Effects of exogenous auxin on pigments and primary metabolite profile of postharvest tomato fruit during ripening

Scientia Horticulturae 219 (2017) 90–97

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Effects of exogenous auxin on pigments and primary metabolite profile of postharvest tomato fruit during ripening Jiayin Li a , Zia Ullah Khan b , Xiaoya Tao a , Linchun Mao a , Zisheng Luo a , Tiejin Ying a,∗ a College of Biosystems Engineering and Food Science, Fuli Institute of Food Science, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang R & D Center for Food Technology and Equipment, Zhejiang University, Hangzhou, People’s Republic of China b Department of Agriculture, Abdul Wali Khan University, Mardan, Khyber-Pakhtunkhwa, Pakistan

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Article history: Received 29 December 2016 Received in revised form 28 February 2017 Accepted 2 March 2017 Keywords: Auxin Fruit quality Primary metabolite profiling Pigment metabolism

a b s t r a c t Auxin is an important plant hormone and plays crucial roles in regulating fruit ripening. The delay of ripening after auxin treatment has been found in tomato and other fleshy fruit. However, the influence of auxin on metabolites alteration during tomato ripening period has not been extensively studied. To investigate the impact of exogenous auxin on tomato fruit quality, pigment metabolism, primary metabolite profiling, and the expression of selected ripening-related transcription factor genes were analyzed. The results showed that exogenous auxin significantly interfered the accumulation and conversion of pigments, total phenolics and flavonoids but did not largely influence the final content of these compounds in full ripe tomato fruit. Dramatic changes on the content of primary metabolites were induced by auxin during tomato ripening period and the alterations were not able to be completely restored at the end of ripening. The contents of citric acid, threonic acid and succinic acid were increased whereas alanine and aspartic acid accumulation was repressed in auxin-treated fruit. The expression patterns of transcription factor genes related to ripening were also disturbed by exogenous auxin. The present study provided an overall insight on how auxin regulates pigment and primary metabolite accumulation during ripening stage and offered useful information for further investigation of auxin impact on fruit quality. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Fruit ripening is a highly complex process, accompanying with massive biological and biochemical changes which lead to the final ripe fruit, and is precisely regulated by plant hormones such as ethylene, abscisic acid and other phytohormones (Kumar et al., 2014; Prasanna et al., 2007). Auxin, a key plant hormone in regulation of cell elongation and division, plays crucial roles in fruit setting and development (Rosquete et al., 2012; Srivastava and Handa, 2005). To date, more and more evidences have revealed that auxin also participates in the regulation of ripening process in its own manner and by interacting with other hormones (Chen et al., 2016; Trainotti et al., 2007; Ziliotto et al., 2012). Fruit ripening process is largely affected by endogenous auxin content. High level auxin has been observed in tomato fruit of ripening-inhibitor (rin) mutant and the decrease of auxin level in fruit might be necessary for triggering ripening process (Liu et al., 2005; Rolle and Chism, 1989) .Transcriptional profiling studies showed that the expression levels of many genes involved

∗ Corresponding author. E-mail address: [email protected] (T. Ying). http://dx.doi.org/10.1016/j.scienta.2017.03.011 0304-4238/© 2017 Elsevier B.V. All rights reserved.

in auxin signaling process significantly changed during ripening period, implying the participation of auxin in ripening process (Kumar et al., 2011; Liu et al., 2011). Beside modulating ripening process, auxin may also influence fruit quality. In apple, sweet berry and grape, the application of exogenous auxin at early stage of fruit development has been found to be able to improve the yield of fruit and change the level of volatile compounds (Bottcher et al., 2011; Yuan and Carbaugh, 2007; Zhang and Whiting, 2011). Recent studies have suggested that silence or overexpression of several auxin response genes, such as SlARF4, SlARF2a and SlIAA27, leads to the conspicuous changes in fruit pigment accumulation, sugar content, phenylpropanoid component and other fruit quality attributes (Bassa et al., 2012; Breitel et al., 2016; Sagar et al., 2013). Chlorophyll degradation, lycopene accumulation and changes of primary (sugar, amino acids, organic acids) and secondary metabolites (phenolics and flavonoids) during ripening mainly contribute the final desirable fruit quality and are under the control of several key ripening-related transcription factors (Klee and Giovannoni, 2011). The metabolism pathway of carotenoid in tomato has been well described and the effect of exogenous auxin on it has also been investigated (Sandmann, 2001; Su et al., 2015). The influence of exogenous auxin on the metabolites alteration during ripening period and tomato fruit final quality, however, has not been exten-

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sively studied. Our previous transcriptome study has suggested that exogenous auxin retards tomato ripening process and interferes the normal expression patterns of many genes involved in metabolism pathway (Li et al., 2016). To investigate the impact of exogenous auxin on tomato fruit quality, pigment metabolism, primary metabolite profiling of tomato fruit through ripening period were analyzed in this study. Transcript levels of the key genes impacted pigment and primary metabolite accumulation were also investigated. 2. Materials and methods 2.1. Plant materials and hormone treatment Four hundreds mature green cherry tomato fruits (cv. Xintaiyang) with uniform size and without injury were collected from a commercial standard greenhouse in Hangzhou, China. Fruits were washed by tap water and drained at room temperature after sterilization with 0.5% sodium hypochlorite aqueous solution, then were randomly divided into two groups and infiltrated with 0.45 mM 2, 4-dichlorophenoxyacetic acid or sterilized water under vacuum (35 KPa) for three minutes. After treatment, fruits were stored in darkness at constant temperature (20 ± 2 ◦ C) and humidity (90 ± 5% RH). Samples of 5 fruits with 3 replicates were randomly taken at 1, 4, 7, 10, 13, 16, 20 and 25 day after treatment during the 25 days of storage for subsequent analyses. 2.2. Measure of chlorophyll and pigment content Chlorophyll was extracted and measured as previously described (Bu et al., 2014; Camejo et al., 2005). Pericarp powders (1 g) pooled from five individual fruits were mixed with 3 mL 80% (v/v) acetone and kept at 4 ◦ C for 12 h. After centrifuging at 6000 × g for 20 min, supernatant was collected and the absorbance at 663, 647 and 470 nm was measured using a UV Spectrophotometer (Shimadzu Corp., Kyoto, Japan). The contents of chlorophyll a and b were calculated according to the equations described by Camejo D et al. (Camejo et al., 2005). Three biological replicates were executed for each pigment measurement. Pigment extraction was based on the method of Se´ırino et al. (Se´ırino et al., 2009). Frozen pericarps were ground into powder (0.5 g) and fully mixed with 6 mol L−1 NaCl aqueous solution (100 ␮L) and n-hexane (50 ␮L) using vortex (30 s). After centrifugation (13200g, 2 min) at 4 ◦ C, the mixture was vortex-mixed (30 s) with dichloromethane (200 ␮L), followed by another vortex homogenization procedure (30s) with ethyl acetate (1000 ␮L). The supernatant was collected after centrifugation (13200g, 2 min, 4 ◦ C) and filtered through a 0.45 ␮m membrane filter before high performance liquid chromatography (HPLC) assay. The assay was conducted according to the method previously described (Bu et al., 2013) using a HPLC with a Shimadzo LC2012A pump (Shimadzo Corp., Tokyo, Japan) and a Zorbas SB-C18 column (silica 5 ␮m, 4.6 mm × 250 mm, Agilent, USA). The mobile phase comprised solvent A of acetonitrile and H2 O (9:1) and solvent B of 100% ethyl acetate. The linear gradient between mobile phase A (acetonitrile/H2 O, v/v = 9:1) and B (ethyl acetate) was as follows: 0% B-100% B, 0–30 min, and the flow rate of mobile phase was 1 mL min−1 . The detection wavelength was 475 nm and the column temperature was 30 ◦ C. The contents of lycopene, ␤-carotene and lutein were calculated according to the standard curve. 2.3. Total phenolics and flavonoids measurement About 1 g pericarps were homogenized with 6 mL of 40% (v/v) ethanol solution, followed by the extraction at 60 ◦ C for 1 h, and then centrifuged for 15 min at 9000g. The supernatant was used

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to determine the concentration of total phenolics and flavonoids according to the methods as previously described (Dewanto et al., 2002). The contents of total phenolics and total flavonoids were expressed as gallic acid equivalents and rutin equivalents, respectively. 2.4. Analysis of metabolite profiling of tomato fruit using Gas Chromatography–Mass Spectrometry Primary metabolite profiling was performed based on the methods previously described (Lisec et al., 2006; Roessner et al., 2001) with slight modifications. Frozen pericarp powder (0.1 g) was vortex-mixed (10 s) with methanol (1.4 mL) and 60 ␮L ribitol (0.2 g L−1 , internal standard) and shaken (300 rpm) at 60 ◦ C for 15 min. Then 1 mL water was added into the mixture and vigorously shaken. After centrifugation (1000g, 4 ◦ C) for 15 min, the supernatant was dried under vacuum. The residue was redissolved and derivatized in 80 ␮L of 20 mg mL−1 methoxyamine hydrochloride in pyridine for 2 h at 40 ◦ C. After that, the mixture was incubated with N,O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA) (80 ␮L) at 40 ◦ C for 0.5 h. The sample was filtered through a 0.45 ␮m filter before analysis by gas chromatography. Sample (1 ␮L) was injected under a split ratio of 25:1 with a HP-5 capillary column (30 m × 0.25 mm × 0.25 ␮m, Agilent Technologies Inc., USA). The analysis of monomer was conducted with the procedures described by Roessner et al. (2001). Values in control samples were normalized to one and the metabolites content in auxin-treated samples represented in relative-value (RV). Three replicates were performed for primary metabolite profiling measurement. 2.5. RNA extraction and assay of quantitative real-time PCR (RT-qPCR) Total RNA was extracted from pooled pericarps of five individual fruit using RNAiso Plus (TaKaRa, Japan) according to the description of protocol in the product manual. Single-strand cDNA was generated from 1 ␮g RNA using PrimeScript RT kit (TaKaRa, Japan). RT-qPCR assay was executed on ABI StepOne RT-PCR System ® (Applied Biosystems, USA) using SYBR Premix Ex TaqTM (TaKaRa, Japan) as described previously (Bu et al., 2013). Primers used in RT-qPCR assay were listed in Table S1. 2.6. Statistics analysis Significance of the data between each samples was analyzed by Student’s t-test and principal component analysis was executed using SPSS version 20.0 (IBM Corp, Armonk, USA). 3. Results 3.1. Effect of exogenous auxin on pigment accumulation of tomato fruit during ripening process The content of Chlorophyll a and b in control sample sharply dropped at 7 day after treatment (DAT) and reached to minimum at 13 DAT (Fig. 1a and b). In auxin-treated sample, this descent process of chlorophyll content was retarded for approximate 3 days (Fig. 1a and b). Lutein content also showed a similar downward trend during ripening stage but no significant difference of it was observed between control and auxin-treated sample (Fig. 1c). Lycopene content was undetectable in control sample until 10 DAT and then rapidly increased in the following period. The content of lycopene was markedly inhibited by exogenous auxin application during the transition from mature green stage to ripening stage. However, lycopene content in control- and auxin-treated sample was almost the same at the end of ripening (Fig. 1d). The content of ␤-carotene

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Fig. 1. Pigments content of fruit in each group during ripening period. Content of (a) chlorophyll a, (b) chlorophyll b, (c) lutein, (d) lycopene and (e) ␤-carotene in control and auxin-treated fruit. Error bars indicate the standard error of three replicates. Asterisks (*) represent significant differences between the control and auxin treatments (Student’s t-test, P < 0.05).

in control sample began to increase at 7 DAT and reached to peak value (75.44 ␮g/g FW) at 13 DAT, following a slightly descent in the rest of storage period (Fig. 1e). The increase of ␤-carotene was also found to be delayed by auxin treatment. The content of ␤-carotene in auxin-applied samples kept in relatively low levels until 10 DAT and reached to the maximum (77.09 ␮g/g FW) at 20 DAT.

3.2. Effect of exogenous auxin on total phenolics and flavonoids content of tomato fruit during ripening process Total phenolic content of tomato fruit both in control and auxin treated group increased continuously during ripening period. In auxin-treated samples, total phenolics content was lower than that in control samples at 7 and 10 DAT (Fig. 2a). With regard

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Fig. 2. Content of total phenolics and flavonoids of fruit in each group. Content of (a) total phenolics and (b) flavonoids in control and auxin-treated fruit. Error bars indicate the standard error of three replicates. Asterisks (*) represent significant differences between the control and auxin treatments (Student’s t-test, P < 0.05).

to total flavonoids, the content of them in control samples began to rapidly rise at 7 DAT and reached to the apex at 13 DAT, followed by a slight decline (Fig. 2b). However, this increase trend was delayed by exogenous auxin treatment. In auxin-treated samples, total flavonoids content was maintained at a relative low level (approximate 400 ␮g/g) until 10 DAT and rose to the maximum within three days (Fig. 2b). At final stage of ripening, no differences of total flavonoids content were observed between the fruit in the two groups although the peak value in auxin-treated samples was lower than that in control fruit (Fig. 2b).

3.3. Effect of exogenous auxin on primary metabolite profiling of tomato fruit during ripening process To gain an overall insight into primary metabolite profiling change stimulated by exogenous auxin during ripening process, samples at 1 (early stage of ripening: mature green), 10 (middle stage of ripening: turning) and 25 DAT (final stage of ripening: red) were selected for metabolite profiling measurement. Forty-one primary metabolite components (15 amino acids, 17 carbohydrates and 9 organic acids and glyceride) in tomato fruit were identified by GC–MS. Principal component analysis was executed on the whole metabolite data. The first three major principal components explained 85.3% of the variance among samples (Fig. 3a). The difference of primary metabolite profiling between control and auxin-treated samples at early (1 DAT) and late (25 DAT) stage was majorly divided by principal components 2, whereas at 10 DAT, it was separated by principal components 1 and 3 (Fig. 3a). The content of most amino acids increased after exogenous auxin treatment at 1 DAT and 7 of them were significantly induced by exogenous auxin, especially aspartic acid (Asp) (RV = 3.40), tryptophan (Trp) (RV = 2.74), asparagine (Asn) (RV = 2.41), phenylalanine (Phe) (RV = 2.04) and glutamic acid (Glu) (RV = 2.03) (Fig. 3b). At 10 DAT, the content of Glu, which was significantly induced by auxin at 1 DAT, decreased in auxin-treated samples, although the contents of the majority of amino acids (9 of 15) were still increased (Fig. 3b). At 25 DAT, the changes of amino acids content between control and auxin-treated samples were diminished. No significant difference in contents of most amino acids was observed between control and auxin-treated samples except alanine (Ala) (RV = 0.59) and Asp (RV = 0.56) (Fig. 3b). Additionally, the contents of branched-chain amino acids valine (Val), leucine (Leu) and isoleucine (Ile) were more likely induced by auxin application at 1 and 10 DAT, and this induction effect was disappeared at 25 DAT (Fig. 3b).

The carbohydrate and relative metabolites were also affected by exogenous auxin application. At 1 DAT, of 17 identified carbohydrate and related metabolites, five (lyxose, galacturonic acid, galactose-1-phosphate, L-threonic acid, glucose 6-phosphate) were significantly induced by auxin (Fig. 3c). However, at 10 DAT, only sucrose and L-threonic acid were induced in auxin-treated samples (Fig. 3c). At 25 DAT, compared with control samples, the content of L-threonic acid still maintained at high level whereas the contents of lactose, allose and D-gluconic acid were significantly descended (Fig. 3c). The accumulation of organic acids was not altered by auxin treatment at 1 DAT except ascorbic acid, the content of which was strongly induced (RV = 2.20) in auxin-treated fruit (Fig. 3d). At 10 DAT, the contents of succinic acid and ascorbic acid in auxin-applied samples were higher than that in control samples (Fig. 3d). Contrarily, citramalic acid content of fruit was significantly reduced by auxin application. At final stage (25 DAT) of ripening, the contents of succinic acid and citric acid were higher in auxin-treated samples whereas the contents of citramalic acid and ascorbic acid were maintained at relative low levels (Fig. 3d). The accumulation of two identified glycerides, mono-stearin and mono-palmitin, was found to be negative regulated by exogenous auxin at 1 and 10 DAT (Fig. 3d). But at 25 DAT, no difference of these two glycerides content was observed in control and auxin-treated sample

3.4. Effect of exogenous auxin on transcription levels of the genes involved in ˇ-carotene metabolism pathway and key ripening-related transcription factor genes Exogenous auxin application inhibited the expression of major genes in ␤-carotene metabolism pathway at early stage of fruit ripening except SlLCY-ˇ and SlLCY-ε. Within first seven days after application, especially at 7 DAT, the expression of SlPSY1 and SlCRTISO were significantly repressed by exogenous auxin (Fig. 4) whereas this repression effect was declined rapidly after 7 DAT and almost vanished during the rest ripening period (Fig. 4). SlPDS1 and SlZDS encode two desaturases (phytoene desaturase and ␨-carotene desaturase, respectively), which catalyze phytoene to neurosporene. The expression of these two genes was also repressed by auxin at 7 DAT. Contrarily, the expression patterns of SlLCY-ˇ and SlLCY-ε were quite different with others. The expression of SlLCY-ˇ was induced by auxin at early (4 DAT) and late stage (25 DAT) of ripening process whereas the transcript levels of SlLCYε in auxin-treated samples showed no significant difference with that in control (Fig. 4)

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Fig. 3. Analysis of primary metabolism profiling of all samples. (a) Principal components analysis of primary metabolism profiling of all samples. Relative levels of (b) amino acids, (c) carbohydrate and (d) organic acid in samples at 1, 10 and 25 DAT. Values in control samples are normalized to one.

The expression of SlRIN and SlCNR, two important ripeningrelative transcription factor, was down-regulated at 4 and 7 DAT in auxin-treated samples (Fig. 5). The transcript levels of SlARF4 and SlAP2a in auxin-treated samples were lower than that in control samples at 7 DAT, and no significant difference of that was observed between the samples in control and auxin application group in other period of ripening (Fig. 5). Distinguished from other transcription factor-encoding genes, the expression of SlGLK2 was induced by auxin, although this induction effect was quite transient (only at 4 DAT) during the whole ripening period (Fig. 5). In addition, the expression of SlGR, another key transcription factor, did not seem to be impacted by exogenous auxin treatment (Fig. 5). 4. Discussions The delay of tomato fruit color transition induced by exogenous auxin treatment has been reported in previous studies and relative high level chlorophyll content was observed in auxin-treated fruit at 96 h after treatment (Li et al., 2016; Su et al., 2015). Lycopene and ␤-carotene are the two most crucial pigment and the final fruit color is mostly depend on the ratio of the two pigments (Rosati et al., 2000). Previous study exhibited that exogenous indoleacetic acid

treatment on mature green tomato markedly inhibited lycopene accumulation but not affected the contents of ␤-carotene and lutein (Su et al., 2015). According to our results, however, the accumulation of ␤-carotene and the expression of SlLCY-ˇ were also inhibited by auxin-treatment (Fig. 1d), which was quite different with the results of Su et al. (Su et al., 2015). These differences may be due to the tomato cultivar used in experiment and the overall coverage of our data of ␤-carotene content throughout the whole ripening period. Key ripening-related transcription factors RIN, GR and CNR are also found to have positive effects on chlorophyll degradation and ␤-carotene synthesis and CLK2 induces chlorophyll accumulation in tomato fruit (Barry et al., 2005; Fujisawa et al., 2013; Manning et al., 2006; Powell et al., 2012). In our results, the expression of SlRIN and SlCNR was inhibited by auxin application whereas SlGLK2 was induced (Fig. 5), implying that auxin might modulate pigment metabolism via regulating the expression of ripening-related transcription factor genes SlRIN, SlCNR and SlGLK2. Besides the transcription factors above, the expression of SlARF4 and SlAP2a, which have been identified to be able to regulate pigment metabolism in fruit tissues (Karlova et al., 2011; Sagar et al., 2013), was down-regulated by auxin at 7 DAT (Fig. 5), indicating the participation of them into auxin-modulated pigment metabolism.

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Fig. 4. Expression changes of the genes involved in ␤-carotene metabolism pathway. The fold change (log2 ratio) of gene expression level induced by exogenous auxin was visualized as heat map. Yellow and red indicates positive regulation, while blue indicates negative regulation. Asterisks (*) represent significant differences between the control and auxin treatments (Student’s t-test, P < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Expression changes of the ripening-related transcription factors genes in the fruit treated with exogenous auxin. Error bars indicate the standard error of three replicates. Asterisks (*) represent significant differences between the control and auxin treatments (Student’s t-test, P < 0.05).

Phenolics and flavonoids, which play essential roles in stress resistance of plant tissues (Dicko et al., 2005; Treutter, 2005), are regarded as beneficial components for human health and contribute greatly to fruit antioxidant ability (Bovy et al., 2007; Martínez-Valverde et al., 2002). Our previous transcriptome data have revealed that the expressions of many genes involved in

flavonoids and phenolics metabolism pathway were differentially regulated by exogenous auxin in tomato fruit (Li et al., 2016). Here we found that auxin treatment significantly reduced the content of total phenolics and flavonoids at middle stage of ripening whereas did not affect the final concentration of them in ripe fruit (Fig. 2). These results support the hypothesis that the impaction of exoge-

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nous auxin on process of fruit ripening is greater than that on characters of ripe fruit. Huge changes on the varieties and contents of primary metabolites took place during fruit ripening period. The starch and other polysaccharides accumulated through fruit development are converted into monosaccharides and oligosaccharides (Klee and Giovannoni, 2011). Significant alteration on sugar metabolism of citrus and grape after application of auxin has been previously reported (Agustí et al., 2002; Bottcher et al., 2012). Based on our metabolite profiling results, exogenous auxin significantly altered the content of carbohydrates and sugar acids in tomato fruit (Fig. 3c). At early (1 DAT) and middle stage (7 DAT), the content of galactose-1-phosphate and threonic acid were the dramatically increased in auxin-treated fruit (Fig. 3c). The increase of these two metabolites may be due to the enhancement of ascorbic acid biosynthesis induced by auxin (Fig. 3d). In plant tissues, galactose1-phosphate is one of the materials for ascorbic acid biosynthesis and threonic acid is a degradation product of ascorbic acid (Zou et al., 2006). High level exogenous auxin treatment induces overproduction of superoxide radicals and stimulates plant tissues to produce more ascorbic acid and other antioxidants to reduce oxidative damage (Teixeira et al., 2007). The content of galacturonic acid, a catabolite of the important cell wall component polygalacturonase, was only induced by auxin treatment at 1 DAT (Fig. 3c). However, our previous study suggested that fruit firmness was not influenced by auxin at 1 DAT, implying that the increment of polygalacturonase activity induced by auxin might be transient. Moreover, auxin treatment altered the contents of several carbohydrates, such as allose and lactose, in final ripe fruit (Fig. 3c). The mechanism of the regulation of carbohydrates metabolism by auxin is still worth to be further investigated. Furthermore, transcription factor gene SlARF4 and SlAP2a have been found to participate in regulation of carbohydrate metabolism in tomato fruit (Chung et al., 2010; Sagar et al., 2013). It might be presumed that auxin regulate carbohydrates metabolism via modulating the expression of related transcription factors. Dramatic increase of free amino acids content has been observed in tomato fruit during ripening period (Sorrequieta et al., 2010). The marked increment of seven free amino acids at 1 DAT in auxintreated fruit (Fig. 3b) suggests that auxin actively participates in regulation of amino acids metabolism. On the other hand, in plant, auxin is mainly biosynthesized from tryptophan and other free amino acids also play important roles in maintaining auxin homeostasis via directly conjugating with auxin (Ljung, 2013). The rapidly increase of Asp, Glu and other amino acids might be due to the quick response to high level exogenous auxin in fruit. When suffering high level auxin stress, plant tissues may synthesize more amino acids to conjugate with the excess auxin. Along with the degradation of exogenous auxin, the content of amino acids in auxin-treated fruit gradually reached to normal level (Fig. 3b). In addition, free amino acids not only contribute to the desirable taste of fruit, but also provide precursors for aroma components. For instance, Glu, Asp and Ala are main donator of amidogen for the biosynthesis of many aromatic compounds and Ala involves in the formation of volatiles in banana fruit (Ardo, 2006; Sorrequieta et al., 2010; Wyllie and Fellman, 2000). The accumulation of Asp and Ala were repressed in auxin-applied fruit at final ripening stage (Fig. 3b), suggesting a long-term effect of auxin on the metabolism of these two amino acids. The changes of free amino acids contents induced by auxin implies that auxin may influence the volatiles of fruit, and the details of it are worth to be further investigated. On the other hand, organic acids content is an important attribute of fruit organoleptic quality and is also maintained at relative high levels in fruit during ripening process (Etienne et al., 2013). Citric acid has been considered as the major organic acid and greatly influences fruit acidity in tomato and other fleshy fruit (Etienne et al., 2013). In

normal tomato fruit ripening process, citric acid content increased and reached to maximum at final ripe stage (Oms-Oliu et al., 2011). In our results, auxin-treated fruit contained more citric acid than that in control fruit at the end stage of ripening (Fig. 3d), indicating auxin application might enhance fruit acidity. The contents of succinic acid and citramalic acid, which were increased in tomato fruit during ripening period (Oms-Oliu et al., 2011), were induced and repressed by auxin treatment, respectively (Fig. 3d). Citramalic acid content has been found to have a high relationship with the “sweetness” attribute of tomato fruit and succinic acid plays as an electron donor in citric acid cycle (Zanor et al., 2009). The changes of organic acids and carbohydrate content evoked by exogenous auxin (Fig. 3b and c) reveal that auxin may increase the sour taste of fruit. 5. Conclusions Present results suggested that exogenous auxin strongly interfered the accumulation and conversion of pigment, total phenolics and flavonoids but not quite influence the final content of these compounds in full ripe tomato fruit. On the other hand, exogenous auxin application also changed the content of primary metabolites during ripening process and these alteration effects was attenuated along with the development of ripening process, though did not completely restored at the end of fruit ripening. Transcript analysis revealed that the regulation on genes expression by exogenous auxin was more potent at early and middle stage of ripening process as compared with the late ripening stage, providing a presumptive explanation for the impermanent impacts of exogenous auxin on pigment and other quality-related components metabolism in tomato fruit. Acknowledgements This work was supported by grants from National Basic Research Program of China (2013CB127101). We would like to thank all members in our lab for their assistance with this study. 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.scienta.2017. 03.011. References Agustí, M., Zaragoza, S., Iglesias, D.J., Almela, V., Primo-Millo, E., Talón, M., 2002. The synthetic auxin 3, 5, 6-TPA stimulates carbohydrate accumulation and growth in citrus fruit. Plant Growth Regul. 36, 141–147. Ardo, Y., 2006. Flavour formation by amino acid catabolism. Biotechnol. Adv. 24, 238–242. Barry, C.S., McQuinn, R.P., Thompson, A.J., Seymour, G.B., Grierson, D., Giovannoni, J.J., 2005. Ethylene insensitivity conferred by the Green-ripe and Never-ripe 2 ripening mutants of tomato. Plant Physiol. 138, 267–275. Bassa, C., Mila, I., Bouzayen, M., Audran-Delalande, C., 2012. Phenotypes associated with down-regulation of Sl-IAA27 support functional diversity among Aux/IAA family members in tomato. Plant Cell Physiol. 53, 1583–1595. Bottcher, C., Harvey, K., Forde, C., Boss, P., Davies, C., 2011. Auxin treatment of pre-veraison grape (Vitis vinifera L.) berries both delays ripening and increases the synchronicity of sugar accumulation. Aust. J. Grape Wine Res. 17, 1–8. Bottcher, C., Boss, P.K., Davies, C., 2012. Delaying Riesling grape berry ripening with a synthetic auxin affects malic acid metabolism and sugar accumulation, and alters wine sensory characters. Funct. Plant Biol. 39, 745–753. Bovy, A., Schijlen, E., Hall, R.D., 2007. Metabolic engineering of flavonoids in tomato (Solanum lycopersicum): the potential for metabolomics. Metabolomics 3, 399–412. Breitel, D.A., Chappell-Maor, L., Meir, S., Panizel, I., Puig, C.P., Hao, Y., Yifhar, T., Yasuor, H., Zouine, M., Bouzayen, M., Granell Richart, A., Rogachev, I., Aharoni, A., 2016. AUXIN RESPONSE FACTOR 2 intersects hormonal signals in the regulation of tomato fruit ripening. PLoS Genet. 12, e1005903. Bu, J.W., Yu, Y.C., Aisikaer, G., Ying, T.J., 2013. Postharvest UV-C irradiation inhibits the production of ethylene and the activity of cell wall-degrading enzymes

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