Fleshy fruits are classically divided into climacteric and nonclimacteric types. It has long been thought that the ripening of climacteric and nonclimacteric fruits is regulated by ethylene and abscisic acid (ABA), respectively. Here, we report that sucrose functions as a signal in the ripening of strawberry (Fragaria × ananassa), a nonclimacteric fruit.
Pharmacological experiments, as well as gain- and loss-of-function studies, were performed to demonstrate the critical role of sucrose in the regulation of fruit ripening.
Fruit growth and development were closely correlated with a change in sucrose content. Exogenous sucrose and its nonmetabolizable analog, turanose, induced ABA accumulation in fruit and accelerated dramatically fruit ripening. A set of sucrose transporters, FaSUT1–7, was identified and characterized, among which FaSUT1 was found to be a major component responsible for sucrose accumulation during fruit development. RNA interference-induced silencing of FaSUT1 led to a decrease in both sucrose and ABA content, and arrested fruit ripening. By contrast, overexpression of FaSUT1 led to an increase in both sucrose and ABA content, and accelerated fruit ripening.
In conclusion, this study demonstrates that sucrose is an important signal in the regulation of strawberry fruit ripening.
Fleshy fruits are important worldwide crops, accounting for a substantial fraction of the world's agricultural output. The regulation of fruit ripening is a key challenge in fleshy fruit production. Fruit ripening involves a series of changes in physiological and biochemical catabolism, which influence a series of fruit quality-related factors, such as flavour, texture, color and aroma. It has long been known that the onset of fleshy fruit ripening can be initiated by a central signal. Fleshy fruits have classically been divided into two categories based on the type of fruit ripening, that is, climacteric and nonclimacteric. Climacteric fruits are characterized by a burst of respiration during ripening and continue to ripen after harvest, whereas nonclimacteric fruits do not (Brady, 1987; Giovannoni, 2001; White, 2002).
Traditionally, it has been thought that the ripening of climacteric fruits is controlled by ethylene (Brady, 1987; Bleecker & Kende, 2000; Giovannoni, 2001; Alexander & Grierson, 2002; White, 2002; Klee & Giovannoni, 2011). In contrast with climacteric fruits, much less is known about the mechanism for the regulation of nonclimacteric fruits. Strawberry is a typical nonclimacteric fruit, which is also one of the most popular fruit crops worldwide. Early studies investigated the role of ethylene in the regulation of strawberry fruit ripening, owing to the pivotal role of ethylene in the regulation of the ripening of climacteric fruits (Hoad et al., 1971). However, subsequent studies suggested that it is auxin, rather than ethylene, that plays an essential role in the regulation of strawberry fruit ripening (Narayanan et al., 1981; Reddy & Poovaiah, 1987; Given et al., 1988; Nam et al., 1999; Aharoni et al., 2002; Mezzetti et al., 2004; Liu et al., 2011). Although the role of auxin in the regulation of strawberry fruit ripening has been of particular interest, in recent years, it has been increasingly suggested that abscisic acid (ABA) may play an important role in the regulation of the development and ripening of nonclimacteric fruits, especially of grape berry (Alexander & Grierson, 2002; Wheeler et al., 2009; Giribaldi et al., 2010; Koyama et al., 2010; Gagné et al., 2011). More recently, Chai et al. (2011) demonstrated that the manipulation of FaPYR1, a gene encoding an ABA receptor, was able to influence the fruit ripening of strawberry. Similarly, Jia et al. (2011) demonstrated that the manipulation of FaNCED1, a gene encoding a key enzyme in the ABA biosynthesis pathway, was also able to manipulate the ripening of strawberry fruit. These studies suggest that ABA plays an important role in the regulation of strawberry fruit ripening.
Sugars have traditionally been regarded as the metabolic resources required for carbon skeleton construction and energy supply in plants. In recent years, however, numerous studies have suggested that sugars may serve as important signals that modulate a wide range of processes in the plant life cycle. Numerous studies have suggested that the effects of sugar on plant growth and development are so universal that sugar signaling may be involved in nearly all fundamental processes, such as embryogenesis, seed germination and early seedling development, vegetative and reproductive growth, senescence and responses to stress stimuli (Koch, 1996; Jang et al., 1997; Lalonde et al., 1999; Sheen et al., 1999; Smeekens, 2000; Loreti et al., 2001; Gibson, 2005; Wind et al., 2010). Nevertheless, most of these studies have focused on the effects of sugars on seed germination and early seedling development. Notably, the isolation and characterization of sugar-insensitive and sugar-hypersensitive mutants in Arabidopsis have revealed intimate connections between sugar and ABA signaling; for example, many sugar mutants turned out to be allelic to known ABA-synthesis and ABA-insensitive mutants, such as ABI3–5, ABF2–4 and ABA1–3 (Smeekens et al., 2000; Finkelstein & Gibson, 2001; Gazzarrini & McCourt, 2001; Rolland et al., 2006). Furthermore, physiological studies have also demonstrated that ABA and sugars often have similar or antagonistic effects on diverse developmental processes (Finkelstein & Gibson, 2001; Rolland et al., 2006). For example, supplementation with exogenous sugars is able to relieve the inhibition of germination by added ABA (Price et al., 2003; Dekkers et al., 2004), whereas glucose is able to inhibit seed germination in Arabidopsis.
It is well known that the sugar content in fleshy fruits is generally quite high. In strawberry fruit, for example, the soluble sugar content may reach nearly 500 mg g−1 DW (Park et al., 2006). Because of this, it has long been thought that sugar is simply a component associated with fruit quality, and it is difficult to imagine that sugar may function as a signal in the regulation of fruit development and ripening. Despite the universal roles of sugar signaling throughout the plant life cycle, it is unknown whether sugar may play a regulatory role in the development and ripening of fleshy fruits. It is well known that sucrose, glucose and fructose are the major components of sugars in plant cells, especially in fruit cells. In the past many years, although sugar signaling in plant cells has been studied extensively, most of these studies have actually laid emphasis on glucose signaling. In contrast with glucose signaling, much less is known about the role of sucrose signaling in plant growth and development. Nevertheless, there is evidence that sucrose may function as a signal that plays a pivotal role in certain processes of plant biology (Chiou & Bush, 1998; Vaughn et al., 2002; Yang et al., 2004; Teng et al., 2005; Martínez-Noël et al., 2009; Dalchau et al., 2011). In this study, we have investigated a possible role of sugar signaling, including that of glucose, fructose and sucrose, in strawberry fruit development and ripening, and have demonstrated that sucrose functions as a signal that plays an important role in the regulation of strawberry fruit ripening.
Materials and Methods
Plant materials and growth conditions
Strawberry plants (Fragaria × ananassa Ducherne, cv Sweet Charlie) were planted in pots (diameter, 230 mm; depth, 230 mm) containing a mixture of nutrient soil, vermiculite and organic fertilizer (7 : 2 : 1, v/v/v). The seedlings were grown in a controlled environment with the following conditions: 25°C : 18°C (day : night), 60% humidity, 12-h photoperiod with a photosynthetic photon flux density (PPFD) of 450 μmol m−2 s−1. Plants were watered daily to the drip point.
To study the effect of different sugars on fruit development, a pharmacological experiment was performed. Fruits of uniform size were selected at stage III and different kinds of sugars, as well as their analogs or control chemicals, were injected with a 1-ml sterile hypodermic syringe at a concentration of 50 mM. For injection, the syringe needle was first pushed into the fruit core through the pedicel, and 200 μl of solution was slowly injected into the fruits. For each treatment, a single injection was performed through the whole process observed. The pharmacological effect on fruit development was recorded by photography and, to more precisely evaluate the pharmacological effect, fruits were sampled and analyzed for a successive change in anthocyanin content after the chemical application.
Determination of ABA, soluble sugar and anthocyanin content
ABA content was determined using the radioimmunoassay (RIA) method, as described by Jia & Davies (2007) with modification. The monoclonal antibody (Mac 252) was obtained from Sigma-Aldrich. Briefly, for RIA analysis, samples were ground to homogeneity at 4°C in a mixture of 80% methanol containing sodium diethyldithiocarbamate trihydrate and quartz sand. The sample mixture was soaked overnight at 4°C and then filtered. The crude extract was adjusted to pH 8.0 with ammonia, and the aqueous phase was concentrated with a rotary film evaporator (RFE) at 35°C in a water bath. The concentrated aqueous phase was frozen and thawed three times, mixed and stirred with polyvinylpolypyrrolidone, and then centrifuged to remove sediment. The supernatant was adjusted to a pH value of 2.5–3.0 and extracted three times with an equal volume of ethyl acetate. The organic phase was combined and evaporated with an RFE at 35°C. The extracts were dissolved in phosphate-buffered saline (pH 6.0). A 50-μl aliquot of ABA extract was mixed with 200 μl of phosphate-buffered saline (pH 6.0), 100 μl of diluted antibody solution and 100 μl of 3H-ABA (8000 cpm) solution. The reaction mixture was incubated at 4°C for 45 min and the bound radioactivity was measured in 50% saturated -precipitated pellets with a liquid scintillation counter. The ABA content was calculated according to a calibration curve. The soluble sugar content determination was performed as described by Jia et al. (2011). Anthocyanin determination was performed as described by Fuleki & Francis (1968).
Experiments with detached fruits
To observe the transient effect of exogenous sucrose on the expression of the genes of interest in strawberry fruit, we adopted the detached-fruit experimental system described by Beruter & Studer (1995). To investigate the association between sucrose-modulated gene expression and ABA signaling, an ABA biosynthesis blocker, nordihydroguaiaretic acid (NDGA), was used at a concentration of 100 μM.
To generate the FaSUT1 overexpression construct, full-length FaSUT1 cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) using the following primers: forward, 5′-GGACTAGTATGCCTACGCCAGAAGCGGACC-3′ (with a SpeI restriction site); reverse, 5′-GGT(A/T)ACCTCATGTGAAAGATCTAGGCTTC-3′ (with a BstEII restriction site). The cDNA was cloned into the SpeI and BstEII restriction sites of the plant expression vector pCAMBIA1304. To generate an intron containing hairpin RNA interference (RNAi) of FaSUT1, a 747-bp fragment near the 5′ end of FaSUT1 cDNA was PCR amplified using the primer pair 5′-CGGGATCCTCTGCTCACTCCCTATGTTC-3′ (with a BamHI restriction site; forward) and 5′-GCTCTAGAAAACCACCCAATCCAGTTT-3′ (with a XbaI restriction site; reverse). The amplified fragment was sequenced and cloned into the XbaI and BamHI restriction sites of the pBI121 vector, and the generated construct was named pBI747. A 335-bp fragment complementary to the 3′ end of the 747-bp fragment described above was PCR amplified from strawberry fruit cDNA using the following primers: 5′-CGGGATCCTTCTTGGCTATGCAACTGGA-3′ (with a BamHI restriction site; forward) and 5′-CGAGCTCAAACCACCCAATCCAGTTT-3′ (with a SacI restriction site; reverse). The 335-bp fragment obtained was sequenced and forward cloned into the BamHI and SacI restriction sites of pBI747; by this means, a hairpin would be produced when this 335-bp fragment is complemented with its complementary sequence at the 3′ end of the 747-bp fragment.
Transfection of strawberry by agroinfiltration
Agrobacterium tumefaciens strain AGL0 (Lazo et al., 1991), containing pCAMBIA-Sut1 or pBI-Sut1i, was grown at 28°C in Luria–Bertani (LB) liquid medium containing 10 mM Mes and 20 μM acetosyringone with appropriate antibiotics. When the culture reached an optical density at 600 nm (OD600) of c. 1.0, Agrobacterium cells were harvested and resuspended in infection buffer (10 mM MgCl2, 10 mM Mes, pH 5.6, 200 μM acetosyringone) and shaken for 2 h at room temperature before being used for infiltration. The Agrobacterium suspension was evenly injected throughout the entire fruit whilst it was still attached to the plant c. 12 d after anthesis, using a sterile 1-ml hypodermic syringe.
Cloning of FaSUTs
Total RNA was extracted from fruit receptacles using an SV Total RNA Isolation System (Promega, Madison, WI, USA) according to the instructions of the system. To identify the sucrose transporters from Fragaria × ananassa, the amino acid sequences of several well-identified and characterized sucrose transporters, such as AY445915 from Malus domestica, NM_001249369 from Glycine max, X69165 from Solanum tuberosum and AF109922 from Pisum sativum, were blasted in the website https://strawberry.plantandfood.co.nz/. Seven sucrose transporters, which were designated as FaSUT1–7, respectively, were identified. The FaSUTs were cloned from cDNA by PCR under the following conditions: 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 2 min, with a final extension of 72°C for an additional 10 min. The primers used are shown in Supporting Information Table S1. The PCR products were ligated into a pMD19-T vector and subsequently transformed into Escherichia coli DH5α. Positive colonies were selected and amplified, and then sequenced by Invitrogen.
Probe preparation and RNA blotting
Digoxigenin (DIG)-labeled probes were synthesized using a PCR DIG Probe Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany) and pMD19-T vector plasmids that contained the cDNA fragments of interest. The (M13+) and (M13−) primers, corresponding to vector sequences adjoining the multiple cloning sites, and the following primers were used: for FaSUT1, forward 5′-TTGCTGTTTCGGTTCTGATC-3′ and reverse 5′-CTTGAACCCAGAGGTAATTC-3′; for FaNCED1, forward 5′-AACCAGTCAAACACTCGCTC-3′ and reverse 5′-AGAATTTGAAGTACTTGAGGTA-3′; for FaBG1, forward 5′-GGTATCGATCAGCAACAAAC-3′ and reverse 5′-ATCCAGTAGAGACCATGCAA-3′. Aliquots of total RNA (15 μg) were separated by electrophoresis on 1% (w/v) agarose gels containing 2.2 M formaldehyde, and then blotted onto nylon membranes (Hybond N+; Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). The filters were hybridized with DIG-labeled DNA probes in high hybridization solution (50% formamide, 2 × sodium chloride/sodium phosphate EDTA (SSPE), 10 mM dithiothreitol (DTT), 1 mg ml−1 herring sperm DNA, 500 μg ml−1 yeast tRNA, 1 mg ml−1 bovine serum albumin (BSA)) overnight in a shaking water bath at 50°C. After hybridization, filters were washed twice in 2 × saline-sodium citrate (SSC) for 15 min at 50°C, twice in 1 × SSC for 15 min at 50°C and twice in 0.1 × SSC for 10 min at 50°C. The membranes were then subjected to immunological detection according to the manufacturer's instructions, using nitroblue tetrazolium/5-bromo-4-chloroindol-3-yl phosphate (NBT/BCIP) stock solution as a chemiluminescent substrate for alkaline phosphatase (Roche Diagnostics).
Functional characterization of FaSUTs
To test whether the cloned FaSUTs indeed exhibit sucrose uptake activity, and also to identify the member of the FaSUT family with the highest activity, the sucrose uptake activity of all FaSUTs was analyzed using the yeast assay system described by Weise et al. (2007). To do this, the open reading frames (ORFs) of FaSUTs were cloned into the yeast expression vector pDR196 (Rentsch et al., 1995). Appropriate restriction sites and a stop codon were introduced into the primers shown in Table S2. The PCR products were cloned into pDR196, yielding pDR196-FaSUT1–7. All of the constructs were confirmed by sequencing. Yeast strain SUSY7/ura3 was transformed with pDR196-FaSUTs, or with the empty vector pDR196 as a control. Solanum tuberosum sucrose transporter 1 (StSUT1) was used as a positive control to test the feasibility of our system.
SYBR real-time PCR
For real-time RT-PCR, the reactions (20 μl) contained 10 μl SYBR Premix ExTaq (Perfect Real Time; Takara Bio Inc., Otsu, Japan), 0.4 μl forward specific primer (10 μM; Sangon, Shanghai, China), 0.4 μl reverse specific primer (10 μM; Sangon) and 2 μl cDNA template. DNA amplification was conducted using Bio-Rad iQ 5 Sequence Detector. The primers used for real-time RT-PCR are shown in Table S3.
Exogenous sucrose modulates strawberry fruit ripening
The whole process of strawberry fruit development, spanning fruit set to ripening, can be divided into three major stages, that is, the green fruit stage, white fruit stage and red fruit stage (Fig. 1). The green and red fruit stages can be divided into several substages, which lack the well-defined boundaries of the major stages. To describe fruit development, we divided the green stage into four substages (Fig. 1a, I–IV). In the first or earliest stage (stage I), the seeds are just fully formed and the receptacle is not yet visible, as a result of the dense distribution of the seeds (see bottom left-hand corner of Fig. 1a). In stage II, the receptacle is just visible, but the fruit is still quite small. In stage III, the fruit begins to expand rapidly and becomes much larger (corresponding to medium-sized fruit). In stage IV, the fruit begins to turn from green to white. Furthermore, we divided the red stage into two substages, that is, the stage at which the fruit has just turned red and the stage at which the fruit is fully red. We named these stages VI and VII, respectively. Thus, as shown in Fig. 1(a), the entire process of strawberry fruit development can be divided into seven stages. Figure 1(b) shows the changes in sugar content during fruit growth and development. The content of all three major sugars, that is, glucose, fructose and sucrose, increased significantly during growth and development. Notably, in the early stages (i.e. stages I–III), the sucrose content was much lower than that of glucose and fructose. Therefore, the increase in sucrose content is much greater than that of glucose and fructose during fruit growth and development.
To further investigate whether sugars regulate strawberry fruit ripening, a pharmacological experiment was performed in which exogenous sucrose, glucose and fructose were introduced by injection into the fruits at the big-green stage (corresponding to the late period of stage III). As shown in Fig. 2(a), the fruits started to turn red only 4 d after sucrose supplementation, and, after 8 d, when the fruits supplied with mannitol were still white, the fruits supplied with sucrose had become fully red. Interestingly, a nonmetabolizable analog of sucrose, turanose, had a similar effect to sucrose on fruit ripening. In addition, glucose was also found to have an obvious effect on fruit ripening. Notably, fructose was not found to have any effect on fruit ripening. To quantitatively evaluate the effect of sugars on fruit ripening, we examined the effect of sugar supplementation on the accumulation of anthocyanin, the pigment that makes strawberries red (Fig. 2b). Consistent with our observations of fruit phenotype during the pharmacological analysis, supplementation of sucrose and its nonmetabolizable analog, turanose, greatly accelerated anthocyanin accumulation. Collectively, these results strongly suggest that sucrose plays an important role in the regulation of strawberry fruit ripening.
The association between sucrose-modulated fruit ripening and ABA signaling
As ABA signaling has been reported to play an important role in strawberry fruit ripening, the modulation of strawberry fruit ripening by sucrose is expected to be closely correlated with ABA signaling. During the seed germination of some plants, it has been proposed that sugar signaling may regulate the expression of the genes involved in ABA metabolism. Thus, to explore the mechanism by which sucrose modulates fruit ripening, we investigated the effect of sucrose and glucose on the expression of the genes encoding the key enzymes in ABA accumulation, that is, 9-cis-epoxycarotenoid dioxygenase (NCED), which controls ABA biosynthesis (Xiong & Zhu, 2003), and β-glucosidase (BG), which releases ABA from its glucose-conjugated state (Lee et al., 2006). A search of the strawberry genome database identified three genes encoding NCED and three encoding BG, which were designated as FaNCED1, FaNCED2 and FaNCED3, and FaBG1, FaBG2 and FaBG3, respectively. A phylogenetic tree of the NCEDs from strawberry and other plants is shown in Fig. S1. A preliminary quantitative RT-PCR analysis showed that exogenous sucrose and glucose modulated the expression of nearly all of the genes involved in ABA accumulation during the green fruit stage, but they did not affect the expression of these genes in the red fruit stage (Fig. S2). Interestingly, although sucrose promoted dramatically the expression of FaNCEDs, it had much less of an effect on the expression of FaBGs. By contrast, although glucose promoted dramatically the expression of FaBGs, it had much less of an effect on the expression of FaNCEDs, suggesting that sucrose- and glucose-modulated gene expression are mediated via two different signaling pathways. In addition, compared with other members of the FaNCED or FaBG family, the effect of sucrose and glucose on the expression of FaNCED1 and FaBG1, respectively, was much more dramatic. As shown in Fig. 3(a), the sucrose-modulated expression of FaNCED1 is rapid, that is, sucrose treatment induced the expression of FaNCED1 within < 0.5 h, suggesting that the sucrose-mediated expression of FaNCED1 is probably a result of direct sucrose signaling rather than a sucrose metabolism-related process. Figure 3(b) shows that the expression of FaNCED1 is quite sensitive to sucrose concentration, that is, sucrose induced significantly the expression of FaNCED1 at a concentration of < 20 mM. A further examination shows that sucrose is able to induce ABA accumulation (Fig. S3). In addition, glucose was found to be capable of inducing ABA accumulation, but, compared with sucrose, the concentration of glucose required to induce ABA accumulation was much higher. These results suggest that physiological concentrations of sucrose play an important role in the regulation of ABA accumulation during strawberry fruit development and ripening.
Given that exogenous sucrose was demonstrated to modulate ABA accumulation and fruit ripening, it was important to determine whether sucrose-modulated fruit ripening is mediated by an ABA-dependent or ABA-independent pathway. To address this question, we examined the effect of an ABA biosynthesis inhibitor, NDGA, on the modulation of sucrose for some ripening-related genes, such as the genes controlling pigmentation (e.g. chalcone synthase, CHS; phenylalanine ammonia-lyase, PAL), flavor formation (e.g. quinone oxidoreductase, QR) and the change in fruit texture (e.g. polygalacturonase, PG; pectate lyase, PT), and sugar metabolism (sucrose synthase, SS; sucrose-phosphate synthase, SPS). As shown in Fig. 4, the blocking of ABA biosynthesis inhibited significantly the sucrose-modulated expression of nearly all of the selected ripening-related genes, except for FaPT1 (a gene involved in pectin metabolism.) and FaNCED1 and FaNCED2 (two genes involved in ABA biosynthesis). This result indicates that sucrose-modulated strawberry fruit ripening could be mediated via both an ABA-dependent and ABA-independent pathway, but that the ABA-dependent pathway probably plays a major role in sucrose-modulated fruit ripening.
Identification and characterization of sucrose transporters in strawberry fruit
Despite the strong evidence that exogenous sucrose modulates fruit ripening, the endogenous sucrose content must be manipulated directly to demonstrate conclusively that sucrose is indeed a signal in the regulation of fruit ripening. As sucrose is known to travel from leaves to fruit via long-distance transport in the phloem system, a sucrose transporter probably plays a pivotal role in the regulation of fruit sucrose content. We searched the database of the strawberry genome and identified seven homologs of the Arabidopsis sucrose transporter AtSUT1; we designated these putative sucrose transporters as FaSUT1–7. The phylogenetic tree of these sucrose transporters and other selected sucrose transporters from plants is shown in Fig. S4. Further experiments showed that the transcripts of all seven putative sucrose transporters were expressed at different stages of fruit development (Fig. 5a). Notably, the expression of FaSUT1 increased dramatically during fruit growth and development (Fig. 5b). To determine whether the putative sucrose transporters indeed have sucrose uptake activity, we performed an activity analysis using a yeast experimental system, and found that all seven members have sucrose uptake activity (Fig. S5). As shown in Fig. 6, the sucrose uptake activity varied greatly between the transporters. For instance, the sucrose uptake activity of FaSUT1 was approximately five-fold higher than that of FaSUT7. The dramatic increase in FaSUT1 expression during strawberry development and its relatively high activity suggest that this protein plays an important role in strawberry fruit growth and development.
Manipulation of FaSUT1 expression alters the progress of fruit ripening
To manipulate the endogenous sucrose content, we generated an intron-containing hairpin RNAi construct that targeted FaSUT1 using a 747-bp fragment of the 3′ part of the FaSUT1 coding region, and placed it under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transient RNAi was performed by injecting the FaSUT1 RNAi construct into the fruit in the middle of the green fruit stage (corresponding to stage III). The empty pBI121 vector was injected as a control. Ten independent RNAi transformants, together with 10 independent controls, were generated. Figure 7(a) presents a representative transformant and its control, showing the effect of FaSUT1 RNAi on the phenotype of the fruit. As shown in this figure, FaSUT1 RNAi led to a significant inhibition of fruit ripening, as reflected by the pale color of the fruit. Although the whole fruit of the empty vector control became fully red, the FaSUT1 RNAi fruits failed to turn red in the region into which the FaSUT1 RNAi construct was introduced. To test whether the inhibition of fruit coloring is indeed correlated with the down-regulation of FaSUT1 expression, we examined the FaSUT1 transcript level in the untransformed receptacle (i.e. the fruit with seeds removed). FaSUT1 RNAi led to a reduction in FaSUT1 mRNA level by > 70% relative to the RNAi control (Fig. 7b,c).
To provide further evidence for the ability of FaSUT1 to regulate fruit ripening, we generated a FaSUT1 overexpression construct by cloning the coding region of FaSUT1 into the restriction site of SpeI and BstEII of the pCAMBIA1304 vector, and placed this region under the control of the CaMV 35S promoter. We transiently expressed FaSUT1 by injecting the FaSUT1 overexpression construct into fruits in the late green fruit stage (corresponding to the late period of stage III or the early period of stage IV), using the empty pCAMBIA1304 vector as a control. As shown in Fig. 8(a), overexpression of FaSUT1 accelerated significantly the progress of fruit ripening. Only 3 d after the introduction of the FaSUT1 overexpression construction into the fruits, they started to become red and, in a further 5 d, they were fully red, whereas the control fruit was just starting to turn red at this point. RNA gel blot in combination with quantitative analysis showed that overexpression of FaSUT1 led to a significant increase in FaSUT1 transcript level (Fig. 8b,c).
Consistent with its influence on the fruit phenotype, the manipulation of FaSUT1 altered strongly the expression of ripening-related genes. As shown in Fig. 9, FaSUT1 RNAi suppressed all of the selected genes involved in fruit ripening, such as FaCHS, FaBG1 and FaQR, whereas it promoted somewhat the expression of FaSS. Notably, FaSUT1 RNAi also suppressed strongly the expression of FaNCED1, a gene encoding the key enzyme in the ABA biosynthesis pathway. By contrast, FaSUT1 overexpression promoted significantly the expression of all of the selected genes involved in fruit ripening, and also of FaNCED1. Collectively, these results demonstrate that FaSUT1 plays an important role in the regulation of strawberry fruit development and ripening.
The effect of manipulation of FaSUT1 expression on the endogenous content of sucrose and ABA
This study aims to demonstrate a role of sucrose in the regulation of strawberry fruit ripening. To elucidate whether FaSUT1-modulated fruit ripening is correlated with a change in sucrose, we analyzed the effect of manipulation of FaSUT1 expression on the endogenous content of sucrose, as well as glucose and fructose. As shown in Fig. 10(a), FaSUT1 RNAi led to a strong decrease in sucrose content relative to the control, but had no significant effect on the fructose and glucose content. By contrast, FaSUT1 overexpression led to a significant increase in sucrose content, but had little effect on the content of glucose and fructose (Fig. 10b). These results suggest that FaSUT1-modulated fruit ripening is the result of its effect on sucrose content.
Modulation of FaSUT1 not only altered the sucrose content, but also affected the ABA content. As shown in Fig. 11, FaSUT1 RNAi caused a significant decrease in ABA content relative to the RNAi control, and, by contrast, FaSUT1 overexpression resulted in a significant increase in ABA content relative to the overexpression control. These results are consistent with those of the pharmacological experiment described above, that is, sucrose was able to modulate dramatically the expression of key enzymes involved in ABA accumulation (Fig. 3). Taken together, these results strongly indicate that sucrose may function as a signal upstream of ABA signaling, and thereby may play a pivotal role in the regulation of strawberry fruit ripening.
It has long been thought that the ripening of fleshy fruits is controlled by a key factor or signal; the ripening of climacteric fruits has been thought to be controlled by ethylene, whereas the ripening of nonclimacteric fruits has been thought to be controlled by ABA (Alexander & Grierson, 2002; Wheeler et al., 2009; Giribaldi et al., 2010; Koyama et al., 2010; Chai et al., 2011; Gagné et al., 2011; Jia et al., 2011). As a plant hormone, the involvement of ABA in fruit development and ripening is not surprising. In contrast with phytohormones, sugar is generally present in a high content and, hence, has been traditionally thought to be a major factor in fruit quality. As such, it is hard to imagine that sugar plays a role in the regulation of fruit ripening. Although it has been increasingly suggested that sugar signaling is involved in nearly all developmental processes of plants, no evidence for a role of sugar signaling in the regulation of fruit development has hitherto been reported. This study demonstrates, for the first time, that a type of sugar, that is sucrose, plays a key role in the regulation of fruit development and ripening.
To demonstrate a role of sucrose in the regulation of strawberry fruit development and ripening, we first performed a pharmacological experiment. As shown in Fig. 2(a), sucrose treatment promoted dramatically fruit ripening. Compared with sucrose, glucose had a lesser effect, although it was also found to have an obvious role in the regulation of fruit development and ripening. If sugar signals only function within cells, it is possible that the different effects of the tested sugars may be a result of the different transport efficiencies from the loading source into cells. In this study, the potential difference among the sugars in their efficiency of long-distance transport was not relevant, as all the sugars were introduced simultaneously into the fruit by injection. If the injected sugars were initially infiltrated into the apoplastic space of fruits, the only concern is a possible difference in the loading rate of the sugars from the apoplastic space into cells. To address this issue, we studied the kinetic process for the loading of sucrose and glucose in fruit tissues (Fig. S6). As shown in Fig. S6(a), the contents of sucrose and glucose increased several-fold immediately after vacuum infiltration, after which the change in sugar content conformed to a typical process of saturation kinetics (Fig. S6b). Given that strawberry fruit development and ripening are relatively long processes (i.e. several weeks), the rapid process of loading (only c. 3 h for saturation), as well as the small difference in loading kinetics between sucrose and glucose, clearly suggests that the different effect observed for the tested sugars was not a result of possible differences in their transport efficiency. It should be noted that the evidence supporting the role of sucrose as a signal not only comes from the observation that exogenous sucrose is capable of promoting fruit ripening (Fig. 2), but also from the observation that its nonmetabolizable structural analog, turanose, is also capable of promoting fruit ripening. In addition, further studies demonstrated that sucrose was capable of modulating the expression of a series of ripening-related genes (Fig. 4) and, likewise, turanose was also capable of modulating the expression of the sucrose-responsive genes (Fig. S7).
Although the pharmacological experiment clearly demonstrated that exogenous sucrose was able to accelerate strongly fruit development and ripening (Fig. 2), to conclusively demonstrate the role of sucrose, we needed to modulate the changes in endogenous sucrose in the fruit. As fruit sucrose is known to be imported from leaves, we identified and characterized sucrose transporters in the fruit (Figs 6, S4, S5). To manipulate the gene encoding sucrose transporter, we adopted the RNAi technique, which has been reported to be a good method for gene manipulation in strawberry fruit (Hoffmann et al., 2006). As shown in Figs 7 and 10, RNAi of FaSUT1 was able to reduce significantly the transcript level of FaSUT1 and the sucrose content. This result suggests that endogenous sucrose indeed plays a role in the regulation of strawberry fruit development and ripening. Sugar signaling has been increasingly suggested to be involved in a wide range of processes in the plant life cycle. Nevertheless, in the past many years, most of the studies on sugar signaling have focused on glucose signaling. There is evidence that sucrose may also function as a signal that plays a pivotal role in certain processes of plant biology (Chiou & Bush, 1998; Vaughn et al., 2002; Yang et al., 2004; Teng et al., 2005; Martínez-Noël et al., 2009; Dalchau et al., 2011). For example, Chiou & Bush (1998) demonstrated that sucrose can act as a signal in assimilate partitioning, and in addition, it has been reported that the expression of certain genes can be specifically modulated by sucrose (Vaughn et al., 2002; Teng et al., 2005). Consistent with these reports, the present study provides strong evidence that sucrose can function as a signal in the regulation of strawberry fruit development and ripening.
As ABA has been suggested to play an important role in the regulation of strawberry fruit ripening (Chai et al., 2011; Jia et al., 2011), it is of particular significance and importance to determine the relationship between ABA and sucrose signaling. As shown in Fig. 3, sucrose was able to regulate the expression of FaNCEDs that encode a key enzyme in the ABA biosynthesis pathway. Importantly, the sucrose-regulated gene expression proved to be a sensitive and rapid process, that is, sucrose could promote significantly the expression of FaNCEDs at a concentration as low as 10 mM, and 50 mM sucrose could promote significantly the expression of FaNCEDs within 0.5 h of application. More importantly, it was found that sucrose and glucose may trigger totally different responses in terms of gene expression; for example, although sucrose promoted dramatically the expression of FaNCED1, it had a lesser effect on the expression of FaBG1. By contrast, although glucose promoted dramatically the expression of FaBG1, it had much less of an effect on the expression of FaNCED1. These findings suggest that sucrose may function as a direct and specific signal involved in the regulation of strawberry fruit ripening.
Sugar is known to be a major component in fruit; the soluble sugar content of strawberry fruit may reach up to c. 500 mg g−1 DW (Kallio et al., 2000; Park et al., 2006). In general, the content of a biologically active molecule, such as a phytohormone, is quite low in cells or tissues. It is not known how sucrose or glucose can function as a signal at such a high content. A possible explanation is that the sucrose or glucose molecule is compartmentalized with its sensors/receptors in the fruit tissue, cells or subcellular spaces. Thus, whether they play a regulatory role as a signal depends on their change in content at action sites (i.e. at sites at which sensors/receptors exist), rather than their total content in whole fruits. A similar phenomenon has been well established for phytohormone signaling. For example, under normal conditions, plant leaves are known to contain high levels of ABA; however, this ABA is not able to make stomata close, as it is compartmentalized in chloroplasts. Under certain conditions (e.g. during specific stages of drought stress), root-derived ABA may be able to regulate stomatal movement, although the amount of root-derived ABA is far less than that normally present in the leaf (Hartung & Radin, 1989; Hartung et al., 2001; Jia & Davies, 2007). As with the long-distance transport of ABA, primary sucrose in fruits is known to come from leaves via long-distance transport through the vascular system (Forney & Breen, 1986; Archbold, 1988; John & Yamaki, 1994); therefore, whether the localization of sucrose transporters is related to sucrose compartmentalization deserves further investigation.
It has been reported that glucose may regulate cell division, whereas sucrose regulates cell expansion and reserve deposition during legume embryogenesis (Borisjuk et al., 2002, 2003). Moreover, the effect of sugar on many developmental processes is concentration dependent (Gibson, 2005). For example, it was found that low concentrations of sucrose (below 50 mM) may stimulate the formation of adventitious roots on the hypocotyls of Arabidopsis, whereas higher concentrations may suppress, rather than stimulate, the formation of adventitious roots (Takahashi et al., 2003). Interestingly, in the present study, we found that changes in sucrose content were closely correlated with phase transitions in the developmental process spanning fruit set to full ripening, that is concentrations of sucrose remained low and relatively stable in the early stages of the green phase and increased sharply when fruits began to enter the white phase (Fig. 1). If we suppose that glucose and sucrose have the same regulatory roles in strawberry fruit as in legume embryogenesis, the close correlation between the change in sucrose content and the transition phase of fruit development also implies that it may be the synergistic action of sucrose and glucose that controls strawberry fruit development and ripening.
Studies have suggested that glucose and ABA often have similar or antagonistic effects on diverse developmental processes in plants (Price et al., 2003; Dekkers et al., 2004). The isolation of glucose-insensitive or -hypersensitive mutants in Arabidopsis revealed that many of the glucose mutants are allelic to known ABA-synthesis and ABA-insensitive mutants. These studies suggest that glucose signaling may be tightly coupled with ABA signaling. Although the close correlation between glucose and ABA signaling has been studied extensively, most of the studies have focused on the effects of glucose and ABA on seed germination and early seedling development. Interestingly, in the present study, we demonstrated that sucrose signaling is also tightly coupled to ABA signaling in strawberry fruit development: for example, exogenous sucrose was found to induce dramatically the expression of FaNCEDs, genes encoding the key enzyme in the ABA biosynthesis pathway. An understanding of the association between sucrose and ABA signaling will greatly enhance our knowledge of the mechanisms that govern fruit development and ripening. To determine whether sucrose signaling is ABA dependent or independent, we examined the expression of the sucrose-responsive genes in association with the blocking of ABA biosynthesis by NDGA. As indicated in Fig. 4, blocking of ABA biosynthesis with NDGA inhibited sucrose-induced expression of some genes, such as FaPG1, FaCHS and FaPAL, whereas it had little effect on FaSPS and FaSS. It appears that the expression of most genes that control directly ripening-related events (i.e. changes in fruit texture, color and flavor) is ABA dependent, and that the expression of others (such as the genes associated with sugar metabolism) is ABA independent. These observations indicate that sucrose signaling-regulated fruit ripening can occur via both ABA-dependent and ABA-independent pathways, whereas the ABA-dependent pathway appears to play a major role in the regulation of fruit ripening.
In the present study, we have demonstrated that, in addition to ABA, sucrose may function as a key signal in the regulation of strawberry fruit ripening. This means that the synergistic action of multiple signals, rather than the activity of a sole signal, is required for the regulation of fruit development and ripening. The requirement for multiple, synergistic signals is probably correlated with the complexity of the mechanism underlying fruit development and ripening. Fruit ripening is the result of an orderly alteration of a series of physiological and biochemical events, such as sugar and acid metabolism, cell wall metabolism, and pigment and flavor metabolism, and each of these metabolic systems or processes is involved in the regulation of a number of genes. It is not known whether each metabolic system is independently regulated by a unique signaling pathway or by a common signaling network. It is unlikely that numerous metabolic systems across a number of developmental stages would be controlled by a sole signal. Thus, it is not surprising that the development and ripening of strawberry fruit may be controlled by multiple signals. Signals other than ABA and sucrose may also be involved in this process. Given that some sucrose transporters play an important role in sucrose accumulation, it is of particular interest to identify the upstream signals that control the sucrose transporters.
In summary (Fig. 12), this study demonstrates that: (1) the sucrose content increases dramatically at a key point in strawberry fruit growth and development (i.e. at the transition from green to white fruit); (2) exogenous sucrose, as well as its nonmetabolizable structural analog, turanose (3-O-α-d-glucopyranosyl-d-fructose), greatly accelerates fruit ripening, whereas glucose and fructose have a much smaller or negligent effect on fruit ripening, suggesting that sucrose-accelerated fruit ripening is not a result of sucrose metabolism; (3) manipulation of the expression of a sucrose transporter alters the endogenous content of sucrose and fruit ripening, that is, overexpression of FaSUT1 leads to an increase in sucrose content and promotes fruit ripening, whereas RNAi-mediated inhibition of FaSUT1 leads to a decrease in sucrose content and delayed fruit ripening, suggesting that endogenous sucrose plays a pivotal role in the regulation of fruit ripening; (4) sucrose regulates the expression of a series of ripening-related genes, as well as of genes involved in sucrose and ABA metabolism, and the process of sucrose-regulated gene expression is sensitive, specific and quick, which is consistent with a signaling behavior; and (5) sucrose induces dramatically the expression of FaNCEDs, genes that encode key enzymes in the ABA biosynthesis pathway, and, further, the manipulation of the endogenous sucrose content alters the endogenous ABA content. Furthermore, the upregulation of most ripening-related genes by sucrose can be arrested significantly by blocking ABA biosynthesis. Collectively, these results strongly suggest that sucrose functions as a signal that acts upstream of the ABA signaling pathway, and thus plays an important role in the regulation of strawberry fruit ripening.