MicroRNAs play an important role in the regulation of strawberry fruit senescence in low temperature

Postharvest Biology and Technology 108 (2015) 39–47

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

MicroRNAs play an important role in the regulation of strawberry fruit senescence in low temperature Xiangbin Xu a,b , Xiuyan Ma b , Huanhuan Lei b , Lili Yin b , Xuequn Shi a, * , Hongmiao Song a,b, * a b

College of Food Science and Technology, Hainan University, Haikou 570228, China College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 12 November 2014 Received in revised form 4 May 2015 Accepted 6 May 2015

To understand the microRNAs (miRNAs)-mediated mechanism of low temperature delaying nonclimacteric strawberry fruit senescence during postharvest period, two small RNA (sRNA) libraries from strawberry fruit stored at low temperature for 24 and 48 h were constructed. A total of 88 known and 1224 novel potential candidate miRNAs were obtained and analyzed. Compared with the expression of miRNAs in strawberry fruit stored for 0 h, 108 miRNAs were up-regulated and 113 were down-regulated in fruit stored at 24 h, and 139 miRNAs were up-regulated and 114 were down-regulated in fruit stored at 48 h. In the process of fruit storage under low temperature, PC-5p-176409_20 repressed abscisic acid (ABA) signaling transduction via the PYR1/PYL1-PP2C-SnRK2 network, miR167 reduced the jasmonic acid (JA) biosynthesis by targeting auxin response factor 8(ARF8), and by which they were involved in delaying fruit senescence. MiR164, miR172, PC-5p-67794_53 and PC-5p-1004_3092 up- or downregulated the expression of their target genes, NAC transcription factors,APETALA2.7 (AP2.7) transcription factor, alpha/beta-hydrolases superfamily protein and glycosyl hydrolase 9B1, respectively, and also involved in delaying fruit senescence under low temperature. These results give valuable information for understanding the role of miRNA in mediating the fruit senescence at low temperature. ã2015 Elsevier B.V. All rights reserved.

Keywords: High-throughput sequencing Fruit senescence MiRNA Abscisic acid Jasmonic acid

1. Introduction MicroRNAs (miRNAs), identified in nearly all eukaryotes, are short (20–24 nucleotides in length), single strand and endogenous non-coding small RNAs (sRNAs) molecules that negatively regulate gene expressions at post-transciptional level (Llave et al., 2002; Bartel 2004). In plants, miRNAs regulate multiple crucial biological processes, such as development, hormone response and stress responses (Jones-Rhoades and Bartel, 2004; Mallory and Vaucheret, 2006; Sunkar et al., 2006; Voinnet 2009). In addition, they are also involved in plant senescence. MiR319 negatively regulates leaf growth and positively regulates leaf senescence by modulating the activity of TCP transcription factors (Schommer et al., 2008). MiR164 prevents premature overexpression of ORE1, a positive regulator of aging-induced cell death and senescence, and regulates the senescence and cell death in Arabidopsis leaves

* Corresponding authors. College of Food Science and Technology, Hainan University, Haikou 570228, China. Tel.: +86 898 6619116. E-mail addresses: [email protected] (X. Shi), [email protected] (H. Song). http://dx.doi.org/10.1016/j.postharvbio.2015.05.006 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.

(Kim et al., 2009). In the climacteric fruit tomato, miR156/157 was found to be complementary to the CNR3’ untranslated-region sequence, which was important for MADS-RIN to induce the transcription of ripening-related genes (Dalmay 2010). To date, 30,424 miRNAs in 206 species (miRbase 20, http://www.mirbase. org/) were identified, but function of most miRNA is still unknown. To totally understand their biological functions in diverse biological processes, the recent development of degradome sequencing provided a new strategy for validating the splicing targets on a whole genome scale, which revolutionized the traditional computational target prediction and was successfully employed on identifying miRNA targets in plants (Addo-Quaye et al., 2008; Xu et al., 2012, 2013). Strawberry [Fragaria  ananassa (Rosaceae Family)] is a very popular fruit, which provides high levels of bioactive compounds in human dies, including vitamin C, vitamin E, b-carotene, and phenolic compounds. However, at ambient temperature, postharvest senescence of strawberry fruit occurs very quickly and their storage time are only 2–3 d, which often seriously affects fruit quality and marketing value, causing huge losses. To maintain quality and extend shelf life, low temperatures (1–4  C) are usually adopted in strawberry fruit storage, by which the senescence can

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be delayed for about 7 d. To further extend strawberry fruit shelf life, it is important to understand the molecular mechanism of low temperature delaying postharvest senescence of strawberry fruit. In the previous work, to investigate the mechanism of postharvest senescence of strawberry fruit at ambient temperature (20  C), two sRNA libraries (fruit stored at 20  C for 0 and 24 h) and one degradome library (mixture of strawberries stored for 0 and 24 h) from strawberry fruit that were constructed and sequenced on an Illumina Hiseq 2500, and many miRNAs involved in fruit senescence were obtained (Xu et al., 2013). Here, in order to further understand the role of miRNAs on delaying senescence of strawberry fruit in response to low temperature (4  C), two other sRNA libraries (fruit stored at 4  C for 24 and 48 h, respectively) from strawberry fruit that were constructed and sequenced. The roles of miRNAs and their targets involved in delaying fruit senescence in response to low temperature (4  C) were analyzed. 2. Materials and methods 2.1. Strawberry fruit materials Strawberry (Fragaria ananassa L. cv. Zhangji) fruit was harvested at commercial maturity from an orchard (a private orchard, the owner of Ms. Ma gave permission to conduct the study on this site) in the Xiasha district, Hangzhou, China, and transported to the laboratory immediately where they were sorted, based on size, without physical injuries or infections. The fruit was put in trays with plastic bags to maintain a relative humidity (about 85%), then stored at 4  C and 20  C for 0, 24, 48 and 72 h. Each treatment contained three replicates of 30 fruit and the entire experiment was repeated twice. 2.2. Determination of fruit senescence Fruit decay and weight loss rate were determined in 30 fruit with three replications, respectively. The senescence was assessed by measuring the extent of decayed area on each fruit, and was determined as: 0, no decay; 1, less than 10% decay; 2, 10–30% decay; 3, more than 30% decay. The decay rate was calculated using the following formula: [(1  N1 + 2  N2 + 3  N3)  100/(3  N)], where N was the total number of fruit measured and N1, N2 and N3 were the number of fruit showing the different degrees of decay. Weight loss rate was calculated by the following formula: (A B)/A  100, where A was the fruit weight just before storage and B was the fruit weight after storage. 2.3. sRNA library construction, sequencing and data analysis The fruit stored at 4  C for 24 and 48 h were collected and frozen in liquid nitrogen immediately, and then stored at 80  C, respectively. The sRNA library construction, sequencing and data analysis were performed according to the methods of Xu et al. (2013). 2.4. Verification of strawberry fruit miRNAs by qRT-PCR To validate the sequencing results, four novel miRNAs and two known miRNAs were assayed in strawberry fruit stored at 4  C for 0, 24 and 48 h by qRT-PCR, respectively. Total RNA was extracted from the strawberry fruit with Trizol reagent following the manufacturer’s instructions (Invitrogen). The total RNA (2.5 mg) was treated with DNase I (TaKaRa, Dalian, China) to remove the genomic DNA and reverse-transcribed using miRNA specific stemloop primers (Appendix D). The forward six primers of six selected miRNAs were designed according to the sequence of miRNA itself, and the universal primer was used as a reverse

primer (Appendix D). 1 mL of cDNA was used as the template in 25 mL PCR reactions which contained 12.5 mL 2  SYBR Premix Ex TaqTM II (TaKaRa, Dalian, China), 1 mL each of forward and reverse primer (10 mM) and 9.5 mL water. Reactions were performed using the iCycler iQ real-time PCR detection system (Bio-Rad). All reactions were performed in triplicate for each sample and actin7 was used as an internal reference. The relative expression level of miRNA was calculated according to the method of Livak and Schmittgen (2001). 2.5. Expression analyses of target genes Total RNA was extracted from strawberry fruit. 0.25 mg of total RNA was treated with RNase-free DNase I (Promega, USA) according to manufacturer’s instructions to remove contaminating DNA. First strand cDNA was synthesized using 1 mL M-MLV reverse transcriptase according to the manufacturer’s protocol (Transgene, Beijing, China). 0.5 mL of cDNA was used as the template in 25 mL PCR reactions which contained 12.5 mL 2  SYBR Premix Ex TaqTM II (TaKaRa, Dalian, China), 1 mL each of forward and reverse primer (10 mM) and 10 mL water. Reactions were performed using the iCycler iQ real-time PCR detection system (Bio-Rad). All reactions were performed in triplicate for each sample. The threshold value was empirically determined based on the observed linear amplification phase of all primer sets. Sample cycle threshold (Ct) values were standardized for each template based on a actin7 control reaction and the comparative Ct method (2–DDCt) was used to determine the relative transcript abundance of each gene (Livak and Schmittgen 2001). The primers of target genes were designed according to their sequence (Appendix D). 2.6. Determination of abscisic acid (ABA) and jasmonic acid (JA) content ABA content was determined by gas chromatography–mass spectroscopy (GC–MS) (Trace 2000 Voyager, Finnigan, ThermoQuest) (Jia et al., 2011). 1 g Flesh tissue (mixture of 15 fruit) was mixed with antioxidant copper reagent, quartz sand, cold methanol (80%) and D3-ABA, ground to homogeneity at 4  C, and soaked at 4  C after mixed with methanol (80%). After soaking for 18 h, the mixture was filtered. The filtrate was collected and combined with 1 mL ammonia water, and was concentrated to an aqueous phase with a vacuum at 35  C. The filtrate was transferred to a centrifuge tube and freeze thawed three times. Then the tubes were melted and centrifuged and placed in refrigerator at 20  C for 18 h. The supernatant was collected by centrifugation after adding polyvinylpolypyrrolidone (PVPP) stirring paste to remove the pigment, and extracted three times with equal volumes of ethyl acetate after adjusting to pH 3.0. The organic phase was frozen to remove water and dried at 35  C, then was separated on a small C18 column after dissolving with acetic acid. 5 mL ammonia water was added to the collected eluate, and dried at 40  C. Finally, diazomethane was added for esterification. The sample was dissolved with acetic acid ethyl ester, transferred to a capillary tube, and concentrated to determine the ABA content by GC–MS. D3-ABA was used as the internal standard. Three replicates were performed for ABA analysis. The JA content was determined according to the methods of Weber et al. (1997), with some modification. Briefly, 1 g flesh tissue (mixture of 15 fruit) was ground to powder and added to 3 mL of ice-cold methanol (80%) containing 100 ng tetrahydro-12-oxo-PDA and 100 ng dihydrojasmonic acid (H2JA) as internal standards. Then transferred to a tube and rotated for 8 h at 4  C. The mixture was filtered. The filtrate was concentrated to an aqueous phase with a vacuum and transferred to a centrifuge tube and freeze-thawed three times. The supernatant was collected by

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centrifugation after adding PVPP stirring paste to remove the pigment, and extracted three times with equal volumes of ethyl acetate after adjusting to pH 3.0 with 2 M HCl. The organic phase was concentrated by vacuum to remove water and dried at 40  C. The sample was dissolved with 5 mL 0.1 M acetic acid and passed through a C18-SepPak classic cartridge (Waters), which had been prewashed with methanol followed by acetic acid. The column was washed with 3 mL of methanol/0.1 M acetic acid (20:80, vol/vol), methanol/0.1 M acetic acid (40:60, vol/vol), and methanol/0.1 M acetic acid (60:40, vol/vol) two times, respectively. The eluent was added 3 mL ammonia water and concentrated by vacuum to remove water and dried at 40  C. Diazomethane 0.5 mL was added to the sample, dried under N2, and redissolved in 1 mL of hexane. JA content was analyzed using GC–MS (Trace 2000 Voyager, Finnigan, ThermoQuest) equipped with a DB-5 quartz capillary column (30 m  0.32 mm (i.d)  0.5 mm). H2JA was used as the internal standard. The endogenous JA content in strawberries is the sum of the endogenous JA and MeJA. Three replicates were performed for JA analysis.

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2.7. Statistical analysis All data were analysed by one-way analysis of variance (ANOVA). Mean separations were performed by Duncan’s multiple range test. 3. Results 3.1. Effect of low temperature on fruit senescence In the present study, the fruit senescence was shown by fruit decay and weight loss rate. As shown in Fig. 1A, nearly all the fruit decayed after 72 h at 20  C storage, but only a few fruit decayed after 72 h at 4  C storage. The fruit decay rate gradually increased with storage time at both 4  C and 20  C. Fruit stored at 20  C presented high decay rate of 18.1, 36.7 and 60.5% after 24, 48 and 72 h storage, but the fruit decay rate was significantly lower at 4  C, which was only 36% after 72 h (Fig. 1B). Similarly, the fruit weight loss rate gradually increased with storage time at both 4  C and

Fig. 1. Senescence changes of fruit stored at 4  C and 20  C. (A) Senescence phenotypic characterization of fruit. The circles indicate the position of fruit decay; (B) Decay and weight loss rate of fruit. The symbols * and ** in the graph show the significant differences at p < 0.05 and p < 0.01, respectively.

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20  C. Total weight loss of fruit stored at 20  C was 2.3, 4.5 and 6.4% after 24, 48 and 72 h storage, respectively, while weight loss of fruit stored at 4  C was only 3.2% after 72 h (Fig. 1B). These results indicated that the low temperature of 4  C storage delayed strawberry fruit senescence. 3.2. Overview of deep-sequencing in sRNA To identify miRNAs which involved in delaying senescence of strawberry fruit in response to low temperature, two independent sRNA libraries were constructed from strawberry fruit stored at 4  C for 24 and 48 h, and sequenced by Illumina Hiseq 2500. A total of 19,675,851 and 20,983,425 raw reads were yielded, respectively (Table 1). After removal of corrupted adapter sequences, reads with length <15 and >25 nt, junk reads, common RNA families (mRNA, rRNA, tRNA, snRNA, snoRNA) and repeats, a total of 18,816,240 and 20,293,492 mappable sRNA sequences were obtained, respectively (Table 1). The cloning frequency of different sized sRNAs (15–32 nt) was similar among the two libraries, and the majority of the sRNAs were 20–24 nt in size, with two major peaks at 21 nt and 24 nt (Fig. 2). This is within the typical size range for Dicer-derived products in which 21 nt sRNAs were the most abundant followed by 24 nt as the second largest percentage representing the class of endogenous sRNA families. 3.3. Identification of miRNAs To identify known miRNAs in the two libraries of strawberry fruit, all the mappable sRNAs were mapped to the currently known plant precursor or mature miRNAs sequences in miRbase (20.0) database. Eighty-eight unique mature miRNAs corresponding to 71 pre-miRNAs with high sequence similarity to the known plant miRNAs were identified (Appendix A). Moreover, by mapping all unique sRNA sequences to the strawberry genome and predicting the secondary structures of the candidate miRNA precursors, 1224 unique novel mature potential candidate miRNAs corresponding to 1157 pre-miRNAs were identified, of which most were 24 nt in length (Appendix B). These data were the same with previous work of Xu et al. (2013), which have been submitted to the Gene Expression Omnibus under the accession number GSE48055. 3.4. Expression profiling of miRNAs in response to low temperature To identify miRNAs involved in delaying senescence of strawberry fruit in response to low temperature, the differential

Fig. 2. Length distribution of mappable counts of sequ-seqs type in two libraries of strawberry fruit.

expression of miRNAs was analyzed using the counts of reads generated from the high-throughput sequencing (Appendix C). The counts of miRNA reads obtained from strawberry fruit stored for 0 h in the previous work of Xu et al. (2013) were used as control. Considering the extremely low abundances might lead to false results, the known and novel mature potential candidate miRNAs with less than 10 norm reads in the two libraries were removed from the expression analysis. Compared with the expression of miRNA in strawberry fruit stored for 0 h, 108 miRNAs were upregulated and 113 were down-regulated in strawberry fruit stored at 4  C for 24 h. There are 18 miRNAs with log2 (24 h/0 h) fold changes higher than 1.5, and 28 miRNAs lower than 0.5, respectively (Fig. 3A). In strawberry fruit stored at 4  C for 48 h, compared with the strawberry fruit stored for 0 h, 139 miRNAs were up-regulated and 114 were down-regulated. There are 34 miRNAs with log2 (48 h/0 h) fold changes higher than 1.5, and 29 miRNAs lower than 0.5, respectively (Fig. 3B). To validate the presence and expression of the identified miRNAs, the differential expression pattern of four novel potential candidate miRNAs, PC-5p-15285_237, PC-5p-1433_2156, PC-3p1366_2253 and PC-3p-5426_650, and two known miRNAs, mdmmiR399i and zma-MIR167j-p3_1ss14CT, were selected for analysis by quantitative realtime PCR (qRT-PCR). As shown in Fig. 4, as the qualitative detection, nearly all the qRT-PCR results were quite

Table 1 Distribution of the sRNA sequences in the two libraries. 24 h Stored fruit

Raw reads 3ADT&length filter Junk reads Rfam mRNA repbase rRNA(45.7%) tRNA(25.4%) snoRNA(6.2%) snRNA(2.4%) other Rfam RNA mappablel reads

48 h Stored fruit

Type

Total

Percentage of total

Unique

Percentage of unique

Total

Percentage of total

Unique

Percentage of unique

NA Sequence type Sequence type RNA class RNA class RNA class RNA class RNA class RNA class RNA class RNA class Sequence type

19675851 94146 64761 2539139 3968408 15779 1334041 1116830 11787 30377 3380026 18816240

100 0.5 0.3 12.9 20.17 0.08 6.78 5.68 0.06 0.15 17.18 95.63

5443657 / / 87450 805086 4470 50580 22086 2260 5306 793238 5110971

100 / / 1.6 14.79 0.082 0.93 0.41 0.042 0.1 14.57 93.89

20983425 98077 58202 2385965 4499476 18274 1261461 1032868 13831 34069 3682276 20293492

100 0.5 0.3 11.37 21.44 0.09 6.01 4.92 0.07 0.16 17.55 99.2

5755904 / / 83570 839224 6208 45386 21114 3012 5988 826340 5449043

100 / / 1.45 14.58 0.11 0.79 0.37 0.05 0.1 14.36 94.67

3ADT&length filter: reads removed due to 3ADT not found and length with <17 nt and >25 nt were removed mRNA: downloaded from http://www.rosaceae.org/projects/ strawberry_genome/v1.0/genes/fvesca_v1.0_genemark_hybrid.fna.gz. Rfam (V 10.0): downloaded from ftp://ftp.sanger.ac.uk/pub/databases/Rfam/9.1/. Repeat (V13.12): downloaded from http://www.girinst.org/repbase/update/index.html. Notes: there is overlap in mapping of reads with mRNA, rRNA, tRNA, snRNA, snoRNA and repeats.

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Fig. 3. Differential expression levels of miRNAs in fruit stored at 4  C for 0, 24 and 48 h. (A) expression levels of miRNAs in fruit stored at 4  C for 24 h compared with that in 0 h. (B) expression levels of miRNAs in fruit stored at 4  C for 48 h compared with that in 0 h.

consistent with the expression pattern of the six miRNAs from high-throughput sequencing. 3.5. Expression of target genes in response to low temperature In the previous work, one degradome library from strawberry fruit have been constructed, and 103 targets (http://www.rosaceae. org/projects/strawberry_genome/v1.0/genes/fvesca_v1.0_genemark_hybrid.fna.gz) cleaved by 74 miRNAs were identified (Xu et al., 2013). Among them, PP2C (gene26316-v1.0-hybrid), ARF8 (gene30394-v1.0-hybrid), AP2.7 (gene25394-v1.0-hybrid), NAC (gene00971-v1.0-hybrid), alpha/beta-hydrolases (gene06057v1.0-hybrid) and glycosyl hydrolase 9B1 (gene06191-v1.0-hybrid) were found to be cleaved by mdm-miR164d_1ss21AC, mdmmiR164e, ptc-miR164f_1ss21TA, mdm-miR167b_R+1_1ss21AC, mdm-miR172g_R+1, PC-5p-67794_53, PC-5p-1004_3092, and

PC-5p-176409_20, respectively, and might involve in fruit senescence (Xu et al., 2013). To understand the roles of these miRNAs in fruit senescence in response to low temperature, the expression of their target genes was analyzed by qRT-PCR. As shown in Fig. 5, compared with that in fruit stored for 0 h, the expression of PP2C was significantly increased in fruit stored at 4  C for 24, 48 and 72 h, but significantly decreased in fruit stored at 20  C for 24 and 48 h. The expression of ARF8 and AP2.7 was increased in fruit at both 4  C and 20  C storage. However, compared with 20  C storage, the 4  C storage significantly retarded the increasing the expression of ARF8 and AP2.7. The expression of NAC gene was significantly upregulated in fruit at low temperature of 4  C for 24, 48 and 72 h, but significantly decreased in fruit stored at 20  C for 24 and 48 h. The expression of alpha/beta-hydrolases and glycosyl hydrolase 9B1 showed the same trend in fruit stored at 4  C and 20  C. They both increased in fruit stored at 4  C for 24, 48, 72 h and in fruit stored at

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Fig. 4. Expression analysis of miRNAs in strawberry fruit stored at 4  C for 0, 24 and 48 h by qRT-PCR. The amount of expression was normalized by the level of actin 7 in qRT-PCR. All reactions of qRT-PCR were repeated three times for each sample. Left indicates the miRNA relative expression tested by qRT-PCR; Right indicates the miRNA relative expression generated from the high-throughput sequencing. The symbols * in the graph show the significant differences at p < 0.05.

20  C for 24, 48 h, but the low temperature of 4  C significantly delayed their expression. 3.6. Phytohormone content analyses To understand the potential role of ABA and JA on the senescence of postharvest strawberry fruit at low temperature, their contents were analyzed using GC–MS. As shown in Fig. 6, compared with fruit stored for 0 h, the ABA content was significantly increased in fruit stored at 4  C for 24, 48 and 72 h. In fruit stored at 20  C for 24 and 48 h, though the ABA content was also increased, but lower than that in fruit stored at 4  C. Compared with fruit stored for 0 h, JA content also significantly increased to high level at 4  C for 72 h. However, compared with fruit stored at 20  C for 24 and 48 h, the content of JA was lower in fruit stored at 4  C, indicated that JA biosynthesis was repressed by the low temperature of 4  C. 4. Discussion ABA, as the important phytohormone, besides the key roles on plant growth and responses to environmental stresses (Leung and Giraudat, 1998; Finkelstein et al., 2002; Himmelbach et al., 2003; Hirayama and Shinozaki, 2007) was also involved in the ripening of climacteric (Chernys and Zeevaart, 2007; Zhang et al., 2009a) and non-climacteric fruit (Rodrigo et al., 2003; Davies et al., 1997; Zhang et al., 2009b), including strawberry fruit (Kano and Asahira, 1981; Manning 1994; Jiang and Joyce, 2003; Jia et al., 2011). In Arabidopsis, PYR1/PYL1-PP2C-SnRK2 is one of the core signaling transduction networks of ABA, in which ABA could promote the interaction of PYR1/PYL1 and PP2C, repress PP2C and activate SnRK2, then transmit the ABA signals (Fujii et al., 2009; Ma et al., 2009; Park et al., 2009). In the previous study, one PP2C gene targeting by the potential candidate miRNA PC-5p-176409_20 has been identified in strawberry fruit by degradome sequencing Xu et al., 2013). In the present study, under low temperature of 4  C for 24, 48 and 72 h, though high content of ABA was detected in strawberry fruit (Fig. 6), the transcription of PP2C was enhanced

(Fig. 5) by the down-regulation of PC-5p-176409_20 (Appendix C), which repressed ABA signal transmitting via the PYR1/PYL1-PP2CSnRK2 signaling network and by which could delay fruit senescence. These may be one of the mechanisms of low temperature delaying strawberry fruit senescence. JA, methyl jasmonate (MeJA), and other derivatives, together known as jasmonates, play key roles in fruit ripening and senescence. JA can function independent of ethylene to enhance lycopene biosynthesis in tomato fruit, and play a central role in promoting fruit ripening (Liu et al., 2012). JA-dependent senescence is defective in the JA-insensitive mutant coronatine insensitive 1 (coi1) (He et al., 2002). Exogenous application of JA could increase expression of senescence associated genes and stimulate leaf senescence (Lim et al., 2007). In plant, the production of JA was regulated by two members of the ARF, ARF6 and ARF8. The ARF6 and ARF8 single mutation delayed flower maturation and leading to the formation of seedless, parthenocarpic fruit (Nagpal et al., 2005). The ARF8 was identified in strawberry fruit and targeted by four members of miR167b, respectively (Xu et al., 2013). In the present study, compared with fruit stored for 0 h, the counts of reads of the four members of miR167b significantly increased in strawberry fruit stored at 4  C for 48 h (Appendix C), by which they repressed the expression of ARF8 (Fig. 5) and decreased the production of JA (Fig. 6), and by which they may involve in delaying the senescence of strawberry fruit under low temperature. The NAC transcription factors [No apical meristem (NAM), Arabidopsis transcription activation factor (ATAF), Cup-shaped cotyledon (CUC)] were one of the largest transcription factor families in plants. They are involved in diverse processes (Olsen et al., 2005; Zhong et al., 2010) and plant senescence (Guo et al., 2004; Buchanan-Wollaston et al., 2005; Yoon et al., 2008; Balazadeh et al., 2010). In Arabidopsis leaf, about 20 NAC transcription factors are differentially regulated during senescence (Guo et al., 2004). Overexpression of the NAP gene, one of Arabidopsis NAC transcription factors, induced the premature senescence. Deficient mutation of the NAP gene, delayed the leaf senescence of Arabidopsis. Expression of the NAP homologs from Oryza sativa and Phaseolus vulgaris restored the delayed leaf senescence in the NAP deficient mutant (Guo and Gan, 2006). Moreover, ANAC092/NAC2/ORE1 gene plays an important role in regulating leaf longevity (Kim et al., 2009) and salt-promoted senescing process (He et al., 2002; Balazadeh et al., 2010). Three members of the miR164 family, mdm-miR164d_1ss21AC, mdmmiR164e and ptc-miR164f_1ss21TA, and their targets NAC domain transcriptional regulator superfamily protein, NAC domain containing protein 87 and NAC domain containing protein 38, were identified in the strawberry fruit (Xu et al., 2013). Compared with fruit stored for 0 h, though no significant change were found in mdmmiR164d_1ss21AC and ptc-miR164f_1ss21TA, the reads of mdmmiR164e significantly decreased in strawberry fruit stored at 4  C for 24 and 48 h (Appendix C), which increased the expression of NAC transcription factor (Fig. 5), and may be involved in delaying the senescence of strawberries under low temperature. APETALA2 (AP2) transcription factors involved plant responses to various biotic and abiotic stresses as well as in plant growth and development (Yamaguchi-Shinozaki and Shinozaki, 2006). They also have been found to play important roles in fruit ripening. In tomato, the AP2a gene negatively regulated fruit ripening (Karlova et al., 2011). In oil palm fruit, the EgAP2.1 expression was induced in mesocarp in response to ethylene and ABA, and involved in fruit ripening (Morcillo et al., 2007). In the previous study, two members of the miR172 family, mdm-miR172l_1ss21GT and mdm-miR172g_R+1 have been identified to target one relative AP2.7 gene in the strawberry fruit, and involved in fruit senescence (Xu et al., 2013). In the present study, compared with fruit stored for 0 h, the reads of mdm-miR172l_1ss21GT and mdm-miR172g_R

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Fig. 5. Expression analysis of target genes in strawberry fruit by qRT-PCR. The symbols * and ** in the graph show the significant differences at p < 0.05 and p < 0.01, respectively.

+1 significantly increased at 24 and 48 h under 4  C storage (Appendix C), by which they repressed the expression of AP2.7 (Fig. 5), and by which they may be involved in delaying strawberry fruit senescence under low temperature.

In plants, cell wall plays important role in controlling cell size and shape, and ensure cell survival. Its structure is complex and contains various components such as polysaccharides, lignin and proteins. Two potential novel miRNAs, PC-5p-67794_53 and

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Fig. 6. The change of ABA and JA content in strawberry fruit stored at 4  C and 20  C. The symbols * in the graph show the significant differences at p < 0.05.

PC-5p-1004_3092, have been identified to target alpha/betahydrolases superfamily protein and glycosyl hydrolase 9B1, respectively, are involved in fruit senescence (Xu et al., 2013). The alpha/beta-hydrolases function as hydrolases, lyases, transferases, hormone precursors or transporters, chaperones or routers of other proteins, and play important role in the degradation of cell wall and fruit ripening (Lenfant et al., 2013). Glycoside hydrolases are common enzymes across all domains of life. They are involved in the metabolism of various carbohydrates containing compounds present in the plant tissues and the degradation and reorganization of cell wall polysaccharides, thereby acting to control fruit softening during ripening (Moctezuma et al., 2003). In the present

study, compared with fruit stored for 0 h, the counts of reads of PC5p-67794_53 and PC-5p-1004_3092 were significantly increased in strawberries stored for 24 and 48 h under 4  C (Appendix C), in which they repressed the expression of alpha/beta-hydrolases and glycosyl hydrolase 9B1, respectively (Fig. 5), and might prevent the degradation of fruit cell wall. This may be also one of the mechanisms of low temperature delaying senescence of strawberry fruit. In conclusion, MiR164e, miR167b, miR172l_1ss21GT, miR172g_R+1, PC-5p-176409_20, PC-5p-67794_53 and PC-5p1004_3092 play an important role in delaying strawberry fruit senescence at low temperature, illustrating their broad prospect in

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