Effect of Riboflavin on the Precocious Maturation of Spine Grape Berries (Vitis davidii Foex)

in HortScience

Spine grape (Vitis davidii Foex) is an important wild plant species in South China. To provide economical and environmentally safe ways to promote the precocious maturation of spine grape berries, the effects of riboflavin were investigated. Riboflavin affected the reactive oxygen species metabolism in spine grape berries by increasing superoxide radical production and the hydrogen peroxide content, and it impaired the activities of the antioxidant enzymes superoxide dismutase and catalase. Riboflavin also induced the upregulated expression of maturation-related genes in advance, and the earlier accumulation of anthocyanin and total soluble solids. Phenological observations revealed that the treated grape berries underwent a color-turning stage 9 days earlier than the control, and the maturation stage occurred 7 days earlier than the control. Thus, riboflavin may significantly promote the precocious maturation of spine grape berries.

Abstract

Spine grape (Vitis davidii Foex) is an important wild plant species in South China. To provide economical and environmentally safe ways to promote the precocious maturation of spine grape berries, the effects of riboflavin were investigated. Riboflavin affected the reactive oxygen species metabolism in spine grape berries by increasing superoxide radical production and the hydrogen peroxide content, and it impaired the activities of the antioxidant enzymes superoxide dismutase and catalase. Riboflavin also induced the upregulated expression of maturation-related genes in advance, and the earlier accumulation of anthocyanin and total soluble solids. Phenological observations revealed that the treated grape berries underwent a color-turning stage 9 days earlier than the control, and the maturation stage occurred 7 days earlier than the control. Thus, riboflavin may significantly promote the precocious maturation of spine grape berries.

Spine grape is a wild grape species in South China that is also called Chinese Bramble grape and Davids Rebe. It belongs to the East Asian Vitis spp. (Meng et al., 2012a) and is mainly distributed in Hunan and Jiangxi Provinces (China). It is rich in anthocyanins, used to produce table grapes, wine, and juice, and loved by local people (Liang et al., 2013; Meng et al., 2012b). However, the maturation stage of spine grape occurs late, which affects its popularization and applications. Fruit maturation is regulated by the various internal and external factors, such as genetic factors, developmental signals, hormones, light, and temperature (Klee and Giovannoni, 2011; Qin et al., 2012; Tian et al., 2013). It is important to find an environmentally friendly and cost-effective way to promote its maturation stage. The use of growth-regulating chemicals and appropriate horticultural manipulations are traditional methods of achieving this aim. Ethylene plays a crucial role in regulating climacteric fruit maturation (Alba et al., 2005), but spine grape is a nonclimacteric fruit and is not sensitive to exogenous ethylene during the ripening process (Causier et al., 2002). However, the production of new regulatory chemicals has presented more possibilities for controlling grape maturation.

Reactive oxygen species (ROS), including superoxide radicals (O2) hydrogen peroxide (H2O2), hydroxyl radicals, and singlet oxygen, play crucial roles as key regulators of growth, development, and defense pathways in plants (Bhattacharjee, 2005; Chen et al., 2012; Mittler et al., 2004). ROS participate in a number of reduction–oxidation (redox) processes, and create a localized oxidative environment that facilitates signaling (Asada, 2006). However, a high level of ROS accumulation can cause cell membrane disintegration and protein oxidation, which can further initiate or promote the aging process (Chan, 2006). High ROS contents can effectively accelerate fruit senescence (Tian et al., 2004, 2013; Wang et al., 2005). The important role of ROS in fruit senescence and fungal pathogenic ability has been recognized in peach fruit (Chan et al., 2007). ROS, particularly H2O2 and O2, play important roles in promoting precocious maturation in muskmelon, tomato, and peach fruit (Jimenez et al., 2002; Lacan and Baccou, 1998; Qin et al., 2009).

Riboflavin, also known as vitamin B2, is an essential cofactor for many metabolic enzymes in multiple cellular processes, such as the citric acid cycle and cellular redox, and plays important roles in regulating plant growth and development (Deng et al., 2014; Taheri and Tarighi, 2010). Riboflavin is also a well-known photo sensitizer and can generate ROS during light exposure (Deng et al., 2014). Exogenous applications of riboflavin promote plant growth and induce resistance to fungal, bacterial, and viral pathogens (Li et al., 2012). Exogenous riboflavin can significantly affect the antioxidant metabolite content and redox homeostasis. The activities of enzymes, such as superoxide dismutase (SOD) and catalase (CAT), and the generation of different types of ROS, such as O2 and H2O2, changed as the level of the riboflavin treatment increased (Deng et al., 2011, 2014). In addition to promoting ROS production, riboflavin treatment also can induce the expression of mature related genes (Liu et al., 2010). Taheri and Tarighi (2011) demonstrated that riboflavin primed the expression of the lipoxygenase (LOX) gene associated with disease resistance and maturation in plants. In riboflavin-treated plants, the upregulation of the phenylalanine ammonia lyase (PAL) gene, a key gene associated with plant disease resistance and anthocyanin synthesis, was demonstrated in grapevine (Boubakri et al., 2013).

The functions of riboflavin as a resistance elicitor or a mediator of resistance signal transduction in plants have been investigated (Boubakri et al., 2013). However, the effects of exogenous riboflavin applications on the precocious maturation of fruit has not previously been reported. Thus, in this study, we examined whether riboflavin could promote the precocious maturation of spine grape.

Materials and Methods

Plant growth and treatment.

Six-year-old spine grape vines were used in this study. Twelve vines were grown at the experimental base of Hunan Agricultural University (Changsha, Hunan Province, China). All spine grape vines were subjected to identical pruning and cultivation practices. At 50 d after full bloom (50 DAB), the berries were treated with 0.5 mmol·L−1 riboflavin containing 0.03% (v/v) Tween-80 at noon on a sunny day. Control berries were treated with distilled water containing 0.03% (v/v) Tween-80. Three clusters (20 berries per cluster) per treatment were randomly collected at 0, 10, 20, 30, 40, 50, and 60 d (50, 60, 70, 80, 90, 100, and 110 DAB, respectively) after treatment. The flesh and peel were separated, and the peel was flash-frozen in liquid nitrogen and stored at −80 °C until further processing.

H2O2 and O2 measurements.

The accumulation of H2O2 and O2 in spine grape peels was determined in accordance with the methods of Deng et al. (2014). The H2O2 content was measured at 560 nm and expressed as nmol·g−1 fresh weight (FW), and the O2 content was measured at 530 nm and expressed as nmol·min−1·g−1 FW.

Antioxidant enzyme activity measurements.

Total protein was extracted in accordance with the methods of Zaka et al. (2002) with modifications. Briefly, fresh grape peel samples were powdered in liquid nitrogen with a precooled mortar and pestle. The needle powder (0.5 g) was extracted in 5 mL ice-cold extraction buffer (0.1-M Tris–HCl pH 7.5, 0.23-M sucrose, 5% polyvinylpyrrolidone, 1-mm ethylene diamine tetraacetic acid (EDTA), 10-mm KCl, 10-mm MgCl2, and 2.5-mm ascorbic acid). The extract was vortexed and then placed on ice for 20 min. Homogenized samples were then centrifuged at 14,000 gn for 15 min at 4 °C, and the resulting supernatants were used for enzyme assays. SOD and CAT activity levels were determined in accordance with the methods of Deng et al. (2014).

Determination of anthocyanin and total soluble solids (TSS) content.

The extraction of anthocyanin from grape peel was performed in accordance with the methods of Liang et al. (2011) with modifications. Briefly, fresh grape peel samples were powdered in liquid nitrogen. Then, 0.5 g powdered samples were placed in 1.5-mL ice-cold extraction buffer (2:28:70, formic acid:water:methanol). The extract was vortexed for 10 min. Then, the extracts were centrifuged at 14,000 gn for 10 min at 4 °C, and the resulting supernatants were used for total anthocyanin assays. Total anthocyanins were determined using a pH differential method described by Cheng and Breen (1991). Results were expressed as mg·g−1 FW. The TSS contents of the flesh was determined using a digital refractometer (PR-1; Atago, Tokyo, Japan) and expressed as a percentage (%).

Expression analysis.

Total RNA was extracted from spine grape peel using a TIANGEN RNA prep Pure Plant Kit (Tiangen Biotech, Beijing, China). First-strand complementary DNA (cDNA) was synthesized from 1 mg of total RNA using a PrimeScript RT reagent kit (TaKaRa Biotechnology Co. Ltd., Dalian, China). The quantitative real-time polymerase chain reaction (PCR) amplifications were carried out in triplicate in 96-well plates, having 20 mL total volumes per well, using SYBR Green PCR Master Mix (TaKaRa Bio-technology Co. Ltd.) in an Applied Biosystems 7500 real-time PCR system (Applied Biosystems, Foster City, CA). The primer sequences specific for the amplification of the cDNA fragments of PAL and LOX are listed by Trouvelot et al. (2008). The expression levels were calculated as 2−∆∆Ct and normalized to the Ct value of VvActin (Sun et al., 2010).

Results

Effects of riboflavin applications on the maturation stage of spine grape berries.

In this experiment, we took photos of spine grape clusters at 60, 80, and 100 DAB (Fig. 1). At 60 DAB, the treated fruit had already entered the coloring period, whereas the control had not. At 80 DAB, in the riboflavin-treated clusters, the berries were mostly red, and a few were even purple, whereas in the control cluster, the berries were mostly light red and a small number of berries was still green. At 100 DAB, the treated cluster was completely purple, whereas the control was still red. As shown in Table 1, the coloring date of riboflavin-treated grape berries occurred 9 d earlier than the control, and the fruit maturation date was ≈7 d earlier than the control.

Fig. 1.
Fig. 1.

Effects of riboflavin applications on spine grape berry maturation. Grape berries development stages at 60, 80, and 100 days after full bloom.

Citation: HortScience horts 54, 9; 10.21273/HORTSCI14192-19

Table 1.

Effects of riboflavin on the maturation stage of spine grape berries.

Table 1.

Effects of riboflavin treatments on total anthocyanin and TSS contents.

To determine whether the riboflavin treatment promoted the precocious maturation of spine grapes, total anthocyanin and TSS contents, as the main criteria for evaluating the maturity of grape fruit, were measured (Fig. 2). As is shown in Fig. 2A, the total anthocyanin contents in peels of grapes treated with riboflavin were greater than that of the control group from 60 DAB to 100 DAB, but there were no significant differences between treatments at 110 DAB. Similarly, the riboflavin treatment of fruit promoted an early increase in the TSS contents in grape fruit, from 60 DAB to 100 DAB, and the TSS contents of grape fruit were significantly greater than in the control.

Fig. 2.
Fig. 2.

Effects of riboflavin treatments on total anthocyanin (A) and total soluble solids (B) contents. Each point represents the means ± SEs of three replicates. *Indicates significant differences at P < 0.05 (least significant difference test). FW = fresh weight.

Citation: HortScience horts 54, 9; 10.21273/HORTSCI14192-19

Effects of riboflavin treatments on ROS metabolism.

Based on previous knowledge regarding the role of riboflavin in peroxidation, experiments were conducted to investigate whether riboflavin is capable of generating ROS for the induction of precocious maturation in spine grapes. O2 and H2O2, which are important ROS molecules, can be generated in plants treated with riboflavin in the light. O2 production in riboflavin-treated fruit was significantly greater than in control berries, and it rapidly increased at 70 DAB and then peaked at 80 DAB. In control berries, O2 production was stable at a low level until 80 DAB, and then it increased, reaching a maximum level in samples collected at 90 DAB (Fig. 3A). As shown in Fig. 3B, after the riboflavin treatment, H2O2 gradually increased, began to rapidly increase at 60 DAB, peaked at 80 DAB, and then rapidly decreased. However, the H2O2 content of the control fruit reached its peak at 90 DAB, ≈10 d later than in the treated fruit.

Fig. 3.
Fig. 3.

Effects of riboflavin treatments on O2 (A) production and the H2O2 (B) content. Each point represents the means ± SEs of three replicates. *Indicates significant differences at P < 0.05 (least significant difference test). FW = fresh weight.

Citation: HortScience horts 54, 9; 10.21273/HORTSCI14192-19

Effects of riboflavin treatments on antioxidant enzyme activity levels.

The activities of antioxidant enzymes, including SOD and CAT, were measured in spine grape berries (Fig. 4). In riboflavin-treated berries, the SOD activity remained relatively low until 70 DAB, and peaked at 80 DAB, which was earlier than the control but the peak was lower. The SOD activity was significantly lower after the riboflavin treatment from 60 DAB to 110 DAB compared with the control berries (Fig. 4A). Similarly, riboflavin significantly restrained the CAT activity in grape berries (Fig. 4B).

Fig. 4.
Fig. 4.

Effects of riboflavin on superoxide dismutase (SOD) (A) and catalase (CAT) (B) activity levels in spine grape berry skins. Each point represents the means ± SEs of three replicates. *Indicates significant differences at P < 0.05 (least significant difference test). Prot = protein.

Citation: HortScience horts 54, 9; 10.21273/HORTSCI14192-19

Effects of riboflavin on PAL and LOX gene expression levels.

To understand the mechanisms involved in the riboflavin-induced precocious maturation of spine grapes, the expression of PAL, as the starting gene of the anthocyanin biosynthetic pathway, and that of LOX, as the main gene associated with fruit maturation and softening, were analyzed (Fig. 5). After the riboflavin treatment, the expression patterns of PAL (Fig. 5A) and LOX (Fig. 5B) genes were similar, and there were no significant differences between the first stage before treatment and the last stage after treatment. However, from 60 DAB to 90 DAB, both PAL and LOX genes showed upregulated expression levels in both treated and control berries, but the expression levels of PAL and LOX genes in treated berries were significantly greater than in the control. At 100 DAB, PAL and LOX genes were downregulated owing to the maturation of the treated berries. However, the expression level of PAL and LOX in control peaks at 100 DAB, but the peak is later and lower than the riboflavin-treated berries.

Fig. 5.
Fig. 5.

Effects of riboflavin on phenylalanine ammonia lyase (PAL) (A) and lipoxygenase (LOX) (B) gene expression levels. Each point represents the means ± SEs of three replicates. *Indicates significant differences at P < 0.05 (least significant difference test).

Citation: HortScience horts 54, 9; 10.21273/HORTSCI14192-19

Discussion

The use of precocious maturation-related cultivation technology is of great significance because it can alter the supply period of grape fruit, which will benefit fruit farmers. To promote the precocious maturation of grape fruit, various methods have been tested, such as hormones, light, temperature, and ROS (Klee and Giovannoni, 2011; Qin et al., 2012; Tian et al., 2013). Riboflavin is a photosensitizer, which can lead to the generation of ROS, such as O2 and H2O2 (Deng et al., 2014). In previous research, we compared the effects of different concentrations of riboflavin on the promotion of fruit maturation and found that 0.5 mmol·L−1 of riboflavin solution had the best effect. In this study, riboflavin was applied to spine grape berries at 50 DAB to promote the precocious maturation of the berries. The riboflavin-treated grape berries entered the color-turning stage 9 d earlier than the control, and the maturation stage occurred ≈7 d earlier than in the control (Table 1, Fig. 1). Our data suggest that riboflavin-mediated ROS production affects the maturation period of spine grape berries.

Total anthocyanin and TSS accumulations are well-known signs of grape berry maturation (Koshita et al., 2011). Here, earlier and quicker rates of increase in total anthocyanin (Fig. 2A) and TSS (Fig. 2B) were observed in riboflavin-treated berries compared with the control, indicating the maturation-related priming effect of riboflavin after treatment. This finding is in agreement with early reports that the accumulation of ROS accelerated fruit senescence (Jimenez et al., 2002; Qin et al., 2009; Tian et al., 2013). In addition, when the treated and control berries were all mature at 110 DAB, there were no significant differences in anthocyanin and TSS contents, which indicated that riboflavin has little effect on anthocyanin and soluble solid contents of grape berry.

Fruit maturation is an oxidative phenomenon accompanied by a pronounced increase in ROS, particularly O2 and H2O2, accumulation (Tian et al., 2013; Warm and Laties, 1982). Riboflavin is involved in peroxidation, which affects the production of ROS. Taheri and Tarighi (2011) reported that the oxidative burst was induced in sugar beet after they were treated with riboflavin. Li et al. (2012) also found a similar result in experiments conducted on pear fruit. H2O2 and O2, as important ROS, which are induced by riboflavin treatment obviously, so only these two ROS were detected in our study. Here, riboflavin treatments resulted in earlier peak values of O2 (Fig. 2A) and H2O2 (Fig. 2B) in berries and increased their contents compared with the control. These results corroborated that O2 and H2O2 are important forms of ROS and essential to the induction of fruit maturation (Tian et al., 2013).

To further investigate the role of riboflavin in promoting grape berry maturation, the activities of SOD, a key enzyme that dismutates O2 into H2O2, and CAT, a key enzyme that degrades H2O2 into water and oxygen (Boubakri et al., 2013), were measured in riboflavin-treated grape berry peels. Riboflavin impaired SOD (Fig. 3A) and CAT activity levels (Fig. 3B). Similarly, Deng et al. (2014) found that riboflavin impaired antioxidant enzyme activities, which may be related to high content of ROS produced by the degradation of riboflavin.

PAL is the starting gene of anthocyanin synthetic pathway, and its expression level is closely related to anthocyanin accumulation. Enhancing the expression of PAL can promote the synthesis of anthocyanins (Qzeki et al., 2010; Wang et al., 2005). Taheri and Tarighi (2010) reported that riboflavin strongly elicited the expression of PAL and LOX genes in rice, and they found a similar pattern in sugar beet (2011). Here, we found that riboflavin could promote the accumulation of anthocyanin in the treated berries earlier than in the control group (Fig. 1A). Moreover, during the 60–90 DAB period, the PAL gene expression (Fig. 5A) and the total anthocyanin content (Fig. 2A) in the treated fruit were greater than those in the control group. PAL gene expression increases at the early ripening stages in fruit, but diminishes at the end of ripening (Pombo et al., 2011). At 100 DAB, the treated fruit were fully mature, total anthocyanin accumulation was completed, and PAL gene expression was downregulated. However, the control group was not fully mature yet; therefore, the PAL gene was upregulated (Fig. 5A) and the total anthocyanin accumulation was still occurring (Fig. 2A). Thus, the expression level of the PAL gene in fruit treated at this point was lower than that of the control group, but the total anthocyanin content was greater than that of the control group.

LOX plays an important role in plant growth, development, and maturation, as well as resistance to mechanical damage, disease, and insect infection (Heitz et al., 1997; Marcelle, 1991). In higher plants, LOX is associated with the synthesis of ethylene, jasmonic acid, and abscisic acid, which contribute to fruit ripening (Parry, 1991). We found similar results (Fig. 5B). Before ripening, the expression of the LOX gene in riboflavin-treated fruit was greater than that of the control. In line with the PAL gene expression pattern, the expression level of the LOX gene decreased in the later stages of fruit ripening (Lin et al., 2018). Consequently, we found that the expression of the LOX gene in treated fruit was lower than that in the control at 100 DAB.

Conclusion

In conclusion, our study showed the ability of riboflavin to promote the precocious maturation of spine grape. Riboflavin induces maturation responses, including O2 and H2O2 accumulation, decreased antioxidant enzyme activity, the upregulated expression of maturation-related genes in advance, and the earlier accumulation of anthocyanin and TSS. We also showed the effects of riboflavin treatments on the coloring and maturation stages of grapes. Our results suggest that riboflavin could be used to promote the maturation stage of spine grape under field conditions. The safety of riboflavin applications and their efficient promotion of maturation could be useful in developing new, simple, and environmentally safe vineyard management strategies.

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Contributor Notes

We thank Lesley Benyon, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.This work was supported by the National Technology System for Grape Industry (CARS-29-ZP-9) and National key research and development program (2018YFD0201300).

These authors contributed equally.

Corresponding author. E-mail: guoshunyang@aliyun.com

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    Effects of riboflavin applications on spine grape berry maturation. Grape berries development stages at 60, 80, and 100 days after full bloom.

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    Effects of riboflavin treatments on total anthocyanin (A) and total soluble solids (B) contents. Each point represents the means ± SEs of three replicates. *Indicates significant differences at P < 0.05 (least significant difference test). FW = fresh weight.

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    Effects of riboflavin treatments on O2 (A) production and the H2O2 (B) content. Each point represents the means ± SEs of three replicates. *Indicates significant differences at P < 0.05 (least significant difference test). FW = fresh weight.

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    Effects of riboflavin on superoxide dismutase (SOD) (A) and catalase (CAT) (B) activity levels in spine grape berry skins. Each point represents the means ± SEs of three replicates. *Indicates significant differences at P < 0.05 (least significant difference test). Prot = protein.

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    Effects of riboflavin on phenylalanine ammonia lyase (PAL) (A) and lipoxygenase (LOX) (B) gene expression levels. Each point represents the means ± SEs of three replicates. *Indicates significant differences at P < 0.05 (least significant difference test).

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