WANG Wen-jun ,Ll Yao ,Ll Fu-hua ,ZENG Kai-fang, ,MlNG Jian,

1 College of Food Science,Southwest University,Chongqing 400715,P.R.China

2 Research Center of Food Storage &Logistics,Southwest University,Chongqing 400715,P.R.China

Abstract The fresh postharvest golden needle mushroom (Flammulina velutipes) sporocarp has a high moisture content and crisp texture,but it still has high physiological activity and respiration,leading to senescence and quality deterioration.Treatments with 1-methylcyclopropene (1-MCP) and polypropylene (PP) crispers were used to study the changes of lignification and softening of F.velutipes during storage. The main findings were as follows:the crisper packaging could effectively prolong the storage time of F.velutipes;either the 1-MCP treatment,crisper packaging or the combination of the two treatments could significantly inhibit the accumulation of lignin and the decreases in the contents of cellulose and pectin,and had certain inhibitory effects on the activities of enzymes involved in lignification and softening including phenylalanine ammonia-lyase (PAL),cinnamyl alcohol dehydrogenase (CAD),cellulase (Cx),pectin methylesterase(PME) and polygalacturonase (PG). Among them,the inhibitory effect of the crisper packaging was higher than the 1-MCP treatment,while the combination of the two treatments was the best. The results of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed that the crisper packaging in combination with the 1-MCP treatment could effectively maintain the integrity and stability of the F.velutipes cellular structure and inhibit the emergence of plasmolysis to prevent cell membrane rupture. The transcription levels showed that the crisper packaging and the combination of the 1-MCP treatment and crisper packing could effectively affect the expression of genes for enzymes related to lignification and softening of F.velutipes. In conclusion,1-MCP and PP crispers could delay the lignification and softening of F.velutipes during storage.

Keywords:Flammulina velutipes,polypropylene crisper,1-MCP,softening and lignification,transcription levels

1.lntroduction

The golden needle mushroom (Flammulina velutipes) is one of the edible fungi with the largest production and the highest consumption levels in China (Yang et al.2012). The fresh postharvest F.velutipes sporocarp has a high moisture content and crisp texture,but it still has high physiological activity and respiration leading to senescence and quality deterioration,such as opening of the lid,stem fibrosis,lignification,weightlessness and browning. Lignification and softening are important signs of quality deterioration,which involve and affect the growth,development,disease resistance and environmental adaptability of edible fungi and various fruits and vegetables. The senescence progress of F.velutipes is closely related to cell wall materials,such as pectin,lignin and cellulose. Lignification is a metabolic process during which plant cell walls accumulate lignin,and the formation of lignification affects the taste and texture of many plant-based foods,such as edible fungi(Li et al.2016),broad beans (Nasar-Abbas et al.2008),bamboo shoots (Zeng et al.2015) and loquat (Xu et al.2015). The degradation of cellulose and pectin in the cell wall during the senescence process leads to softening of the fruit and vegetable tissues. The lignification process involves three steps:biosynthesis,transport and polymerization of lignin monomers. Research on the enzymes involved in the lignification process has been progressing in various edible fungi. The essence of fruit softening is that the structure of the colloidal layer changes in cells,and then cells are separated,and cell wall substances begin to degrade. Fruit softening is closely related to the activity of cell wall degrading enzymes,especially polygalacturonase (PG) and cellulase (Cx),and the activities of pectin methylesterase(PME) and other cell wall degrading enzymes (Ben-Arie et al.1996).

The 1-methylcyclopropene (1-MCP) is widely used to delay the ripening process of fruits and vegetables and prolong their storage period,especially the respiratory climacteric varieties (Watkins et al.2006). For nonclimacteric fruits,1-MCP treatment has proven to be effective in preservation at a low effective concentration.1-MCP has a simple chemical structure,and no toxicity or unpleasant odor,but it has good stability and can be synthesized easily. Polypropylene crisper,also known as PP plastic crisper,is a plastic container made of food-grade raw materials. PP material has a lower thermal deformation temperature (100°C),transparency,glossiness and rigidity point,but its impact strength is high. The crisper has high temperature resistance,strong durability and sealing ability. It can be used repeatedly,which can save resources and protect the environment.1-MCP has been widely used as a preservative in fruits,vegetables and flowers,while polypropylene crispers have also been widely used in the storage of fruits and vegetables. Many studies have proven that the influence of 1-MCP treatment is affected by temperature and materials. Polypropylene crispers also have different storage effects due to the varying properties of fruits and vegetables. However,few studies have reported the effects of 1-MCP and polypropylene crisper on the properties of an edible fungus,such as F.velutipes.

In this study,F.velutipes was treated with 1-MCP,polypropylene crisper and the combination of the two.Lignin,pectin,cellulose and the related enzyme activities were determined,and the microstructure was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Transcriptional techniques were used to study the regulation and mechanism of 1-MCP treatment combined with the polypropylene crisper on the lignification and softening of F.velutipes. The process of applying the polypropylene crisper and 1-MCP to the preservation of F.velutipes would be easy to operate and popularize. The results of this study provide a theoretical basis for the storage and packaging of fresh F.velutipes and other fungi.

2.Materials and methods

2.1.1-MCP and polypropylene crisper

Flammulina velutipes were harvested from Beibei(Chongqing,China),showing a fresh light-yellow color,and transported to the laboratory as soon as possible.The F.velutipes without mechanical damage and with a bright color and integral caps,were used as the experimental materials,and precooled at 4°C for 8 h.

In the experiment,polypropylene crispers (PP crisper)of 294 mm×210 mm×215 mm and 4 mm thickness were used. Each crisper could store 2 kg of F.velutipes. The thickness of the polyethylene (PE) cling film was 0.01 mm.The 1-MCP preservative paper was purchased from Taiwan Lytone Company (Taiwan,China). We determined the most effective concentration of 1-MCP was 5.625 μL L-1(1.5 sheets of 1-MCP preservative paper) in a previous experiment.

2.2.Treatment and sampling

Fresh F.velutipes were divided into four experimental groups. The F.velutipes was placed into ordinary plastic trays and packed with ordinary PE cling film as the control group (Control). 1-MCP preservative papers were placed in the crisper and sprayed evenly with and a small amount of distilled water. The cover of the crispers was sealed and the crispers were stored in an environment of 4°C for 10 h;then ventilated,and the 1-MCP preservative papers were taken out. Finally,the F.velutipes was packed with ordinary PE cling film as the 1-MCP treatment group(1-MCP) and placed in the polypropylene crisper as the 1-MCP treatment combined with polypropylene crisper group (1-MCP+Crisper). The F.velutipes was directly placed in the polypropylene crisper as the polypropylene crisper treatment group (Crisper). Three replicates (2 kg F.velutipes per replicate) were prepared for each treatment. All of the F.velutipes were stored at 4°C.

Physiological and biochemical indexes of F.velutipes were determined every 6 d,and samples were prepared.The tissues of F.velutipes (about 10 cm in length) was quickly cut into small pieces. Each treatment had three replicates,with 5 g per replicate,and the experiment was conducted twice. All tissue samples were immediately frozen in liquid nitrogen and stored at -80°C until further use.

2.3.Measurements of lignin,pectin and cellulose in F.velutipes during storage

Lignin,pectin and cellulose contents were determined by the assay kits of Shanghai Cablebridge Biotechnology Co.,Ltd.(Shanghai,China) following the manufacturer’s instructions. The contents of lignin and cellulose were expressed on a dry weight basis;pectin content was expressed on a fresh weight basis,and all measurements were done in triplicate.

2.4.Scanning electron microscope (SEM)

The F.velutipes tissues were collected and placed in vials containing 3.5% (v/v) glutaraldehyde in 0.1 mol L-1phosphate buffered saline (pH 7.4). The tissues were kept in this solution for 48 h for fixation and then washed twice with 0.1 mol L-1phosphate buffered saline. The samples were dehydrated in an each dilution of an ethanol series (30,50,70,85 and 95%,v/v),ending with absolute ethanol twice. Then,the ethanol was replaced with tertiary butyl alcohol. After dehydration,the tissues were dried using a vacuum drying oven with a vacuum pressure of ＞0.08 kPa for 2 h at 65°C. Finally,the specimens were sputter-coated with gold in an ion coater for 2 min. All samples were viewed in a JEOL JSM-6510LV SEM (JEOL,Tokyo,Japan) operating at 25 kV at 5 000× magnification.

2.5.Transmission electron microscopy (TEM)

Tissue samples of F.velutipes (2 mm×1 mm) were fixed in vials containing 3.5% (v/v) glutaraldehyde and washed with 0.1 mol L-1phosphate buffered saline (pH 7.2). Samples were fixed with 1% osmium diacid and washed with 0.1 mol L-1phosphate buffered saline,and dehydrated in each dilution of an ethanol series(35,45,60,70,85 and 95%,v/v). Then,samples were treated with propylene oxide,embedded in Spur resin,polymerized at 20°C for 8 h,removed,repaired and sliced on an ultra-thin slicing machine,stained with uranium dioxide acetate-lead citrate,and observed and photographed with a Hitachi H-7560 TEM (Hitachi Ltd.,Tokyo,Japan).

2.6.Measurements of softening and lignification related enzymes of F.velutipes

The phenylalanine ammonia-lyase (PAL) activity was quantified with assay kits purchased from Shanghai Cablebridge Biotechnology Co.,Ltd.,using a Multiskan Spectrum Microplate Spectrophotometer (BioTek Instruments,Inc.,United States). The specific enzyme activity was expressed on a fresh weight basis as units (U)g-1. One unit of PAL activity was defined as the enzyme amount per g of tissue,per mL reaction system that causes a change of 0.05 in the absorbance at 290 nm per min under specified conditions. The assays were performed with three replicates. For cinnamyl alcohol dehydrogenase (CAD) analysis,1 g frozen tissue powder was added to a 1.5-mL centrifugal tube with 1 mL pre-cooled 0.1 mmol L-1phosphate buffer saline(pH 6.25) containing 15 mmol L-1mercaptoethanol,2%polyethylene glycol and 0.1 g polyvinyl pyrrolidone (PVP).The suspensions were centrifuged at 18 000 r min-1for 20 min. An aliquot of supernatant with a volume of 200 μL was added to 800 μL of reaction solution containing 10 mmol L-1NADP and 5 mmol L-1trans-cinnamic acid,then placed in a 37°C water bath for 30 min. Finally,after stopping the reaction with 1 mmol L-1HCl solution,the absorbance at 340 nm was determined. Phosphate buffer saline with a volume of 200 μL was added to 800 μL of the reaction solution as a control (Goffner et al.1992).

The cellulase (Cx),pectin methylesterase (PME)and polygalacturonase (PG) activities were determined with assay kits purchased from Shanghai Cablebridge Biotechnology Co.,Ltd.,using a Nano Drop 2000 microspectrophotomer (Implen,Inc.,München,Germany)according to the manufacturer’s instructions. One unit of cellulase activity was defined as the enzyme amount per g of tissue liberating 1 μg of reducing sugar per min at 37°C and the data were expressed on a fresh weight basis.One unit of PME activity was defined as the enzyme amount per g of tissue decomposition of pectin to produce 1 mg galacturonic acid per hour under the conditions of 50°C and pH 3.5,and the data were expressed on a fresh weight basis. One unit of PG activity was defined as the enzyme amount of per g of tissue decomposition of pectin acid to produce 1 mg galacturonic acid per hour under the conditions of 40°C and pH 6.0,and the data were expressed on a fresh weight basis. All the measurements were done in triplicate.

2.7.Total RNA isolation and lllumina sequencing

Samples at 0,6 and 12 d post treatment were selected for RNA-Seq assay. Three biological replicates for each treatment were used for RNA isolation,library preparation and sequencing assay. Total RNA from F.velutipes was extracted and purified using the RNAprep Pure Plant Kit (DP441,TIANGEN,China) according to the manufacturer’s handbook. RNA-Seq was performed at Beijing Novogene Bioinformatics Technology Co.,Ltd.(Beijing,China). Total RNA quality,purity,and RNA integrity were assessed using 1% agarose gel electrophoresis,NanoPhotometer®spectrophotometer(IMPLEN,CA,USA) (OD260/280),Qubit®RNA Assay Kit with a Qubit®2.0 fluorometer (Life Technologies,CA,USA),and the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies,CA,USA),respectively.

Briefly,after passing the quality test for total RNA,the mRNA was enriched using magnetic beads attached to Oligo (dT). Sequencing libraries were generated using NEBNext®UltraTM RNA Library Prep Kit for Illumina®(NEB,USA) following the manufacturer’s recommendations. For the raw sequence data,the lowquality reads were removed to obtain the clean reads.The fragments per kilobase of exon per million fragments mapped (FPKM) was the unit of measurement for relative assessment of gene expression levels (Trapnell et al.2012). Differential expression analysis of two conditions/groups was performed using the DESeq R package(version 1.10.1). DESeq provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The differential expression of genes related to softening and lignification enzymes was evaluated.

2.8.Statistical analysis

The experiments were performed using a completely randomized design. The statistical analyses for contents of lignin,pectin and cellulose,and the related enzyme activities,were analyzed via a one-way analysis of variance (ANOVA),followed by Duncan’s multiple-range tests at P＜0.05 (SPSS Inc.,Chicago,IL,USA). The figures were drawn using GraphPad Prism 7 Software(GraphPad Software,Inc.,San Diego,USA) and Adobe Photoshop CS6 (Adobe Systems Incorporated,Inc.,San Jose,USA). Those genes satisfying the criteria for both|log2(FoldChange)|＞1 and P-value＜0.05 were considered to be differentially expressed.

3.Results

3.1.Effects of 1-MCP treatment and the polypropylene crisper on lignin,pectin and cellulose contents of F.velutipes

As shown in Fig.1-A,the accumulation of lignin by F.velutipes was inhibited by 1-MCP treatment and the polypropylene crisper. In the control group and 1-MCP treatment group,the lignin accumulation reached a maximum at 12 d. However,the accumulation of lignin reached a maximum at 24 d in the polypropylene crisper group and the 1-MCP treatment combined with crisper group,and then gradually decreased. From 12 d on,the lignin content of the control group was significantly higher than those of the other three treatment groups,and the differences among the three treatment groups were significant (P＜0.05).

As shown in Fig.1-B,the pectin contents of each group decreased throughout the storage period. The pectin contents of the control group and the 1-MCP group decreased rapidly until 6 d,then decreased relatively slowly. The pectin contents of the crisper group and the 1-MCP treatment combined with the crisper group decreased at a slower rate during the storage period. The pectin contents of the crisper group and the 1-MCP combined with the crisper treatment group were significantly lower than that of the control group (P＜0.05)in the storage period. When storage time reached 14 d,the pectin contents of the 1-MCP treatment group,the crisper group and the 1-MCP treatment combined with crisper group had decreased by 40.1,24.7 and 24.2%,respectively.

The cellulose contents of each experimental group of F.velutipes decreased with time during storage,as shown in Fig.1-C. The cellulose contents of the 1-MCP group,crisper group and the 1-MCP combined with crisper group decreased by 37.4,24.0 and 19.3% at 18 d,respectively.It was significantly lower than that of the control group(P＜0.05). The cellulose content of the control group decreased linearly. The cellulose contents decreased by 32.6 and 29.2% in the crisper group and the 1-MCP combined with crisper group at 30 d.

Fig.1 Effects of 1-MCP treatment and the polypropylene crisper on lignin (A),pectin (B) and cellulose content (C) of Flammulina velutipes. Control,placed into ordinary plastic trays and packed with ordinary PE cling film;Crisper,directly placed in the polypropylene crisper;1-MCP,treated with 1-MCP and packed with ordinary PE cling film;1-MCP+Crisper,treated with 1-MCP and placed in the polypropylene crisper. Data are expressed as the means of triplicate assays. Vertical bars represent the standard errors of the means. The asterisks (＊)represent significant differences (P＜0.05) between the treated group and the control on each storage day.

3.2.Scanning electron microscopy (SEM)

The microstructures of F.velutipes tissues were observed by SEM during storage. The results are shown in Fig.2-A. Fresh F.velutipes cells had good expansibility and a uniform cell wall structure. The parenchyma of thecontrol group and the 1-MCP treatment group collapsed and separated,and the cells of the control group contracted and deformed after 6 d of storage. The tissue structure of the control group was severely damaged,the cells were shrivelled and deformed,the intercellular layer became larger and the cells were paralyzed after 12 d of storage. However,the tissues of the crisper group and the 1-MCP treatment combined with crisper group maintained a relatively plump state at 6 and 12 d. The tissues of crisper group were slightly deformed at 12 d.

3.3.Transmission electron microscope (TEM)

The microstructure of F.velutipes during storage by TEM is shown in Fig.2-B. The cells of F.velutipes were plump and the cell wall and internal structures were complete at 0 d. The cells in the control group and the 1-MCP treatment group began to deform,and cells in the control group began to burst at 6 d. In the control group,the shapes of cells were seriously deformed,the number of ruptured cells increased,and the leakage of intracellular contents was apparent at 12 d,but the 1-MCP treatment group had not yet displayed serious deformation. In contrast,the cells of the crisper group and the 1-MCP treatment combined with crisper group maintained a relatively plump state at 6 d,and the cell structure was clear inside. Only cell enlargement was observed,but no leakage of intracellular contents.

Fig.2 Effects of 1-MCP treatment and the crisper on the microscopic structure of tissues and cells of Flammulina velutipes evaluated by scanning electron microscopy (SEM) (A) and transmission electron microscopy (TEM) (B). Control,placed into ordinary plastic trays and packed with ordinary PE cling film;1-MCP,treated with 1-MCP and packed with ordinary PE cling film;Crisper,directly placed in the polypropylene crisper;1-MCP+Crisper,treated with 1-MCP and placed in the polypropylene crisper.

3.4.Effect of 1-MCP treatment and crisper on softening and lignification-related enzyme activities in F.velutipes

Fig.3-A shows that the PAL activity in all groups initially increased but subsequently decreased. In the control group and the 1-MCP treatment group,the highest PAL activity value was reached at 6 d. The PAL activity of the crisper group and the 1-MCP treatment combined with crisper group reached the peak at 12 d. The PAL activities of the 1-MCP treatment group,the crisper group and the combination treatment group were 12.945,11.261 and 10.285 U g-1at 18 d,respectively,which were significantly lower than that of the control group (P＜0.05).The PAL activities of the crisper and 1-MCP treatment combined with crisper groups were 9.753 and 8.955 U g-1,with a significant difference between the two groups(P＜0.05).

Fig.3-B shows that the change trend of CAD activity in the control group was similar to that of the 1-MCP treatment group during storage,that is,the activity of CAD reached its peak at 6 d,and then decreased. However,the CAD of the crisper group and the 1-MCP treatment combined with crisper group remained relatively stable during storage. The CAD activity increased slowly at first,reached a small peak after 12 d,and then decreased slowly. The peak values of CAD activity in the control,1-MCP treatment,crisper and combination treatmentgroups were 84.33,65.61,54.17 and 49.17 U mg-1,respectively,and the highest values of the control group were significantly higher than those of other groups(P＜0.05).

The Cx activity of the control group and the 1-MCP treatment group increased steadily during storage,while the Cx activity of the crisper group and the 1-MCP treatment combined with crisper group increased slowly in the early period of storage,reached their peak values at 24 d,then decreased to 39.28 and 25.25 U g-1at 30 d,respectively (Fig.3-C).

The PME activity of the control group and the 1-MCP treatment group increased gradually during storage. In contrast,the activity of the crisper group and the 1-MCP combined with crisper group remained stable during the whole storage period,with a slight growth trend (Fig.3-D).From 12 d,the activity of PME in the control group was significantly higher than that of the other treatment groups(P＜0.05). There was no significant difference between the crisper group and the 1-MCP treatment combined with crisper group during the whole storage period.

The PG activity of the control group and the 1-MCP treatment group increased rapidly after 6 d. The crisper group and the 1-MCP treatment combined with crisper group showed a slight trend of rising,and then declining(Fig.3-E). The activities of PG in the 1-MCP group,the crisper group and the 1-MCP treatment combined with crisper group were 2.472,1.194 and 1.043 mg h-1g-1at 18 d,respectively,which were 77.0,37.2,and 32.5% of the values of the control group,respectively.

Fig.3 Effect of 1-MCP treatment and the crisper on phenylalanine ammonia-lyase (PAL;A),cinnamyl alcohol dehydrogenase(CAD;B),cellulase (Cx;C),pectin methylesterase (PME;D)and polygalacturonase (PG; E) enzyme activities of Flammulina velutipes. Control,placed into ordinary plastic trays and packed with ordinary PE cling film;Crisper,directly placed in the polypropylene crisper;1-MCP,treated with 1-MCP and packed with ordinary PE cling film;1-MCP+Crisper,treated with 1-MCP and placed in the polypropylene crisper. Values are mean value±SD (n=3). The asterisks (＊) represent significant differences (P＜0.05) between the treated group and the control on each storage day.

3.5.Expression of enzyme genes involved in the softening and lignification processes of F.velutipes

Four PAL enzyme related genes were identified by gene annotation,and the gene IDs were Cluster-5646.17904(F v PA L 1),C l u s t e r-5 6 4 6.1 7 9 0 6 (F v PA L 2),Cluster-5646.17907 (FvPAL3) and Cluster-5646.54724(FvPAL4) (Fig.4). Among them,the genes FvPAL1,FvPAL2 and FvPAL3 were similar to the PAL gene sequence of Plagiochasma appendiculatum,and FvPAL4 was similar to the PAL gene sequence of Ephedra sinica.The FvPAL2 and FvPAL4 had the highest expression levels in the control group and each treatment group.The expression levels of FvPAL2 and FvPAL4 in the control group and each treatment group peaked at 6 d.Compared with the control group,both the FvPAL2 and FvPAL4 genes of each of the treated groups were down-regulated. The FvPAL3 was expressed only in the control group,and the 1-MCP group at 6 and 12 d,while no expression was observed in the crisper group or the 1-MCP treatment combined with crisper group.The FvPAL1 was only expressed in the control group at 6 and 12 d,but not in the other three treatment groups.This indicated that the 1-MCP treatment,crisper and the combination of the two down-regulated FvPAL1,and the crisper and 1-MCP combined with the crisper treatments could down-regulate FvPAL3.

Fig.4 Phenylalanine ammonia-lyase (PAL) related gene FPKM (fragments per kilobase of exon per million fragments mapped)values. Control,placed into ordinary plastic trays and packed with ordinary PE cling film;Crisper,directly placed in the polypropylene crisper;1-MCP,treated with 1-MCP and packed with ordinary PE cling film;1-MCP+Crisper,treated with 1-MCP and placed in the polypropylene crisper. Values are mean±SD (n=3). The letters a-c indicate significant differences at the 0.05 level on each storage day.

Two genes related to the Cx enzyme were found by gene annotation,and the gene IDs were Cluster-5646.15691 and Cluster-5646.15692. These two genes are similar to the Cx gene sequence of Lingulodinium polyedrum. As shown in Fig.5,the 1-MCP,crisper,and combination treatments all showed downregulation of the FvCx1 (Cluster-5646.15691) and FvCx2(Cluster-5646.15692) genes.

Fig.5 Cellulase (Cx) related gene FPKM (fragments per kilobase of exon per million fragments mapped) values. Control,placed into ordinary plastic trays and packed with ordinary PE cling film;Crisper,directly placed in the polypropylene crisper;1-MCP,treated with 1-MCP and packed with ordinary PE cling film;1-MCP+Crisper,treated with 1-MCP and placed in the polypropylene crisper. Values are mean±SD (n=3). The letters a-d indicate significant differences at the 0.05 level on each storage day.

Six genes related to the PME enzyme were found by gene annotation,and the gene IDs were FvPME1(Cluster-5646.3904),FvPME2 (Cluster-5646.3906),F v P M E 3 (C l u s t e r-5 6 4 6.4 5 3 0 0),F v P M E 4(Cluster-5646.45302),FvPME5 (Cluster-5646.45304),and FvPME6 (Cluster-6267.0) (Fig.6). Among them,the FvPME1,FvPME2,FvPME3,FvPME4 and FvPME5 genes were similar to the PME gene sequence of Vitis riparia,and FvPME6 was similar to the PME gene sequence of Phytophthora capsici. Among them,FvPME1 and FvPME6 were expressed only in the control group and the 1-MCP treatment group,but not in the crisper group or the combined 1-MCP treatment with crisper group,and FvPME1 was only expressed in the control group at 6 d. This indicated that the crisper and 1-MCP treatments can significantly down-regulate the FvPME1 and FvPME6 genes. However,FvPME3,FvPME4 and FvPME5 were expressed in the control group and in each treatment group,and the combined 1-MCP with crisper treatment can significantly down-regulate the FvPME3,FvPME4 and FvPME5 genes. Compared with the control group at 0 d,there were two differential gene expression up-regulations (FvPME4 and FvPME5)and one differential gene expression down-regulation(FvPME3) in the control group at 6 d;the expressions of the three differentially expressed genes in the 1-MCP treatment group were up-regulated;the two differential gene expressions of the crisper group were downregulated (FvPME3 and FvPME4),and one differential gene expression was up-regulated (FvPME5);and there was one differential gene expression down-regulation(FvPME4) in the combination treatment group. The expressions of the three differentially expressed genes in the control group and the 1-MCP treatment group were up-regulated;while two differentially expressed genes were down-regulated (FvPME4 and FvPME5) in the crisper group and the combination treatment group at 12 d.Unlike the other genes,the FvPME2 gene was only expressed in the control group at 12 d,indicating that each treatment could promote the expression level of FvPME2 gene.

Fig.6 Pectin methylesterase (PME) related gene FPKM (fragments per kilobase of exon per million fragments mapped) values.Control,placed into ordinary plastic trays and packed with ordinary PE cling film;1-MCP,treated with 1-MCP and packed with ordinary PE cling film;Crisper,directly placed in the polypropylene crisper;1-MCP+Crisper,treated with 1-MCP and placed in the polypropylene crisper. Values are mean±SD (n=3). The letters a-c indicate significant differences at the 0.05 level on each storage day.

Four genes related to the PG enzyme were found by gene annotation,and the gene IDs were FvPG1(Cluster-5646.15543),FvPG2 (Cluster-5646.15546),F v P G 3 (C l u s t e r-5 6 4 6.1 5 5 4 8),a n d F v P G 4(Cluster-5646.51803) (Fig.7). These four genes were similar to the Medicago truncatula PG gene sequence.The FvPG1 was expressed in the control group and each treatment group during storage. The expression level of FvPG2 increased with the storage time. In contrast,the expression level of FvPG3 decreased in each group with the prolongation of storage time,and FvPG4 was not expressed in the control group or any of the treatment groups at 0 d.

Fig.7 Polygalacturonase (PG) related gene FPKM (fragments per kilobase of exon per million fragments mapped) values. Control,placed into ordinary plastic trays and packed with ordinary PE cling film;1-MCP,treated with 1-MCP and packed with ordinary PE cling film;Crisper,directly placed in the polypropylene crisper;1-MCP+Crisper,treated with 1-MCP and placed in the polypropylene crisper. Values are mean±SD (n=3). The letters a-c indicate significant differences at the 0.05 level on each storage day.

4.Discussion

The respiration and transpiration of F.velutipes are vigorous after harvest,and many kinds of physiological activities are intensified. Lignification and softening are associated with the accumulation and metabolism of cell wall materials such as lignin,cellulose and pectin.The occurrence of lignification is closely related to the production and accumulation of lignin,and its process is involved in the phenylpropanoid metabolic pathway(Schoch et al.2001;Luo et al.2008;Fornalé et al.2010).In this experiment,the activities of key enzymes (PAL and CAD) involved in the synthesis of lignin were significantly inhibited by 1-MCP and crisper treatment,which led to the slow accumulation of lignin in F.velutipes. PAL linkes primary metabolism and phenylpropanoid metabolism,catalyzes the first step of phenylpropanoid metabolism,and is also a key enzyme related to the rate-limiting in the phenylalanine metabolic pathway. The increase in cucumber lignin content is significantly associated with the up-regulation of gene expression levels encoding PAL during storage (Wu et al.2016). The process of lignification of apricot fruit is accompanied by an increase in the expression level of CAD genes (Zhang et al.2017). The apple softening process is delayed,and the expression of its PG gene is also inhibited (Johnston et al.2002;Costa et al.2010). The Cx enzyme is closely related to the initiation of ripening and softening of fruits(Elhassan et al.2016). It has also been found that a PME gene in tomato reached its maximum expression level after softening.

The softening of edible fungi is mainly caused by the degradation of the pectin in the microfibril interfilaments and the intercellular compartment of the sporocarp,the dissolution of the cellulose material,and the separation of the cells. An increase of cellulase activity causes the cellulose to degrade,thereby destroying the cellulose microfibril-hemicellulose-pectin“crosslinked structure”of the cell wall,and the pectin enzyme decomposes the pectin,resulting in the softening. The main function of PME is to catalyze the demethanol of galacturonic acid,and PG acts on the demethanol-based polygalacturonic acid (Luo et al.2006). The process of PME is the premise of PG (Dahodwala et al.1974). PME catalyzes the production of demethylated pectin,endo-PG dissociates exo-PG (Strand et al.1976),and enhances the matrix of exo-PG (Cooper and Wood 1975). Then endo-PG and exo-PG combine to dissolve cell wall pectin,resulting in softening. In the experiments conducted here,it was found that the activities of Cx,PME and PG in the control group and the 1-MCP treatment group increased.However,there was no obvious upward trend (P＜0.05)in the crisper group or the 1-MCP treatment combined with crisper group,the activities were low during storage,which significantly inhibited the degradation of cellulose and pectin. The 1-MCP treatment did not inhibit the activities of Cx,PME and PG of F.velutipes. However,the PME and PG activities of the 1-MCP group were still significantly lower than those of the control group in the late stage of storage. This was also consistent with the effect of the 1-MCP treatment group on inhibiting the softening effect of F.velutipes much less than those of the crisper group and the 1-MCP treatment combined with crisper group.

SEM and TEM were used to observe the tissue morphology of F.velutipes. During the processes of browning,lignification and softening of F.velutipes,the cell structure also changed significantly. The F.velutipes gradually deteriorated,with quality deterioration,the cell wall deformed and began to degrade,and plasmolysis occurred. The cell membrane ruptured,which was accompanied by the outflow of the contents in the control group. The 1-MCP treatment,the crisper treatment and their combination treatment could maintain the integrity of the F.velutipes cell structure. In particular,the crisper group and the 1-MCP treatment combined with crisper group of F.velutipes still maintained great cell morphology at 12 d. The cell state at that point was not significantly(P＜0.05) different from the fresh F.velutipes at 0 d,which intuitively reflected that all the treatments could effectively control the lignification and softening of F.velutipes.

The 1-MCP treatment,crisper treatment and their combination influenced the lignification and softening of F.velutipes.,not only in delaying the accumulation of lignin,maintaining the pectin and cellulose contents,and significant inhibiting the related enzyme activities,but also in the effects on expression levels of PAL,Cx,PME and PG related genes. The crisper and 1-MCP combined with crisper treatments could significantly inhibit the expression levels of FvPAL,FvCx,FvPME and FvPG. The 1-MCP treatment also significantly inhibited the expression levels of the FvPAL2 and FvPG2 genes. The expression of CAD-related genes was not screened in the transcriptome data measured,probably due to the low expression level of FvCAD in F.velutipes or the absence of FvCAD expression during the pre-storage period.

The results of this study also showed that the three treatments did not inhibit the expression levels of certain genes of FvPOD,FvPME and FvPG,and their expression levels were even higher than those of the control and 1-MCP-treated samples. There are other possible mechanisms for the relationships between the expression levels of these genes,the activities of enzymes and the lignification of F.velutipes. Further studies are needed to clarify these relationships.

5.Conclusion

The combination of the 1-MCP treatment and polypropylene crisper could significantly control the softening and lignification of F.velutipes. This method could inhibit the accumulation of lignin and the decreases of cellulose and pectin,and has certain inhibitory effects on the activities of enzymes involved in lignification and softening. The crisper packaging and the combination of the 1-MCP treatment and crisper packing could effectively affect the expression of genes for enzymes related to lignification and softening of F.velutipes. These results will be of great reference value in the commercial application of the preservation of F.velutipes in the future.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (31471576),and the Chongqing Science and Technology Commission,China(cstc2015shmszx80019).

Declaration of competing interest

The authors declare that they have no conflict of interest.