Quality Attributes and Microstructure of Cell Walls in ‘Suli’ Plum Fruit (Prunus salicina Lindl.) during Softening

Abstract

The quality attributes and cell wall materials of ‘Suli’ plums stored at 4 °C and 25 °C with 80%–90% relative humidity were investigated. Rapid changes in firmness, soluble solids content (SSC), titratable acidity (TA), and decay rate at 25 °C were evaluated. These changes were suppressed in the plums stored at 4 °C. Modification of cell walls in the ‘Suli’ plums was investigated by Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM). FTIR and AFM indicated that low temperature significantly maintained cell wall integrity, as well as suppressed the conversion of protopectin and degradation of pectin during storage. The results confirmed that the microstructures and composition of the cell wall in ‘Suli’ plums were closely related to firmness, which is considered the main index of fruit softening.

Introduction

Plums (Prunus salicina Lindl.) are widely distributed all over Asia because of their flavor and taste. These fruits are rich in carbohydrates, organic acids, cellulose, proteins, and vitamins (particularly, vitamin C). However, plums have a short post-harvest shelf life because of softening. Reduced firmness limits the sales of plums, restricting their presence in the market to the main production area. Therefore, the mechanisms underlying the softening of plums need to be clarified, and a solution has to be determined.

Fruit softening is a multifaceted process. Numerous studies show that fruit softening is closely associated with the alteration of components and microstructure of cell walls (Brummell et al., 2004; Rosli et al., 2004). Pectin and cellulose are currently considered the building blocks of the fruit cell wall, with pectin significantly affecting rigidity and being widely present in the middle lamella of the cell wall. Pectins are complex structural domains composed of homogalacturonans, type I rhamnogalacturonans, and type II rhamnogalacturonans (Bonnin et al., 2002). These structural units are integrated by (1→4)-α-D-GalA (galacturonic acid) and linkages (Round et al., 2010; Paniagua et al., 2014; Willats et al., 2001). During fruit ripening, polysaccharides in flesh cell walls exhibit depolymerization and solubility, thereby changing the morphology and profile of pectin structural domains (Vicente et al., 2005). Despite these numerous reports, the existence of pectin in the cell wall of softening plums has rarely been explored.

Conventional analysis of enzymatic activity fails to reflect the structural transformation of fruit pectins. Variations in pectin content and molecular composition also provide insufficient information about the structure of pectin (Missang et al., 2001). The association between the texture changes and the cell wall pectin has been demonstrated (Manganaris et al., 2008). Furthermore, atomic force microscopy (AFM) has been successfully applied in component analysis of fruit cell walls, such as pectins from unripe tomatoes (Kirby et al., 2008;); the branching of pectin from marsh cinquefoil (Ovodova et al., 2006); and quantitative analysis of pectin molecules (Yang et al., 2006). In the aforementioned reports, water-soluble pectin (WSF), chelate-soluble pectin (CSF), and sodium carbonate-soluble (SSF) pectin were analyzed by AFM and found to be related to fruit softening (Brummell et al., 2006). The depolymerization and solubility of pectins during softening were observed by AFM, which exerts a probe-broadening effect on the cantilever (Decho., 1999; Adams et al., 2003). Further understanding of the application of different spectroscopic techniques is needed to improve the characterization of the cell wall.

Owing to its capacity for rapid testing, high sensitivity, and simplicity of sample preparation, Fourier transform infrared (FTIR) microspectroscopy can provide microanalysis and nondestructive testing. This method has been proved to provide accurate compositional and structural information on cell walls is suitable for studying the topochemistry of main components at the cellular level (Simón et al., 2004; Barron et al., 2005; Bichara et al., 2016; Abidi et al., 2014). In addition, only a small number of samples are needed to accomplish FTIR detection. Therefore, the application of FTIR spectroscopy would elucidate the microstructural changes in the cell wall of plums after storage.

In the current study, the ‘Suli’ plum was selected as a research object from a widely planted area in Guizhou Province, China for its more softing properties than other varieties. This study aimed to identify modifications in the quality attributes and the microstructures of cell walls in ‘Suli’ plums during softening. The firmness, physicochemical properties, and contents of the water-soluble fraction (WSF), chelate-soluble fraction (CSF), and sodium carbonate-soluble fraction (SSF) were also examined and quantified. The degradation mechanism of the plum cell wall was proposed. Two storage conditions were chosen for comparative research. The results are expected to contribute to a theoretical guide for plum producers and distributors.

Materials and Methods

Fruit materials    Firm ‘Suli’ plums were harvested at their maturity stage in the morning from an orchard in Qingzhen, Guiyang, China. The plums were immediately transported for 95 km to the storage and processing laboratory after harvest. The plums without physical injuries or visual blemishes/infections were selected following the principle of uniform shape, size, and color (Pan et al., 2018). Ten plums were used for initial quality attribute and pectin extraction. For the experiment, about 1 000 fruits were selected and then divided into two treatment groups. The two groups of plum were placed into plastic baskets. One group was directly stored at 25 °C in an area with 80%–90% relative humidity (the average monthly temperature and RH at the time of local plum harvest). The other group was pre-cooled to 4 °C overnight in a refrigerator prior to storage in an area with 80%–90% relative humidity. Correlation analysis was performed periodically during storage, except for the decay rate. Separate sets of plums in each group were counted to determine the decay rate. Each treatment was performed in triplicate.

Firmness, titratable acidity, soluble solids content, and decay rate    Firmness was measured using an FTC TMS-TOUCH texture analyzer (FTC Inc., Sterling, USA) as described by Luo (2010), with slight modifications. The flesh of plums without skin and stem was used. Ten fruits were measured individually for each analysis with the following parameters: load cell = 25 kg, probe = 5 mm diameter aluminum cylinder, test speed = 1 mm/s, and deformation = 75%. Firmness was defined as the maximum penetration force (N) during tissue breakage.

Titratable acidity (TA) as percent malic acid was assayed using an indicator to titrate 20 mL of diluted juice (10.0 g of mashed plum was diluted to 100 mL with distilled water) with 0.1 mol L⁻¹ NaOH. Phenolphthalein was selected to indicate the endpoint of titration. Titration was ended when the solution changed its color from lucency to pink without fading in 30 s. The soluble solids content (SSC) was analyzed using a refractometer (LB10T, Guangdong, China) at 25 °C. The results for SSC were expressed in% (Xin et al., 2010). The ‘Suli’ plums that showed visible symptoms of disease, regardless of severity, were considered rotten. These plums were counted, and the result was expressed as a percentage of the total number (Sharma et al., 2016).

Cell wall material extraction and content    Cell wall materials (CWMs) were extracted three times for each sampling (5 plums per extraction) by using the method described by Chen et al (2011), with slight modifications. Fresh flesh without skin (10 g) was boiled in 200 mL ethanol (80%, v/v) for 20 min after being ground. Ethanol was then removed by filtration. The solid residue was immersed in 50 mL dimethyl sulfoxide: H₂O (9:1, v/v) for 12 h in an environment set at 4 °C. The residue was subsequently transferred to a 2:1 chloroform–ethanol (v/v) solution for 10 min. The mixed liquor was then washed with 200 mL acetone with the use of a filter. The residue was used as a cell wall material. Additional CWMs were extracted for scanning electron microscopy (SEM) and FTIR.

Three different cell wall substances were obtained by stepwise extraction, depending on their solubility in different solutions. The CWMs from each group were suspended and stirred in 10 mL distilled water for 4 h at room temperature. The solution was centrifuged at 8000 g for 10 min by using a refrigerated centrifuge at 4 °C. The supernatant was collected, and the residual precipitation was subjected to reiteration extraction two more times. The supernatant obtained after centrifugation with three times as much force was then mixed and then designated as WSF. The residual pellet was extracted three times with 10 mL of 0.05 mol L⁻¹ cyclohexane-trans-1,2-diamine tetra-acetate (CDTA) under the same extraction conditions. The slurry was centrifuged to obtain a chelate-soluble fraction (CSF). The insoluble pellet after CSF extraction was further extracted in 10 mL of 0.05 mol L⁻¹/0.002 mol L⁻¹ Na₂CO₃/CDTA to obtain SSF.

The carbazole colorimetric method was used to determine the contents of the three fractions, with slight modifications (Liu et al., 2009). About 1 mL of the sample was mixed with 6 mL of sulfuric acid (98% w/w) in a test tube and then cooled using tap water; the mixture was then held for 20 min in a boiling water bath and again cooled to room temperature. The mixture was added with 0.2 mL of carbazole ethanol solution. Subsequently, the solution was stored in a dark place for 30 min at room temperature. A UV-2102 spectrophotometer (Unico Instrument Co. Ltd., Shanghai, China) was used to analyze the absorbance at 540 nm, with galacturonic acid as the standard. The results were expressed in g kg⁻¹ on a fresh-weight basis (Keutgen and Pawelzik, 2007).

Scanning electron microscopy and Fourier transform infrared spectroscopy    The CWMs were dried in a desiccator with discoloration silica gel at room temperature to remove the organic and inorganic solvents. The dried CWMs were transferred to an aluminum SEM stub and then fixed with a conductive adhesive tape. Gold powder with a thickness of 25 nm was sprayed on the surface of the samples (E1010, Hitachi, Tokyo, Japan). CWMs were examined and photographed using an S-3400N scanning electron microscope (Hitachi, Tokyo, Japan).

A Nicolet 6700 spectrometer with a resolution of 0.1 cm⁻¹ was used to obtain FTIR spectra (wavelength coverage, 400–4 000 cm⁻¹). Tablets, including 2 mg of cell walls and KBr (1:100 p/p) for FTIR spectroscopy, were prepared usingthe Graseby Specac Manual Hydraulic Press. All spectra were baseline-corrected and normalized with EZ OMNIC (Thermo Fisher Scientific Inc., USA). The data obtained by FTIR spectroscopy were exported in the *.xls format. The FTIR spectral map was established by Originpro 8.0 (OriginLab Corporation., USA). With reference to the method by Dong (2017), semi-quantitative analysis of the FTIR spectra was conducted as follows: The relative absorbance of a characteristic absorption peak was selected as the base absorbance at 2922 and 1739 cm⁻¹ (designated as AC-H and ACOOR, respectively). The ratio of absorbance of every other characteristic peak was calculated.

Atomic force microscopy    AFM of CWMs was conducted as described in a previous study (Cao et al., 2018; Cybulska et al., 2015), with slight modifications. AFM (SPA400, Seiko, Tokyo, Japan) testing activities were administered at 25 °C in an environment with 30%–40% RH. A clean mica sheet surface was loaded with 10 mg samples. The specimens were then dried and kept in a desiccator with discoloration silica gel at room temperature. The mica surface loaded with samples were scanned using the tapping mode with a Si₃N₄ scanner. The resolution of AFM was 0.01 nm vertically and 0.2 nm horizontally. The scanning frequency was about 0.5–2.0 Hz, and the scan size was set at 5 µm × 5 µm. Each sample was imaged for three times.

Images obtained by AFM were analyzed using AFM software (WSxM 4.0 Beta 5.2) offline. Noise from the samples had to be reduced using the flatten function in the software. The bright and dark parts represented the peaks and troughs of the sample, respectively. Sample roughness was analyzed using several functions in the AFM software.

Statistical analysis    Determination of the TA, SSC, decay rate, and three fraction contents of the CWMs was conducted in triplicate, and evaluation of firmness was performed in six replicates. Three AFM and FTIR images for each sample were analyzed. The data were statistically analyzed using SPSS ver. 19.0 for Windows (SPSS Inc., Chicago, USA). ANOVA with Tukey's post-hoc test was used to determine the statistical significance of the differences (*P < 0.05) in firmness, TA, SSC, decay rate, three fraction contents, and semi-quantitative data from the FTIR images of the samples. The results were expressed as mean ± standard deviation.

Results

Firmness and physicochemical properties    Firmness is the most important index used to reflect the storage characteristics of a fruit. The mechanical properties of a fruit are typically examined by texture profile analysis. The firmness of the ‘Suli’ plums remained high during storage at 4 °C, whereas that of the plums stored at 25 °C declined rapidly from 12 N to 0.6 N (Fig. 1A). The plums softened progressively in the first 9 d at 4 °C; however, the firmness of the plums markedly increased at the last stage of storage, which is probably attributable to the shrinkage of pulp tissue caused by the loss of water in the last stage of plums storage. Based on the abnormal phenomenon, the hypothesis that the plums stored at 4 °C are more prone to tissue shrinkage due to water loss than to softening was also confirmed (Fig. 2). Significant difference in firmness between the two storage temperatures was indicated (*P < 0.05).

Fig. 1.

Firmness (A), SSC content (B), TA content (C) and Decay rate (D) of ‘Suli’ plum fruit during storage at 4 and 25 °C. Firmness were from 6 repeated results. SSC, TA and Decay rate were triplicate measured. The vertical bar indicates the standard error.

Fig. 2.

Schematic image of ‘Suli’ plum pectin degradation after storage.

By contrast, no significant differences in SSC and TA contents between both groups were found (Figs. 1B and 1C). The SSC content of the ‘Suli’ plums repeatedly increased with fruit ripening; however, the TA content exhibited a fluctuating downward trend. The SSC in the ‘Suli’ plums at 25 °C was 7.23% at the initial stage of storage and rose to 10.76% at the end of storage. On Day 3 of storage, SSC rapidly rose to the highest level (14.60%) and then lost about 40% within 1 d. Within the entire shelf life, the SSC in plums at 25 °C only increased by about 3.53%. Similar to SSC, TA decreased from 1.02% to 0.99%. TA fluctuated between 0.72% and 1.10% during the eight-day period. After the ripening period (8 d), TA decreased by 3% relative to its value at the initial stage. The SSC and TA of the ‘Suli’ plums at 4 °C exhibited a stable development trend. Several studies have demonstrated that SSC is positively correlated to TA, indicating that the sugar content in fruit is proportional to the free organic acid content (Saliba-Colombani et al., 2001). Owing to the conversion of starch and other polysaccharides, SSC increased, particularly at the storage temperature of 25 °C. TA as a substrate was involved in respiratory metabolism, leading to its decreased levels (Javanmardi and Kubota, 2006).

The changes, which indicate the deterioration of plums, mainly manifest as flesh translucency, plant pathogen infection, and dehydration (Bal, 2013). As shown in Fig. 1D, low temperatures can delay the deterioration of the ‘Suli’ plums. In the current study, the plums stored at 25 °C decayed after 1 d, whereas those at 4 °C decayed after 3 d. At the end of the shelf life, the decay rates of the plums were 34.8% at 25 °C and 6.0% at 4 °C. The deterioration of the plums stored at 25 °C mainly manifested as flesh translucency. Meanwhile, the plums stored at 4 °C became dehydrated (Fig. 2), indicating that storage at 4 °C significantly suppressed the deterioration of plums (*P < 0.05).

Changes in cell wall fractions    Fruit ripening is usually accompanied by pectin solubilization and depolymerization (Kurz et al., 2008). The contents of the three cell wall fractions are listed in Table. 1. The WSF content increased from 0.40 g kg⁻¹ to 1.05 g kg⁻¹ at 25 °C (Day 8) and to 0.55 g kg⁻¹ at 4 °C (Day 15). The CSF content was 0.20 g kg⁻¹ at harvest, and this decreased to 0.04 g kg⁻¹ at 25 °C and to 0.15 g kg⁻¹ at 4 °C. Meanwhile, SSF content decreased from 0.50 g kg⁻¹ to 0.18 and 0.42 g kg⁻¹.

Table 1. Content of water-soluble fraction (WSF), chelator-soluble fraction (CSF), sodium carbonate-soluble fraction (SSF) of ‘Suli’ plum fruit during softening on the 0^th day, 8^th day (25 °C) and 15^th day (4 °C).

Group	WSF (g kg−1)	CSF (g kg−1)	SSF (g kg−1)
0th day	0.40±0.025c	0.20±0.006a	0.50±0.008a
8th day (25 °C)	1.05±0.020a	0.04±0.012b	0.18±0.017c
15th day (4 °C)	0.55±0.024b	0.15±0.025a	0.42±0.023b

Note: Each value is expressed as mean ± SE (n = 3). Different letters in column represent statistically significant differences (*P < 0.05).

On the basis of the results (Table 1), the WSF content of the ‘Suli’ plums stored at 25 °C markedly increased (*P < 0.05) but slightly changed at 4 °C. The two storage temperatures, particularly 25 °C, significantly increased the degradation of CSF and SSF (*P < 0.05). Previous reports have shown that WSF content is related to flesh translucency, which increases the mobility of the intercellular material and facilitates the formation of pectin–sugar gel complexes (Brummell et al., 2004). CSF and SSF maintain the structural integrity of the cell walls and texture properties in plums (Lara et al., 2004; Renard and Ginies, 2009). On the basis of the results, the transformation of insoluble-water fraction into WSF was evidently promoted in the ‘Suli’ plums at 25 °C but was considerably suppressed at 4 °C. Similar correlations were reported for other plums (Pan et al., 2018).

FTIR analysis of CWM in ‘Suli’ plums    Typical FTIR spectra of CWMs from the ‘Suli’ plums under different storage temperatures are listed in Table 2, which exhibits different vibrations with assignments. Several characteristic absorption peaks were detected (Fig. 3).

Table 2. Assignments of the main vibrations (cm⁻¹) in the FTIR spectra of CWM in ‘Suli’ plums (at harvest, 4 °C, 25 °C).

Vibrations			Assignment	Comments
At harvest	4 °C	25 °C
3 347.29 ± 0.16	3 349.26 ± 0.07	3 350.03 ± 0.65	-OH,-NH stretching	Protein, Carbohydrate
2 922.30 ± 0.03	2 922.83 ± 0.02	2 923.77 ± 0.08	C-H stretching	Protein, Cellulose, pectin
1 739.27 ± 0.05	1 738.27 ± 0.14	1 738.17 ± 0.01	C=O stretching from -COOR	Pectin
1 630.54 ± 0.01	1 630.75 ± 0.06	1 631.42 ± 0.04	-C=O stretching from -CO-NH	Amide I
1 548.75 ± 0.01	1 548.84 ± 0.09	1 548.86 ± 0.14	N-H deformation and C-N stretching	Amide II
1 368.55 ± 0.13	1 368.57 ± 0.01	1 369.20 ± 0.15	-COO− stretching	Pectin
1 316.53 ± 0.08	1 316.73 ± 0.03	1 317.64 ± 0.03	Interaction between C-N stretching and C-N-H in-plane bending of protein	Amide III
1 011.01 ± 0.05	1 011.11 ± 0.07	1 011.35 ± 0.02
951.56 ± 0.10	951.25 ± 0.08	951.04 ± 0.04

Note: Each value is expressed as mean ± SE (n = 3).

Fig. 3.

Fourier Transform Infrared (FTIR) spectra from cell wall material (CWM) in ‘Suli’ plums (at harvest, 4 °C, 25 °C).

The broadest absorption peak around 3349 cm⁻¹ was mainly ascribed to OH, and NH stretching oscillations existed in protein and carbohydrates (Yang and Yen, 2002). The hydrogen bonding between OH groups have a broad absorption band, and the characteristic absorption position of N-H stretching is also assigned in region 3580–3200 cm⁻¹ (Karnik et al., 2016). However, the characteristic absorption peak did not appear in the expected N-H stretching region. Therefore, information on proteins is not available in the region. The peak around 3349 cm⁻¹ mainly occurred from O-H stretching of the -COOH perssad existed in the galacturonic acid backbone. The vibration near 2922 cm⁻¹ was attributed to C-H stretching and could arise from protein, cellulose, and pectin (Abidi et al., 2008). The 1800–800 cm⁻¹ region of the raw spectra was mainly associated with protein and pectin (Akerholm et al. 2004; Ciolacu et al. 2011). The FTIR spectrum with a lower shoulder at 1739 cm⁻¹ was considered as cell wall pectin. The band was raised by the C=O stretching vibration from carbonyl ester (COOR) (Alonso-Simóna et al., 2011). Information on the amide bond in proteins can be read in an FTIR spectrum, mainly contributed by the amide I band and amide II band. The amide I absorption located around 1630 cm⁻¹ was basically ascribed to the C=O stretching vibration from CO-NH in the spectrum. Amide II mainly occurs in the 1570–1515 cm⁻¹ region, but these characteristic peaks are difficult to distinguish. Another typical protein absorption located at 1316 cm⁻¹ was ascribed to C-N-H in plane bending vibrations and C-N stretching vibrations. Typically, a relatively small peak is around 1368 cm⁻¹ related to amounts of carboxylic acid (COO⁻). Many researchers hold that the polysaccharide fingerprint region is mainly distributed in the 1200–900 cm⁻¹ region (Liu et al., 2014). A very strong infrared peak occurred near 1011 cm⁻¹ generally attributed to the C-O stretching vibration, which explains that the band is a common peak associated with sugars. Meanwhile, the other band occurred at 951 cm⁻¹ only as an unknown peak.

The results obtained by semi-quantitative analysis of the FTIR spectra of CWMs from the ‘Suli’ plums (at harvest; 4 °C, 25 °C) are listed in Table. 3. The analysis showed certain differences in the composition and structure of the cell walls in the ‘Suli’ plum under different storage temperature. The differences in three functional groups distributed at 3 349, 1 548, and 1 011 cm⁻¹ were statistically significant after storage at 25 °C (Table. 3). No significant difference was indicated after storage at 4 °C. In accordance with the qualitative and quantitative analysis, the difference was mainly reflected in the location and/or intensity of fruit ripening. As previously mentioned, the band at 3 347 cm⁻¹ shifted to 3 349 and 3 350 cm⁻¹ after storage at 4 °C and 25 °C. The band was presented in the original spectra of CWMs in plums at harvest; meanwhile, semi-quantitative analysis indicated an enhancement in intensity. The other two functional groups were mainly related to amide II and sugars. The peak intensity of N-H absorbance at 1548 cm⁻¹ from CWMs in plums at 4 °C evidently weakened compared with the CWMs in plums at harvest and increased at 25 °C. The peak intensity at 1011 cm⁻¹ rapidly strengthened from 25 °C groups. The result indicated that low temperature surpressed the conversion of protopectin and the degradation of pectin.

Table 3. Semi-quantitative analysis of FTIR spectra of CWM in ‘Suli’ plums (at harvest, 4 °C, 25 °C).

Functional	A/A (C-H)			A/A (COOR)
	At harvest	4 °C	25 °C	At harvest	4 °C	25 °C
-OH; -NH	3.26a ± 0.08	3.30a ± 0.03	5.29b ± 0.7	3.46a ± 0.05	4.02a ± 0.03	5.90b ± 0.06
C-H	1.00 ± 0.00	1.00 ± 0.00	1.00 ± 0.00	1.06 ± 0.01	1.22 ± 0.06	1.12 ± 0.07
-COOR	0.94 ± 0.03	0.82 ± 0.04	0.90 ± 0.03	1.00 ± 0.00	1.00 ± 0.00	1.00 ± 0.00
-CO-NH (C=O)	1.79a ± 0.05	1.96a ± 0.01	2.83b ± 0.07	1.91a ± 0.05	2.38b ± 0.01	3.16c ± 0.04
-CO-NH (N=H)	0.74a ± 0.08	0.58a ± 0.06	1.05b ± 0.06	0.79a ± 0.02	0.70a ± 0.07	1.18b ± 0.11
-COO-	1.87 ± 0.07	1.59 ± 0.02	1.91 ± 0.01	1.99 ± 0.07	1.93 ± 0.08	2.14 ± 0.02
C-N; C-N-H	2.02 ± 0.06	1.80 ± 0.08	2.29 ± 0.02	2.14 ± 0.01	2.19 ± 0.03	2.56 ± 0.05
	10.37a ± 0.13	9.40a ± 0.09	13.39b ± 0.13	11.02a ± 0.03	11.44a ± 0.05	14.94b ± 0.04
	5.22 ± 0.05	5.02 ± 0.05	7.25 ± 0.08	5.55 ± 0.07	6.11 ± 0.01	8.09 ± 0.9

Note: Each value is expressed as mean ± SE (n = 3). Different letters in row represent statistically significant differences at *P < 0.05.

Effect of storage temperature on the microstructure of CWM.    Notably, firmness plays an irreplaceable role in presenting the quality attributes of plums during the post-harvest period (Tessmer et al., 2016). The microstructural changes in the CWMs of the ‘Suli’ plums during softening were observed by SEM (Figure 4). The CWMs of plums at harvest exhibited structural integrity and had a conducting tissue and a smooth pellicle. The CWMs were highly structured, maintained in accordance with the high firmness values of the plums presented in the two groups. The CWMs at 4 °C (Day 15) appeared slightly distorted with a vanishing conducting tissue. Regardless, no obvious changes were observed, compared with the CWMs of plums at harvest (Figures 4A and 4B). Differences were observed in the plums stored at 25 °C on Day 8, with firmness values close to 0 N. The differences suggested that the microstructure of the CWMs changed significantly, as observed by SEM (Fig. 4C). Microstructural damage was evident in the CWMs of plums on Day 8 of storage at 25 °C, indicating a disordered group with a vanishing conducting tissue and a shrinking pellicle. The obtained results show that softening of plums is closely related to the spatial structural changes in CWM.

Fig. 4.

Microstructure images of cell wall material (CWM) in ‘Suli’ plums at harvest (A and a), 4 °C on the 15^th day (B and b) and 25 °C on the 8^th day (C and c).

The atomic force microscopy (AFM) can provide very high resolution morphological images of various sample surface by scanning these samples surface with a probe. Fig. 5 shows the AFM images of the CWMs of fresh plums, the plums on Day 15 of storage at 4 °C, and plums on Day 8 of storage at 25 °C. The effects of storage temperature (4 °C and 25 °C) can be determined by comparison of data in this figure. The color bar legends at the right of the images show the full height of the samples scanned. Images of fresh plums are shown in Fig. 5A. The figure reveals only several peaks/points, which is consistent with the SEM images (Fig. 4A) in which CWMs are shown to have a smooth and integrated surface. The image reveals that the cell wall has a good extended network. The peak height of the CWMs of plums stored at 4 °C gradually decreased, and more aggregates were formed (Figs. 5B and b). The peak height distribution of the group stored at 4 °C in Fig. 5B decreased in range from 0 to 88.11 nm. Meanwhile, the peaks was no longer sharp. Figs. 5C and c show the microstructure of CWMs from plums on Day 8 of storage at 25 °C. When Comparison of the images of the other two groups reveals the collapse of the smooth surface. Several peaks and troughs formed, and the height difference between the two reached 171.39 nm. Roughness analysis can provide indicators in Table 4, such as RMS roughness, roughness average, average height, surface skewness and surface kurtosis. The RMS roughness of the CWMs from fresh plums on Day 8 of storage at 25 °C gradually increased (5.92, 8.56, and 18.22 Rq). The average height of CWMs in plums stored at 25 °C was 89.46 nm, that of plums stored at 4 °C was only 29.55 nm, and that of fresh plums was 30.35 nm. These results indicate that the lower the temperature, the slower the degradation of the CWMs in plums, which is consistent with the results of FTIR and comparison of firmness. The results of AFM indicate that roughness can effectively reflect the softening degree of ‘Suli’ plums.

Fig. 5.

AFM images of cell wall material (CWM) from ‘Suli’ plums at fresh fruit (A and a), 4 °C on the 15^th day (B and b) and 25 °C on the 8^th day (C and c). Typical plane and 3-dimensional images were represented in upper and lower case letters, respectively. Scan area 5.00 µm. × 5.00 µm.

Table 4. The roughness analysis of ‘Suli’ plum fruit during softening on the 0^th day, 8^th day (25 °C) and 15^th day (4 °C).

Group	RMS (nm)	Roughness average (nm)	Average height (nm)	Surface skewness (nm)	Surface kurtosis (nm)
At harvest	5.92a ± 1.5	2.29a ± 0.59	30.35a ± 1.86	5.59a ± 0.59	62.21a ± 10.56
4 °C	8.56a ± 0.26	4.73a ± 0.00	29.55a ± 11.22	2.48b ± 0.40	13.53b ± 0.28
25 °C	18.22b ± 0.44	14.02b ± 0.48	89.46b ± 2.30	−0.09c ± 0.11	3.97c ± 0.04

Note: Each value is expressed as mean±SE (n=3). Different letters in column represent statistically significant differences (P < 0.05).

Discussion

Compared with other varieties, ‘Suli’ plums taste sweet and crisp and cost more. ‘Suli’ plums are supplied in large volumes around July each year, causing a reduction in price. Moreover, increased ambient temperature promotes the softening of ‘Suli’ plums. Plums that show signs of softening have to be sold at a low price to avoid further losses, consequently dampening enthusiasm for growing plums. Therefore, maintaining the quality attributes of ‘Suli’ plums is an important concern.

In this study, two patterns for abnormal softening could be modeled in the ‘Suli’ plums during storage at 4 °C and 25 °C, ignoring fruit maturity. The research focused on the relationship between the composition and nanostructure of the cell wall, as well as the quality attributes of plums. On the basis of the results, variations in quality attributes at storage temperatures of 4 °C and 25 °C after the entire storage period were evident: plums with an increased decay rate rapidly softened at 25 °C and were suppressed at 4 °C. Meanwhile, changes in SSC and TA in plums at 25 °C were more obviously. For the cell wall fractions in plums, the WSF content increased at 25 °C, which was confirmed by the rapid development of translucency (Candan et al., 2011). The study also confirmed the high association between firmness and CSF or SSF content (Ponce et al., 2010). The innovation in this study was the application of FTIR and AFM to analyze cell walls in ‘Suli’ plums. Fruit cell walls include various polymers (e.g. polysaccharides and cellulose). Each component contains particular spectral information from molecular self-assembly or interacting with neighboring molecules in FTIR (Sene et al., 1994). The widely accepted bonding types in fruit cell walls are hydrogen bonding and ester carbonyl, which are typically located near 3 430 and 1 740 cm⁻¹ respectively (Liu et al., 2014; Kamnev et al., 1998). The results obtained by FTIR spectroscopy indicated that the storage temperature markedly affected the position of absorption bands and the intensity of hydrogen bonding. However, possibly owing to overlapping bands, no significant differences were found in the absorption peak and strength of pectin. Regardless, the spectral results suggested that storage temperature altered the pattern of interaction among the cell wall components of the ‘Suli’ plums. For the application of AFM in cell walls, the morphological features and roughness analysis of CWMs were emphasized in this study. On the basis of the results, the CWMs extracted from the ‘Suli’ plums stored at 4 °C exhibited excellent spatial framing, extensibility, and flatness, whereas the plums stored at 25 °C showed the opposite attributes. Roughness analysis was consistent with the SEM images. This study on cell walls illustrated that increased storage temperature promoted the degradation of cell wall components and collapse of the cell wall structure.

In summary, microstructural evaluation by AFM, combined with FTIR analysis, indicated that the quality attributes of ‘Suli’ plums were related to the microscopic features of CWMs, particularly pectins. These results were also supported by previous studies, such as that by Zdunek (2014). Regardless, the details explaining the changes in the cell wall of ‘Suli’ plums have yet to be completely understood. The higher susceptibility of ‘Suli’ plums to softening than that of other varieties remains undetermined. Future studies should explore the metabolic pathways of relevant enzymes that lead to softening of plums.

Conclusions

The CWMs, including pectin, semicellulose, and cellulose play an important role in fruits, which act as a cytoskeleton and maintain the shape of the fruit. The properties, particularly texture, are closely associated with CWMs. AFM and FTIR can be used to analyze the softening of plums. Smoothness and reduced roughness are closely associated with the firmness and freshness of plums. The results of this study offer an insight into the degradation mechanism of softened ‘Suli’ plums and provides a theoretical direction for determining the relationship between quality attributes and the cell wall microstructure in fruits.

Acknowledgements    The authors thank the Guizhou Provincial Forestry department, Guiyang, China for providing funding for this study.

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