Glucosinolates and metabolism

CHAPTER 4

Glucosinolates and metabolism Ibrahim Guillermo Castro-Torres1, Víctor Alberto Castro-Torres2, Minerva Hernández-Lozano3, Elia Brosla Naranjo-Rodríguez4, Miguel Ángel Domínguez-Ortiz5 1

Colegio de Ciencias y Humanidades, Plantel Sur, Universidad Nacional Autónoma de México (UNAM), Ciudad de México, México; 2Instituto de Química, UNAM, Ciudad de México, México; Facultad de Química Farmacéutica Biológica, Universidad Veracruzana, Xalapa de Enríquez, Veracruz, México; 4Departamento de Farmacia, Facultad de Química, UNAM, Ciudad de México, México; 5 Instituto de Ciencias Básicas, Universidad Veracruzana, Xalapa de Enríquez, Veracruz, México 3

Contents 4.1 Myrosinase, a key enzyme in glucosinolates metabolism 4.2 Human metabolism of glucosinolates 4.3 Gut metabolism 4.3.1 Isothiocyanates 4.3.2 Nitriles 4.4 Hepatic metabolism 4.5 Future perspectives 4.6 Plant metabolism of glucosinolates 4.6.1 Sulfur metabolism 4.6.2 Metabolism 4.6.3 REDOX regulation of metabolism 4.6.4 Transport in glucosinolate metabolism 4.7 Conclusion References Further reading

108 109 109 109 112 116 117 118 119 122 127 128 132 133 141

The metabolism of glucosinolates (GSLs) has been a topic mostly investigated in plants compared with humans. GSLs have many functions as secondary metabolites, and this has been one of the reasons why they are studied frequently in plants. With the advance of scientific research, the effect of GSLs on human diseases was investigated, for this reason it was relevant to study their pharmacokinetics and particularly their metabolism. In this chapter, updated trends about the human and vegetable metabolism of GSLs are discussed, citing the chemical changes that occur in secondary metabolites due to the effect of myrosinase and liver enzymes.

Glucosinolates: Properties, Recovery, and Applications ISBN 978-0-12-816493-8 https://doi.org/10.1016/B978-0-12-816493-8.00004-4

Copyright © 2020 Elsevier Inc. All rights reserved.

107

108

Glucosinolates: Properties, Recovery, and Applications

4.1 Myrosinase, a key enzyme in glucosinolates metabolism Myrosinase was first discovered by Bussy (1939) in mustard seeds, and later in all GSL-containing plants (Pessina et al., 1990). This enzyme, termed b-thioglucoside glucohydrolase or sinigrase EC: 3.2.1.147, is an Sglycosidase which catalyzes the hydrolysis of thioglucosides such as GSLs into D-glucose and an unstable aglucone (Wittstock et al., 2016). Spontaneous rearrangement of the aglucone leads to formation of metabolites such as isothiocyanate, thiocyanates, nitriles, epithionitriles, and oxazolidine-2thione, which are formed depending on the type of GSL, environmental conditions (e.g., pH, temperature, metal ions, and ascorbic acid), and the presence of specifier proteins (Oliviero et al., 2018). Myrosinase is composed of two identical 55e65 kDa polypeptides which are heavily glycosylated resulting in a native molecular weight of 120e150 kDa of the dimeric proteins. This structure is maintained through tetrahedral coordination of a Zn2þ by two conserved residues of each monomer (His 56 and Asp 70 in the Sinapis alba myrosinase) containing various thiol, sulfide groups, and 18% carbohydrates (Wittstock et al., 2016). The primary sequence of this enzyme from cabbage (Brassica oleracea) consists of 546 amino acids including a glycoside hydrolase family 1 (Glyco_hydro_1) domain (41e522 amino acids). Classical myrosinases are characterized by a glutamine (Q) residue, essential for binding to the glucose of aliphatic GSLs, while atypical myrosinases have two basic residues by glutamic acid which can cleave the bond between sulfur and glucose. In the plants, GSL coexists with myrosinase but in different compartments: GSL are stored in vacuoles of GSL-accumulating cells and myrosinase is located in the cytoplasm (Krul et al., 2002) of specialized GSL-free idioblast cells termed myrosin cells, which are present in seeds, roots, stems, leaves, and flowers in the main types of plant tissues (Oliviero et al., 2018). Atypical myrosinases differ from classical in terms of their subcellular localization because they are in peroxisomes and endoplasmic reticulum bodies, respectively (Krul et al., 2002; Shirakawa and Hara-Nishimura, 2018). When the cell structure of plant is disrupted by chewing or chopping, myrosinases were expressed hydrolyzing GSLs (Wittstock et al., 2016). Myrosinase has been identified also in insects (Hemiptera and Coleoptera), fungi (Pessina et al., 1990; Oliviero et al., 2018), and human gut microbiota (e.g., Bacteroides, Peptostreptococcus, Enterococcus, and Escherichia). Sequence analyses suggest that insect and bacterial myrosinases evolved independently from plant myrosinases and

Glucosinolates and metabolism

109

their physiological roles are still largely unknown (Leoni et al., 1997). The human gut microbiota myrosinase can also hydrolyze GSL provided by intake of Brassicales vegetables although to a lesser extent, which may influence their health effects in humans (Oliviero et al., 2018).

4.2 Human metabolism of glucosinolates In this review, a little investigated aspect of GSL metabolism is included, referring to human metabolism. This topic has very little information and is limited to the study of the small intestine, which is the site where GSLs are metabolized. The investigations carried out are associated with the study in experimental animals or cell lines, due to the ethical considerations of research in human beings. There is little information about the biotransformation of GSLs in the liver, so this topic becomes more summarized. We will consider discussing the role of different bacteria that metabolize GSLs and that are found in the small intestine, as well as the influence of some proteins with functions similar to those of myrosinase.

4.3 Gut metabolism GSLs have different chemical structures and their degradation products in the intestine will depend on this (Hanschen et al., 2014). An important factor is the activity or inactivity of the myrosinase enzyme, which is associated with the type of cooking of the vegetables or with their natural consumption (Rungapamestry et al., 2006). Most people consume cooked vegetables, and this action produces denaturation of myrosinase (Rungapamestry et al., 2007). It is important to know that denaturation can occur at different degrees, depending on the cooking temperature, be it with water, steam, or microwaves (Conaway et al., 2000). Myrosinase can be inactivated on exposure to temperatures over 60 C and its activity will determine the amount of hydrolysis products of the GSLs (Angelino et al., 2015). Isothiocyanates are the major products of the hydrolysis of GSLs by myrosinase. Nitriles can also be produced by the degradation of GSLs.

4.3.1 Isothiocyanates The metabolism of these compounds is the one that has been studied the most. Isothiocyanates are absorbed in the small bowel and colon and are detectable in human urine 2e3 h after consumption of cruciferous vegetables (Johnson, 2002).

110

Glucosinolates: Properties, Recovery, and Applications

Myrosinases are thioglucoside glucohydrolases that catalyze the initial step of bioactivation of GSLs, the hydrolysis of their thioglucosidic bond (Rask et al., 2000). These enzymes are usually composed of two identical 55e65 kDa polypeptides which are heavily glycosylated resulting in a native molecular weight of 120e150 kDa of the dimeric proteins (Bjorkman and Janson, 1972), and its structure consists in a dimeric structure and is maintained through tetrahedral coordination of a Zn2þ by two conserved residues of each monomer (Burmeister et al., 1997). Myrosinases accept GSLs of different structural types as substrates but differ in their affinity to individual GSLs and the efficiency of their conversion. Goodman suggested that there was no convincing evidence for the presence of significant myrosinase activity in mammalian cells (Goodman et al., 1959). Nevertheless, numerous studies have suggested that in fact this activity derives from the gut microflora. The bacterial microflora of the human colon also expresses myrosinase activity. Significant quantities of isothiocyanate metabolites are excreted in the urine of healthy human volunteers after eating brassica vegetables, even when myrosinase has been completely inactivated by cooking (Getahun and Chung, 1999). This is clear confirmation that bacterial myrosinase activity can cause the breakdown of GSLs in the distal gut, leading to release of isothiocyanates into the fecal stream (Fig. 4.1) (Krul et al., 2002). In addition, it has been shown that myrosinase has a role in the liver metabolism and not only as a substrate for the intestinal bacteria, because its activity is related to the same process in plants, where it has been found that isothiocyanates are obtained by-products in the metabolism of GSLs, which are inducers of phase 2 enzymes but are also substrates for glutathione transferases, which are phase 2 enzymes (Zhang et al., 1995). The activity of myrosinase in the production of isothiocyanates in humans has been proven because these chemical compounds react with glutathione; the enzymecatalyzed nucleophilic attack of the sulfhydryl group of glutathione on the central carbon of the isothiocyanate group results in the formation of

Figure 4.1 Glucosinolates are hydrolyzed by myrosinase to form isothiocyanates.

Glucosinolates and metabolism

111

Figure 4.2 Isothiocyanates are eventually converted into mercapturic acids by the sequential action of glutathione transferase, g-glutamyltranspeptidase, cysteinylglycinase, and N-acetyltransferase.

glutathione dithiocarbamates that are modified by a sequence of enzymatic reactions, leading ultimately to the formation of N-acetylcysteine dithiocarbamates, also known as mercapturic acids, which have been detected in the human urine (Fig. 4.2) (Tate, 1980). We refer to the glutathione adduct and its subsequent degradation products collectively as dithiocarbamates. Therefore, several studies in humans have reported the urinary excretion of N-acetylcysteine derivatives when oral GSLs were administered. The excretions were substantial, accounting for 30%e67% of the doses administered in 8e24 h (Zhang and Talalay, 1994). The glutathione adduct and its four sequential metabolic products are excreted in the urine, but the N-acetylcysteine mercapturic acids predominate (Zhang and Talalay, 1994). The evidence of the functioning of intestinal myrosinase in humans has been verified with studies of bioavailability of glucosinolates, for example, Verkerk conducted a study where glucoraphanin was administered in the absence of vegetal myrosinase; passage through the upper digestive tract had little impact on glucoraphanin recovery (Verkerk et al., 2009). These result in a drop in pH stomach due to a loss of glucoraphanin in gastric digestion

112

Glucosinolates: Properties, Recovery, and Applications

with a further 7%e28% loss when GSL was recovered from the small intestine. These studies supporting the possibility that GSLs hydrolysis can occur by the intestinal myrosinase showed that when subjects were given a cooked broccoli meal, low levels of sulforaphane metabolites still appeared in plasma and urine, in the absence of vegetal myrosinase. Interestingly, the low level of urinary isothiocyanate metabolites was delayed, compared with metabolites from uncooked broccoli, suggesting prolonged transit time (to the lower gut) before hydrolysis (Verkerk et al., 2009). This was further supported by a study where subjects were given antibiotics and an enema before a heated broccoli sprouts extract, in an attempt to remove the microbiota myrosinase activity. These subjects produced even fewer urinary sulforaphane metabolites than without the antibiotics and enema (Shapiro et al., 2006). In other studies, which support the activity of myrosinase in the human intestinal microbiota, when 150 mmol sulforaphane was administered, sulforaphane appeared in the portal blood within 15 min and maintained a blood level of 150 mM for an hour. In contrast, when the same dose was given as glucoraphanin, sulforaphane reached the plasma level of 1/10 concentration (9e12 mM) and not until 1 hour. The cecum had been ligated proximally and distally before dosing, so clearly the enzymatic activity microbiota was responsible for hydrolysis (Lai et al., 2010). In particular, it can be summarized that the activity of myrosinase in the plants tissue is deactivated, e.g., by excess cooking, then enzymes with myrosinase activity coproduced by intestinal bacteria present in the human distal gut are capable of hydrolyzing the ingested GSLs. Human gut bacterial metabolism is poorly understood and highly variable among individuals, with some being high and others low metabolizers of GSLs into isothiocyanates (Luang-In et al., 2014; Li et al., 2011). Unfortunately, there are not a large number of studies about the functioning and metabolic activity of myrosinase in humans; however, with the previous review, it can be preliminarily concluded that the activity of said enzyme is mainly related to the functioning of the intestinal microbiota in humans. However, further studies at the intestinal level are required to correctly elucidate the participation of myrosinase in the metabolism of GSLs.

4.3.2 Nitriles Cruciferous plants contain some proteins other than myrosinase, which have not been identified in the human intestine, such as nitrile specifier

Glucosinolates and metabolism

113

Figure 4.3 The myrosinase system allows the hydrolysis of glucosinolates to isothiocyanates; however, nitriles can also be produced as shown in this figure, where from a certain glucosinolate are generated cyanoepithiopropane and butenitrile.

protein, epithiospecifier protein, and thiocyanate-forming protein (Kuchernig et al., 2012). These molecules also intervene in the metabolism of GSLs, converting them into nitriles, epithionitriles, and thiocyanates, but most of the research has been done on vegetables (Fig. 4.3). The metabolism of GSLs in the intestine will depend on the presence of bacteria that contain this type of enzymes, and there are no studies where they have characterized in the small intestine, the three proteins mentioned above. The closest information is obtained in the intestine of experimental animals. In one study, the hydrolysis of sinigrin by enzymes other than myrosinase was analyzed. Sinigrin (allyl-glucosinolate or 2-propenyl-glucosinolate) is a natural aliphatic GSL present in plants of the Brassicaceae family (Kuchernig et al., 2012). The sinigrin was incubated in a solution with rat intestinal microbiota, after 12 h it was demonstrated that more than 64% of the initial amount of GSL was degraded, producing different substances, such as allyl isothiocyanate, allyl cyanide, and 1-cyano-2,3-epithiopropane (Lu et al., 2011). The main degradation product was the allyl isothiocyanate, identified by high-performance liquid chromatography and gas chromatographyemass spectrometry (Lu et al., 2011). In different plants, the epithiospecifier protein is responsible for carrying out this type of degradation of the sinigrin, so it is believed that this molecule is present in the microbiota of the rat and probably in the human gut. In the small intestine, the action of sulfatases is what allows the degradation of GSLs; these enzymes have been identified in many bacteria while

114

Glucosinolates: Properties, Recovery, and Applications

few have been cloned and characterized (Gadler and Faber, 2007). Some bacteria that can metabolize GSLs to desulfoglucosinolates are Escherichia coli VL8, Lactobacillus agilis R16, and Enterococcus casseliflavus CP1. As discussed above, the chemical structure of GSLs will determine the enzymatic action of plants or bacteria, some species of bacteria cannot hydrolyze certain GSLs to produce nitriles, for example, L. agilis R16 and E. casseliflavus CP1 cannot metabolize glucoraphanin, but there are other bacteria that do this work. The in vitro and in vivo models to quantify the degradation products of GSLs have many disadvantages because the samples must be treated with great sensitivity to be able to analyze them with spectroscopic or spectrometric techniques. In one study, the three bacteria mentioned above (E. casseliflavus CP1, L. agilis R16, and E. coli VL8) were used to study the metabolism of three GSLs: sinigrin, glucotropaeolin, and gluconasturtiin. After 24 h of incubation, gluconasturtiin was metabolized into different nitriles and isothiocyanates by E. coli and E. casseliflavus. L. agilis produced only nitriles. More than 80% of GSLs were metabolized by all bacteria (Luang-In et al., 2016). In this study, it was shown that pH is a very important factor that can intervene in the intestinal metabolism of GSLs because L. agilis and E. coli produced an acid pH in their culture media after metabolizing GSLs. E. casseliflavus produced alkaline media. The hydrolysis products of the GSLs were formed at a pH between 3.0 and 7.5 (Luang-In et al., 2016). The protein most associated with the degradation of GSLs to nitriles is epithiospecifier protein, which acts by the myrosinase system (Kuchernig et al., 2012). The activity of this protein was shown to be dependent on ferrous ions (Fe2þ) (Kuchernig et al., 2012). When the side chain of a GSL contains a double bond, the protein promotes a chemical reaction between the sulfur of the thioglucoside bond and the double bond, to form a tyranny ring (Wittstock and Burow, 2007). The plant Arabidopsis thaliana has been studied, from which a gene that codes for epithiospecifier protein was isolated, it was demonstrated that this protein not only promotes the formation of epionitriles but also the formation of simple nitriles from a great variety of GSLs (Kissen and Bones, 2009). In an experimental study, different A. thaliana proteins were isolated and these proteins were expressed in E. coli by means of genetic engineering. The recombinant proteins were used to study the degradation of GSLs to nitriles. Proteins showed nitrile-specifier activity on 2-propenylglucosinolate and

Glucosinolates and metabolism

115

benzylglucosinolate; however, these proteins modify their activity depending on the concentration of iron (II) ions and pH (Kong et al., 2012). The importance of this research is based on the fact of using a bacterium that abounds in the intestinal flora of the human being, so that the in vitro results obtained can probably be extrapolated to humans, only that they need more research. This study leaves open an important field, which will allow us to work with isolated bacteria directly from the intestinal flora, to analyze if they can express the proteins of certain vegetables and can contribute to the intestinal metabolism of GSLs. In the previous study, we could notice that iron ions are very important to determine the type of substance in which GSLs are degraded. The presence of these substances can favor the hydrolysis of GSLs to nitriles and not to isothiocyanates. Other bacteria of the intestinal flora capable of metabolizing GSLs are as follows: Enterococcus faecalis, Enterococcus faecium, and certain Peptostreptococcus spp. and Bifidobacterium spp (Li et al., 2011; Palop et al., 1995). Bacteroides thetaiotaomicron has been identified in the rat microflora as a strain capable of metabolizing sinigrin; this bacterium was also identified in human feces (Elfoul et al., 2001; Mullaney et al., 2013). Lactobacillus plantarum KW30 and Lactococcus lactis ssp. lactis KF147 also have the ability to metabolize glucoraphanin and glucoerucin in sulforaphane and erucine. According to the studies cited so far, the intestinal microflora is the main producer of myrosinase in humans, so in therapies that need intestinal washes or treatment with antibiotics, the microflora can be destroyed and the hydrolysis of GSLs is completely eliminated, altering its metabolism (Tian et al., 2018). Recently, a clinical study on GSL metabolism was published, which represents an important advance in this area of knowledge, because much of the research is reported in preclinical models. In the clinical study, the consumption of broccoli and radish daikon in its raw and cooked states was analyzed. The intake of the vegetables was between 100 and 200 g daily for 16 days. The results were compared with control subjects. On day 17, the urine of the patients was analyzed by means of mass spectrometry, to detect the presence of the degradation products of the GSLs, mainly sulforaphane. This metabolite was detected in the urine, indicating that it was metabolized efficiently by the intestinal flora; however, the study did not consider any data on hepatic metabolism (Charron et al., 2018).

116

Glucosinolates: Properties, Recovery, and Applications

4.4 Hepatic metabolism This research area is one of the least explored in the metabolism of GSLs. The few works that are published were in experimental animals; however, they are an important premise to infer possible effects on human metabolism. At the end of the 1980s, the first indications of hepatic metabolism of GSLs were published. In an experimental work, groups of rats fed a diet rich in GSLs were studied (Nugon-Baudon et al., 1990). These metabolites produced some toxic effects in animals, such as increased size of the liver, kidneys, and thyroid gland; however, the GSL diet produced an increase in important enzymes involved in liver detoxification, so it is concluded that GSLs continue to be metabolized by the hepatic route, after passing through intestinal hydrolysis and the metabolites produced can contribute to the toxic effects that were observed in rats. In another experimental study in rats, it was demonstrated that after ingestion of different proportions of cabbage, the hepatic detoxification activity is induced; in this experimental work, it was demonstrated that metabolites such as diindolylmethane and indole-3-acetonitrile acted directly in the liver, after having carried out an intestinal hydrolysis. It was also shown that GSLs were metabolized in their first path through the small intestine and reached the liver, where they probably were not further metabolized (McDanell et al., 1987). It is hypothesized that the main breakdown products of GSLs, i.e., isothiocyanates, are those that exert chemopreventive effects in the liver (Abdull Razis et al., 2010). These metabolites have been isolated in cell cultures and their chemical structure remains intact after demonstrating the therapeutic effects. This clearly indicates that after intestinal metabolism, at least the isothiocyanates do not undergo modifications in their molecular structure. Many authors considered that only GSL derivatives were those that possessed the therapeutic properties; however, in an experimental study performed in hepatocyte cultures, it was shown that intact GSLs induce many important liver functions. Abdull Razis and coworkers demonstrated that glucoraphanin and glucoerucine increased the O-dealkylations of methoxy- and ethoxyresorufin, which are markers for cytochrome (CYP) P1 activity and elevated the apoprotein levels of microsomal CYP1A1, CYP1A2, and CYP1B1; therefore, these GSLs are modulators of certain liver enzymes that play an important role in cellular metabolism. In Fig. 4.4, we summarize the intestinal and hepatic metabolism of two GSLs.

Glucosinolates and metabolism

117

Figure 4.4 The human metabolism of glucosinolates starts from the moment in which the metabolites are ingested from some cruciferous. The figure shows the examples of glucoraphasatin and glucoraphanin, which are metabolized at the level of the small intestine via the myrosinase, an enzyme found within the bacterial flora. The metabolites that are produced are rafasatin and sulforabano, which are transported through the portal circulation and reach the liver, where it has been shown that they do not undergo modifications in the chemical structure; on the contrary, they will exert different therapeutic effects.

4.5 Future perspectives The metabolism of GSLs in vegetables has been investigated extensively, compared with metabolism in humans. The biogenetic routes for the synthesis and degradation of GSLs, mainly isothiocyanates, thiocyanates, and nitriles, are clearly established in plants. The investigation of the human metabolism requires the analysis of those intestinal bacteria that are able to degrade the GSLs, but the studies are still limited, because they are associated with the analysis of the intestinal microbiota of experimental animals, existing certain limitations. GSLs are present in the diet of many people, due to the consumption of cruciferous and it is shown that some hydrolysis products such as isothiocyanates exert antioxidant properties and inhibit cell proliferation. Because of these important effects, it is necessary to study the

118

Glucosinolates: Properties, Recovery, and Applications

pharmacokinetics of these secondary metabolites, focusing on their metabolism due to the probability of generating more substances that enhance the therapeutic effects or that may generate toxic effects. Associated with the latter, the limitation of investigating bacterial metabolism is that many isothiocyanates can exert toxicity on microorganisms and for this reason cannot be detected with spectroscopic or spectrometric techniques. It has been found that during bacterial metabolism there is more production of nitriles than isothiocyanates. In vegetables, GSLs are degraded by myrosinase as a defense mechanism against many factors, for this reason bacteria can cause toxicity. In future investigations, human enterocytes expressed in knockout animals could be studied, to ensure that the bacteria and proteins analyzed are those that abound in the human microbiota, or to prepare enterocyte cell lines, which can carry out the metabolism of GSLs. Other fields of study are those related to the search for proteins capable of metabolizing GSLs that are not necessarily found in the intestinal flora but are present inside nonbacterial cells and that have the ability to hydrolyze the metabolites. The hepatic metabolism of GSLs is another area of great interest, which has not been considered. It is taken into account that in the small intestine it is the first point of absorption of nutrients; however, many substances can cross the brush border of the enterocyte and enter the portal circulation to reach the liver. In this organ, multiple metabolic processes are carried out and it is a site that contains hundreds of enzymes and proteins responsible for the catabolic reactions. The analysis of GSLs in urine and feces has been reported and these are very important guidelines. The analysis in urine has shown that different isothiocyanates are eliminated and this may depend on the intestinal flora of each person, but also on the hepatic metabolism, until now not investigated. In the liver, phase II biochemical reactions can be promoted, with the aim of returning the water-soluble metabolites so that they can be eliminated in the urine. The GSLs or hydrolysis products could be being modified in the liver, which requires a detailed investigation. The presence of isothiocyanates has also been demonstrated in feces, which is more associated with intestinal microbiota. The human metabolism of GSLs remains a topic of interest for the scientific community.

4.6 Plant metabolism of glucosinolates GSLs were discovered in vegetables, which is why most of the research is in this area. In the plant metabolism, a numerous pathway of biosynthesis and

Glucosinolates and metabolism

119

degradation of GSLs are reported. Plant research of GSLs has left important schemes of the routes of secondary metabolism, as well as the participation of the myrosinase and the bacteria that are also found in vegetables. The metabolism of GSLs can be studied without restriction in vegetables, considering that cruciferous plants are not in danger of extinction and are part of the diet of many population groups in the world. The spectroscopic and spectrometric tools have allowed elucidating the structure of each isolated GSL and inferring its degradation to isothiocyanates, thiocyanates, or nitriles during metabolism. Complementary to the study of plant metabolism are the computational tools, which will allow to infer routes of possible degradation, or even biochemical interactions of the GSLs with future pharmacological targets.

4.6.1 Sulfur metabolism The knowledge about sulfur metabolism is very important for the study of GSL metabolism, because these substances contain at least two sulfur atoms in their chemical structure, one that is part of the thioglucosidic bond and another in the sulfate group. However, a GSL may contain a third additional sulfur atom derived from methionine and incorporated into the side chains, as is the case with aliphatic GSLs (Huseby et al., 2013). Therefore, as Falk discusses, the total sulfur content in the plant directly affects the biosynthesis of GSLs in vegetables (Falk et al., 2007). The metabolism of sulfur is related to the assimilation of different sulfates and both pathways are regulated by two groups of transcription factors called MYB3 (Borevitz et al., 2000); the first formed by the factors MYB28, MYB76, and MYB29, which receive the alternative name of high aliphatic GSLs (HAG1 and HAG3) and involved mainly in the biosynthesis of aliphatic GSLs (Gigolashvili et al., 2007; Hirai et al., 2007; Sønderby et al., 2007), whereas the second group of transcribers include MYB51, MYB122, and MYB34 called high indolic GSLs (HIG1, HIG2, HIG3) or also called ATR1, which affect the biosynthesis of indolic GSLs (Celenza et al., 2005; Malitsky et al., 2008). In addition to the MYB transcription factors, GSL synthesis is regulated by the transcription factor OBP2 belonging to the DOF family (DNA-binding one-finger transcription factor) identified in Arabidopsis, which is inducible in herbivores and methyl jasmonate (Skirycz et al., 2006). Levy adds that the regulation of GSL levels in plants is also related to the expression of the calmodulin-binding protein (IQD), because it produces alterations in the concentration of GSLs (Levy et al., 2005). When there is a reduction in

120

Glucosinolates: Properties, Recovery, and Applications

sulfur, the biosynthesis of GSLs is affected and is controlled by SLIM1 (sulfur limitation-1), an ethylene-insensitive3-like transcription factor, which is a central regulator in the plant response to the low bioavailability of sulfur (Maruyama-Nakashita et al., 2006). The association between GSL biosynthesis and sulfur metabolism has been studied since the 1970s. Underhill and colleagues reported that the final step in the synthesis of the GSL nucleus is the sulfation reaction of the desulfo-GS12 precursors (Underhill et al., 1973) (Fig. 4.5). Sulfation is catalyzed by the SOT sulfotransferases responsible for transferring the activated sulfate group of 30 -phosphoadenosine-50 -phosphosulfate (PAPS) to the hydroxylated substrate. The PAPS is synthesized from the sulfate in two steps dependent on ATP (Underhill et al., 1973) (Fig. 4.5). Sulfate is first adenylated by ATP sulfurylase to adenosine-50 -phosphosulfate (APS), which is then phosphorylated by APS kinase (APK) to PAPS. APS is phosphorylated by APK to PAPS for the synthesis of GSLs and other sulfated compounds, or it is reduced by APS reductase (APR) to sulfite. Sulfite is reduced to sulfide by sulfite reductase (SiR). Sulfide is incorporated into O-acetylserine (OAS) by OAS thiolyase (OAS-TL) to form cysteine. The importance of PAPS for the synthesis of GSLs was demonstrated in Arabidopsis by finding a very low concentration of GSLs in altered plants in two of the four APK genes (Mugford et al., 2009). Several studies have shown that GSLs are regulated not only by nutrition with sulfates but also other nutrients affect their synthesis and accumulation (Gerendas et al., 2009; Hirai et al., 2004). The effects of nitrogen limitation are highly dependent on the sulfur supply; with adequate sulfate availability, plants accumulate aliphatic GSLs, possibly as storage of excess sulfur (Li et al., 2007). Interestingly, the nitrogen used for plant nutrition affects the accumulation of GSLs; the synthesis is greater in plants that grow in

Figure 4.5 The metabolism of sulfur is intimately linked to the metabolism of sulfur, because when this element is converted into salts, such as sulfates, these can be a source of sulfur atoms or donors of functional groups for the assembly of the final structure of glucosinolate.

Glucosinolates and metabolism

121

ammonium than in nitrates (Marino et al., 2016). Similarly, phosphate deficiency leads to an increase in the synthesis and accumulation of GSLs (Pant et al., 2005). On the other hand, the Mugford studies have shown that the availability of PAPS has a great influence on the synthesis of GSLs, even under normal conditions (Mugford et al., 2009). The analysis of Arabidopsis mutants with decreased PAPS production revealed a strong reduction in GSL contents, approximately by 80%. In the study of these mutants, the concentration of GSLs and the sulfate of 12-hydroxyjasmonate were reduced approximately five times in the plants apk1 and apk2. The reduction in GSLs resulted in an increase in transcription levels for the genes involved in GSL biosynthesis and the accumulation of desulfated precursors (Mugford et al., 2009). It also led to large alterations in sulfur metabolism: the concentration of sulfate and thiols increased in plants apk1 and apk2. The data indicate that the APK1 and APK2 isoforms of the APK play an important role in the synthesis of secondary sulfated metabolites and are required for normal growth rates (Mugford et al., 2009). The analysis of other mutants of the APKs revealed that the PAPS is an essential compound, because the loss of APK1, APK3, and APK4 was lethal; however, this is not caused by the lack of GSLs, because other mutants without these compounds are viable (Mugford et al., 2010). In addition, the authors conclude that the biosynthesis of APS compartmentally in plastids and cytosol does not affect the synthesis of GSLs, because these plants were viable, but the phenotype of the mutants in which APS occurred in the plastids (apk3) or in cytosol (apk1, apk2, apk4), it can only be explained when a PAPS transport is carried out through the plastid envelope (Mugford et al., 2010). The interaction between the APK and the APS reductase is responsible not only for the supply of PAPS for the synthesis of GSLs but also for the division of sulfur between the primary and secondary metabolism. The reduced synthesis of GSLs in the apk1 and apk2 mutants resulted in an increased flow through primary reductive assimilation and the accumulation of reduced sulfur, glutathione, and cysteine compounds (Mugford et al., 2011). The transfer of sulfate from PAPS to the desulfoglucosinolates results in the formation of a second product, 50 -phosphoadenosine-30 -phosphate (PAP) (Xiong et al., 2001). PAP is rapidly metabolized to AMP and phosphate by the action of the enzyme called Fiery1 (FRY1). FRY1 has been found in numerous genetic studies for diverse phenotypes, which have rarely been mechanically understood, because the enzyme dephosphorylates two types of substrates, dinucleotide phosphates or inositol phosphates, which participate in the regulation of a large number of development processes

122

Glucosinolates: Properties, Recovery, and Applications

(Chan et al., 2013). Inositol is considered a very important cellular signal (Estavillo et al., 2011). It seems that there is an intimate relationship between PAP and FRY1, which is genetically regulated to prevent the accumulation of some substance and interfere in the synthesis of GSLs (Lee et al., 2012). PAPS induces a high production of PAP, and this must be regulated by FRY1, so if this enzyme is deactivated, it would be expected that PAP accumulates within the cellular compartments and as such interfere in the production of GSLs (Lee et al., 2012); The analysis of the mutants apk1 and apk2 has revealed that FRY1 is crucial in the metabolic pathway of GSLs and can be regulated positively so as not to affect the biosynthetic pathway (Mugford et al., 2009).

4.6.2 Metabolism To study the metabolism of GSLs, it is important to consider that they are metabolites that comprise at least 120 anionic thioglucosides with welldefined structures, the chemical structure is very important in the biosynthetic routes, because each step is specifically controlled by some enzyme (Grubb et al., 2004; Halkier and Gershenzon, 2006). The majority of GSL-producing plants are members of the order of the Brassicales (formerly known as Capparales), which includes the cruciferous families, such as Brassicaceae, Capparaceae, and Caricaceae. The Brassicales comprise more than 350 genera and 3000 species. Of the hundreds of crucifer species investigated, all are able to synthesize GSLs, which have been identified in more than 500 species of diverse noncruciferous dicotyledonous angiosperms, highlighting the unique genus Drypetes producer of the family Euphorbiaceae (it has not been shown that any other member of this large family of plants contains them) and the monogenic Moringaceae family, which contains 13 species that biosynthesize only one family of GSLs (Grubb et al., 2004). Within the plant cells, coexist the GSLs and the myrosinase and when there is some damage to the plant tissue, for example, when the herbivores are fed, the cells release the enzyme and hydrolyze the GSLs in different products, which have shown many biological activities (Rask et al., 2000; Barth and Jander, 2006). These degradation products act as toxic metabolites against microorganisms, insects, and herbivorous animals that interact with the plant, it can be considered that GSLs are defense metabolites (Barth and Jander, 2006; Lambrix et al., 2001). Fig. 4.6 shows the chemical structure of a GSL, which is hydrolyzed by the action of myrosinase, which acts directly on the thioglucoside bond, releasing one

Glucosinolates and metabolism

123

Figure 4.6 Glucosinolates can be hydrolyzed by the action of myrosinase, in different degradation products, among which the most important are isothiocyanates and nitriles.

molecule of glucose and another of aglycone. The aglycone, in turn, can generate different products depending on its chemical structure, pH, iron ions concentration, epitiospecific protein, and the epitiospecific modifier (Kutz et al., 2002). The different chemical changes that occur during the vegetal metabolism of GSLs have been studied in in vivo models, to facilitate the precision of the chemical products that originate (Maruyama-Nakashita et al., 2003). The most important experiments have shown dynamic changes in the structure of the GSLs during the development of seedlings and how the structure is modified before factors such as pH, temperature, and light (Celenza et al., 2005). Other aspects studied are the variation in the concentration of GSLs and myrosinase in the day and night or the modification of the enzymatic activity before the deficiency of sulfur (Hanschen et al., 2018). A. thaliana is a cruciferae in which the metabolism of GSLs has been studied extensively. It was the first vegetable in which the biosynthetic pathway of these secondary metabolites was elucidated (Kroymann et al., 2003). Within the biosynthesis of GSLs, there are proteins that intervene in chemical reactions where the functional groups of their amino acids in the side chain are modified, oxidative decarboxylations are carried out, and three genes have been characterized that can regulate the elongation of the chain of the GSL

124

Glucosinolates: Properties, Recovery, and Applications

(Graser et al., 2000). In the plant metabolism of GSLs, three phases are involved mainly, in the first, the elongation of the amino acid chain is carried out, in which methylene groups are inserted, especially in the side chain. In the second phase, the conversion of the amino acid combination to the general structure of GSL is carried out, and finally, in the third phase, chemical modifications are carried out in the side chain of the metabolite (Wittstock and Halkier, 2002). Most GSLs are synthesized from methionine, which is modified by the sequential addition of one to nine methylene groups in its side chain (Fig. 4.7). The route is initiated by the transamination of methionine to form the corresponding keto-acid 2-oxo, which is then extended by a methylene group in a three-step cycle, consisting of a condensation with acetyl-CoA, isomerization, and oxidative decarboxylation (Hull et al., 2000). The newly formed keto-acid 2-oxo can be transformed into the corresponding methionine derivative or can undergo additional cycles of chain elongation. The pathway is similar to the incorporation of a single methylene group that occurs in leucine biosynthesis (Mikkelsen et al., 2000). The conversion of methionine involves the formation of

Figure 4.7 This figure shows the path of elongation that occurs in the chain of methionine to give continuous biosynthesis of glucosinolate.

Glucosinolates and metabolism

125

intermediates that are common to the chemical structure of GSL. In the plant metabolism, the synthesis phase of the GSL nucleus is very important, where the two initial steps of the biosynthetic pathway are catalyzed by the cytochrome P450 enzymes belonging to the CYP79 and CYP83 families (Fig. 4.8). In the metabolic pathway, five gene products of cytochrome P450 (CYP79A2, CYP79B2, CYP79B3, CYP79F1, and CYP79F2) have been shown to catalyze the conversion of phenylalanine, tryptophan, or short- and long-chain methionine substrates to the corresponding aldoxins (Hansen et al., 2001). These five products catalyze the production of virtually all GSLs in A. thaliana. The aldoxins continue the metabolism with the help of other cytochromes to form S-alkylthiohydroxy acids, which are cleaved to thiohydroxy acids, through the enzyme carbon-sulfur lyase. Subsequently, glycosylation is continued by S-glucosyl transferase and chemical modifications are made in the functional groups of the side chains, which leads to the structural diversity that can be observed in GSLs (Bak et al., 1999; Mikkelsen et al., 2004). The formation of GSLs generates an intermediate called acinitro, which is unstable and is conjugated with the thiol group of cysteine, through the carbon atom (Petersen et al., 2002; Piotrowski et al., 2004). This step is catalyzed by cytochromes of family 83. In CYP83A1 and CYP83B, complexes have been identified in Arabidopsis (Bak et al., 2001; Naur et al., 2003). The biochemical characterization of recombinant CYP83A1 and CYP83B1 shows that both enzymes can metabolize all the aldoximes tested (Hansen and Kliebensteindj Halkier, 2007). However, CYP83A1 has a high

Figure 4.8 Biosynthesis of the glucosinolate nucleus.

126

Glucosinolates: Properties, Recovery, and Applications

affinity for aliphatic aldoximes, whereas CYP83B1 prefers indole-3acetaldoxime and aromatic aldoximes as substrates (Kroymann et al., 2001). Recently, a flavin-monooxygenase system has been identified, which catalyzes the conversion of methylthioalkyl GSLs into methylsulfinylalkyl GSLs; however, the enzymes that catalyze the methylation of indole GSLs have not yet been fully characterized (Fig. 4.9) (Textor et al., 2007). The level and composition of GSLs in plants are determined in part by fluctuations in the genetic makeup of plants; nevertheless, the most precise advances have focused on the investigation of Arabidopsis, where natural populations have been used to elucidate the genetic bases of the metabolism of GSLs (Kliebenstein et al., 2001). The analysis of the place of the quantitative traits of GSLs in the Arabidopsis ecotypes revealed two main loci that control the variations of most aliphatic GSLs (Textor et al., 2007). This locus located in the upper part of chromosome 5 encodes several methylthioalkyl malate synthases (MAM), which control the length of the side chain of different aliphatic GSLs derived from methionine.22.23. MAM1 catalyzes the condensation in the first and second elongation cycle of the methionine side chain to form dihomomethionine. MAM2 catalyzes only the first chain to generate homomethionine. MA3 catalyzes the six

Figure 4.9 Biochemical model to represent the biosynthesis of glucosinolates.

Glucosinolates and metabolism

127

cycles of elongation of the methionine chain and produces short- and longside chain methionines (Kliebenstein et al., 2001). A second quantitative locus analysis located at the top of chromosome 4 is an alkenyl/hydroxypropyl (AOP) locus. This locus has three alleles (a helical protein (OHP)), each of which controls a type of side-chain modification, the ALK allele produces alkenyl side chains, OHP produces hydroxyalkyl side chains, and the null allele produces side chains of methilsulfinil in the A. thaliana Ecotype Columbia, AOP is composed of two genes in tandem, AOP2 and AOP3, the first that produces alkenyl and the last one that directs the production of hydroxyalkyl GSLs (Kliebenstein et al., 2001).

4.6.3 REDOX regulation of metabolism The common mechanisms in posttranscriptional regulation in GSL biosynthesis are carried out through oxidation and reduction, because in the primary and secondary metabolism, we find enzymes involved in the elongation of methionine, which use these chemical reactions (He et al., 2009). An example of this process is the isopropylmalate dehydrogenase enzyme, which catalyzes the oxidation and decarboxylation of the MAM reaction products and produces elongated methionine by a single methylene unit. In the initial stage of aliphatic GSL biosynthesis, methionine is deaminated to the corresponding 2-oxo acid, i.e., 4-methylthio-2oxobutanoic acid, by the branched-chain aminotransferases BCAT3 and BCAT4 (Fig. 4.10) (Schuster et al., 2006; Knill et al., 2008). The methylthioalkyl malate synthases MAM1 and MAM3 catalyze the condensation of 2-oxo acid with acetyl CoA to produce a substituted 2-malate (2-(20 methylthioethyl) malate) derivative (Textor et al., 2004, 2007). The 2malate derivative is isomerized to a 3-malate derivative that undergoes an oxidative decarboxylation to produce a 2-oxo acid extended by a methylene group (Field et al., 2004). The enzymes that catalyze the isomerization and oxidation reactions are not yet characterized. If extended 2-oxo acid is transaminated by BCAT3 to form the corresponding elongated methionine (homomethionine), it enters the GSL pathway to produce C3 GSLs (Field et al., 2004). A second enzyme in the synthesis pathway that is regulated by redox reactions is the APK (Ravilious et al., 2012). It has been shown that the APS enzyme has an inhibitory effect because to form an efficient ternary complex, ATP has to first bind and create an optimal configuration of the active site (Bick et al., 2001). The binding of APS by itself does not trigger these necessary conformational changes and has an inhibitory effect on the

128

Glucosinolates: Properties, Recovery, and Applications

Figure 4.10 The reactions of oxide reduction, as well as the respective enzymes that catalyze these processes have an important participation on the methionine and the metabolism of glucosinolates.

reaction (Kopriva and Koprivova, 2004). The activity of APS reductase is also regulated by redox reactions; the activity increases with oxidation and decreases with reduction, compared with the regulation of the APK. This allows us to understand that changes in the redox environment in the plastids can direct the flow of sulfur to the primary or secondary metabolism; that is why under oxidized conditions, reduced compounds, such as glutathione, would be favored, because they help the detoxification of reactive oxygen species. On the contrary, in environments of reduced species, the sulfur will flow toward PAPS and toward the synthesis of GSLs and other secondary compounds (Kopriva and Koprivova, 2004).

4.6.4 Transport in glucosinolate metabolism In the metabolism of GSLs, as well as any substance, it is very important to address the issue of transport, because once they are synthesized, they need specific mechanisms to reach the cells or specific sites where they can be used. To study the transport of substances it is very important to consider the chemical structure, due to the polarity, many metabolites are insoluble in water and require specific transportation mechanisms (Kopriva and Gigolashvili, 2016). Previously, it was commented that the metabolism of GSLs has been studied extensively in A. thaliana, the same has happened with the study of the transport of metabolites, this plant has been the one

Glucosinolates and metabolism

129

that has revealed the mechanisms of action (Schuster et al., 2006). The onset of GSL metabolism occurs when methionine undergoes some chemical reactions and needs to enter the chloroplast (Gigolashvili and Kopriva, 2014). When the amino acid has been converted into the 4-methylthio-2oxobutanoate derivative, the BAT5 transporter (bile acid transporter 5) transports it to the interior of the chloroplast (Jensen et al., 2014). This transporter was named this way, because it was believed that plants synthesized and transported bile acids (Jensen et al., 2014); however, it was discovered that this is incorrect, because bile acids are synthesized in mammals from cholesterol, a substance that has not been identified in the vegetables. BAT5 contributes to the transport of keto acids through the chloroplast membrane (Gigolashvili et al., 2009). In the biosynthesis of aliphatic GSLs, the importation of two keto acids is required for the lengthening of the methionine chain and this transporter has the main role of importing those keto acids (Furumoto et al., 2011). On the other hand, there is a molecule called PAPS, which is responsible for donating sulfate groups to GSLs to assemble their chemical structure (Hirschmann et al., 2014). If the GSLs are sulfated, they can be transported through the vegetable to reach key sites where they can be used, on the contrary, if they are sulfated they accumulate in the same synthesis site to be used as defense metabolites (Mugford et al., 2010). The main group that requires PAPS to carry out the sulfation is in the chloroplast and needs to be transported to the cytosol, which will be the site where the sulfation reactions will take place (Piotrowski et al., 2004). The transporter that does this is called PAPS transporter 1 (PAPST1) and is considered a universal sulfate donor (Gigolashvili et al., 2012). Once this carrier carries the specific component that needs PAPS to carry out the sulfation of the desulfoglucosinolates, these must be stored in the vacuoles of the plant or in the S cells, which are cells identified in A. thaliana, as storage sites of sulfur (Jørgensen et al., 2015). Precisely, these cells are lysed when herbivores damage plant tissue, releasing a large concentration of GSLs that are hydrolyzed by the action of myrosinase. These cells have come to be called mustard pumps (Koroleva et al., 2010). On the other hand, the presence of transporters of the ABC superfamily, which have been identified in plant vacuoles, has also been reported (Petrussa et al., 2013). ABC transporters have been characterized for being in the membranes of cells and for having a broad specificity for their substrates (Bednarek et al., 2009). When GSLs are catabolized to some defense product, the transporters must expel them outside the cell (Fuchs et al., 2016; Lu et al., 2015). The transporter PDR8 of the ABC family has

130

Glucosinolates: Properties, Recovery, and Applications

been identified as the one in charge of exporting biologically active products that come from the degradation of GSLs (Lu et al., 2015). This transporter was identified for the first time in A. thaliana (Fuchs et al., 2016). In this plant, it was discovered that PDR8 mainly transports a GSL derivative called 4-methoxy-indole-3-ylmethylglucosinolate (4MOI3M) (Lipka et al., 2005). Another important topic is the transport of GSLs at long distances, for example, to the seeds of vegetables, where the presence of these secondary metabolites has been demonstrated (Brudenell et al., 1999). Research on the transport of GSLs has been possible, thanks to the fact that the metabolites can be radiolabeled and, with this, their route is determined from the leaves to the seeds, transported by the phloem (NourEldin and Halkier, 2013). The long-distance transporters that have been identified and characterized are called transporters for GSLs 1 and 2 (GTR1 and GTR2) (Andersen and Halkier, 2014). The GSLs can be transported in the leaves through plasmodesmata, later they reach storage cells in the external defensive perimeters, close to the vacuoles. GTR transporters influence the transit of GSLs, not only through the phloem to travel long distances but through the same leaf of the plant (Jørgensen et al., 2015). The transport and metabolism of GSLs can be affected during plant growth and as such by the influence of plant hormones, which are a group of naturally occurring, organic substances which influence physiological processes at low concentrations (Davies and Davies, 2010). Because vegetal organisms experience a great range of biotic and abiotic environmental stresses such as pathogen infection, herbivore damage, mechanical injury, mineral nutrition, and seasonal temperature fluctuation, they produce some defensive hormones that act as positive regulators coordinating their activity (Stotz et al., 2011). These substances include jasmonic acid, abscisic acid, ethylene, salicylic acid, and nitric oxide. Much of what is known about specific defense hormones has come from the study of agricultural crops (Zhang et al., 2014). For instance, jasmonic acid induces the synthesis of proteinase inhibitors which deter insect feeding, whereas salicylic acid participates in the systemic acquired resistance response, where an older leaf’s pathogenic attack promotes the development of resistance in younger leaves (Davies and Davies, 2010). Plant responses with a chemical defense in two levels: constitutive (through phytoanticipins) and inductive (through phytoalexins). The first is the amount of the defense that vegetal express under usual environmental conditions, whereas the second is the amount of additional allocation to defense produced in response to an environmental signal associated with a

Glucosinolates and metabolism

131

greater need for defense populations. Thus, more attack would express greater constitutive allocation to protection, whereas groups that face infrequent injuries are predicted to have lower constitutive levels (Zhang et al., 2014). In summary, plant defenses depend on constitutive and induced production of phytoanticipins and phytoalexins, respectively (Stotz et al., 2011). But nevertheless, some phytopathogens can present capable enzymes to detoxify the phytoanthropins or phytoalexins produced by your host (Pedras and Ahiahonu, 2005). In the specific case of Brassicaceae as Arabidopsis and Capparaceae plant families (Bednarek et al., 2009), the primary chemical defense compounds include to GSLs, relevant phytoanticipins thioglucosides (Stotz et al., 2011) that are biosynthesized from amino acids. These compounds play an antioxidative role, participating in the resistance to insect pests, pathogens in plants (Abuyusuf et al., 2018), and general herbivores through their hydrolyzed products that occur in wounds or the tissues attack (Bednarek et al., 2009). In addition to these biological effects, GSLs are responsible for the smells and flavors characteristic of the Brassicaceae (Abuyusuf et al., 2018). There are more of 96 GSLs in the Brassicaceae family, most are unique to certain species and genera (Anjum et al., 2011). Arabidopsis synthesizes up to 40 different GSLs (Jeschke et al., 2017), but the most intensely studied breakdown products are the isothiocyanates, formed through a spontaneous rearrangement of the GSL aglucone in the absence of supplementary proteins. GSL-derived isothiocyanates are very reactive and have been shown to be toxic to bacteria, fungi, nematodes, and insects. Experimental studies indicate that GSLs pathway may act in a pathogendependent defense (Wittstock et al., 2016) at the contact site, which blocks the penetration of nonhost fungal pathogens (Khare et al., 2017). GSLs commonly grouped into three classes based on their precursors: aliphatic, derived from methionine or other aliphatic amino acids; indolic, derived from tryptophan; and benzenic, derived from phenylalanine or tyrosine (Jeschke et al., 2017). Aliphatic and indolic GSLs are the two most important types of GSL (Stotz et al., 2011). To function requires their tissue damage-triggered activation by myrosinases compartmentalized either in specialized myrosin cells in the phloem parenchyma or in stomata cells. Depending on temperature, basal levels of root GSLs have been reported to show a circadian pattern that reflects the vegetal’s ability to anticipate herbivore and pathogen attack. The concentration of GSLs varies depending of the species, the organ, and the state in which the plant is (Malik et al., 2010), likewise, it is appreciated a reduction by age (Ludvick-

132

Glucosinolates: Properties, Recovery, and Applications

Müller et al., 2000). In the Brassicaceae, the signaling molecules can change the profile of GSLs. By example, pathways mediated by jasmonate, salicylic acid, and ethylene accumulate GSLs in a tissue differential way depending on the concentration of these compounds. It has also been described that the use of fertilizers, with reduced nitrogen content and high sulfur, promotes an accumulation of indole, aliphatic, and aromatics GSLs (Jahangir et al., 2009). pH of the soil and humidity are crucial for the formation of GSLs because the isothiocyanates are produced in neutral pH in plants with a lot of watering (Bones and Rossiter, 2006). On the other hand, phytoalexins are found in plants in low concentration before some type of stress occurs, but once the plant responds to a stimulus, they increase their synthesis quickly (Taiz and Zeiger, 2010). 44 types of phytoalexin are known (Ahuja et al., 2012), included brassilexin, metoxibrassinin, and camalexin (Pedras and Ahiahonu, 2005). Camalexin or 3thiazole-2-yl-indole is a sulfur-containing tryptophan-derived secondary metabolite from A. thaliana, considered to be the major inducible phytoalexin involved in biotic response as antimicrobial. ABC transporter ABCG34 mediates the secretion of camalexin from the epidermal cells to the surface of leaves and thereby confers resistance to several infections. Many genetic approaches confirmed that camalexin plays a positive role in plant resistance to necrotrophic fungi such as Alternaria brassicicola, Botrytis cinerea, and Plectosphaerella cucumerina in a dose-dependent manner. Camalexin has also been reported to play a defensive role against the hemibiotrophic fungus Leptosphaeria maculans and Phytophthora brassicae, an oomycete (Lemarie et al., 2015), and its accumulation also inhibits the growth of virulent strains of Pseudomonas syringae. Because camalexin is found typically in plants under attack by pathogens, it is often used as an indicator of plant pathogeninduced stress (Zhang et al., 2014) and contributing at the postinvasive stage of immunity (Frerigmann et al., 2016). A common point that connects the GSLs and camalexin pathways is the origin and mode of incorporation of the organic sulfur atom(s) present in these molecules; studies developed in A. thaliana refer GSH as the sulfur donor (Bednarek et al., 2009).

4.7 Conclusion In this chapter, the most relevant aspects of GSL metabolism were summarized, addressing the metabolism in humans and plants, discussing the main experimental studies that have been carried out, preclinical and clinical.

Glucosinolates and metabolism

133

The essence of a review article or book chapter is to inform the most innovative of the subject and not try to repeat everything that has already been published; however, the human metabolism of GSLs is a little investigated topic, so it represents an important area of opportunity, and in our chapter we report the most relevant from a critical point of view. In contrast, the plant metabolism has been widely studied, so we specify data that may serve future researchers of metabolism to further amplify this research. The plant metabolism of GSLs has been described with greater amplitude and the emblematic model of this study is A. thaliana. In this plant, we have been able to identify different proteins involved in the biosynthetic pathway of GSLs, as well as transcriptional regulators of metabolism. The new research horizons should point to the analysis of metabolism in other types of cruciferous, to find the protein diversity that is involved in the biosynthesis of GSLs, from investigating new enzymes, new transcription factors, and enzymatic complexes to developing models in vitro with cell culture of cruciferous plants. The field of genetics and genomics are revolutionizing these investigations, with the aim of being able to investigate which molecules regulate the concentration of GSLs in different seasons of the year and before the plant response to herbivores or different pests. In this part of the metabolism, it is also important to use the computer tools because the molecular design can predict the behavior of all the substances involved in the metabolism of GSLs.

References Abdull Razis, A.F., Bagatta, M., De Nicola, G.R., Iori, R., Ioannides, C., 2010. Intact glucosinolates modulate hepatic cytochrome P450 and phase II conjugation activities and may contribute directly to the chemopreventive activity of cruciferous vegetables. Toxicology 277 (1e3), 74e85. Abuyusuf, Md, Arif Hasan, K.R., Hoy-Taek, K., Rafiquil, I., Jong-In, P., Ill-Sup, N., 2018. Altered glucosinolate profiles and expression of glucosinolate biosynthesis genes in ringspot-resistant and susceptible cabbage lines. International Journal of Molecular Sciences 19, 2833. Ahuja, I., Kissen, R., Bones, A.M., 2012. Phytoalexins in defense against pathogens. Trends in Plant Science 17, 73e90. Andersen, T.G., Halkier, B.A., 2014. Upon bolting the GTR1 and GTR2 transporters mediate transport of glucosinolates to the inflorescence rather than roots. Plant Signaling and Behavior 9, e27740. Angelino, D., Dosz, E.B., Sun, J., Hoeflinger, J.L., Van Tassell, M.L., Chen, P., Harnly, J.M., Miller, M.J., Jeffery, E.H., 2015. Myrosinase-dependent and -independent formation and control of isothiocyanate products of glucosinolate hydrolysis. Frontiers of Plant Science 6, 831.

134

Glucosinolates: Properties, Recovery, and Applications

Anjum, N.A., Gill, S.S., Ahmad, I., Pacheco, M., Duarte, A.C., Umar, S., Khan, N.A., Pereira, M.E., 2011. The plant family Brassicaceae: an introduction. In: Anjum, N.A., Ahmad, I., Pereira, M.E., Duarte, A.C., Umar, S., Khan, N.A. (Eds.), The Plant Family Brassicaceae. Springer, The Netherlands. Bak, S., Olsen, C.E., Petersen, B.L., Møller, B.L., Halkier, B.A., 1999. Metabolic engineering of p-hydroxybenzylglucosinolate in Arabidopsis by expression of the cyanogenic CYP79A1 from Sorghum bicolor. The Plant Journal 20, 663e671. Bak, S., Tax, F.E., Feldmann, K.A., Galbraith, D.W., Feyereisen, R., 2001. CYP83B1, a cytochrome P450 on the metabolic branch paint in auxin and indole glucosinolate biosynthesis in Arabidopsis. The Plant Cell 13, 101e111. Barth, C., Jander, G., 2006. Arabidopsis myrosinases TGG1 and TGG2 have redundant function in glucosinolate breakdown and insect defense. The Plant Journal 46, 549e562. Bednarek, P., Pislewska-Bednarek, M., Svatos, A., Schneider, B., Doubsky, J., Mansurova, M., Humphry, M., Consonni, C., Panstruga, R., Sanchez-Vallet, A., Molina, A., Schulze-Lefert, P., 2009. A glucosinolate metabolism pathway in living plant cells mediates broad-spectrum antifungal defense. Science 323 (5910), 101e106. Bick, J.A., Setterdahl, A.T., Knaff, D.B., Chen, Y., Pitcher, L.H., Zilinskas, B.A., Leustek, T., 2001. Regulation of the plant-type 50-adenylyl sulfate reductase by oxidative stress. Biochemistry 40, 9040e9048. Bjorkman, R., Janson, J.C., 1972. Studies on myrosinases. 1. Purification and characterization of a myrosinase from white mustard seed (Sinapis alba L.). Biochimica et Biophysica Acta 276, 508e518. Bones, A.M., Rossiter, J.T., 2006. The enzymatic and chemically induced decomposition of glucosinolates. Phytochemistry 67, 1053e1067. Borevitz, J.O., Xia, Y., Blount, J., Dixon, R.A., Lamb, C., 2000. Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. The Plant Cell 12 (12), 2383e2394. Brudenell, A., Griffiths, H., Rossiter, J., Baker, D., 1999. The phloem mobility of glucosinolates. Journal of Experimental Botany 50, 745e756. Burmeister, W.P., Cottaz, S., Driguez, H., Iori, R., Palmieri, S., Henrissat, B., 1997. The crystal structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of an Sglycosidase. Structure 5, 663e675. Celenza, J.L., Quiel, J.A., Smolen, G.A., Merrikh, H., Silvestro, A.R., Normanly, J., Bender, J., 2005. The Arabidopsis ATR1 Myb transcription factor controls indolic glucosinolate homeostasis. Plant Physiology 137, 253e262. Chan, K.X., Wirtz, M., Phua, S.Y., Estavillo, G.M., Pogson, B.J., 2013. Balancing metabolites in drought: the sulfur assimilation conundrum. Trends in Plant Science 18, 18e29. Charron, C.S., Vinyard, B.T., Ross, S.A., Seifried, H.E., Jeffery, E.H., Novotny, J.A., 2018. Absorption and metabolism of isothiocyanates formed from broccoli glucosinolates: effects of BMI and daily consumption in a randomised clinical trial. British Journal of Nutrition 120 (12), 1370e1379. Conaway, C.C., Getahun, S.M., Liebes, L.L., Pusateri, D.J., Topham, D.K.W., BoteroOmary, M., Chung, F.L., 2000. Disposition of glucosinolates and sulforaphane in humans after ingestion of steamed and fresh broccoli. Nutrition and Cancer 38 (2), 168e178. Davies, P.J., 2010. The plant hormones: their nature, occurrence, and functions. In: Davies, P.J. (Ed.), Plant Hormones. Springer, Dordrecht, pp. 1e15. Elfoul, L., Rabot, S., Khelifa, N., Quinsac, A., Duguay, A., Rimbault, A., 2001. Formation of allyl isothiocyanate from sinigrin in the digestive tract of rats monoassociated with a human colonic strain of Bacteroides thetaiotaomicron. FEMS Microbiology Letters 197, 99e103.

Glucosinolates and metabolism

135

Estavillo, G.M., Crisp, P.A., Pornsiriwong, W., Wirtz, M., Collinge, D., Carrie, C., Pogson, B.J., 2011. Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis. The Plant Cell 23, 3992e4012. Falk, K.L., Tokuhisa, J.G., Gershenzon, J., 2007. The effect of sulfur nutrition on plant glucosinolate content: physiology and molecular mechanisms. Plant Biology (Stuttgart). 9 (5), 573e581. Field, B., Cardon, G., Traka, M., Botterman, J., Vancanneyt, G., Mithen, R., 2004. Glucosinolate and amino acid biosynthesis in Arabidopsis. Plant Physiology 135, 828e839. Frerigmann, H., Pislewska-Bednarek, M., Sánchez-Vallet, A., Molina, A., Glawischnig, E., Gigolashvili, T., Bednarek, P., 2016. Regulation of pathogen-triggered tryptophan metabolism in Arabidopsis thaliana by MYB transcription factors and indole glucosinolate conversion products. Molecular Plant 9 (5), 682e695. Fuchs, R., Kopischke, M., Klapprodt, C., Hause, G., Meyer, A.J., SchwarzlVander, M., Lipka, V., 2016. Immobilized subpopulations of leaf epidermal mitochondria mediate PEN2-dependent pathogen entry control in Arabidopsis. The Plant Cell 28. TPC2015T20100887-RA. Furumoto, T., Yamaguchi, T., Ohshima-Ichie, Y., Nakamura, M., Tsuchida-Iwata, Y., Shimamura, M., Westhoff, P., 2011. A plastidial sodium-dependent pyruvate transporter. Nature 476, 472e475. Gadler, P., Faber, K., 2007. New enzymes for biotransformations: microbial alkyl sulfatases displaying stereo- and enantioselectivity. Trends in Biotechnology 25 (2), 83e88. Gerendas, J., Podestat, J., Stahl, T., Kubler, K., Bruckner, H., Mersch-Sundermann, V., Muhling, K.H., 2009. Interactive effects of sulfur and nitrogen supply on the concentration of sinigrin and allyl isothiocyanate in Indian mustard (Brassica juncea L.). Journal of Agricultural and Food Chemistry 57, 3837e3844. Getahun, S.M., Chung, F.L., 1999. Conversion of glucosinolates to isothiocyantes in humans after ingestion of cooked watercress. Cancer Epidemiology, Biomarkers & Prevention 8, 447e451. Gigolashvili, T., Kopriva, S., 2014. Transporters in plant sulfur metabolism. Frontiers of Plant Science 5, 442. Gigolashvili, T., Berger, B., Mock, H.P., Müller, C., Weisshaar, B., Flügge, U.I., 2007. The transcription factor HIG1/MYB51 regulates indolic glucosinolate biosynthesis in Arabidopsis thaliana. The Plant Journal 50, 886e901. Gigolashvili, T., Yatusevich, R., Rollwitz, I., Humphry, M., Gershenzon, J., Flügge, U.I., 2009. The plastidic bile acid transporter 5 is required for the biosynthesis of methioninederived glucosinolates in Arabidopsis thaliana. The Plant Cell 21 (6), 1813e1829. Gigolashvili, T., Geier, M., Ashykhmina, N., Frerigmann, H., Wulfert, S., Krueger, S., Fleugge, U.I., 2012. The Arabidopsis thylakoid ADP/ATP carrier TAAC has an additional role in supplying plastidic phosphoadenosine 50-phosphosulfate to the cytosol. The Plant Cell 24, 4187e4204. Goodman, I., Fouts, J.R., Bresnick, E., Menegas, R., Hitchings, G.H.A., 1959. Mammalian thioglycosidase. Science 130, 450e451. Graser, G., Schneider, B., Oldham, N.J., Gershenzon, J., 2000. The methionine chain elongation pathway in the biosynthesis of glucosinolates in Eruca sativa Brassicaceae. Archives of Biochemistry and Biophysics 378, 411e419. Grubb, C.D., Zipp, B.J., Muller, J.L., Masuno, M.N., Molinski, T.F., Abel, S., 2004. Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis. The Plant Journal 40, 893e908. Halkier, B.A., Gershenzon, J., 2006. Biology and biochemistry of glucosinolates. Annual Review of Plant Biology 57, 303e333.

136

Glucosinolates: Properties, Recovery, and Applications

Hanschen, F.S., Lamy, E., Schreiner, M., Rohn, S., 2014. Reactivity and stability of glucosinolates and their breakdown products in foods. Angewandte Chemie International Edition in English 53 (43), 11430e11450. Hanschen, F.S., Pfitzmann, M., Witzel, K., Stützel, H., Schreiner, M., Zrenner, R., 2018. Differences in the enzymatic hydrolysis of glucosinolates increase the defense metabolite diversity in 19 Arabidopsis thaliana accessions. Plant Physiology and Biochemistry 124, 126e135. Hansen, B.G., Kliebensteindj Halkier, B.A., 2007. Identification of a flavin-monooxygenase as the S-oxygenating enzyme in aliphatic glucosinolate biosynthesis in Arabidopsis. The Plant Journal 50, 902e910. Hansen, C.H., Wittstock, U., Olsen, C.E., Hick, A.J., Pickett, J.A., Halkier, B.A., 2001. Cytochrome P450 CYP79F1 from Arabidopsis catalyzes the conversion of dihomomethionine and trihomomethionine to the corresponding aldoximes in the biosynthesis of aliphatic glucosinolates. Journal of Biological Chemistry 276, 11078e11085. He, Y., Mawhinney, T.P., Preuss, M.L., Schroeder, A.C., Chen, B., Abraham, L., Chen, S., 2009. A redox-active isopropylmalate dehydrogenase functions in the biosynthesis of glucosinolates and leucine in Arabidopsis. The Plant Journal 60, 679e690. Hirai, M.Y., Yano, M., Goodenowe, D.B., Kanaya, S., Kimura, T., Awazuhara, M., Saito, K., 2004. Integration of transcriptomics and metabolomics for understanding of global responses to nutritional stresses in Arabidopsis thaliana. Proceeding of the National Academy of Sciences of the United States of America 101, 10205e10210. Hirai, M.Y., Sugiyama, K., Sawada, Y., Tohge, T., Obayashi, T., Suzuki, A., Araki, R., Sakurai, N., Suzuki, H., Aoki, K., Goda, H., Nishizawa, O.I., Shibata, D., Saito, K., 2007. Omics-based identification of Arabidopsis Myb transcription factors regulating aliphatic glucosinolate biosynthesis. Proceedings of the National Academy of Sciences of the United States of America 104, 6478e6483. Hirschmann, F., Krause, F., Papenbrock, J., 2014. The multi-protein family of sulfotransferases in plants: composition, occurrence, substrate specificity, and functions. Frontiers of Plant Science 5, 556. Hull, A.K., Vij, R., Celenza, J.L., 2000. Arabidopsis cytochrome P450s that catalyze the first step of tryptophan-dependent indole-3-acetic acid biosynthesis. Proceedings of the National Academy of Sciences of the United States of America 97, 2379e2384. Huseby, S., Koprivova, A., Lee, B.R., Saha, S., Mithen, R., Wold, A.B., Bengtsson, G.B., Kopriva, S., 2013. Diurnal and light regulation of sulphur assimilation and glucosinolate biosynthesis in Arabidopsis. Journal of Experimental Botany 64 (4), 1039e1048. Jahangir, M., Abdel-Farid, I.B., Kim, H.K., Choi, Y.H., Verpoorte, R., 2009. Healthy and unhealthy plants: the effect of stress on the metabolism of Brassicaceae. Environmental and Experimental Botany 67 (1), 23e33. Jensen, L.M., Halkier, B.A., Burow, M., 2014. How to discover a metabolic pathway? an update on gene identification in aliphatic glucosinolate biosynthesis, regulation and transport. Biological Chemistry 395, 529e543. Jeschke, V., Kearney, E.E., Schramm, K., Kunert, G., Shekhov, A., Gershenzon, J., Vassão, D.G., 2017. How glucosinolates affect generalist Lepidopteran larvae: growth, development and glucosinolate metabolism. Frontiers of Plant Science 8, 1995. Johnson, I.T., 2002. Glucosinolates: bioavailability and importance to health. International Journal for Vitamin and Nutrition Research 72 (1), 26e31. Jørgensen, M.E., Nour-Eldin, H.H., Halkier, B.A., 2015. Transport of defense compounds from source to sink: lessons learned from glucosinolates. Trends in Plant Science 20, 508e514. Khare, D., Choi, H., Un Huh, S., Bassin, B., Kim, J., Martinoia, E., Hoon Sohn, K., Paek, K.H., Lee, Y., Arabidopsis, 2017. ABCG34 contributes to defense against necrotrophic pathogens by mediating the secretion of camalexin. Proceedings of the National Academy of Sciences 712e720.

Glucosinolates and metabolism

137

Kissen, R., Bones, A.M., 2009. Nitrile-specifier proteins involved in glucosinolate hydrolysis in Arabidopsis thaliana. Journal of Biological Chemistry 284 (18), 12057e12070. Kliebenstein, D.J., Gershenzon, J., Mitchell-Olds, T., 2001. Comparative quantitative trait loci mapping of aliphatic, indolic and benzylic glucosinolate production in Arabidopsis thaliana leaves and seeds. Genetics 159, 359e370. Knill, T., Schuster, J., Reichelt, M., Gershenzon, J., Binder, S., 2008. Arabidopsis branchedchain aminotransferase 3 functions in both amino acid and glucosinolate biosynthesis. Plant Physiology 146, 1028e1039. Kong, X.Y., Kissen, R., Bones, A.M., 2012. Characterization of recombinant nitrilespecifier proteins (NSPs) of Arabidopsis thaliana: dependency on Fe(II) ions and the effect of glucosinolate substrate and reaction conditions. Phytochemistry 84, 7e17. Kopriva, S., Gigolashvili, T., 2016. Chapter five. Glucosinolate synthesis in the context of plant metabolism. Advances in Botanical Research 80, 99e124. Kopriva, S., Koprivova, A., 2004. Plant adenosine 50-phosphosulphate reductase: the past, the present, and the future. Journal of Experimental Botany 55, 1775e1783. Koroleva, O.A., Gibson, T.M., Cramer, R., Stain, C., 2010. Glucosinolate-accumulating Scells in Arabidopsis leaves and flower stalks undergo programmed cell death at early stages of differentiation. The Plant Journal 64, 456e469. Kroymann, J., Textor, S., Tokuhisa, J.G., Falkk, L., Bartram, S., Gershenzon, J., MitchellOlds, T., 2001. A gene controlling variation in Arabidopsis glucosinolate composition is part of the methionine chain elongation pathway. Plant Physiology 127, 1077e1088. Kroymann, J., Donnerhacke, S., Schnabelrauch, D., Mitchell-Olds, T., 2003. Evolutionary dynamics of an Arabidopsis resistance quantitative trait locus. Proceedings of the National Academy of Sciences of the United States of America 100, 14587e14592. Krul, C., Humblot, C., Philippe, C., Vermeulen, M., van Nuenen, M., Havenaar, R., Rabot, S., 2002. Metabolism of sinigrin (2-propenyl glucosinolate) by the human colonic microflora in a dynamic in vitro large-intestinal model. Carcinogenesis 23 (6), 1009e1016. Kuchernig, J.C., Burow, M., Wittstock, U., 2012. Evolution of specifier proteins in glucosinolate-containing plants. BMC Evolutionary Biology 12, 127. Kutz, A., Müller, A., Hennig, P., Kaiser, W.M., Piotrowski, M., Weiler, E.W., 2002. A role for nitrilase 3 in the regulation of root morphology in sulphur-starving Arabidopsis thaliana. The Plant Cell 30, 95e106. Lai, R.H., Miller, M.J., Jeffery, E., 2010. Glucoraphanin hydrolysis by microbiota in the rat cecum results in sulforaphane absorption. Food and Function 1, 161e166. Lambrix, V., Reichelt, M., Mitchell-Olds zon, J., 2001. The Arabidopsis epithiospecifier the hydrolysis of glucosinolates to nitriles plusia ni herbivory. The Plant Cell 13, 2793e2807. Lee, B.R., Huseby, S., Koprivova, A., Chetelat, A., Wirtz, M., Mugford, S.T., Kopriva, S., 2012. Effects of fou8/fry1 mutation on sulfur metabolism: is decreased internal sulfate the trigger of sulfate starvation response? PLoS One 7, e39425. Lemarie, S., Robert-Seilaniantz, A., Lariagon, C., Lemoine, J., Marnet, N., Levrel, A., Jubanoult, M., Manzanares-Daleux, M.J., Gravot, A., 2015. Camalexin contributes to the partial resistance of Arabidopsis thaliana to the biotrophic soilborne protist Plasmodiophora brassicae. Frontiers of Plant Science 6, 1e10. Leoni, O., Iori, A., Palmieri, S., Esposito, E., Menegatti, E., Cortesi, R., Nastruzzi, C., 1997. Myrosinase-generated isothiocyanate from glucosinolates: isolation, characterization and in vitro antiproliferative studies. Bioorganic and Medicinal Chemistry 5 (9), 1799e1806. Levy, M., Wang, Q., Kaspi, R., Parrella, M., Abel, S., 2005. Arabidopsis IQD1, a novel calmodulin-binding nuclear protein, stimulates glucosinolate accumulation and plant defense. The Plant Journal 43, 79e96.

138

Glucosinolates: Properties, Recovery, and Applications

Li, S., Schonhof, I., Krumbein, A., Li, L., Stutzel, H., Schreiner, M., 2007. Glucosinolate concentration in turnip (Brassica rapa ssp. rapifera L.) roots as affected by nitrogen and sulfur supply. Journal of Agricultural and Food Chemistry 55, 8452e8457. Li, F., Hullar, M.A., Beresford, S.A., Lampe, J.W., 2011. Variation of glucoraphanin metabolism in vivo and ex vivo by human gut bacteria. British Journal of Nutrition 106, 408e416. Lipka, V., Dittgen, J., Bednarek, P., Bhat, R., Wiermer, M., Stein, M., Landtag, J., Brandt, W., Rosahl, S., Scheel, D., Llorente, F., Molina, A., Parker, J., Somerville, S., Schulze-Lefert, P., 2005. Pre- and postinvasion defenses both contribute to nonhost resistance in Arabidopsis. Science 310 (5751), 1180e1183. Lu, M., Hashimoto, K., Uda, Y., 2011. Rat intestinal microbiota digest desulfosinigrin to form allyl cyanide and 1-cyano-2,3-epithiopropane. Food Research 44 (4), 1023e1028. Lu, X., Dittgen, J., Pislewska-Bednarek, M., Molina, A., Schneider, B., Svatos, A., Doubský, J., Schneeberger, K., Weigel, D., Bednarek, P., Schulze-Lefert, P., 2015. Mutant allele-specific uncoupling of PENETRATION3 functions reveals engagement of the ATP-binding cassette transporter in distinct tryptophan metabolic pathways. Plant Physiology 168 (3), 814e827. Luang-In, V., Narbad, A., Nueno-Palop, C., Mithen, R., Bennett, M., Rossiter, J.T., 2014. The metabolism of methylsulfinylalkyl- and methylthioalkyl-glucosinolates by a selection of human gut bacteria. Molecular Nutrition & Food Research 58, 875e883. Luang-In, V., Albaser, A.A., Nueno-Palop, C., Bennett, M.H., Narbad, A., Rossiter, J.T., 2016. Glucosinolate and desulfo-glucosinolate metabolism by a selection of human gut bacteria. Current Microbiology 73 (3), 442e451. Ludvick-Müller, J., Krishna, P., Forreiter, C., 2000. A glucosinolate mutant of Arabidopsis is thermosensitive and defective in cytosolic Hsp90 expression after heat stress. Plant Physology 123, 949e958. Malik, M.S., Riley, M.B., Norsworthy, J.K., Bridges Jr., W., 2010. Glucosinolate profile variation of growth stages of wild radish (Raphanus raphanistrum). Journal of Agricultural and Food Chemistry 58, 3309e3315. Malitsky, S., Blum, E., Less, H., Venger, I., Elbaz, M., Morin, S., Eshed, Y., Aharoni, A., 2008. The transcript and metabolite networks affected by the two clades of Arabidopsis glucosinolate biosynthesis regulators. Plant Physiology 148, 2021e2049. Marino, D., Ariz, I., Lasa, B., Santamaria, E., Fernandez-Irigoyen, J., Gonzalez-Murua, C., Aparicio Tejo, P.M., 2016. Quantitative proteomics reveals the importance of nitrogen source to control glucosinolate metabolism in Arabidopsis thaliana and Brassica oleracea. Journal of Experimental Botany 67, 3313e3323. Maruyama-Nakashita, A., Inoue, E., Watanabe-Takahashi, H., 2003. Transcriptome profiling genes in Arabidopsis reveals global effects multiple metabolic pathways. Plant Physiology 123, 597e605, 2003. Maruyama-Nakashita, A., Nakamura, Y., Tohge, T., Saito, K., Takahashi, H., 2006. Arabidopsis SLIM1 is a central transcriptional regulator of plant sulfur response and metabolism. The Plant Cell 18, 3235e3251. McDanell, R., McLean, A.E., Hanley, A.B., Heaney, R.K., Fenwick, G.R., 1987. Differential induction of mixed-function oxidase (MFO) activity in rat liver and intestine by diets containing processed cabbage: correlation with cabbage levels of glucosinolates and glucosinolate hydrolysis products. Food and Chemical Toxicology 25 (5), 363e368. Mikkelsen, M.D., Hansen, C.H., Wittstock, U., Halkier, B.A., 2000. Cytochrome P450 CYP79B2 from Arabidopsis catalyzes the conversion of tryptophan to indole-3acetaldoxime, a precursor of indole glucosinolates and indole-3- acetic acid. Journal of Biological Chemistry 275, 33712e33717.

Glucosinolates and metabolism

139

Mikkelsen, M.D., Naur, P., Halkier, B., 2004. Arabidopsis mutants in the C-S lyase of glucosinolate biosynthesis establish a critical role for indole-3-acetaldoxime in auxin homeostasis. The Plant Journal 37, 770e777. Mugford, S.G., Yoshimoto, N., Reichelt, M., Wirtz, M., Hill, L., Mugford, S.T., Nakazato, Y., Noji, M., Takahashi, H., Kramell, R., Gigolashvili, T., Flügge, U.I., Wasternack, C., Gershenzon, J., Hell, R., Saito, K., Kopriva, S., 2009. Disruption of adenosine-50 -phosphosulfate kinase in Arabidopsis reduces levels of sulfated secondary metabolites. The Plant Cell 21, 910e927. Mugford, S.G., Matthewman, C.A., Hill, L., Kopriva, S., 2010. Adenosine-50phosphosulfate kinase is essential for Arabidopsis viability. FEBS Letters 584, 119e123. Mugford, S.G., Lee, B.R., Koprivova, A., Matthewman, C., Kopriva, S., 2011. Control of sulfur partitioning between primary and secondary metabolism. The Plant Journal 65, 96e105. Mullaney, J.A., Kelly, W.J., McGhie, T.K., Ansell, J., Heyes, J.A., 2013. Lactic acid bacteria convert glucosinolates to nitriles efficiently yet differently from Enterobacteriaceae. Journal of Agricultural and Food Chemistry 61, 3039e3046. Naur, P., Petersen, B.L., Mikkelsen, M.D., Bak, S., Rasmussen, H., Olsen, C.E., Halkier, B.A., 2003. CYP83A1 and CYP83B1, two nonredundant cytochrome P450 enzymes metabolizing oximes in the biosynthesis of glucosinolates in Arabidopsis. Plant Physiology 133, 63e72. Nour-Eldin, H.H., Halkier, B.A., 2013. The emerging field of transport engineering of plant specialized metabolites. Current Opinion in Biotechnology 24, 263e270. Nugon-Baudon, L., Rabot, S., Szylit, O., Raibaud, P., 1990. Glucosinolates toxicity in growing rats: interactions with the hepatic detoxification system. Xenobiotica 20 (2), 223e230. Oliviero, T., Verkerk, R., Dekker, M., 2018. Food isothiocyanates from Brassica vegetables-effects of processing, cooking, mastication, and digestion. Molecular Nutrition & Food Research e1701069. Palop, M.L., Smiths, J.P., Tenbrink, B., 1995. Degradation of sinigrin by Lactobacillus agilis strain R16. International Journal of Food Microbiology 26, 219e229. Pant, B.D., Pant, P., Erban, A., Huhman, D., Kopka, J., Scheible, W.R., 2005. Identification of primary and secondary metabolites with phosphorus status-dependent abundance in Arabidopsis, and of the transcription factor PHR1 as a major regulator of metabolic changes during phosphorus limitation. Plant, Cell and Environment 38, 172e187. Pedras, M.S.C., Ahiahonu, P.W.K., 2005. Metabolism and detoxification of phytoalexins and analogs by phytopathogenic fungi. Phytochemistry 66, 391e411. Pessina, A., Thomas, R.M., Palmieri, I.S., Luisi, P.L., 1990. An improved method for the purification of myrosinase and its physicochemical characterization. Archives of Biochemistry and Biophysics 280, 383e389. Petersen, B.L., Chen, S.X., Hansen, C.H., Olsen, C.E., Halkier, B.A., 2002. Composition and content of glucosinolates in developing Arabidopsis thaliana. Planta 214, 562e571. Petrussa, E., Braidot, E., Zancani, M., Peresson, C., Bertolini, A., Patui, S., Vianello, A., 2013. Plant flavonoids-biosynthesis, transport and involvement in stress responses. International Journal of Molecular Sciences 14 (7), 14950e14973. Piotrowski, M., Schemenewitz, A., Lopukhina, A., Muller, A., Janowitz, T., Weiler, E.W., Oecking, C., 2004. Desulfoglucosinolate sulfotransferases from Arabidopsis thaliana catalyze the final step in the biosynthesis of the glucosinolate core structure. Journal of Biological Chemistry 279, 50717e50725. Rask, L., Andréasson, E., Ekbom, B., Eriksson, S., Pontoppidan, B., Meijer, J., 2000. Myrosinase: gene family evolution and herbivore defense in Brassicaceae. Plant Molecular Biology 42, 93e113.

140

Glucosinolates: Properties, Recovery, and Applications

Ravilious, G.E., Nguyen, A., Francois, J.A., Jez, J.M., 2012. Structural basis and evolution of redox regulation in plant adenosine-50-phosphosulfate kinase. Proceedings of the National Academy of Sciences of the United States of America 109, 309e314. Rungapamestry, V., Duncan, A.J., Fuller, Z., Ratcliffe, B., 2006. Changes in glucosinolate concentrations, myrosinase activity, and production of metabolites of glucosinolates in cabbage (Brassica oleracea Var. capitata) cooked for different durations. Journal of Agricultural and Food Chemistry 54 (20), 7628e7634. Rungapamestry, V., Duncan, A.J., Fuller, Z., Ratcliffe, B., 2007. Effect of cooking brassica vegetables on the subsequent hydrolysis and metabolic fate of glucosinolates. Proceedings of the Nutrition Society 66 (1), 69e81. Schuster, J., Knill, T., Reichelt, M., Gershenzon, J., Binder, S., 2006. Branched-chain aminotransferase4 is part of the chain elongation pathway in the biosynthesis of methionine-derived glucosinolates in Arabidopsis. The Plant Cell 18, 2664e2679. Shapiro, T.A., Fahey, J.W., Dinkova-Kostova, A.T., Holtzclaw, W.D., Stephenson, K.K., Wade, K.L., Ye, L., Talalay, P., 2006. Safety, tolerance, and metabolism of broccoli sprout glucosinolates and isothiocyanates: a clinical phase I study. Nutrition and Cancer 55, 53e62. Shirakawa, M., Hara-Nishimura, I., 2018. Specialized vacuoles of myrosin cells: chemical defense strategy in brassicales plants. Plant and Cell Physiology 59 (7), 1309e1316. Skirycz, A., Reichelt, M., Burow, M., Birkemeyer, C., Rolcik, J., Kopka, J., Zanor, M.I., Gershenzon, J., Strnad, M., Szopa, J., Mueller-Roeber, B., Witt, I., 2006. DOF transcription factor AtDof1.1 (OBP2) is part of a regulatory network controlling glucosinolate biosynthesis in Arabidopsis. The Plant Journal 47, 10e24. Sønderby, I.E., Hansen, B.G., Bjarnholt, N., Ticconi, C., Halkier, B.A., Kliebenstein, D.J., 2007. A systems biology approach identifies a R2R3 MYB gene subfamily with distinct and overlapping functions in regulation of aliphatic glucosinolates. PLoS One 2, e1322. Stotz, H.U., Sawada, Y., Shimada, Y., Hirai, M.Y., Sasaki, E., Krischke, M., Brown, P.D., Saito, K., Kamiya, Y., 2011. Role of camalexin, indole glucosinolates, and side chain modification of glucosinolate-derived isothiocyanates in defense of Arabidopsis against Sclerotinia sclerotiorum. The Plant Journal 67 (1), 81e93. Tate, S.S., 1980. Enzymes of mercapturic acid formation. In: Jakoby, W.B. (Ed.), Enzymatic Basis of Detoxication, vol. II. Academic Press, New York, pp. 95e120. Taiz, L., Zeiger, E., 2010. Plant Physiology, fifth ed. The Benjamin/Cummings, Redwood City,California. USA. Textor, S., Bartram, S., Kroymann, J., Falk, K.L., Hick, A., Pickett, J.A., Gershenzon, J., 2004. Biosynthesis of methionine-derived glucosinolates in Arabidopsis thaliana: recombinant expression and characterization of methylthioalkylmalate synthase, the condensing enzyme of the chainelongation cycle. Planta 218, 1026e1035. Textor, S., De Kraker, W., Hause, B., Gershenzon, J., Tokuhisa, J.G., 2007. MAM3 catalyzes the formation of all aliphatic glucosinolate chain lengths in Arabidopsis. Plant Physiol 144, 60e71. Tian, S., Liu, X., Lei, P., Zhang, X., Shan, Y., 2018. Microbiota: a mediator to transform glucosinolate precursors in cruciferous vegetables to the active isothiocyanates. Journal of the Science of Food and Agriculture 98 (4), 1255e1260. Underhill, E.W., Wetter, L.R., Chisholm, M.D., 1973. Biosynthesis of glucosinolates. Biochemical Society Symposia 38, 303e326. Verkerk, R., Schreiner, M., Krumbein, A., Ciska, E., Holst, B., Rowland, I., De, S.R., Hansen, M., Gerhauser, C., Mithen, R., Dekker, M., 2009. Glucosinolates in Brassica vegetables: the influence of the food supply chain on intake, bioavailability and human health. Molecular Nutrition and Food Research 53 (2), S219. Wittstock, U., Burow, M., 2007. Tipping the scales–specifier proteins in glucosinolate hydrolysis. IUBMB Life 59 (12), 744e751.

Glucosinolates and metabolism

141

Wittstock, U., Halkier, B.A., 2002. Glucosinolate research in the Arabidopsis era. Trends in Plant Science 7, 263e270. Wittstock, U., Kurzbach, E., Herfurth, A.M., Stauber, E.J., 2016. Glucosinolate breakdown. Advances in Botanical Research 80, 126e159. Xiong, L., Lee, B., Ishitani, M., Lee, H., Zhang, C., Zhu, J.K., 2001. FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis. Genes and Development 15, 1971e1984. Zhang, Y., Talalay, P., 1994. Anticarcinogenic activities of organic isothiocyanates: chemistry and mechanisms. Cancer Research 54, 1976e1981. Zhang, Y., KoIm, R.H., Mannervik, B., Talalay, P., 1995. Reversible conjugation of isothiocyanates with glutathione catalyzed by human glutathione transferases. Biochemical and Biophysical Research Communications 206, 748e755. Zhang, N., Lariviere, A., Tonsor, S.J., Traw, M.B., 2014. Constitutive camalexin production and environmental stress response variation in Arabidopsis populations from the Iberian Peninsula. Plant Science 225, 77e85.

Further reading Chen, S., Andreasson, E., 2001. Update on glucosinolate metabolism and transport. Plant Physiology and Biochemistry 39, 743e758. Madsen, S.R., Kunert, G., Reichelt, M., Gershenzon, J., Halkier, B.A., 2015. Feeding on leaves of the glucosinolate transporter mutant gtr1gtr2 reduces fitness of Myzus persicae. Journal of Chemical Ecology 41, 975e984. Mazumder, A., Dwivedi, A., du Plessis, J., 2016. Sinigrin and its therapeutic benefits. Molecules 21 (4), 416.