Update on the Current Understanding of Biosynthesis

International Journal of Agriculture and Food Science Technology. ISSN 2249-3050 Volume 4, Number 1 (2013), pp. 37-50 © Research India Publications http://www.ripublication.com

Update on the Current Understanding of Biosynthesis, Biology and Transport of Glucosinolates in Brassica plants Astha Singh* and Matthew Hall Faculty of Agriculture and Environment, The University of Sydney, Australia. * Corresponding author’s email: [email protected]

Abstract Glucosinolates are secondary metabolites mostly found in the family Brassicaceae. These compounds are extensively associated with economically important plants, and may provide protective capacity against cellular oxidative stresses. Although known to be present in plants for over 100 years, interest and research about glucosinolate formation, function, and potential benefits to human health has taken place in recent times. Glucosinolates are simple organic compounds consisting of a core molecule combined with different side chains, which contribute to their diversity and subsequently determine the nature of hydrolysis products. Injured or affected plant tissue brings the compartmentalised enzyme myrosinase into contact with the localised glucosinolate, which separates the glucose group from the parent glucosinolate. These compounds have been shown to have the potential to contribute to human nutritional wellbeing, as plants high in glucosinolates are known to possess anti-carcinogenic and anti-microbial properties. Developments in the understanding of glucosinolate transport have recently progressed. This work has shown that changes in the biochemistry of the parent glucosinolate are possible at the site of damage, without the sole reliance of long distance phloem transport. The purpose of this review paper is to provide an update on recent developments which have taken place in the understanding of glucosinolate biosynthesis, biology and transport. Key Words: Glucosinolates, Regulation, Biochemistry, Biosynthesis, Myrosinase, Bio-fumigation, Defence, Signalling, Plant Defence.

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Astha Singh and Matthew Hall

Introduction Plants are known to synthesize thousands of individual compounds referred to as secondary metabolites, which have been shown to have defensive properties through the use of toxins and/or chemical feeding deterrents, and can be effective against a wide range of herbivores and pathogens (Burow et al., 2006; Kim and Jander, 2007; Wink, 1988). The major role of glucosinolates in plants is thought to be as chemical signal providers, communicating a range of information resulting in both defensive and attractant signals. Glucosinolates are the inert storage form of a two-phase plant defence system in which biologically active compounds such as isothiocynate, nitrile, and thiocyanate are produced by the compartmentalized enzyme myrosinase (Bones and Rossiter, 1996; Burow et al., 2009). This defence system is only initiated when myrosinase is brought into contact with the localized glucosinolate, this mostly occurs after plant tissue damage. Some specialist insects have evolved to take advantage of these compounds, triggering ovipositing and have adapted digestion processes or behaviours which eliminate their toxicity. The transport of glucosinolates in plants has previously been shown to occur from source to sink via diffusion in the phloem, however more recently it has been shown that the modification of glucosinolate type with the induction of specific enzymes is able to alter the structure in plant parts. This eliminates the requirement for transport of glucosinolates to the site of cellular damage as previously believed. This also indicates that although long distance transport occurs via phloem, the modification of the parent glucoside is possible at the site of damage without the requirement for long distance phloem transport. Human interest in glucosinolates is primarily due to their anti-carcinogenic properties and potential to contribute to dietary phyto-nutrient consumption, thereby improving human health and wellbeing. Alternatively, the investigation of glucosinolates is also focused on the understanding and manipulation of their expression in plants to improve commercial cropping systems against pathogenic infection (Halkier and Gershenzon, 2006; Larkin and Griffin, 2007). The improved utilization of glucosinolate containing plants in this context may decrease the dependency on chemical control measures and promote the use of natural alternatives like biofumigation.

Importance Glucosinolates remain intact unless they are brought into direct contact with the compartmentalized enzyme myrosinase, this can occur due to a multitude of variables including disruption of cells by pests, food processing, or mastercation (Bridges et al., 2002; Redovnikovic et al., 2008). These compounds coexist in plants as they are excluded from direct contact with each other; glucosinolates are stored in the vacuoles of so-called S-cells, while myrosinase is ill separated in immediately adjacent cells. Once plant tissue disruption occurs glucosinolates are released at the site of damage and are hydrolysed by myrosinase (Redovnikovic et al., 2008). This process releases glucose and subsequent breakdown products, which are potent inducers of phase II enzymes which are responsible for achieving protection against carcinogenesis, mutagenesis, electrophiles and reactive oxygen species (Fahey et al., 1997).

Update on the Current Understanding of Biosynthesis, Biology and Transport of 39 Glucosinolates are broken down in the human digestive system in two ways, either by plant myrosinase in the small intestine or by bacterial myrosinase in the colon (Kristensen et al., 2007). The importance of postharvest handling of agricultural commodities is currently a major limiting factor leading to the net loss of glucosinolates from the farm gate to the consumers table. The presence and understanding of these secondary metabolites is of direct benefit to humans in two ways: firstly, from an agricultural context through improved farming systems; and secondly, from a medicinal perspective through improved utilization of compounds beneficial for human health. These two broad areas of interest will help to dictate further research strategies and findings. Research in recent years has improved our knowledge significantly with regards to biosynthetic pathways, and evolutionary understanding liked to these relevant pathways. The majority of this information has been generated as a direct result of the completion of the Arabidopsis genome project. Through this detailed and extensive project much knowledge has been acquired in regards to glucosinolates and the ways in which these compounds alter structure and source of formation at differing times of the plant life cycle, or in response to a multitude of possible abiotic and biotic stresses. Most plant species from the Brassicaceae family have the distinct taste which is primarily a consequence of their isothiocyanate hydrolysis products (Halkier and Gershenzon, 2006). Epidemiological studies have suggested that Brassica vegetables possess protective properties against carcinogenic compounds, notably the effect which they have against lung, alimentary tract, and breast cancers (Johnson, 2002; Neuhouser et al., 2003; Pledgie-Tracy et al., 2007). In comparison, glucosinolates are also known for their toxic effects in both humans and animals when consumed in high amounts and when not excreted in sufficient quantities. However, in non-toxic concentrations they can induce phase I and II enzymes, resulting in enzyme inhibition, modification of hormone metabolism and protection against cellular oxidative degradation (Fahey and Talalay, 1999; Petri et al., 2003). This biophysical outcome is what has largely fueled the recent interest and subsequent research into how these compounds interact in humans. Approximately 120 glucosinolates have been identified in plants. They represent a group of nitrogen and sulphur rich compounds which are derived from glucose (Troyer et al., 2001; Prakash and Gupta, 2012). These compounds are found exclusively in plants of the Brassicaceae and other related families (Brown and Morra, 1997). The chemical structure of glucosinolates is such that there is a central carbon atom which is bound via a sulphur atom to the thioglucose group that makes a sulphated ketoxine and via a nitrogen atom to a sulphate group. An additional a side group is also bound to the central carbon atom, allowing for differences in glucosinolate type to occur (Chew, 1988; Lambrix et al., 2001; Rask et al., 2000).

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Beneficial Glucosinolates According to Troyer et al. (2001) broccoli sprouts are a rich source of glucosinolates and isothiocynates which induce phase II detoxication enzymes, boost antioxidant status and protect against chemically induced cancer (Shapiro et al., 2001). Hong and Kim (2008) showed that the glucosinolate beta-Phenylethyl isothiocyanate inhibited the growth of human-derived hepatoma cell line (HepG2) in a concentrationdependent manner, assessed by the MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5diphenyltetrazolium bromide) method. Beta-Phenylethyl isothiocyanate also has antimicrobial properties against food-borne pathogens like Vibrio parahaemolyticus, Staphylococcus aureus and Bacillus cereus. Historically glucosinolates were regarded as a predicament, due to their chemical nature, but now beneficial effects such as anticarcinogenesis and anti-microbial activities have brought these compounds into focus (Andreasson, 2000; Shapiro et al., 2001; Hong and Kim, 2008). Another advantage of glucosinolates in human health is given by the cancer prevention research program, Washington, US, that implied that – increased consumption of Brassica vegetables has reduced prostate cancer risks. Indirectly the involvement of glucosinolates which cause the induction and upregulation of the GST (glucosinolate) gene, provides protection against prostate carcinogenesis (Kristal and Lampe, 2002). To date, the collective investigation of glucosinolates has focused on aspects which are of direct benefit to humans, such as their cancer preventative characteristics, crop protection potential, and biological fumigation properties for agricultural purposes (Halkier and Gershenzon, 2006).

Components of Glucosinolates Similar to cyanogenic glucosides, glucosinolate side chains are derived from amino acids and produce intermediate compounds called aldoximes (Poulton, 1990). The glucose molecule conjugates with the aglycone due to the S-glycosyltransferase, which has been charecterised (GrootWassink et al., 1994; Reed et al., 1993). Previously the biosynthesis of glucosinolates was not well understood (Ettlinger and Kjaer, 1968) but the above findings suggest that glucosinolate biosynthesis is evolutionarily related to cyanogenic glucosides synthesis. There are reports that sulphotransferases also has a role in the biosynthesis of glucosinolates (Klein and Papenbrock, 2008), which is dependent on monooxygenases and peroxidises during biosynthesis (Halkier, 1999). The aglycones of the three types of glucosinolates (aliphatic, indolic and benzenic) all derive from amino acids; L-alanine, L-leucine, L-isoleucine, L-valine, L-tyrosine, L-phenylalanine and L-tryptophan; and also from several chain-elongated homologous of methionine (Halkier, 1999; Rask et al., 2000).

Update on the Current Understanding of Biosynthesis, Biology and Transport of 41

Glucosinolate Formation In rapeseed (Brassica napus L.) glucosinolates are present in different concentrations depending on the developmental stage of the plant (Clossais-Besnard and Larher, 1991; Fieldsend and Milford, 1994a; Fieldsend and Milford, 1994b); specific time of the day (Rosa, 1997); environmental conditions (Bouchereau et al., 1996) and different stress conditions like: pathogen attack (Doughty et al., 1991), wounding (Bodnaryk, 1992), jasmonate treatment (Bodnaryk, 1992) and salicylic acid treatment (Kiddle et al., 1994). These different conditions cause the accumulation of glucosinolates, for example – wounding and methyl-jasmonate treatment causes induction of indolyl glucosinolate whereas benzenic glucosinolates are induced by salicylic acid. So during rapid growth periods of the plant additional glucosinolates often accumulate (Clossais-Besnard and Larher, 1991). Moreover in fully expanded leaves the concentration of glucosinolates is lower than in younger leaves (Porter et al., 1991). Glucosinolates have been under considerable debate with regards to their location, if their sub cellular localisation is followed, we can better understand their involvement in growth, development and defence (Kelly et al., 1998). Understanding the biosynthetic pathway of glucosinolate formation is complicated as glucosinolates are not localised and are water soluble. This is because; in-situ localisation of glucosinolates does not contain the substance that is used in localisation. If conventional fixation and dehydration procedures are followed, analysis can be done using enzyme-linked immunosorbent assay (ELISA) and raising antibodies against glucosinolates (Kelly et al., 1998). Research suggests that vacuolar localisation of glucobrassicin and sinigrin is possible (Grob and Matile, 1979; Helmlinger et al., 1983), meaning that different types of glucosinolates perform differently in the plants.

Glucosinolates and Myrosinase Environmental factors such as light, nutrition, fungal infection, injury and herbivore attack are able to revise the glucosinolate pattern significantly. A change of the glucosinolate profile by several environmental factors has brought forward different theories regarding their potential roles in the plant. For instance some studies using several glucosinolate biosynthesis mutants suggested that glucosinolate metabolism may play a role in plant development. However, the most accepted theory is that the glucosinolate-myrosinase system is involved in defence against herbivores and pathogens (Redovnikovic et al., 2008). Myrosinase is an enzyme found in plants and bowel microflora and it hydrolyses glucosinolates to form isothiocynates (Shapiro et al., 2001). Myrosinase is also known as thioglucosidase and is an intermediate product between the glucosinolate and derivative formations. The glucosinolate-myrosinase system is believed to be a part of plant defence against insects and pathogens (Pontoppidan, 2001; Wielanek and Urbanek, 2006). It is also a transmission stage of the formation of isothiocynates, so it may help in studies related to elucidation of the biosynthetic pathway of glucosinolates.

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Biosynthetic Pathways of Glucosinolates Significant research on biosynthesis of glucosinolates has shown that amino acids are added with methylene groups into their side chains, they get re-configured to give the basic structure of glucosinolates and then the initially formed glucosinolates are modified by secondary transformations (Halkier and Gershenzon, 2006). There are three phases in the course of glucosinolate biosynthesis: firstly, methylene groups are inserted into side chains of aliphatic and aromatic amino acids; secondly, the amino acid is altered to produce the core structure of the glucosinolate; and thirdly, the initially formed glucosinolate is modified by secondary transformations. Dixon (2001) has shown that through a series of steps amino acids are formed into glucosinolates followed by the formation of L-Phenylalanine ammonia lyase (PAL) (Fig 1).

Fig 1. Pathway of glucosinolate biosynthesis from amino acid, tryptophan. Activity of Phenylalanine ammonia lyase (PAL) and formation of cinnamic acid (Reprinted by permission from Macmillan Publishers Ltd: [Nature] (Dixon, 2001), copyright (2001). Salicylic acid affects the synthesis of glucosinolates, it increases the accumulation of glucosinolates and is more specific than that observed due to a fungal infection or herbivore attack (Kiddle et al., 1994). This response was shown by applying salicylic acid soil drench to rapeseed plants and then observing the levels of glucosinolates in the leaves. In addition, it has recently been reported by Byun et al. (2009) that cold stress genes related to salicylic acid and glucosinolate synthesis were detected as an early response to cold stress. As amino acids are the precursors of glucosinolates treatment of the roots with amino acid precursors of glucosinolate or/and cysteine biosynthesis increased levels of glucosinolate production, combinations of phenylalanine with cysteine (for gluconasturtiin and glucotropaeolin) and methionine with o-acetylserine (for glucoiberverin) were found the most effective in a study by Wielanek et al. (2009). Mewis et al. (2006) reported that both glucosinolate content and gene expression data

Update on the Current Understanding of Biosynthesis, Biology and Transport of 43 indicate that salicylate and ethylene signalling limit some jasmonate mediated responses to herbivore attack in Arabidopsis. Recombinant branched-chain amino transferase 4 showed high efficiency with methionine and its derivatives and the corresponding 2-oxo acids, suggesting its participation in the chain elongation pathway of Met-derived glucosinolate biosynthesis (Schuster et al., 2006). Grubb et al. (2004) suggest the primary involvement of UDP-glucose: thiohydroximate Sglucosyltransferase in glucosinolate biosynthesis in the Arabidopsis glucosynthesis pathway. It has been reported that the amino acid methionine-derived glucosinolates belong to a class of plant secondary metabolites that serve as chemoprotective compounds in plant biotic defense reactions and also exhibit strong anticancerogenic properties beneficial to human health. In a screen for the trans-activation potential of transcription factors toward glucosinolate biosynthetic genes, it has been identified that the HAG1 (High Aliphatic Glucosinolate 1, also referred to as MYB28) gene as a positive regulator of aliphatic methionine-derived glucosinolates (Gigolashvili et al., 2007). There is also some evidence about the relationship between jasmonates and production of glucosinolates (Kubicka and Zadernowski, 2007). In regards to aliphatic and indole glucosinolates, it has been reported that the concentration of nitrogen containing tryptophan derived indole glucosinolate was greater with increased nitrogen supply; however sulphur containing methionine derived aromatic and aliphatic glucosinolates decreased with increasing nitrogen and low sulphur contents. This response indicates that there is a direct relationship between biosynthesis and the availability of nitrogen and sulphur compounds (Li et al., 2007).

Biological Functions A wide range of studies on glucosinolates have illustrated that they posses toxic, growth inhibition and feeding deterrent signals to a wide variety of organisms (Brader et al., 2001; Burow et al., 2006; Kim and Jander, 2007). The induction of indole glucosinolates are known to be strongly associated with biotic stress factors resulting from a range of sources including; fungal pathogen attack (Brader et al., 2001; Doughty et al., 1991; Kirkegaard et al., 2001), bacterial infection (van Dam et al., 2009), insect feeding (Ahuja et al., 2010) and generalist or specialist herbivores (Kim and Jander, 2007). The biological function of glucosinolates in plants is therefore wide ranging and has been partly established thus far, the primary purpose of these compounds is to act as signalling mediators to communicate as part of a wider plant defence system. Alternatively humans have identified that low levels of consumption of these compounds may be beneficial to health and wellbeing (Johnson, 2002).

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Transport of Glucosinolates in Plants The glucosinolate-myrosinase system in Brassica plants is involved in multiple aspects of plant development and defence responses. The hydrolysis of glucosinolates by myrosinase has been extensively shown to result in a diverse range of substances which have varied biological functions and additionally are also precursors for plant hormones (Halkier and Gershenzon, 2006). The transport of glucosinolates in plant tissues is of significant interest as it provides evidences for its biosynthesis. Glucosinolates have been reported in all parts of Brassica plants, but the concentration ranges greatly throughout the plant life cycle (Steinbrenner et al., 2012). Chen and Halkier (2000) have also shown the radiolabeled phydroxybenzylglucosinolate (p-OHBG) and sucrose is transported via phloem; both tracers were co-applied to the tip of detached leaves and collected in phloem exudates at the petioles. Similarly this relationship between glucosinolate content in the leaves pre and post flowering has been shown to differ in Arabidopsis (Brown et al., 2003). Phloem acts as an essential delivery system to heterotrophic plant tissue delivering important plant resources such as, photo-assimilates, amino acids, and signalling molecules (Chen and Halkier, 2000). Observations made by Lykkesfeldt and Moller (1993) establish that benzylglucosinolate is synthesized in the leaves of Indian cress (Tropaeolum majus L.), but were found to accumulate in other organs such as the seeds indicating that glucosinolates are transported in the plant. Brudenell et al. (1999) found that glucosinolates and desulphoglucosinolates both have physicochemical properties allowing for phloem mobility. The diversity of glucosinolates was found to be the highest in organs such as seeds and fruits, which may have arisen in an attempt to improve the defensive potential of these plant parts (Brown et al., 2003). The major seed glucosinolates have been identified in Arabidopsis as being methylthioalkyl with side chains and benzoate ester substituents. Younger rosette leaves have been shown to contain higher concentrations of glucosinolates than older leaves; as a consequence of this the centre of the rosette appears to be better protected from herbivores and pathogens than the older leaves (Brown et al., 2003).

Further Investigation Although significant improvements in the understanding of glucosinolates have occurred in recent years, there still remains much research into how plants transport and utilize these compounds. Further understanding of the physiological process with which plants synthesize glucosinolates will further benefit their usefulness to agricultural systems and medicinal pursuits. The literature suggests that some glucosinolates may hold health benefits for humans, but the results are not conclusive. Toxic and bitter tasting glucosinolates were previously removed from Brassica vegetables by breeding and other genetic techniques. Beneficial glucosinolates should be incorporated into commercial varieties to provide higher nutritional content. Glucosinolates contribute to the flavour

Update on the Current Understanding of Biosynthesis, Biology and Transport of 45 of vegetables, serve as plant defence compounds, and possess anti-carcinogenic and anti-microbial abilities. Molecular and metabolic tools have been found effective and could be used as a prospective means to generate higher glucosinolate containing crops (Halkier and Gershenzon, 2006). It has been shown that indole glucosinolate levels in plants can be increase through the use of mechanical wounding (Bodnaryk, 1992; Pontoppidan et al., 2001) or the application of methyl jasmonate (Bodnaryk, 1994; Kliebenstein et al., 2007; Mewis et al., 2006). The artificial induction of plant defence systems and the subsequent increase in glucosinolate levels illustrates that the formation and deployment of these compounds can be both site specific and also more general (Kim and Jander, 2007). Literature does not suggest much about the interaction of glucosinolates with plant diseases and the effect that infection has on their levels. A disease response may result in a change in the concentration of glucosinolates, this response has been found between infection, resistance and glucosinolate modification. Glucosinolates are important and useful compounds found in many agricultural crops, their biosynthesis, biology and transport in plants is complex and ever changing. If we are to utilize these compounds commercially or for health purposes then it is important that these aspects be further clarified.

References [1]

[2]

[3] [4] [5] [6]

[7]

[8]

Ahuja, I., Rohloff, J., Bones, A.M. (2010). Defence mechanisms of Brassicaceae: implications for plant-insect interactions and potential for integrated pest management. A review. Agronomy for Sustainable Development 30: 311-348. Andreasson, E. (2000). Structural and functional studies of the myrosinaseglucosinolate system in Arabidopsis thaliana and Brassica napus, p.15-25. PhD thesis, Department of Plant biology, Swedish University of Agricultural sciences. Bodnaryk, R.P. (1992). Effects of wounding on glucosinolates in the cotyledons of oilseed rape and mustard. Phytochemistry 31: 2671-2677. Bodnaryk, R.P. (1994). Potent effect of jasmonates on indole glucosinolates in oilseed rape and mustard. Phytochemistry 35: 301-305. Bones, A.M., Rossiter, J.T. (1996). The myrosinase-glucosinolate system, its organisation and biochemistry. Physiologia Plantarum 97: 194-208. Bouchereau, A., Clossais-Besnard, N., Benasoud, A., Leport, L., Renard, M. (1996). Water stress effects on rapeseed quality. European Journal of Agronomy 5: 19-30. Brader, G., Tas, E., Palva, E.T. (2001). Jasmonate-dependent induction of indole glucosinolates in Arabidopsis by culture filtrates of the nonspecific pathogen Erwinia carotovora. Plant Physiology 126: 849-860. Bridges, M., Jones, A.M.E., Bones, A.M., Hodgson, C., Cole, R., Bartlet, E., Wallsgrove, R., Karapapa, V.K., Watts, N., Rossiter, J.T. (2002). Spatial

46

[9]

[10]

[11] [12]

[13]

[14] [15]

[16] [17]

[18] [19]

[20] [21]

[22]

Astha Singh and Matthew Hall organization of the glucosinolate-myrosinase system in Brassica specialist aphids is similar to that of the host plant. Proceedings of the Royal Society B 269: 187–191. Brown, P.D., Morra, M.J. (1997). Control of soil-borne plant pests using glucosinolate-containing plants, p. 167-231. In: D.L. Sparks (ed.). Advances in Agronomy. Elsevier Academic Press Inc, San Diego. Brown, P.D., Tokuhisa, J.G., Reichelt, M., Gershenzon J. (2003). Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana. Phytochemistry 62: 471-481. Brudenell, A.J.P., Griffiths, H., Rossiter, J.T., Baker, D.A. (1999). The phloem mobility of glucosinolates. Journal of Experimental Botany 50: 745-756. Burow, M., Markert, J., Gershenzon, J., Wittstock, U. (2006). Comparative biochemical characterization of nitrile-forming proteins from plants and insects that alter myrosinase-catalysed hydrolysis of glucosinolates. FEBSJournal 273: 2432-2446. Burow, M., Losansky, A., Muller, R., Plock, A., Kliebenstein, D.J., Wittstock, U. (2009). The genetic basis of constitutive and herbivore-induced ESPindependent nitrile formation in Arabidopsis. Plant Physiology 149: 561-574. Byun, Y-J., Kim, H-J., Lee, D-H. (2009). LongSAGE analysis of the early response to cold stress in Arabidopsis leaf. Planta 229: 1181-1200. Chen, S., Halkier, B.A. (2000). Characterization of glucosinolate uptake by leaf protoplasts of Brassica napus. Journal of Biological Chemistry 275: 22955-22960. Chew, F.S. (1988). Biological effects of glucosinolates. ACS Symposium Series 380:155-181. Clossais-Besnard, N., Larher, F. (1991). Physiological role of glucosinolates in Brassica napus. Concentration and distribution pattern of glucosinolates among plant organs during a complete life cycle. Journal of the Science of Food and Agriculture 56: 25-38. Dixon, R.A. (2001). Natural products and plant disease resistance. Nature 411: 843-847. Doughty, K.J., Porter, A.J.R., Morton, A.M., Kiddle, G., Bock, C.H., Wallsgrove, R. (1991). Variation in the glucosinolate content of oilseed rape (Brassica napus L.) leaves. II. Response to infection by Alternaria Brassicae (Berk) sacc. Annals of Applied Biology 118: 469-477 Ettlinger, M.G., Kjaer, A. (1968). Sulfur compounds in plants. Recent Advances in Phytochemistry. 1: 59–144. Fahey, J.W., Talalay, P. (1999). Antioxidant functions of sulforaphane: a potent inducer of phase II detoxication enzymes. Food and Chemical Toxicology 37: 973-979. Fahey, J.W., Zhang, Y., Talalay, P. (1997). Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proceedings of the National Academy of Sciences of the United States of America 94: 10367-10372.

Update on the Current Understanding of Biosynthesis, Biology and Transport of 47 [23] Fieldsend, J., Milford, G.F.J. (1994a). Changes in glucosinolates during crop development in single-low and double-low genotypes of winter oilseed rape (Brassica napus). 1. Production and distribution in vegetative tissues and developing pods during development and potential role in the recycling of sulfur within the crop. Annals of Applied Biology 124: 531-542. [24] Fieldsend, J., Milford, G.F.J. (1994b). Changes in glucosinolates during crop development in single-low and double-low genotypes of winter oilseed rape (Brassica napus). 2. Profiles and tissue-water concentrations in vegetative tissues and developing pods. Annals of Applied Biology 124: 543-555. [25] Gigolashvili, T., Yatusevich, R., Berger, B., Muller, C., Flugge, U.I. (2007). The R2R3-MYB transcription factor HAG1/MYB28 is a regulator of methionine-derived glucosinolate biosynthesis in Arabidopsis thaliana. Plant Journal 51: 247-261. [26] Grob, K., Matile, P.H. (1979). Vacuolar location of glucosinolates in horseradish root cells. Plant Science Letters 14: 327-335. [27] GrootWassink, J.W.D., Reed, D.W., Kolenovsky, A.D. (1994). Immunopurification and immunocharacterization of the glucosinolate biosynthetic enzyme thiohydroximate s-glucosyltransferase. Plant Physiology 105: 425-433. [28] Grubb, D.C., Zipp, B.J., Ludwig-Muller, J., Masuno, M.N., Molinski, T.F., Abel, S. (2004). Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis. The Plant Journal 40: 893908. [29] Halkier, B.A. (1999) Glucosinolates, p. 193-223. In: R. Ikan (ed.). Naturally occurring Glycosides. John Wiley and Sons, Chichester. [30] Halkier, B.A., Gershenzon, J. (2006). Biology and biochemistry of glucosinolates. Annual Review of Plant Biology 57: 303-333. [31] Helmlinger, J., Rausch, T., Hilgenberg, W. (1983). Localization of newly synthesized indole-3-methylglucosinolate (=glucobrassicin) in vacuoles from horseradish (Armoracia rusticana). Physiologia Plantarum 58: 302-310. [32] Hong, E., Kim, G-H. (2008). Anticancer and antimicrobial activities of βphenylethyl isothiocyanate in Brassica rapa L. Food Science and Technology Research 14: 377-382. [33] Johnson, I.T. (2002). Glucosinolates: bioavailability and importance to health. International Journal for Vitamin and Nutrition Research 72: 26-31. [34] Kelly, P.J., Bones, A., Rossiter, J.T. (1998). Sub-cellular immunolocalization of the glucosinolate sinigrin in seedlings of Brassica juncea. Planta 206: 370377. [35] Kiddle, G.A., Doughty, K.J., Wallsgrove, R.M. (1994). Salicylic acid-induced accumulation of glucosinolates in oilseed rape (Brassica napus L.) leaves. Journal of Experimental Botany 45: 1343-1346. [36] Kim, J.H., Jander, G. (2007). Myzus persicae (green peach aphid) feeding on Arabidopsis induces the formation of a deterrent indole glucosinolate. The Plant Journal 49: 1008-1019.

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[37] Kirkegaard, J.A., Rebetzke, G.J., Richards, R.A. (2001). Inheritance of root glucosinolate content in canola. Australian Journal of Agricultural Research 52: 745-753. [38] Klein, M., Papenbrock, J. (2008). Sulfotransferases and their role in glucosinolate biosynthesis, p. 149-166. In: N. Khan, S. Singh, S. Umar (eds.). Sulfur assimilation and abiotic stress in plants. Springer, Berlin, Heidelberg. [39] Kliebenstein, D.J., D'Auria, J.C., Behere, A.S., Kim, J.H., Gunderson, K.L., Breen, J.N., Lee, G., Gershenzon, J., Last, R.L., Jander, G. (2007). Characterization of seed-specific benzoyloxyglucosinolate mutations in Arabidopsis thaliana. The Plant Journal 51: 1062-1076. [40] Kristal, A.R., Lampe, J.W. (2002). Brassica vegetables and prostate cancer risk: a review of the epidemiological evidence. Nutrition and Cancer 42: 1-9. [41] Kristensen, M., Krogholm, K.S., Frederiksen, H., Bugel, S.H., Rasmussen, S.E. (2007). Urinary excretion of total isothiocyanates from cruciferous vegetables shows high dose-response relationship and may be a useful biomarker for isothiocyanate exposure. European Journal of Nutrition 46: 377-382. [42] Kubicka, E., Zadernowski, R. (2007). Enhanced jasmonate biosynthesis in plants and possible implications for food quality. Acta Alimentaria 36: 455469. [43] Lambrix, V., Reichelt, M., Mitchell-Olds, T., Kliebenstein, D.J., Gershenzon, J. (2001). The Arabidopsis epithiospecifier protein promotes the hydrolysis of glucosinolates to nitriles and influences Trichoplusia ni herbivory. The Plant Cell 13: 2793-2807. [44] Larkin, R.P., Griffin, T.S. (2007). Control of soilborne potato diseases using Brassica green manures. Crop Protection 26: 1067-1077. [45] Li, C.X., Sivasithamparam, K., Walton, G., Salisbury, P., Burton, W., Banga, S.S., Banga, S., Chattopadhyay, C., Kumar, A., Singh, R., Singh, D., Agnohotri, A., Liu, S.Y., Li, Y.C., Fu, T.D., Wang, Y.F., Barbetti, M.J. (2007). Expression and relationships of resistance to white rust (Albugo candida) at cotyledonary, seedling, and flowering stages in Brassica juncea germplasm from Australia, China, and India. Australian Journal of Agricultural Research 58: 259-264. [46] Lykkesfeldt, J., Moller, B.L. (1993). Synthesis of benzylglucosinolate in Tropaeolum majus L. (isothiocyanates as potent enzyme inhibitors). Plant Physiology 102: 609-613. [47] Mewis, I., Tokuhisa, J.G., Schultz, J.C., Appel, H.M., Ulrichs, C., Gershenzon, J. (2006). Gene expression and glucosinolate accumulation in Arabidopsis thaliana in response to generalist and specialist herbivores of different feeding guilds and the role of defense signaling pathways. Phytochemistry 67: 2450-2462. [48] Neuhouser, M.L., Patterson, R.E., Thornquist, M.D., Omenn, G.S., King, I.B., Goodman, G.E. (2003). Fruits and vegetables are associated with lower lung cancer risk only in the placebo arm of the beta-carotene and retinol efficacy trial (CARET). Cancer Epidemiology, Biomarkers & Prevention 12: 350-358.

Update on the Current Understanding of Biosynthesis, Biology and Transport of 49 [49] Petri, N., Tannergren, C., Holst, B., Mellon, F.A., Bao, Y., Plumb, G.W., Bacon, J., O'Leary, K.A., Kroon, P.A., Knutson, L., Forsell, P., Eriksson, T., Lennernas, H., Williamson, G. (2003). Absorption/metabolism of sulforaphane and quercetin, and regulation of phase II enzymes, in human jejunum in vivo. Drug Metabolism and Disposition 31: 805-813. [50] Pledgie-Tracy, A., Sobolewski, M.D., Davidson, N.E. (2007). Sulforaphane induces cell type-specific apoptosis in human breast cancer cell lines. Molecular Cancer Therapeutics 6: 1013-1021. [51] Pontoppidan, B. (2001). Studies of the glucosinolate-myrosinase system in relation to insect herbivory on oilseed rape (Brassica napus) and in Arabidopsis thaliana, p.55-60. PhD thesis, Department of Plant biology, Swedish University of Agricultural sciences [52] Porter, A.J.R., Morton, A.M., Kiddle, G., Doughty, K.J., Wallsgrove, R.M. (1991). Variation in the glucosinolate content of oilseed rape (Brassica napus L.) leaves. 1. Effect of leaf age and position. Annals of Applied Biology 118: 461-467. [53] Poulton, J.E. (1990). Cyanogenesis in plants. Plant Physiology 94: 401-405. [54] Prakash, D., Gupta, C. (2012). Glucosinolates: the phytochemicals of nutraceutical importance. Journal of Complementary & Integrative Medicine 9: Article 13. [55] Rask, L., Andreasson, E., Ekbom, B., Eriksson, S., Pontoppidan, B., Meijer, J. (2000). Myrosinase: gene family evolution and herbivore defense in Brassicaceae. Plant Molecular Biology 42: 93-113. [56] Redovnikovic, I.R., Glivetic, T., Delonga, K., Vorkapic-Furac, J. (2008). Glucosinolates and their potential role in plant. Periodicum Biologorum 110: 297-309. [57] Reed, D.W., Davin, L., Jain, J.C., Deluca, V., Nelson, L., Underhill, E.W. (1993). Purification and properties of UDP-glucose: thiohydroximate glucosyltransferase from Brassica napus L. seedlings. Archives of Biochemistry and Biophysics 305: 526-532. [58] Rosa, E.A.S. (1997). Daily variation in glucosinolate concentrations in the leaves and roots of cabbage seedlings in two constant temperature regimes. Journal of the Science of Food and Agriculture 73: 364-368. [59] 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: 2664-2679. [60] Shapiro, T.A., Fahey, J.W., Wade, K.L., Stephenson, K.K., Talalay, P.(2001). Chemoprotective glucosinolates and isothiocyanates of broccoli sprouts: metabolism and excretion in humans. Cancer Epidemiology, Biomarkers & Prevention 10: 501-508. [61] Steinbrenner, A.D., Agerbirk, N., Orians, C.M., Chew, F.S. (2012). Transient abiotic stresses lead to latent defense and reproductive responses over the Brassica rapa life cycle. Chemoecology 22: 239-250.

50


[62] Troyer, J.K., Stephenson, K.K., Fahey, J.W. (2001). Analysis of glucosinolates from broccoli and other cruciferous vegetables by hydrophilic interaction liquid chromatography. Journal of Chromatography 919: 299-304. [63] van Dam, N.M., Tytgat, T.O.G., Kirkegaard, J.A. (2009). Root and shoot glucosinolates: a comparison of their diversity, function and interactions in natural and managed ecosystems. Phytochemistry Reviews 8: 171-186. [64] Wielanek, M., Urbanek, H. (2006). Enhanced glucotropaeolin production in hairy root cultures of Tropaeolum majus L. by combining elicitation and precursor feeding. Plant Cell, Tissue and Organ Culture 86: 177-186. [65] Wielanek, M., Krolicka, A., Bergier, K., Gajewska, E., Sklodowska, M. (2009). Transformation of Nasturtium officinale, Barbarea verna and Arabis caucasica for hairy roots and glucosinolate-myrosinase system production. Biotechnology Letters 31: 917-921. [66] Wink, M. (1988). Plant-breeding: importance of plant secondary metabolites for protection against pathogens and herbivores. Theoretical and Applied Genetics 75: 225-233.