- Email: [email protected]
The methyl jasmonate accelerates the strawberry fruits ripening process
Scientia Horticulturae 249 (2019) 250–256
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
The methyl jasmonate accelerates the strawberry fruits ripening process ⁎
T
Yanli Han, Cen Chen, Zhiming Yan, Jing Li, Yuanhua Wang
Jiangsu Vocational College of Agriculture and Forestry, No. 19, Wenchang East Road, Jurong, Jiangsu, 212400, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Strawberry Methyl jasmonic acid (MeJA) Gene expression Gene overexpression Fruit ripening
In this study we analyzed changes in jasmonic acid (JA) content during the development of the octoploid strawberry cultivar (Fragaria × ananassa Duch. "Benihoppe"). Here, strawberry fruits were treated with different concentrations of methyl jasmonic acid (MeJA, 50 μM, 100μM, 230 μM, 400 μM), respectively, to identify the optimal concentration of MeJA in promotion fruit maturation. We also examined the expression of genes linked to fruit ripening, as well as physiological changes that occurred after MeJA treatment. Using transient gene expression analyses, we performed that key genes in the jasmonic acid biosynthesis pathway, including FaAOC and FaAOS, were overexpressed in fruit, and we further studied their effects on fruit maturation. The results showed that endogenous JA content in the strawberry fruit increased sharply from the small fruit stage to the white fruit stage, but declined after the fruit had ripened, reaching a minimum when fully ripened. MeJA treatment can promote the development and maturation of strawberry fruit, and we found that the optimal concentration to promote maturation was 230 μM. MeJA treatment was associated with increased expression of genes involved in pigment metabolism, sugar metabolism, fruit softening, and hormone metabolism, as well as increases in JA, anthocyanin, and sugar content. Moreover, MeJA treatment was associated with decreased fruit hardness. Overexpression of FaAOC and FaAOS were also found to accelerate strawberry fruit maturation.
1. Introduction Methyl jasmonic acid (MeJA) is an endogenous plant hormone that is a member of the jasmonate family (JAs) (Pei et al., 2017; Ba et al., 2016; Cao et al., 2014; Browse, 2009). Studies have demonstrated that jasmonic acid (JA) plays an important role in the growth and development of plants, as well as in the regulation of fruit development (Kausch et al., 2012; Król et al., 2015). In addition, JA is also involved in plant stress response and disease resistance, and many studies have demonstrated that JA plays a role in disease resistance in many fruit species, including blueberries, grapes, mango, banana, pear, and others (Kucuker et al., 2014; Huang et al., 2015; Yao and Tian, 2005; Andi et al., 2018; Zhao et al., 2016, 2013; Xu et al., 2015; Zhang et al., 2009). Strawberry (Fragaria×ananassa Duch.) is a common fruit with considerable nutritional and economic value. Its short growth and fruiting period makes it easy to study growth processes and fruit development, therefore it is used as a model plant for studies of fruit trees. Previous research has shown that the shelf life of strawberries harvested after MeJA treatment is prolonged. MeJA has also been shown to inhibit strawberry fruit decay as well as reduce the occurrence of gray mold after harvest (Saavedra et al., 2016; Li et al., 2006). Moreover, some studies demonstrated that JA promotes the maturation and
⁎
development of strawberry fruit during ripening (Concha et al., 2013a, b; Preuß et al., 2014). Current research has focused on the effects of JA on stress tolerance, postharvest physiology, postharvest disease, and fruit quality (Ellis and Turner, 2001; Breithaupt et al., 2006; Wang et al., 2010; Gonzalez-Aguilar et al., 2000; Lorenzo et al., 2003). However, recent studies have noted that the mechanisms by which JA regulates strawberry fruit development have not been clearly investigated. Thus, in this study, we show the changes in endogenous JA content during strawberry fruit development, and get the optimal MeJA concentration for fruit ripening. We determine the effects of MeJA on the expression of ripening associated genes. Additionally, we measured the role of the JA biosynthetic pathway genes FaAOC and FaAOS during strawberry development process. This study may provide new ideas for future research on the MeJA in promotion maturation of strawberry fruit. 2. Material and methods 2.1. Plant material The octoploid strawberry cultivar Fragaria×ananassa Duch.
Corresponding author. E-mail address: [email protected] (Y. Wang).
https://doi.org/10.1016/j.scienta.2019.01.061 Received 10 April 2018; Received in revised form 11 January 2019; Accepted 16 January 2019 0304-4238/ © 2019 Published by Elsevier B.V.
Scientia Horticulturae 249 (2019) 250–256
Y. Han et al.
2.4. Effects of MeJA on strawberry fruit development
"Benihoppe" was used as the experimental plant on which all experiments were performed. The development of the strawberry cv. 'Benihoppe' fruit was broken down into seven stages according to the time after flowering; these include: the small fruit (SF; 8–10 days after flowering), middle fruit (MF; 12–15 days after flowering), big fruit (BF; 15–18 days after flowering), white fruit (WF, 18–23 days after flowering), turning fruit (TF; 22–26 days after flowering), partly red fruit (PF; 25–28 after flowering), and red fruit (RF; 28–33 days after flowering) stages (Fait et al., 2008). Because the development of strawberry fruits may be affected by environmental factors, including temperature, humidity, and light, the stages were defined as time ranges and were calculated based on the specific time of fruit development in the experiment. These experiments took place from Dec. 2014 to Mar. 2016 in an elevated strawberry planter located in the greenhouse of the AgriExpo Garden at the Jiangsu Polytechnic College of Agriculture and Forestry. During growth, the day and night temperatures were 20–23 °C and 10–15 °C, respectively. Test plants grew well, without any diseases or insect pests present.
MeJA liquid (Sigma), with a stock concentration of 4.6 mol/L, was diluted in sterile water to prepare working solutions of different concentrations for our experiments. Based on previous tests of MeJA on strawberry fruit development, fruits close to the MF stage (12 days after flowering) were selected for MeJA sensitivity experiments. Different concentrations of MeJA (50 μM, 100 μM, 230 μM, and 400 μM) were injected via fruit stalks using 1 ml syringe. These injections included 100 μl of MeJA for each application, and one application was administered every other day and three times in total. This experiment was repeated three times, 30 fruits were treated each time. Since the MeJA solution was prepared using sterile water, sterile water without MeJA was used for the control group. The surface changes of each strawberry fruit were photographed for observation every day after the first injection, and these photographs were scored together 13 days after the injection to identify the optimal concentration of MeJA. 2.5. Effects of MeJA on physiological indices of strawberry fruit Sucrose content was measured by HPLC as described by Wang et al. (2016). Anthocyanin content was calculated according to the following formula: Anthocyanin (mg/g) = 0.1 × (A530 −0.25 × A650) / 0.462, where anthocyanin (mg/g FW) stands for total anthocyanin content, A530 for absorption at a wavelength of 530 nm, and A650 for absorption at a wavelength of 650 nm (Fuleki and Francis, 1968a; 1968b). Three measurements were performed for each biological replicate. Flesh firmness was measured after the removal of fruit skin on three sides of each fruit using a KM-model fruit hardness tester (Fujihara); flesh firmness was recorded in N. Fruit compression for each fruit was measured three times, and the average of the maximum force was used.
2.2. RNA extraction, cDNA synthesis and qPCR RNA was then obtained from fruit exposed to MeJA to determine the effects of MeJA on gene expression using qPCR. A MeJA solution with the optimal MeJA concentration (230 μM, as determined using the above method) was prepared and injected into strawberry fruit at the MF stage 12 days after flowering. Again the administration was given once every other day, three times in total. This experiment also was repeated three times, with 30 fruit injected in each replicate, and sterile water was again used in the control group. Fruits were harvested 15 days after the first injection and the flesh and seeds of the fruit were separated. Total RNA from the fruits was extracted using the TaKaRa RNA extraction kit (TaKaRa, Otsu, Japan). Genomic DNA was removed by a 15 min incubation at 37 °C with RNase-Free DNase (TaKaRa, Otsu, Japan) followed by an RNA Clean Purification Kit (BioTeke, Beijing, China). The purity and integrity of RNA was analyzed by agarose gel electrophoresis and by absorbance (A260:A230 and A260:A280) ratios. To generate first-strand cDNA, 1 μg of total RNA was reverse-transcribed using a universal primer (5′-AAGCAGTGGTATCAA CGCAGAG TAC(T)30VN-3′ (where N = A, C, G, or T; V = A, G, or C) supplied with the SMART™ RACE cDNA Synthesis Kit (TaKaRa) according to the manufacturer’s protocol. For real-time qPCR, reactions (20 μl) contained 10 μl SYBR Premix ExTaq (TaKaRa), 0.4 μl forward-specific primer, 0.4 μl reverse-specific primer, 2 μl cDNA template, and 7.2 μl ddH2O. This mixture was placed in an iQ5 Sequence Detector (Bio-Rad, Hercules, CA), and DNA amplification was conducted using the following thermocycling program: 95 °C for 2 min, followed by 40 cycles at 94 °C for 20 s, 53 °C for 20 s, and 72 °C for 30 s A melt protocol followed to determine amplicon specificity: this was performed using 71 cycles increasing from 60 to 95 °C at increments of 0.5 °C per cycle for 30 s. The primers used for qPCR are listed in Table S2.
2.6. Overexpression of JA biosynthesis genes FaAOS and FaAOC genes in strawberry fruit In order to assess the effect of JA on strawberry fruit maturation, JA biosynthesis genes of FaAOS and FaAOC were used to be studied. The full-length sequences of both genes were isolated from strawberry and were cloned into the Gateway plant expression vector pK7WG2D. We used this vector to create two destination vectors, pK7WG2D-FaAOS and pK7WG2D-FaAOC. Both vectors, as well as a blank pK7WG2D vector, were transformed into Agrobacterium EHA105. Transformed Agrobacterium were streaked on LB solid medium and were cultured. A single colony was then selected and inoculated into LB liquid medium with 20 μM acetosyringone, and this mixture was cultured at 28 °C with shaking. Agrobacteria were collected when the OD600 reached 1.0, and were re-suspended in an infection buffer (10 mM MgCl2, 10 mM MES, 20 μM acetosyringone). After shaking at room temperature for 2 h, the suspension was injected into strawberry fruit. 1 ml of the suspension was injected into fruit 14 days after flowering via fruit stalks using 1 ml syringe. This inoculation contained 50 μl of bacterial suspension, and was performed once every other day for 3 times in total. Changes in strawberry phenotype were recorded by photos, which were taken every day after injection until the fruits turned fully red. 30 fruits were injected and this experiment was repeated three times for each vector.
2.3. Determination of JA content during strawberry fruit development 3. Results Ten fruits for each stage were sampled from three groups of strawberry plants growing in the experimental planter. The flesh and seeds of the fruit were separated immediately after harvest. The flesh was then frozen with liquid nitrogen and JA content was analyzed. The JA content was measured by homogenizing all fruits at a given stage, and 200 mg of the homogenate was removed to determine JA content (Kausch et al., 2012). This procedure was performed in triplicate for each stage and the mean values for all stages were obtained. Statistical tests were performed using SPSS.
3.1. JA content in strawberry fruit at different development stages Our results showed that JA content at the GF stage was very low, although it quickly increased with fruit development and rapidly increased afterward, reaching a peak at the WF stage. Thereafter, the JA content slowly reduced as the fruit coloured, reaching minimal levels when the fruits turned fully red (Fig. 1). This preliminary result indicated that JA content changes during strawberry fruit development, 251
Scientia Horticulturae 249 (2019) 250–256
Y. Han et al.
Fig. 1. Jasmonic acid content at different strawberry fruit development stages.
and appears to fluctuate in concert with fruit developmental stage.
other concentrations. The strongest ripening-promoting effect was observed at a JA concentration of 230 μM. Fruit ripening occurred about 5 days earlier in response to injection with 230 μM MeJA, relative to the control condition. These results further demonstrate that MeJA enhances the maturation and development of strawberry fruit.
3.2. Effects of different concentrations of MeJA on fruit ripening process From Fig. 2, it can be seen that JA treatments of different concentrations varied in terms of their effects on ripening. Although all treatments promoted strawberry fruit ripening to different degrees relative to the control, the difference between the effects of JA at 50 μM and 100 μM were not clear. Moreover, a JA concentration of 400 μM started to inhibit fruit ripening, at least compared with the effects of
3.3. Effect of MeJA on the expression of ripening-related genes in strawberry fruit In order to understand the nature of advanced fruit ripening, we
Fig. 2. Effects of different concentrations of MeJA on strawberry fruit ripening. 252
Scientia Horticulturae 249 (2019) 250–256
Y. Han et al.
Fig. 4. Effects of jasmonic acid application on sugar metabolism-related gene expression. Asterisks indicate statistically significant differences at p < 0.05, as determined by a Student’s t-test.
metabolism genes promotes sugar synthesis and transport. We conclude that exogenous MeJA treatment may promote the accumulation of sugar in strawberry fruit, thus accelerating fruit maturation. Fruit hardness also changes during fruit ripening. Ripe strawberry fruit is much softer than it is before maturation, so changes in the expression of genes related to fruit softening during fruit ripening is an important index to measure fruit ripening. According to Concha et al. (2013a, b), genes affecting fruit ripening and softening including FaEXP1, FaEXP2, FaEXP3, FaEXP4, FaEXP5, FaCEL1, FaCEL2, FaXYL1, FaPE, FaPL, and FaPG. These genes play an important role in fruit ripening and softening. Our results show that most of these genes were upregulated after MeJA treatment relative to the control; for example, FaPL was upregulated more than 5-fold (Fig. 5). Thus, our results suggest that exogenous MeJA treatment can promote the expression of genes associated with fruit softening. This ostensibly results in softer fruits, and therefore advances fruit ripening. The most important hormone pathway affecting fruit ripening is the ABA pathway. The application of exogenous MeJA may therefore affect the expression of genes involved in both the JA and ABA synthesis and metabolic pathways. We examined the expression of genes encoding four key enzymes in the JA synthesis pathway, including FaLOX, FaAOC, FaAOS, and FaOPDA1, as well as key genes involved in the ABA synthesis pathway, including FaNCED1, FaNCED2, FaNCED3, and FaAAO. Our results showed that the expression of FaLOX, FaAOC, FaAOS, and FaOPDA1 were somewhat upregulated after MeJA treatment (Fig. 6), and that the expression of
Fig. 3. Effects of jasmonic acid application on anthocyanin biosynthesis-related gene expression. Asterisks indicate statistically significant differences at p < 0.05, as determined by a Student’s t-test.
examined the expression of ripening metabolism-related genes to explain how JA promotes the ripening of strawberry fruit, as well as which genes may be affected by accelerating the fruit ripening process. First, we wanted to quantify differences in the expression of genes related to pigment metabolism, since the most obvious changes in fruit ripening are changes in colour. Based on previous work quantifying the expression of 6 genes involved in strawberry pigment metabolism (Table S1), including FaCHS, FaCHI, FaDFR, FaF3H, FaANS, and FaUFGT, it was found that the expression levels of FaCHS, FaCHI, FaF3H, and FaUFGT were all up-regulated during fruit ripening, but the greatest increases were observed in the expression of FaCHS and FaCHI, which increased about 5- to 8-fold for each gene (Fig. 3). This suggests that MeJA treatment may result in increased expression of some pigment metabolism-related genes, and that FaCHS and FaCHI may be the two genes most strongly affected by the advanced ripening of strawberry fruit in response to exogenous MeJA. In addition to upregulated genes, other genes such as FaDFR and FaANS, were found to be downregulated, which may indicate that MeJA inhibits the expression of these two genes, although the specific reasons for this downregulation need to be explored further. Flavour is an important indicator of strawberry fruit quality, and sugar content is the most important factor determining fruit flavor. Therefore, the expression of 10 sugar metabolism-related genes (Table S1), including FaSS, FaSPS1, FaSPS2, FaSUT1, FaSUT2, FaSUT3, FaSUT4, FaSUT5, FaSUT6, and FaSUT7, was examined. As shown in Fig. 4, increased MeJA was associated with increased expression for most of these genes, especially the sucrose synthase genes FaSPS1 and FaSPS2. These two genes play important roles in sucrose synthesis, indicating that MeJA treatment can promote the upregulation of sucrose synthase genes, thereby promoting sucrose synthesis and subsequently causing the accumulation of more sugar in strawberry fruit as well as accelerating fruit ripening. Gene expression results for the sucrose transporter family, including FaSUT1, FaSUT2, FaSUT3, FaSUT4, FaSUT5, FaSUT6, and FaSUT7, demonstrated that the expression of transporter genes was upregulated after sucrose synthesis. Thus, sucrose transport in strawberry fruit was also enhanced. Among these genes, the expression of FaSS was downregulated because it degrades sucrose in the sugar metabolism pathway. Taken together, our results showed that MeJA inhibits the expression of FaSS as well as the degradation of sugar, whereas the increased expression of other sugar
Fig. 5. Effects of jasmonic acid application on fruit softening-related gene expression. Asterisks indicate statistically significant differences at p < 0.05 as determined by a Student’s t-test. 253
Scientia Horticulturae 249 (2019) 250–256
Y. Han et al.
Fig. 6. Effects of jasmonic acid application on hormone metabolism-related gene expression. Asterisks indicate statistically significant differences at p < 0.05, as determined by a Student’s t-test.
FaAOC and FaAOS showed the strongest change in relative expression. This indicates that FaAOC and FaAOS are important for the JA synthesis pathway, as well as for strawberry fruit ripening. This result is not consistent with the results of several previous studies (Concha et al., 2013a, b; Preuß et al., 2014). This inconsistency may be caused by different concentrations of exogenous MeJA and by the different strawberry varieties used. Among the genes involved in ABA metabolism, the functions of FaNCED1, FaNCED2, and FaNCED3 in the promotion of fruit ripening in strawberry have been reported, although few studies on their relationship to JA have been published. The results of our analysis may also indicate that exogenous MeJA can promote upregulation of FaNCED1, FaNCED2, and FaNCED3, with a 10-fold increase in relative expression in the MeJA treatment than that in the control fruit. This suggests that JA and ABA somehow interact during strawberry fruit ripening. However, a detailed mechanism of this process should be studied further in the future.
Fig. 7. Effects of jasmonic acid application on strawberry fruit physiological indices. a. Jasmonic acid content; b. Anthocyanin content; c. Sucrose content; d. Fruit hardness. Asterisks indicate statistically significant differences at p < 0.05, as determined by a Student’s t-test.
et al., 2012) Meantime, other reports mainly focus on the function of MeJA in postharvest preservation of fruits and prevention of postharvest diseases (Matsui et al., 2004). However, there are few studies on the effect of jasmonic acid on fruit ripening and development, especially on the molecular mechanism of fruit ripening and development. Our research will provide a new idea for the role of jasmonic acid in fruit ripening. In this study, the endogenous JA content at different developmental stages was analyzed to determine the effect of JA on the ripening of strawberry fruit. Our results showed that JA content increases rapidly with fruit development, but then decreases sharply, reaching the lowest level when the fruits are fully ripe. Therefore, JA may have a very important role in the initiation of fruit ripening, and its content begins to decline in red fruits at the late ripening stage(Kondo, 2006). Jia et al. (2016) also showed a simaler results in grape, the JA content increased rapidly in the initiation of fruit ripening stage. At the later stage during fruit development, the JA content decreasd. At this stage the function of JA as an initiator of ripening is no longer needed, and this may be related to the function of JA in disease resistance (Agrawal et al., 2003; Zhang et al., 2005). Different concentrations of MeJA were used to treat strawberry fruits and the results showed that exogenous MeJA can also promote the ripening of strawberry fruit. Although different degrees of promotion were observed in response to different concentrations of JA, an overall promoting effect was consistently observed. This may indicate that JA is involved in fruit ripening, and this conclusion is consistent with research findings in grapes and apples (). Studies of MeJA-treated Kyoho grapes showed that MeJA caused increased anthocyanin content, promoted accelerated colouring, and increased the sugar-acid ratio in the fruit (D’Onofrio et al., 2018; Schaller, 2001). MeJA treatment has also been shown to accelerate colouring of apple fruits (Rudell et al., 2002). We found that the optimital concentrations of MeJA applied in strawberry is 230 μM. Moreover, jasmonic acid and methyl jasmonate are harmless to our body, which can be applied during fruit ripening and even after fruit ripening without causing harm to human body. Jasmonic acid will play an increasingly important role in fruit ripening and development in the future. We will do more in-depth research on this basis. Exogenous MeJA treatment of strawberry fruit revealed differences
3.4. Effects of MeJA on physiological indices during strawberry fruit ripening The results of analysis of JA, anthocyanin, and soluble sugar contents, as well as fruit hardness after exogenous MeJA treatment, indicated that exogenous MeJA treatment can increase JA, anthocyanin, and soluble sugar contents and fruit firmness (Fig. 7). These results are consistent with our gene expression results. This further demonstrated that MeJA treatment can promote the ripening of strawberry fruit. 3.5. Overexpression of FaAOC and FaAOS in strawberry fruit According to our gene expression analysis, we found that FaAOC and FaAOS are important for the JA synthesis pathway. Therefore, these two genes were overexpressed in strawberry fruit using transient transfection. The results showed (Fig. 8) that the transient expression of FaAOC and FaAOS in fruit also accelerated fruit ripening by 3–5 days compare to the control fruit. We conclude that JA is of great importance for the ripening of strawberry fruit. 4. Discussion Many studies have shown that jasmonic acid can induce the expression of defense genes in plants, and plant react quickly when it is injured to acquire the defense ability against foreign diseases. (Pei et al., 2017; Ba et al., 2016; Cao et al., 2014; Browse, 2009; Kaushc 254
Scientia Horticulturae 249 (2019) 250–256
Y. Han et al.
Fig. 8. FaAOS and FaAOC overexpression accelerated strawberry fruit ripening.
Funding
in the relative expression of four kinds of genes related to fruit ripening, including pigment metabolism genes, sugar metabolism genes, fruit ripening and softening-related genes, and hormone synthesis pathwayrelated genes (Zhao et al., 2013; Xu et al., 2015; Zhang et al., 2009). These changes suggest that JA plays an important role in the ripening of fruit by affecting multiple genes associated with fruit ripening rather than by a simple function. FaAOS and FaAOC genes play an important role in the jasmonic acid synthesis pathway (Kondo et al., 2007; Ashish et al., 2015). The results showed that the expression of these two genes also changed significantly after treatment with exogenous MeJA. Using overexpression lines of strawberry, we found that FaAOC and FaAOS may enhance the accelerated maturation of fruit by ˜3–5 days relative to a control condition. Therefore, FaAOS and FaAOC genes play a key role in the jasmonic acid synthesis pathway of strawberry fruit.This finding is consistent with previous results, indicating that MeJA can accelerate fruit colouring in strawberry. Based these results, we conclude that JA may promote the development and ripening of strawberry fruit, and that this finding may provide new directions for research on the regulation of strawberry fruit quality. A number of studies have shown that jasmonic acid is beneficial to the storage and preservation of fruits. Studies have shown that jasmonic acid treatment can inhibit strawberry fruit decay and prolong fruit shelf life (Gansser et al., 1997; Garrido-Bigotes et al., 2018). The effects of methyl jasmonate treatment on fruit colouring were also studied. Most of these studies focused on fruit disease resistance and shelf life extension after harvest, but there was little research on whether jasmonate could play a role in fruit development. In this study, jasmonic acid was used to regulate the coloring, aroma, softening, glycolic acid synthesis and hormone-related genes of strawberry fruit. The results showed that jasmonic acid could regulate the coloring, softening, glycolic acid synthesis and hormone-related gene expression, thus promoting fruit coloring and softening and achieving early fruit ripening. However, it was found that jasmonic acid did not regulate aroma synthesis genes significantly, and the reasons and mechanisms need to be further studied.
This work was supported by the National Natural Science Fund of China (31701898), the Natural Science Fund of Jiangsu Province, China (BK20160567), the Natural Science Fund of Jiangsu Provincial Department of Education, China (16KJB210016). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2019.01.061. References Agrawal, G.K., Jwa, N.S., Shibato, J., Han, O., Iwahashi, H., Rakwal, R., 2003. Diverse environmental cues transiently regulate OsOPR1 of the “octadecanoid pathway” revealing its importance in rice defense/stress and development. Biochem. Biophys. Res. Co. 310 (4), 1073–1082. Andi, S.A., Gholami, M., Ford, C.M., 2018. The effect of methyl jasmonate and light irradiation treatments on the stilbenoid biosynthetic pathway in Vitis vinifera cell suspension cultures. Nat. Prod. Res. 32 (8), 909–917. Ashish, R., Jyothilakshmi, V., Hitendra, K.P., Alok, P., Ramesh, P., Axel, M., Ramesh, V.S., 2015. Upregulation of jasmonate biosynthesis and jasmonate-responsive genes in rice leaves in response to a bacterial pathogen mimic. Funct. Integr. Genomics 15, 363–373. Ba, L.J., Kuang, J.F., Chen, J.Y., Lu, W.J., Ma, J.A.Z., 2016. Attenuates the MaLBD5Mediated transcriptional activation of jasmonate biosynthesis gene MaAOC2 in regulating cold tolerance of banana fruit. J. Agric. Food Chem. 64 (4), 738–745. Breithaupt, C., Kurzbauer, R., Lilie, H., Schaller, A., Strassner, J., Huber, R., Macheroux, P., Clausen, T., 2006. Crystal structure of 12-oxophytodienoate reductase 3 from tomato Self-inhibition by dimerization. P. Natl. Acad. Sci. USA 103 (39), 14337–14342. Browse, J., 2009. The power of mutants for investigationg jasmonate biosynthesis and signaling. Phytochemistry 70 (13-14), 1539–1546. Cao, S., Cai, Y., Yang, Z., Joyce, D.C., Zheng, Y., 2014. Effect of MeJA treatment on polyamine, energy status and anthracnose rot of loquat fruit. Food Chem. 145, 86–89. Concha, C.M., Figueroa, N.E., Poblete, L.A., Oñate, F.A., Schwab, W., Figueroa, C.R., 2013a. Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit. Plant Physiol. Biochem. 70, 433–444. Concha, C.M., Figueroa, N.E., Poblete, L.A., Onate, F.A., Schwab, W., Figueroa, C.R., 2013b. Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit. Plant Physiol. Biochem. 9 (70), 433–443. D’Onofrio, C., Matarese, F., Cuzzola, A., 2018. Effect of methyl jasmonate on the aroma of Sangiovese grapes and wines. Food Chem. 242, 352–361. Ellis, C., Turner, J.G., 2001. The Arabidopsis mutant cev1 has constitutively active jasmonate and ethylene signal pathways and enhanced resistance to pathogens. Plant
Conflict of interest The authors declare that they have no conflict of interests. 255
Scientia Horticulturae 249 (2019) 250–256
Y. Han et al.
Ichinose, Y., 2004. Structure and expression of 12-oxophytodienoate reductase (subgroup I) genes in pea, and characterization of the oxidoreductase activities of their recombinant products. Mol. Genet. Genom. 271 (1), 1–10. Pei, T., Ma, P., Ding, K., Liu, S., Jia, Y., Ru, M., Dong, J., Liang, Z., 2017. SmJAZ8 acts as a core repressor regulating JA-induced biosynthesis of salvianolic acids and tanshinones in Salvia miltiorrhiza hairy roots. J. Exp. Bot. https://doi.org/10.1093/jxb/ erx484. Preuß, A., Augustin, C., Figueroa, C.R., Hoffmann, T., Valpuesta, V., Sevilla, J.F., Schwab, W., 2014. Expression of a functional jasmonic acid carboxyl methyltransferase is negatively correlated with strawberry fruit development. J. Plant Physiol. 171 (15), 1315–1324. Rudell, D.R., Mattheis, J.P., Fan, X., 2002. Methyl jasmonate enhances anthocyanin accumulation and modifies production of phenolics and pigments in Fuji apples. J. Am. Soc. Hortic. Sci. 127 (3), 435–441. Saavedra, G.M., Figueroa, N.E., Poblete, L.A., Cherian, S., Figueroa, C.R., 2016. Effects of preharvest applications of methyl jasmonate and chitosan on postharvest decay, quality and chemical attributes of Fragaria chiloensis fruit. Food Chem. 190, 448–453. Schaller, F., 2001. Enzymes of the biosynthesis of octadecanoid-derived signalling molecules. J. Exp. Bot. 52 (354), 11–23. Wang, K., Jin, P., Shang, H., Zheng, Y., 2010. Effect of methyl jasmonate in combination with ethanol treatment on postharvest decay and antioxidant capacity in Chinese bayberries. J. Agr. Food Chem. 58 (17), 9597–9604. Wang, L.F., Qi, X.X., Huang, X.S., Xu, L.L., Jin, C., Wu, J., Zhang, S.S., 2016. Overexpression of sucrose transporter gene PbSUT2 from Pyrus bretschneideri, enhances sucrose content in Solanum lycopersicum fruit. Plant Physiol. Biochem. 105, 150–161. Xu, Y.Y., Li, H., Lin, J., Li, X.G., Chang, Y.H., 2015. Isolation and characterization of Calcineurin B-like gene (PbCBL1) and its promoter in birch-leaf pear (Pyrus betulifolia Bunge). Genet. Mol. Res. 14 (4), 16756–16770. Yao, H.J., Tian, S.P., 2005. Effects of pre- and post-harvest application of salicylic acid or methyl jasmonate on inducing disease resistance of cherry fruit in storage. Postharvest Biol. Tcehnol. 35, 253–262. Zhang, J., Simmons, C., Yalpani, N., Crane, V., Wilkinson, H., Kolomiets, M., 2005. Genomic analysis of the 12-oxo-phytodienoic acid reductase gene family of Zea mays. Plant Mol. Biol. 59 (2), 323–343. Zhang, H.Y., Ma, L.C., Mark, T., Xu, H.X., Dong, Y., Jiang, S., 2009. Methyl jasmonate enhances biocontrol efficacy of Rhodotorula glutinis to postharvest blue mold decay of pears. Food Chem. 117 (4), 621–626. Zhao, M.L., Wang, J.N., Shan, W., Fan, J.G., Kuang, J.F., Wu, K.Q., Li, X.P., Chen, W.X., He, F.Y., Chen, J.Y., Lu, W.J., 2013. Induction of jasmonate signalling regulators MaMYC2s and their physical interactions with MaICE1 in methyl jasmonate-induced chilling tolerance in banana fruit. Plant Cell Environ. 36 (1), 30–51. Zhao, N., Lin, H., Lan, S., Jia, Q., Chen, X., Guo, H., Chen, F., 2016. VvMJE1 of the grapevine (Vitis vinifera) VvMES methylesterase family encodes for methyl jasmonate esterase and has a role in stress response. Plant Physiol. Biochem. 102, 125–132.
Cell 13 (5), 1025–1033. Fait, A., Hanhineva, K., Beleggia, R., Dai, N., Rogachev, I., Nikiforova, V.J., Fernie, A.R., Aharoni, A., 2008. Recnfiguration of the achene and receptacle metabolic networks during Strawberry fruit development. Plant Physiol. 148 (2), 730–750. Fuleki, T., Francis, F.J., 1968a. Quantitative methods for anthocyanins. 1. Extraction and determination of total anthocyanin in cranberries. J. Food Sci. 33, 72–78. Fuleki, T., Francis, F.J., 1968b. Quantitative methods for anthocyanins. 2. Determination of total anthocyanin and degradation index for cranberry juice. J. Food Sci. 33, 78–82. Gansser, D., Latza, S., Berger, R.G., 1997. Methyl jasmonates in developing strawberry fruit (Fragaria×ananassa Duch. Cv. Kent). J. Agric. Food Chem. 45, 2477–2480. Garrido-Bigotes, A., Figueroa, P.M., Figueroa, C.R., 2018. Jasmonate metabolism and its relationship with abscisic acid during strawberry fruit development and ripening. J. Plant Growth Regul. 37, 101–113. Gonzalez-Aguilar, G.A., Fortiz, J., Cruz, R., Beaz, R., Wang, C.Y., 2000. Methyl jasmonate reduces chilling injury and maintains postharvest quality of mango fruit. J. Agr. Food Chem. 48 (2), 515–519. Huang, X., Li, J., Shang, H., Meng, X., 2015. Effect of methyl jasmonate on the anthocyanin content and antioxidant activity of blueberries during cold storage. J. Sci. Food Agric. 95 (2), 337–343. Jia, H., Zhang, C., Pervaiz, T., Zhao, P., Liu, Z., Wang, B., et al., 2016. Jasmonic acid involves in grape fruit ripening and resistant against botrytis cinerea. Funct. Integr. Genomics 16 (1), 79–94. Kausch, K.D., Sobolev, A.P., Goyal, R.K., Fatima, T., Laila-Beevi, R., Saftner, R.A., Handa, A.K., Mattoo, A.K., 2012. Methyl jasmonate deficiency alters cellular metabolome, including the aminome of tomato (Solanum lycopersicum L.) fruit. Amino Acids 42 (23), 843–856. Kondo, S., 2006. The role of jasmonates in fruit color development and chilling injury. Acta Hortic. 727, 45–56. Kondo, S., Yamada, H., Setha, S., 2007. Effect of jasmonates differed at fruit ripening stages on 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase gene expression in pears. J. Am. Soc. Hortic. Sci. 132, 120–125. Król, P., Igielski, R., Pollmann, S., Kępczyńska, E., 2015. Priming of seeds with methyl jasmonate induced resistance to hemi-biotroph Fusarium oxysporum f.sP. Lycopersici in tomato via 12-oxo-phytodienoic acid, salicylic acid, and flavonol accumulation. J. Plant Physiol. 179, 122–132. Kucuker, E., Ozturk, B., Celik, S.M., Aksit, H., 2014. Pre-harvest spray application of methyl jasmonate plays an important role in fruit ripening, fruit quality and bioactive compounds of Japanese plums. Sci. Hortic. 176 (2), 162–169. Li, D.P., Xu, Y.F., Sun, L.P., Liu, L.X., Hu, X.L., Li, D.Q., Shu, H.R., 2006. Salicylic acid, ethephon, and methyl jasmonate enhance ester regeneration in 1-MCP-treated apple fruit after long-term cold storage. J. Agric. Food Chem. 54 (11), 3887–3895. Lorenzo, O., Piqueras, R., Sanchez-Serrano, J.J., Solano, R., 2003. Ethylene response factor1 integrates signals from ethylene and jasmonate pathways in plant defense. Plant Cell 15 (1), 165–178. Matsui, H., Nakamura, G., Ishiga, Y., Toshima, H., Inagaki, Y., Toyoda, K., Shiraishi, T.,
256
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
The methyl jasmonate accelerates the strawberry fruits ripening process ⁎
T
Yanli Han, Cen Chen, Zhiming Yan, Jing Li, Yuanhua Wang
Jiangsu Vocational College of Agriculture and Forestry, No. 19, Wenchang East Road, Jurong, Jiangsu, 212400, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Strawberry Methyl jasmonic acid (MeJA) Gene expression Gene overexpression Fruit ripening
In this study we analyzed changes in jasmonic acid (JA) content during the development of the octoploid strawberry cultivar (Fragaria × ananassa Duch. "Benihoppe"). Here, strawberry fruits were treated with different concentrations of methyl jasmonic acid (MeJA, 50 μM, 100μM, 230 μM, 400 μM), respectively, to identify the optimal concentration of MeJA in promotion fruit maturation. We also examined the expression of genes linked to fruit ripening, as well as physiological changes that occurred after MeJA treatment. Using transient gene expression analyses, we performed that key genes in the jasmonic acid biosynthesis pathway, including FaAOC and FaAOS, were overexpressed in fruit, and we further studied their effects on fruit maturation. The results showed that endogenous JA content in the strawberry fruit increased sharply from the small fruit stage to the white fruit stage, but declined after the fruit had ripened, reaching a minimum when fully ripened. MeJA treatment can promote the development and maturation of strawberry fruit, and we found that the optimal concentration to promote maturation was 230 μM. MeJA treatment was associated with increased expression of genes involved in pigment metabolism, sugar metabolism, fruit softening, and hormone metabolism, as well as increases in JA, anthocyanin, and sugar content. Moreover, MeJA treatment was associated with decreased fruit hardness. Overexpression of FaAOC and FaAOS were also found to accelerate strawberry fruit maturation.
1. Introduction Methyl jasmonic acid (MeJA) is an endogenous plant hormone that is a member of the jasmonate family (JAs) (Pei et al., 2017; Ba et al., 2016; Cao et al., 2014; Browse, 2009). Studies have demonstrated that jasmonic acid (JA) plays an important role in the growth and development of plants, as well as in the regulation of fruit development (Kausch et al., 2012; Król et al., 2015). In addition, JA is also involved in plant stress response and disease resistance, and many studies have demonstrated that JA plays a role in disease resistance in many fruit species, including blueberries, grapes, mango, banana, pear, and others (Kucuker et al., 2014; Huang et al., 2015; Yao and Tian, 2005; Andi et al., 2018; Zhao et al., 2016, 2013; Xu et al., 2015; Zhang et al., 2009). Strawberry (Fragaria×ananassa Duch.) is a common fruit with considerable nutritional and economic value. Its short growth and fruiting period makes it easy to study growth processes and fruit development, therefore it is used as a model plant for studies of fruit trees. Previous research has shown that the shelf life of strawberries harvested after MeJA treatment is prolonged. MeJA has also been shown to inhibit strawberry fruit decay as well as reduce the occurrence of gray mold after harvest (Saavedra et al., 2016; Li et al., 2006). Moreover, some studies demonstrated that JA promotes the maturation and
⁎
development of strawberry fruit during ripening (Concha et al., 2013a, b; Preuß et al., 2014). Current research has focused on the effects of JA on stress tolerance, postharvest physiology, postharvest disease, and fruit quality (Ellis and Turner, 2001; Breithaupt et al., 2006; Wang et al., 2010; Gonzalez-Aguilar et al., 2000; Lorenzo et al., 2003). However, recent studies have noted that the mechanisms by which JA regulates strawberry fruit development have not been clearly investigated. Thus, in this study, we show the changes in endogenous JA content during strawberry fruit development, and get the optimal MeJA concentration for fruit ripening. We determine the effects of MeJA on the expression of ripening associated genes. Additionally, we measured the role of the JA biosynthetic pathway genes FaAOC and FaAOS during strawberry development process. This study may provide new ideas for future research on the MeJA in promotion maturation of strawberry fruit. 2. Material and methods 2.1. Plant material The octoploid strawberry cultivar Fragaria×ananassa Duch.
Corresponding author. E-mail address: [email protected] (Y. Wang).
https://doi.org/10.1016/j.scienta.2019.01.061 Received 10 April 2018; Received in revised form 11 January 2019; Accepted 16 January 2019 0304-4238/ © 2019 Published by Elsevier B.V.
Scientia Horticulturae 249 (2019) 250–256
Y. Han et al.
2.4. Effects of MeJA on strawberry fruit development
"Benihoppe" was used as the experimental plant on which all experiments were performed. The development of the strawberry cv. 'Benihoppe' fruit was broken down into seven stages according to the time after flowering; these include: the small fruit (SF; 8–10 days after flowering), middle fruit (MF; 12–15 days after flowering), big fruit (BF; 15–18 days after flowering), white fruit (WF, 18–23 days after flowering), turning fruit (TF; 22–26 days after flowering), partly red fruit (PF; 25–28 after flowering), and red fruit (RF; 28–33 days after flowering) stages (Fait et al., 2008). Because the development of strawberry fruits may be affected by environmental factors, including temperature, humidity, and light, the stages were defined as time ranges and were calculated based on the specific time of fruit development in the experiment. These experiments took place from Dec. 2014 to Mar. 2016 in an elevated strawberry planter located in the greenhouse of the AgriExpo Garden at the Jiangsu Polytechnic College of Agriculture and Forestry. During growth, the day and night temperatures were 20–23 °C and 10–15 °C, respectively. Test plants grew well, without any diseases or insect pests present.
MeJA liquid (Sigma), with a stock concentration of 4.6 mol/L, was diluted in sterile water to prepare working solutions of different concentrations for our experiments. Based on previous tests of MeJA on strawberry fruit development, fruits close to the MF stage (12 days after flowering) were selected for MeJA sensitivity experiments. Different concentrations of MeJA (50 μM, 100 μM, 230 μM, and 400 μM) were injected via fruit stalks using 1 ml syringe. These injections included 100 μl of MeJA for each application, and one application was administered every other day and three times in total. This experiment was repeated three times, 30 fruits were treated each time. Since the MeJA solution was prepared using sterile water, sterile water without MeJA was used for the control group. The surface changes of each strawberry fruit were photographed for observation every day after the first injection, and these photographs were scored together 13 days after the injection to identify the optimal concentration of MeJA. 2.5. Effects of MeJA on physiological indices of strawberry fruit Sucrose content was measured by HPLC as described by Wang et al. (2016). Anthocyanin content was calculated according to the following formula: Anthocyanin (mg/g) = 0.1 × (A530 −0.25 × A650) / 0.462, where anthocyanin (mg/g FW) stands for total anthocyanin content, A530 for absorption at a wavelength of 530 nm, and A650 for absorption at a wavelength of 650 nm (Fuleki and Francis, 1968a; 1968b). Three measurements were performed for each biological replicate. Flesh firmness was measured after the removal of fruit skin on three sides of each fruit using a KM-model fruit hardness tester (Fujihara); flesh firmness was recorded in N. Fruit compression for each fruit was measured three times, and the average of the maximum force was used.
2.2. RNA extraction, cDNA synthesis and qPCR RNA was then obtained from fruit exposed to MeJA to determine the effects of MeJA on gene expression using qPCR. A MeJA solution with the optimal MeJA concentration (230 μM, as determined using the above method) was prepared and injected into strawberry fruit at the MF stage 12 days after flowering. Again the administration was given once every other day, three times in total. This experiment also was repeated three times, with 30 fruit injected in each replicate, and sterile water was again used in the control group. Fruits were harvested 15 days after the first injection and the flesh and seeds of the fruit were separated. Total RNA from the fruits was extracted using the TaKaRa RNA extraction kit (TaKaRa, Otsu, Japan). Genomic DNA was removed by a 15 min incubation at 37 °C with RNase-Free DNase (TaKaRa, Otsu, Japan) followed by an RNA Clean Purification Kit (BioTeke, Beijing, China). The purity and integrity of RNA was analyzed by agarose gel electrophoresis and by absorbance (A260:A230 and A260:A280) ratios. To generate first-strand cDNA, 1 μg of total RNA was reverse-transcribed using a universal primer (5′-AAGCAGTGGTATCAA CGCAGAG TAC(T)30VN-3′ (where N = A, C, G, or T; V = A, G, or C) supplied with the SMART™ RACE cDNA Synthesis Kit (TaKaRa) according to the manufacturer’s protocol. For real-time qPCR, reactions (20 μl) contained 10 μl SYBR Premix ExTaq (TaKaRa), 0.4 μl forward-specific primer, 0.4 μl reverse-specific primer, 2 μl cDNA template, and 7.2 μl ddH2O. This mixture was placed in an iQ5 Sequence Detector (Bio-Rad, Hercules, CA), and DNA amplification was conducted using the following thermocycling program: 95 °C for 2 min, followed by 40 cycles at 94 °C for 20 s, 53 °C for 20 s, and 72 °C for 30 s A melt protocol followed to determine amplicon specificity: this was performed using 71 cycles increasing from 60 to 95 °C at increments of 0.5 °C per cycle for 30 s. The primers used for qPCR are listed in Table S2.
2.6. Overexpression of JA biosynthesis genes FaAOS and FaAOC genes in strawberry fruit In order to assess the effect of JA on strawberry fruit maturation, JA biosynthesis genes of FaAOS and FaAOC were used to be studied. The full-length sequences of both genes were isolated from strawberry and were cloned into the Gateway plant expression vector pK7WG2D. We used this vector to create two destination vectors, pK7WG2D-FaAOS and pK7WG2D-FaAOC. Both vectors, as well as a blank pK7WG2D vector, were transformed into Agrobacterium EHA105. Transformed Agrobacterium were streaked on LB solid medium and were cultured. A single colony was then selected and inoculated into LB liquid medium with 20 μM acetosyringone, and this mixture was cultured at 28 °C with shaking. Agrobacteria were collected when the OD600 reached 1.0, and were re-suspended in an infection buffer (10 mM MgCl2, 10 mM MES, 20 μM acetosyringone). After shaking at room temperature for 2 h, the suspension was injected into strawberry fruit. 1 ml of the suspension was injected into fruit 14 days after flowering via fruit stalks using 1 ml syringe. This inoculation contained 50 μl of bacterial suspension, and was performed once every other day for 3 times in total. Changes in strawberry phenotype were recorded by photos, which were taken every day after injection until the fruits turned fully red. 30 fruits were injected and this experiment was repeated three times for each vector.
2.3. Determination of JA content during strawberry fruit development 3. Results Ten fruits for each stage were sampled from three groups of strawberry plants growing in the experimental planter. The flesh and seeds of the fruit were separated immediately after harvest. The flesh was then frozen with liquid nitrogen and JA content was analyzed. The JA content was measured by homogenizing all fruits at a given stage, and 200 mg of the homogenate was removed to determine JA content (Kausch et al., 2012). This procedure was performed in triplicate for each stage and the mean values for all stages were obtained. Statistical tests were performed using SPSS.
3.1. JA content in strawberry fruit at different development stages Our results showed that JA content at the GF stage was very low, although it quickly increased with fruit development and rapidly increased afterward, reaching a peak at the WF stage. Thereafter, the JA content slowly reduced as the fruit coloured, reaching minimal levels when the fruits turned fully red (Fig. 1). This preliminary result indicated that JA content changes during strawberry fruit development, 251
Scientia Horticulturae 249 (2019) 250–256
Y. Han et al.
Fig. 1. Jasmonic acid content at different strawberry fruit development stages.
and appears to fluctuate in concert with fruit developmental stage.
other concentrations. The strongest ripening-promoting effect was observed at a JA concentration of 230 μM. Fruit ripening occurred about 5 days earlier in response to injection with 230 μM MeJA, relative to the control condition. These results further demonstrate that MeJA enhances the maturation and development of strawberry fruit.
3.2. Effects of different concentrations of MeJA on fruit ripening process From Fig. 2, it can be seen that JA treatments of different concentrations varied in terms of their effects on ripening. Although all treatments promoted strawberry fruit ripening to different degrees relative to the control, the difference between the effects of JA at 50 μM and 100 μM were not clear. Moreover, a JA concentration of 400 μM started to inhibit fruit ripening, at least compared with the effects of
3.3. Effect of MeJA on the expression of ripening-related genes in strawberry fruit In order to understand the nature of advanced fruit ripening, we
Fig. 2. Effects of different concentrations of MeJA on strawberry fruit ripening. 252
Scientia Horticulturae 249 (2019) 250–256
Y. Han et al.
Fig. 4. Effects of jasmonic acid application on sugar metabolism-related gene expression. Asterisks indicate statistically significant differences at p < 0.05, as determined by a Student’s t-test.
metabolism genes promotes sugar synthesis and transport. We conclude that exogenous MeJA treatment may promote the accumulation of sugar in strawberry fruit, thus accelerating fruit maturation. Fruit hardness also changes during fruit ripening. Ripe strawberry fruit is much softer than it is before maturation, so changes in the expression of genes related to fruit softening during fruit ripening is an important index to measure fruit ripening. According to Concha et al. (2013a, b), genes affecting fruit ripening and softening including FaEXP1, FaEXP2, FaEXP3, FaEXP4, FaEXP5, FaCEL1, FaCEL2, FaXYL1, FaPE, FaPL, and FaPG. These genes play an important role in fruit ripening and softening. Our results show that most of these genes were upregulated after MeJA treatment relative to the control; for example, FaPL was upregulated more than 5-fold (Fig. 5). Thus, our results suggest that exogenous MeJA treatment can promote the expression of genes associated with fruit softening. This ostensibly results in softer fruits, and therefore advances fruit ripening. The most important hormone pathway affecting fruit ripening is the ABA pathway. The application of exogenous MeJA may therefore affect the expression of genes involved in both the JA and ABA synthesis and metabolic pathways. We examined the expression of genes encoding four key enzymes in the JA synthesis pathway, including FaLOX, FaAOC, FaAOS, and FaOPDA1, as well as key genes involved in the ABA synthesis pathway, including FaNCED1, FaNCED2, FaNCED3, and FaAAO. Our results showed that the expression of FaLOX, FaAOC, FaAOS, and FaOPDA1 were somewhat upregulated after MeJA treatment (Fig. 6), and that the expression of
Fig. 3. Effects of jasmonic acid application on anthocyanin biosynthesis-related gene expression. Asterisks indicate statistically significant differences at p < 0.05, as determined by a Student’s t-test.
examined the expression of ripening metabolism-related genes to explain how JA promotes the ripening of strawberry fruit, as well as which genes may be affected by accelerating the fruit ripening process. First, we wanted to quantify differences in the expression of genes related to pigment metabolism, since the most obvious changes in fruit ripening are changes in colour. Based on previous work quantifying the expression of 6 genes involved in strawberry pigment metabolism (Table S1), including FaCHS, FaCHI, FaDFR, FaF3H, FaANS, and FaUFGT, it was found that the expression levels of FaCHS, FaCHI, FaF3H, and FaUFGT were all up-regulated during fruit ripening, but the greatest increases were observed in the expression of FaCHS and FaCHI, which increased about 5- to 8-fold for each gene (Fig. 3). This suggests that MeJA treatment may result in increased expression of some pigment metabolism-related genes, and that FaCHS and FaCHI may be the two genes most strongly affected by the advanced ripening of strawberry fruit in response to exogenous MeJA. In addition to upregulated genes, other genes such as FaDFR and FaANS, were found to be downregulated, which may indicate that MeJA inhibits the expression of these two genes, although the specific reasons for this downregulation need to be explored further. Flavour is an important indicator of strawberry fruit quality, and sugar content is the most important factor determining fruit flavor. Therefore, the expression of 10 sugar metabolism-related genes (Table S1), including FaSS, FaSPS1, FaSPS2, FaSUT1, FaSUT2, FaSUT3, FaSUT4, FaSUT5, FaSUT6, and FaSUT7, was examined. As shown in Fig. 4, increased MeJA was associated with increased expression for most of these genes, especially the sucrose synthase genes FaSPS1 and FaSPS2. These two genes play important roles in sucrose synthesis, indicating that MeJA treatment can promote the upregulation of sucrose synthase genes, thereby promoting sucrose synthesis and subsequently causing the accumulation of more sugar in strawberry fruit as well as accelerating fruit ripening. Gene expression results for the sucrose transporter family, including FaSUT1, FaSUT2, FaSUT3, FaSUT4, FaSUT5, FaSUT6, and FaSUT7, demonstrated that the expression of transporter genes was upregulated after sucrose synthesis. Thus, sucrose transport in strawberry fruit was also enhanced. Among these genes, the expression of FaSS was downregulated because it degrades sucrose in the sugar metabolism pathway. Taken together, our results showed that MeJA inhibits the expression of FaSS as well as the degradation of sugar, whereas the increased expression of other sugar
Fig. 5. Effects of jasmonic acid application on fruit softening-related gene expression. Asterisks indicate statistically significant differences at p < 0.05 as determined by a Student’s t-test. 253
Scientia Horticulturae 249 (2019) 250–256
Y. Han et al.
Fig. 6. Effects of jasmonic acid application on hormone metabolism-related gene expression. Asterisks indicate statistically significant differences at p < 0.05, as determined by a Student’s t-test.
FaAOC and FaAOS showed the strongest change in relative expression. This indicates that FaAOC and FaAOS are important for the JA synthesis pathway, as well as for strawberry fruit ripening. This result is not consistent with the results of several previous studies (Concha et al., 2013a, b; Preuß et al., 2014). This inconsistency may be caused by different concentrations of exogenous MeJA and by the different strawberry varieties used. Among the genes involved in ABA metabolism, the functions of FaNCED1, FaNCED2, and FaNCED3 in the promotion of fruit ripening in strawberry have been reported, although few studies on their relationship to JA have been published. The results of our analysis may also indicate that exogenous MeJA can promote upregulation of FaNCED1, FaNCED2, and FaNCED3, with a 10-fold increase in relative expression in the MeJA treatment than that in the control fruit. This suggests that JA and ABA somehow interact during strawberry fruit ripening. However, a detailed mechanism of this process should be studied further in the future.
Fig. 7. Effects of jasmonic acid application on strawberry fruit physiological indices. a. Jasmonic acid content; b. Anthocyanin content; c. Sucrose content; d. Fruit hardness. Asterisks indicate statistically significant differences at p < 0.05, as determined by a Student’s t-test.
et al., 2012) Meantime, other reports mainly focus on the function of MeJA in postharvest preservation of fruits and prevention of postharvest diseases (Matsui et al., 2004). However, there are few studies on the effect of jasmonic acid on fruit ripening and development, especially on the molecular mechanism of fruit ripening and development. Our research will provide a new idea for the role of jasmonic acid in fruit ripening. In this study, the endogenous JA content at different developmental stages was analyzed to determine the effect of JA on the ripening of strawberry fruit. Our results showed that JA content increases rapidly with fruit development, but then decreases sharply, reaching the lowest level when the fruits are fully ripe. Therefore, JA may have a very important role in the initiation of fruit ripening, and its content begins to decline in red fruits at the late ripening stage(Kondo, 2006). Jia et al. (2016) also showed a simaler results in grape, the JA content increased rapidly in the initiation of fruit ripening stage. At the later stage during fruit development, the JA content decreasd. At this stage the function of JA as an initiator of ripening is no longer needed, and this may be related to the function of JA in disease resistance (Agrawal et al., 2003; Zhang et al., 2005). Different concentrations of MeJA were used to treat strawberry fruits and the results showed that exogenous MeJA can also promote the ripening of strawberry fruit. Although different degrees of promotion were observed in response to different concentrations of JA, an overall promoting effect was consistently observed. This may indicate that JA is involved in fruit ripening, and this conclusion is consistent with research findings in grapes and apples (). Studies of MeJA-treated Kyoho grapes showed that MeJA caused increased anthocyanin content, promoted accelerated colouring, and increased the sugar-acid ratio in the fruit (D’Onofrio et al., 2018; Schaller, 2001). MeJA treatment has also been shown to accelerate colouring of apple fruits (Rudell et al., 2002). We found that the optimital concentrations of MeJA applied in strawberry is 230 μM. Moreover, jasmonic acid and methyl jasmonate are harmless to our body, which can be applied during fruit ripening and even after fruit ripening without causing harm to human body. Jasmonic acid will play an increasingly important role in fruit ripening and development in the future. We will do more in-depth research on this basis. Exogenous MeJA treatment of strawberry fruit revealed differences
3.4. Effects of MeJA on physiological indices during strawberry fruit ripening The results of analysis of JA, anthocyanin, and soluble sugar contents, as well as fruit hardness after exogenous MeJA treatment, indicated that exogenous MeJA treatment can increase JA, anthocyanin, and soluble sugar contents and fruit firmness (Fig. 7). These results are consistent with our gene expression results. This further demonstrated that MeJA treatment can promote the ripening of strawberry fruit. 3.5. Overexpression of FaAOC and FaAOS in strawberry fruit According to our gene expression analysis, we found that FaAOC and FaAOS are important for the JA synthesis pathway. Therefore, these two genes were overexpressed in strawberry fruit using transient transfection. The results showed (Fig. 8) that the transient expression of FaAOC and FaAOS in fruit also accelerated fruit ripening by 3–5 days compare to the control fruit. We conclude that JA is of great importance for the ripening of strawberry fruit. 4. Discussion Many studies have shown that jasmonic acid can induce the expression of defense genes in plants, and plant react quickly when it is injured to acquire the defense ability against foreign diseases. (Pei et al., 2017; Ba et al., 2016; Cao et al., 2014; Browse, 2009; Kaushc 254
Scientia Horticulturae 249 (2019) 250–256
Y. Han et al.
Fig. 8. FaAOS and FaAOC overexpression accelerated strawberry fruit ripening.
Funding
in the relative expression of four kinds of genes related to fruit ripening, including pigment metabolism genes, sugar metabolism genes, fruit ripening and softening-related genes, and hormone synthesis pathwayrelated genes (Zhao et al., 2013; Xu et al., 2015; Zhang et al., 2009). These changes suggest that JA plays an important role in the ripening of fruit by affecting multiple genes associated with fruit ripening rather than by a simple function. FaAOS and FaAOC genes play an important role in the jasmonic acid synthesis pathway (Kondo et al., 2007; Ashish et al., 2015). The results showed that the expression of these two genes also changed significantly after treatment with exogenous MeJA. Using overexpression lines of strawberry, we found that FaAOC and FaAOS may enhance the accelerated maturation of fruit by ˜3–5 days relative to a control condition. Therefore, FaAOS and FaAOC genes play a key role in the jasmonic acid synthesis pathway of strawberry fruit.This finding is consistent with previous results, indicating that MeJA can accelerate fruit colouring in strawberry. Based these results, we conclude that JA may promote the development and ripening of strawberry fruit, and that this finding may provide new directions for research on the regulation of strawberry fruit quality. A number of studies have shown that jasmonic acid is beneficial to the storage and preservation of fruits. Studies have shown that jasmonic acid treatment can inhibit strawberry fruit decay and prolong fruit shelf life (Gansser et al., 1997; Garrido-Bigotes et al., 2018). The effects of methyl jasmonate treatment on fruit colouring were also studied. Most of these studies focused on fruit disease resistance and shelf life extension after harvest, but there was little research on whether jasmonate could play a role in fruit development. In this study, jasmonic acid was used to regulate the coloring, aroma, softening, glycolic acid synthesis and hormone-related genes of strawberry fruit. The results showed that jasmonic acid could regulate the coloring, softening, glycolic acid synthesis and hormone-related gene expression, thus promoting fruit coloring and softening and achieving early fruit ripening. However, it was found that jasmonic acid did not regulate aroma synthesis genes significantly, and the reasons and mechanisms need to be further studied.
This work was supported by the National Natural Science Fund of China (31701898), the Natural Science Fund of Jiangsu Province, China (BK20160567), the Natural Science Fund of Jiangsu Provincial Department of Education, China (16KJB210016). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2019.01.061. References Agrawal, G.K., Jwa, N.S., Shibato, J., Han, O., Iwahashi, H., Rakwal, R., 2003. Diverse environmental cues transiently regulate OsOPR1 of the “octadecanoid pathway” revealing its importance in rice defense/stress and development. Biochem. Biophys. Res. Co. 310 (4), 1073–1082. Andi, S.A., Gholami, M., Ford, C.M., 2018. The effect of methyl jasmonate and light irradiation treatments on the stilbenoid biosynthetic pathway in Vitis vinifera cell suspension cultures. Nat. Prod. Res. 32 (8), 909–917. Ashish, R., Jyothilakshmi, V., Hitendra, K.P., Alok, P., Ramesh, P., Axel, M., Ramesh, V.S., 2015. Upregulation of jasmonate biosynthesis and jasmonate-responsive genes in rice leaves in response to a bacterial pathogen mimic. Funct. Integr. Genomics 15, 363–373. Ba, L.J., Kuang, J.F., Chen, J.Y., Lu, W.J., Ma, J.A.Z., 2016. Attenuates the MaLBD5Mediated transcriptional activation of jasmonate biosynthesis gene MaAOC2 in regulating cold tolerance of banana fruit. J. Agric. Food Chem. 64 (4), 738–745. Breithaupt, C., Kurzbauer, R., Lilie, H., Schaller, A., Strassner, J., Huber, R., Macheroux, P., Clausen, T., 2006. Crystal structure of 12-oxophytodienoate reductase 3 from tomato Self-inhibition by dimerization. P. Natl. Acad. Sci. USA 103 (39), 14337–14342. Browse, J., 2009. The power of mutants for investigationg jasmonate biosynthesis and signaling. Phytochemistry 70 (13-14), 1539–1546. Cao, S., Cai, Y., Yang, Z., Joyce, D.C., Zheng, Y., 2014. Effect of MeJA treatment on polyamine, energy status and anthracnose rot of loquat fruit. Food Chem. 145, 86–89. Concha, C.M., Figueroa, N.E., Poblete, L.A., Oñate, F.A., Schwab, W., Figueroa, C.R., 2013a. Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit. Plant Physiol. Biochem. 70, 433–444. Concha, C.M., Figueroa, N.E., Poblete, L.A., Onate, F.A., Schwab, W., Figueroa, C.R., 2013b. Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit. Plant Physiol. Biochem. 9 (70), 433–443. D’Onofrio, C., Matarese, F., Cuzzola, A., 2018. Effect of methyl jasmonate on the aroma of Sangiovese grapes and wines. Food Chem. 242, 352–361. Ellis, C., Turner, J.G., 2001. The Arabidopsis mutant cev1 has constitutively active jasmonate and ethylene signal pathways and enhanced resistance to pathogens. Plant
Conflict of interest The authors declare that they have no conflict of interests. 255
Scientia Horticulturae 249 (2019) 250–256
Y. Han et al.
Ichinose, Y., 2004. Structure and expression of 12-oxophytodienoate reductase (subgroup I) genes in pea, and characterization of the oxidoreductase activities of their recombinant products. Mol. Genet. Genom. 271 (1), 1–10. Pei, T., Ma, P., Ding, K., Liu, S., Jia, Y., Ru, M., Dong, J., Liang, Z., 2017. SmJAZ8 acts as a core repressor regulating JA-induced biosynthesis of salvianolic acids and tanshinones in Salvia miltiorrhiza hairy roots. J. Exp. Bot. https://doi.org/10.1093/jxb/ erx484. Preuß, A., Augustin, C., Figueroa, C.R., Hoffmann, T., Valpuesta, V., Sevilla, J.F., Schwab, W., 2014. Expression of a functional jasmonic acid carboxyl methyltransferase is negatively correlated with strawberry fruit development. J. Plant Physiol. 171 (15), 1315–1324. Rudell, D.R., Mattheis, J.P., Fan, X., 2002. Methyl jasmonate enhances anthocyanin accumulation and modifies production of phenolics and pigments in Fuji apples. J. Am. Soc. Hortic. Sci. 127 (3), 435–441. Saavedra, G.M., Figueroa, N.E., Poblete, L.A., Cherian, S., Figueroa, C.R., 2016. Effects of preharvest applications of methyl jasmonate and chitosan on postharvest decay, quality and chemical attributes of Fragaria chiloensis fruit. Food Chem. 190, 448–453. Schaller, F., 2001. Enzymes of the biosynthesis of octadecanoid-derived signalling molecules. J. Exp. Bot. 52 (354), 11–23. Wang, K., Jin, P., Shang, H., Zheng, Y., 2010. Effect of methyl jasmonate in combination with ethanol treatment on postharvest decay and antioxidant capacity in Chinese bayberries. J. Agr. Food Chem. 58 (17), 9597–9604. Wang, L.F., Qi, X.X., Huang, X.S., Xu, L.L., Jin, C., Wu, J., Zhang, S.S., 2016. Overexpression of sucrose transporter gene PbSUT2 from Pyrus bretschneideri, enhances sucrose content in Solanum lycopersicum fruit. Plant Physiol. Biochem. 105, 150–161. Xu, Y.Y., Li, H., Lin, J., Li, X.G., Chang, Y.H., 2015. Isolation and characterization of Calcineurin B-like gene (PbCBL1) and its promoter in birch-leaf pear (Pyrus betulifolia Bunge). Genet. Mol. Res. 14 (4), 16756–16770. Yao, H.J., Tian, S.P., 2005. Effects of pre- and post-harvest application of salicylic acid or methyl jasmonate on inducing disease resistance of cherry fruit in storage. Postharvest Biol. Tcehnol. 35, 253–262. Zhang, J., Simmons, C., Yalpani, N., Crane, V., Wilkinson, H., Kolomiets, M., 2005. Genomic analysis of the 12-oxo-phytodienoic acid reductase gene family of Zea mays. Plant Mol. Biol. 59 (2), 323–343. Zhang, H.Y., Ma, L.C., Mark, T., Xu, H.X., Dong, Y., Jiang, S., 2009. Methyl jasmonate enhances biocontrol efficacy of Rhodotorula glutinis to postharvest blue mold decay of pears. Food Chem. 117 (4), 621–626. Zhao, M.L., Wang, J.N., Shan, W., Fan, J.G., Kuang, J.F., Wu, K.Q., Li, X.P., Chen, W.X., He, F.Y., Chen, J.Y., Lu, W.J., 2013. Induction of jasmonate signalling regulators MaMYC2s and their physical interactions with MaICE1 in methyl jasmonate-induced chilling tolerance in banana fruit. Plant Cell Environ. 36 (1), 30–51. Zhao, N., Lin, H., Lan, S., Jia, Q., Chen, X., Guo, H., Chen, F., 2016. VvMJE1 of the grapevine (Vitis vinifera) VvMES methylesterase family encodes for methyl jasmonate esterase and has a role in stress response. Plant Physiol. Biochem. 102, 125–132.
Cell 13 (5), 1025–1033. Fait, A., Hanhineva, K., Beleggia, R., Dai, N., Rogachev, I., Nikiforova, V.J., Fernie, A.R., Aharoni, A., 2008. Recnfiguration of the achene and receptacle metabolic networks during Strawberry fruit development. Plant Physiol. 148 (2), 730–750. Fuleki, T., Francis, F.J., 1968a. Quantitative methods for anthocyanins. 1. Extraction and determination of total anthocyanin in cranberries. J. Food Sci. 33, 72–78. Fuleki, T., Francis, F.J., 1968b. Quantitative methods for anthocyanins. 2. Determination of total anthocyanin and degradation index for cranberry juice. J. Food Sci. 33, 78–82. Gansser, D., Latza, S., Berger, R.G., 1997. Methyl jasmonates in developing strawberry fruit (Fragaria×ananassa Duch. Cv. Kent). J. Agric. Food Chem. 45, 2477–2480. Garrido-Bigotes, A., Figueroa, P.M., Figueroa, C.R., 2018. Jasmonate metabolism and its relationship with abscisic acid during strawberry fruit development and ripening. J. Plant Growth Regul. 37, 101–113. Gonzalez-Aguilar, G.A., Fortiz, J., Cruz, R., Beaz, R., Wang, C.Y., 2000. Methyl jasmonate reduces chilling injury and maintains postharvest quality of mango fruit. J. Agr. Food Chem. 48 (2), 515–519. Huang, X., Li, J., Shang, H., Meng, X., 2015. Effect of methyl jasmonate on the anthocyanin content and antioxidant activity of blueberries during cold storage. J. Sci. Food Agric. 95 (2), 337–343. Jia, H., Zhang, C., Pervaiz, T., Zhao, P., Liu, Z., Wang, B., et al., 2016. Jasmonic acid involves in grape fruit ripening and resistant against botrytis cinerea. Funct. Integr. Genomics 16 (1), 79–94. Kausch, K.D., Sobolev, A.P., Goyal, R.K., Fatima, T., Laila-Beevi, R., Saftner, R.A., Handa, A.K., Mattoo, A.K., 2012. Methyl jasmonate deficiency alters cellular metabolome, including the aminome of tomato (Solanum lycopersicum L.) fruit. Amino Acids 42 (23), 843–856. Kondo, S., 2006. The role of jasmonates in fruit color development and chilling injury. Acta Hortic. 727, 45–56. Kondo, S., Yamada, H., Setha, S., 2007. Effect of jasmonates differed at fruit ripening stages on 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase gene expression in pears. J. Am. Soc. Hortic. Sci. 132, 120–125. Król, P., Igielski, R., Pollmann, S., Kępczyńska, E., 2015. Priming of seeds with methyl jasmonate induced resistance to hemi-biotroph Fusarium oxysporum f.sP. Lycopersici in tomato via 12-oxo-phytodienoic acid, salicylic acid, and flavonol accumulation. J. Plant Physiol. 179, 122–132. Kucuker, E., Ozturk, B., Celik, S.M., Aksit, H., 2014. Pre-harvest spray application of methyl jasmonate plays an important role in fruit ripening, fruit quality and bioactive compounds of Japanese plums. Sci. Hortic. 176 (2), 162–169. Li, D.P., Xu, Y.F., Sun, L.P., Liu, L.X., Hu, X.L., Li, D.Q., Shu, H.R., 2006. Salicylic acid, ethephon, and methyl jasmonate enhance ester regeneration in 1-MCP-treated apple fruit after long-term cold storage. J. Agric. Food Chem. 54 (11), 3887–3895. Lorenzo, O., Piqueras, R., Sanchez-Serrano, J.J., Solano, R., 2003. Ethylene response factor1 integrates signals from ethylene and jasmonate pathways in plant defense. Plant Cell 15 (1), 165–178. Matsui, H., Nakamura, G., Ishiga, Y., Toshima, H., Inagaki, Y., Toyoda, K., Shiraishi, T.,
256