Expression analysis of candidate cell wall-related genes associated with changes in pectin biochemistry during postharvest apple softening

Postharvest Biology and Technology 112 (2016) 176–185

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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio

Expression analysis of candidate cell wall-related genes associated with changes in pectin biochemistry during postharvest apple softening Sunny George Gwanpuaa , Ifigeneia Mellidoua , Jelena Boeckxa , Clare Kyomugashob , Niels Bessemansa , Bert E. Verlindenc , Maarten L.A.T.M. Hertoga , Marc Hendrickxb , Bart M. Nicolaia,c , Annemie H. Geeraerda,* a Division of Mechatronics, Biostatistics and Sensors (MeBioS), Department of Biosystems (BIOSYST), KU Leuven, W. de Croylaan 42, Bus 2428, B-3001 Leuven, Belgium b Laboratory of Food Technology and Leuven Food Science and Nutrition Research Centre (LFoRCe), Department of Microbial and Molecular Systems (M2S), KU Leuven, Kasteelpark Arenberg 22, PB 2457, B-3001 Leuven, Belgium c Flanders Centre of Postharvest Technology, W. de Croylaan 42, B-3001 Leuven, Belgium

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 March 2015 Received in revised form 22 September 2015 Accepted 25 September 2015 Available online 9 October 2015

The property of apple fruit (Malus
domestica Borkh.) of being able to retain their postharvest firmness to some extent, is an important aspect in many breeding programs. A clear identification of genes responsible for softening is therefore imperative. Polygalacturonase1 has been identified in the literature as the main gene that regulates softening in fruits, including apple. The aim of this work was to identify the role of other key cell wall-related genes in apple. Candidate genes were selected within alpha-larabinofuranosidase (a-AF), beta-galactosidase (b-GAL), endo-polygalacturonase (PG), and pectin methylesterase (PME) gene families, by quantifying their expression in firm and soft ‘Jonagold’ apples, using qRT-PCR. Expression levels of candidate genes were further investigated in ‘Jonagold’ and ‘Granny Smith’ apples with or without 1-MCP treatment, stored for 6 months. 1-MCP treated fruit showed very low levels of ethylene production and no significant loss in firmness when placed under normal ripening conditions. During ripening in non-1-MCP treated ‘Jonagold’ and ‘Granny Smith’ apples, the expression of MdPG1,Mdb-GAL1, Mdb-GAL2, and Mda-AF2 were upregulated by several folds, and showed positive correlations to softening and ethylene production. Softening was accompanied by polyuronide solubilisation and a significant loss in side chain neutral sugars. Mdb-GAL2 expression levels during ripening of the untreated fruit were comparable to that of MdPG1. Correlation of Mda-AF2 to softening was not as high as for MdPG1 and Mdb-GAL2, while all candidate MdPME genes were downregulated during softening. This suggests that in addition to MdPG1, Mdb-GAL2 may play a central role in apple fruit softening. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Apple (Malus
domestica Borkh.) fruit softening Gene expression Polygalacturonase Beta-galactosidase

1. Introduction Enzyme-induced cell wall modifications are thought to be the main cause of flesh softening during ripening in many fruit. While both cell wall pectin and hemicellulose may undergo modifications during fruit softening (Fischer and Bennett, 1991; Chin et al., 1999; Yoshioka et al., 1992; Brummell, 2006; Bennett and Labavitch, 2008), loss in flesh firmness during ripening of apple is mainly due to changes in cell wall pectin polysaccharides, with very little

* Corresponding author. Tel.: +32 16320591. E-mail address: [email protected] (A.H. Geeraerd). http://dx.doi.org/10.1016/j.postharvbio.2015.09.034 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.

changes occurring in the hemicellulose fraction (Yoshioka et al., 1992; Fischer and Bennett, 1991). As recently as the 1980s, polygalacturonase (PG) was believed to be the sole enzyme responsible for cell wall changes during softening, due to strong correlations between PG activity and softening in tomato (Crookes and Grierson, 1983). However, molecular genetic studies have led to suggestions that, although responsible for a major component of pectin depolymerisation and associated solubilisation, PG is neither required nor sufficient for fruit softening (Smith et al., 1990). This has led to investigations of other cell wall-related enzymes, such as pectate lyases, pectin methylesterases (PME), b-galactosidases (b-GAL), and a-L-arabinofuranosidases (a-AF) (Fischer and Bennett, 1991; Goulao et al., 2007; Rose et al., 1998;

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Brummell and Harpster, 2001). Additionally, some non-pectolytic enzymes and proteins have been shown to be involved in reorganizing the cellulose–xyloglucan network, such as, xyloglucan endotransglucosylase/hydrolase and expansin (Atkinson et al., 2009; Rochange and McQueen-Mason, 2000; Cosgrove, 2002). High expression of genes encoding expansin has been reported in ripe apple fruit (Costa et al., 2008; Wakasa et al., 2003), but a study by Trujillo et al. (2011) failed to relate apple texture with the expression of the expansin gene. This is contrary to tomato in which suppression or overexpression of the ripening-related expansin gene, Exp1, was correlated to softening and cell wall modifications (Brummell et al., 1999), or in banana in which the expression of an expansin gene, MaExp1, was upregulated when unripe banana was treated with ethylene, and suppressed in unripe banana treated with 1-MCP (Trivedi and Nath, 2004). Studies using transgenic tomato plant materials have shown that no single enzyme can be considered as solely responsible for fruit softening (Tieman et al., 1992; Giovannoni et al., 1989). Nevertheless, the relative importance of different enzymes may vary among fruit depending on the main mechanism of softening. For example, tomato fruit undergo extensive pectin depolymerisation during softening and show very high PG activity (Brummell and Labavitch, 1997; Dellapenna et al., 1990; Huber and Lee, 1989). Contrarily, it has been reported that although there is pectin solubilisation during normal ripening in apple fruit, there is hardly any pectin depolymerisation, with very low PG activity (Yoshioka et al., 1992), although Atkinson et al. (2012) recently showed that downregulation of the PG1 gene in apples resulted in less firmness degradation, and a larger molecular weight of the chelator-soluble pectin (CSP) fraction. Banana and strawberry fruit have been reported to have very low levels of PG, but high pectate lyase activities, and polyuronide solubilisation and depolymerisation during ripening (Marín-Rodríguez et al., 2002). Also, transgenic strawberry plants with antisense PL gene were much firmer, and showed a lower degree of in vitro cell wall swelling, than control fruit at the fully ripe stage (Jiménez-Bermúdez et al., 2002). However, PL activity was undetected in ripe ‘Jonagold’ apples (Gwanpua et al., 2014). It is likely that within the plethora of cell wall-modifying enzymes, different enzymes are central in the process of cell wall disassembly during softening of different types of fruits. Identification of key cell wall-related genes in apple fruit could assist in breeding programs having a high firmness as a desirable trait. For this purpose, a complete analysis of the expression pattern of different candidate cell wall-related genes in apple fruit, and the associated polyuronide modifications during softening is needed. Most of the current literature linking the expression of cell wallrelated genes to softening in apples have focused on the well characterised polygalacturonase1 (MdPG1) (Costa et al., 2010; Atkinson et al., 2012; Nybom et al., 2012). Some authors reported other cell wall-related genes to be expressed during apple fruit ripening (Goulao et al., 2008; Hiwasa et al., 2003; Ross et al., 1994), but most of these studies were based on the Arabidopsis genome, and their correlation to softening and modifications in cell wall biochemistry has not been fully investigated. Concerted action of PME and PG could be responsible for pectin backbone depolymerisation, while b-GAL and a-AF would mainly induce changes in the pectin side chains. By using sequencing information from the apple genome (Velasco et al., 2010) and transcriptome (Mellidou et al., 2014), the aim of this work was to identify the most important softening-related genes within the families of genes encoding PG, a-AF, b-GAL, and PME, by studying their expression during softening of ‘Jonagold’ and ‘Granny Smith’ apple, under different postharvest ripening conditions, while keeping in mind the wall polysaccharide changes that accompany ripening and softening in these apples. The relevance of ethylene in regulation of the

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expression of cell wall-related genes, and pectin modifications was investigated by using 1-methylcyclopropene (1-MCP), an agent known to block ethylene receptors (Watkins, 2006). 2. Materials and methods 2.1. Plant materials and storage experiments Apples (Malus
domestica Borkh.) cv ‘Jonagold’ and ‘Granny Smith’ were harvested at commercial maturity. ‘Jonagold’ apples were harvested in 2013 from an orchard in Rotselaar, Belgium, while ‘Granny Smith’ apples were harvested in 2013, from an orchard in Piemonte, Italy. After harvest, the fruit were immediately cooled to 1  C, and transported to the laboratory. Fruits of both cultivars were stored at 1  C under controlled atmosphere conditions (CA) for 6 months, with and without 1MCP treatment, and sampling was done at the end of storage and after 7 d subsequent exposure to shelf life conditions (18  C under regular air). The CA condition for both cultivars consisted of 1 kPa O2 and 3 kPa CO2. The application of 1-MCP (SmartFreshTM, AgroFresh Inc., Spring House, PA, USA) was done immediately after cooling, following the manufacturer’s guidelines, by exposing the fruit to 625 ppb 1-MCP in airtight containers for 24 h. At each sampling point, 20 fruit were used as biological replicates for the assessment of firmness, while four replicates were used for ethylene production measurements, quantifications of cell wall-related gene expression, and characterisation of cell wall pectin. For screening of cell wall-related candidate genes, ‘Jonagold’ apples were stored under CA or regular air at 1  C for 2 months. 2.2. Evaluation of flesh firmness and ethylene production Flesh firmness and ethylene production was measured as described in Gwanpua et al. (2012). In brief, firmness was measured using an LRX Universal Testing Machine (Lloyd Instruments, UK), equipped with a load cell of 500 N, to which a self-cutting cylindrical plunger with a surface of 1 cm2 was attached. The firmness was taken as the maximum force (N) needed to penetrate the fruit to a depth of 8 mm, while traveling at a constant speed of 8 mm s1. Two measurements were taken on the equator, 180 apart, and the average was taken as the firmness value. To measure ethylene production, an apple was enclosed in a jar of 1.7 L and flushed for 3 h with humidified air. The inlet and outlet of the jars were then closed and 3 mL gas samples were withdrawn from the jars and analysed by injecting into a CompactGC (Interscience, Louvain-la-Neuve, Belgium) gas chromatograph. Ethylene standards ranging from 50 ppb to 50 ppm were used for calibration. After 3 h a second sample was taken from the jars and analysed. The ethylene production rate was calculated from the difference between the two readings. 2.3. Screening of candidate cell wall-related genes in the apple genome As a first step in identifying key softening-related genes, cell wall-related genes whose expression have been associated with softening in different apple cultivars, as reported in literature, were selected to represent candidate genes. Within the PG gene family, MDP0000326734, referred in literature as MdPG1, has been shown by several authors to be closely linked to flesh softening (Wakasa et al., 2006; Longhi et al., 2013; Mann et al., 2008; Atkinson et al., 2012). Two Malus b-GAL genes have so far been associated with apple fruit softening, MDP0000416548 (Mann et al., 2008; Ross et al., 1994) and MDP0000127542 (Atkinson et al., 2012; Ireland et al., 2014). Only two a-AF genes were obtained from BLAST

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searching on the apple reference genome assembly (Velasco et al., 2010) using the well characterised protein sequences of Malus Alpha-L-arabinofuranosidase, Q7
9G7. Both of these a-AF genes, Malus ID MDP0000055078 and MDP0000256049, have been reported in ripe apple fruit (Goulao et al., 2008; Nobile et al., 2011; Mann et al., 2008). PME genes have seldom been associated with softening in apples. A PME gene was reported to be upregulated by ethephon treatment in ‘Fuji’ and ‘Golden Delicious’ apples (Wei et al., 2010). The MdPME primers used in that study corresponded to MDP0000196867. To identify additional cell wall-related genes expressed in fruit tissue, RNA-Seq data from ‘Braeburn’ apples stored for four months under CA conditions were used (Mellidou et al., 2014). It should be noted that while the genetic regulation of cell wall disassembly in ‘Braeburn’ apples may not be the same as in ‘Jonagold’ and other apple cultivars, the RNA-Seq database published by Mellidou et al. (2014) is, at the moment, the best available regarding postharvest storage of apples. Moreover, previously reported softening-related genes (discussed above) were shown to be highly expressed in this RNA-Seq data (Supplementary Table S1). Based on their normalised expression values (RPKM, reads per kilobase of exon per million mapped reads), a set of cell wall-related genes (within the gene families encoding PG, PME, a-AF, and b-GAL) expressed in fruit tissues during postharvest storage (RPKM > 1) was selected (Supplementary Table S1). Specific primers designed for these genes are given in Supplementary Table S2. A further screening of the candidate genes was performed by quantifying their expression in ‘Jonagold’ apples stored in CA or regular air for 2 months using qRT-PCR. 2.4. Real-time quantitative PCR (qRT-PCR) The transcriptional profiles of softening-related cell wallrelated genes identified by RNA-Seq were analysed by real-time quantitative PCR (qRT-PCR) using the SYBR Green I technology on a Rotor Gene Q (Qiagen GmbH, Hilden, Germany), as previously described (Mellidou et al., 2012). Total RNA was extracted from fruit cortex samples. Ground tissue samples (300 mg) were homogenised in 800 mL of extraction buffer containing cetyl-trimethyl-ammonium bromide, according to Gasic et al. (2004). The mixture was incubated at 65  C for 10 min with occasional mixing by inversion. Chloroform (800 mL) was added and mixed by inversion, and the mixture was centrifuged at 14,000 rpm for 10 min at room temperature. The supernatant was mixed with a half volume of ethanol, and loaded and washed through the RNeasy mini kit column (Qiagen). The purity of total RNA extracted was determined as the 260/280 nm or 260/230 nm ratio using NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA). Purified RNA was reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen) following manufacturer's protocol. All qRT-PCR reactions contained 1 mL of cDNA template (500 ng/mL), 7.5 mL of Absolute QRT-PCR SYBR Green Mix (ABgene1 Ltd., Epsom, UK), and 1 mL of 0.25 mM primer pairs, in a final volume of 15 mL. Primers were designed using the Primer3 web tool (http://bioinfo.ut.ee/primer3/) and verified against the Malus predicted consensus gene set (Velasco et al., 2010) using the BLAST function of the Genome Database for Rosaceae (Supplementary Table S3). The cycling conditions were as follows: denaturation step at 95  C for 10 min, followed by 45 cycles of denaturation at 95  C for 20 s, annealing at 63  C for 20 s, and extension at 72  C for 20 s. A melting curve analysis was performed to confirm the specificity of the primer pairs on the amplification product, ranging from 55  C to 95  C, with temperature increasing in steps of 0.5  C/s. The criteria of acceptance for reaction efficiency ranged from 90 % to 115 %, with an R2 > 0.95. The relative quantification of expression levels was performed using the

comparative cycle threshold method (Pfaffl, 2001). All qRT-PCR expression data were normalised against actin (MDP0000886327), which is commonly used as a housekeeping gene in apple (e.g. Espley et al., 2007; Ireland et al., 2014). 2.5. Cell wall pectin extraction and fractionation Cell wall material was extracted using the cold alcohol insoluble solids method proposed by Renard (2005), with some modifications. While it is common practice during cell wall extraction to inactivate endogenous cell wall enzymes by treatment with either phenol buffer, boiling ethanol, or phenol–acetic acid–water, Renard (2005) showed that in apple fruit the variability in the yield and composition of cell wall materials is statistically insignificant when compared to extraction in cold ethanol. “Precautions were taken during cell wall extraction and fractionation to minimise modifications of cell wall pectin by endogenous enzymes. About 30 g of small pieces of apple cortex tissue was frozen in liquid nitrogen, crushed in frozen state and immediately homogenised in 192 mL 95% (v/v) ethanol using a mixer (Buchi mixer B-400, Flawil, Switzerland). The suspension was filtered (Machery-Nagel MN 615 Ø 90 mm) and the residue was again homogenised in 96 mL 95% (v/v) ethanol. After another filtration step, the residue was homogenised in 96 mL acetone. The alcohol insoluble residue (AIR) was obtained by drying the final residue overnight at 40  C. The following day, the AIR was dropped into boiling water for 5 min to obtain the water extractable pectin (WEP). The mixture was filtered, and the filtrate was taken as the WEP. This procedure to extract WEP was chosen rather than extraction by stirring in ambient water for 6 h after pre-treatment with hot ethanol (as is commonly done by other researchers in the fruit ripening field), because the fraction yield was much higher (Supplementary Fig. S1). Moreover, we observed that extraction in ambient water led to a more pronounced reduction in the pectin DM, possibly due to PME action (Supplementary Fig. S2). The chelator extractable pectin (CEP) was obtained from the residue by stirring in 0.05 M cyclohexane-trans-1,2-diamine tetra-acetic acid for 6 h at 28  C. Finally, the Na2CO3 extractable pectin (NEP) was extracted by stirring the resulting residue in a 0.05 M Na2CO3 solution, containing 0.02 M NaBH4 for 22 h at 4  C, followed by a further extraction for 6 h at 28  C. The uronic acid content of the AIR and of the different pectin fractions were separately measured spectrophotometrically at 520 nm, following complete hydrolysis by concentrated sulphuric acid, based on the method described by Blumenkrantz and Asboe-Hansen (1973). The residue obtained after extraction of the NEP, which is predominantly hemicellulose and cellulose, was not further analysed since it has been reported that very little changes occur in the hemicellulose and cellulose fractions during ripening in apples (Yoshioka et al., 1992; Percy et al., 1997). Lyophilised samples of the different pectin fractions were obtained following dialysis for 48 h (molecular weight cut-off of 12–14 kDa) against demineralised water, to minimise the presence of small co-solutes. The CEP fraction was initially dialysed against 0.1 M NaCl for 24 h followed by dialysis against demineralised water for another 24 h. 2.6. Determination of the degree of methylation of pectin using Fourier transform infra-red (FT-IR) spectroscopy The pectin degree of methylation (DM) was determined using FT-IR spectroscopy. A sample from the dry material (AIR or WEP) was firmly compacted to ensure smooth surfaces, and to remove entrapped air. No pH adjustment was needed, since all samples had pH between 6.0 and 6.5. 100 scans were run per sample placed on the sample holder of the FT-IR (Shimadzu FTIR-8400S, Japan) and the transmittance was recorded at wavenumbers from

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Table 1 The expression of selected candidate cell wall-related genes, based on their expression in ‘Jonagold’ apples stored at 1  C under controlled atmosphere (CA) and at 1  C under regular air (RA) for 2 months. S.E. is the standard error of the mean of four biological replicates. Expression was quantified using real-time quantitative PCR, and are normalised with the expression of actin gene. Gene family

Malus ID

CA stored (S.E.) (firm apples)

RA stored (S.E.) (soft apples)

Naming selected candidate genes

Mda-AF

MDP0000055078 MDP0000256049

0.46 (0.15) 2.7 (0.6)

0.88 (0.62) 4.7 (1.9)

Mda-AF1 Mda-AF2

Mdb-GAL

MDP0000416548 MDP0000030527 MDP0000188563a MDP0000265046 MDP0000151981 MDP0000127542 MDP0000202465

6.6 (1.9) 0.14 (0.10) n.a. 0.42 (0.14) 0.27 (0.20) 47 (13) 0.00

14 (4) 0.00 n.a. 0.00 0.00 102 (38) 0.00

Mdb-GAL1

MDP0000236404 MDP0000168416 MDP0000326734 MDP0000943790

0.00 0.19 (0.11) 1.5 (0.5) 1.2 (0.5)

0.00 0.03 154 (16) 2.2 (1.4)

MDP0000231962 MDP0000168682 MDP0000836165 MDP0000686191 MDP0000162904 MDP0000200773 MDP0000196867

0.00 0.00 0.87 (0.19) 2.3 (1.4) 0.06 (0.02) 0.00 0.05 (0.02)

0.00 0.00 0.15 2.9 (2.6) 0.10 (0.09) 0.00 0.03 (0.02)

MdPG

MdPME

a

Mdb-GAL2

MdPG1 MdPG2

MdPME1 MdPME2

MdPME3

The expression was so low that it could not be quantified.

4000 cm1 to 400 cm1, at a resolution of 4 cm1. The spectra were converted into absorbance mode prior to base line correction and reading of the absorption at the maxima of both peaks. The obtained ratio (R) between the intensity of the peak situated at 1740 cm1 (due to ester carbonyl group (C¼O) stretching) to the combined intensities of the peak at 1740 cm1 with the peak at 1600 cm1 (due to carboxylate group (COO ) stretching) (Szymanska-Chargot and Zdunek, 2013) was used to calculate the DM based on the calibration line DM = 136.86
R + 3.987 for the WEP fraction and DM = 1.0405
R  3.9096 for AIR (Kyomugasho et al., 2015).

2.8. Data analysis Significant differences (p < 0.05) between means were investigated using full factorial ANOVAs, with 1-MCP treatment, cultivar, and time being the main factors. A multiple comparison was carried out between all factor level combinations using Turkey's Honest Significant Difference (HSD) test in JMP 11 statistical software (SAS Institute, Cary, NC, USA). For each variable analysed, significant differences were estimated in common for all means within both cultivars, with and without 1-MCP treatment, and over all time points.

2.7. Neutral sugar analysis The neutral sugar content of the different pectin fractions was analysed as described in Gwanpua et al. (2014). Briefly, the lyophilised samples were completely hydrolysed in 4 M trifluoroacetic acid at 110  C for 1.5 h. After cooling in an ice bath, trifluoroacetic acid was removed from the digested sample by evaporating under nitrogen at 45  C. The samples were then diluted to 0.1% w/v, and the different neutral sugars were quantified by high-performance anion exchange chromatography using a Dionex system (DX600), equipped with a GS50 gradient pump, a CarboPacTM PA20 column, a CarboPacTM PA20 guard column, and an ED50 electrochemical detector (Dionex, Sunnyvale, USA). Prior to sample injection, the system was equilibrated for 5 min using 100 mM NaOH, and for an additional 5 min using 4 mM NaOH. Samples (10 mL) were injected and eluted for 20 min at a flow rate of 0.5 mL/min with 4 mM NaOH at 30  C, followed by column regeneration (for 10 min) using 500 mM NaOH. Commercial neutral sugar standards at varying concentrations (1–10 mg L1) were used as external standards for identification and quantification. To correct for degradation of the monosaccharides during the acid hydrolysis step, mixtures of the sugar standards were subjected to the aforementioned hydrolysis conditions, and the peak areas were compared to those of untreated standard mixtures (Houben et al., 2011).

Fig. 1. Changes in ethylene production rates (A) and fruit flesh firmness (B) of ‘Jonagold’ (JG) and ‘Granny Smith’ (GS) apples, with and without 1-MCP treatment, at the end 6 months storage, followed by exposure to ambient shelf life conditions for 7 d. The bars are the means (30 replicates for firmness loss and four for ethylene production) and the error bars are the standard errors of the means. Means with the same letters are not significantly different (p = 0.05). CA denotes controlled atmosphere.

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Correlation analysis between cell wall-related gene expression, softening (firmness loss), and ethylene production rates was performed using Pearson's correlation coefficient using Matlab (The Mathworks Inc., Natick, USA). 3. Results 3.1. Candidate genes for regulation of cell wall disassembly ‘Jonagold’ apples stored for 2 months at 1  C under CA experienced a firmness decline of 3.8 N, while those stored at 1  C under regular air had a firmness loss of 26 N. The qRT-PCR results of the expression of the screened candidate cell wall-

related genes in fruit stored under these two conditions are shown in Table 1. Both Mda-AF genes were upregulated in the fruit, although Mda-AF1 had a much lower expression. Within the MdbGAL gene family, MDP0000416548 and MDP0000127542 were upregulated, and will be further referred to as Mdb-GAL1 and Mdb-GAL2, respectively. Two MdPG genes were overexpressed in the soft fruit: the well-studied MdPG1 (MDP0000326734), and MDP0000943790. This second MdPG gene was named MdPG2. Within the MdPME gene family, MDP0000836165 and MDP0000686191 were expressed in both CA and air stored fruit. Although MDP0000196867 had very low expression in apples stored both under CA and regular air, it was included as a candidate MdPME gene because its expression has

Fig. 2. The evolution of the expression of candidate cell wall-related genes in ‘Jonagold’ (JG) and ‘Granny Smith’ (GS) apples, with and without 1-MCP treatment, at the end 6 months storage, followed by exposure to ambient shelf life conditions for 7 d. The values represent the relative mRNA abundance normalised against actin expression. The bars are the means of four individual fruit measurements, while the error bars are the standard errors of the means. Means with the same letters are not significantly different (p = 0.05). CA denotes controlled atmosphere.

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previously been associated with ripening in apples (Wei et al., 2010). These three PME genes were named MdPME1, MdPME2, and MdPME3 respectively. The nine screened cell wall-related genes (Mda-AF1, Mda-AF2, MdPG1, MdPG2, Mdb-GAL1, Mdb-GAL2, MdPME1, MdPME2, and MdPME3) were considered as candidate genes for apple softening, and their expression were further investigated. 3.2. Expression analysis of softening-related candidate genes during storage under different conditions In order to determine whether any of the candidate cell wallrelated genes/enzymes might be considered to be key contributors to cell wall changes and softening in ‘Jonagold’ and ‘Granny Smith’ apples, we analysed gene expression in samples that had been stored in air or CA, with or without 1-MCP treatment. Correlations of ethylene synthesis, fruit softening, specific wall pectin changes, and expression of the selected candidates were then developed. The firmness and the ethylene production rates in these fruit are shown in Fig. 1. At the end of storage, 1-MCP treated fruit had lower rates of ethylene production than untreated fruit for ‘Jonagold’ apples, but no significant difference between treated and nontreated fruit for ‘Granny Smith’ was observed. Moreover, the firmness at the end of storage for 1-MCP treated fruit was not significantly different from that of the untreated fruit for both the ‘Jonagold’ and ‘Granny Smith’ apples. However, after 7 d of ambient shelf life exposure, the ethylene production rates for the 1-MCP treated fruit remained low, and were not significantly different from values at the end of storage, while there was a climacteric rise in ethylene production for the non-treated fruit. This was correlated to the extent of softening, whereby the firmness of

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the 1-MCP treated fruit at the end of 7 d exposure to ambient shelf conditions was not significantly different from the firmness at the end of storage, whereas for the untreated fruit, there was a rapid loss in firmness after 7 d of shelf life exposure. The results of the expression of the cell wall-related candidate genes in the 1-MCP and non-1-MCP treated fruit are shown in Fig. 2. The expression of all tested potentially softening related cell wall-related genes was not significantly different for 1-MCP and non-1-MCP treated fruit for both ‘Jonagold’ and ‘Granny Smith’ at the end of storage, except for MdPME1, for which the 1-MCP treated ‘Granny Smith’ apples had a significantly lower expression than the untreated fruit. After 7 d subsequent exposure to ambient shelf life conditions, there was a several fold increase in the expression of MdPG1, Mdb-GAL1 and Mdb-GAL2 in the untreated fruit, while they remained at very low levels for the 1-MCP treated fruit, comparable to the values at the end of storage. Also, there was a significant increase in the expression of Mda-AF2 for the untreated ‘Jonagold’ apples at the end of 7 d exposure to shelf life conditions. Moreover, MdPME1 expression was significantly downregulated in the untreated ‘Granny Smith’ apples when the fruit were transferred to ambient shelf life conditions. There was no obvious significant pattern in the expression of the other cell wall-related genes between the 1-MCP and non-1-MCP treated fruit, nor between the fruit at the end of 6 months storage and after 7 d subsequent exposure to ambient shelf life conditions. 3.3. Correlation analysis between cell wall-related gene and flesh softening To identify genes whose expression were correlated to softening, and possible co-expression patterns between the

Fig. 3. Pearson correlations of cell wall-related gene expression, ethylene production, firmness loss in ‘Jonagold’ (A) and ‘Granny Smith’ apples (B), and associated p-values (C and D, respectively). The heat map is described as positive values set to red colour and negative values set to blue colour. Correlations with p < 0.05 are indicated by *, and p < 0.01 by **.

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different genes, a correlation analysis was performed between all the candidate cell wall-related genes, the loss in firmness, and the rate of ethylene production. The result of the correlation plots for both ‘Jonagold’ and ‘Granny Smith’, with the associated p-values is shown in Fig. 3. MdPG1, Mdb-GAL1, Mdb-GAL2, and Mda-AF2 were strongly positively correlated to loss in firmness and ethylene production rate for both apple cultivars. The correlations were generally stronger in ‘Jonagold’ apples, with correlation coefficients between 0.95 and greater than 0.99, and the associated pvalues being less than 0.05 in all cases, except for Mda-AF2. The other candidate cell wall-related genes were poorly correlated to softening and ethylene production, with some genes showing weak negative correlations. MdPG1, Mdb-GAL1, Mdb-GAL2, and Mda-AF2 were strongly positively correlated to each other, suggesting possible coregulation of these genes. MdPG1 was negatively correlated to MdPG2 in both apple cultivars. All three MdPME genes were strongly positively correlated to each other in ‘Granny Smith’ apples, but no clear co-expression pattern was observed in ‘Jonagold’ apples. This suggests that the three MdPME genes may be co-regulated in ‘Granny Smith’ apples.

3.4. Changes in cell wall pectin polysaccharides during ripening in 1MCP treated and non-treated ‘Jonagold’ apples Cell wall pectin for the ‘Jonagold’ apples at the end of 6 months storage and after subsequent exposure to ambient shelf conditions for 7 d, for both 1-MCP treated and non-treated ‘Jonagold’ and ‘Granny Smith’ apple, was characterised. Fig. 4 shows the results of the proportion of different pectin fractions, and the galactose and arabinose contents of the different pectin fractions, at the end of storage and after 7 d of shelf life exposure for both 1-MCP treated and non-treated fruit. The water extractable pectin (WEP) fraction showed no significant effect of the 1-MCP treatment, and did not change when the apples were exposed to 7 d shelf life. Similarly, the chelator extractable pectin (CEP) and the Na2CO3 extractable pectin (NEP) fractions were not significantly different in the 1-MCP treated and untreated fruit. The side chain galactose and arabinose content in the WEP fraction was not significantly different between the 1-MCP treated and non-treated apples, although there appeared to be more in the untreated fruit after 7 d shelf life exposure. No differences in galactose and arabinose content of the side chain was observed in the CEP fractions. In

Fig. 4. Pectin characterisation of cell wall materials extracted from ‘Jonagold’ apples stored for 6 months at 1  C under controlled atmosphere (CA), with and without 1-MCP treatment, followed by exposure to ambient shelf life for 7 d. Pectin was fractionated into water extractable pectin (WEP), chelator extractable pectin (CEP), and sodium carbonate extractable pectin (NEP) fractions. The side chain neutral sugars contents for galactose (Gal) and arabinose (Ara) for the different fractions are also shown. The bars are the means of four individual fruit measurements, while the error bars are the standard errors of the means. Means with the same letters are not significantly different (p = 0.05).

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the NEP fraction, the galactose and arabinose content was significantly higher in the 1-MCP treated fruit at the end of storage, but no additional loss in these sugar was observed after shelf life exposure in both 1-MCP treated and non-treated apples. No clear softening-related losses were observed for other side chain neutral sugars (fucose, rhamnose, glucose, mannose, and xylose: Supplementary Fig. S3). The degree of methylation (DM) for both the Alcohol Soluble Residue (AIR) and the WEP fraction is shown in Fig. 5. There was no significant difference in the DM of AIR for both 1-MCP treated and non-treated apples, both at the end of storage and following shelf life exposure. The DM of the WEP was lower in the non-treated apples at the end of storage, but not after shelf life exposure. 4. Discussion 4.1. MdPG1 is the main softening-related gene, but beta-galactosidase could be key regulator of enzyme-induced cell wall disassembly during apple softening Most studies on the role of cell wall enzymes in fruit softening have focused on the role of PG because of its very high expression and activities in tomato, the model plant for fleshy fruit development and ripening (Sitrit and Bennett, 1998; Sheehy et al., 1988). In apples, several studies have shown that MdPG1 is the main cell wall-related gene that regulates fruit softening. Costa et al. (2010) used QTL analysis to show that MdPG1 expression was associated with fruit firmness in apples, a finding that was later validated by Longhi et al. (2013). Also, Atkinson et al. (2012) showed that PG1-suppressed ‘Royal Gala’ apples were firmer than wild-type ‘Royal Gala’ apples after ripening. This study confirmed that MdPG1 is indeed a key regulator of apple softening, since its expression was highest amongst all candidate genes, and showed the strongest correlations with softening. The effect of 1-MCP treatment, which inhibits ethylene production by preventing ethylene from binding to its receptors, on the expression of cell

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wall-related genes was most profound on MdPG1. Additionally, ‘Granny Smith’ apples, which softened to a lesser extent than ‘Jonagold’ apples during shelf life exposure of the non-1-MCPtreated fruit, had a lower expression of MdPG1. While MdPG1 is clearly a key regulator of softening, Mdb-GAL1 and Mdb-GAL2 were shown to be highly expressed in soft fruit, particularly in ‘Granny Smith’, where the expression of Mdb-GAL2 was even higher than MdPG1. Another important aspect was the extensive loss in side chain neutral sugars (galactose and arabinose) that were observed in the untreated fruit for both ‘Jonagold’ and ‘Granny Smith’. In an earlier study it was found that loss of neutral sugars from the side chain of RG-I is closely related to ripening (Gwanpua et al., 2014). Other studies have also shown that the loss of side-chain RG-I branching, potentially by the action of side-chain enzymes like b-GAL and a-AF, is correlated with loss of firm texture in apples (Redgwell et al., 1997; Peña and Carpita, 2004; Ng et al., 2015). In a comparative study between two apple cultivars with different softening rates, ‘Royal Gala’ and ‘Scifresh’, higher content of cell wall Gal, concomitant with lower b-GAL activity and more intense immunolabelling of RG-I galactan side chains was measured in the slower softening ‘Scifresh’ (Ng et al., 2015). The expression of both Mdb-GAL1 and Mdb-GAL2 was upregulated during ripening, and was significantly higher in untreated fruit, compared to 1-MCP treated ‘Jonagold’ and ‘Granny Smith’ fruit. Ireland et al. (2014) showed that ethylene treatment of apple fruit in which the ACC oxidase1 gene was suppressed resulted in an increased expression of Mdb-GAL2 (which they referred to as BGAL101). Downregulation of a tomato Mdb-GAL gene (TBG4) resulted in a decrease in exo-galactanase activity (15% of wild-type levels), with 50% reduction in galactose loss just before ripening, and up to 40 % reduction in softening later in ripening (Smith et al., 2002). This reduction in softening was much higher than that obtained in earlier studies where a 99% suppression of PG activity in tomato did not result in any significant reduction in softening (Langley et al., 1994; Smith et al., 1990). To the best of our knowledge, no study has investigated the effect of downregulation

Fig. 5. The degree of methylation (DM) of the alcohol insoluble residues (AIR), and water extractable pectin (WEP) fraction of ‘Jonagold’ apples stored for 6 months at 1  C under controlled atmosphere (CA), with and without 1-MCP treatment, followed by exposure to ambient shelf life for 7 d. The bars are the means of four individual fruit measurements, while the error bars are the standard errors of the means. Means with the same letters are not significantly different (p = 0.05).

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of Mdb-GAL genes on apple texture evolution. Considering the fact that increase in cell wall porosity by side chain enzyme is essential for the activity of PG (Brummell and Harpster, 2001), it would be interesting to use QTL or transgenic studies, to investigate the relevance of Mdb-GAL2 on apple fruit softening. Significant loss of RG-I side-chain arabinose also occurred during softening, but the correlation of Mda-AF2 to softening was not as strong as those shown by MdPG1, Mdb-GAL1 and Mdb-GAL2. It is possible that the loss of side-chain arabinose that occurs during apple softening is mediated by other enzymes having a-AFlike activities. For example, Nobile et al. (2011) identified a glycoside hydrolase gene (which encodes a protein with a-AF activity), which they called MdAF3, to be associated with mealiness in apples. 4.2. Softening in apple fruit is associated with downregulation of PME Like in several other studies (Gwanpua et al., 2014; Billy et al., 2008; Massiot et al., 1996), no significant change in DM was observed during softening. Although MdPME1, MdPME2, and MdPME3 were more expressed at the end of storage in the untreated fruit, particularly in ‘Granny Smith’, all three genes were rapidly downregulated during softening when the fruit were placed under shelf life conditions. Downregulation of PME during ripening could be one mechanism through which the plant prepares itself for softening, as extensive demethylation may strengthen the cell wall. While demethylation of the pectin homogalacturonan backbone by PME enhances the action of PG, the resulting free carboxyl groups may form Ca2+ cross-bridges, strengthening intercellular adhesion (Jarvis, 1984; Carpita and Gibeaut, 1993). Heat treated apples were shown to have lower degree of methylation, and were firmer than the control fruit during storage (Klein et al., 1995). The decrease in PME during ripening does not in any way downplay the involvement of this enzyme in cell wall disassembly, but suggests its role may be more relevant during fruit development, in which newly synthesised pectin polysaccharides with very high DM are demethylated as they are incorporated into the cell wall’s polysaccharide network. High PME activities were measured in expanding fruit of ‘Scifresh’ and ‘Royal Gala’, but the activities rapidly decreased in the mature and ripe fruit (Ng et al., 2013). Similar observations were made in ‘Mondial Gala’ apples (Goulao et al., 2007). The high DM of pectin in both the AIR and WEP fraction should not preclude the action of PG, since plant homogalacturonan is thought to be de-esterified in blocks, as plant PMEs are processive  and Kohn, 1984). Therefore, regions of (Catoire, 1998; Markovie zero methyl esterification would be present and available for PG action, unless where they were aggregated into junction zones (Ngouémazong et al., 2012; Sénéchal et al., 2014). Pectin solubilisation was observed, and could be related to hydrolysis of pectin by PG. In a separate study, Gwanpua et al. (2014) showed that depolymerisation of water soluble polyuronide is observed in apples after extensive softening. Polyuronide depolymerisation has often been associated with the later stages of softening (Rose et al., 1998). 5. Conclusions Cell wall metabolism during fruit softening is a complex process and is unlikely to be regulated by a single cell wall-related gene. While PG has received the most attention, it was shown in this study that other cell wall-related genes, specifically the b-GAL genes, may also play an important role in regulating apple fruit softening. This may be of much importance during early ripening,

where solubilisation of polyuronide occurs in the absence of significant polyuronide depolymerisation, but with extensive loss of side chain galactose and arabinose. Although PME has been reported to be involved in cell wall disassembly, no candidate gene within the PME gene family was regulated during softening in ‘Jonagold’ and ‘Granny Smith’ apples, nor was there any significant demethylation. In addition to the well-studied MdPG1 gene, MdbGAL1, Mdb-GAL2, and Mda-AF2 are important genetic markers for apple fruit softening that should be considered in future transgenic and QTL studies to better understand apple fruit softening, particularly Mdb-GAL2. Acknowledgements This publication has been produced with the financial support of the European Union (grant agreement FP7/2007-2013—Frisbee), and the European COST Action FA1106 (‘QualityFruit’). The opinions expressed in this document do not by any means reflect the official opinion of the European Union or its representatives. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. postharvbio.2015.09.034. References Atkinson, R.G., Johnston, S.L., Yauk, Y.K., Sharma, N.N., Schröder, R., 2009. Analysis of xyloglucan endotransglucosylase/hydrolase (XTH) gene families in kiwifruit and apple. Postharvest Biol. Technol. 51, 149–157. Atkinson, R.G., Sutherland, P.W., Johnston, S.L., Gunaseelan, K., Hallett, I.C., Mitra, D., Brummell, D.A., Schroder, R., Johnston, J.W., Schaffer, R.J., 2012. Downregulation of polygalacturonase 1 alters firmness, tensile strength and water loss in apple (Malus
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