Increased Glucosinolate Production in Brassica oleracea var. italica

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Increased Glucosinolate Production in Brassica oleracea var. italica Cell Cultures Due to Coronatine Activated Genes Involved in Glucosinolate Biosynthesis P. J. Sánchez-Pujante, A. B. Sabater-Jara, S. Belchí-Navarro, M. A. Pedreño, and L. Almagro* Department of Plant Biology, Faculty of Biology, University of Murcia, Campus de Espinardo, E-30100 Murcia, Spain

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S Supporting Information *

ABSTRACT: In this work, the effect of different elicitors and culture conditions on the production of glucosinolates in broccoli cell cultures was studied. The results showed that 0.5 μM coronatine was the best elicitor for increasing glucosinolate production (205-fold increase over untreated cells after 72 h of treatment). Furthermore, the expression levels of some genes related to the biosynthetic pathway of glucosinolates as well as three Myb transcription factors also have been studied. The highest glucosinolate levels found in coronatine-treated cells were closely correlated with the highest gene expression levels of Cyp79b2, Cyp83b1, St5a, Myb51, and Myb122 after 6 h of treatment. The data shown in this study provide new insight into the key metabolic steps involved in the biosynthesis of glucosinolates, which will be of use for future applications of metabolic engineering techniques in broccoli. KEYWORDS: broccoli suspension-cultured cells, coronatine, gene expression, glucosinolate

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family) were found in Arabidopsis thaliana.14 It is known that Myb34, Myb51, and Myb122 genes play a key role in the transcriptional regulation of indole glucosinolates,15 while Myb28, Myb29, and Myb76 genes play a role in the regulation of aliphatic glucosinolates.16−18 Previous works have indicated that dominant mutants or lines overexpressing Myb genes enhanced both the indole glucosinolate biosynthesis and the expression levels of some genes related to glucosinolate biosynthesis.17−19 Some authors have suggested that glucosinolate biosynthesis increases in response to herbivore attack, environmental factors, signaling molecules, elicitors, or pathogens.20,21 This increment could be partially mediated by hormones related with defense responses such as ethylene, jasmonic acid, and salicylic acid.21,22 Many studies using plant in vitro cultures showed that methyl jasmonate (MJ) is able to induce the biosynthesis of secondary metabolites.23−26 In fact, MJ induced a significant increase in both indole glucosinolate production and gene expression of Cyp79b2 and Cyp79b3 in A. thaliana.20 Moreover, coronatine (Cor) is a toxin produced by the phytopathogenic bacterium Pseudomonas syringae,27 and it acts as an analogue of jasmonate derivatives in plants.28 However, although there is much evidence that jasmonic acid or MJ increases the production of secondary metabolites in plants,29,30 little is known about the effect of Cor on the production of secondary metabolites. In recent studies, it was shown that Cor enhanced the biosynthesis of secondary metabolites in different plant suspension-cultured cells (SCC).31,32

INTRODUCTION Broccoli (Brassica oleracea L. var. italica) is a popular vegetable whose consumption has increased worldwide due to its high nutritive value.1 Broccoli has a wide range of benefits for human health because it is a good source of high-value natural products.2 Some of the beneficial effects of broccoli have been attributed to phenolic compounds and glucosinolates because they have antioxidant activity.3 Broccoli is also known as a protein-rich vegetable, even though there are few studies related with this topic.1 Epidemiological studies indicated that the intake of broccoli might decrease the risk for lung, stomach, colon, and rectal cancers, probably as a result of the glucosinolates and derivative products it contains.4 Glucosinolates are secondary metabolites that mainly belong to the Brassicaceae family.5 Their chemical structure is formed by β-thioglycoside N-hydroxysulfates bound to a sulfur β-Dglucopyranose, and they differ in the side chain derived from amino acids. These glucosinolates are classified into three different groups: aromatic, aliphatic, or indole glucosinolates.6 Glucosinolates are precursors of isothiocyanates, which are formed by the action of myrosinase, and they have been suggested to present antitumoral and antioxidant activities.6−8 Myrosinase is released and glucosinolate hydrolysis occurs when the plant cells are injured.9 The biosynthetic pathway of glucosinolates has been characterized using different genetic and biochemical methods.10,11 There is evidence of the involvement of P450 enzymes (CYP79B2, CYP79B3, CYP83B1, CYP79F1, CYP79F2, CYP79F3, and CYP79F4) as responsible for catalyzing the conversion of tryptophan12,13 to the corresponding aldoximes, which are then transformed to indole glucosinolates (Figure 1). On the other hand, some MYB transcription factors involved in the biosynthetic pathway of glucosinolates (R2R3MYB © XXXX American Chemical Society

Received: September 3, 2018 Revised: December 13, 2018 Accepted: December 13, 2018

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DOI: 10.1021/acs.jafc.8b04298 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Metabolic pathway involved in the biosynthesis of indole glucosinolates. CYP79B2, CYP79B3, CYP83B1, CYP81F1, CYP81F2, CYP81F3, and CYP81F4: cytochrome P450; GSTF9 and GSTF10: phi glutathione S-transferases; SUR1: carbon−sulfur lyase; UGT74B1: uridine diphosphate glycosyltransferase; ST5a: sulfotransferase; IGMT1, IGMT2: indole glucosinolate methyltransferase; MYB34, MYB51, and MYB122: R2R3MYB transcription factors. Calli and SCC were maintained under the same conditions described for in vitro plants. Moreover, we analyzed the cell growth of broccoli SCC by measuring the fresh weight (FW) in g/L and the conductivity of the extracellular culture medium in mS/cm for 14 days, because cell growth remained constant from day 9 to day 14. In addition, several growth kinetic parameters in these broccoli SCC, such as Vexp, which is the biomass increment during the linear stage of the exponential phase, and Ta, which is the time where it is necessary to carry out a subculture, were calculated. Elicitation of Broccoli Suspension-Cultured Cells. The experiments of elicitation were performed using 7-day-old broccoli SCC. The broccoli cells were incubated in 30 mL of culture medium as described earlier (Murashige and Skoog basal salt mixture, casein hydrolysate, Morel vitamins, sucrose, benzyladenine, and naphthaleneacetic acid) using a high cell density (200 g of FW/L), and these elicited SCC were maintained under the same temperature and photoperiod conditions described above, in a rotary shaker at 110 rpm. Treatments were carried out in triplicate by adding 50 mM cyclodextrins (CD), 100 μM MJ, 17 mM NaCl, or 1 μM Cor, separately or in combination with the culture medium, and leaving it for 168 h (7 days). This elicitation time was chosen as the standard time for the production of glucosinolates in broccoli SCC as described in previous works using other plant SCC.26,35 Moreover, to analyze the effect of different concentrations of Cor on glucosinolate production, broccoli SCC were treated with 0.5, 1, and 2 μM Cor for 6, 24, 48, 72, 144, 168, and 216 h using MS basal salt mixture,33 30 g/L sucrose, and a cell density of 200 g of FW/L. To increase the glucosinolate production, different sucrose concentrations were also analyzed. For that, broccoli cells were transferred into culture medium that contained 15, 30, or 45 g/L (which means 43.8, 87.6, and 131.5 mM, respectively) sucrose at the beginning of the experiments using MS basal salt mixture33 and a cell density of 200 g of FW/L. Furthermore, we also performed elicitation experiments using two different Murashige and Skoog basal salt mixtures: MS basal salt mixture (MS)33 or MS modified (MSM) containing 1/2 MS

Because of the health benefits of glucosinolates, it is interesting to increase their production by alternative sources to conventional crops. In this sense, plant in vitro cultures turn out to be a promising system to obtain glucosinolates because they are independent of climatic and edaphic conditions. Moreover, the use of plant cell cultures does not suppose any interaction with the ecosystem because these cell cultures are obtained under sterile conditions and they do not compete with conventional crops, mainly destined for the food industry.11 In this work, we analyzed the effect of different elicitors and conditions on the glucosinolate production, as well as the expression profile of several genes related to their biosynthetic pathway, including Myb genes involved in their transcriptional regulation in broccoli SCC.

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MATERIALS AND METHODS

Plant Material. B. oleracea L. var. italica cv. Chronos plants were germinated in vitro to obtain sterile explants. First, seeds of broccoli were disinfected with ethanol (70%, 1 min) followed by treatment with 1.5% sodium hypochlorite solution for 10 min, which contained 0.1% Tween 20. The seeds were then transferred into tubes containing the medium of germination consisting of Murashige and Skoog basal salt mixture,33 250 mg/L casein hydrolysate, Morel vitamins,34 30 g/L sucrose, and 6.2 g/L agar. In vitro plants were grown under a 16 h light and 8 h dark photoperiod with a light intensity of 18.4 W/m2 at 25 °C. Broccoli calli were obtained from hypocotyl derived from 20-dayold in vitro plants. Callus induction was carried out using the medium described earlier containing a hormonal dose of 1 mg/L naphthaleneacetic acid and 5 mg/L benzyladenine. Finally, broccoli SCC were initiated as described by Almagro et al.,32 using the culture medium described earlier for calli in the absence of plant agar, and they were maintained in a rotary shaker at 110 rpm, subculturing every 7 days. B


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Journal of Agricultural and Food Chemistry macroelements. These experiments were carried out using 0.5 or 1 μM Cor for 72 h in broccoli SCC using a cell density of 200 g of FW/ L. Finally, the effect of cell density on total glucosinolate levels was analyzed using four initial cell densities (50, 100, 200, and 300 g of FW/L), in the absence or in the presence of 0.5 or 1 μM Cor for 72 h using MS basal salt mixture33 and 30 g/L sucrose. During the experiments, cell viability was evaluated as described by Duncan and Widholm.36 Extraction and Quantification of Glucosinolates. Glucosinolates were extracted as described by Helliń et al.37 from 200 mg of freeze-dried cells. Four glucosinolates (glucobrassicin, 4-hydroxyglucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin) were identified and quantified using a high-performance liquid chromatograph mass spectrometer (HPLC/MS) as described by Helliń et al.37 Glucobrassicin was quantified with respect to its corresponding standard while neoglucobrassicin, 4-hydroxyglucobrassicin, and 4metoxyglucobrassicin were extracted from rapeseed and quantified with respect to glucobrassicin as described by Helliń et al.37 RNA Isolation, cDNA Synthesis, and Real-Time Quantitative Polymerase Chain Reaction. Total RNA was obtained using TRIZOL reagent following the manufacture’s recommendations. The concentration of RNA was determined optically using a NanoDrop ND-2000 (NanoDrop Technologies, Inc., U.S.A.). To check the integrity of RNA, we carried out a 1.5% agarose gel electrophoresis, which was stained with RedSafe (iNtRON Biotechnology, Korea). First-strand cDNA was synthesized as described by Almagro et al.32 The primers (table in Supporting Information) were used to perform quantitative real-time polymerase chain reaction (qRT-PCR) experiments in a QuantStudio 5 Flex (Applied Biosystems, Spain) as described by Almagro et al.32 Primers were designed with OligoAnalyzer 3.1 software (Supporting Information), and the efficiency reaction for each primer pair was assessed as described by Miras-Moreno et al.38 The expression values were normalized with respect to Actin (GenBank: FJ969844.1) gene, which was used as a housekeeping gene. The expression levels of Myb122, Myb51, Myb34, Cyp79b2, Cyp83b1, St5a, and Gstf 9 genes were analyzed at 6, 24, 48, and 72 h after treatment with 0.5 μM Cor. Statistical Analysis. For each experiment, three independent biological replicates were used. Statistical analysis was performed using the SPSS package (SPSS, Inc., Chicago, U.S.A.) version 22.0. A two-way analysis of variance (ANOVA) and Tukey HSD posthoc test were carried out. P values 0.05). By contrast, Myb34 transcription levels increased

Figure 5. Effect of different concentrations of coronatine (Cor) on cell growth of broccoli SCC treated for 216 h. Data are the means of three independent replicates ± SD.

Cor, significant differences in cell growth were detected compared with control SCC. The cell biomass increased from 13 g of DW/L to 41 g of DW/L in control SCC after 216 h of incubation, whereas in the presence of Cor, a slight increase in biomass (from 12.7 to 21 g of DW/L) was observed at the end of the experiment (216 h). In addition, a decrease in cell viability was observed in elicitor-treated cells (Supporting Information figure), with the lowest levels being reached after 216 h of treatment with 2 μM Cor. In agreement with our results, other works showed that Cor decreased the cell growth of plant cells.53 Taurino et al.,48 Onrubia et al.,31 and Almagro et al.32 observed that Cor caused a decrease in cell growth in V. E


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Figure 6. Relative expression of Cyp79b2 (A), Cyp83b1 (B), Gstf 9 (C), and St5a (D) genes in broccoli SCC treated with 0.5 μM coronatine (Cor) at 6, 24, 48, and 72 h of treatment. Levels of transcripts were calculated using the Actin gene as internal control. Values are given as the mean ± SD of one experiments with three replicates. F-values from two-way ANOVA significant at the 99.9% (***), 99% (**), or 95% (*) level of probability.

compared with control plants.20 In our study, the increase in glucosinolates (Figure 4) and the expression levels of genes involved in their biosynthetic pathway (Figure 6) suggested that these genes might be regulated by MYB34, MYB51, and MYB122 transcription factors in Cor-treated cells. More specifically, we observed a high expression of Myb34 gene, as occurs in A. thaliana plants in the presence of jasmonate.60 Therefore, Cor and MJ seem to activate the cell-signaling pathways that trigger the induction of Myb34, Myb51, and Myb122, and these, in turn, regulate genes involved in indole glucosinolate biosynthesis and production. Effect of Sucrose Concentration, Basal Medium, and Cell Density on Total Glucosinolate Content in Broccoli Suspension-Cultured Cells Elicited with Coronatine. The composition of the culture medium is important for enhancing the production of secondary metabolites in plant in vitro cultures. In fact, both the growth and production of secondary metabolites are influenced by many of the constituents of the culture medium.61 In the case of

exponentially, with the highest levels being detected at 72 h of cultivation (>320-fold increase over control SCC; Figure 7C). In this study, all the genes analyzed showed a significant increase in the gene expression after the elicitation with Cor (Figures 6 and 7). In general terms, high expression of genes under methyl jasmonate treatment is associated with high levels of glucosinolate production. 39,40,49 In fact, the application of jasmonic acid leads to an increase in both some Myb genes and glucosinolate biosynthesis genes in A. thaliana plants.59 Our results suggested that both indole glucosinolate biosynthesis and the transcription factor genes are markedly upregulated by Cor, triggering a higher biosynthesis of indole glucosinolates. Similar results were presented by Yi et al.,57 who observed that MJ, whose function is similar to that of Cor in plants, enhanced the expression of Myb34, Myb51, and Myb122 genes, with the expression levels being 2-fold higher in cabbage than in broccoli and kale plants. In A. thaliana plants, MJ also induced some Cyp genes that increased the biosynthesis of indole glucosinolates 3- to 4-fold F


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Figure 7. Relative expression of transcription factors Myb51 (A), Myb122 (B), and Myb34 (C) in broccoli SCC treated with 0.5 μM coronatine (Cor) at 6, 24, 48, and 72 h of treatment. Levels of transcripts were calculated using the Actin gene as internal control. Values are given as the mean ± SD of one experiments with three replicates. F-values from two-way ANOVA significant at the 99.9% (***), 99% (**), or 95% (*) level of probability.

glucosinolates, the modification of the culture medium is important because it alters the production of these compounds in plant in vitro cultures.39,62 In this work, we have analyzed the effects of three concentrations of sucrose on glucosinolate production in broccoli SCC after 72 h of treatment, because in previous experiments (Figure 4) the optimal time for the production of glucosinolates was fixed at 72 h (3 days). In these experiments, 6 g of fresh cells were transferred into 30 mL of culture medium, which contained 15, 30, or 45 g/L sucrose as well as 0.5 and 1 μM Cor (Table 1). As can be observed in Table 1, the production of glucosinolates is dependent on the sucrose concentration in the culture medium. As expected, the total glucosinolate content was influenced not only by the sucrose concentration but also by the presence of 0.5 and 1 μM Cor, as well as by the interaction of both factors (p < 0.001). Although the maximal levels of glucosinolates using 0.5 μM Cor were reached in the presence of 45 g/L sucrose (15.34 ± 1.32 mg/g of DW), no significant differences were observed when the culture medium was supplemented with 30 g/L sucrose. Therefore, the optimal conditions were those when broccoli SCC were treated with 0.5 μM Cor and using 30 g/L sucrose (13.62 ± 0.32 mg/g of DW) because the carbon source concentration is an important parameter in terms of the economy of the process. These results indicated that sucrose concentration present in the culture medium is important because it is the energy source for plant cells, although it may also be involved in other physiological processes regulating the production of secondary metabolites.26 In previous works, two mechanisms have been associated with the effect of sugars on plant cells: an altered cellular metabolism and osmotic stress.63 On the other hand, it would be interesting to verify whether changes in basal nutrients of the culture medium lead to differences in glucosinolate production in broccoli SCC. In this sense, it is known that low levels of nitrogen and phosphorus enhanced the flavonol production in A. thaliana and tomato seedlings.64 Furthermore, Lattanzio et al.65 also showed that Origanum vulgare shoot cultures produced more phenolic compounds when these shoot cultures were grown in a halfstrength MS medium. However, our results indicated that, when broccoli SCC were grown in a culture medium that

Table 1. Effect of Sucrose Concentration, Basal Salt Mixture, and Cell Density on Total Glucosinolate Content in Broccoli SCC Elicited with Different Concentrations of Coronatine (Cor) for 72 ha total glucosinolate content (mg/g of DW) 0.5 μM Cor

1 μM Cor

11.930 ± 0.168 b 13.621 ± 0.321 a,b 15.348 ± 1.326 a

12.604 ± 0.659 b 11.897 ± 0.141 b 15.451 ± 0.457

13.621 ± 0.321 a 8.152 ± 0.982 b

11.897 ± 0.141 8.897 ± 2.787

11.797 ± 1.100 b 13.905 ± 0.902 a 9.812 ± 0.624 b 10.526 ± 0.006 b F-value

8.803 ± 0.685 c 13.905 ± 0.506 a 10.327 ± 0.393 b 9.614 ± 0.081 c

control [sucrose] (g/L) 15 0.155 ± 0.041 30 0.134 ± 0.038 45 0.184 ± 0.055 basal medium MS 0.134 ± 0.038 MSM 0.158 ± 0.064 cell density (g of FW/L) 50 0.167 ± 0.097 100 0.268 ± 0.113 200 0.239 ± 0.050 300 0.146 ± 0.009

treatment (A) culture condition (B) A×B

[sucrose] (g/L)

basal medium

cell density (g of FW/L)

2582.827*** 14.088***

149.395*** 7.688**

1647.915*** 28.251***

13.612***

24.138***

12.062***

a

Different letters denote statistically significant differences between different conditions in each elicitor treatment according to Tukey’s test (p < 0.05). F-values from two-way ANOVA significant at the 99.9% (***), 99% (**), or 95% (*) level of probability. DW, dry weight; FW, fresh weight; MS, Murashige and Skoog basal medium; MSM, MS basal medium that contains the macronutrients reduced by half.

contained the reduced salt mixture (MSM), there was a decrease in the amount of glucosinolates (p < 0.01) at all the concentrations of Cor used (Table 1). Therefore, a high concentration of macronutrients in the culture medium would lead to a high production of glucosinolates in broccoli SCC treated with Cor. G


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As regards the cell density used in these experiments, the only statistically significant differences were observed when broccoli SCC were treated with 0.5 or 1 μM Cor using an initial cell density of 100 g/L (Table 1). These results could be explained by the fact that in this work broccoli SCC started with different cell densities were elicited with the same amount of Cor, and therefore, the number of interactions between the receptors of cell membranes and the elicitor will be higher when the cell amount is low. This was also found in V. vinifera SCC elicited with CD and MJ, where the maximal production of trans-resveratrol was found using low cell densities.26 However, the production of glucosinolates decreased using a cell density of 50 g/L (11.79 ± 1.10 mg/g of DW) compared to 100 g/L (13.90 ± 0.90 mg/g of DW) in the presence of 0.5 μM Cor (Table 1). This could be due to the decreased cell growth when broccoli SCC were initiated with the lowest cell density used (50 g/L). In fact, inoculum density is a key parameter that can alter biomass accumulation, growth, and the production of metabolites in plant cell cultures.66−69 In this sense, some authors analyzed the ajmalicine production from Catharanthus roseus cell cultures using different inoculum densities, and they observed that the production of ajmalicine was significantly lower when low cell densities were used.69 In conclusion, from the elicitors analyzed in this work, Cor is the best treatment for increasing the levels of glucosinolates. Although cell growth decreased in the presence of Cor, the production of glucosinolates increased 205- and 185-fold in the presence of 0.5 and 1 μM Cor, respectively, at 72 h of treatment compared to control conditions. Hence, Cor is an efficient elicitor for increasing the biosynthesis of glucosinolates in broccoli SCC. In addition, to increase the glucosinolate content using our broccoli cell line, the optimal time for the production of these compounds was 72 h of elicitation. Moreover, the amount of sucrose, basal salt medium, and cell density used seemed to be limiting factors in the glucosinolate production process, with the maximal levels of glucosinolates being reached when broccoli SCC were treated with 0.5 μM Cor and grown in a MS basal medium with 30 g/L sucrose using a low cell density (100 g/L). The study of the expression profile of genes related to biosynthetic pathway of glucosinolates provides new insights into the key metabolic steps that could be used in metabolic engineering. Taken together, the results suggest that glucosinolate production using plant in vitro cultures treated with Cor could be considered as an alternative procedure to their extraction from whole plant.

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This work has been supported by the Ministerio de Economiá y Competitividad (no. BIO2017-82374-R) and Fundación Seneca-Agencia de Ciencia y Tecnologiá de la Región de Murcia (no. 19876/GERM/15). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED Cor, coronatine; CD, cyclodextrins; CYP, cytochrome P450; DW, dry weight; FW, fresh weight; MJ, methyl jasmonate; MS, MS basal salt mixture; MSM, MS modified; SCC, plant suspension-cultured cells

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b04298. Primer pairs used for real-time quantitative PCR and cell viability in broccoli SCC treated with coronatine (PDF)

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REFERENCES

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*E-mail: [email protected]. Phone: +34868884904. Fax: +34868883963. ORCID

L. Almagro: 0000-0001-8393-2943 H


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Increased Glucosinolate Production in Brassica oleracea var. italica

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