Changes in Cuticular Wax Composition of Two Blueberry Cultivars

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Changes in Cuticular Wax Composition of Two Blueberry Cultivars During Fruit Ripening and Postharvest Cold Storage Wenjing Chu, Haiyan Gao, Hangjun Chen, Wei-Jie Wu, and Xiangjun Fang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05020 • Publication Date (Web): 28 Feb 2018 Downloaded from http://pubs.acs.org on March 1, 2018

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Journal of Agricultural and Food Chemistry

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Changes in Cuticular Wax Composition of Two Blueberry Cultivars During

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Fruit Ripening and Postharvest Cold Storage

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WENJING CHU,† HAIYAN GAO,†,* HANGJUN CHEN,† WEIJIE WU,† AND XIANGJUN FANG†

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†

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of Post-Harvest Handling of Fruits, Ministry of Agriculture; Key Laboratory of Fruits

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and Vegetables Postharvest and Processing Technology Research of Zhejiang

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Province, 298 Middle Desheng Road, Hangzhou 310021, China

Food Science Institute, Zhejiang Academy of Agricultural Science; Key Laboratory

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*To whom correspondence should be addressed. Telephone: (+86) 571-86406661;

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Fax (+86) 571-86404378; E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Cuticular wax plays an important role for the quality of blueberry fruits.

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In this study, the cuticular wax composition of two blueberry cultivars, ‘Legacy’

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(Vaccinium corymbosum) and ‘Brightwell’ (V. ashei), were examined during fruit

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ripening and postharvest cold storage. The results showed that wax was gradually

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deposited on the epidermis of blueberry fruits, and the content of major wax

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compounds, except that for diketones, increased significantly during fruit ripening.

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The total wax content was 2-fold greater in ‘Brightwell’ blueberries than that in

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‘Legacy’ blueberries during fruit ripening. The total wax content of both cultivars

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decreased during 30 d of storage at 4 °C, and the variation of cuticular wax

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composition was cultivar-dependent. The content of diketones decreased significantly

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in ‘Legacy’ blueberries, while the content of triterpenoids and aliphatic compounds

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showed different fold changes in ‘Brightwell’ blueberries after 30 d of storage at 4 °C.

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Overall, our study provided a quantitative and qualitative overview of cuticular wax

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compounds of blueberry fruits during ripening and postharvest cold storage.

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KEYWORDS: Cuticular wax; blueberry; fruit ripening; cold storage; gas

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chromatography-mass spectrometry

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INTRODUCTION

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Blueberry fruits contain high concentrations of bioactive components, such as

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procyanidins, anthocyanins, flavonols and chlorogenic acid (1-3). These components

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have antimicrobial, antioxidant, anti-cancer, antidiabetic and cardio-protective

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activities. Blueberry is a popular fruit worldwide for their high nutritional value and

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health benefits. However, they are prone to postharvest decay, physical damage,

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softening, water loss and shriveling. Cuticular wax is a hydrophobic coat which

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consists of cutin and wax and surrounds the fruit epidermis. It creates an effective

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barrier against water loss, ultraviolet radiation and microbial invasion (4,5), and plays

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an important role in the postharvest quality of fruits. Previous studies in sweet cherry

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have shown that the postharvest quality and storability characteristics of fruits, such

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as weight loss, firmness and susceptibility to physiological disorders, are greatly

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affected by the chemical composition, structure and properties of the cuticle (6,7).

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Mukhtar and others (8) also reported that water loss and shelf life is negatively

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correlated with the wax content in European plum. Moggia and others (9) found that

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softening rates in blueberryare highly relevant to the content of ursolic acid in

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cuticular wax at harvest.

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Cuticular wax is a conspicuous white layer which is formed during fruit ontogeny

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in a wide range of fruits such as blueberry, grapes and plum (10). The biosynthesis of

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cuticular wax begins with C16 and C18 fatty acid synthesis in the plastids of

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epidermal cells. These molecules are then transported to the cytoplasm and further

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elongated to very-long-chain (VLC) fatty acids by the fatty acid elongase complex in

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the endoplasmic reticulum. The chain lengths of the VLC fatty acids range from C20

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to C34. They are converted either into corresponding alkanes, aldehydes, secondary

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alcohols and ketones or into primary alcohols and wax esters (11,12). The wax load

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and chemical composition vary widely among different species, cultivars and tissues,

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and are affected by ripening stages and environmental conditions (13–15). Liu and

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others (11) compared two cultivars of Citrus sinensis, ‘Newhall’ and ‘Glossy

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Newhall’, and found that their wax loads varied considerably during fruit ripening.

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Wu and others (12) observed significant differences in the chemical composition of

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cuticular wax in three Asian pear cultivars, ‘Kuerle’, ‘Xuehua’ and ‘Yuluxian’, during

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storage. Pensec and others (16) reported that high levels of triterpenoids existed in the

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cuticular wax of young grapes, but gradually decreased with a slight increase of

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neutral triterpenoids during fruit ripening.

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In our previous studies, we determined the chemical composition and morphology

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of cuticular wax in the mature fruit of nine blueberry cultivars (17) and investigated

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the effects of cuticular wax on the concentrations of bioactive compounds, activity of

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antioxidant enzymes and other quality indices during cold storage (18). Moggia and

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others (9) also explored the triterpenoid fraction of cuticular wax and its effect on the

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postharvest fruit behavior in blueberries. However, only limited information is

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available regarding the compositional changes in cuticular wax during fruit ripening

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and cold storage, and thus, the underlying mechanisms for this remain unclear.

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Therefore, the objective of this study was to assess the chemical composition of

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cuticular wax in two most widely cultivated highbush and rabbiteye blueberry

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cultivars in China, ‘Legacy’ (Vaccinium corymbosum) and ‘Brightwell’ (V. ashei),

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during ripening and cold storage. The results from this study are important in

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understanding changes in the development and chemical composition of cuticular wax,

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and its effect on the quality of blueberry fruits.

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MATERIALS AND METHODS

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Blueberry Samples. Blueberry fruits of two cultivars, ‘Legacy’ (V.corymbosum) and

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‘Brightwell’ (V.ashei), were respectively hand-picked at three different phenological

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stages (T1, green fruit; T2, red fruit; and T3, dark blue fruit) from a local orchard in

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Zhejiang Province, China in June 2015. Blueberry fruits were harvested using

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polyvinyl chloride gloves, placed in plastic baskets, and transported to the laboratory

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by a refrigerated boxcar (10 °C) within 2 h after harvest. Fruits which were of

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uniform size and color and showed no signs of physical damage were used for

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analysis. Fruits at three mature stages (T1, T2 and T3) were analyzed immediately

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after harvest for examining changes of wax content during fruit ripening. In addition,

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fruits at mature stage T3 were used of the storage study, and were subpackaged and

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stored at 4 °C and 90% relative humidity for 30 d.

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Chemicals and Standards. All solvents and reagents used for analysis and extraction

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were of analytical grade. Authentic standards of β-amyrin, α-amyrin and lupeol were

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purchased from Sigma-Aldrich (St. Louis, MO, USA). Oleanolic acid, ursolic acid

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and n-tetracosane were purchased from J&K Scientific (Beijing, China).

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Determination of Fruit Surface Area. The fruit surface area was determined as

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described by Chu et al. (17). The equatorial and polar diameters of each fruit were

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measured using a digital Verniercaliper (Guanglu Measuring Instrument, Guilin,

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China), and the fruit surface area was calculated according to the equation shown

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below:

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Surface area = πd2,

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Where d is the average of the equatorial and polar diameters of each fruit.

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Extraction and

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chromatography-mass spectrometry (GC-MS) analysis were performed as described

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by Chu et al. (17) with minor modifications. In brief, 20 fruits were submerged in 20

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ml chloroform and gently shaken for 1 min at 25 °C. The extraction was performed in

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duplicate and the two extracts were combined and filtered, 100µL (1µg/µL)

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n-tetracosane, an internal standard, was added to the combined and filtered extract.

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The extract containing internal standard was evaporated at 50 °C under a stream of

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nitrogen. The total wax extraction was repeated in triplicate for each sample. Extracts

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were

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bis-N,O-(trimethylsilyl)trifluoroacetamide (BSTFA; Alfa) for 40 min at 70 °C. Then,

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the remaining BSTFA was evaporated under a stream of nitrogen, and each sample

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was re-dissolved in chloroform. Wax compounds were analyzed by a Finnigan gas

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chromatograph (Trace GC Ultra) equipped with a DB-1 MS capillary column (30 m ×

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0.25 mm, i.d. ×0.25 µm; Agilent Technologies, Santa Clara, CA, USA) and attached

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to a Finnigan mass spectrometer (Trace DSQ). GC-MS conditions were as follows:

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column temperature, 2 min at 70 °C, increase from 70 °C to 200 °C at a rate of

incubated

Analysis of

in

Cuticular Wax. Wax extraction and

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µL

pyridine

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and

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gas

µL


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20 °C/min, 2 min at 200 °C, increase from 200 °C to 310 °C at a rate of 3 °C/min, and

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30 min at 310 °C; injection at 250 °C; MS transfer line at 250 °C; ion source at

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270 °C; carrier gas, helium (1.0 mL/min); 10:1 split ratio; EI 70 eV; and m/z 50–650.

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Wax compounds were identified either by the NIST11 MS Library or by comparing

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their mass spectra and retention times with those of generic standards. Triterpenoids

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were quantified using the external standard method based on calibration curves, while

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VLC aliphatic compounds were quantified by comparison with known amounts of the

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internal standard tetracosane (17). The results were expressed as an absolute (per unit

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of fruit surface area, µg/cm2) and relative (the percentage of each compound

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compared to the total wax content, %) value.

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Determination of Quality Parameters. Twenty blueberry fruits were randomly

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selected to measure the surface color (lightness, L*; change of greenness to redness, a*;

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and change of blueness to yellowness, b*) using the chromameter CR-400 (Konica

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Minolta Sensing, Tokyo, Japan). Firmness of blueberry fruit was measured using a

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TA.XT Plus Texture Analyzer (Stable Micro Systems Ltd., Godalming, U.K.) with a

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5 mm diameter stainless steel probe. Each fruit which peel was removed before

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determination was equatorially compressed by 5 mm distance at a speed of 1 mm/s

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with a 5-g trigger force, and the maximum force (N) was recorded. The weight loss 8


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was determined by weighing 100 fruits before and after the storage period and

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expressed as a percentage of the initial weight. The decay incidence was evaluated by

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the number of decayed fruits relative to the total number of fruits (100 fruits). Fruits

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with visible fungal growth or bacterial lesions on the surface were considered to be

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decayed.

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Total soluble solids (TSS) and titratable acidity (TA) were assessed in fruit juice

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obtained from 20 fruits. TSS was determined using a digital hand-held refractometer

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(Atago PAL-1, Tokyo, Japan). TA was determined by titration with 0.05 M NaOH to

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an end-point pH of 8.2, and the results were expressed as a percentage of citric acid.

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Statistical Analysis. All experiments were performed at least in triplicate, and the

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results were expressed as means ± standard deviation. One-way analysis of variance

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(ANOVA) in conjunction with Duncan’s multiple-range test was performed to

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identify statistically significant differences among the treatment levels at P < 0.05. All

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analyses were carried out using SPSS 21 (IBM, Armonk, NY, USA).

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RESULTS AND DISCUSSION

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Changes in Fruit Quality and Wax Content during Ripening. The quality

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parameters and cuticular wax contents of ‘Legacy’ and ‘Brightwell’ blueberries at 9



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three different phonological stages are shown in Table 1. The average weight,

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diameter, surface area per fruit, a*, TSS and TSS/TA significantly (P < 0.05)

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increased, whereas L*, b*, firmness and TA significantly (P < 0.05) decreased, with

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fruit ripening in both cultivars. The amounts of cuticular wax increased by 38.61% in

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‘Legacy’ blueberries and by 23.43% in ‘Brightwell’ blueberries from T1 to T3. The

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total wax content increased continuously during fruit ripening. This observation was

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supported by in the findings in several previous studies of orange (11), tomato (19)

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and pear (20). Within each phenological stage, no significant differences were

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observed in TSS, TA, and the tested physical parameters including average weight,

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diameter, surface area per fruit and skin color, between the two cultivars, whereas the

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total wax content in ‘Brightwell’ blueberries was 2-fold greater than that in ‘Legacy’

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blueberries.

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Changes in Wax Composition during Ripening. Triterpenoids. Triterpenoids are

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the primary cuticular wax compounds in grape (16), peach (21) and blueberry (9,17).

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As shown in Figure1, a significant (P < 0.05) increase in the content of triterpenoids

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was observed from T1 to T3 in both cultivars, suggesting that the biosynthesis of

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triterpenoids occurred during fruit ripening. However, changes in the relative content

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of triterpenoids were different between the two cultivars during fruit ripening. The

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relative content of triterpenoids did not change significantly from T1 to T3 in the

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‘Legacy’ blueberries but increased significantly (P < 0.05) in the ‘Brightwell’

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blueberries (Figure 2A). The relative content of triterpenoid acids increased

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significantly (P < 0.05), whereas that of triterpenoid alcohols decreased significantly

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(P < 0.05), during fruit ripening in both cultivars (Table 2). The deposition pattern of

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triterpenoids in cuticular wax differs among fruit species due to genetic and

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environmental factors (15). Peschel and others (22) reported that the content of

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triterpenes decreased during fruit ripening in sweet cherry. A decrease in the content

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of triterpenoids was also observed during fruit ripening in grape (16). However,

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triterpenoids continuously accumulated in cuticular wax during fruit ripening in

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tomato (19,23) and orange (24). We found that the contents of triterpenoids increased

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significantly (P < 0.05) during fruit ripening in both blueberry cultivars (Figure 1);

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and the primary triterpenoid was oleanolic acid in ‘Legacy’ blueberries and ursolic

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acid in ‘Brightwell’ blueberries (Table 2).

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VLC aliphatic compounds. VLC aliphatic compounds, including primary alcohols,

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alkyl esters, aldehydes, alkanes, secondary alcohols, ketones and fatty acids, have

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chain lengths ranging from C20 to C34 (14). As shown in Figure 1 and Table 2,

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diketones, aldehydes, primary alcohols, fatty acids and alkanes were identified in the

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cuticular wax of both cultivars, and the dominant VLC aliphatic compounds were

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β-diketones. However, hentriacontane-10,12-dione was found only in ‘Legacy’

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blueberries and tritriacontane-12,14-dione was found only in ‘Brightwell’ blueberries.

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No significant differences were identified in the absolute content of diketones among

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the three different phenological stages in both cultivars (Figure 1), whereas the

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relative content of diketones decreased significantly (P < 0.05) from T1 to T3 (Figure

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2B). The content of aldehydes, primary alcohols, fatty acids and alkanes increased

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from T1 to T3 in both cultivars (Figure 1), indicating that these wax compounds

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gradually accumulated during blueberry fruit ripening. However, a different

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deposition rate was found among the different wax classes during fruit ripening. In

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‘Legacy’ blueberries, the relative content of aldehydes, primary alcohols and fatty

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acids increased by 2.71-fold (from 2.616 to 7.092%), 4.75-fold (from 1.036 to

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4.917%), and 2.66-fold (from 1.319 to 3.511%), respectively, from T1 to T3, whereas

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the relative content of alkanes decreased by 1.17-fold (from 1.726 to 1.475%). In

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‘Brightwell’ blueberries, the relative content of all VLC compounds showed different

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fold increases from T1 to T3 (Figure 2C–F).

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Changes in Fruit Quality and Wax Content during Cold Storage. Changes in the

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quality attributes of ‘Legacy’ and ‘Brightwell’ blueberries harvested at T3 during cold

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storage are shown in Table 3. The average weight, diameter and surface area per fruit

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of both cultivars did not change significantly after 30 d of storage at 4 °C. However,

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the total wax content decreased significantly (P < 0.05) with storage times in both

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cultivars, and similar results were obtained in previous studies involving sweet cherry

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(6), Asian pear (12) and apple (25).

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The weight loss increased significantly (P < 0.05) with the increasing storage time

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in both cultivars; however, it was more in ‘Brightwell’ blueberries than in ‘Legacy’

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blueberries (Table 3). To a large extent, the postharvest weight loss in fruits is

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attributed to water loss, and postharvest water loss in fruits is restricted by cuticular

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wax (26). However, the postharvest water loss is not related to the total wax content

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whereas closely related to lipid composition of the cuticle (27,28). Previous studies

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showed that the high ratio of n-alkanes to triterpenoids in cuticular wax decreases the

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water loss rate during postharvest storage of pepper, peach and sweet cherry (6,21,27).

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Moggia and others (9) also found that ursolic acid content in cuticular wax was highly

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correlated with weight loss in the blueberry cultivars ‘Duke’ and ‘Brigitta’. We found

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that weight loss was negatively correlated with the ratio of n-alkanes to triterpenoids

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(r = -0.82) in ‘Legacy’ blueberries and also with the contents of primary alcohols and

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fatty acids (r = -0.96) in ‘Brightwell’ blueberries. In blueberries, the intracuticular

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wax layer, which is composed of aliphatic compounds (29), functions as a

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transpiration barrier and thus, the weight loss is determined by the content of alkanes,

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primary alcohols and fatty acids.

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Cuticular wax is one of the major barriers against fungal infections and can inhibit

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the conidial germination and mycelial growth of some fungi such as Alternaria

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alternata (20). Gabler and others (30) reported that wax concentration was positively

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correlated with resistance to mold in grape. We found that the decay incidence

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increased significantly (P < 0.05) after 15-30 d of storage in both cultivars; however,

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the decay incidence was 40.43% lower in ‘Brightwell’ blueberries than that in

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‘Legacy’ blueberries after 30 d of storage. This may be partially due to the higher

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cuticular wax content in ‘Brightwell’ blueberries (Table 3).

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Changes in Wax Composition during Postharvest Cold Storage. Changes in the

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cuticular wax composition of ‘Legacy’ and ‘Brightwell’ blueberries during cold

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storage are shown in Figure3 and Table 4. Two triterpenoid alcohols, amyrin and

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lupeol, and two triterpenoid acids, oleanolic and ursolic acids, were identified in the

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relative content of triterpenoids in both cultivars. However, large differences were

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detected between ‘Legacy’ and ‘Brightwell’ blueberries for all of the identified

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compounds. Similar results were also reported by Moggia and others (9). During cold

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storage, the absolute and relative content of triterpenoids did not change significantly

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in ‘Legacy’ blueberries. However, the absolute content of triterpenoids decreased

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from 123.01 µg/cm2 to 108.36 µg/cm2, whereas the relative content increased from

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68.69 to 73.67% after 30 d of storage in ‘Brightwell’ blueberries. This was probably

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due to a significant (P < 0.05) increase in the relative amount of ursolic acid.

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Cultivar-related differences have also been observed in the content of triterpenoids in

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cuticular wax during postharvest storage in peach and sweet cherry (6,21).

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A total of 34 aliphatic compounds, including diketones (15.44–18.77%), aldehydes

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(C24-C32, 1.36–7.09%), primary alcohols (C22-C30, 2.25–4.92%), fatty acids

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(C16-C30, 1.69–3.51%), and alkanes (C23-C31, 0.89–1.47%), were detected in both

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cultivars (Supporting information; Table 4). In ‘Legacy’ blueberries, the content of

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diketones decreased from 16.43 µg/cm2 to 14.10 µg/cm2 during the entire 30 d storage

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period. No significant change was identified in the content of other aliphatic

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compounds during cold storage (Figure 3). In ‘Brightwell’ blueberries, significant

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decreases in the contents of aliphatic compounds were observed during the tested 30 d

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storage period. Specifically, the contents of diketones, aldehydes, primary alcohols,

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fatty acids and alkanes decreased by 34.6, 36.36, 32.4, 37.9 and 22.4%, respectively

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(Figure 3).

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Overall, our results showed that cuticular wax was gradually deposited on the

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epidermis of blueberry during fruit ripening, and played a vital role in postharvest

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storage quality of blueberries possibly due to reducing the water loss and infection

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susceptibility. The major wax compounds included triterpenoids, aldehydes, fatty

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acids, primary alcohols and alkanes. Also noted was that the total wax contents and

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compounds might differ in different blueberry cultivars and decrease during storage.

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The results not only provided aquantitative and qualitative overview of cuticular wax

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compounds of blueberries at different phenological stages and postharvest storage

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periods, but also warranted additional research in investigate how wax components

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may be accumulated during blueberry ripening, and how wax components may

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contribute to postharvest storage quality of blueberries.

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ACKNOWLEDGEMENT

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This work was supported by the National Natural Science Foundation of China (Grant

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No. 31772042, 31471635), the National Science & Technology Support Program of

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China (Grant No. 2015BAD16B06) and the National Special Fund for Agro-scientific

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Research in the Public Interest, China (Grant No.201303073).

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Supporting Information.

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Table S1 and S2 list the wax constituents (relative %) identified on ‘Legacy’ and

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‘Brightwell’ blueberries at phenological stage and after cold storage, respectively.

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This material is available free of charge via the Internet at http://pubs.acs.org.

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A.; Jetter, R. Composition of cuticular waxes coating flag leaf blades and peduncles

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of Triticum aestivum cv. Bethlehem. Phytochem. 2016, 130, 182-192.

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(15) Vichi, S.; Cortés-Francisco, N.; Caixach, J.; Barrios, G.; Mateu, J.; Ninot, A.;

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Romero, A. Epicuticular wax in developing olives (Olea europaea) is highly

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dependent upon cultivar and fruit ripeness. J. Agric. Food. Chem. 2016, 64,

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5985-5994.

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Bertsch, C.; Chong, J.; Szakiel, A. Changes in the triterpenoid content of cuticular

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waxes during fruit ripening of eight grape (Vitis vinifera) cultivars grown in the Upper

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Rhine Valley. J. Agric. Food. Chem. 2014, 62, 7998-8007.

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(17) Chu, W.; Gao, H.; Cao, S.; Fang, X.; Chen, H.; Xiao, S. Composition and

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morphology of cuticular wax in blueberry (Vaccinium spp.) fruits. Food Chem. 2017,

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219, 436-442.

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developmental pattern of tomato fruit wax accumulation and its impact on cuticular

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transpiration barrier properties: effects of a deficiency in β-ketoacyl-coenzyme A

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synthase (LeCER6). Plant Physiol. 2007, 144, 1667-1679.

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(20) Li, Y.; Yin, Y.; Chen, S.; Bi, Y.; Ge, Y. Chemical composition of cuticular

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waxes during fruit development of Pingguoli pear and their potential role on early

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events of Alternaria alternata infection. Funct. Plant Biol. 2014, 41, 313-320.

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(21) Belge, B.; Llovera, M.; Comabella, E.; Graell, J.; Lara, I. Fruit cuticle

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composition of a melting and a nonmelting peach cultivar. J. Agric. Food. Chem.

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2014, 62, 3488-3495.

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(22) Peschel, S.; Franke, R.; Schreiber, L.; Knoche, M. Composition of the cuticle of developing sweet cherry fruit. Phytochem. 2007, 68, 1017-1025.

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(23) Kosma, D. K.; Parsons, E. P.; Isaacson, T.; Lü, S.; Rose, J. K. C.; Jenks, M. A.

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Fruit cuticle lipid composition during development in tomato ripening mutants.

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Physiol. Plant. 2010, 139, 107-117.

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J.; Deng, X.; Cheng, Y. Regulation of cuticle formation during fruit development and

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ripening in 'Newhall' navel orange (Citrus sinensis Osbeck) revealed by

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transcriptomic and metabolomic profiling. Plant Sci. 2016, 243, 131-144.

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(25) Dong, X.; Rao, J.; Huber, D. J.; Chang, X.; Xin, F. Wax composition of 'Red

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Fuji' apple fruit during development and during storage after 1-methylcyclopropene

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treatment. Hortic. Environ. Biotechnol. 2012, 53, 288-297.

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(26) Lara, I.; Belge, B.; Goulao, L. F. The fruit cuticle as a modulator of postharvest quality. Postharvest Biol. Technol. 2014, 87, 103-112.

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fruit post-harvest water loss in an advanced backcross generation of pepper

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(Capsicum sp.). Physiol. Plant. 2012, 146, 15-25.

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(28) Parsons, E. P.; Popopvsky, S.; Lohrey, G. T.; Alkalai-Tuvia, S.; Perzelan, Y.;

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Bosland, P.; Bebeli, P. J.; Paran, I.; Fallik, E.; Jenks, M. A. Fruit cuticle lipid

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composition and water loss in a diverse collection of pepper (Capsicum). Physiol.

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Plant. 2013, 149, 160-174.

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Riederer, M. Tomato fruit cuticular waxes and their effects on transpiration barrier

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properties: functional characterization of a mutant deficient in a very-long-chain fatty

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acid β-ketoacyl-CoA synthase. J. Exp. Bot. 2004, 55, 1401-1410.

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(30) Gabler, F. M.; Smilanick, J. L.; Mansour, M.; Ramming, D. W.; Mackey, B. E.

383

Correlations of morphological, anatomical, and chemical features of grape berries

384

with resistance to Botrytis cinerea. Phytopathol. 2003, 93, 1263-1273.

385

386

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387

Figure Captions

388

Figure 1. Content of major wax compounds identified in ‘Legacy’ (A) and

389

‘Brightwell’ (B) blueberries at three phenological stages (T1, T2, and T3). Different

390

letters indicate significant differences among phenological stages within each class of

391

wax compounds at P < 0.05.

392 393

Figure 2. Relative content of triterpenoids (A), diketones (B), aldehydes (C), primary

394

alcohols (D), fatty acids (E) and alkanes (F) in ‘Legacy’ and ‘Brightwell’ blueberries

395

at three phenological stages (T1, T2, and T3). Different letters indicate significant

396

differences among phenological stages within each cultivar at P < 0.05.

397 398

Figure 3. Content of major wax compounds identified in ‘Legacy’ (A) and

399

‘Brightwell’ (B) blueberries after 0 d, 15 d, and 30 d of storage at 4°C. Different

400

letters indicate significant differences among storage periods within each class of wax

401

compounds at P < 0.05.

402 403 404

23



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405

Tables

406

Table 1. Quality Attributes of Fresh ‘Legacy’ and ‘Brightwell’ Blueberries at Three Phenological Stages (T1, T2 and T3) Legacy Parameter

T1

Weight per fruit (g) Diameter (mm) Fruit surface (cm2/fruit)

T3

T1

T2

T3

0.97 ± 0.13c

1.45 ± 0.10 b

1.76 ± 0.12 a

1.05 ± 0.16 c

1.52 ± 0.10 b

1.81 ± 0.11 a

11.21 ± 0.56 c

12.06 ± 0.54 b

13.08 ± 0.39 a

11.29 ± 0.61 c

12.34 ± 0.69 b

13.17 ± 0.57 a

3.96 ± 0.39 c

4.58 ± 0.41 b

5.37 ± 0.32 a

4.01 ± 0.44 c

4.79 ± 0.53 b

5.46 ± 0.47 a

43.84 ± 4.80 b 32.23 ± 1.93 d

65.03 ± 3.96 a

46.29 ± 4.84 b

35.01 ± 2.95 c

*

L

62.96 ± 4.22 a

a*

-11.67 ± 5.10 d

11.64 ± 2.99 b

0.43 ± 0.78 c

-10.42 ± 2.80 d

14.53 ± 3.26 a

0.62 ± 1.77 c

*

28.85 ± 4.73 a

2.05 ± 3.54 c

-5.03 ± 0.84 d

24.50 ± 3.12 b

2.96 ± 3.12 c

-4.63 ± 0.89 d

Firmness (N)

5.12 ± 0.61 b

2.57 ± 0.36 c

1.20 ± 0.21 d

5.58 ± 1.00 a

2.89 ± 0.35 c

1.36 ± 0.21 d

o

TSS ( Brix)

8.17 ± 0.25 c

9.33 ± 0.25 b

11.63 ± 0.15 a

8.13 ± 0.31 c

9.70 ± 0.20 b

11.97 ± 0.21 a

TA (%)

2.42 ± 0.04 a

1.89 ± 0.07 b

0.59 ± 0.01 c

2.44 ± 0.09 a

1.84 ± 0.05 b

0.57 ± 0.01 c

3.37 ± 0.08 d

4.95 ± 0.13 c 19.61 ± 0.22 b

3.34 ± 0.14 d

5.27 ± 0.21 c

21.10 ± 0.72 a

145.12 ± 10.39 b 164.63 ± 8.45 a

179.10 ± 4.83 a

b

TSS/TA 2

Total wax content (µg/cm )

407

T2

Brightwell

63.17 ± 3.44 d 73.65 ± 1.96 cd

87.60 ± 4.60 c

TSS, total soluble solids; TA, titratable acidity. Different letters indicate significant differences within each row at P < 0.05.

408

24


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409

Table 2. Relative Content (%) of Triterpenoids and Diketones in Fresh ‘Legacy’ and ‘Brightwell’ Blueberries at Three Phenological Stages (T1,

410

T2 and T3)a Legacy

Brightwell

T1

T2

T3

T1

T2

T3

β-amyrin

16.772 ± 1.176 a

12.526 ± 1.241 b

11.125 ± 0.294 b

4.474 ± 0.122 a

3.811 ± 0.250ab

3.666 ± 0.232 b

α-amyrin

2.145 ± 0.258 a

1.846 ± 0.158 a

1.954 ± 0.114 a

10.399 ± 0.058 a

9.082 ± 0.267 b

8.763 ± 0.250 b

Lupeol

5.782 ± 0.159 a

5.482 ± 0.586 a

4.031 ± 0.185 b

4.601 ± 0.186 a

3.783 ± 0.287ab

2.969 ± 0.346 b

26.932 ± 0.591 b

28.740 ± 0.401 a

29.845 ± 0.013 a

9.530 ± 0.243 b

10.736 ± 0.369 a

10.922 ± 0.313 a

5.551 ± 0.330 b

8.470 ± 0.994 a

8.881 ± 0.029 a

37.999 ± 0.812 c

40.368 ± 0.604 b

42.372 ± 0.216 a

Hentriacontane-10,12-dione

24.896 ± 0.522 a

20.423 ± 1.611 b

18.775 ± 0.570 b

nd

Tritriacontane-12,14-dione

nd

nd

nd

19.468 ± 0.495 a

Triterpenoids

Oleanolic acid Ursolic acid

Diketones nd 17.138 ± 0.597 b

nd 15.444 ± 0.458 c

411

a

412

means ± standard deviation of three replicates. Different letters indicate significant differences within each row and cultivar at P < 0.05.

Relative content (%) was regarded as percentage of each compound compared to the total wax content. nd, non-detectable. Values represent

413

25



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414 415

Table 3. Quality Attributes of ‘Legacy’ and ‘Brightwell’ Blueberries after 0 d, 15 d and 30 d of Storage at 4 °C Parameter

Legacy

Brightwell

0d

15 d

Weight per fruit (g)

1.76 ± 0.12 ab

1.74 ± 0.10 ab

1.71 ± 0.10 b

1.81 ± 0.11 a

1.79 ± 0.11 a

1.75 ± 0.11 ab

Diameter (mm)

13.08 ± 0.39 a

13.11 ± 0.41 a

12.95 ± 0.54 a

13.17 ± 0.57 a

13.12 ± 0.55 a

12.89 ± 0.44 a

5.37 ± 0.32 a

5.40 ± 0.34 a

5.27 ± 0.44 a

5.46 ± 0.47 a

5.41 ± 0.46 a

5.22 ± 0.36 a

87.60 ± 4.60 c

65.61 ± 6.17 d

80.41 ± 5.71 cd

179.10 ± 4.83 a

138.23 ± 9.32 b

147.07 ± 6.10 b

Weight loss (%)

-

1.55 ± 0.12 d

3.80 ± 0.12 b

-

1.86 ± 0.09 c

4.19 ± 0.18 a

Decay incidence (%)

-

3.82 ± 1.29 c

15.7 ± 1.58 a

-

2.85 ± 1.26 c

11.18 ± 1.83 b

Fruit surface (cm2/fruit) 2

Total wax content (µg/cm )

30 d

0d

15 d

30 d

416

Different letters indicate significant differences within each row at P < 0.05. Blueberries used in this storage study were harvested at T3

417

phenological stage.

418

419

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420


Table 4. Relative Content (%) of Wax Compounds in ‘Legacy’ and ‘Brightwell’ Blueberries after 0 d, 15 d and 30 d of Storage at 4 °Ca Legacy

Brightwell

0d

15 d

30 d

Triterpenoids

55.837 ± 0.323 a

55.421 ± 0.411 a

56.987 ± 0.955 a

β-amyrin

11.125 ± 0.294 a

10.965 ± 0.492 a

11.766 ± 0.826 a

3.666 ± 0.232 a

3.548 ± 0.141 a

4.013 ± 0.057 a

α-amyrin

1.954 ± 0.114 a

1.291 ± 0.074 b

1.699 ± 0.073 a

8.763 ± 0.250 a

8.597 ± 0.104 a

9.273 ± 0.313 a

Lupeol

4.031 ± 0.185 a

3.099 ± 0.476 a

3.678 ± 0.879 a

2.969 ± 0.346 a

2.698 ± 0.120 a

3.133 ± 0.077 a

29.845 ± 0.013 a

31.404 ± 0.733 a

30.846 ± 1.245 a

10.922 ± 0.313 a

10.842 ± 0.615 a

10.491 ± 0.508 a

Ursolic acid

8.881 ± 0.029 a

8.662 ± 0.101 a

8.998 ± 0.415 a

42.372 ± 0.216 b

46.162 ± 0.935 a

46.756 ± 0.265 a

Diketones

18.775 ± 0.570 a

18.200 ± 0.744 a

17.581 ± 1.206 a

15.444 ± 0.458 a

13.020 ± 0.252 b

12.300 ± 0.371 b

Hentriacontane-10,12-dione

18.775 ± 0.570 a

18.200 ± 0.744 a

17.581 ± 1.206 a

nd

nd

nd

Tritriacontane-12,14-dione

nd

nd

nd

15.444 ± 0.458 a

13.020 ± 0.252 b

12.300 ± 0.371 b

Oleanolic acid

0d

15 d

68.692 ± 0.665 b 71.847 ± 1.467 ab

30 d 73.667 ± 0.691 a

Aldehydes

7.092 ± 0.446 a

6.160 ± 0.243 a

6.178 ± 0.241 a

1.359 ± 0.088 a

1.073 ± 0.097 b

1.056 ± 0.049 b

Primary alcohols

4.917 ± 0.392 a

5.210 ± 0.061 a

4.957 ± 0.442 a

2.247 ± 0.219 a

2.419 ± 0.244 a

1.856 ± 0.185 a

Fatty acids

3.511 ± 0.166 a

3.538 ± 0.251 a

3.227 ± 0.094 a

1.695 ± 0.240 a

1.481 ± 0.095 a

1.286 ± 0.046 a

Alkanes

1.475 ± 0.033 a

1.483 ± 0.049 a

1.417 ± 0.062 a

0.893 ± 0.039 a

0.812 ± 0.020 a

0.844 ± 0.011 a

421

a

422

means ± standard deviation of three replicates. Different letters indicate significant differences within each cultivar at P < 0.05. Blueberries used

Relative content (%) was regarded as percentage of each compound compared to the total wax content. nd, non-detectable. Values represent

27



423

in this storage study were harvested at T3 phenological stage.

28


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424

Figures

425 426 427 428 429 430 431

Figure 1.

432 433 434 435 436 437 438 439 440 441

29



442 443 444 445 446 447 448 449 450 451 452 453 454

Figure 2.

455 456 457 458 459

30


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460 461 462 463 464 465 466

Figure 3.

467 468 469

31



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Changes in Cuticular Wax Composition of Two Blueberry Cultivars

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