of oxidative
497
stresses in Chinese cabbage. Plant Biotechnology Reports. 2010, 4, 243-251
498
(44) Neill, S. J.; Bright, J.; Desikan, R.; Hancock, J. T.; Harrison, J. T.; Wilson, I. Nitric oxide evolution and
499
perception. J. Exp. Bot. 2008, 59, 25-35.
500
(45) Xu, M.; Dong, J.; Zhang, M. Cold-induced endogenous nitric oxide generation plays a role in chilling
501
tolerance of loquat fruit during postharvest storage. Postharvest Biol. Tec. 2012, 65. 5-12.
502
(46) Hussain, S. S.; Ali, M.; Ahmad, M. Polyamines: natural and engineered abiotic and biotic stress tolerance in
503
plants. Biotechnol Adv. 2011, 29, 300-311.
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Journal of Agricultural and Food Chemistry
504
(47) Hyun, T. K.; Eom, S. H.; Jeun, Y. C. Identification of glutamate decarboxylases as a γ-aminobutyric acid
505
(GABA) biosynthetic enzyme in soybean. Industrial Crop. Prod. 2013, 49, 864-870.
506
(48) Cao, S.; Cai, Y.; Yang, Z. MeJA induces chilling tolerance in loquat fruit by regulating proline and
507
γ-aminobutyric acid contents. Food Chem. 2012, 133, 1466-1470.
508
(49) Merodio, C.; Muñoz, M. T.; Cura, B. D. Effect of high CO2 level on the titres of γ-aminobutyric acid, total
509
polyamines and some pathogenesis-related proteins in cherimoya fruit stored at low temperature. J. Exp. Bot. 1998,
510
325, 1339-1347.
511
.
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FIGURE CAPTIONS
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Figure 1. Effect of exogenous NO on CI incidence (A), MDA content (B) and
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electrical leakage (C) of bamboo shoots during storage at 1 °C. Values are presented
515
as means ± SD (n = 3).
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Figure 2. Effect of exogenous NO on NOS content (A), NO activity (B) of bamboo
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shoots during storage at 1 °C. Values are presented as means ± SD (n = 3).
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Figure 3. Effect of exogenous NO on Put (A), Spd (B) and Spm content (C) of
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bamboo shoots during storage at 1 °C. Values are presented as means ± SD (n = 3).
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Figure 4. Effect of exogenous NO on ADC (A), ODC (B), DAO (C) and PAO activity
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(D) of bamboo shoots during storage at 1 °C. Values are presented as means ± SD (n
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= 3).
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Figure 5. Effect of exogenous NO on proline content (A), OAT (B), P5CS (C) and
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PDH activity (D) of bamboo shoots during storage at 1 °C. Values are presented as
525
means ± SD (n = 3).
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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).
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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GRAPHIC FOR TABLE OF CONTENTS
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