Changing surface wax compositions and related gene expression in three cultivars of Chinese pear fruits during cold storage

Materials

The XH, YL, and YLX pear fruits were all harvested at commercial maturity from Zhao County, Hebei Province, China on September 15, September 24, and August 18, 2015, respectively. They were transported to a laboratory within 2 h of the harvest. Fruit with uniform size and no visible mechanical defects were selected and stored at 0 °C for 270 days.

Total wax extraction was performed at day 0, 45, 90, 180, and 270 of cold storage. Two pieces of peel were sampled along the symmetrical part of the long axis of the fruit. These portions were rapidly frozen in liquid nitrogen and stored at −80 °C for RNA extraction.

Total wax extraction

Each fruit was dipped into a succession of chloroform solutions for 30 s. Then extract solutions were mixed and added into 400 µL 0.5 g L⁻¹ n-tetracosane (Accustandard Inc, New Haven, CT, USA) as the internal standard. The extracting solutions were then evaporated under a gentle flow of nitrogen gas until the samples were completely dry (Wang et al., 2014). Total wax extraction contained three replicates with five fruits for each replicate.

Wax determination

Dried wax extracts were re-dissolved in 400 µL pyridine at 50 °C for 30 min, and then were added into 400 µL bis-N, O-trimethylsilyl trifluoroacetamide (TCI, Tokyo, Japan) for derivative reaction at 60 °C for 40 min. The mixture solution was completely dried under a slow nitrogen flow. The dried wax extract was last resolved in 2.5 mL chloroform and filtered through a 0.22 µm aperture filter before determination by gas chromatography-mass spectrometry (GC-MS) (DSQ II; Thermo Fisher Scientific, Waltham, MA, USA). A DB-1 capillary (30 m * 0.25 mm ID * 0.25 µm film thickness; Agilent Technology Inc, Palo Alto, CA, USA) was used to separate the wax components. Helium was used as the carrier gas at a rate of 1.0 mL per min. The temperature conditions were as follows: the initial temperature started at 70 °C and was increased to 200 °C at the rate of 10 °C per min and was held for 1 min; the temperature was elevated to 300 °C at the rate of 4 °C per min and was held for 20 min. The mass spectrum program settings: EI ion source (70 eV). The mass-to-charge ratio ranged from 50–650; the temperature of the inlet, ion source, and transmission was 250 °C, 300 °C, and 250 °C, respectively. A qualitative analysis of the wax components was conducted according to the NIST 09 library, and the quantitative calculations were carried out by the internal standard method (Yang et al., 2017). The wax content was expressed as µg cm⁻².

RNA extraction and qRT-PCR analysis of wax related genes

Total RNA extraction was conducted according to the CTAB method described by Gasic, Hernandez & Korban (2004). A single chain of cDNA was synthetized using the RT Reagent kit with gDNA Eraser (TaKaRa Biomedicals, Dalian, China). SYBR* Premix Ex Taq™ (TaKaRa Biomedicals, Dalian, China) was used for the qRT-PCR analysis of wax related genes. Actin2 was used as the endogenous reference. The quantity analysis of the gene expression was based on the formula 2^−ΔΔCT (Livak & Schmittgen, 2001). The expression values of detected genes in YL at day 0 were all labeled as ‘1’ to standardize the expression of these genes in the other two varieties. Melting curves were completed at the end of the amplification reaction and they were used to test specificity of primers.

The primers of the detected genes are listed in Table S1. The primers of LACS1, KCS1L, KCS6, CER10, ABCG11, ABCG12, CER1L, CAC3, and CAC3L were designed by OMIGA 2.0. The primers of ACT2 and WSD1L refer to Li et al. (2017) and Yang et al. (2017), respectively. The other primers refer to Wu et al. (2017).

Statistical analysis

Three replicates were conducted for all of the assays and the values were expressed as means ± standard error. Data were analyzed by one-way ANOVA, and were compared according to Duncan (α = 0.05) and LSD (p < 0.05) using SPSS Statistics 22 (IBM, Armonk, NY, USA). Cluster relationships, the dimension reduction of the wax compositions, and the correlation analysis of the wax compositions and wax-related gene expression were completed using cluster heat maps, principal component analysis (PCA), and heat maps in Origin 2017 (Origin Lab, Northampton, MA, USA).

Variations in wax composition on fruit surface in three pear cultivars during cold storage

Fruit surface waxes contained aliphatic compounds and triterpenoids in all three pear cultivars. Triterpenoids were dominant in the wax compositions of XH and YL, while aliphatic compounds were predominant in the wax of YLX (Fig. S1 and Table 1). The higher proportions of alkenes, fatty acids, and esters were found in the surface wax of YLX at a later storage stage (Fig. S1), and the contents of total alkanes, total alkenes, total fatty acids, and total esters were all highest on day 90 in YLX among the three pear cultivars (Table 1). YL showed the highest content of total aldehydes on day 90 (Table 1). The total triterpenoid content in the three cultivars all showed an upward trend before day 90; however, the amounts of triterpenoid in XH and YL were higher than in YLX during cold storage (Table 1). In addition, the content of total fatty alcohols in XH reached the peak on day 180 and was higher than YLX on day 180 (Table 1).

In order to further identify the differences in waxes among the three varieties during storage, PCA results showed that the cumulative variance contribution rate of PC1 (49.9%) and PC2 (36.3%) was 86.2%. This could represent the seven wax components used to evaluate the effects of variety and storage time on the surface wax of three pear cultivars (Fig. 1). Fatty alcohols, triterpenoids, and aldehydes possessed the load on the positive axis of PC1 and PC2, however, alkenes, esters, fatty acids, and alkanes occupied the load on the positive axis of PC1 and the negative axis of PC2 (Fig. 1). The three pear cultivars were divided into two groups at a 95% confidence: wax composition, such as alkenes, esters, fatty acids, and alkanes reflected the surface wax characteristics of YLX, while fatty alcohols, triterpenoids, and aldehydes represented the surface wax features of XH and YL (Fig. 1).

Surface wax profiles and variations among three pear cultivars during cold storage

Alkanes with C₂₁–C₃₁ chains, three fatty acids, two esters, aldehydes with C₂₄–C₃₄ chains, fatty alcohols with C₁₆–C₂₈ chains, and seven triterpenoids were found in the three pear cultivars (Figs. S2–S7). The waxes were mainly composed of C₂₉ alkane, three fatty acids, two esters, three aldehydes (C₃₀, C₃₂ and C₃₄), three fatty alcohols (C₂₀, C₂₂ and C₂₄), and three triterpenoids (Fig. 2 and Figs. S2–S7). The contents of C₂₉ alkane, the three fatty acids, and two esters all peaked on day 90 and were highest in YLX compared with the other two varieties (Fig. 2). The YL cultivar recorded the two highest aldehyde contents of C₃₀ and C₃₂ before day 90 and on day 90 of cold storage (Fig. 2). The content of the three fatty alcohols (C₂₀, C₂₂ and C₂₄) in XH all showed an upward trend before day 90, and peaked on day 90 (Fig. 2). In addition, the contents of the three main triterpenoids all reached peak values on day 90 in all three pear cultivars. They were higher in XH and YL than in YLX (Fig. 2).

Thirty-five wax compositions were clustered into two categories (Fig. S8). Each component of the alkanes, fatty acids, and esters all belonged to one category, while each compound of aldehydes (except for the composition C₃₃), fatty alcohols, and triterpenoids (except for [(3β,5α)-4,4-dimethylcholesta-8,24-dien-3-yl] oxy) were classified into another category (Fig. S8).

Expression pattern of wax related genes in three pear cultivars during cold storage

The expression levels of the fifteen genes (LACS1, KCS2, KCS6, FDH, KCS20, GL8, CER10, CER60, LTPG1, LTP4, ABCG12, CER1L, CAC3, CAC3L, and DGAT1L) in the peel of YLX all peaked at day 45, and were higher than those in XH and YL (Figs. 3 and 4). Figures 3 and 4 showed the increasing trend of the relative expression abundance of 10 genes (KCS2, FDH, GL8, CER60, LTPG1, LTP3, CER1L, CAC3, CAC3L, DGAT1L) in the peel of YL prior to day 45. In addition, the expression levels of the transcripts of nine genes (LACS1, FDH, KCS20, CER10, CER60, LTPG1, LTP4, CER1L, CAC3L) in the peel of XH were unchanged; however, only the expression abundances of another three genes (GL8, CAC3, and DGAT1L) in XH increased before day 45. The expression levels of WSD1L all showed a rising trend before day 45, but no significant differences were found at day 45 among three pear cultivars (Fig. 4).

Correlation analysis between wax composition content and related gene expression levels in three pear cultivars

A positive correlation existed between the mRNA expression of three genes (LTPG1, LTP4, and CAC3L) and the content of alkanes, alkenes, fatty acids and esters in the waxes (p < 0.05 or p < 0.01) (Fig. 5). The expression levels of LACS1, KCS2, FDH, KCS20, CER10, CER1L, and CAC3 showed a positive correlation with alkane, fatty acid, and ester content at p < 0.05 or p < 0.01(Fig. 5). The expression levels of KCS1L, KCS6, and ABCG12 were only positively related to the content of alkanes, and the expression patterns of GL8 and DGAT1L showed a positive correlation with the contents of alkanes and esters at p < 0.05 or p < 0.01, respectively (Fig. 5). In addition, the transcription levels of the 20 detected genes, except for LACS2, were negatively correlated with the contents of aldehydes, fatty alcohols, and triterpenoids in varying degrees (Fig. 5).