Biosynthesis and nutritious effects

CHAPTER 2

Biosynthesis and nutritious effects Quan V. Vo1 1

Department of Natural Sciences, Quang Tri Teachers Training College, Quang Tri Province, Viet Nam

Contents 2.1 Introduction 2.2 Biosynthesis of glucosinolates 2.2.1 General biosynthesis of GLs 2.2.1.1 The synthesis of GLs in plants 2.2.1.2 Side-chain modifications

2.2.2 Biosynthesis of indole glucosinolates 2.2.3 Biosynthesis of aliphatic glucosinolates 2.3 The effects of plant in vitro culture conditions on biosynthesis and levels of GLs 2.3.1 The effects of phytohormones 2.3.2 Effects of level of cell differentiation 2.3.3 The effects of elicitors 2.3.4 The effects of metabolic engineering strategies 2.4 Effects of nutrition and other factors on levels of glucosinolates in plants 2.4.1 The effects of potassium 2.4.2 The effects of sulfur 2.4.3 The effects of nitrogen 2.4.4 The effects of other factors 2.5 Conclusion Acknowledgments References

47 48 48 50 51 52 53 54 54 54 55 56 58 58 60 62 63 64 64 64

2.1 Introduction In recent years, beside the significant development of total synthesis of artificial glucosinolates (GLs) (Rollin and Tatibouët, 2011; Vo et al., 2018, 2013a, 2013b, 2014), the GL biosynthetic pathway has attracted much attention for all of the areas including genetic regulation, biosynthetic pathway, controlled transcription factors, and references therein (Grubb and Abel, 2006; Halkier, 1999; Halkier and Du, 1997; Halkier and Gershenzon, Glucosinolates: Properties, Recovery, and Applications ISBN 978-0-12-816493-8 https://doi.org/10.1016/B978-0-12-816493-8.00002-0

Copyright © 2020 Elsevier Inc. All rights reserved.

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Glucosinolates: Properties, Recovery, and Applications

2006; Ishida et al., 2014; Mikkelsen et al., 2002; Mithen, 2001; Sanchez-Pujante et al., 2017; Sønderby et al., 2010; Wink, 2011; Yan and Chen, 2007). It is generally observed that most studies about the biosynthesis of GLs focus on three different types (aliphatic, indole, and aromatic) of important GLs that depend on the amino acid nature. In which, the typical natural amino acids including leucine, valine, methionine, isoleucine, and alanine were responsible for the biosynthesis of aliphatic GLs; tryptophan was responsible for the biosynthesis of indole GLs, and the aromatic GLs were supposed to be formed from phenylalanine or tyrosine. Although the biosynthetic pathway of aliphatic and indole GLs is well known, that of aromatic GLs remains a largely open question (Liu et al., 2016; Sanchez-Pujante et al., 2017).

2.2 Biosynthesis of glucosinolates 2.2.1 General biosynthesis of GLs The biosynthesis of GLs includes three independent steps (Figs. 2.1 and 2.2). Firstly, the synthesis of chain-elongated amino acids; secondly, the synthesis of GLs which include three minor steps: aldoxime formation, formation of thiohydroximic acids, and formation of desulfo-GLs and GLs; and, finally, modifications of the side chain (Halkier and Du, 1997; Halkier and Gershenzon, 2006; Mikkelsen et al., 2002; Nour-Eldin and Halkier, 2009). The chain elongation reaction occurs in the various extended methionine and phenylalanine precursor amino acids, but it has not been observed in the biosynthesis of indole GLs (Ishida et al., 2014; SanchezPujante et al., 2017). The mechanism of this process is similar to the formation of leucine from acetate and valine (Strassman and Ceci, 1963). First, the deamination of the amino acid produces an a-keto acid, which is then extended by condensation with acetyl-CoA. After that, a rearrangement of

Figure 2.1 Chain elongation of amino acid.

Biosynthesis and nutritious effects

49

Figure 2.2 Biosynthetic pathways of cyanogenic glucosides and glucosinolates (Mikkelsen et al., 2002).

the hydroxyl group occurs prior the decarboxylation of the dicarboxy acid that forms a chain-elongated a-keto acid. Finally, the chain-elongated amino acid is produced in a transamination reaction (Mikkelsen et al., 2002; Robin et al., 2016; Sanchez-Pujante et al., 2017; Fig. 2.1). The data

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Glucosinolates: Properties, Recovery, and Applications

of studies in 14C-labeled and 15N-labeled have confirmed the pathways in the biosynthesis of GLs (Chisholm and Wetter, 1964; Graser et al., 2001, 2000; Matsuo and Yamazaki, 1964; Mithen and Campos, 1996; Schuster et al., 2006; Underhill et al., 1973). 2.2.1.1 The synthesis of GLs in plants The first step in synthesis of GLs is aldoxime formation. The formation of the aldoxime structure by the action of cytochromes P450 (CYP) from the CYP79 family is the first common step of the precursor amino acids (Figs. 2.2 and 2.3; Bak et al., 1998; Chen et al., 2003; Halkier et al., 2002; Hansen et al., 2001; Hull et al., 2000; Kutz et al., 2002; Mikkelsen et al., 2000, 2002; Nafisi et al., 2006; Naur et al., 2003; Selmar, 2005; Weis et al., 2014). The studies have shown that recombinant CYP79A2 was able to convert L-[14C]phenylalanine into the (E)- and (Z) isomers of phenylacetaldoxime (Mikkelsen et al., 2002). Both CYP79B2 and CYP79B3 were heterologously expressed in Escherichia coli and shown to convert tryptophan into indole-3-acetaldoxime (Hull et al., 2000; Mikkelsen et al., 2000). The heterologous expression of CYP79F1 in E. coli showed that the recombinant enzyme was able to convert homo-, dihomo-, trihomo-, tetrahomo-, pentahomo-, and hexahomomethionine into the respective (E)- and (Z) isomers of corresponding aldoximes (Hansen et al., 2001), whereas the enzyme CYP79F2 was only able to convert long-chain methioninederived amino acids such as penta- and hexahomomethionine into the corresponding aldoximes (Mikkelsen et al., 2002). The second step is the formation of thiohydroximic acids. The aldoximes are converted into GLs by a sequence of reactions shown in Fig. 2.2. In the first step, the oxidation of the aldoxime is accelerated by the catalysts CYP83A1 or/and CYP83B1 (Bak et al., 2001; Hansen et al., 2001a). The

Figure 2.3 An overview of the endogenous and exogenous CYP79s used in metabolically engineered Arabidopsis plants (Mikkelsen et al., 2002).

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51

aldoxime is first oxidized to produce an aci-nitro compound or a nitrile oxide compound, which functions as the acceptor for the thiol donor (Ettlinger and Kjaer, 1968). After that, the thiohydroximic acid is formed by cleavage of the S-alkylthiohydroximate in a reaction thought to be catalyzed by a CeS lyase (Fig. 2.2; Wallsgrove and Bennett, 1995). While CYP83B1 was shown to have a high affinity for tryptophan- and phenylalanine-derived aldoximes (Bak and Feyereisen, 2001), it has a very low affinity to metabolize the aliphatic aldoximes (Mikkelsen et al., 2002). At the same time, CYP83A1 has high affinity for the aliphatic aldoximes, but it does not affect aromatic aldoximes and has a low affinity for indole-3acetaldoxime (Bak and Feyereisen, 2001). However, it was demonstrated that CYP83A1 and CYP83B1 were not redundant in the plant under normal physiological conditions (Mikkelsen et al., 2002). The last step is the formation of desulfo-GLs and GLs. The thiohydroximic acid is then S-glucosylated by a soluble uridine 50 -diphosphoglucuronic acid (UDPG): thiohydroximate glucosyltransferase to yield a desulfo-GL that is subsequently converted into the GL by a 30 -phosphoadenosine-50 -phosphosulfate (PAPS): desulfoglucosinolate sulfotransferase (Fig. 2.2; Halkier and Du, 1997; McWalter et al., 2004; Mikkelsen et al., 2002). In the case of biosynthesis of indole GLs, most of steps for the formation of GL structure are similar to that described for the aliphatic GLs, except for the elongation of tryptophan by introducing methylene group (Robin et al., 2016; Sanchez-Pujante et al., 2017). 2.2.1.2 Side-chain modifications Secondary modifications of the side chain may occur following the formation of the basic GL (Fig. 2.2). These include oxidation, methoxylation, desaturation, hydroxylation, sulfation, and glucosylation. The studies demonstrated that the diversity is the result of genetic variation in only three major loci: Gsl-oxid, Gsl-alk, and Gsl-oh, despite the great variation in aliphatic side-chain structures (Giamoustaris and Mithen, 1996; Gigolashvili et al., 2007; Mithen et al., 1995). Thus, other GLs are formed from the basic GLs. To control the biosynthesis and metabolism of GLs in plants, several transcription factors have been studied (Brader et al., 2001; Celenza et al., 2005; Frerigmann and Gigolashvili, 2014; Gigolashvili et al., 2009; Hirai et al., 2007; Ishida et al., 2014; Kliebenstein et al., 2002; Levy et al., 2005; Malitsky et al., 2008; Mewis et al., 2005; Mikkelsen et al., 2003; Skirycz et al., 2006; Sønderby et al., 2007). Most of the studied transcription factors, the MYBs, IQDs, and Dofs, were mainly regulated for the

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Glucosinolates: Properties, Recovery, and Applications

biosynthesis and metabolism of indole and aliphatic GLs (Sanchez-Pujante et al., 2017), while the regulation of biosynthesis of aromatic GLs remain unknown and need further investigation.

2.2.2 Biosynthesis of indole glucosinolates Biosynthesis of indole GLs has been of interest in recent years. Studying biosynthesis of GLs may lead to an understanding of how GLs are established in plants. From that understanding, it may be possible to set up chemical syntheses of GLs and study the bioassays of the intermediates in the hydrolysis of GLs (Pedras and Okinyo, 2008). It was found that the biosynthesis of indole GLs was complex, but by studying the metabolites of labeled compounds, the biosynthesis of glucobrassicin and neoglucobrassicin could be derived from L-tryptophan (Andersen and Muir, 1966; Bednarek et al., 2009; Dombrecht et al., 2007; Gigolashvili et al., 2007; Ludwig-Müller et al., 1999; Mikkelsen et al., 2000; Pedras et al., 2010, 2008; Pfalz et al., 2009; Piotrowski et al., 2004; Rausch et al., 1983; Schlaeppi et al., 2010; Schuster et al., 2006; Siemens et al., 2008; Stotz et al., 2011; Sugawara et al., 2009). The effects of chemicals on the biosynthesis of indole GLs were reported by Jirácek et al. (1971, 1974a,b). The first study was into the effects of alloxan, Na2MoO4, and MnCl2 on the amounts of glucobrassicin and neoglucobrassicin in rape seedlings. It was shown that alloxan at 102 and 104 M caused inhibition of glucobrassicin and neoglucobrassicin formation, especially in 2-day-old germinating seeds. In older seedlings, alloxan did not affect the content of glucobrassicin, but caused serious inhibition of neoglucobrassicin formation: the decrease was 50%e84% (P < .05) at all concentrations studied. In contrast, Na2MoO4 102 M did not cause significant change in the content of glucobrassicin and neoglucobrassicin, but at 103 M concentration it caused a large increase of GB content (þ385%, P < .01) as well as neoglucobrassicin content (þ60%, P < .01) in 4-day-old rape seedlings (Jirácek et al., 1971). From the results, it was clear that alloxan in both cases exerted mainly an inhibiting effect on indole GL biosynthesis. Molybdate in younger intact seedlings stimulated the formation of both GLs especially on the fourth day; later, its effect was not significant. Following this study, the effects of zinc and copper on the content and the biosynthesis of glucobrassicin and neoglucobrassicin in etiolated rape seedling was reported (Jirácek et al., 1974a,b). It was shown that zinc ions at chronic long-term application increased the

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53

glucobrassicin and neoglucobrassicin levels in seedlings, whereas in the shortterm, zinc ions increased the levels of both GLs except for the concentration of zinc ions around 104 to 105 M. The zinc ions did exhibit a specific effect on neoglucobrassicin biosynthesis, on membrane permeability as against sulfate ions, and on the incorporation of sulfur into proteins (Jirácek et al., 1974a). On the other hand, copper ions increased the levels of glucobrassicin and neoglucobrassicin in the concentration range of 5  104 to 105 M. Furthermore, Cu2þ in higher concentration increased the uptake of sulfate ions by hypocotyl segments, and lower concentrations increased the incorporation of 35S from 35SO2 cek et al., 1974a,b). 4 into proteins (Jirá

2.2.3 Biosynthesis of aliphatic glucosinolates As typical natural GLs, biosynthesis of aliphatic GLs has been concerned in many studies (Binder, 2010; Chen et al., 2003; Chisholm and Matsuo, 1972; Davila et al., 2017; Hansen et al., 2001b; Kong et al., 2016; Magrath et al., 1994; Meenu et al., 2015; Miao et al., 2013; Mucha et al., 2015; Niu, 2008; Yatusevich, 2008; Zhang et al., 2015a). Following the general biosynthesis of GLs pathways, the important steps in the biosynthesis of aliphatic GLs are side-chain elongation and side-chain modification (Chisholm and Matsuo, 1972; Magrath et al., 1994), thus most of the studies focused on the modulation biosynthetic genes of aliphatic GLs. The study on the genetic regulation of side-chain length by analyzing recombinant populations of Brassica napus and Arabidopsis thaliana demonstrated that the presence or absence of propyl GLs was regulated by a single locus (Gsl-pro), whereas butyl and pentyl GLs were regulated by two other loci (Gsl-elong-C and Gslelong-A). However, the transformation of a B. napus line with the Gsl-elongAr gene (or a Brassica homologue) could affect the biosynthesis and reduce the total amount of aliphatic GLs (Magrath et al., 1994). The study over the conversion of amino acids into oximes in the biosynthesis of aliphatic GLs indicated that the biosynthesis of long-chain aliphatic GLs could be regulated by CYP79F1 and CYP79F2, whereas the biosynthesis of short-chain aliphatic GLs was controlled by CYP79F1 (Chen et al., 2003). To enhance the biosynthesis of GLs, many metabolic engineering studies have been carried out on whole plants, callus, cell cultures, or hairy root cultures and in both vitro and vivo conditions. The following part will highlight the effects of in vitro culture conditions and nutrition in biosynthesis and levels of GLs of plants in both plant in vitro cultures and whole plants.

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Glucosinolates: Properties, Recovery, and Applications

2.3 The effects of plant in vitro culture conditions on biosynthesis and levels of GLs 2.3.1 The effects of phytohormones It is claimed that phytohormones could affect the biosynthesis of GLs and result in impacting levels of GLs in plants (Cargnel et al., 2014; Kastell et al., 2013b; Kim et al., 2013; Sanchez-Pujante et al., 2017). Kim et al. reported that the levels of some indole and aliphatic GLs such as neoglucobrassicin, hydroxyglucobrassicin, glucobrassicin, 4-methoxyglucobrassicin, gluconapin, glucoerucin, gluconasturtiin, and glucoraphanin could be induced by low concentration of indole-3-acetic acid (Kim et al., 2013). That was shown that in the presence of indole-3-acetic acid (0.1 mg/L), the highest levels of these GLs reached 1.6-fold more GLs than those found in controlled hairy root cultures of Brassica oleracea var. Italica. At the same time, the total levels of GLs increased by 2 mmol/g DW with the addition of about 0.5 mg/L kinetin, whereas the addition of dichlorophenoxyacetic acid and its derivatives could reduce total levels of GLs in hairy root cultures of B. oleracea var. Italica and Sinapis alba (Kastell et al., 2013b; Kim et al., 2013).

2.3.2 Effects of level of cell differentiation Several studies showed that the level of cell differentiation played a fundamental role in the profile of obtained GLs (Alvarez et al., 2008; Kastell et al.2013a, 2013b; Wielanek and Urbanek, 1999). Alvarez et al. reported that the GL profiles in plants could be affected by source of cell cultures. The study in cell cultures derived from hypocotyls and hypocotyl tissues of A. thaliana showed that while cell cultures were able to produce seven different GLs after 3 days of cultivation, A. thaliana hypocotyls produced 16 different GLs (Alvarez et al., 2008). In which, the amount of glucoraphanin in hypocotyls was fourfold higher than that found in cell cultures, while the levels of indole GLs in cell cultures, particularly in 4-hydroxyglucobrassicin, were until 15-fold higher than those detected in hypocotyls tissues. In term of total levels of GLs, cell cultures produced higher total levels of GLs (1406.2  134.3 nmol/g FW) than hypocotyls did (700.2  71.1 nmol/g FW) (Alvarez et al., 2008). The studies also showed that the levels of GLs were affected by the level of cell differentiation. Study in Tropaeolum majus indicated that the amount of glucotropaeolin in hairy root cultures was higher than that found callus, cell cultures, and leaves of whole plants (Wielanek and Urbanek, 1999),

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55

whereas study in A. thaliana concluded that the total levels of GLs in leaves were higher than in roots, callus, and hairy root cultures (Kastell et al., 2013a). Kastell et al. reported that the cell differentiation could affect kind of GLs (Kastell et al., 2013b). The study showed that indole and aliphatic GLs could be found in the highest concentration in Brassica rapa leaves, whereas S. alba leaves contained mainly aromatic GLs. Surprising, B. rapa and S. alba hairy root cultures contained only indole GLs including hydroxyglucobrassicin, glucobrassicin, and neoglucobrassicin.

2.3.3 The effects of elicitors Using elicitors, which is recently paid much attention, is one of the most important strategies to control the biosynthesis and levels of GLs in plants (Adio et al., 2011; Augustine and Bisht, 2015a,b; Chiu et al., 2018; Falk et al., 2014; Kastell et al., 2018; Pickett and Khan, 2016; Yi et al., 2016). Most of studies have claimed that jasmonic acid, methyl jasmonate, salicylic acid, and their derivative are key elicitors to increase levels of GLs in plants due to the production of secondary metabolites (Almagro et al., 2014; Alvarez et al., 2008; Kastell et al., 2018, 2013a, 2013b; Natella et al., 2016; Tassoni et al., 2005). The studies showed that the treatment of methyl jasmonate could increase the levels of GLs in A. thaliana cell cultures and T. majus hairy root cultures compared with nonelicited cultures (Alvarez et al., 2008; Wielanek and Urbanek, 1999). At the same time, the optimal level of jasmonic acid to obtain the highest levels of GLs in S. alba hairy root cultures were at around 100 mM during 14 days. In which, neoglucobrassicin was a main production (about 9 mmol/g DW). However, the optimal jasmonic acid treating concentration to produce the highest levels of GLs in B. rapa hairy root cultures was at about 50 mM, during 14 days (about 75 mmol/g DW) (Kastell et al., 2013b). Consideration in the total levels of GLs has indicated that the presence of jasmonic acid or/and methyl jasmonate could increase the total amounts of GLs, some kind of plants such as B. rapa, B. rapa ssp. Pekinensis, Brassica juncea, and Eruca sativa (Augustine and Bisht, 2015a,b; Kastell et al., 2018, 2013b; Padilla et al., 2007; Zang et al., 2015). In the case of individual GLs, the study in Arabidopsis and E. sativa plants showed that in the presence of methyl jasmonate or jasmonic acid only indole GL levels could be increased (Kastell et al., 2018; Natella et al., 2016), whereas the correlation between these elicitors with aromatic and aliphatic GLs is needed further investigation. The study of the effects of other elicitors including sucrose,

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Glucosinolates: Properties, Recovery, and Applications

mannitol, NaCl, 1-aminocyclopropane-L-carboxylic acid, and salicylic acid on levels of GLs in plants showed that sucrose and mannitol could significantly increase the total levels of GLs, whereas the effects of the other elicitors were not clear and not associated to any statistically significant changes of total GLs content (Natella et al., 2016). Thus, it is generally observed that using elicitors to control the levels of GLs in plants is value method to enhance amount of GLs. Although the effects of jasmonic acid and methyl jasmonate on the levels of indole GLs are clear, correlation between other elicitors and aromatic and aliphatic GLs as well as individual GLs is unknown and needs further investigation.

2.3.4 The effects of metabolic engineering strategies Metabolic engineering is known as a key method to modify GL profiles and decrease unwanted compounds in the biosynthesis of GLs. Thus, this strategy has gained much attention in recent years (Miao et al., 2017; Pfalz et al., 2011). There are various approaches, which have been applied for engineering GL in Brassica crops, including modulation of GL biosynthesis genes, ablation of myrosin cells, and redirection of metabolic flux 3(Augustine et al., 2013; Borgen et al., 2010; Bulgakov et al., 2016; Chavadej et al., 1994; Chung et al., 2016; Liu et al., 2011, 2012; Niimi et al., 2015; Park et al., 2011; Qian et al., 2015; Zang et al., 2009, 2008b; Zhang et al., 2015b). It was shown that the biosynthesis of indole and aliphatic GLs could be regulated by certain metabolic engineering strategies. Study in the effects of CYP79B2, CYP79B3, and CYP83B1 genes on the levels of indole GLs in Chinese cabbage demonstrated that single CYP79B3 or CYP83B1 did not affect the profiles of indole GLs, whereas the contents of glucobrassicin, 4-methoxyglucobrassicin, and 4hydroxyglucobrassicin significantly increased in the coexpressing CYP79B2 or CYP79B3 with CYP83B1. Noticing that, the effects of overexpressing all three genes were no better than that of overexpressing the two genes (He et al., 2000; Kim and Jander, 2007; Osbourn, 1996; Zang et al., 2008a,b; Zeng et al., 2003). However, Zang et al. reported that the change of levels of indole GLs in Chinese cabbage hairy roots did not follow a similar trend (decreasing neoglucobrassicin, increasing 4methoxyglucobrassicin, but unchanging glucobrassicin) in transgenic plants (Zang et al., 2009). Redirection of metabolic flux, which is used to manipulate indole GL contents, is also a useful strategy. It was shown that the level of indole GLs oilseeds could be reduced to only 3% of that in wild

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57

type (Chavadej et al., 1994). Moreover, the total levels of indole GLs of the whole plants were also decreased after transformation; thus, the transport engineering is recommended to block the transport of GLs from pod wall into seeds in Arabidopsis (Nour-Eldin and Halkier, 2013). In terms of aliphatic GLs, study of the effects of Arabidopsis MAM1, CYP79F1, and CYP83A1 on the levels of aliphatic GLs in Chinese cabbage indicated that the concentrations of gluconapin and glucobrassicanapin increased in the MAM1 transgenic line M1-1, whereas the amounts of all aliphatic GLs were elevated in the CYP83A1 transgenic line A1-1 (Zang et al., 2008a). The effects of F1-1 line on levels of GLs were different from the F1-2 and F1-3 lines, whereas the F1-2 and F1-3 lines could reduce amount of gluconapin and glucobrassicanapin. The F1-1 line increased levels of gluconapoleifein, 4-methoxyglucobrassicin, and glucobrassicin. Moreover, only the level of aliphatic GLs could be enhanced by overexpressing BnMAM1 or BnCYP83A1, but the overexpressing BnUGT74B1 in B. napus could increase the levels of both indole and aliphatic GLs (Zang et al., 2015). The GL composition and content in oilseed B. napus has been successfully manipulated by silencing the Brassica homologues of Arabidopsis aliphatic GL pathway genes including MAM and GS-ALK (Liu et al., 2011, 2012). It was shown that the levels of progoitrin and gluconapin were decreased after silencing of the MAM gene family in B. napus canola and B. napus rapeseed, in contrast that of 2-propenyl GL was increased. In which, the progoitrin content was reduced by 65% in seeds of B. napus transgenic plants by silencing of the GS-ALK gene family (Liu et al., 2012). The aliphatic GLs were also regulated by using the transcription factors which can regulate the expression of multiple genes related to GL metabolic pathway (Miao et al., 2017). Augustine et al. reported that the silencing of BjMYB28 in B. Juncea could reduce the levels of aliphatic GLs by the expression of aliphatic GL biosynthetic genes (Augustine et al., 2013). Noticing that, the manipulation of single synthetic gene seems to be less effective than that of transcription factors in controlling metabolic pathways in plants (Braun et al., 2001; Capell and Christou, 2004); thus, most of studies focused on the effects of heterologous transcription factors in biosynthesis of GLs (Bovy et al., 2002; Ray et al., 2003; Shin et al., 2006). However, the manipulation of exogenous CYP79 homologues was still applied successfully in Arabidopsis (Bak et al., 1999; Brader et al., 2006). In terms of glucoraphanin, one of the healthiest benefits of GLs, the aim for metabolic engineering methods was to enhance the concentration of glucoraphanin but bring down the level of undesirable one-gluconapin.

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Glucosinolates: Properties, Recovery, and Applications

Thus, the fragment of antisense AOP2 gene, which was responsible for the conversion of glucoraphanin into gluconapin, was transformed to Chinese kale to disrupt the conversion (Hansen et al., 2008; Neal et al., 2010; Qian et al., 2015). As a result, the level of glucoraphanin in Chinese kale significantly increased, whereas that for gluconapin seems to be unchanged compared with the wild-type plants. Silencing of the GS-ALK gene family could increase the level of glucoraphanin of B. napus (Liu et al., 2012). As a key method to modify GL profiles, metabolic engineering strategies should be continuously investigated to control metabolic pathways in plants and enhance desirable natural compounds.

2.4 Effects of nutrition and other factors on levels of glucosinolates in plants 2.4.1 The effects of potassium Different studies have shown that K deficiency may be able to increase total GL levels in plants (Almuziny et al., 2017; Troufflard et al., 2010; Van Dam et al., 2009). A study over two important GL types (indole and aliphatic, methylsulfinylalkyl, and methylthioalkyl) showed that while the total concentrations of these GL types were little affected by K deficiency in the roots, these figures for the shoots were approximately two times higher in K-deficient plants than in control plants with indole GLs increasing more strongly than aliphatic GLs. Besides, the significant increases in several individual compounds, particularly neoglucobrassicin, glucohirsutin, and glucoraphanin, led to the rise of total shoot GL contents. Furthermore, the K deficiency increased the levels of glucoraphanin, one of the most potentially useful GLs in both shoot wild type and Coi1e16 of A. thaliana plants (Troufflard et al., 2010). A study on the influence of K starvation on some typical natural GLs showed that the K deficiency affects differently each GLs as well as parts of plants. For example, the amounts of some methylsulfinylalkyl GLs (glucoraphanin, glucoalyssin, glucohesperin, glucosibarin) declined in roots by K starvation, whereas these for shoots raised (Table 2.1). The changes of GL levels in plants by K starvation relates to the effects of K on the biosynthesis of GLs that was investigated in the effects of K starvation on the biosynthesis of GLs in A. thaliana plants by measured GL levels to establish their dependence on Coi1 signaling. The observed increase of indole GLS in K-deficient plants corresponds to transcriptional upregulation of CYP79B2 and CYP79B3dthe enzymes

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Table 2.1 The effect of K deficiency on the levels of GLs in root and shoot of wild type and Coi1e16 Arabidopsis thaliana plants (Troufflard et al., 2010). Wild-type Coi1-16 plants plants Families

Name of GLs

Root

Shoot

Shoot

Methylsulfinylalkyl GLs

Glucoiberin Glucoraphanin Glucoalyssin Glucohesperin Glucosibarin Glucohirsutin 4-Methylthiobutyl glucosinolate 5-Methylthiopentyl glucosinolate 7-Methylthioheptyl glucosinolate 8-Methylthiooctyl glucosinolate Glucobrassicin 4-Hydroxylglucobrassicin 4-Methoxiglucobrassicin Neoglucobrassicin

þ     þ 

þ þ þ þ þ þ þ

þ þ þ þ þ þ 



þ

þ













  þ þ

þ þ þ þ

þ þ þ 

Methylthioalkyl GLs

Indole GLs

þ, increase; , decrease.

that catalyze the first step of GLs biosynthesis from tryptophan (Hull et al., 2000)dwhereas the synthesis of aliphatic GLS from chain-elongated methionine is catalyzed by enzymes encoded by CYP79F1 and CYP79F2, which have different but overlapping specificity and expression patterns within the plant (Figs. 2.1 and 2.2; Troufflard et al., 2010). The low K status reduced the level of enzyme CYP79F1 but increased the rate of CYP79F2; thus, the overall and specific influences on aliphatic GLs are difficult to predict and need further identification. Almuziny et al. claimed that K limitation makes plants more susceptible to pathogens and diseases, but the effect of K on GL myrosinase and defenses is likely to be indirect. Plants might exhibit increased sensitivity to methyl jasmonate under K-deficient conditions (Troufflard et al., 2010), which could be partially responsible for the observed increase in sinigrin under this treatment. Compared with aliphatic GLs, the indole GL composition responds more rapidly to simulated herbivory and could

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potentially act as an effective, immediate, herbivore deterrent following pest damage (Almuziny et al., 2017). Forieri et al. reported that the K deficiency did not affect total Fe or S content and the OAS level in A. thaliana roots, whereas the Kedeficiency response caused significant increase in the content of total amino acids, apart from Ala and Asp, and of reduced sulfur-containing metabolites. Furthermore, the K deficiency-induced alterations of the phytohormone system were conserved between roots and shoots such as the increase in hormones including abscisic acid, salicylic acid, and jasmonic acid. However, the response to K deficiency was shown not to be part of this cross talk, providing evidence for the specificity of the Fe and S network (Forieri et al., 2017). It is undoubted that potassium plays a fundamental role in the GL biosynthesis process and the levels of GLs as well as the quantities of GLs in plants. The K deficiency could rise the amounts of hormones or/and enzymes such as abscisic acid, salicylic acid, jasmonic acid, CYP79B2, CYP79B3, and CYP79F2 in Brassica; as a result, there is an increase in total GL content and the level of some helpful GLs (glucoraphanin, glucobrassicin, neoglucobrassicin, and 4-methoxiglucobrassicin) as well.

2.4.2 The effects of sulfur The relationship between S deficiency and the biosynthesis of GLs in plants has been concerned in many studies (Aarabi et al., 2016; Ahmad et al., 2007; Aires et al., 2006, 2007; Almuziny et al., 2017; Assefa et al., 2013; Attoa et al., 2003; Badenes-Perez et al., 2010; Bloem et al., 2006, 2007; Borpatragohain et al., 2016; De Pascale et al., 2007; Li et al., 2007; Maruyama-Nakashita et al., 2003, 2006; Maruyama et al., 2009; Salac et al., 2006, 2005; Schnug et al., 1995). Biosynthetic and metabolomic studies of GLs have indicated that there is a simultaneous occurring in the activation of sulfate and assimilation and S limitation (SLIM) could reduce the levels of GLs in plants (Hirai et al., 2003; Maruyama-Nakashita et al., 2003; Nikiforova et al., 2003). The study of S-deficient conditions in Arabidopsis has demonstrated that S limitation is a key factor which regulates the genes involved in sulfate uptake and S assimilation and affects not only GL biosynthesis but also sulfate uptake (Borpatragohain et al., 2016). Under S limitation, the S assimilation and sulfate uptake from the root are enhanced by the upregulation of SULTRs genes (SULTR1;1, SULTR1;2, SULTR3;4, and SULTR4;2) (Takahashi et al., 2011), microRNAs

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(miR395) (Allen et al., 2005; Bonnet et al., 2004; Buhtz et al., 2010; Frerigmann, 2016; Jagadeeswaran et al., 2014; Jones-Rhoades and Bartel, 2004; Kawashima et al., 2011, 2009; Koprivova and Kopriva, 2014, 2016a, 2016b; Liang et al., 2010; Liang and Yu, 2010; Matthewman et al., 2012; Sunkar et al., 2007), as well as the GSL hydrolysis enzyme (myrosinase) (Fig. 2.4; Frerigmann and Gigolashvili, 2014; Takahashi et al., 2011), whereas key enzymes such as MAM1, MAML, CYP79B2/B3, CYP83B1, GST, BCAT, MYB34, SOTs, CeS lyase, GST, and AOPs are responsible for the downregulation in the SLIM1 mutants under S limitation (Hirai et al., 2005; Lewandowska and Sirko, 2008; Maruyama-Nakashita et al., 2006; Nikiforova et al., 2003, 2005). The study on individual families of GLs has demonstrated that the effects of S deficiency on the biosynthesis of GLs vary following the phases of GL biosynthesis. For example, study on biosynthesis of aliphatic GL in Arabidopsis varies, in the transgenic lines of distinct MYBs, expression level of MYB28 can be induced, whereas MYB29 and MYB76 expression level were positively correlated with S concentrations. That may be explained by the negative effects of S limitation on the R2R3-MYBs enzymes (Frerigmann and Gigolashvili, 2014; Takahashi et al., 2011). However, the expression level of MYB28, MYB34, MYB51, and MYB122, which target indole GLs, was not affected by S limitation (Li et al., 2013). Studies showed that the uptake of sulfate from the soil can be increased by the

Figure 2.4 Effects of S on GL accumulation in Brassica crops (Borpatragohain et al., 2016).

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hydrolysis of indole GLs under S stress, as a result of the enhancing lateral root formation by inducing the synthesis of auxin (indole-3-acetic acid) (Borpatragohain et al., 2016). The relationship between S limitation and increase in accumulation of indole-3-acetic acid is not fully studied; however, the hydrolysis of indole GLs may be associated with genes that regulate the tryptophan biosynthesis, the activation of a GSL hydrolysis, and/or the over zexpression of nitrilases (Frerigmann and Gigolashvili, 2014; Hirai et al., 2005a, 2005b, 2004; Kutz et al., 2002; Nikiforova et al., 2003, 2005). The investigation of sulfur fertilizer on the level of GLs on plants, e.g., Brassica, indicated that total level of GLs and individual GL concentrations was increased with increasing S supply, particularly in aromatic and aliphatic GLs (Almuziny et al., 2017; Li et al., 2007). Overall, this appears to suggest that increase in GL content typically was observed under increasing S supply. Hence, S supply should be controlled to maximize sulfate uptake, assimilation, and utilization to improve GL levels in plants and reduce wasting S in fertilizers.

2.4.3 The effects of nitrogen Nitrogen is known as a forming atom of GLs, thus effects of nitrogen on biosynthesis and concentration of GLs in plants have been paid much attention (Almuziny et al., 2017; Barickman et al., 2009; Bilsborrow et al., 1993; Chen et al., 2006; Fabek et al., 2012; Fayyaz Ul et al., 2007; Fismes et al., 2000; Genard et al., 2017; Gerendas et al., 2009; Kawashima et al., 2009; Kim et al., 2002; Klein et al., 1980; Kopsell et al., 2007, 2014; Li et al., 2007; Schonhof et al., 2007; Textor and Gershenzon, 2009). Several studies have demonstrated that N deficiency could generally reduce the quality of GLs on plants (Schonhof et al., 2007; Textor and Gershenzon, 2009); however, the effects are not clear for all GLs and kind of plants and generally have been inconclusive and still unknown (Bones et al., 2015). For example, N supplementation had no effect on the levels of GLs in T. majus (Bloem et al., 2007), while it reduced the levels of GLs in broccoli florets (Schonhof et al., 2007), and increased the concentration of GLs in canola seeds and broccoli leaf tissues (Omirou et al., 2009). In which, the impact of nitrogen fertilization on the levels of indole GLs was higher than that of aliphatic GLs (Omirou et al., 2009). That may be explained by the presence of nitrogen in the biosynthesis of indole GLs (Mikkelsen et al., 2002). Practically, the investigation of the nitrogen fertilization impact on concentration of GLs has been carried out in the combination of the effects

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of sulfur (Chen et al., 2006; Chun et al., 2017; Kim et al., 2002; Kopsell et al., 2007; Li et al., 2007; Omirou et al., 2009; Schonhof et al., 2007; Textor and Gershenzon, 2009). It was shown that to maximize concentration of GLs in Broccoli crop, the ratio of N:S supplying should not be higher than 10:1 (Schonhof et al., 2007). Our knowledge about the effects of N deficiency on biosynthesis and the cellular metabolism of GLs is limited. Most of the conducted studies focus only on some typical GLs, e.g., sinigrin (Attaran et al., 2014; Broeckling et al., 2006; Fallovo et al., 2011; Liang et al., 2006; Schonhof et al., 2007), this issue for other important GLs such as glucoraphanin, indole GLs, and aromatic GLs is still unknown.

2.4.4 The effects of other factors The effects of phosphorus (P) and other factors on biosynthesis and concentration of GLs on plants have been concerned in some studies (Yang et al., 2009). Yang et al. reported the effects of P supply and light intensities in pak choi plants (Yang et al., 2009). It was shown that, under normal light intensity and under P deficiency, the level of total GLs was significantly increased (by 164%), the ratio of aliphatic GLs to total GLs increased from 58% to 67%, but that did not affect the ratio of indole GLs to total GLs. In contrast, under low light intensity condition, the effects of P supply level were slightly on the concentration of total GLs and the ratios of aliphatic, aromatic, and indole GLs to total GLs. In term of individual GLs, the impacts of P deficiency were different. Under natural daylight intensity, concentrations of gluconapin, glucobrassicanapin, glucobrassicin, neoglucobrassicin, 4methoxyglucobrassicin, and gluconasturtiin by P deficiency. While under P limitation, the concentrations of gluconapin, glucobrassicanapin, glucobrassicin and neoglucobrassicin decreased but that for glucobrassicin and 4methoxyglucobrassicin and neoglucobrassicin increased. The physiological and molecular mechanisms for the effects of P supply on biosynthesis and level of GLs in plants need further identification. However, study on the biosynthesis of GLs indicated that P deficiency could increase gene expression and enhance activity of UDP-glucose pyrophosphorylase (Ciereszko et al., 2001, 2005), that is usually used for the producing of desulfo-GLs from thiohydroximic acids (Mikkelsen et al., 2002). Hence, P limitation should enhance the concentration of GLs in plants (Ciereszko et al., 2001, 2005; Wittstock and Halkier, 2002; Yang et al., 2009).

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Effects of other factors on biosynthesis and level of GLs in plants have been also paid much attention in recent years. Most of studies focused on the improvement of the levels of total or/and individual GLs in the presence of other factors including mineral fertilizers (Se, Mo, Cu, Fe, Zn, .), high light intensities, and carbon dioxide levels (Antonious, 2015; Balik et al., 2006; Barickman et al., 2014; Braziene et al., 2012; Bybordi, 2011; Charron et al., 2001; Ebrahimian et al., 2017; Lim et al., 2016; Liu et al., 2009; Paudel et al., 2016; Yang et al., 2009, 2015). However, the metabolic pathways which connect the suppling factors with the level of GLs have not been fully explored.

2.5 Conclusion The biosynthesis of GLs and nutrition effects on the biosynthetic pathway and level of GLs in plants were discussed. It was shown that there are three main steps in the synthesis of GLs in plants. While the transcriptional regulation, cofactors, and derived intermediates for the biosynthesis of aliphatic and indole GLs are well described, these for aromatic GLs have not been fully understood. It is generally observed that the increase in S supply could rise the concentration of GLs in plants, whereas P, N, and K deficiencies may increase levels of GLs. The applications of phytohormones, cell differentiation elicitors, and metabolic engineering strategies for controlling the biosynthetic pathway of GLs have received considerable achievements. However, the effects of plant in vitro culture conditions and fertilizers on the levels of GLs are different for GL families as well as individuals and need further investigation.

Acknowledgments The researched is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 104.06-2018.308.

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