Abscisic acid induces differential expression of genes involved in wound-induced suberization in postharvest tomato fruit

HAN Xue-yuan, MAO Lin-chun, LU Wen-jing, TAO Xiao-ya, WEl Xiao-peng, LUO Zi-sheng

College of Biosystems Engineering and Food Science/Fuli Institute of Food Science/Zhejiang Key Laboratory of AgroFood Processing, Zhejiang University, Hangzhou 310058, P.R.China

Abstract Fruit wounding occurred at harvest and transportation requires rapid suberization as a major part of the healing process to prevent infection and desiccation. The focus of this work was to explore the mediation of abscisic acid (ABA) on woundinduced suberization and to determine expression profiles of specific genes involved in wound-induced suberization in tomato fruit. The measurements of weight loss and fruit flrmness suggested wound-induced suberization started at 2 d after wounding. The suberization process with the accumulation of suberin polyphenolics (SPP) and polyaliphatics (SPA)observed through autofluorescence microscopy and Sudan IV staining was accelerated by ABA. Expressions of SlPAL5 and Sl4CL involved in the synthesis of SPP reached the highest at 4 and 8 d after wounding following ABA application,respectively. Associated with SPA biosynthesis, SlLACS1 and SlLACS2 showed the most abundant transcripts at 8 and 6 d in ABA group, respectively. Transcript levels including SlKCSs, SlCYP86B1, SlFAR3, and SlGPATs were up-regulated at 2 d after wounding by ABA. Activities of polyphenol oxidase and lipoxygenase were also enhanced during wound-induced suberization following ABA application. The results in this study proved that ABA accelerated the wound-induced suberization progress by increasing the transcript levels of relevant genes in postharvest tomato fruit.

Keywords: tomato, abscisic acid, wound-induced suberization, genes

1. lntroduction

Physical wounding is often the case occurred at harvest,handling, transportation and storage of fruit. Therefore,wound-healing plays an important role in maintaining postharvest quality and shelf life of fruit. Complete healing requires a complexly structural and physiological progress starting with a rapid oxidation at the wound surface (Razem and Bernards 2003), and followed by the establishment of a functional suberin system (Dastmalchiet al. 2015). In addition, the formation of wound-healing layer was reported to involve hormone signaling, as well (Cooksonet al. 2013;Taoet al. 2016). The phytohormone abscisic acid (ABA) was associated to the wound-healing of potato tuber (Lulaiet al.2008; Kumaret al. 2010) and tomato fruit (Taoet al. 2016).

A naturally occurring suberization phenomenon of apple surface and analogous suberization due to cuticular deflciency in tomato skin were mediated by genes expression program (Kawaharaet al. 2013; Lashbrookeet al. 2015, 2016). So, wound-induced suberin formation of tomato fruit would be triggered by the differential genes expression spatially and temporally. According to related literature (Ranathungeet al. 2011; Frankeet al. 2012),the key enzymes of suberin biosynthetic pathway was introduced and functionally evaluated as followed.

The induction of phenylalanine ammonialyase (PAL) is crucial in the initial step of the phenylaprapanoid metabolism and the production of various aromatics associated with the suberin polyphenolics (SPP) (Joos and Hahlbrock 1992;Bernards 2002). 4-Coumarate ligase (4CL) is another important enzyme which catalyzes the reaction as the production of caffeoyl-CoA, feruloyl-CoA, andp-coumaroyl-CoA in this pathway, and these acyl-CoAs are speculated to contribute to link SPP and suberin polyaliphatics (SPA)domain (Kolattukudy 2001; Pollardet al. 2008).

When SPP is assembled in a programmed module into the walls of the parenchyma cells near the wound, SPA precursors are synthesized by elongation, hydroxylation and omega oxidation of fatty acids modifled by series of suberin biosynthetic enzymes. First, the general step for metabolizing fatty acids is to convert free fatty acids to corresponding fatty acyl-coenzyme A thioesters by long chain acyl-CoA synthetases (LACSs), which are required to activate intermediates such as C16 and C18 fatty acids(Schnurr and Shockey 2004; Luet al. 2009). The suberin polymer is featured by the composition of long chain and very-long-chain fatty acids (Pollardet al. 2008). The β-ketoacyl-CoA synthases (KCSs) belonging to the fatty acid elongase complex regulate the elongation of long-chain fatty acyl-CoAs (Pollardet al. 2008). Literature indicated that KCS6 and KCS20 were responsible for suberin precursors to extend by C28 or longer chain (Paulet al. 2006; Serraet al.2009). The ω-hydroxy acids and α,ω-dicarboxylic acids are two major components of the suberin polymer. CYP86 subfamily belonging to cytochrome P450 monooxygenases catalyzes the hydroxylation of the ω-position methyl group of fatty acids (Compagnonet al. 2009; Molinaet al. 2009;Vishwanathet al. 2015).

Long-chain fatty acids are catalyzed to be fatty alcohols by fatty acyl reductases (FARs) as important constitutes of suberin. InArabidopsis, there was a family of eight FAR genes recognized by Rowland and Domergue (2012).FAR1,FAR4andFAR5of these genes are referred to the generation of fatty alcohols in suberin (Domergueet al.2010; Vishwanathet al. 2015). By the T-DNA insertion,there have shown chain-length speciflc decrease in primary alcohols from mutant lines of each gene:far1decreased in C22 fatty alcohol,far4decreased in C20 fatty alcohol andfar5decreased in C18 fatty alcohol in suberin composition(Domergueet al. 2010).

With bothsn-2 acyltransferase and phosphatase activities, glycerol-3-phosphate acyltransferase GPAT4 and GPAT6 are special bifunctional enzymes contributing to the production ofsn-2 monoacylglycerol (Yanget al.2010). GPAT4 and GPAT6 are strongly inclined to C16:0 and C18:1 ω-oxidized acyl-CoAs than longer or unmodifled acyl chains aliphatics (Yanget al. 2010). Remarkably,mutation of single geneGPAT6led to obvious changes in pericarp genes transcript levels related to lipid biosynthesis(Petitet al. 2016).

Oxidation reaction has been suggested to have roles in suberization (Quirogaet al. 2000, 2001; Lucenaet al.2003) and ligniflcation (Whettenet al. 1998; Lewis 1999).The accumulation of both aromatic and aliphatic suberin in potato was correlated to enhancement of oxidase activities(Espelie and Kolattukudy 1985). The linkage of cell walls and the deposit of ligniflcation on plant tissues occurred partly because of the enhanced activity of polyphenol oxidases (PPO) (Gillet al. 2010). Lipoxygenase (LOX)catalyzes the polyunsaturated fatty acids to be oxidized as a result of generating superoxide anions, hydroperoxides,and peroxy radicals (Gardner 1980), which are supposed to be signal molecules of wound-healing (Tenhakenet al.1995). LOX also exhibited wound dependent increases in activity and related to suberization (Bernardset al. 2004).

This research focused to explore the mediation of ABA on wound-induced suberization and to determine quantitative expression proflles of wound-induced genes within healing time course to reveal the differential association of speciflc genes with wound-induced suberization following ABA application in tomato fruit.

2. Materials and methods

2.1. Plant materials

At green immature stage (about 25 d after anthesis), cherry tomato (Solanum lycopersicumvar.cerasiforme Xintaiyang)without physical injuries or infections were harvested on the basis of size and color from a greenhouse (20–25°C,70–85% relative humidity) located at Xiaoshan District,Hangzhou, China. Surface-disinfection of harvested fruit was conducted with 0.5% (v/v) solution of sodium hypochlorite for 3 min, followed by washing with sterile deionized water and drying at room temperature.

2.2. Fruit wounding and experimental treatments

Two wounds of about 10 mm diameter and 1.5 mm deep dimension were carried out with a sterilized scalpel on each fruit equator. Then, the wounded fruit was evenly divided into three groups and each group was put into a vacuum dryer (SHZ-D III, Mingyuan Instrument Co., Ltd., China)containing correspondingly treating solution to vacuumize(0.07 MPa, 3 min) at room temperature. The treating solutions were deionized water (control), 0.5 mmol L–1ABA(abscisic acid, ≥90%, Aladdin Industrial Inc., China) and 0.1 mmol L–1FLD (fluridone, an inhibitor of ABA biosynthesis,99.8%, Fluka Analytical, Germany), respectively. ABA and FLD concentrations and vacuuming conditions used in this experiment were based on results of preliminary experiment(Taoet al. 2016). After treatments, fruit were air-dried and put in darkness for wound-healing in a 20°C and 90%humidity chamber (HWS, Ningbo Southeast Instrument Co., Ltd., China).

2.3. Measurements of weight loss and fruit firmness

Weight loss was determined by weighing fruits at the start of the experiment and at indicated intervals according to Van Dijket al. (2006). This approach allowed indirect measurement of water evaporation loss. Every ten wounded fruits as a replicate were weighed and recorded.After weighing, tomatoes were put back to original healing conditions and then re-weighed at indicated intervals. The weight loss percentage was calculated as following:

W0is the average weight of the flrst batch (three replicates) at 2 d and Wtis the average weight of the same batch attd.

Fruit flrmness of each individual tomato was measured at two wounds using the TA.XT2i texture analyzer (Stable Microsystems Texture Technologies Inc., UK). Every ten wounded fruits as a replicate were measured. The cylinder probe with 4-mm diameter penetrated the sample with a test speed of 1 mm s–1and a uniform force 5.0 g to a depth of 2 mm. The maximum peak force (g) was recorded as kg cm–2.

2.4. Suberization determination

The accumulations of SPP and SPA on parenchyma cell walls were determined and regarded as estimable criteria of suberization formation of healing layer. Isolate healing sections with a scalpel from wound-healing layer at the indicated time and then dehydrate sections in 20, 30 and 40% (m/v) sucrose solution for 20 min, orderly and respectively. The sections were then embedded into medium(Surgipath?(Leica) FSC 22?, Leica Biosystems, USA) and cooled to –15°C. By a cryostat microtome (CM1950, Leica Microsystems Co., Germany), longitudinal sections of 10 μm thickness were prepared. The rating of suberization were separately determined through the accumulation of SPP with autofluorescence and SPA staining by Sudan IV (Sigma Chemical Co.) on suberizing layer from longitudinal sections in triplicate, using methods described by Kesanakurtiet al.(2012) and Taoet al. (2016). A Zeiss LSM780 confocal light microscope (Carl Zeiss Inc., Thornwood, NY, USA)conflgured for epifluorescence illumination and white light,was performed to microscopy. According to Lozano-Baenaet al. (2007) and Lulai and Neubauer (2014), phenolic compounds were determined by autofluorescence under laser excitation at 488 nm. And the accumulation of SPA stained by Sudan IV was observed under white light. Digital images were collected with a Leica DFC450 camera (Leica Microsystems Co., Germany). Healing sections were also maintained at –80°C for following molecular experiments.

Quantiflcation of fluorescence intensity by densitometry with mean fluorescence density was analyzed and staining area percentage with Sudan IV was calculated by ImageJ software (W.S.Rasband, National Institutes of Health, USA).

2.5. RNA extraction, cDNA synthesis and quantitative real-time PCR analysis

Trizol reagent method was applied to extract total RNA from healing sections and measure the absorbance value of RNA extraction at 260 nm to determine RNA contents.The PrimeScriptTMRT reagent Kit with gDNA Eraser (Perfect Real Time, TaKaRa Bio Inc., Dalian, China) was applied to reverse transcribe the flrst-strand cDNA from DNase-treated RNA according to the manufacturer’s instructions. The cDNA was used as template for real-time PCR (qRT-PCR).

Gene transcriptional levels were measured by real-time PCR on a CFX96-TouchTMDeep Well Sequence Detection system (Bio-Rad Laboratories, Inc., CA, USA). SYBR?PremixEx TaqTMII (Tli RNaseH Plus) (TaKaRa Bio Inc.,Dalian, China) was used to amply the cDNA. And SYBR-green fluorescence was used to monitor the ampliflcation of the target genes every cycle. Gene-speciflc primers, SYBR?PremixEx TaqTMII (Tli RNaseH Plus) and the template were mixed to obtain the PCR reaction (25 μL) mix. The thermal cycling procedure was 95°C for 2 min followed by 95°C for 5 s, 60°C for 30 s and for 40 cycles. Melting curves were obtained through recording the absorbance every 0.5°C from 60 to 90°C. As a PCR cycle where a statistically signiflcant increase of reporter fluorescence flrstly was detected, the Ct(threshold cycle) was recorded to indicate the starting copy numbers of the target gene. Relative quantitation of transcript levels was determined using the 2–ΔΔCTcalculation method (Livak and Schmittgen 2001) and was presented as fold change relative to the distilled water (control) treatment at 2 d which was normalized to a value of one.Actinwas selected as reference gene and each gene was analyzed in triplicate. All the primers for qRT-PCR analysis are listed in Table 1.

Table 1 Genes and primers sequence used for qRT-PCR expression analysis

2.6. Measurements of LOX and PPO activity

The crude enzyme of lipoxygenase (LOX) was extracted according to Aghdamet al. (2014). Healing sections (0.2 g)of each replicate was ground under low temperature with 1.0 mL of 50 mmol L–1Tris–HCl (pH 8.0), containing 0.5 mmol L–1phenylmethylsulfonyl fluoride, 10 mmol L–1KCl and 500 mmol L–1sucrose. Then, homogenize the extracts and centrifuge at 4°C at 12 000×g for 10 min. The supernatants were collected for LOX activity assay according to ELISA Kit of plant lipoxygenase (Shanghai Enzyme-linked Biotechnology Co., Ltd., China).

Polyphenol oxidase (PPO) enzyme was extracted according to Liuet al. (2007) with some modiflcations.Section sample (0.2 g) was ground under low temperature with 1.0 mL of 100 mmol L–1sodium phosphate buffer(pH 6.5) containing 1% (v/v) Triton X-100 and 4% (w/v) of polyvinyl polypyrrolidone (PVPP). Then, homogenize the extracts and centrifuge at 4°C at 15 000×g for 30 min and the supernatant was used for enzyme assay in accordance with ELISA Kit of plant polyphenol oxidase.

2.7. Statistical analysis

This study was conducted as a randomized block design with 3 harvests and 3 blocks of tomato fruits each harvest corresponding to three different treatment. Unless indicated,data presented represent the mean and the standard error(mean±SE). The statistical signiflcance of the differences among samples was analyzed by Duncan test atP=0.05 signiflcance level using the version 20.0 of SPSS (IBM Corp, Armonk, USA). WhenP≤0.05, a difference was regarded to be statistical signiflcance and expressed with different letters.

3. Results

3.1. Weight losing rate and fruits firmness on wounds

The weight losing rate was considered as water evaporation indicator from wounds. The difference among treatments was observed from the 2nd d and more obvious at 4 d.Furthermore, ABA treatment with the lowest losing rate slowed water evaporation (Fig. 1-A). Weight loss was more in FLD and control and continued through 8 d after wounding.

Fig. 1 The trend lines of weight losing rate (A) and fruit flrmness (B) in water, abscisic acid (ABA) and fluridone (FLD) treated groups, respectively.

Fruit flrmness on wound sites was also an important index for healing development, which decreased gradually with healing-storage (Fig. 1-B). First, with storage time, the flrmness of the whole fruit was inevitably reduced by ripening and water losing. But the difference in decline rate among treatments was observed from the 2nd d. And the downtrend after 6 d has not been as fast as that in the flrst 5 d.Furthermore, ABA group relieved more wound softening compared to other two groups.

3.2. Observation of wound-induced suberization

The progress of wound-induced suberization was illustrated by the separate accumulations of SPP (Fig. 2) and SPA(Fig. 3) on healing layerviaobserving longitudinal sections.The initial phase of suberization involved in the deposition of SPP on cell walls neighboring wounds surface (Fig. 2).Four days after wounding, especially in ABA group, intensity of SPP accumulations was visibly strong and fluorescence area had also extended to contiguous cell walls. At 8 d, SPP had accumulated around the entire layer of parenchyma cell walls on the wounds and density reached 0.10 A.U. in ABA group (Fig. 2-M). ABA treatment promoted more SPP accumulation than water and FLD treatments.

Relatively mild accumulations of SPA on the wounds began at 2 d and became contiguous on the outer parenchyma cell walls 4 d after wounding. By 6 d, SPA accumulations became discernible on the cell walls when the Sudan IV staining area was 2.70% in ABA group while 1.90% in control (Fig. 3-M). Afterwards, SPA accumulation on newly formed wound-healing layer turned slow with a horizontal asymptotic pattern in staining area.

Considering above physiological parameters and histological observation, wound-healing or suberin layer of tomato fruit has mainly formed within 8 d after wounding.Both SPP and SPA began accumulating gradually from the 2nd d, and obviously increasing accumulation of SPP and SPA appeared 4 and 6 d after wounding, respectively. The ABA-treated fruit showed quicker healing process and more SPP and SPA than control and FLD.

3.3. Transcript levels of SlPAL5 and Sl4CL

PALand 4CL are the rate-limiting enzymes of phenylalanine pathway and initiate the monomers production for SPP accumulation. Wounding induced a near 3-fold increase of basal level inSlPAL5transcript in control group by 4 d after wounding, and the expression was further up-regulated by 4.55 fold in ABA group. This rise was followed by a gradual decline from 6 d and decreased to the initial level at 8 d(Fig. 4-A).Sl4CLencoding 4-coumarate ligase catalyzes phenolic acids into corresponding acyl-CoA esters for crosslinking among suberin monomers. The transcription ofSl4CLpresented up-regulation upon wounding and got the maximum at 8 d, especially in ABA group (Fig. 4-B).SlPAL5expression showed a greater change range and appeared a peak earlier thanSl4CL. In addition, ABA promoted the transcriptions ofSlPAL5andSl4CLin a certain extent during wound-healing period.

3.4. Transcript levels of genes associated with SPA synthesis

SlLACS1andSlLACS2encoding long chain acyl-CoA synthetases contribute to activating intermediates such as C16 fatty acids in suberin synthesis.SlLACS1andSlLACS2had the different expression patterns among treatments(Fig. 5-A and B). Two dyas after wounding, the expression ofSlLACS1was down-regulated in ABA and FLD group.Afterwards, expression ofSlLACS1was up-regulated by ABA until reaching the maximum of 2.82 fold at 8 d. The transcription ofSlLACS2was also enhanced by ABA, except at 4 d after wounding. Wound induced greater change ofSlLACS2thanSlLACS1and ABA deflnitely accelerated their transcriptions especially at 6 and 8 d, respectively. In contrast, transcription level ofSlLACS1was suppressed obviously by FLD.

Fig. 2 Autofluorescence and density of suberin polyphenolics (SPP) accumulation at wound-healing layer of tomato fruit in water,abscisic acid (ABA) and fluridone (FLD) treated groups, respectively. The values are expressed as mean±SE of triplicate. Vertical bars indicate standard errors. Bars=200 μm.

SlKCS6andSlKCS20encoding β-ketoacyl-CoA synthase control the length of fatty acyl-CoAs. The transcript level ofSlKCS6increased during wound-healing period, especially in ABA group at 2 and 8 d when it was 2.51 and 2.29 fold as much as the basal level. ABA always promotedSlKCS6transcription during wound-healing period (Fig. 5-C).ABA treatment tremendously motivated the expression ofSlKCS20by 5-fold upon wounding at 2 d. However,the expression ofSlKCS20maintained at low levels after wounding, although the expression in ABA group was higher than control and FLD (Fig. 5-D).

SlCYP86B1encoding fatty acyl ω-hydroxylase catalyzes hydroxylation of the ω-position (terminal position) methyl group of aliphatics. ABA induced moreSlCYP86B1transcription especially at 2 d after wounding. Afterwards,the expression did not continue to increase, instead it showed little changes from 4 d after wounding. However,FLD depressed the expression ofSlCYP86B1which maintained a constant low level (Fig. 5-E).

FAR3 encoding a fatty acyl reductase catalyzes fatty acids to fatty alcohol as an essential constitutor in suberin.In Fig. 5-F, ABA considerably promoted the transcription ofSlFAR32 d after wounding. In the next few days, the transcription ofSlFAR3stayed at lower levels, but ABA showed up-regulation influence. The expression in FLD group was inhibited and always displayed the lowest level.

SlGPAT4andSlGPAT6encoding glycerol-3-phosphate acyltransferase generate speciallysn-2 monoacylglycerol monomer. Transcriptional level ofSlGPAT4was similar withSlGPAT6during wound-healing period (Fig. 5-G and H). Expressions of bothSlGPAT4andSlGPAT6were tremendously promoted by ABA 2 d after wounding (7.37 and 5.53 fold, respectively). However, this rapid increase expression ofSlGPAT4andSlGPAT6was followed by a near 5- and 4-fold decrease. In contrast, FLD also displayed down-regulation in expressions of bothSlGPAT4andSlGPAT6.

Fig. 3 The suberin polyaliphatics (SPA) accumulation stained by Sudan IV of wound-healing layer on tomato fruit in water, abscisic acid (ABA) and fluridone (FLD) treated groups, respectively. The values are expressed as mean±SE of triplicate. Vertical bars indicate standard errors. Bars=100 μm.

Fig. 4 The transcription levels of SlPAL5 and Sl4CL during wound-healing period in water, abscisic acid (ABA) and fluridone (FLD)treated groups, respectively. The values are expressed as mean±SE of triplicate. Vertical bars indicate standard errors. Different letters show signiflcantly difference between samples by Duncan’s test at P≤0.05.

3.5. Transcript levels and enzymatic activity of PPO and LOX

Fig. 5 The expression of genes related to suberin aliphatics during wound-healing period in water, abscisic acid (ABA) and fluridone(FLD) treated groups, respectively. Vertical bars indicate standard errors. Different letters show signiflcantly difference between samples by Duncan’s test at P≤0.05.

Fig. 6 The changes of SlPPO (A) and SlLOX (B) expression and their enzymatic activities (C and D) in tomato fruit during woundhealing. The values are expressed as mean±SE of triplicate. Vertical bars indicate standard errors. Different letters show signiflcant difference between samples by Duncan’s test at P≤0.05. FW, fresh weight.

The expression pattern ofSlPPOwas similar withSlLOX(Fig. 6-A and B). Their expressions reached the highest levels at 4 d and obviously enhanced by ABA, then gradually declined. Changes in the enzymatic activity of PPO in the wound-healing tissue were presented in Fig. 6-C, which was consistent with the expression ofSlPPO. Compared to the level at 2 d, there was a signiflcant increase in PPO activity at 4–6 d after wounding (maximum 99.18 U g–1FW). Similar withSlLOXexpression, LOX activity showed a rapid increase upon wounding, especially in ABA treatment(Fig. 6-D). However, the transcript levels and enzymatic activities of PPO and LOX all declined to a much lower level 8 d after wounding than the initial value at 2 d.

4. Discussion

Hormones play regulatory role in the overall wound response(Suttleet al. 2013). The phytohormone ABA known mainly for its effect on fruit ripening plays an essential role in adaptive responses to various stresses. It was considered as an important signal regulator for wound response because of its effect on the activation of several defense and suberization genes in wounded tomato fruit. Moreover, we applied inhibitor (FLD) of ABA to further explore the effects of ABA on wound-induced suberization. ABA positively enhanced the activity and transcript of enzymes and genes associated with suberin, although accurate regulatory mechanism has not been made. The metabolism and signal transduction of ABA would be bound up in promoting suberin biosynthesis.

Suberin is a kind of lipid polyester occurred in barrier tissues of plants, in which two building blocks (SPP and SPA) were identifled as key structures (Lendzian 2006).Lashbrookeet al. (2016) proposed that the induction of suberization in tomato fruit required a certain extent of injury, rather than the phenomenon in cutin deflcient(cd1,cd2andcd3) (Isaacsonet al. 2009). In this work, due to the excision of cutin and its replacement with suberin,changes in gene transcriptional levels occurred in suberized fruit surface. Through parallel coexpression comparison analysis of transcriptome datasets from multitissues and several species, Lashbrookeet al. (2016) found several coexpression genes of suberin formation, and these genes were mainly associated with fatty acid synthesis,phenylalanine metabolism, extracellular polymerization,and transcriptional regulation. Candidate genes of SPP and SPA biosynthesis of tomato fruit were also collected from the synthesis pathway of suberin in related researches (Soleret al. 2007; Ranathungeet al. 2011; Lashbrookeet al. 2016).

PAL has long been recognized as a very important enzyme in wound-healing and particularly wound-induced suberization. Our previous study also reported its distinct increase in activity during wound-healing (Taoet al. 2016).The wound-inducible increase inSlPAL5transcription would be the prompt need of phenols as part of healing layer.4CL as an essential ligase plays role in the later stage of phenylalanine metabolism, which catalyzes ferulic acid,cinnamic acid and caffeic acid into corresponding acyl-CoA esters those routed for assembly and polymerization into the SPP (Mintz-Oronet al. 2008). Lashbrookeet al. (2016)also suggested 4CL may be speciflc to suberin formation inArabidopsisseeds. In this research, the prompt increase inSlPAL5expressionand constant up-regulationofSl4CLcould be related with the accumulation of SPP monomers of tomato fruit after wounding. The rapid induction ofSlPAL5andSl4CLalso contributed to the initiation of SPP autofluorescence.

In previous reports, chemical components of SPA in wounded tomato fruit included longer chainn-alkanes (C16 to C25), alkanoic acids (C14 to C22), ω-hydroxy acids and α, ω-dicarboxylic acids (Taoet al. 2016). LACSs, KCSs,CYP86B1, FARs and GPATs play roles in the synthesis of SPA as descripted by Frankeet al. (2012) and Vishwanathet al. (2015). In this study,SlLACS1was mediated fluctuant andSlLACS2was obviously up-regulated, which might suggest their contribution to the production and acylation of long chain fatty acids during wound-healing of tomato fruit.Chemical component analyses of the mutants ofLACS2gene indicated additional actions in suberin synthesis (Li-Beissonet al. 2013).AtKCS2andAtKCS20, two β-ketoacyl-CoA synthases ofArabidopsis thaliana, were referred to elongation of C20 acyl precursors in suberin (Frankeet al.2009; Leeet al. 2009).StKCS6of potato tuber was shown to be considerably expressed in suberizing periderm (Serraet al. 2009). Similarly,SlKCS6andSlKCS20were also detected to be induced by wound-induced suberization in tomato fruit, and their relatively stable up-regulation transcription would consolidate the accumulation of SPA in healing layer of tomato fruit.

Some studies about mutant and transgene, such asArabidopsis cyp86b1mutant (Compagnonet al. 2009),transgenic expression ofTaFAR5of bread wheat (Triticum aestivum) in tomato (Wanget al. 2015), overexpression ofArabidopsis GPAT4 andGPAT8(Liet al. 2007), andArabidopsisgpat5mutant (Beissonet al. 2007), further demonstrated the effects ofCYP86B1,FARsandGPATson suberization. In tomato fruit, wound induced rapid expression ofSlCYP86B1,SlFAR3,SlGPAT6, andSlGPAT4although declined later,whichindicated their possible roles in wound-induced suberization of tomato fruit.SlCYP86B1could initiate the generation of ω-hydroxy acids and α,ω- dicarboxylic acids during wound-healing in tomato fruit.The general components of suberin include saturated fatty alcohols of C18, C20 and C22 chain lengths (Schreiberet al. 2005; Domergueet al. 2010). The rapid expression ofSlFAR3might be for generation of fatty alcohols of tomato suberin. GPATs catalyze acyl transfer reactions to produce monoacylglycerols that could be regarded as the initial suberin model (Vishwanathet al. 2015).

The PPO in plants catalyzes oxidation of phenols to quinones in O2-dependent manner, which has been considered as a defence response (Subramanianet al.1999). The biosynthesis of polyphenols and the process of suberization at wound sites require the increase in PPO activity (Rhodes and Wooltorton 1978). The free radicals produced by LOX were also very active and contributed to ligniflcation, which represented a protection for plants(Gillet al. 2010). Regardless of their supposed effect on the polymerization in suberin accumulation, these reactive oxygen species (ROS) up-regulated defense-accociated genes serving possibly as signaling molecules to initiate suberization. At the initial stage before healing-layer completion, the transcriptional response and activities of PPO and LOX increased rapidly responding to wounding.With completion of healing-layer, the oxidation process gradually reduced and their activities also decreased accordingly.

The enzymatic activity and gene transcript of signaling enzymes in common are often rapidly enhanced (Hirt 1999).Interestingly, the transcript levels ofSlKCS20,SlCYP86B1,SlFAR3,SlGPAT4, andSlGPAT6reached the maximal peak 2 d after wounding in an ABA-dependent manner. This result indicated that the response of these genes to wounding of tomato fruit could be a kind of quick and temporary process as a kind of initial signal, and then their metabolic products were gradually deposited to constitute suberins.

5. Conclusion

Through physiological traits and histological observation,our study showed that ABA accelerated the progress of wound-induced suberization in postharvest tomato fruit.The measurements of weight loss and fruit flrmness suggested wound-induced suberization was likely to start at 2 d after wounding. Autofluorescent accumulation of SPP developed from scattered fragments to contiguous modules, flnally extended around integrated cell walls. Prior to SPA accumulation, obvious accumulation of SPP began at 2 d after wounding, gradually deposited and covered the superflcial cells. Collectively, the assembly of SPP and SPA was promoted following ABA application. In addition to the SPP and SPA accumulation, there were notable alterations in transcript levels evaluated in time terms to gain insights into the formation of suberin in tomato fruit. The expression proflles of genes considered to be speciflc to suberin biosynthesis were found to be up-regulated as a whole in response to wounding following ABA application, includingSlPAL5,Sl4CL,SlLACS1,SlLACS2,SlKCS6,SlKCS20,SlCYP86B1,SlFAR3,SlGPAT4,andSlGPAT6. So it was speculated that ABA promoted the accumulation and integrity of wound suberin in tomato fruitviaenhancing the transcript levels of related genes. The differentials in their expression proflles were also the primary demonstration of measurable changes of biological flux as wound-healing development. Evaluation about expression proflles of wound-induced genes that mediate suberin formation would help in understanding the wound-healing process in tomato fruit. In addition, the wound-induced increase in activities of LOX and PPO also suggested roles supporting healing and wound-induced suberization of tomato fruit.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (31372113) and the National Basic Research Program of China (973 program, 2013CB127101).