Introduction

Strawberry coloration has been considered as a vital factor in determining the fruit quality and market value. As previously reported, strawberry coloration is attributed to accumulation of anthocyanins that belong to flavonoids, a widely distributed class of phenolic compounds (Crecente-Campo et al. 2012). Anthocyanins are the water-soluble pigments responsible for vivid red, blue, and purple colors of many plants, which are conducive to attract pollinators, disperse seeds, and tolerate various biotic and abiotic stress (Zhang et al. 2014). Moreover, anthocyanins are able to reduce risk of obesity, diabetes, inflammation, cancer, and other chronic diseases in many cell-line studies, animal models and human clinical trials. These health-beneficial effects can be more or less associated with the antioxidant property and free-radical scavenging ability of these compounds (He and Giusti 2010; Pojer et al. 2013).

Anthocyanins consist of an anthocyanidin aglycone bound to one or more glycosyl moieties. At least 23 anthocyanidins and 500 anthocyanins have been characterized. The formation of all these anthocyanin derivatives is primarily based on six common types of anthocyanidins, namely delphinidin (Dp), peonidin (Pn), pelargonidin (Pg), petunidin (Pt), malvidin (Mv), and cyanidin (Cy) (Castañeda-Ovando et al. 2009). Additionally, it has been investigated that Cy as the common aglycone was found in over 82% of examined fruits and berries (Jaakola 2013). However, Pg anthocyanins occupied the most of proportion and Cy derivatives took up a very small percentage in strawberry pigment (da Silva et al. 2007).

The anthocyanin biosynthetic pathway has been almost entirely elucidated, and most of the structural genes encoding the enzymes responsible for each step have been identified including phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA ligase (C4L), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′5′-hydroxylase (F3′5′H), dihydroflavonol 4-reduc-tase (DFR), anthocyanidin synthase (ANS), and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT), which were coordinately modulated by a stable ternary MBW complex consisting of MYB/bHLH/WD40 transcription factors in most species (Petroni and Tonelli 2011). As is well-known that MYB components from the protein complex are primarily responsible for anthocyanin biosynthesis and they could combine or not with bHLH and WD40 to regulate downstream gene expression in this pathway, like MYBP1 in maize (Grotewold et al. 1994), IbMYB1 in sweet potato (Mano et al. 2007), and PavMYB10.1b in sweet cherry (Jin et al. 2016).

In addition to genetic factor, environmental elements, such as light, temperature, and nutrient depletion, are also able to affect anthocyanin production in plant. It has been documented that red organ coloration is more prominent and remarkable in cooler seasons or regions (Lin-Wang et al. 2011; Man et al. 2015; Mori et al. 2005, 2007; Ubi et al. 2006), which suggests that there is a temperature-specific influence and increases concerns about the effect of ongoing climate warming on red organ coloration (Ibáñez et al. 2010; Sugiura and Yokozawa 2004). Thus, low-temperature stimulation is a potential method to increase anthocyanin content. Basically, this is achieved through a process of regulating the expression of related structural genes and MBW regulatory complex. Increasing evidences have pointed out that MYBs specifically modulate anthocyanin biosynthesis, but the function of bHLH and WD40 in this pathway is intricate. During exposure to low temperature, BoPAP1 encoding a MYB transcription factor was significantly induced and might play a critical role in activating the anthocyanin structural genes (C4H, F3H, DFR, ANS and UFGT) for the accumulation of abundant anthocyanins in the purple kale (Zhang et al. 2012). Apple fruit generally requires low temperature to accumulate anthocyanin, which is related to low temperature inducing expression of MdMYBA specifically binding to ANS (Ban et al. 2007). On the contrary, heat condition could cause a dramatic reduction of both anthocyanin concentration and transcript levels of the structural genes by rapidly decreasing expression of MYB10 in apple (Lin-Wang et al. 2011). Previously, different results about the impact of temperature on strawberry coloration were displayed. Anthocyanin content increased as the ratio of maximum-to-minimum temperature became higher in ripe strawberries from ‘Earliglow’ and ‘Kent’ cultivars (Wang and Zheng 2001), and high air temperature can lead to poor fruit coloration in ‘Sachinoka’ (Matsushita and Ikeda 2016). However, Ferreyra et al. (2007) concluded that there was no significant difference between summer and winter concerning anthocyanin content in strawberry cultivar “Selva”. Therefore, the mechanism by which low-temperature stress affects anthocyanin accumulation in strawberry is not yet clear. Here, anthocyanin contents and the expression levels of structural and regulatory genes were assayed to elucidate the cause of fruit coloration under low temperature.

Materials and methods

Plant materials and treatments

The cultivated octoploid strawberry (Fragaria × ananassa Duch. cv. Toyonoka) was used in this study. Seedlings in approximately 2-L plastic pots containing the mixture with perlite, garden soil, and nutrient soil (1:2:2, v/v/v) were grown in Sichuan Agricultural University greenhouse. The low-temperature treatment was conducted once the fruit turned white. The potted strawberries with white fruit were divided into two groups and moved to growth chambers set to either a low temperature (4 °C) or control regimen (25 °C) at 75% relative humidity, 100 µmol m−2 s−1 light intensity for 16 h per day. Fruit samples were harvested at 0, 6, 12, 24, 48, and 72 h after treatment. Subsequently, they were frozen in liquid nitrogen and immediately stored at − 80 °C until needed.

Anthocyanin and proanthocyanidin content determination

Approximately 1.0 g of the fine powder was homogenized in 5 ml 1% (v/v) HCl:MeOH solvent overnight at 4 °C in the dark. After the mixture was centrifuged at 5000×g for 20 min, the upper aqueous phase was collected in a brown volumetric flask. Then, the solid layer was repeatedly extracted with the fresh extractant until no more material dissolved. Finally, the supernatant fractions were pooled and set to the final volume of 10 ml. 1 ml of the resulting supernatant was filtered using the Millipore filter with a 0.45 µm nylon membrane. Quantitative determination of anthocyanin content was performed using Agilent 1260 HPLC system (Agilent, USA) installed with ZORBAX SB-C18 column (150 × 4.6 mm, 5 µm) and variable wavelength detectors (VWD). The mobile phases consisted of A (ultra-pure water), B (acetonitrile), C (formic acid). Separation was achieved by a linear gradient elution at the flow rate of 1 ml min−1 and column temperature of 30 °C. The injection volume was 10 µl, and gradient elution conditions were as follows: 0 min, 100% A; 13 min, 78% A + 20% B + 2% C; 20 min, 58% A + 40% B + 2% C; 25 min, 100% A. The anthocyanins monitored at 520 nm were quantified by constructing the standard curve with pelargonidin-3-glucoside (Sigma-Aldrich, USA).

The content of proanthocyanidin was colorimetrically analyzed by improved DMAC (4-Dimethylaminocinnamaldehyde) method (Prior et al. 2010). 0.5–1.0 g of the fine powder from frozen fruit was soaked in 20 ml extraction buffer containing acetone, deionized water, and acetic acid (150:49:1 v/v/v) and then mixed well by shaking for 1 h. After 20 min centrifugation at 10,000×g at 12 °C, the upper aqueous phase was used for proanthocyanidin determination at 640 nm.

O2 − production rate and SOD activity assay

The strawberry powder (∼ 0.5 g) was extracted using 5 ml 50 mM pre-cooling potassium phosphate buffer (PH 7.8) containing 1% (w/v) of polyvinylpolypyrrolidone (PVP), and then the homogenate was spun at 10,000×g for 10 min at 4 °C. The supernatant liquor was employed to detect O2− (superoxide anion) production rate and SOD (superoxide dismutase) activity. O2− production rate was measured by following Cai et al. (2006) method. SOD (EC 1.15.1.1) activity was determined according to the nitroblue tetrazolium (NBT) method (Ali et al. 2013; Beauchamp and Fridovich 1971), and one unit of SOD enzymatic activity (U) was defined as the amount of enzyme that caused a 50% inhibition of NBT photoreduction rate.

Total RNA isolation and first strand cDNA synthesis

The improved CTAB (cetyltrimethylammonium bromide) method as described by Chen et al. (2012) was adopted to extract total RNA from harvested fruit samples. RNA quality and yield were evaluated by spectrophotometric measurement as well as 1% agarose gel electrophoresis analysis. Then 1 µg of total RNA from each sample was reverse-transcribed to cDNA according to the protocol of PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Japan). All cDNAs were diluted tenfold prior to use in qRT-PCR (quantitative real-time RT-PCR) reactions.

Quantitative real-time RT-PCR

The 96-well plates were used to perform qRT-PCR reactions on the CFX96 real-time PCR system (Bio-Rad, USA). PCR amplification was done in a 10 µl total volume containing 5 µl SYBR Premix (Takara, Japan), 0.4 µl each primer (0.4 µM), 1 µl diluted cDNA and 3.2 µl RNase-free water. PCR procedure was set with three-step cycling conditions: 3 min pre-denaturation at 95 °C, followed by 40 cycles of 10 s denaturation at 95 °C, 30 s annealing at 60 °C and 15 s polymerization at 72 °C. The specificity of primer amplification was assessed by inserting the melting curve ranging from 65 to 95 °C (increment 0.5 °C/5 s) after the final cycle and the potential reagent contamination was checked by no template added in the mixture. Primers listed in Table 1 were designed using Primer Premier 5.0 software.

Table 1 Primers used for qRT-PCR
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Results

Anthocyanin and proanthocyanidin content

Standard curve was obtained using the major anthocyanin in strawberry (pelargonidin-3-glucoside) as reference compound to analyze the samples (Fig. S1). The amount of anthocyanin in control samples at 25 °C showed a stable accumulation and slow increase, while it could be induced quickly by the low temperature (4 °C), and kept a higher level from 12 to 48 h, and subsequently had a sharp decrease showing the value close to the control (Fig. 1a). The change of proanthocyanidin content presented a fluctuant trend in both regimens. Eventually, proanthocyanidin content in low temperature was lower, nevertheless it was very significantly higher than the control samples in the medium-term treatment (Fig. 1b).

Fig. 1
figure 1

Anthocyanin (a) and proanthocyanidin (b) content in strawberry fruits treated at 4 °C and 25 °C. Each value in the histogram represents mean ± standard error from three biological experiments (n = 3). Asterisks indicate significant differences based on one-way analysis of variance in SPSS 23.0 followed by the Dunnett t test (*P < 0.05; **P < 0.01)

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O2 − production rate, SOD enzyme activity

Low temperature stress could cause the accumulation of superoxide anion (O2−). As shown in Fig. 2a, the production rate of O2− was upregulated by low temperature from 0 to 48 h, though there was no significant difference between two regimes except 24 h. In the late treatment (48–72 h), the production rate of O2− was higher in the control samples. In addition, SOD activity of strawberry fruit in 25 °C chamber reached the peak at 24 h and was higher than samples in low temperature (Fig. 2b), which might be the reason why the production rate of O2− was remarkably lower in the control samples at that time.

Fig. 2
figure 2

O2− production rate (a) and SOD activity (b) in strawberry fruits treated at 4 °C and 25 °C. Each value in the histogram represents mean ± standard error from three biological experiments (n = 3). Asterisks indicate significant differences based on one-way analysis of variance in SPSS 23.0 followed by the Dunnett t test (*P < 0.05; **P < 0.01)

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Expression pattern of anthocyanin biosynthetic genes

Expression profiles of genes involving the anthocyanin biosynthetic pathway were investigated in strawberry fruits exposed to two different temperatures through the quantitative real-time RT-PCR technology. Results were illustrated in Fig. 3, which reported that most of genes except CHS, ANS, UFGT and ANR showed a lower transcript level in the low temperature treatment, but almost had no significant difference compared to control samples at 25 °C. The ANS and UFGT expressions were remarkably induced by low temperature, and displayed the similar pattern as anthocyanin concentration. The transcript levels of CHS and ANR in low temperature was higher just during the late course of treatment. In addition, expression pattern of all genes was substantially similar between two regimens during the whole time course of treatment.

Fig. 3
figure 3

Expression patterns of structural genes involving anthocyanin biosynthesis in strawberry fruits treated at 4 °C and 25 °C. Each value in the histogram represents mean ± standard error from three biological experiments (n = 3). Asterisks indicate significant differences based on one-way analysis of variance in SPSS 23.0 followed by the Dunnett t test (*P < 0.05; **P < 0.01)

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Expression pattern of transcriptional factor MYB genes

The MYBs is one of the largest families of transcription factors (TFs) in plant, which have been documented to implicate in the regulation of diverse biological processes. In this work, we only detected the expression profiles of three MYB genes that have been reported they participated in the anthocyanin biosynthesis (Fig. 4). Transcript abundance of MYB10 had the similar trend in two treatments. Namely, it experienced a decrease and then increase in change, and happened once again during the entire time course. Notwithstanding the higher expression level of MYB10 in strawberries exposed to low temperature, there was no significant difference in two groups. We can see MYB1 transcript level had an increase after a slight drop and peaked at 24 h in 4 °C samples; however, the peak value was significantly lower than that in 25 °C. Subsequently, the expression level of MYB1 began to decline in both regimes, while it rose again at 72 h in low temperature. MYB5 transcripts had decreased sharply in late period and the value was close to each other under two different temperatures.

Fig. 4
figure 4

Expression patterns of MYB transcription factors involving anthocyanin biosynthesis in strawberry fruits treated at 4 °C and 25 °C. Each value in the histogram represents mean ± standard error from three biological experiments (n = 3). Asterisks indicate significant differences based on one-way analysis of variance in SPSS 23.0 followed by the Dunnett t test (*P < 0.05; **P < 0.01)

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Discussion

Strawberry coloration which depends on anthocyanin production and accumulation is one of the most important determinates of fruit quality. As is well-known that anthocyanins have antioxidant capability and have been considered as therapeutic agents due to their beneficial health effects including preventing or lowering the risk of cardiovascular disease, inflammation, diabetes, and cancer (Miguel 2011). However, anthocyanins are difficult to be applied as food dyes because they are extremely unstable and easily degraded in the isolated form (Giusti and Wrolstad 2003). In addition, both external and internal factors can affect anthocyanins biosynthesis and accumulation. Environmental factors, especially temperature, show great influence on these processes. It has been reported that high temperature usually caused inferior accumulation of anthocyanin in comparison to low temperature. With an increase of night temperature, the growth rate of grape berries was accelerated; however, accompanied by the poorer coloration (Mori et al. 2005). After grape berries were exposed to high air temperature (35 °C), the anthocyanin concentration decreased to about half that of control berries (Mori et al. 2007). Red-fleshed fruits of Malus crabapple lost color gradually during hot summer. Researchers have found that high temperature highly reduced the biosynthetic and accumulated potential of anthocyanins and triggered reactive oxygen species (ROS) generation. Owing to the coupled influence of high temperature and low oxygen, the transcription of specific POD (peroxidase) genes was upregulated. Increased POD and H2O2 (hydrogen peroxide) activities gave rise to sequentially-coupled oxidation of anthocyanin pigment and consequently cause color loss in fruit (Rehman et al. 2017). Niu et al. (2017) concluded that the anthocyanin accumulation in plum fruit depended on the counterbalance between its synthesis and degradation at the high temperature. In apples such as ‘Akibae’, ‘Tsugaru’, and ‘Tsugaru Hime’ fruits, anthocyanin production was significantly enhanced under the lower-temperature treatments (15 > 20 °C > 25 °C > 30 °C), whereas the increase of anthocyanin content in ‘Akane’ fruit was similar at 15, 20 °C and 25 °C treatments, indicating that different cultivars might be have different sensitivity to temperature (Honda et al. 2014). Compared with high temperature, generally, the lower temperature can potentially increase anthocyanin content by maintaining or even increasing expression levels of related genes, and stabilizing physiological attributes (Man et al. 2015; Rehman et al. 2017; Xie et al. 2012). In our study, low temperature (4 °C) could significantly enhance the anthocyanin content in a short term. Meanwhile, the production rate of O2− increased rapidly. However, the SOD showed lower activity in comparison to control samples exposed 25 °C during this period. In lettuce, anthocyanin content was increased under low-temperature treatment, while POD activity decreased (Boo et al. 2011). Those suggested that anthocyanin possibly played an important role to scavenge O2− in response to low temperature. Therefore, the production rate of O2− began to slow down at 48 h during low-temperature treatment in our study.

Several evidences showed that temperature affected anthocyanin production by modulating the genes involved in anthocyanin biosynthetic pathway. Here, we investigated the expression of PAL, CHS, F3H, DFR, ANS, UFGT, LAR, and ANR as structural genes, and of MYB10, MYB5 and MYB1 as regulatory genes. The results showed most of structural genes except ANS and UFGT had no significant difference between two temperature regimes. The expression level of ANS and UFGT was higher in low temperature (4 °C) and followed a similar tendency as anthocyanin content. It has been extensively reported that the expression of late-stage genes (DFR, ANS and UFGT) in anthocyanin biosynthetic pathway at a high level was critical for anthocyanin accumulation. UFGT was the limiting factor of anthocyanin accumulation and pericarp coloration in a given red litchi cultivar (Wei et al. 2011). Besides, absence of UFGT leaded to white grape cultivars (Kobayashi et al. 2001). Silencing of LDOX in apple and pomegranate caused a shift in the profile of intermediate flavonoids and blocked anthocyanin biosynthesis (Ben-Simhon et al. 2015; Szankowski et al. 2009). Jasmonate-induction of anthocyanin accumulation in Arabidopsis accomplished through significantly up-regulating the ‘late’ genes DFR, LDOX, and UF3GT (Shan et al. 2009). This close relationship between the late-stage genes and anthocyanin content was also demonstrated in other strawberry cultivars (Almeida et al. 2007). After the expression level of DFR was down-regulated by RNAi technology, the strawberry fruit color became pale. Meanwhile, metabolites were diverted to the quercetin-glycoside biosynthesis pathway when the anthocyanin biosynthesis was hindered (Lin et al. 2013).

In the past decades, numerous studies have indicated that expression of structural genes in anthocyanin biosynthetic pathway is controlled by MYB–bHLH–WD40 ternary complex. The MYB proteins are believed to be key components in the activation of specific gene expression pattern (Jaakola 2013). MYB10 was illustrated to positively regulate anthocyanin synthesis in strawberry through overexpression and suppression assay (Kadomura-Ishikawa et al. 2015a), while FaMYB1 was suggested to negatively modulate anthocyanin biosynthesis in the strawberry fruit using the same verification method (Kadomura-Ishikawa et al. 2015b). Our data showed higher expression of MYB10 was exerted in low-temperature treatment, but had no significant difference in comparison to control. In addition, MYB1 expression was diametrically opposite to the anthocyanin content. These results agreed with above reports and also indicated that low temperature might regulate the MYB10 and MYB1 expression to affect anthocyanin accumulation. MYB5 was speculated to fine tune both proanthocyanidin biosynthesis during early fruit development and anthocyanin biosynthesis during strawberry ripening through combining with the negative regulator MYB1 (Aharoni et al. 2001; Schaart et al. 2013). In this work, expression level of MYB5 lacked the remarkable differences between two temperature regimes and therefore could not tell its relationship with anthocyanin accumulation under low temperature.

Taken together, low temperature induced anthocyanin production in strawberry, which contributed to relieve the oxidative damage. The induction of anthocyanin content in strawberry exposed to low temperature might be a result from an increase of MYB10, ANS and UFGT or a decrease of MYB1 at the expression level.

Author contribution statement

HRT and YTZ conceived this project and designed the experiment. YL and WJH collected samples and measured the physiological indexes. YTZ performed the qRT-PCR, analyzed the data and wrote this paper. BS and QC provided technical support and helped to analyze the data. HRT supervised the analysis and critically revised the manuscript.