In plants, β–glucosidases (BG) have been implicated in developmental and pathogen defense, and are thought to take part in abscisic acid (ABA) synthesis via hydrolysis of ABA glucose ester to release active ABA; however, there is no genetic evidence for the role of BG genes in ripening and biotic/abiotic stress in fruits. To clarify the role of BG genes in fruit, eight Fa/FvBG genes encoding β–glucosidase were isolated using information from the GenBank strawberry nucleotide database. Of the Fa/FvBG genes examined, expression of FaBG3 was the highest, showing peaks at the mature stage, coincident with the changes observed in ABA content. To verify the role of this gene, we suppressed the expression of FaBG3 via inoculation with Agrobacterium tumefaciens containing tobacco rattle virus carrying a FaBG3 fragment (RNAi). The expression of FaBG3 in FaBG3-RNAi-treated fruit was markedly reduced, and the ABA content was lower than that of the control. FaBG3-RNAi-treated fruit did not exhibit full ripening, and were firmer, had lower sugar content, and were pale compared with the control due to down-regulation of ripening-related genes. FaBG3-RNAi-treated fruit with reduced ABA levels were much more resistant to Botrytis cinerea fungus but were more sensitive to dehydration stress than control fruit. These results indicate that FaBG3 may play key roles in fruit ripening, dehydration stress and B. cinerea fungal infection in strawberries via modulation of ABA homeostasis and transcriptional regulation of ripening-related genes.
The β–glucosidases (BG) are involved in plant developmental and pathogen defense processes (Esen, 1993; Kleczkowski and Schell, 1995). Recently, considerable progress has been made in elucidating the functions of BGs in activation of plant hormones, including abscisic acid (ABA) (Lee et al., 2006), and in chemical plant defense responses against pathogens (Morant et al., 2008). However, little is known about the contribution of BGs to the adjustment of ABA homeostasis during fruit ripening.
Abscisic acid plays a crucial role in fruit development and ripening, as well as in adaptive responses to biotic/abiotic stresses (Zhang et al., 2009a,b; Sun et al., 2010; Seymour et al., 2013). In higher plants, ABA is produced from the cleavage product of C40 carotenoids via an indirect pathway (Tan et al., 1997; Iuchi et al., 2001; Kato et al., 2006). Recently, multiple signal transduction pathways of ABA in plants have become fairly well-established (Shen et al., 2006; Ma et al., 2009; Pandey et al., 2009; Park et al., 2009). ABA levels are increased via two biosynthetic mechanisms. One mechanism involves de novo biosynthesis of ABA (Marin et al., 1996; Audran et al., 1998; Sun et al., 2013). The other biosynthetic process involves one-step hydrolysis of Glc-conjugated ABA by a BG (Dietz et al., 2000; Lee et al., 2006). ABA-glucose ester is the most abundant conjugated form of ABA (Hansen and Dörffling, 1999), and its synthesis requires a glucosyltransferase, whereas its dissociation from ABA requires BGs. Both types of enzyme have been identified in plants (Xu et al., 2002), and some are up-regulated in the context of environmental stresses (Dietz et al., 2000). Recently, a major BG has been detected in the cytosol. This BG catalyzes the hydrolysis of ABA glucose ester into active ABA to rapidly adjust ABA levels (Sauter et al., 2002). Loss of AtBG1 leads to defects in ABA-mediated responses (Lee et al., 2006). These studies indicate that BG may contribute to ABA homeostasis in plants, and suggest that a complex regulation mechanism is required for ABA accumulation. Like whole plants, fruits encounter many types of environmental stresses such as dehydration, and stress may have negative effects on fruit development and plant reproduction. Fruits have systems in place that help them tolerate dehydration (Kondo et al., 2009). Under stress conditions, cellular ABA levels drastically increased, which in turn activates ABA-mediated signaling or ABA-responsive gene expression and adaptive responses (Himmelbach et al., 2003). However, it remains unknown how ABA levels are finely controlled at the transcriptional level in fruit. Direct genetic/molecular evidence regarding the role of BG genes in fruit has not yet been reported.
Upon pathogen attack, infected plant cells generate signaling molecules to initiate defense mechanisms in surrounding cells to limit pathogen spread. The role of ABA, which participates in several processes, is supported by well-documented observations and molecular characterization (Audenaert et al., 2002). Increased endogenous ABA levels were observed in response to infection with viruses, bacteria and fungi (Whenham et al., 1986; Kettner and Dőrffling, 1995). To date, the role of BGs in disease resistance in fruit has not been reported.
Strawberry is one of the most abundant wild fruits worldwide, and is also widely used as a research model to study non-climacteric fruits (Kano and Asahira, 1981; Manning, 1994; Thomas, 2006; Jia et al., 2011). In this study, we examined the role of FaBG3 in strawberry fruit ripening and biotic/abiotic stress resistance using a tobacco rattle virus (TRV)-induced gene silencing technique (Hoffmann et al., 2006).
Gene isolation, phylogenetic analysis, and expression analysis of Fa/FvBGs
Eight BG cDNA sequences from two strawberry species (Fragaria ananassa and Fragaria vesca) were isolated and designated BG1–BG8 (Figure 1). Based on multiple protein sequence alignments (Figure S8, and Tables S2 and S3), most of the functional residues or domains were well-conserved within the gene family. According to phylogenetic analysis, the BG proteins cluster into three sub-classes (BG sub-class I: FaBG4, FaBG5, FvBG6, FvBG7 and FvBG8; BG sub-class II: FaBG1 and FaBG2; BG sub-class III: FaBG3) (Figure 1). The dynamic process of strawberry fruit development is clearly divided into seven stages (Figures 2 and 4a). Among the eight genes examined, BG6 and BG8 were barely detectable, while expression of the other six BG genes varied greatly during fruit development and ripening (Figure 2a). Compared with the other genes, FaBG3 was expressed at the highest levels, and its expression increased rapidly from de-greening until full ripening, consistent with the changes observed in endogenous ABA content. The next highest expression levels were observed for FaBG2, FaBG5 and FvBG7. Expression of these genes was highest at the small green and large green stages, followed by a decreasing expression pattern during fruit development. BG1 and BG4 were expressed in trace amounts, and no drastic changes were observed during fruit development. As shown in Figure 2(b), at the white stage, all five BG genes (FaBG1, FaBG2, FaBG3, FaBG5 and FaBG7) (Figure 2b) were more highly expressed in the achenes than in the cortex or stele, and expression of FaBG3 was higher in all tissues of the achene, cortex and stele than that of the other BG genes. Expression of FaBG3 and FaBG2 and total BG activity were promoted by exogenous ABA treatment (Figure S5, Methods S1 and S4, and Appendix S1) and dehydration in fruit (Figure S3 and Method S2), but this gene was down-regulated in leaves in response to dehydration (Figure S4 and Method S3). As FaBG3 was highly expressed during ripening and was induced by ABA, we targeted this gene for subsequent reverse genetic analysis.
Phenotypes of strawberry fruits attached to plants with suppressed FaBG3 expression
We next used the TRV vector to suppress the expression of FaBG3 (Figure 3). Thirty fruits attached to ten independent greenhouse-grown plants were injected with the FaBG3-RNAi TRV vector. Ten plants and their fruits were evaluated 6 days after RNAi-treatment (DAT). FaBG3-RNAi fruits did not completely ripen and failed to reach to the normal size. Compared with the control fruit, the FaBG3-RNAi fruit at the pink stage was firmer and paler in color, and had a lower soluble sugar content and a weaker aroma (Figure 4b,c). The water loss rate was determined primarily by measuring fruit weight loss. The water loss rate of freshly picked fruit was 0%. However, after dehydration, the water loss in FaBG3-RNAi fruit was more rapid than that of the control. For example, the water loss rate was 53% in FaBG3-RNAi fruit at 3 days after dehydration treatment, while that of the control was only 48% (Figures 4d,e and 8o). Moreover, we performed a comparative assay to determine the level of resistance of FaBG3-RNAi and wild-type fruits. As we wished to use assay conditions that would result in a moderately aggressive infection, inoculation solutions containing 105 and 107 of Botrytis cinerea spores μl−1 were selected (Figure S6). This infection process allowed us to detect both increases and decreases in disease severity. The B. cinerea spore solution produced a moderate number of spreading B. cinerea lesions in control detached fruits. As shown in Figure 4(f–k), FaBG3-RNAi fruits infected with 105 spores μl−1 did not exhibit B. cinerea lesions at 4 days after inoculation (Figure 4j), while the fruit appeared lightly lesioned in response to infection using 107 spores μl−1 (Figure 4k). By contrast, the control fruit was lightly or heavily lesioned in response to infection by 105 or 107 spores μl−1 of B. cinerea (Figure 4g,h) at 4 days after inoculation. Therefore, FaBG3-RNAi fruits appeared to be much more resistant to B. cinerea than control fruits.
Suppression of FaBG3 by RNAi alters the expression of genes involved in metabolism and signaling of ABA and ethylene
As shown in Figure 5, at 6 DAT, the expression of FaBG3 was markedly down-regulated in FaBG3-RNAi fruits, while other BG genes were up-regulated. The ABA content in FaBG3-RNAi fruit was lower than that in control fruit at 6 DAT. The expression of FaSS (encoding sucrose synthase) in FaBG3-RNAi fruit was higher than in the control, while the expression of FaSPS (encoding sucrose phosphate synthase) and FaSUT1 (encoding a sucrose transporter) was not significantly different from that in control fruit at 6 DAT (Figure 5f). In FaBG3-RNAi fruit, the expression of FaQR (encoding quinine oxidoreductase) was down-regulated (Figure 5b), while the expression of FaNCED2 (encoding a 9-cis-epoxycarotenoid dioxygenase) was slightly up-regulated, and the expression of FaCYP707A1 (encoding 8′-hydroxylase) was markedly up-regulated, compared with the control (Figure 5e). We examined the expression of five genes encoding proteins in the anthocyanin biosynthesis pathway, and found that expression of FaCHS (encoding chalcone synthase), FaF3H (encoding flavonoid-3–hydroxylase) and FaUFGT (UDP Glc-flavonoid 3-O-glucosyl transferase) was down-regulated, while the expression of FaCHI (encoding chalcone isomerase) and FaDFR (encoding dihydroflavo-nol-4–reductase) was not significantly different from control levels (Figure 6). In addition, we examined the exp-ression of genes encoding cell-wall hydrolases. In FaBG3-RNAi fruit, genes encoding polygalacturonase (FaPG), expansin (FaEXP) and β–xylosidase (FaXYL) were down-regulated at 6 DAT compared with the control, while expression of genes encoding pectin methylesterase (FaPE) and β–galactosidase (FaGAL1) was no different from control fruits (Figure 6b). Finally, in the FaBG3-RNAi fruit, ethylene production was slightly down-regulated at 6 DAT compared with the control (Figure 7). Quantitative real-time PCR analysis showed that expression of FaETR2 (involved in the ethylene response) and FaACS1 [encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase] was consistently up-regulated in the FaBG3-RNAi fruit compared with the control, while FaACO2 (encoding ACC oxidase) was down-regulated compared with the control at the pink stage (Figure 7).
Dehydration of FaBG3-RNAi-treated fruits
Both control and FaBG3-RNAi fruits were harvested at 6 DAT (Figure 4d,e). The fruits were then incubated in the laboratory (20°C, 50% relative humidity). As shown in Figure 8, expression of FaBG3 was markedly down-regulated in FaBG3-RNAi fruits 3 days after dehydration compared with the control, while FaBG2 and FaBG5 were up-regulated. The ABA content in FaBG3-RNAi fruits was lower than that of the control fruit at 3 days after dehydration (Figure 8n). The weight loss rate in FaBG3-RNAi fruits was higher than that of control fruit at 3 days after dehydration (Figure 8o). The expression of FaNCED genes (FaNCED2 and FaNCED3) and FaCYP707A1 was down-regulated in the FaBG3-RNAi fruits (Figure 8a–c). Moreover, in the FaBG3-RNAi fruit, ethylene production and expression of FaACS1, FaACO2 and FaETR2 were lower than those of control fruit at 3 days after dehydration (Figure 8g–i). In addition, genes involved in abiotic stress, such as FaRD genes, FaCBL and FaDREB1B, were also down-regulated at 3 days after dehydration compared to control fruit (Figure 8j–m).
Susceptibility of FaBG3-RNAi-treated fruit to B. cinerea
Fruits harvested at 6 DAT with FaBG3-RNAi were infected by B. cinerea and then placed into a container (plastic case) at 20°C, 95% relative humidity. As shown in Figure 9, the ABA content in FaBG3-RNAi fruit was lower than that in control fruit at 5 DAI with B. cinerea. In addition, anthocyanin accumulation was lower than that of the control, but phenylalanine ammonia lyase activity was higher. Moreover, the content of phenolic compounds, including quercetin, epicatechin, kaempferol, catechin and isorhamnetin, was higher in FaBG3-RNAi fruit than in the control fruit at 5 DAI, but there was no difference in chlorogenic acid content between control and transgenic fruit (Figure 9i and Figures S9 and S10). Expression of five genes encoding cell-wall hydrolases (FaPG, FaEXP, FaXYL, FaPE and FaGAL) was down-regulated in FaBG3-RNAi fruit at 5 DAI compared with the control (Figure 10a). The amount of ethylene released in fruit infected with B. cinerea was slightly higher than that in control fruit (Figure 10b).
FaBG3 is involved in fruit ripening in strawberry
Strawberry, a non-climacteric fruit, is referred to as an enlarged receptacle with achenes. The respiratory burst does not exist in strawberry, and ethylene appears to have a less pivotal impact on ripening, while ABA may play a central regulatory role as a signaling molecule during fruit ripening (Archbold, 1988; Jia et al., 2011). Exogenous ABA or indole-3-acetic acid treatments restore the development of de-achened receptacles, indicating that both ABA and auxin in the achenes regulate the development of strawberry fruit (Kano and Asahira, 1981; Ji et al., 2012) (Figure S2).
β–glucosidase is believed to take part in ABA synthesis via hydrolysis of ABA-glucose ester to release active ABA. We determined that the expression of FaNCED2 was threefold higher than that of FaBG3 during fruit ripening (Figure S1 and Appendix S1), which suggests that de novo synthesis by FaNCED2 may be the key pathway that determines ABA levels, while reactivation of ABA by FaBG3 may also regulate ABA levels in fruit (Figure 2). The result obtained in this study coincided with previous reports in other fruits such as grape (Vitis vinifera) (Sun et al., 2011; Zhang et al., 2013), watermelon (Citrullus lanatus) (Li et al., 2012), melon (Cucumis melo) (Sun et al., 2012a,b; ) and cucumber (Cucumis sativus) (Wang et al., 2013), in which BG gene expression levels peaked when fruit began to ripen. Among the Fa/FvBG genes examined, FaBG3 was highly expressed, and expression of this gene correlated well with changes in ABA levels, which may play an important role in fruit ripening. Therefore, we further verified the function of FaBG3 using pTRV-RNAi (Figure 3). The results showed that FaBG3-RNAi-treated fruits had a lower level of FaBG3 transcription and lower ABA levels than the control, indicating that the ABA content in fruits is partly dependent on the function of FaBG3. In other words, in addition to de novo synthesis of ABA by FaNCED2, the reactivity of ABA with FaBG3 may play a role in adjusting ABA level during fruit ripening (Figure 4). Not only is FaBG3 involved in ABA homeostasis, but this gene also participates in modulation of transcriptional levels of ripening-related genes. Suppressing the expression of FaBG3 down-regulates the expression of a series of genes that are related to ripening and fruit quality, including genes involved in cell-wall catabolism, the anthocyanin synthesis pathway, aroma-related genes and sugar metabolism (Molina-Hidalgo et al., 2013), resulting in transgenic fruit that does not fully ripen (Figures 5 and 6). Therefore, FaBG3 plays an important role in ripening and quality formation in strawberry fruit. Also, the ABA content in achenes decreased rapidly during the germination process. The ABA content is determined by the action of three pathways, including de novo synthesis (FaNCED2), catabolism (FaCYP707A1) and reactivation (FaBG3; Figure S7), which suggests that ABA produced by FaBG3 may also be required for seed germination in strawberry.
Response of FaBG3-RNAi fruit to dehydration stress
It is well-known that plant cells possess an adversity information system include ‘stress recognition’ and ‘defense signaling’. When plants encounter environmental stresses, they launch a variety of reactions through signal transduction, thereby avoiding the impact or harm caused by the environmental stress (Finkelstein et al., 2002; Deluc et al., 2009). In the adversity information system, dehydration-responsive element binding protein and calcineurin B-like protein are two extremely important signaling proteins that play important roles in plant growth. Whether an adversity information system exists in fruit, and the function of such a system in fruit, have not yet been determined.
In this study, we analyzed the expression patterns of genes responsive to dehydration stress in fruit and vegetative tissue. After dehydration stress, significant increases in both content of ABA and transcription of ABA-related genes were detected in the pulp (Figure S3 and Appendix S1). Expression of FaBG genes and CYP707A1 reached peak values 12 h after dehydration treatment, while FaNCED2 expression and ABA content continuously increased, reaching maximum levels at 24 h after dehydration treatment in dehydrated pulp. It is noteworthy that, after dehydration stress treatment, expression of FaBG genes increased more rapidly than FaNCED2 expression, suggesting that production of ABA by BGs is more rapid than production of ABA by FaNCED2 through a lengthy and complex de novo biosynthetic pathway. In addition, the major gene responsible for fruit development and the responses to dehydration may not be the same gene. For example, among the FaBG genes, it appears that FaBG3 plays a role in responses to dehydration stress in fruit (Figure 8e) but not in leaves (Figure S4E and Appendix S1). We found that the expression patterns of the FaBG genes were different in the ripening process and in the response to dehydration, which indicates that BG genes may play different roles under these two conditions. Under dehydration stress conditions, the ABA level increased significantly via BG activity using two distinct mechanisms, i.e. induction of BG transcripts and post-translational activation (Lee et al., 2006). During dehydration stress, expression of the BG genes increased (Figures S3 and S4), leading to rapid increases in ABA levels in response to dehydration (Iuchi et al., 2001; Seo and Koshiba, 2002). Therefore, BGs are important, as they provide fruit with rapidly increasing ABA levels that enable them to respond to dehydration stress, despite the presence of intact de novo biosynthetic pathways.
FaBG3-RNAi fruit was more sensitive to dehydration stress than the control fruit. FaBG3-RNAi fruit exhibited down-regulated expression of FaRD genes, FaCBL and FaDREB1B, together with a variety of ABA-deficient phenotypes, including failure to fully close the stomates of sepals and more rapid water loss in both sepals and fruit compared with the control. Fruits, like whole plants, are subjected to ever-changing environmental conditions, and, accordingly, require constant fine-tuning of the active pool of ABA in response to the severity and duration of the stress. Future studies are required to identify the site of action of the BGs and the transporter of the BG enzyme in strawberry fruit.
FaBG3-RNAi exhibits reduced susceptibility to B. cinerea in strawberry fruit
FaBG3-RNAi fruits are more resistant to B. cinerea than control fruits, indicating that BG-dependent defense is a potential defense mechanism against B. cinerea (Figure 4f–k). The plant cell wall may serve as an effective physical barrier to pathogens, but it is also a matrix where many proteins involved in pathogen perception are delivered. By breaching the cell wall, a pathogen potentially reveals itself to the plant and activates plant responses, setting off events that may halt or limit its advance (Dario Cantu et al., 2008). Ripening fruit becomes increasingly susceptible to opportunistic pathogens. The expression of genes encoding cell-wall hydrolases, such as FaBG, FaEX and FaXYL, was largely down-regulated in FaBG3-RNAi fruits, indicating that cell-wall integrity may be an important reason for the higher levels of resistance to B. cinerea in the FaBG3-RNAi fruits than in the control fruits (Figure 10). In addition, we observed an increase in phenylalanine ammonia lyase activity in FaBG3-RNAi fruits 5 days after infection with B. cinerea, which resulted in an increase in phenolic substances; this response was not observed in control fruits. Perhaps due to the reduced accumulation of anthocyanins, precursors of the flavonoid pathway were shunted to the phenylpropanoid pathway, leading to large increases in the levels of phenolic compounds. Fruit polyphenols have multiple biological activities, including antioxidant and antimicrobial effects (Canter and Ernst, 2004). Phenylalanine ammonia lyase activity and its production are thought to be partially repressed by endogenous ABA present in fruits. Application of exogenous ABA generally increases the susceptibility of plants to fungal pathogens (Ward et al., 1989). A correlation between phenylalanine ammonia lyase activity and resistance to B. cinerea was previously described in bean (Vigna angularis) (De Meyer et al., 1999a) and soybean (Glycine max (L.)Merr.) plants (Ward et al., 1989). Our results show that ABA interacts with the BG-dependent disease response. In addition, ethylene release was higher in FaBG3-RNAi fruits than in the control, and also plays a role in the increased resistance to B. cinerea (Figure 10). This result is similar to those of a previous report (Herde et al., 1999), in which aminocyclopropane carboxylate levels (the direct precursor of ethylene) were twofold higher in ABA-negative tomato plants than in wild-type plants (Sharp et al., 2000). Our results suggest that interaction may occur between ABA-induced defenses and the pathway of reactivation of ABA by BG.
In conclusion, the present work examined the role of Fa/FvBG genes in strawberry fruit. The results indicate that FaBG3 may play key roles in fruit ripening and B. cinerea fungal infection of strawberry fruits via modulation of ABA homeostasis and transcriptional regulation of ripening-related genes.
In silico analysis
The BG cDNA sequences of Arabidopsis, Zea mays and Oryza sativa were obtained from the National Center for Biotechnology Information nucleotide database (http://www.ncbi.nlm.nih.gov/nucleotide/) as described previously (Zouhar et al., 2001; Lee et al., 2006; Opassiri et al., 2006; Xu et al., 2012). Sequence-based homology searches were performed in the National Center for Biotechnology Information database using the consensus sequence of BG gene family members in Arabidopsis, Z. mays and O. sativa to identify homologous expressed sequence tags (ESTs) or cDNAs in strawberry (http://blast.ncbi.nlm.nih.gov/). The open reading frames (ORF) and protein translation sequences of the homologs were determined using DNAMAN (www.lynnon.com).
Deduced amino acid sequences of Fa/FvBGs were aligned with the homologous proteins in Arabidopsis, Z. mays and/or V. vinifera using ClustalX 2.0.12 software (www.clustal.org) with the default setting. The alignment results were edited and marked using BOXSHADE 3.21 software (http://www.ch.embnet.org/software/BOX_form.html). The phylogenetic trees were constructed using the neighbor-joining method in MEGA 4.0.2 software (www.megasoftware.net) with the bootstrap analysis setting at 1000 replicates to evaluate the reliability of phylogenetic groups. The tree files were viewed and edited using MEGA 4.0.2 software.
Construction of the viral vector and agroinoculation
The pTRV1 and pTRV2 virus-induced gene silencing vectors (described by Liu et al., 2002) were kindly provided by Y.–L. Liu (School of Life Science, Tsinghua University, Beijing, China). A 456 bp cDNA fragment of FaBG3 was amplified using primers 5′-CGGGAATTCTTGCTGAAAGGATCGTTTGACTT-3′ (sense) and 5′-CGGGAGCTCGC--CAAACCGAACAGTGTAACCTA-3′ (antisense). The amplified fragment (from spans bp 1024 to 1476) was cloned into EcoRI/SacI-digested pTRV2. Agrobacterium tumefaciens strain GV3101 containing pTRV1, pTRV2 and the pTRV2 derivative pTRV2-FaBG3 was used for RNAi. Thirty fruits from ten independent plants grown in greenhouse were selected for inoculation, and each basal pedicel was injected with the FaBG3-RNAi TRV vector at the white stage. The fruits were evaluated 6 days after treatment.
Strawberry (Fragaria × ananassa) cv. Albion plants were grown under standard greenhouse conditions with a 16 h photoperiod. Fruits were classified into seven developmental stages: small green (SG, approximately 7 days after anthesis), large green (LG, approximately 14 days after anthesis), bright green (BG, approximately 18 days after anthesis), white (W, approximately 21 days after anthesis), turning (T, approximately 23 days after anthesis), pink ripening (PR, approximately 25 days after anthesis) and red (R, approximately 27 days after anthesis). Ten fruits were randomly selected at each stage. Achenes and receptacles were separated using the tip of a scalpel blade. Pulp is synonymous with receptacle. All samples were frozen in liquid nitrogen and stored at −80°C prior to analysis.
Quantitative real-time PCR analysis
Total RNA was isolated from strawberry samples using the hot borate method (Wan and Wilkins, 1994). Genomic DNA was eliminated using an RNase-free DNase I kit (Takara, www.takara.com.cn) according to the manufacturer's instructions. The quality and quantity of every RNA sample were assessed by agarose gel electrophoresis. The cDNA was synthesized from total RNA using the PrimeScript™ RT reagent kit (Takara) according to the manufacturer's instructions. Primers used for real-time PCR, designed using Primer 5 software ((http://www.premierbiosoft.com/), are listed in Table S1. Actin was used as an internal control gene, and the stability of its expression was tested in preliminary studies (Figure S6). All primer pairs were tested by PCR. The presence of a single product of the correct size for each gene was confirmed by agarose gel electrophoresis and double-strand sequencing (Invitrogen, www.invitrogen.com). The amplified fragment of each gene was sub-cloned into the pMD18–T vector (Takara), and used to generate standard curves by serial dilution. The real-time PCR was performed using a Rotor-Gene 3000 system (Corbett Research, http://www.qiagen.com/Corbett/Welcome.aspx?CountryID=CN) with SYBR Premix Ex Taq™ (Takara). Each 20 μl reaction contained 0.8 μl of primer mix (containing 4 μm of each forward and reverse primer), 1.5 μl cDNA template, 10 μl SYBR Premix Ex Taq™ (2 ×) mix and 7.7 μl water. Reactions were performed under the following conditions: 95°C for 30 sec (one cycle), 95°C for 15 sec, 60°C for 20 sec and 72°C for 15 sec (40 cycles). Relative fold expression changes were calculated using the relative two standard curves method with Rotor-Gene 6.1.81 software (Invitrogen).
Infection with B. cinerea
B. cinerea fungus was grown on strawberry fruit at 25°C, 95% relative humidity. After 15 days, spores were washed from the fruit with distilled water containing 0.01% v/v Tween 20. After removing mycelial debris, spores were counted and added to the inoculation solution at the proper concentration. The FaBG3-RNAi strawberry fruits were incubated in inoculation solution containing 105 or 107 spores μl−1 for 5 min. Then the strawberry fruits were incubated on paper at 25°C under 95% relative humidity. The control fruits were treated with sterile water. Incubators were covered with plastic film to guarantee a relative humidity of 95–100%. Five days after inoculation, infection was evaluated by counting the number of spreading lesions on each fruit.
Phenolic compound analysis
Phenolic compounds in fruits were analyzed at 5 days after B. cinerea infection. Both control and FaBG3-RNAi-treated fruits were sampled at 5 days after infection. Powdered samples (2.5 g) were extracted with 40 ml pre-cooled methanol containing 1% 2,6-di-tert-butyl-4–methyl-phenol for 30 min at ambient temperature (ultrasonic extraction). The extract was then centrifuged at 5500 g for 5 min at 4°C, and the supernatant was saved. This step was repeated twice. All supernatants were collected and evaporated to 5 ml at 44°C using a rotary evaporator, water was added to 15 ml, and the pH was adjusted to 7.00 for the neutral phenolic compounds or 2.00 for the acidic phenolic compounds using 1 M NaOH or 1 M HCl, respectively. Then the solution was extracted using 15 ml ethyl acetate by stirring for 5 min. All the ethyl acetate phases were collected and evaporated to dryness at 44°C and dissolved in 5 ml 100% v/v methanol for HPLC analysis. The solution was filtered through a 0.22 μm filter prior to injection into the HPLC system. An Agilent (www.home.agilent.com) 1200 HPLC was used for determination of phenolic compounds. An Agilent Eclipse XDB-C18 column (4.6 × 150 mm, 5 μm) was used. The column temperature was 25°C, and the flow rate was 0.8 ml min−1. The solvents were 0.15% v/v phosphoric acid (solvent A) and 100% v/v methanol (solvent B). Elution was by means of a changeable gradient of solvent B: 0–5 min at 20%, 5–20 min from 20–39%, 20–35 min from 39–20%, 35–95 min from 20–90%, and 95–100 min from 90–20%. The wavelength detected was 280 nm.
Determination of anthocyanin content
Anthocyanin concentration was measured by extracting receptacle surface of equal weight (five replications) with 1% HCl methanol and determining the absorbance at 530 and 657 nm. The formula A = A530–0.25 A657 was used to compensate for the contribution of chlorophyll and its degradation products to the absorption at 530 nm (Rabino and Mancinelli, 1986). The anthocyanin concentration was a relative value, and we set A =0.01 equal to 1 unit.
Determination of ABA content
For ABA extraction, 1.0 g of pulp was ground in a mortar and homogenized in extraction solution (80% v/v methanol). Extracts were centrifuged at 10 000 g for 20 min. The supernatant was eluted through a Sep-Pak C18 cartridge (Waters, www.waters.com) to remove polar compounds, and then stored at −20°C for ELISA. The stepwise procedure for indirect ELISA of ABA was as follows. Each well of a microtiter plate was pre-coated with ABA–BSA conjugate diluted in coating buffer by the manufacturer (ELISA kit for ABA, College of Agronomy and Biotechnology, China Agricultural University). To each well was added 50 ml of standard or sample in assay buffer (8.0 g NaCl, 0.2 g KH2PO4, 2.96 g Na2HPO4·12 H2O, 1.0 ml Tween 20 and 1.0 g gelatin, added to 1000 ml water), followed by 50 ml of ABA antibody (Invitrogen) diluted 1:2000 in assay buffer. The plates were incubated for 0.5 h at 37°C and then washed four times with scrubbing buffer (which contained the same ingredients as assay buffer, but without the gelatin). Anti-mouse IgG coupled to alkaline phosphatase (100 ml of a 1:1000 dilution) was added to each well, and the plates were incubated for 0.5 h at 37°C. The plates were washed as above, and then 100 ml of a 1–2 mg ml−1 solution of o–phenylenediamine substrate and 0.04% by volume of 30% v/v hydrogen peroxide in substrate buffer (5.10 g C6H8O7·H2O, 18.43 g Na2HPO4·12 H2O and 1.0 ml Tween 20, added to 1000 ml water) were added to each well. After 10–15 minutes, 50 ml of 2.0 mol l−1 H2SO4 was added to each well to terminate the reaction. The absorbance was read at 490 nm using a Thermo Electron (Labsystems) Multiskan MK3 (Pioneer, www.pioneerbiomed.com). The concentration of ABA in a sample was calculated from logit B/B0-transformed standard curve data, where B and B0 are the absorbance values in the presence and absence of the competing antigen, respectively.
Determination of ethylene production
Ethylene production from the fruit was measured by enclosing three fruits in 1.0 liter airtight containers for 2 h at 20°C, withdrawing 1 ml of the headspace gas, and injecting it into a gas chromatograph (Agilent model 6890N) fitted with a flame ionization detector and an activated alumina column. Flesh tissues from each fruit were frozen in liquid nitrogen and stored at −80°C until use.
This work was supported by the National Basic Research Program of China (2009CB119000). This work was also partially supported by the Chinese Universities Scientific Fund (2013YJ001). We would like to thank Y.–L. Liu (School of Life Science, Tsinghua University, Beijing, China) for the pTRV vectors. We would like to thank the native English speaking scientists of Elixigen Company for editing our manuscript.