Effects of exogenous abscisic acid on the expression of citrus fruit ripening-related genes and fruit ripening

Scientia Horticulturae 201 (2016) 175–183

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Effects of exogenous abscisic acid on the expression of citrus fruit ripening-related genes and fruit ripening Xiaohua Wang, Wu Yin, Juxun Wu, Lijun Chai ∗∗ , Hualin Yi ∗ Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China

a r t i c l e

i n f o

Article history: Received 2 September 2015 Received in revised form 2 December 2015 Accepted 13 December 2015 Keywords: Fruit ripening NDGA ABA Sugars and acids

a b s t r a c t Despite exogenous abscisic acid (ABA) treatment is a widely-used effective method to prove its role in plants, this method has been limitedly explored in citrus fruits. Here, we examined the effects of exogenous ABA and nordihydroguaiaretic acid (NDGA) on the color index H value and the contents of soluble sugars, organic acids and endogenous hormones in the fruits of Citrus reticulata Blanco cv. Ponkan. Additionally, the expression profiles of genes involved in the ABA, sugar and organic acid metabolism and signal transduction pathway in fruits were also investigated. The results indicated that exogenous ABA could accelerate fruit coloring, significantly decrease the organic acid content, and affect the ripening of citrus fruit, while treatment with NDGA could restrain fruit coloring and acid degradation. Exogenous ABA treatment produced minor effect on the accumulation of sugars but largely regulated the expression of most sugar- and acid-related genes, indicating a coordinate interaction between the ABA signaling pathway and sucrose signaling pathway to regulate citrus fruit ripening. Especially, CsSUC 3 plays a vital role in accumulation of sugars during the last ripening stage of citrus fruit, which could be induced by ABA treatment. The transcript levels of CsACO1 and CsNADP-IDH in ABA-treated fruits were significantly higher than in the control fruits during the late ripening stages, which was conducive to the degradation of organic acids. Moreover, ABA could restrict its own accumulation at 14 DAY not only by inducing the expression of ABA 8 -hydroxylase 1 but also by reducing the expression of CsNCED1, and the transcriptional levels of other ABA-related genes were largely triggered by exogenous ABA. Overall, ABA is a positive regulator of ripening and could be used to regulate citrus fruit ripening effectively. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The fleshy fruits have been divided into two groups with contrasting ripening patterns. Climacteric fruits (such as tomato, banana, peach and apple) show a burst in ethylene release and an increase in respiration rate during ripening, but not the nonclimacteric fruits (such as citrus, strawberry and cherry; White, 2002; Leng et al., 2014). Ethylene remains the most explored hormone owing to its predominant role in the ripening of climacteric fruits (Alexander and Grierson, 2002; Kevany et al., 2007; Liu et al., 2015), while abscisic acid (ABA) seems to have a stronger role in

Abbreviations: ABA, abscisic acid; NDGA, nordihydroguaiaretic acid; DAY, days after the first treatment; DAF, days after flowering; qRT-PCR, quantitative real-time polymerase chain reaction. ∗ Corresponding author. Fax: +86 27 87282010. ∗∗ Corresponding author. E-mail addresses: [email protected] (X. Wang), [email protected] (W. Yin), [email protected] (J. Wu), [email protected] (L. Chai), [email protected] (H. Yi). http://dx.doi.org/10.1016/j.scienta.2015.12.024 0304-4238/© 2015 Elsevier B.V. All rights reserved.

the ripening of non-climacteric fruits (Jia et al., 2011; Wang et al., 2013; Nicolas et al., 2014). So far, numerous studies have been performed about the role of ABA in the regulation of fruit ripening. A rapid increase of ABA content was found during fruit ripening in both non-climacteric (Romero et al., 2012a; Wang et al., 2013) and climacteric fruits, prior to the peak of ethylene (Zhang et al., 2009a). Recently, Zhang et al. (2009b) found that the exogenous ABA could increase endogenous ABA levels both in pulp and seed, inducing the expression of ethylene-related genes and promoting tomato fruit ripening. However, the treatment with NDGA showed the opposite results, delaying fruit ripening. Additionally, the application of ABA accelerated fruit coloring and ripening in strawberry. Silencing a 9-cis-epoxycarotenoid dioxygenase gene (FaNCED1) encoding a key ABA synthesis enzyme, using TRV- mediated VIGS, caused a decrease in ABA content and uncolored fruits. More importantly, this uncolored phenotype could be rescued by exogenous ABA (Jia et al., 2011). The ABA-deficient mutant fruit provides an ideal experimental system to explore the role of ABA in the regulation of fruit ripening. Pinalate, a spontaneous fruit-specific ABA-deficient mutant from the ‘Navelate’ orange (Citrus sinensis L. Osbeck), shows

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a delay on fruit degreening (Rodrigo et al., 2003). Another ABAdeficient mutant, high-pigment 3 (hp3), was obtained in tomato. The lack of ABA leads to an abnormal increase of the plastid compartment size and the accumulation of 30% more carotenoids in the mature fruit (Galpaz et al., 2008). These results show that ABA plays a crucial role in the regulation of fruit ripening. Recently, the underlying molecular basis of this control in fruits has attracted more and more attention. Two different ABA receptors from strawberry, FaCHLH/ABAR and FaPYR1 (Chai et al., 2011; Jia et al., 2011), and one receptor from grape, VvPYL1 (Li et al., 2012), were determined to play positive roles in ABA-mediated fruit ripening. Jia et al. (2013b) found that up- and down- regulation of FaABI1 (the type 2C protein phosphatase) expression levels delayed and advanced fruit ripening, suggesting its negative role in strawberry fruit ripening. More recently, another signal component, FaSnRK2.6, functioning downstream of PYR/PYL and PP2Cs, was also found to be a negative regulator during strawberry fruit development and ripening (Han et al., 2015). In addition, a transcription factor, VvABF2, has been proven to be involved in several ABA-mediated ripening-related pathways, positively regulating grape fruit ripening process (Nicolas et al., 2014). These results provide some insights into the molecular mechanism of fruit ripening regulatory networks in response to ABA. Despite great advances in the research on the role of ABA in the regulation of fruit ripening, little information is available regarding the ABA-mediated ripening controlling mechanism in citrus. Currently, the approach to explore the regulation of fruit ripening is very limited in citrus. Specific citrus materials, such as spontaneous late-ripening mutants and color mutants, are usually used in the study of citrus ripening (Rodrigo et al., 2003; Liu et al., 2006a). In the present work, an experiment with a widespread application prospect in the actual production was conducted on Jinshuigan (Citrus reticulata Blanco cv. Ponkan). We examined the effects of exogenous ABA and NDGA on the fruit ripening-related quality and endogenous hormones content. The expression profiles of genes involved in the ABA, sugar and organic acid metabolism and signal transduction pathway in fruits treated with ABA and NDGA treatment were also investigated.

2.2. Peel color measurement The citrus peel color of each fruit was measured at three evenly distributed equatorial sites using the CIELAB color system of a MINOLTA CR-400 chromameter (Japan). Eight fruits were measured for each group, twenty-four sites (3 × 8) in total. H values represent hue angle, and if a > 0 and b > 0, H = tan−1 (b/a); if a < 0 and b > 0, H = 3.14 + tan−1 (b/a) (a, ±yellow/green; b, ±red/blue). H values vary from 0 to 3.14, standing for purple (0), orange red (0.39), red (0.78), orange (1.17), yellow (1.57), yellow–green (2.09), green (2.61) and blue (3.14), respectively. 2.3. Analysis of soluble sugars and organic acids The soluble sugar and organic acid composition and concentrations were determined by using an Agilent 6890 N gas chromatography as described by Bartolozzi et al. (1997) with minor modifications. Briefly, 3 g of frozen powder was suspended in 12 ml of chilled 80% methanol in a 75 ◦ C water bath for 15 min. After a 45 min ultrasonic extraction and 4000 × g centrifugation for 10 min, the supernatant was collected in a 50 ml volumetric flask. This extraction procedure was repeated two times. Next, each volumetric flask was supplemented with 1 ml internal standard (0.025 g ml−1 phenyl-␤-d-glucopyranoside, 0.025 g ml−1 methyl␣-d-glucopyranoside). Finally, the solution was diluted with 80% ethanol to a final volume of 50 ml and a 0.5 ml of this solution was vacuum concentrated and derivatized for GC analysis. Three replicates were conducted for each sample. 2.4. Determination of endogenous hormone (ABA, JA and IAA) Content For endogenous hormone extraction, D6 -ABA, H2 JA and D5 -IAA (Icon Isotopes) were used as internal standards for ABA, JA and IAA, respectively, which aimed to calibrate samples. The other procedures were completely followed as described in the report of ABA quantification (Wu et al., 2014). Additionally, the reaction monitoring conditions (Q1/Q3) of JA and IAA and their icon isotopes were described by Pan et al. (2010). High-performance liquid chromatography (HPLC) was used for qualitative and quantitative analysis. Four replicates were performed for each sample.

2. Materials and methods

2.5. RNA extraction and qRT-PCR expression analyses

2.1. Plant material and ABA treatment

The pulps sampled from three different trees were mixed and 3 g of the material was used for total RNA extraction according to the procedures described previously (Liu et al., 2006b). After the total RNA with high quality and integrity was extracted, the firststrand cDNA was synthesized using a PrimeScriptTM RT Reagent kit with gDNA Eraser (TaKaRa) according to the manufacturer’s instructions. Several genes involved in the fruit ripening process, such as genes in ABA metabolism and signal transduction pathways and soluble sugars and organic acids metabolism pathways, were selected for quantitative real-time PCR (qRT-PCR). A part of the gene-specific primers were referred to our previous reports (Wu et al., 2014; Zhang et al., 2014). Sequences of other primer pairs were listed in Supplementary Table S1. The differential expression of the selected genes was validated by using a QuantStudioTM 7 Flex Real-Time PCR System (Applied Biosystems), with the reference of actin gene according to Liu et al. (2007). Each well contained 10 ␮l of the reaction mixture consisting of primers diluted in SYBR Green PCR Master Mix (Applied Biosystems) and cDNA in double-distilled water (ddH2 O). Reactions were set with an initial incubation at 50 ◦ C for 2 min and 95 ◦ C for 5 min, and then 40 cycles at 95 ◦ C for 15 s, 58 ◦ C for 15 s and 72 ◦ C for 30 s. Four technical replications

Jinshuigan (Citrus reticulata Blanco cv. Ponkan) used in this study was cultivated in the Fruit and Tea Institute of Hubei Agricultural Science Academy, Hubei Province, China. Fruits before color breaker stage (190 DAF) in nine grown trees were divided into three groups, treated with 500 ␮M ABA, 500 ␮M nordihydroguaiaretic acid (NDGA), and distilled water as a control, respectively. The treatment was repeated every week to ensure high ABA levels during fruit ripening, till the fruits were fully colored. The optimal treatment concentration and efficacy duration of ABA and NDGA for citrus were selected based on the results of a pretest shown in Fig.S1 in the supplementary material. Samples were harvested at four ripening stages, breaker (BK), colored (C), full colored (FC), and full ripening (FR), which were defined according to the previous report (Romero et al., 2012a), corresponding to 0, 14, 26 and 35 days after the first treatment (DAY), respectively (Fig. 1A) . For each stage, 8 fruits were sampled from each tree, and a total of 72 fruits (9 × 8) were collected from 9 trees. After separating from the peels, the pulps were rapidly frozen in liquid nitrogen and kept at −80 ◦ C for further analysis.

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Fig. 1. (A) Phenotypic characterization of citrus fruits applied with 500 ␮M ABA, 500 ␮M NDGA and distilled water (control) during fruit ripening process, which was divided into the following four stages: breaker (BK), colored (C), full colored (FC), and full ripening (FR), corresponding to 0, 14, 26, 35 d after the first treatment (DAY), respectively. Red boxes represent indicate the harvest time and the green arrow indicates time axis. (B) Changes in the peel color of control and ABA/NDGA treated-fruits. The error bars represent SE (n = 24). Letters indicate statistically significant differences (P < 0.05) analyzed using Student’s t-test. No marked letter means no significant difference (P < 0.05) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

were performed. The output data were analyzed by the instrument on-board QuantStudioTM Real-Time PCR Software v1.1. 3. Results 3.1. Effect of exogenous ABA and NDGA treatments on the contents of soluble sugars, organic acids, and endogenous hormones during fruit ripening In this study, the fruits did not show any visible differences in the phenotype by 500 ␮M ABA or NDGA treatments (Fig. 1A). But the H value of ABA-treated fruit was lower than that of the control fruit, and the NDGA treatment had the opposite result, suggesting that ABA treatment accelerates citrus fruit coloring (Fig. 1B). Exogenous ABA and NDGA were observed to have minor effects on the content of soluble sugars but significant effects on the levels of organic acids and the ratio of TSS to TOA. Fruits treated with ABA had a lower organic acid content and a higher TSS/TOA ratio during ripening, while fruits treated with NDGA showed an opposite trend at 14 DAY, suggesting that exogenous ABA could promote the degradation of organic acids (Fig. 2). The level of endogenous ABA in the control fruit reached the peak at 14 DAY, while that of the fruits treated with ABA or NDGA reached a relative lower peak at 26 DAY. Compared with the control, a typical decrease was observed in the ABA content in the pulp of fruits treated with exogenous ABA or NDGA at 14 DAY, but the level in the ABA-treated fruits was still significantly higher than that treated with NDGA, as with the pre-test results (Supplementary Fig. S1B). Additionally, ABA or NDGA treatment had less effect on the levels of JA and IAA in citrus fruits (Fig. 3). 3.2. Transcriptional regulation of ABA-related genes in response to treatments with exogenous ABA and NDGA Fig. 5 The ABA measurement data indicated that exogenous ABA could significantly affect the ABA content in the fruit. To provide direct genetic evidence for the role of exogenous ABA in the regulation of ABA metabolism and signaling, the transcript levels of 18 genes involved in ABA metabolism and signal transduction pathways were analyzed (Fig. 4). The 9-cis-epoxycarotenoid dioxygenase (CsNCEDs) gene and the abscisic-aldehyde oxidase (CsAAO) were key genes in the ABA biosynthesis, and ABA 8 -hydroxylase 1 was involved in the degradation pathway. The expression patterns of most genes were the same in fruits no matter which treatment was used, but the expression levels were greatly influenced by

ABA or NDGA treatment (Fig. 4). Compared with the control, the expression of CsNCED1 at 14 DAY was repressed by ABA treatment and induced by NDGA treatment, while ABA 8 -hydroxylase 1 was up-regulated by both treatments at 14 DAY. In addition, the transcript levels of CsNCED1, CsAAO, CsABI2, CsHAB1 and CsHAB2 were lower at 14 DAY and higher at 26 DAY in response to ABA treatment, a delay in expression compared with the control, which was consistent with the change pattern of ABA content in citrus under ABA treatment (Fig. 3 and 4). In the control fruits, the expression patterns of CsPP2C 25 and CsABI2 were the same, which reached minimums at the stage of 14 DAY and 26 DAY, but the lowest peaks were delayed or advanced for a stage by ABA treatment, respectively. It was noteworthy that NDGA treatment markedly reduced the expression levels of CsPP2C 25, CsPP2C 56 and CsSnRK2s leading to a lower content of endogenous ABA in NDGA-treated fruits (Fig. 4B). Fig. 6A gave an overview of the relative expression levels of ABA-related genes in control and ABA/NDGA-treated fruits, which was more intuitive to compare gene expression differences with a gradient color from green to red. Overall, exogenous ABA and NDGA significantly triggered the transcript levels of most ABA-related genes. 3.3. Expression profiles of genes in the sugar and organic acid metabolism and signal transduction pathway after treatment with exogenous ABA and NDGA treatments To further investigate the regulation role of ABA in sugar and acid metabolism, the expression profiles of the genes in the sugar and organic acid metabolism and signal transduction pathways were analyzed (Fig. 5). Interestingly, the results of qRT-PCR showed that sugar-related genes, such as CsSPS, CsSS, CsSUC1, CsSUC3, were significantly regulated by ABA or NDGA treatments, while the content of fructose, glucose and sucrose displayed limited differences (Fig. 2 and 5A). The ABA or NDGA treatments had the similar effect on the expression patterns of sucrose synthase (CsSS) and sucrosephosphate (CsSPS) compared with the control. The expression of CsSUC3 remained at a lower level at the first three stages, but reached a much higher level at the final ripening stage in both control and ABA/NDGA-treated fruits, suggesting that CsSUC3 functions mainly in the post-maturation stage of citrus fruit. A pattern of upregulation in this gene was observed at 35 DAY for both ABA and NDGA treatments, and exogenous ABA had a greater effect than NDGA (Fig. 5A). Since the concentration of organic acids is related to their synthesis and degradation rate (Chen et al., 2013), the expression levels of genes in the acid metabolism pathway were investigated

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Fig. 2. Effect of ABA, NDGA and distilled water (control) treatment on the content of soluble sugars and organic acids in the flesh of citrus. The error bars represent SE (n = 3). Letters indicate statistically significant differences (P < 0.05) analyzed using Student’s t-test. No marked letter means no significant difference (P < 0.05).

Fig. 3. Effect of ABA, NDGA and distilled water (control) treatment on content of abscisic acid (ABA), jasmonic acid (JA) and auxin (IAA) in the flesh of citrus. The error bars represent SE (n = 4). Letters indicate statistically significant differences (P < 0.05) analyzed using Student’s t-test. No marked letter means no significant difference (P < 0.05).

(Fig. 5B). Most genes exhibited similar expression patterns, such as CsPFK3, CsNADP-ME1, CsCSY2, CsATCS and CsGAD4. Their expression levels were higher in control fruits at 14 DAY, but higher in NDGA-treated fruits at 26 and 35 DAY, which were similar to the cross type presented in the contents of malic acid and citric acid (Fig. 2). The cytosolic Aconitase (CsACO1) and NADP(+)-isocitrate dehydrogenase (CsNADP-IDH) are like switches, whose activities control the accumulation and reduction of citric acid toward fruit maturation (Sadka et al., 2000a; Sadka et al., 2000b); therefore, we analyzed CsACO1 and CsNADP-IDH and found the transcript levels of these genes fluctuated in response to ABA and NDGA treatments. The expression of these two genes in ABA-treated fruits was significantly higher at 26 DAY than in control fruits, which was conducive to the degradation of organic acids (Fig. 2 and 5B). We also drew the heatmap of sugar- and acid- related genes in control and ABA/NDGA- treated fruits (Fig. 6B). The expression levels of most sugar-related genes were up-regulated during the fruit

ripening process (the squares were deepening red), with the constant accumulation of fructose, glucose and sucrose (Fig. 2), while the expression levels of most acid-related genes decreased firstly and then increased, or remained almost constant throughout the four stages in the control and ABA/NDGA fruits, with the color at the middle stages being greener than that at the first and last stages. Taken together, these results indicated that exogenous ABA and NDGA could regulate sugar accumulation and acid degradation at the transcription level.

4. Discussion Exogenous ABA is the most widely-used and effective method to prove its role in fruits and leaves or roots of plants (Nicolas et al., 2014; Han et al., 2015). However, this method has been limitedly explored in citrus fruits, probably due to the thick peel and epicuticular wax (Storey and Treeby, 1994). Romero et al. (2012b)

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Fig. 4. Effect of ABA, NDGA and distilled water (control) treatment on transcript levels of the genes in the ABA metabolism (A) and signal transduction pathways (B) in the pulp of citrus. The error bars represent SE (n = 4). Letters indicate statistically significant differences (P < 0.05) analyzed using Student’s t-test. No marked letter means no significant difference (P < 0.05).

found that Pinalate, a spontaneous fruit-specific ABA-deficient mutant from the Navelate orange, could largely accumulate ABA by exogenous 1 mM ABA treatment and reach a slightly higher

phytohormone level than the wild type. Nevertheless, in the preliminary test of this study, we found the optimal ABA concentration for citrus is 500 ␮M (Supplementary Fig. S1). The NDGA was used

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Fig. 5. Effect of ABA, NDGA and distilled water (control) treatment on transcript levels of sugar- (A) and organic acid- (B) related genes in the pulp of citrus. The error bars represent SE (n = 4). Letters indicate statistically significant differences (P < 0.05) analyzed using Student’s t-test.

as a negative control due to its permeating speed and its recognized ability to block ABA accumulation (Creelman et al., 1992). The results in our study confirmed that immature green and broken citrus fruits were sensitive to NDGA, and endogenous ABA biosynthesis could be remarkably reduced by NDGA treatment, but not completely suppressed (Supplementary Fig. S1B and Fig. 3). As described in a previous study (Zhang et al., 2009b), the function mechanism of NDGA is to either regulate the expression of ABA biosynthesis-related genes or inhibit the synthase activities directly. The transcriptions of CsNCED s and CsAAO were not obviously restrained by NDGA treatment (Supplementary Fig. S1C and Fig. 4), suggesting that the blocked accumulation of ABA resulted from the direct suppression of NDGA on synthase activities instead of its regulation at the transcriptional level.

4.1. ABA is a positive regulator of Citrus fruit ripening Fruit ripening involves well-orchestrated coordination of several regulatory steps, leading to subtle changes in two primary metablolic and physiological traits, including accumulation of carotenoids and sugars, and degradation of chlorophyll and organic acid, and other traits, such as cell wall modifications associated with fruit softening and synthesis of volatiles (Kumar et al., 2014). It is widely acknowledged that the application of exogenous ABA enhances the accumulation of several metabolites and degradation of other metabolites involved in fruit ripening, thereby accelerating fruit ripening (Wheeler et al., 2009; Koyama et al., 2010; Wang et al., 2013), which is consistent with the result from the present study

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Fig. 6. Gene cluster analysis of ABA- (A), sugar- and organic acid- (B) related genes in the fruits of citrus treated with ABA, NDGA and distilled water (control). Number 0, 14, 26 and 35 above the heat map represent the increase/decrease of gene expression in different days after the first time treatment. All data are obtained from qRT-PCR. Each square indicates the relative gene expression level. Green depicts a lower level and red a higher level compared the 0 DAY. The heat map was drawn by HemI (Heatmap Illustrator, version 1.0) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

in that the exogenous ABA could promote the process of citrus fruit ripening. Sugar is generally considered to be an important indicator of fruit quality and its content could be as high as 130 mg g−1 FW in common varieties of citrus fruits (Wu et al., 2014; Zhang et al., 2014), which is similar to our result (Fig. 2). Recent works on strawberry revealed that application of sucrose could stimulate fruit ripening and accumulation of ABA and promote the expression of FaNCEDs (Jia et al., 2011, 2013a). Treatment with sucrose and ABA could induce the onset of grape’s ripening including color changes, softening, and gene expression, proving their role in regulating fruit ripening (Gambetta et al., 2010). Additionally, Wu et al. (2014) found that the increase pattern of sucrose was closely related to phase transitions in the developmental process of citrus fruit. These results suggest that sucrose can also play the regulatory role as a signal in fruit ripening. Thus, we hypothesized that ABA treatment has the same impact on sucrose signaling pathway. In the present study, we found that exogenous ABA largely regulated the expression of most genes in sucrose metabolism and signal transduction pathways (Fig. 5A). Particularly, CsSUC 3 plays a vital role in the accumulation of sugars during the last ripening stage of citrus fruit and ABA application could enhance this function (Fig. 5A). These findings indicate that there may be a coordinate interaction between the ABA signaling pathway and sucrose signaling pathway to regulate citrus fruit ripening. 4.2. Effects of exogenous ABA and NDGA on ABA content and expression profiles of ABA-related genes in Citrus fruits The endogenous ABA of Citrus, a non-climacteric fruit, is gradually accumulated during fruit development and rapidly increased at breaker or ripening stage (Romero et al., 2012a). Similarly, we observed that the endogenous ABA of the citrus fruit pulp was constantly increased and reached a peak before full ripening in our study (Fig. 3). In addition to ABA, a lot of other hormones are also involved in the regulation of fruit ripening, such as IAA (Manning, 1994; Bottcher et al., 2010) and JA

(Ziosi et al., 2008; Concha et al., 2013). Recently, the crosstalk between ABA and JA pathway has received much attention and de Ollas et al. (2013) found that JA accumulation preceded the increase in ABA levels, which was necessary for the subsequent ABA increase in roots under stress, but not the opposite. Results in this work showed that a transient increase at 14 DAY in JA levels was concomitant to the accumulation of ABA, indicating an interaction between ABA and JA at the biosynthetic level during citrus fruit ripening. The levels of JA and IAA did not respond to the exogenous treatment with ABA and NDGA in the same way, suggesting the changes in endogenous levels of JA and IAA might act upstream of the ABA pathway and both took part in citrus fruit ripening process (Fig. 3). Recently, numerous studies have centered on the ABA signal transduction pathway (Ma et al., 2009; Pandey et al., 2009; Park et al., 2009), and one core ABA signaling pathway has been proposed, including ABA-PYR/PYL perception, PYR/PYL-PP2Cs interaction, inhibition of the negative regulation of PP2Cs, and activation of SnRK2s, leading to the ABA response (Fujii et al., 2009). Certainly, there is a negative-feedback regulatory mechanism to maintain the homeostasis of ABA levels and modulate the ABA response by up-regulating the PP2Cs and down-regulating PYR/PYL genes (Santiago et al., 2009), which is consistent with the finding in the present study that CsPYL4 was down-regulated, and several members of the PP2Cs (such as CsPP2C 56, CsAHG1) were notably up-regulated in both control and ABA/NDGA treated-fruits during the fruit ripening (Fig. 4B). In fruits, the endogenous ABA content is determined by the pathways of biosynthesis and catabolism, with the key genes being NCED1 and ABA 8 -hydroxylase 1, respectively (Zhang et al., 2009a; Wu et al., 2014). Therefore, changes in the dynamic balance of these two processes may result in alteration of ABA level. The relationship between exogenous ABA treatment and endogenous ABA level is a complicated regulatory relationship instead of a simply positive or negative correlation (Fig. 3). In this study, we found that ABA could restrict its own accumulation at 14 DAY not only by inducing the expression of ABA 8 -hydroxylase 1 but also by reducing the expression of CsNCED1,

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which was contrary to the result of NDGA treatment (Figs. 3 and 4A). The aforementioned observations imply a negative feedback regulation in ABA biosynthesis and catabolism just as in the ABA signaling pathway (Melcher et al., 2009). However, the underlying molecular mechanism remains to be further elucidated. 5. Conclusion In the present study, exogenous ABA was found to accelerate fruit coloring (by reducing peel H value), significantly decrease organic acid content, and influence the expression of genes in sugar and organic acid metabolism and signal transduction pathway, thus promoting the process of citrus fruit ripening. Furthermore, exogenous ABA could affect the level of endogenous ABA and the expression of ABA-related genes in citrus fruits. This field trial offers some practical insights to the regulation of citrus fruit ripening. Acknowledgments This work was funded by the National Modern Citrus Industry System (CARS-27), the Ministry of Education Innovation Team (IRT13065), and National Natural Science Foundation of China (No. 31301760). We thank the Fruit and Tea Institute of Hubei Agricultural Science Academy, Hubei, China, for supply of samples. 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.2015. 12.024. References Alexander, L., Grierson, D., 2002. Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening. J. Exp. Bot. 53, 2039–2055. Bartolozzi, F., Bertazza, G., Bassi, D., Cristoferi, G., 1997. Simultaneous determination of soluble sugars and organic acids as their trimethylsilyl derivatives in apricot fruits by gas-liquid chromatography. J. Chromatogr. A 758, 99–107. Bottcher, C., Keyzers, R.A., Boss, P.K., Davies, C., 2010. Sequestration of auxin by the indole-3-acetic acid-amido synthetase GH3-1 in grape berry (Vitis vinifera L.) and the proposed role of auxin conjugation during ripening. J. Exp. Bot. 61, 3615–3625. Chai, Y.M., Jia, H.F., Li, C.L., Dong, Q.H., Shen, Y.Y., 2011. FaPYR1 is involved in strawberry fruit ripening. J. Exp. Bot. 62, 5079–5089. Chen, M., Xie, X., Lin, Q., Chen, J., Grierson, D., Yin, X., Sun, C., Chen, K., 2013. Differential expression of organic acid degradation-related genes during fruit development of navel oranges (Citrus sinensis) in two habitats. Plant Mol. Biol. Rep. 31, 1131–1140. Concha, C.M., Figueroa, N.E., Poblete, L.A., Onate, F.A., Schwab, W., Figueroa, C.R., 2013. Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit. Plant Physiol. Biochem. 70, 433–444. Creelman, R.A., Bell, E., Mullet, J.E., 1992. Involvement of a lipoxygenase-like enzyme in abscisic acid biosynthesis. Plant Physiol. 99, 1258–1260. de Ollas, C., Hernando, B., Arbona, V., Gomez-Cadenas, A., 2013. Jasmonic acid transient accumulation is needed for abscisic acid increase in citrus roots under drought stress conditions. Physiol. Plant. 147, 296–306. Fujii, H., Chinnusamy, V., Rodrigues, A., Rubio, S., Antoni, R., Park, S.Y., Cutler, S.R., Sheen, J., Rodriguez, P.L., Zhu, J.K., 2009. In vitro reconstitution of an abscisic acid signalling pathway. Nature 462, 660–664. Galpaz, N., Wang, Q., Menda, N., Zamir, D., Hirschberg, J., 2008. Abscisic acid deficiency in the tomato mutant high-pigment 3 leading to increased plastid number and higher fruit lycopene content. Plant J. 53, 717–730. Gambetta, G.A., Matthews, M.A., Shaghasi, T.H., Mcelrone, A.J., Castellarin, S.D., 2010. Sugar and abscisic acid signaling orthologs are activated at the onset of ripening in grape. Planta 232, 219–234. Han, Y., Dang, R., Li, J., Jiang, J., Zhang, N., Jia, M., Wei, L., Li, Z., Li, B., Jia, W., 2015. SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE2. 6, an Ortholog of OPEN STOMATA1, is a negative regulator of strawberry fruit development and ripening. Plant Physiol. 167, 915–930. Jia, H.F., Chai, Y.M., Li, C.L., Lu, D., Luo, J.J., Qin, L., Shen, Y.Y., 2011. Abscisic acid plays an important role in the regulation of strawberry fruit ripening. Plant Physiol. 157, 188–199.

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