Postharvest Application of Oligochitosan and Chitosan Reduces Calyx

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Post-harvest Application of Oligochitosan and Chitosan Reduces Calyx Alterations of Citrus Fruit Induced by Ethephon Degreening Treatment Lili Deng, Baofeng Yin, Shixiang Yao, Weihao Wang, and Kaifang Zeng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02534 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 14, 2016

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Journal of Agricultural and Food Chemistry

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Post-harvest Application of Oligochitosan and Chitosan Reduces

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Calyx Alterations of Citrus Fruit Induced by Ethephon Degreening

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Treatment

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Lili Deng †, §, Baofeng Yin †, Shixiang Yao †, §, Weihao Wang †, §, Kaifang Zeng †, §, *

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†

College of Food Science, Southwest University, Chongqing 400715, PR China

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§

Chongqing Engineering Research Center of Regional Food, Chongqing 400715, PR

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China

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* Corresponding author

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Address: College of Food Science, Southwest University, Chongqing 400715, PR

12

China

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Tel.: +86 23 68250374

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Fax: +86 23 68250374.

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E-mail address: [email protected].

16 17 18 19 20 21 22

ACS Paragon Plus Environment


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ABSTRACT: In the present study, we investigated whether the post-harvest application of

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oligochitosan and chitosan could be used as potential alternatives to 2,4-dichlorophenoxyacetic

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acid (2,4-D) treatment to prevent calyx senescence of mandarin fruits induced by degreening

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treatment. The results of scanning electron microscopy indicated that the ethephon degreening

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treatment could accelerate the formation of pedicel abscission layers. Treatments with 15 g kg−1

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oligochitosan, 5 g kg−1 chitosan, and 50 mg kg−1 2,4-D significantly suppressed the formation of

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pedicel abscission layers of ethephon degreening fruit and inhibited the browning of the calyx.

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These two treatments delayed the degradation of protopectin, cellulose and lignin. Inhibition of

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the increase in the abscisic acid (ABA) content was also observed in these two treatments. In

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conclusion, these two treatments, particularly 15 g kg−1 oligochitosan, could be potentially used

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as alternatives to 2,4-D to improve calyx alterations induced by the ethephon degreening

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treatment in mandarin fruits.

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KEYWORDS:

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dichlorophenoxyacetic acid (2,4-D); calyx abscission

mandarin;

ethephon

degreening;

oligochitosan;

37 38 39 40 41 42 43 44


chitosan;

2,4-

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INTRODUCTION

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Citrus fruit do not have a clearly defined point of maturity. Colour is not an adequate indicator of

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maturity because the appearance of citrus fruit primarily depends on sufficiently low night

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temperatures. Early season citrus varieties, such as “Clemenules” mandarins (Citrus clementina

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Hort. ex Tan), “Navelina” oranges (Citrus sinensis L.) of Valencia (Spain) and “Wase satsuma”

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mandarins (Citrus unshiu Marc.) of Chongqing (China), reach acceptable internal maturity

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standards for marketing while their peel is still green.1 Moreover, to avoid winter frost, several

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citrus varieties that are grown near the northern border of the citrus production area, such as

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Ponkan (Citrus reticulata Blanco) of Quzhou (China), must be harvested early before their peels

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are fully coloured.2 Both early season and early harvested citrus varieties have a poor external

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colouration. To achieve uniform external colouration in these fruits, a post-harvest degreening

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treatment (either through ethylene fumigation or ethephon dipping) is widely conducted as a

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post-harvest practice to promote peel degreening in early season and early harvested citrus

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varieties when declining field temperatures have not yet induced sufficient natural colour

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development.3 Ethylene-degreening treatment promotes the degradation of chlorophyll and

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accumulation of carotenoids with orange and yellow colours2 and thus improves the external

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colour of these fruits. In addition, reflecting the high cost of building degreening rooms,

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ethephon degreening has been widely used in China.

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However, the application of ethylene during such treatments is associated with calyx

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senescence (drying, browning, and abscission) in fruits.4 Ethylene increased the activities of

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polygalacturonic acid enzyme (PG) and cellulase (Cx) at the abscission zone of fruits,5 thereby

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promoting the browning and abscission of the calyx. Numerous studies have been conducted on



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the development of new degreening methods to reduce the use of ethylene and the exposure time

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of fruits to ethylene.1, 6-8

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2,4-dichlorophenoxyacetic acid (2,4-D), a synthetic auxin (plant growth regulator), has been

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used as a preharvest treatment to reduce fruit splitting as well as the premature drop of mandarin

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fruit 9 and also has been used as a postharvest packhouse treatment to retard calyx abscission (to

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repress

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ethylene-degreening treatment, thereby maintaining the quality of citrus fruits.10-12 2,4-D reduces

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PG and Cx activities and increases the lignin and water contents of fruit peels.5,13 Currently,

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European Union and US legislations have restricted the use of 2,4-D in the citrus post-harvest

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industry because of concerns over its effects on human health and environmental safety.

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Therefore, determining an alternative to this auxin in controlling calyx senescence is necessary.

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Several efforts have been focused on identifying a new growth regulator,14 but the use of 2,4-D

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cannot sufficiently be replaced.

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Both chitosan (poly-β-(1,4)-d-glucosamine), a deacetylated form of chitin and a natural

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antimicrobial compound, and oligochitosan, prepared by the enzymatic hydrolysis of

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deacetylated chitosan polymers, delay the senescence of fruits.15,16 Oligochitosan also promotes

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the accumulation of the hydroxyproline-rich glycoprotein content and delays the degradation of

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protopectin.15,17 In the present study, we investigated whether the post-harvest application of

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oligochitosan and chitosan could serve as a potential alternative to 2,4-D treatment to prevent

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calyx senescence in mandarin fruits induced by degreening treatment. Moreover, the

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mechanisms that underlie this process were elucidated.

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MATERIALS AND METHODS

postharvest

decay)

or

calyx

senescence,

occurring


as

a

consequence

of

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Fruits and treatments. Wase satsuma mandarin fruits were harvested from a commercial

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orchard in Beibei, Chongqing, China, in mid-September at the green mature stage. The fruits

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were harvested in the morning and immediately transported to the laboratory. Disease- and

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damage-free fruits of uniform size, colour, and maturity were selected and used in the present

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study.

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According to previous experiments (unpublished), harvested navel orange fruit showed the

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best induction of disease resistance to green mould, blue mould and anthracnose when inoculated

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for 24 h after chitosan or oligochitosan treatment. Therefore, ethephon degreening treatment was

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conducted at 24 h after the chitosan or oligochitosan treatments. As 2,4-D is typically used as a

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dip, drench or in a wax10, 2,4-D was used together with ethephon. The fruits were subjected to

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the following treatments. (1) Control (ethephon treatment): fruits were dipped into

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sterile-distilled water for 1 min, air-dried for 24 h, and subsequently immersed into 1000 mg L−1

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ethephon solution for 1 min; (2) ethephon + 2,4-D treatment: fruits were dipped into

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sterile-distilled water for 1 min, air-dried for 24 h, and subsequently immersed in 1000 mg L−1

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ethephon solution (with 50 mg kg−1 2,4-D) for 1 min; (3) 15 g kg−1 oligochitosan + ethephon

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treatment: fruits were dipped into 15 g kg−1 oligochitosan (molecular weight: 1,500-2,000 Da,

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purchased from Jinan Haidebei Marine Bioengineering Co., Ltd., Shandong, China) for 1 min,

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air-dried for 24 h, and subsequently immersed into 1000 mg L−1 ethephon solution for 1 min; and

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(4) 5 g kg−1 chitosan + ethephon treatment: fruits were dipped into 5 g kg−1 chitosan (90% degree

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of deacetylation, purchased from Jinan Haidebei Marine Bioengineering Co., Ltd., Shandong,

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China, dissolved in 1.0% glacial acetic acid, pH was adjusted to 5.4 with 1 mol L−1 NaOH) for 1

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min, air-dried for 24 h, and subsequently immersed in 1000 mg L−1 ethephon solution for 1 min.



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After natural drying at room temperature for approximately 3 h, all fruits were individually

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packaged in plastic bags (polyethylene; area = 15 mm × 15 mm; thickness = 0.015 mm) and

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incubated at 20 °C and 85–90% RH for colour and biochemical analyses.

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Browning is the main calyx alteration for ethephon degreening fruit; therefore, the severity of

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browning in citrus fruit during ethephon degreening was investigated in the present study (10

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days). Fruit colour measurement and sampling were conducted after 0, 2, 4, 6, 8, and 10 d of

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storage. The initiation time of ethephon treatments was marked as 0 d in storage. Each sampling

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comprised a total of thirty fruits for each treatment, separated into 3 replicates, with ten fruits for

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each replicate. After measurement of the peel colour, the peel near the stem end (0.01 m) from

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each fruit was collected, combined, frozen in liquid nitrogen, ground into a fine powder, and

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stored at −80 °C for analysis of structure-related compounds and enzyme activities.

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Peel colour measurement. The procedure was adapted from Lim et al.18 The peel colour was

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measured using a Hunter Lab UltraScan Pro colourimeter using EasyMatch QC software (Hunter

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Associate Laboratory Inc., Reston, VA, U.S.A.) with thirty fruits per treatment, and two

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measurements were obtained on the equatorial zone of each fruit. We adopted the CIE 1976

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L*a*b* colour scale, where L* indicates the level of lightness or darkness (0 for black, 100 for

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white), a* represents the level of redness (positive values) or greenness (negative values), and b*

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represents the level of yellowness (positive values) or blueness (negative values). Hue angle and

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chroma were calculated according to previously reported methods.19

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Chlorophyll and total carotenoid extraction and quantification. The frozen ground

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material of flavedo was used for total chlorophyll and carotenoid extraction, and flavedo

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pigments were extracted using acetone (with 0.1‰ BHT). The absorbance of acetone extracts


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was measured at 662, 645, and 470 nm. The total carotenoids and total Chl (a + b) contents were

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calculated according to the method of Lichtenthaler and Wellburn,20 and the Chl (a + b) content

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was determined after summing the Chl a and Chl b values. Each sample was extracted at least

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two times. Here, Chl a = 11.75 A662 - 2.350 A645; Chlb = 18.61 A645 - 3.960 A662; and Cx+c (total

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carotene) =1000 A470 - 2.270 Ca - 81.4 Cb/227.

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Calyx quality assessment. The calyx quality of thirty fruits per treatment was visually

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evaluated according to the method of Knight et al.21 with certain modifications. During the

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ethephon degreening process (10 d), no full calyx abscission was observed. This evaluation of

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calyx alterations involved the evaluation of each fruit for a browning area over the calyx. The

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browning severity was assessed using a scoring system: 1 (nil), 2 (slight), 3 (medium), and 4

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(high). The browning index was calculated using the formula: Index = ∑[(scale value × number

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of the fruit within each scale)/(total number of fruit × the highest scale)].

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Measurement of the abscisic acid (ABA) content. ABA extraction and analysis were

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performed according to the methods of Ma et al. and Kondo et al.22,23 with certain modifications.

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Frozen skin samples (1.0 g) were homogenized in 10 mL of cold 80% (v/v) methanol and

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extracted at 4 °C for 6 h with occasional shaking. The extract was centrifuged at 10,000 × g for

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15 min at 4 °C, and the residue was washed twice with 2.5 mL of cold 80% (v/v) methanol and

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centrifuged as indicated above. All of the supernatants were collected and concentrated to an

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aqueous solution in vacuo, and the aqueous phase was adjusted to pH 8.0 with 1 mol L-1 NaOH

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and extracted three times with ethyl acetate (v/v = 2:1) and three times with chloroform (v/v =

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2:1). The aqueous phase was centrifuged at 10,000 × g for 15 min at 4 °C, and the supernatant

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was adjusted to pH 3.0 with 1 mol L-1 HCl and extracted twice with an equal volume proportion



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of ethyl acetate. The ethyl acetate extract was subsequently concentrated to dryness and

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redissolved in 2 mL of methanol. After filtration through a 0.22-µm membrane filter, the filtrate

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was stored in a sealed amber vial until high-performance liquid chromatography (HPLC)

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analysis.

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A 20-µL volume of the extracted sample was subsequently analysed via HPLC using an

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Agilent Zorbax C-18 column (250 mm × 5.0 mm, 4.6 µm; Agilent Technologies, Inc., U.S.A.)

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connected to a UV detector set at 254 nm. The elution solvent was methanol/acetate buffer (pH

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3.0) (60/40, v/v) at a flow rate of 0.5–0.8 mL min-1. The duration of the analysis was 20 min. The

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separated ABA was identified after comparing the observed retention times with standards (0.2 g

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L-1). To quantify ABA, the ranges of concentrations were determined where the linearity between

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the peak area and amounts of eluted ABA occurred.

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Sample preparation for scanning electron microscopy (SEM). Samples for SEM were

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prepared according to the method of Agusti et al. with slight modifications.24 Rind pieces

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(5 mm × 3 mm × 3 mm) from the exposed face of the fruit were vacuum-infiltrated for 10 min in

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2.5% glutaraldehyde in 50 mmol L−1 phosphate buffer (pH 7.2) prior to fixing the samples in the

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same solution overnight at 4 °C. The material was subsequently rinsed several times using the

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same buffer, dehydrated in a graded ethanol series (30%, 50%, 70%, 85%, and 95%) for

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approximately 10 min, and dehydrated twice with 100% ethanol for approximately 10 min.

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Subsequently, ethanol was replaced with a graded isoamyl acetate series (50%, 70%, 80%, 90%,

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and 95%) for approximately 10–15 min and dehydrated twice with 100% tert-butyl alcohol for

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approximately 10–15 min. The material was dried at the critical point in a vacuum drying oven

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and mounted on SEM stubs. The mounted material was coated with gold using a Polaron E-6100


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sputter coater. SEM was performed on a HITACHI S-3400N electron microscope at an

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acceleration voltage of 10–15 kV.

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Measurements of pectin contents and related enzyme activities. The water-soluble pectin

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and protopectin contents were determined by carbazole–vitriol colourimetry.25 PG and pectin

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methylesterase (PME) activities were assessed according to the method of Deng et al.17 One unit

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of polygalacturonase was defined as the amount of enzyme that liberates 1 mg of galacturonic

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acid per h per g fresh weight. One unit of PME was defined as the amount that yields 1 mM of

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CH3O- per g fresh weight per h.

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Measurements of cellulase (Cx) activity. Cx activity analysis was conducted according to the

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methods of Abu-Goukh et al. (2003) and Gusakov et al.26,27 with slight modifications. A 1.0 g

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portion of the peel was homogenized in 5 mL of 100 mmol L−1 sodium acetate buffer [pH 6.0,

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with 0.2% sodium dithionite (Na2S2O4) and 1% polyvinyl (PVP)] for 20 min at 4 °C, and the

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

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enzyme extraction to assay the activities of Cx. Cx activity was determined after measuring the

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reducing groups released from carboxymethyl cellulose. The concentration of the reducing

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groups was determined with D-glucose as the standard. The reaction mixture contained 1.5 mL of

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10 g L-1 carboxymethyl cellulose and 0.50 mL of enzyme solution (0.50 mL of buffer in the

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control). After incubation at 37 °C for 1 h, the solution was analysed for reducing groups using

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3,5-dinitrosalicylic acid reagent at 540 nm. Cx activity was determined as units, with one unit

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being defined as the amount of the enzyme that catalyses the formation of 1.0 g of reducing

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groups per second per kilogram of original fresh weight.

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Measurements of lignin content and related enzyme activities. The lignin contents were



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determined according to the method of Deng et al.,28 with the lignin contents expressed as OD280

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kg−1. Cinnamate 4-hydroxylase (C4H) was determined according to the method of Lamb and

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Rubery.29 Approximately 1.0 g of the peel was homogenized in 5.0 mL of ice-cold phosphate

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buffer (200 mmol L−1, pH 7.5, with 5 mmol L−1 β-mercaptoethanol), and the homogenate was

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centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was collected as an enzyme extract

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to assay the activities of C4H. Enzyme extract (50 µL) was incubated with 2 mL of 50 mmol L−1

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phosphate buffer, 1 mL of 2 mmol L−1 trans-cinnamic acid, 100 µL of 0.5 mmol L−1 NADP-Na2,

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and 100 µL of 0.5 mmol L−1 G-6PNa2. The mixtures were incubated for 1 h at 37 °C, and the

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reaction was stopped with 200 µL of 6 mol L−1 HCl. Blanks with reaction mixtures were

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incubated without enzyme extract. One unit of enzyme activity was defined as the amount of

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enzyme resulting in a single change in the absorbance per second at 340 nm. 4 - Coumarate:

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Coenzyme A Ligase (4CL) was determined according to the method of Knobloch and

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Hahlbrock30 with certain modifications. Approximately 2.0 g of the peel was homogenized in 4.0

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mL of ice-cold extraction solution [pH 8.9, with 50 mmol L-1 Tris-HCl, 5 mmol L−1

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β-mercaptoethanol, 5 mmol L−1 EDTA, 5 mmol L−1 vitamin C, 10 µmol L−1 leupeptin, 1 mmol

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L−1 PMSF, 0.15% PVP (w/v), and 30% glycerinum] to stabilize the enzyme. After ultrasonic

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irradiation (2 min), the homogenate was filtered through four layers of cheesecloth and

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centrifuged at 12,000 × g for 20 min at 4 °C. The supernatant was collected as an enzyme extract

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to assay the activities of 4CL. The enzyme extract (0.8 mL) was incubated with 2.2 mL of

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reaction buffer (with 5 mmol L−1 p-coumaric acid, 50 mmol L−1 ATP, 1 mmol L−1 CoA-SH, 15

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mmol L−1 MgSO4·7H2O), and the mixtures were incubated for 10 min at 40 °C. One unit of

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enzyme activity is defined as the amount of enzyme resulting in a single change in absorbance


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per second at 333 nm.

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Statistical analysis. Experiments were performed using a completely random design. All

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statistical analyses for this experiment were performed using SPSS 17.0 (SPSS Inc., Chicago, IL,

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USA). The data are expressed as the means ± SD. The data were analysed using one-way

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ANOVA to determine differences in treatments. Mean separations were performed using

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Duncan’s multiple range tests. Differences at P < 0.05 were considered significant. All

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experiments were replicated three times.

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RESULTS

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Effects of oligochitosan, chitosan, and 2,4-D treatments on the peel colour of

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ethephon-treated Wase satsuma mandarin fruits. Ethephon accelerated the loss of green

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colour and the development of a deeper orange colour. As shown in Fig. 1, the onset of

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colour-break occurred on the 4th day after the degreening treatment. The peel turned completely

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yellow in ethephon-treated fruits on the 6th day after harvest. Fruit colour can be quantitatively

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described according to the lightness (L*), hue angle (H°), and chroma (C*). Ethephon can

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accelerate the decrease in H°, whereas chroma, L*, a*, and b* sharply increased during

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degreening (Table 1). However, compared with ethephon-treated fruits, the 2,4-D treated fruits

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presented a poor colour and basically exhibited a lower a* and a*/b* and higher H° at the early

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time of degreening process (3-4 day after ethephon treatment). During the first 3 d after

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treatment, 15 g kg−1 oligochitosan and 5 g kg−1 chitosan delayed changes in a*, b*, a*/b*, and H°

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to a certain extent, whereas these colour indices reached similar levels with ethephon-treated

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fruits at a later storage time. These results indicate that 15 g kg−1 oligochitosan and 5 g kg−1

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chitosan did not negatively influence the colour change of ethephon-treated fruits (P > 0.05).



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Effects of oligochitosan, chitosan, and 2,4-D treatments on the contents of chlorophylls

244

and total carotenoids. As shown in Figs. 2A to 2B, the Chl a and Chl b contents of

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ethephon-treated fruits markedly decreased after 2 d of storage at 20°C. In addition, 15 g kg−1

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oligochitosan, 5 g kg−1 chitosan, and 2,4-D treatments delayed the decrease in Chl a and Chl b

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resulting from ethephon treatment during the first 2 d. However, the contents of Chl a, Chl b, and

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Chl (a + b) reached similar levels as those of ethephon-treated fruits on day 4 of storage (P >

249

0.05). Moreover, 5 g kg−1 chitosan treatment appeared to exhibit a certain inhibitory effect on the

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decrease of Chl b. The contents of total carotenoids sharply increased after degreening treatment.

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However, the contents of total carotenoids in ethephon, 5 g kg−1 chitosan, and 2,4-D treatments

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did not exhibit a significant difference during the experimental period, whereas 15 g kg−1

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oligochitosan treatment was more effective in promoting the accumulation of carotenoids in

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ethephon-treated fruits, which improved the peel colour (Fig. 2D). At 6, 8, and 10 d after

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treatment, the total carotenoid contents in the 15 g kg−1 oligochitosan-treated fruits were 6.22%,

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12.00%, and 14.98% higher than those in ethephon-treated fruits, whereas the total carotenoid

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contents in 2,4-D-treated fruits were 11.80% lower than in ethephon-treated fruits on day 10 after

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treatment. These results can partially explain the differences in the previously described colour

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symptoms.

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Effects of oligochitosan, chitosan, and 2,4-D treatments on the calyx browning of

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ethephon-treated Wase satsuma mandarin fruits during storage. Calyx browning is the

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primary characteristic accelerated by degreening treatment. As shown in Fig. 3, the browning

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index in ethephon-treated fruits increased markedly during the experimental period, but no

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significant difference was observed in the four treatments during the early stage. At a later


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storage stage (8 d), 15 g kg−1 oligochitosan and 5 g kg−1 chitosan could significantly reduce the

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incidence of browned calyxes compared with that in the control (P < 0.05). The browning

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indices of the fruits treated with 15 g kg−1 oligochitosan, 5 g kg−1 chitosan, and 2,4-D were

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10.10%, 12.12%, and 5.05% lower, respectively, than those of ethephon-treated fruits on day 8

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after treatment. These results indicated that a suitable concentration of oligochitosan and chitosan

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is useful for controlling calyx browning caused by degreening.

271

Effects of oligochitosan, chitosan, and 2,4-D treatments on the ABA content of

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ethephon-treated Wase satsuma mandarin fruits during storage. As shown in Fig. 4, the

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ABA contents of mandarin fruits sharply increased after ethephon treatment, subsequently

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peaking on day 4. The inhibition effect on the increase in ABA content was observed in three

275

treatments. Among the treatments, 15 g kg−1 oligochitosan exhibited an effect similar to that of

276

2,4-D. The ABA contents of ethephon-treated fruits were 2.11, 2.19, and 1.89 times higher than

277

those of the fruits treated with 15 g kg−1 oligochitosan, 5 g kg−1 chitosan, and 2,4-D, respectively,

278

on day 4 after treatment. However, no significant difference (P > 0.05) was observed in the three

279

treatments.

280

Effects of oligochitosan, chitosan, and 2,4-D treatments on the ultrastructure of the stem

281

end of ethephon-treated Wase satsuma mandarin fruits during storage. The results of SEM

282

indicated that ethylene degreening could accelerate the formation of pedicel abscission layers

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(Fig. 5). Treatments with 15 g kg−1 oligochitosan, 5 g kg−1 chitosan, and 2,4-D significantly

284

suppressed the formation of pedicel abscission layers in ethephon-degreened fruits. As shown in

285

Fig. 5, an evident abscission zone was observed in ethephon-treated fruits after 4 d of storage at

286

20 °C; however, no abscission zone was detected in fruits treated with 15 g kg−1 oligochitosan, 5



287

g kg−1 chitosan, and 2,4-D. After 10 d of storage, ethylene significantly increased (P < 0.05) the

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width of the abscission zone, and separation also occurred in some areas of the zone. Treatments

289

with 50 mg kg−1 2,4-D could delay the formation of the abscission zone with a significant effect,

290

and the inhibition levels of 15 g kg−1 oligochitosan and 5 g kg−1 chitosan were superior to those

291

of 2,4-D treatments. Besides, on day 10, significant difference can be observed among the gaps

292

between the calyx and the fruit. Almost no junction was observed in ethephon degreened fruit,

293

while in 2,4-D treated fruit, junction still can be well seen, in 5 g kg−1 chitosan or 15 g kg−1

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oligochitosan treated fruit, the junction parts were wider than that in ethephon or 2,4-D treated

295

fruit. These results indicated that 15 g kg−1 oligochitosan and 5 g kg−1 chitosan treatments

296

exerted significant effects on reducing calyx alterations of citrus fruits induced after degreening

297

treatment compared with 2,4-D treatments.

298

Effects of oligochitosan, chitosan, and 2,4-D treatments on the metabolism of

299

ethephon-treated Wase satsuma mandarin fruits during storage. A decrease in the

300

protopectin concentration was observed in all of the treatments, whereas ethephon promoted the

301

degradation of protopectin (Fig. 6A). At 10 d after the ethephon degreening treatment, the

302

protopectin content decreased to 51.8% of the harvest. Treatments with 15 g kg−1 oligochitosan,

303

5 g kg−1 chitosan, and 2,4-D significantly inhibited the degradation of protopectin (P < 0.05),

304

with 15 g kg−1 oligochitosan exhibiting the best inhibition effect among the treatments. At 4 d

305

after the ethephon degreening treatment, the protopectin contents of 15 g kg−1 oligochitosan, 5 g

306

kg−1 chitosan, and 2,4-D treatments were 29.9%, 20.7%, and 26.4% higher, respectively, than

307

those of the control fruits. At 10 d after the ethephon degreening treatment, the protopectin

308

contents of 15 g kg−1 oligochitosan, 5 g kg−1 chitosan, and 2,4-D treatments were 33.6%, 30.7%,


Page 14 of 39

Page 15 of 39


309

and 33.8% higher, respectively, than those of the control fruits.

310

In addition, a sharp increase in water-soluble pectin contents was observed with decreasing

311

protopectin (Fig. 6B). Treatments with 15 g kg−1 oligochitosan, 5 g kg−1 chitosan, and 2,4-D also

312

demonstrated significant inhibition of the increase in water-soluble pectin (P < 0.05). The

313

water-soluble pectin contents of ethephon-treated fruits were 1.44, 1.24, and 1.26 times those of

314

the fruits treated with 15 g kg−1 oligochitosan, 5 g kg−1 chitosan, and 2,4-D, respectively, at 10 d

315

after the treatment. However, no significant difference (P > 0.05) was observed in these three

316

groups.

317

As shown in Fig. 6C, the activity of PG increased sharply after the ethephon treatment. At 8 d

318

after the ethephon degreening treatment, the activity of PG increased to 4.51 times that of the

319

harvest. During the later stage of storage, the activity of PG in ethephon-treated fruits was

320

significantly higher than those in the other three treatments. The PG activity in ethephon-treated

321

fruits were 1.28, 1.36, and 1.30 times higher than those in the fruits treated with 15 g kg−1

322

oligochitosan, 5 g kg−1 chitosan, and 2,4-D, respectively, on the 10th day after treatment (P
0.05) was observed between 5 g kg−1 chitosan and 2,4-D

324

treatments, whereas the inhibition effect of 15 g kg−1 oligochitosan was markedly higher than

325

that of the 2,4-D treatment. The PG activities in the 2,4-D-treated fruits were 15.3%, 23.4%, and

326

23.0% lower than those in the control fruits at 6, 8, and 10 d, respectively, whereas the PG

327

activities in 15 g kg−1 oligochitosan-treated fruits were 18.1%, 27.5%, and 21.6% lower than

328

those in the control fruits at 6, 8, and 10 d, respectively.

329

A significant increase in the PME activity was also observed in ethephon-treated fruits (P
0.05) was observed in the fruits treated with 15 g kg−1 oligochitosan and 2,4-D. The PME

336

activity in the 5 g kg−1 chitosan-treated fruits was slightly higher than those in the 15 g kg−1

337

oligochitosan- and 2,4-D-treated fruits, but remained significantly lower than that of

338

ethephon-treated fruits.

339

Effects of oligochitosan, chitosan, and 2,4-D treatments on the cellulase (Cx) activity of

340

ethephon-treated Wase satsuma mandarin fruits during storage. As shown in Fig. 7, the

341

activity of Cx in the abscission area increased during degreening, and ethephon promotes this

342

process. Cx activity during the 6th day was 1.40 times that during harvest in ethephon-treated

343

fruits and 1.09, 1.06, and 1.14 times that in the 15 g kg−1 oligochitosan-, 5 g kg−1 chitosan-, and

344

2,4-D-treated fruits, respectively. No significant difference (P > 0.05) was observed among the

345

three treatments.

346

Effects of oligochitosan, chitosan, and 2,4-D treatments on lignin content and the C4H

347

and 4CL activities of ethephon-treated Wase satsuma mandarin fruits during storage.

348

During degreening, the lignin content of degreening fruits exhibited a similar change with

349

cellulose (Fig. 8A). A peak value was observed in the 15 g kg−1 oligochitosan-, 5 g kg−1 chitosan-,

350

and 2, 4-D-treated fruits. The lignin contents of 15 g kg−1 oligochitosan-, 5 g kg−1 chitosan-, and

351

2,4-D-treated fruits were 1.25, 1.37, and 1.24 times, respectively, that of ethephon-treated fruits

352

at day 2 after treatment. 2,4-D treatment significantly promoted the accumulation and delayed the


Page 16 of 39

Page 17 of 39


353

degradation of lignin. The lignin contents of 2,4-D treated fruits were 20.0%, 44.5%, and 21.8%

354

higher than those of ethephon-treated fruits on days 4, 6, and 8, respectively. Treatments with 15

355

g kg−1 oligochitosan and 5 g kg−1 chitosan demonstrated effects similar to that of 2,4-D, with 5 g

356

kg−1 chitosan exhibiting better results than 2,4-D treatment. The lignin contents of 5 g kg−1

357

chitosan-treated fruits were 9.75% and 9.83% higher than those of 2,4-D treated fruits at 2 d and

358

4 d, respectively, but no significant difference (P > 0.05) was observed in the two treatments at a

359

later degreening stage.

360

The C4H activity increased during degreening, peaking at a peak value at 6−8 d after

361

treatment (Fig. 8B). Treatment with 15 g kg−1 oligochitosan, 5 g kg−1 chitosan, and 2,4-D

362

significantly increased the activity and peak value of C4H. The peak values of the C4H activities

363

in 15 g kg−1 oligochitosan-, 5 g kg−1 chitosan-, and 2,4-D-treated fruits were 1.85, 1.47, and 1.4

364

times, respectively, that of ethephon-treated fruits (P < 0.05).

365

As shown in Fig. 8C, the 4CL activity in ethephon-treated fruits decreased during degreening.

366

The fruits treated with 2,4-D exhibited no significant difference with ethephon-treated fruits,

367

whereas the 4CL activities in 15 g kg−1 oligochitosan and 5 g kg−1 chitosan treatments increased

368

within a short period after the degreening treatment. The 4CL activities in 15 g kg−1

369

oligochitosan- and 5 g kg−1 chitosan-treated fruits were 50.4% and 50.3% higher than those in

370

ethephon-treated fruits 2 d after degreening, with the activity of 15 g kg−1 oligochitosan- treated

371

fruits exceeding that of ethephon-treated fruits during the entire degreening process.

372

These results indicated that 15 g kg−1 oligochitosan, 5 g kg−1 chitosan, and 2,4-D treatments

373

can promote lignin generation in ethephon-degreened fruits and delay the degradation of lignin

374

during late storage by increasing the activities of C4H and 4CL.



375

DISCUSSION

376

Treatment with ethylene to induce degreening enhances the abscission process (e.g., drying,

377

browning, and abscission), fungal penetration, and weight loss of citrus fruits.4,31 A significant

378

increase in disease incidence using ethylene at concentrations above those required for

379

degreening might also be associated with the activities of abscission enzymes, PG, and Cx.31 The

380

problems resulting from ethylene were further validated in the present study. The results of SEM

381

indicated that the ethephon degreening treatment could accelerate the formation of pedicel

382

abscission layers.

383

2,4-D has been widely used to retard the problems resulting from the ethylene degreening

384

treatment.11,12 2,4-D reduces the PG and Cx activities

5

385

contents in fruit peel.13 Previous studies have indicated that the control of stem end rot using

386

2,4-D is achieved by maintaining an intact and viable button that prevents fungus penetration.32

387

The effects of 2,4-D on the aspects of degreening citrus fruits were also further validated in the

388

present study. The results indicated that 2,4-D treatment delays the degradation of protopectin

389

and cellulose and the formation of lignin by regulating the activities of related enzymes (PG,

390

PME, Cx, 4CL, and C4H) and decreasing the ABA content at stem ends. Consequently, the

391

abscission of the stem end is delayed.

and increases the lignin and water

392

The effective regulation of ABA and structurally related compounds is of considerable

393

importance in controlling stem end abscission. Richardson and Cowan observed that the ABA

394

levels in navel and Valencia flavedo reached a maximum value along with the onset of a colour

395

break and that subsequent declines in the levels of ABA were correlated with the expression of

396

full colour development.33 Similar changes were observed in the present experiment. The colour


Page 18 of 39

Page 19 of 39


397

break and peak value of ABA in ethephon-treated fruits were both observed on day 4. 2,4-D

398

treatment retarded chlorophyll breakdown and delayed the ethylene-induced accumulation of

399

free ABA, consistent with the results of Brisker et al. 34

400

Compared with 2,4-D treatments, the problems resulting from the ethephon degreening

401

treatment of citrus fruits can also be partially resolved using 15 g kg−1 oligochitosan and 5 g kg−1

402

chitosan. The SEM results indicate that 15 g kg−1 oligochitosan and 5 g kg−1 chitosan delay the

403

formation of the pedicel abscission layer. Moreover, the abscission layer areas of these two

404

treatments are smaller than that of the 2,4-D treatment. The inhibition of these treatments of the

405

activities of PME, PG, and Cx significantly inhibit the degradation of propectin and cellulose,

406

and their promotion effects on C4H and 4CL activities stimulate the accumulation of lignin.

407

Similar to the 2,4-D treatment, the inhibition of ABA increase was also observed in these two

408

treatments. These results provide an adequate explanation for the findings of the SEM

409

experiment and improved control effect for calyx browning.

410

However, compared with ethephon-treated fruits, 2,4-D-treated fruits exhibited a relatively

411

poor colour, with a significantly lower chroma, higher H*, and lower accumulation of

412

carotenoids in peel at the early time of degreening process (3-4 day after ethephon treatment),

413

whereas 15 g kg−1 oligochitosan and 5 g kg−1 chitosan demonstrated no negative effect on the

414

colour formation of ethephon-degreened citrus fruits. Treatment with 15 g kg−1 oligochitosan

415

even promoted the accumulation of carotenoids.

416

Taken together, these findings suggest that the post-harvest application of 15 g kg−1

417

oligochitosan and 5 g kg−1 chitosan, particularly 15 g kg−1 oligochitosan, can be a potential

418

alternative to 2,4-D treatment to prevent calyx senescence of mandarin fruits induced by



419

ethephon degreening treatment.

420

ABBREVIATIONS

421

2,4-D, 2,4-dichlorophenoxyacetic acid; SEM, scanning electron micrographs; ABA, abscisic acid;

422

PG, polygalacturonic acid enzyme; Cx, cellulase; PME, pectin methylesterase; C4H, cinnamate

423

4-hydroxylase; 4CL, 4 - Coumarate: Coenzyme A Ligase.

424

ACKNOWLEDGEMENTS

425

This work was funded by the National Natural Science Foundation of China (Grant No.

426

31401540), the Fundamental Research Funds for the Central Universities (Grant No.

427

SWU113047), Chongqing Postdoctoral Science Foundation funded project (Grant No.

428

Xm2014106), and the Projects in the National Science & Technology Pillar Program during the

429

Twelfth Five-year Plan Period (2015BAD16B07).

430

REFERENCE

431

(1). Sdiri, S.; Navarro, P.; Monterde, A.; Benabda, J.; Salvador, A. New degreening treatments to improve the

432

quality of citrus fruit combining different periods with and without ethylene exposure. Postharvest Biol.

433

Technol. 2012, 63, 25-32.

434

(2). Zhou, J. Y.; Sun, C. D.; Zhang, L. L.; Dai, X.; Xu, C. J.; Chen, K. S. Preferential accumulation of

435

orange-colored carotenoids in Ponkan (Citrus reticulata) fruit peel following postharvest application of

436

ethylene or ethephon. Sci Hortic. 2010, 126, 229-235.

437 438 439 440

(3). Sdiri, S.; Navarro, P.; Ben Abda, J.; Monterde, A.; Salvador, A. Antioxidant activity and vitamin C are not

affected by degreening treatment of Clementine mandarins. Acta Horticulturae. 2010, 934, 893-900.

(4). Cohen, E. The effect of temperature and relative humidity during degreening on the coloring of Shamouti

orange fruit. J. Hort. Sci. 1978, 53, 143-146.


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(5). Brown, G. E.; Burns, J. K. Enhanced activity of abscission enzymes predisposes oranges to invasion by

Diplodia natalensis during ethylene degreening. Postharvest Biol. Technol. 1998, 14, 217-227.

(6). Barry, G. H.; van Wyk, A. A. Low-temperature cold shock may induce rind colour development of ‘Nules

Clementine’mandarin (Citrus reticulata Blanco) fruit. Postharvest Biol. Technol. 2006, 40, 82-88.

445

(7). Tietel, Z.; Weiss, B.; Lewinsohn, E.; Fallik, E.; Porat, R. Improving taste and peel color of early-season

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Satsuma mandarins by combining high-temperature conditioning and degreening treatments. Postharvest

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Biol. Technol. 2010, 57, 1-5.

448

(8). Ma, G.; Zhang, L.; Kato, M.; Yamawaki, K.; Kiriiwa, Y.; Yahata, M.; Ikoma, Y.; Matsumoto, H. Effect of the

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combination of ethylene and red LED light irradiation on carotenoid accumulation and carotenogenic gene

450

expression in the flavedo of citrus fruit. Postharvest Biol. Technol. 2015, 99, 99-104.

451 452 453 454

(9). Stander, O. P.; Theron, K. I.; Cronjé, P. J. Foliar 2, 4-D Application after Physiological Fruit Drop Reduces

Fruit Splitting of Mandarin. HortTechnol. 2014, 24, 717-723.

(10). Cronjé, P. J. R.; Crouch, E. M.; Huysamer, M. Postharvest calyx retention of citrus fruit. In V International

Postharvest Symposium. 2004, 682, 369-376.

455

(11). Ferguson, L.; Ismail, M.; Davies, F.; Wheaton, T. Pre- and postharvest gibberellic acid and 2,

456

4-dichlorophenoxyacetic acid applications for increasing storage life of grapefruit. Proceedings of Florida

457

State Horticultural Society. 1982, 95, 242-245.

458 459

(12). Carvalho, C. P.; Salvador, A.; Navarro, P.; Monterde, A.; Martínez-Jávega, J. M. Effect of auxin treatments

on calyx senescence in the degreening of four mandarin cultivars. HortScience. 2008, 43, 747-752.

460

(13). Ma, Q. L.; Ding, Y. D.; Chang, J. W.; Sun, X. H.; Zhang, L.; Wei, Q. J.; Deng, X. X. Comprehensive

461

insights on how 2, 4-dichlorophenoxyacetic acid retards senescence in post-harvest citrus fruits using

462

transcriptomic and proteomic approaches. J. Exp. Bot. 2014, 65, 61-74.



463

(14). Sdiri, S.; Navarro, P.; Salvador, A. Postharvest application of a new growth regulator reduces calyx

464

alterations of citrus fruit induced by degreening treatment. Postharvest Biol. Technol. 2013, 75, 68-74.

465

(15). Zeng, K. F.; Deng, Y. Y.; Ming, J.; Deng, L. L. Induction of disease resistance and ROS metabolism in navel

466 467 468 469 470

oranges by chitosan. Sci Hortic. 2010, 126, 223-228.

(16). Yin, H.; Zhao, X. M.; Du, Y. G. Oligochitosan: a plant diseases vaccine e a review. Carbohydr. Polym. 2010,

82, 1-8.

(17). Deng, L. L.; Zhou, Y. H.; Zeng, K. F. Pre-harvest spray of oligochitosan induced the resistance of harvested

navel oranges to anthracnose during ambient temperature storage. Crop Prot. 2015, 70, 70-76.

471

(18). Lim, P. K.; Jinap, S.; Sanny, M.; Tan, C. P., Khatib, A. The Influence of deep frying using various vegetable

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oils on acrylamide formation in Sweet Potato (Ipomoea batatas L. Lam) chips. J. Food Sci. 2013, 79:

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T115-T121

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(19). McGuire, R. G. Reporting of objective color measurements. HortScience. 1992, 27, 1254-1255.

475

(20). Lichtenthaler, H. K.; Wellburn, A. R. Determinations of total carotenoids and chlorophylls a and b of leaf

476 477 478

extracts in different solvent. Biochem. Soc. Trans. 1983, 603, 591-593.

(21). Knight, T. G.; Klieber, A.; Sedgley, M. Structural basis of the rind disorder oleocellosis in Washington navel

orange (Citrus sinensis L. Osbeck). Ann. Bot. 2002, 90, 765-773.

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(22). Ma, Z.; Ge, L.; Lee, A. S.; Yong, J. W. H.; Tan, S. N.; and Ong, E. S. Simultaneous analysis of different

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classes of phytohormones in coconut (Cocos nucifera L.) water using high-performance liquid

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chromatography and liquid chromatography-tandem mass spectrometry after solid-phase extraction. Anal.

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Chim. Acta. 2008, 610, 274-281.

483 484

(23). Kondo, S.; Tomiyama, H.; Rodyoung, A.; Okawa, K.; Ohara, H.; Sugaya, S.; Terahara, N.; Hirai, N.

Abscisic acid metabolism and anthocyanin synthesis in grape skin are affected by light emitting diode (LED)


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irradiation at night. J Plant Physiol. 2014, 171, 823-829.

(24). Agustí, M.; Almela, V.; Juan, M.; Alferez, F.; Tadeo, F. O.; Zacarías L. Histological and physiological

characterization of rind breakdown of 'Navelate' sweet orange. Ann. Bot. 2001, 88, 415-422.

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(25). Manganaris, G. A.; Vasilakakis, M.; Mignani, I.; Diamantidis, G.; Tzavella-klonari, K. The effect of

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preharvest calcium sprays on quality attributes, physicochemical aspects of cell wall components and

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susceptibility to brown rot of peach fruits (Prunus persica L. cv. Andross). Sci. Hortic. 2005, 107, 43-50.

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(26). Abu-Goukh, Abu-Bakr A.; Hind, A. Bashir. Changes in pectic enzymes and cellulase activity during guava

492 493 494 495 496 497 498 499 500 501 502 503 504 505 506

fruit ripening. Food Chem. 2003, 83, 213-218.

(27). Gusakov, A. V.; Kondratyeva, E. G.; Sinitsyn, A. P. Comparison of two methods for assaying reducing

sugars in the determination of carbohydrase activities. Int J Anal Chem. 2011, 1-4

(28). Deng, L. L.; Zeng, K. F.; Zhou, Y. H.; Huang, Y. Effects of postharvest oligochitosan treatment on

anthracnose disease in citrus (Citrus sinensis L. Osbeck) fruit. Eur Food Res Technol. 2015, 240, 795-804.

(29). Lamb, C. J.; Rubery, P. H. A spectrophotometric assay for transcinnamic acid 4 - hydroxylase activity. Anal.

Biochem. 1975, 68, 554-561.

(30). Knobloch, K. H.; Hahlbrock, K. Isoenzymes of p-Coumarate: CoA Ligase from cell suspension cultures of

glycine max. Eur. J. Biochem. 1975, 52, 311-320.

(31). Barmore, C. R.; Snowden, S. E.; Brown, G. E. Endopolygalacturonase from Valencia oranges infected with

Diplodia natalensis. Phytopathology. 1984, 74, 735-737.

(32). DeWolfe, T. A.; Erickson, L. C.; Brannaman, B. Retardation of Alternaria rot in stored lemons with 2, 4-D.

Proceedings of the American Society for Horticultural Science. 1959, 74, 364-367.

(33). Richardson, G. R.; Cowan, A. K. Abscisic acid content of Citrus flavedo in relation to colour development. J.

Hortic. Sci. 1995, 70, 769-773.



507 508

(34). Brisker, Hananya E.; Eliezer, E. Goldschmidt.; Raphael, Goren. Ethylene-induced formation of ABA in

citrus peel as related to chloroplast transformations. Plant Physiol. 1976, 58, 377-379.

509


Page 24 of 39

Page 25 of 39


510

Figure captions

511

Fig. 1. Colour symptoms of “ethephon-degreened mandarin fruits” stored at room

512

temperature (20 °C)

513

Wase satsuma mandarin samples were subjected to the following treatments: (1) control

514

(ethephon treatment, 1000 mg L−1), (2) ethephon + 50 mg kg−1 2,4-D treatment, (3) 15 g kg−1

515

oligochitosan + ethephon treatment, and (4) 5 g kg−1 chitosan + ethephon treatment. All fruits

516

were individually packaged and incubated at 20 °C and 85%–90 % RH for 10 d.

517 518

Fig. 2. Effects of oligochitosan, chitosan, and 2,4-D treatments on the Chl a content (A), Chl

519

b content (B), Chl a + b content (C), and carotenoid content (D) of ethephon-treated Wase

520

satsuma mandarin fruits during storage at 20 °C.

521


522


523


524

were individually packaged and incubated at 20 °C and 85%–90 % RH for 10 d. Each data point

525

represents the mean of three replicates. Bars indicate standard errors.

526 527

Fig. 3. Effects of oligochitosan, chitosan, and 2,4-D treatments on the browning index of

528

ethephon-treated Wase satsuma mandarin fruits during storage at 20 °C.

529


530


531




532


533

represents the mean of three replicates. Bars indicate the standard errors. The values for the same

534

day followed by different letters are significantly different according to Duncan’s multiple range

535

tests at P < 0.05.

536 537

Fig. 4. Effects of oligochitosan, chitosan, and 2,4-D treatments on the ABA content of

538


539


540


541


542


543


544 545

Fig. 5. Effects of oligochitosan, chitosan, and 2,4-D treatments on the ultrastructure of the

546

calyx abscission zone of ethephon-treated Wase satsuma mandarin fruits (×40) during

547

storage at 20 °C.

548


549


550


551

were individually packaged and incubated at 20 °C and 85%–90 % RH for 10 d.

552 553

Fig. 6. Effects of oligochitosan, chitosan, and 2,4-D treatments on the protopectin content


Page 26 of 39

Page 27 of 39


554

(A), water-soluble pectin content (B), polygalacturonase activity (C) and pectin

555

methylesterase activity (D) of ethephon-treated Wase satsuma mandarin fruits during

556

storage at 20 °C.

557


558


559


560


561


562 563

Fig. 7. Effects of oligochitosan, chitosan, and 2,4-D treatments on the cellulase activity of

564


565


566


567


568


569


570 571

Fig. 8. Effects of oligochitosan, chitosan, and 2,4-D treatments on the lignin content (A),

572

cinnamate-4-hydroxylase activity (B), and 4-coumarate: coenzyme A ligase activity (C) of

573


574


575




576


577


578

569 represents the mean of three replicates. Bars indicate standard errors.

579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597


Page 28 of 39

Page 29 of 39


Table 1. Analysis of peel colour after four degreening treatments

598

Days after degreening treatment (d)

0

L*

a*

b*

a* / b*

Chroma

3

6

9

Ethephon (ETH)(1000 mg L−1)

41.90±0.90 a

62.26±0.76 a

70.90±0.27 a

67.97±0.31 a

ETH+50 mg kg-1 2,4-D

41.90±0.90 a

59.99±0.71 b

68.67±0.62 a

69.39±0.38 a

ETH+15 g kg−1 Oligochitosan

41.90±0.90 a

60.86±1.27 ab

70.00±0.17 a

70.00±1.40 a

ETH+5 g kg−1 Chitosan

41.90±0.90 a

60.19±0.67 b

70.92±2.15 a

68.72±1.74 a

Ethephon (ETH) (1000 mg L−1)

−9.82±0.43 a

−1.13±0.13 a

11.04±0.67 a

15.28±1.06 a


−9.82±0.43 a

−3.83±0.10 c

8.50±0.51 b

14.59±0.69 a


−9.82±0.43 a

−2.82±0.19 b

11.04±0.44 a

15.70±1.14 a


−9.82±0.43 a

−2.62±0.09 b

10.59±1.13 a

15.52±0.38 a


26.83±0.80 a

56.44±0.95 a

69.03±1.95 a

67.73±2.40 a


26.83±0.80 a

53.04±2.26 b

66.86±1.09 a

68.98±1.67 a


26.83±0.80 a

53.80±1.22 ab

67.37±1.69 a

68.18±1.42 a


26.83±0.80 a

53.30±0.85 ab

67.74±1.03 a

67.04±0.65 a


−0.367±0.026 a

−0.02±0.002 a

0.160±0.005 a

0.225±0.008 ab


−0.367±0.026 a

−0.072±0.002 c

0.127±0.005 b

0.211±0.005 b


−0.367±0.026 a

−0.053±0.003 b

0.164±0.003 a

0.230±0.012 a


−0.367±0.026 a

−0.050±0.002 b

0.156±0.014 a

0.231±0.004 a


28.58±0.62 a

56.45±0.95 a

69.90±2.03 a

69.44±2.57 a


28.58±0.62 a

53.18±2.26 a

67.40±1.15 a

70.50±1.78 a


28.58±0.62 a

53.88±1.23 a

68.27±1.73 a

69.96±1.64 a



Hue

Page 30 of 39


28.58±0.62 a

53.37±0.84 a

68.57±1.19 a

68.82±0.71 a


110.14±1.34 a

91.15±0.12 c

80.92±0.30 b

77.30±0.43 ab


110.14±1.34 a

94.14±0.11 a

82.76±0.31 a

78.07±0.27 a


110.14±1.34 a

93.01±0.19 b

80.70±0.17 b

77.05±0.64 b


110.14±1.34 a

92.82±0.14 b

81.13±0.79 b

76.97±0.21 b

599

* Values of the same day followed by different letters are significantly different according to Duncan’s multiple

600

range test at P < 0.05.

601 602 603 604 605 606 607 608 609 610 611 612 613 614 615


Page 31 of 39


616 617

Figure 1

618

619 620 621 622 623 624 625 626 627 628



Page 32 of 39

629 630

Figure 2

631

180

ETH

ETH+2,4-D

ETH+COS

ETH+CTS

120

A Chlorophyll b content (mg kg-1 )

Chlorophyll a content (mg kg-1 )

240

120

60

0

B

98 76 54 32 10

0

2

4 6 Storage time (d)

8

10

0

350

2


8

70

D Carotenoid content (mg kg-1 )

Chlorophyll(a+b) content (mg kg-1 )

C 280

210

140

70

0

63

56

49

42

35 0

632

10

2


8

10

0

2

633 634 635 636 637 638 639 640



8

10

Page 33 of 39


641 642

Figure 3

643 3.60

ETH ETH+2，4-D

Browning index

3.20

ETH+1.5%COS ETH+0.5%CTS

a a

b ab

c

bc c

2.80

2.40

a a a a

2.00 4

644

8 Storage time (d)

10

645 646 647 648 649 650 651 652 653 654 655 656 657 658


c


659 660

Figure 4

661

Content of abscisic acid (g kg-1)

2.0

ETH ETH+2,4-D ETH+COS ETH+CTS

1.6 1.2 0.8 0.4 0.0 0

662

2


8

663 664 665 666 667 668 669 670 671 672 673 674 675 676


10

Page 34 of 39

Page 35 of 39


677 678

Figure 5

679

680 681 682



Page 36 of 39

683 684

Figure 6

685 16.0

A

ETH+2,4-D

Contents of protopectin (mg kg-1 )

12.8

ETH+COS ETH+CTS

10.6

8.4

6.2

13.0

10.0

7.0

4.0

4.0 0

2


8

0

10 1.1

6.0

Pectin methylesterase activity (mg kg-1 s-1 )

4.0 3.0 2.0

2


8

10

2


8

10

D

C

5.0 Polygalacturonase activity (mg kg-1 s-1 )

B

ETH Contents of water-soluble pectin (mg kg-1 )

15.0

0.9

0.7

0.5

1.0 0.3

0.0 0

686

2


8

10

0

687 688 689 690 691 692 693 694


Page 37 of 39


695 696

Figure 7

697 1.60

cellulase activity (µg kg-1 s-1)

1.45

1.30

1.15

1.00 0

698

2


8

699 700 701 702 703 704 705 706 707 708 709 710 711 712


10


713 714

Figure 8

715

Contents of lignin (OD280 kg-1)

2000

ETH ETH+2,4-D ETH+COS ETH+CTS

A

1650

1300

950

600 0

2


8

10

2


8

10

2


8

10

Cinnamate-4-hydroxylase activity (U kg-1 s-1)

3.5

B 2.9 2.3 1.7 1.1 0.5 0

4-Coumarate:Coenzyme A Ligase (U kg-1 s-1)

0.30

C

0.24 0.18 0.12 0.06 0.00 0

716 717 718 719 720 721


Page 38 of 39

Page 39 of 39


722

Graphic for table of contents (TOC graphic)

723 724


Postharvest Application of Oligochitosan and Chitosan Reduces Calyx

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