Phytoalexins from the Poaceae: Biosynthesis, Function and Prospects in Food Preservation

Food Research International 52 (2013) 167–177

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Food Research International

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Review Phytoalexins from the Poaceae: Biosynthesis, function and prospects in food preservation

Chukwunonso E.C.C. Ejike a,b, Min Gong c, Chibuike C. Udenigwe c,⁎ a Department of Food Science, University of Guelph, Ontario, N1G 2W1, Canada b Department of Biochemistry, College of Natural and Applied Sciences, Michael Okpara University of Agriculture, Umudike, PMB 7267, Umuahia, Abia State, Nigeria c Health and Bio-products Research Laboratory, Department of Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, Nova Scotia, B2N 5E3, Canada article info abstract

Article history: Phytoalexins are compounds synthesized by plants in response to extrinsic stress such as microbial attack and Received 15 January 2013 physical injury. Some members of the Poaceae, including cereal crops rice, maize and sorghum, are known to Accepted 9 March 2013 produce substantial amounts of structurally diverse groups of phytoalexins. However, no phytoalexin has been identiﬁed in other cereals such as wheat and barley although they possess certain phytoalexin biosynthetic Keywords: genes. Phytoalexins identiﬁed in the cereal food Poaceae include the momilactones, oryzalexins, phytocassanes Phytoalexin and sakuranetin from rice; the kauralexins and zealexins from maize; and the 3-deoxyanthocyanidins from sor- Poaceae Food preservation ghum. These phytoalexins are known to exhibit considerable antimicrobial activities against a wide array of Antimicrobial pathogenic fungi and bacteria. Despite their prospects for use as naturally derived antimicrobial agents, there Elicitor is scarcity of information on the application of these inducible compounds in the food system. Since food wastage due to spoilage microorganisms constitutes a challenge in global food security, these phytoalexins can potentially be utilized as sustainable natural antimicrobial food preservatives considering the abundance of their sources. This paper reviews the chemistry, biosynthesis and antimicrobial activities of phytoalexins from the cereal food Poaceae, and highlights their potential application in food preservation. © 2013 Elsevier Ltd. All rights reserved.

Contents

1. Introduction ...... 168 2. Promoting food security through food preservation ...... 168 3. Brief history of phytoalexins ...... 168 4. Phytoalexins and the Poaceae ...... 169 4.1. Rice ...... 169 4.2. Maize ...... 169 4.3. Wheat ...... 169 4.4. Barley ...... 170 4.5. Sorghum ...... 171 5. Chemistry and antimicrobial properties of phytoalexins produced by the Poaceae ...... 171 5.1. Terpenoid phytoalexins ...... 171 5.1.1. Diterpenoid phytoalexins ...... 171 5.1.2. Sesquiterpenoid phytoalexins ...... 172 5.2. Flavonoid phytoalexins ...... 173 5.2.1. Sakuranetin ...... 173 5.2.2. 3-Deoxyanthocyanidins ...... 173 6. Biosynthesis of phytoalexins produced by the Poaceae ...... 173 6.1. Terpenoid phytoalexins ...... 173 6.2. Flavonoid phytoalexins ...... 174 6.2.1. Sakuranetin ...... 174 6.2.2. 3-Deoxyanthocyanidins ...... 174

⁎ Corresponding author. Tel.: +1 902 843 6625; fax: +1 902 843 1404. E-mail address: [email protected] (C.C. Udenigwe).

7. ProspectsofphytoalexinsfromthePoaceaeinfoodpreservation...... 174 8. Conclusion and future direction ...... 175 Acknowledgment ...... 175 References ...... 175

1. Introduction One neglected component of ensuring food security is reducing losses and wastages. Food losses refer to the reduction in the quantity The challenge of food security remains a major global obstacle facing of edible food at any point in the food supply chain, that is, the produc- mankind. Although food production has increased in the last few de- tion, postharvest and processing stages. Losses occurring at the retail cades, the progress made in improving the quantity of food produced and consumption stages – end of the food supply chain – are rather annually may not ensure global food security in the future with enor- called “food waste” (Julian & Barthel, 2010). Food wastages are found mous food losses and wastages (Gustavsson, Cederberg, Sonesson, more in developed countries while food spoilage occurs more in devel- van Otterdijk, & Meybeck, 2011). As part of current initiatives to oping countries. It is estimated that about 33% of the food produced enhance food security, proper preservation methods are required to globally (about 1.3 billion tonnes per year) is wasted before or after it ensure that the best food quality and shelf-life are attained. Recently, gets to the final consumer (FAO, 2012a; Gustavsson et al., 2011). It is there has been a drive towards the use of “natural” food preservatives, further projected that halving the amount of food wasted globally especially as synthetic preservatives raise health-related concerns, and (assuming 30% is currently wasted) would provide 25% of current pro- as properties of foods other than their nutritional and sensory attributes duction by 2050 thereby enhancing future food security (Government are recognized (Corbo et al., 2009). In this direction, plant-derived Office for Science, London, 2011). Reducing food losses and wastages, compounds with antimicrobial activity are now increasingly explored in order to guarantee food security requires among other things, for use in the preservation and enhancement of food quality. In light efficient processing and preservation techniques that guarantee food of these developments, there is a need to discover sustainable means quality and improve shelf-life. Considering that microorganisms and of producing structurally diverse natural antimicrobial agents, and to enzymes are responsible for a large proportion of food spoilage and overcome microbial resistance that can possibly be encountered with losses (Turgis, Vu, Dupont, & Lacroix, 2012), the development of safe the use of specific antimicrobial preservatives. and natural antimicrobial food additives and enzyme inhibitors as Phytoalexins have been studied for more than a century, and there components of systems that can enhance the effectiveness of preserva- exists a large body of knowledge on these inducible plant defense com- tion while maintaining product quality and safety would help elongate pounds, which are mostly antimicrobial. Plants in the family Poaceae shelf-life and improve food security (see Tajkarimi, Ibrahim, & Cliver, are about the most geographically widespread plants on earth, and 2010 for a detailed review on food-derived antimicrobial agents). they have been reported to produce a variety of structurally diverse Therefore, we foresee a role for stress-induced phytoalexins from the phytoalexins (Peters, 2006). Moreover, progress has been made in cereal food Poaceae as natural antimicrobial agents for food preserva- genetically modifying plants to make them produce, or enhance their tion (Fig. 1) due to their structural diversity, abundance and wide global production of, certain desirable phytoalexins (Großkinsky, van der distribution of their food sources. Graff, & Roitsch, 2012). However, there is scarcity of literature information on the potential use of phytoalexins as antimicrobial agents in the 3. Brief history of phytoalexins food system. This paper reviews the current state of knowledge on the chemistry and biosynthesis of phytoalexins from the five most Phytoalexins are a diverse group of low molecular weight secondary important cereals produced globally – rice, maize, wheat, barley and metabolites that exhibit antimicrobial activity, and are momentarily sorghum – and draws attention to their potential use and prospects as generated by either endogenous or exogenous signal molecules (biotic natural antimicrobial food preservatives. and abiotic stressors) called elicitors. They are an important part of the plant's defense gamut (Pedras, Yaya, & Glawischnig, 2011; Schmelz 2. Promoting food security through food preservation et al., 2011) and their antimicrobial effect is targeted to a variety of plant pathogens. The French botanist Noel Bernard, working more Food security is defined as a “situation that exists when all people, at all times, have physical, social, and economic access to sufficient, safe, Cereal Poaceae and nutritious food that meets their dietary needs and food preferences for an active and healthy life” (FAO, 2002a). Food production has Rice Sorghum Maize increased in the last half century, such that the number of people who are food-insecure has been tremendously reduced, despite the Elicitors Elicitors continued growth of the world's population (FAO, 2006). Despite the advances made in food production and distribution, food insecurity re- Momilactones, 3-Deoxyanthocyanidins Kauralexins, mains a major challenge, especially in developing countries. Approxi- oryzalexins, zealexins phytocassanes, PHYTOALEXINS mately 800 million people (one in six of the world's population) are sakuranetin food-insecure. Of this number, 35% reside in South Asia; 30% in East Asia; 22.5% in sub-Saharan Africa; and the other 12.5% live in Latin America, Middle East and North Africa (Pinstrup-Andersen, Antimicrobial activity Pandya-Lorch, & Rosegrant, 2001). This is even more worrisome when placed in the context of future population growth dynamics, where the bulk of population growth would occur in developing countries (Evans, Inhibition of food- 2009). These challenges require that the way food is produced, stored, Antimicrobial agent in food preservation? borne pathogen? processed, distributed, and accessed (the entire food supply or “farm to ” fork chain) be assessed and appropriately reviewed (Godfray et al., Fig. 1. Prospects of elicitor-induced cereal Poaceae phytoalexins as antimicrobial com- 2010). pounds for food preservation. C.E.C.C. Ejike et al. / Food Research International 52 (2013) 167–177 169 than a century ago, discovered that the tubers of Orchis morio and Spielmeyer et al., 2004). Thus, it appears plausible that more specialized Himantoglossum hircinum, both orchids, acquired resistance to further labdane-related diterpenoid metabolism will be widespread through- fungal attack after they had been infected by Rhizoctonia repens out the Poaceae. Some members of the family however produce flavo- (Grayer & Kokubun, 2001; Stoessl & Arditti, 1984). Though Bernard noid phytoalexins. showed that the fungal inhibitor was diffusible, the chemical structures of the compound would take a few more decades to be elucidated. 4.1. Rice The name phytoalexins was first used by Müller and Börger (1940) who observed the above phenomenon in potato tubers infected by Rice (Oryza sativa L.)iscultivatedinwarmclimateareasandisan the oomycete Phytophthora infestans.Theydefined phytoalexins as important food source. It provides about 20% of the total direct human “chemical compounds produced by a living cell as a result of invasion food-energy intake worldwide and is also the predominant staple food by a parasite”.Thisdefinition has changed over the years as new knowl- in many developing countries especially in Asia (FAO, 2004). Rice edge about phytoalexins has been acquired. For example, Van Etten, production, in milled terms, increased steadily over the years and Mansfield, Bailey, and Farmer (1994) insisted that the definition should reached 486.8 million tonnes in 2012 (FAO, 2012b). Furthermore, the reflect that phytoalexins had to be synthesized de novo to distinguish relatively small size of the rice genome, approximately 430 Mb, the them from phytoanticipins, which are compounds that increase in con- ease of its transformation, the extensive genetic sequence information centration under microbial stress although they exist prior to the stress. available for it, coupled with its importance as a food crop make rice a Paxton's (1980) definition of phytoalexins as “low molecular weight, model system and most studied member of the Poaceae. Rice has been antimicrobial compounds that are both synthesized and accumulated studied extensively, especially with respect to its metabolism including in plants after exposure to microorganisms or abiotic agents” however the production of phytoalexins in response to extrinsic stress (Kikuchi et appears to be the most acceptable within the phytoalexin research al., 2003; Peters, 2006; Tamogami, Rakwal, & Kodama, 1997). community. Currently, phytoalexins are defined mostly based on their In rice, 15 phytoalexins that include 14 diterpenoid phytoalexins production and functions, and not by chemical structures or biosynthetic and one flavonoid phytoalexin have been identified following treat- origin (Grayer & Kokubun, 2001). ment with elicitors such as chitin oligosaccharide (biotic) and in leaves The first characterized phytoalexin, pisatin, was isolated and infected with the blast fungus Magnaporthe grisea (biotic) or irradiated reported in 1960 from Pisum sativum after infection with Sclerotinia with UV light (abiotic) (Kodama, Miyakawa, Akatsuka, & Kiyosawa, fructicola and seven other facultative and obligate plant pathogenic 1992; Kodama, Suzuki, Miyakawa, & Akatsuka, 1988, Koga et al., fungi (Cruickshank & Perrin, 1960). Since then, phytoalexins have 1995). Other known elicitors of phytoalexin production in rice are been found in at least 75 host plants (both in the Gymnospermae cerebrosides and xylanase protein from Trichoderma viride,and and Angiospermae), including cruciferous vegetables, soybean, garlic, ethylene-inducing xylanase in rice-cultured cells. These elicitors are tomato, beans, potatoes, and cereals, suggesting that a wide variety of known to bring about a variety of defense responses in rice (Kurusu plants may be rich sources of these compounds. Species differences et al., 2010; Okada et al., 2009). From the prism of their hydrocarbon and preferences in the accumulation of these secondary metabolites precursors, rice phytoalexins can be classified into different groups abound. The most important phytoalexins are currently thought namely oryzalexins, phytocassanes, momilactones and sakuranetin to be derived from the shikimate (phenylpropanoid), isoprenoid (Okada et al., 2009)asshowninFig. 2. The major intermediates in (terpenoid) and alkaloid forming pathways, exemplified by the the biosynthesis of the four major groups of diterpene phytoalexins stilbenes, sesquiterpenes and camalexins, respectively (Großkinsky oryzalexins A–F, phytocassanes A–E, momilactones A and B and et al., 2012; Iriti & Faoro, 2009). Phytoalexin research has evolved oryzalexin S are ent-sandaracopimaradiene, entcassa-12,15-diene, over the years not only to cover defense against plant pathogens 9β-pimara-7,15-diene and stemar-13-ene, respectively (Cartwright, and pests but also extends to human health promotion (Boue et al., Langcake, Pryce, Leworthy, & Ride, 1981; Toyomasu et al., 2008) where- 2009; Holland & O'Keefe, 2010; Jahangir, Kim, Choi, & Verpoorte, as sakuranetin belongs to the flavonoids. 2009; Nwachukwu, Luciano, & Udenigwe, in press; Udenigwe & Aluko, 2012). 4.2. Maize

4. Phytoalexins and the Poaceae Maize (Zea mays L) is thought to be “the largest crop on earth” with over 810 million tonnes annual seed harvest for 2009 (FAOSTAT, The Poaceae or grass family covers substantial surface area of the 2010). It forms the staple food for many populations of the world es- earth's land mass. Cereal crop plants, the most important members pecially in the Americas and Africa. Maize is cultivated worldwide of the Poaceae, provide the bulk of the world's caloric intake (FAO, and finds application in a variety of industries including animal farm- 2005). It is estimated that the world's cereal production reached ing, cosmetics and biofuel production (IFBC, 1990). Maize produces approximately 2.282 billion tonnes in 2012, while there will be a labdane-type diterpenoid phytoalexins termed kauralexins as well projected 1.3% rise in cereal consumption in 2013 (FAO, 2012b). Rice as acidic sesquiterpenoids known as zealexins (Fig. 3). Elicitors of and wheat are the two most important cereals for direct human con- phytoalexin biosynthesis in maize include various food-grade and sumption; however maize, wheat and rice together accounted for 87% pathogenic microorganisms such as Aspergillus flavus, Aspergillus of all grain production worldwide and 43% of all food calories over a sojae, Cochliobolus heterostrophus, Colletotrichum sublineolum, Fusarium decade ago (Dyson, 1996). Barley is grown largely in high altitudes, graminearum, Ostrinia nubilalis, Rhizopus microspores and Ustilago mainly as animal feed, while sorghum is a sturdy pro-poor cereal that maydis (Huffaker et al., 2011; Schmelz et al., 2011). is grown in warm ecological zones. For the purpose of the present review, our discussions will be focused on prospects of phytoalexins 4.3. Wheat in these most important cereal crops. Members of the Poaceae produce largely diterpenoid phytoalexins. Wheat (Triticum spp.) is the third most produced cereal crop, after The committed step in labdane-related diterpenoid biosynthesis is cat- maize and rice. Its production for 2012 was approximately 659.4 mil- alyzed by labdadienyl/copalyl diphosphate synthase, CPS (Wu et al., lion tonnes (FAO, 2012b). However, though wheat and rice provide 2012). Molecular phylogenetic analyses based on characterization of the bulk of the direct caloric intake for the global human population maize, barley, wheat and rice CPS suggest that the expansion and func- (Dyson, 1996), wheat is ranked as the most important food grain tional diversification of the CPS gene to more specialized metabolism because of the wide array of products derived from it. Wheat flour may have occurred early in cereals (Harris et al., 2005; Peters, 2006; is used to bake breads, cakes, cookies; to make pastas and noodles; 170 C.E.C.C. Ejike et al. / Food Research International 52 (2013) 167–177

Momilactone A Momilactone B

Oryzalexin A Oryzalexin B Oryzalexin C Oryzalexin D

Oryzalexin F Oryzalexin S Oryzalexin E

Phytocassane A Phytocassane B Phytocassane C

Phytocassane E Phytocassane D

Sakuranetin

Fig. 2. Structures of known rice phytoalexins momilactones, oryzalexins, phytocassanes and sakuranetin.

and to produce alcoholic beverages by fermentation. The husk of the 2004). It is a short-season early-maturing grain that grows in a variety grain or bran that is usually separated during milling is a source of of environmental conditions, but more in high altitude tropical important phytochemicals. Wheat contains expanded CPS and kaurene and sub-tropical areas with low temperatures (Nevo, 1992). Barley synthase-like (KSL) gene families that encode the diterpene synthases, falls within the Bambusoideae–Ehrhartoideae–Pooideae clade of the known to act sequentially. Diterpene synthases are the distinguishing Poaceae and is more closely related to rice than maize. Barley can be feature of labdane-related diterpenoid biosynthesis. Unfortunately, no used in various applications within the food system especially as diterpene phytoalexins has been identiﬁed from wheat. However, the animal/human food and in brewing malts, and has also been used to UV-inducible transcription of the Triticum aestivum CPS (TaCPS) 1 produce starch (OECD, 2004). Barley has not been shown to produce and TaCPS2 in wheat suggests that both genes potentially play a role labdane-related diterpenoids other than gibberelic acid. However, chro- in phytoalexin biosynthesis (Wu et al., 2012; Zhou et al., 2012). mosome mapping in barley, wheat, and rice with barley gene probes show that barley (just like wheat) contains multiple copies of CPS, 4.4. Barley KSL, and kaurene oxidase-like genes. The barley CPS and KSL genes were found to be placed in regions of the chromosome known to be Barley (Hordeum vulgare L.) is one of the ﬁrst domesticated cereals syngeneic with the rice labdane-related diterpenoid gene clusters and an important cereal crop in the world (Akar, Avci, & Dusunceli, encoding diterpene synthases (Spielmeyer et al., 2004; Wu et al., C.E.C.C. Ejike et al. / Food Research International 52 (2013) 167–177 171

Kauralexin A1 Kauralexin A2 Kauralexin A3

Kauralexin B1 Kauralexin B2 Kauralexin B3

Zealexin A2 Zealexin A1

Zealexin B1 Zealexin A3

Fig. 3. Structures of known maize isoprenoid phytoalexins kauralexins and zealexins.

2012). Therefore, although no labdane-related diterpenoid phyto- luteolin and apigenin have also been identiﬁed as phytoalexins alexins have yet been reported in barley, it appears likely that the in pathogen-infected sorghum (Du, Chu, Wang, Chu, & Lo, 2010; production of these secondary metabolites in a defense-related Puttalingaiah, Shetty, Shetty, Neergaard, & Jørgensen, 2009). manner may also occur in the plant. In general, it is thought that the labdane-related diterpenoid phytoalexins are widely produced in the 5. Chemistry and antimicrobial properties of phytoalexins Poaceae in response to external stress factors (Peters, 2006). produced by the Poaceae

4.5. Sorghum 5.1. Terpenoid phytoalexins

The yearly production and harvesting area of sorghum (Sorghum Most of the terpenoid phytoalexins found in the Poaceae are bicolor) have reached 60 million tonnes and 44 million hectares, respec- diterpenoid phytoalexins. However, sesquiterpenoid phytoalexins tively (Deb,Bantilan,Roy,&Rao,2004). Sorghum is grown for grain, for- have been reported in some species such as maize. Terpenoid phyto- age, syrup and sugar mainly in the tropics, subtropics and warm alexins are thought to act in concert with a plethora of other plant de- temperate regions of the world, and the stems and fibers also find indus- fenses such as antimicrobial proteins (Huffaker et al., 2011). trial uses. Sorghum requires much less water than many other members of the Poaceae grown for food, and has a remarkable ability to produce a crop under low levels of inputs and adverse stress conditions, little won- 5.1.1. Diterpenoid phytoalexins der it is known as “the camel amongst crops” (FAO, 2002b). Moreover, sweet sorghum is an important raw material in bioethanol production 5.1.1.1. Phytocassanes. Phytocassane A to D are produced upon elicita- (Carpita & McCann, 2008; Zheng et al., 2011). Sorghum synthesizes a dis- tion by fungal pathogen Magnaporthe grisea and isolated from rice tinctive class of flavonoid phytoalexins known as 3-deoxyanthocyanidins stems infected with Rhizoctonia solani (Koga et al., 1995). Phytocassane upon elicitation by pathogens (Lo, de Verdier, & Nicholson, 1999). The de E, which is induced by the potato pathogen Phytophthora infestans novo biosynthesis of 3-deoxyanthocyanidin phytoalexins in sorghum is a also shows antifungal property against Magnaporthe grisea (Koga light-independent defense response (Weiergang, Hipskind, & Nicholson, et al., 1997). These studies indicated that the antifungal activities of

1996). The 3-deoxyanthocyanidins are structurally related to aglycones phytocassane B, C and E (EC50 values, 4–7 μg/mL) are about four-folds of anthocyanins, and include apigeninidin, luteolinidin, diosmetinidin, stronger than the activities of phytocassane A and D. This higher anti- tricetinidin and columnidin (Fig. 4). Sorghum bicolor L. is considered the fungal activity was attributed to the β-hydroxyl group in C-1 position only dietary source of 3-deoxyanthocyanins, which are present in large of phytocassane B, C and E (Fig. 2), which can form intramolecular quantities (up to 10 mg g−1) in the bran of some sorghum cultivars hydrogen bond with the carbonyl group in position C-11 (Koga et al., (Awika, Rooney, & Waniska, 2004). Moreover, ﬂavone compounds 1997). 172 C.E.C.C. Ejike et al. / Food Research International 52 (2013) 167–177

Apigeninidin Luteolinidin

Diosmetinidin

Tricetinidin Columnidin

Fig. 4. Structures of some ﬂavonoid phytoalexins 3-deoxyanthocyanidines in sorghum.

5.1.1.2. Momilactones. Momilactones are potent phytoalexins and enzyme synthesized as an aftermath of fungal attack; kauralexins allelochemicals, and have been found only in rice and the moss A1, A2, A3, B1, B2 and B3 (Fig. 3) have been reported in maize Hypnum plumaeforme (for a detailed review, see Kato-Noguchi, (Schmelz et al., 2011). Based on the study, kauralexins were found 2011). Momilactones A (3-oxo-9β-pimara-7,15-dien-19,6β-olide) to be elicited by mechanical damage due to Ostrinia nubilalis stem and B (3,20-epoxy-3α-hydroxy-9β-pimara-7,15-dien-19,6β-olide) herbivory, exposure to pectinase, infection with maize fungal patho- were first isolated from rice husks as growth inhibitors (Kato et al., gen Colletotrichum graminicola, but more importantly by Rhizopus 1973; Takahashi, Kato, Tsunagawa, Sasaki, & Kitahara, 1976). They microsporus, which was reported to strongly induce higher concen- were later found as phytoalexins in the leaves and straw of rice treated trations of all six kauralexins more than any of the other elicitors. with 2,2-dichloro-3,3-dimethylcyclopropane carboxylic acid before Kauralexin B3 reduced significantly the growth of R. microsporus infection by Magnaporthe grisea (Cartwright, Langcake, Pryce, even at doses as low as 10 μg/mL whereas kauralexin A3 and B3 accu- Leworthy, & Ride, 1977, Cartwright et al., 1981; Kodama et al., 1988; mulation each greatly inhibited the growth of C. graminicola that Lee et al., 1999). In addition to biotic (Magnaporthe grisea) and abiotic causes anthracnose stalk rot; the maize phytoalexins also exhibited (physical and chemical treatments) elicitors, several other elicitors of potent anti-feedant activity against O. nubilalis (Schmelz et al., 2011). these diterpenoid phytoalexins have been identified including cerebroside, β-glucooligosaccharides chitin, N-acetylchitoheptaose and chitosan (Agrawal et al., 2002; Koga et al., 2006; Okada et al., 2009; 5.1.2. Sesquiterpenoid phytoalexins Shimizu et al., 2008; Umemura, Ogawa, Koga, Iwata, & Usami, 2002; Zealexins are acidic sesquiterpenoids that are present in maize. Yamaguchi et al., 2000). Momilactones A and B exert antifungal activity Zealexin accumulation is usually subsequent to a strong induction of toward Magnaporthe oryzae which causes rice blast, Botrytis cinerae, the genes that code for the terpene synthases TPS6 and TPS11 Fusarium solani, Colletrotichum gloesporides and Fusarium oxysporum (Kollner et al., 2008). The above terpene synthases are closely related although the antifungal activity of momilactone B is thought to be and both also generate β-macrocarpene from farnesyl pyrophosphate better than that of momilactone A (Fukuta et al., 2007). This discrepan- as the dominant product; the hydrocarbon skeleton of the zealexins cy could be due to the additional six-membered ring or the hydroxyl and β-macrocarpene are considerably similar (Kollner et al., 2008). group, which seem to be the only additional features in the structure Sesquiterpenoid phytoalexins are also found in abundance in of momilactone B (Fig. 2). Moreover, both phytoalexins also possess other plant families. For instance, gossypol, capsidiol, and rishitin, antibacterial activity against Escherichia coli, Pseudomonas ovalis, Bacillus all sesquiterpenoid phytoalexins, are important elicitor-induced pumilus and Bacillus cereus (Fukuta et al., 2007). defenses in cotton (Gossypium hirsutum) and solanaceous plants (Delannoy et al., 2005; Desjardins, Gardner, & Weltring, 1992; 5.1.1.3. Oryzalexins. Like momilactones, oryzalexins A, B and C were Stoessla, Stothersb, & Ward, 1976). Although 14 fungal-induced acidic initially isolated from Magnaporthe grisea infected leaf tissues of rice sesquiterpenoids are detectable in maize, only zealexins A1, A2, and (Akatsuka et al., 1985). However, the biosynthesis and accumulation of A3 (Fig. 3) have been isolated in sufficiently large amounts with a oryzalexin A, B and C have also been shown to be elicited by UV radiation high degree of purity that is required for bioassays (Huffaker et al., and jasmonic acid (Kato, Kodama, & Akatsuka, 1994; Tamogami, Mitani, 2011). Kodama, & Akatsuka, 1993). When tested for antifungal activity against The most potent elicitors of zealexins are mycotoxigenic fungi Magnaporthe grisea spore germination, the oryzalexins were shown to such as Ustilago maydis and Fusarium graminearum (Basse, 2005; be weaker (EC50 values, 20–136 μg/mL) than momilactones A and B Doehlemann et al., 2008; Huffaker et al., 2011). Physical injuries caused with EC50 values of 15 and 3 μg/mL, respectively, and the additional by feeding tunnels created by O. nubilalis larvae also elicit zealexin accu- lactone moiety in the momilactones was suggested to be the active mulation (Huffaker et al., 2011). Zealexin A1 was found to significantly functionality (Koga, 2003). Among the oryzalexins, oryzalexin D seems impede the growth of R. microsporus, A. flavus and F. graminearum while to have the best antifungal activity and this could possibly be due to zealexin A3 inhibited the growth of A. flavus and F. graminearum but not the hydroxyl group at C-3 and C-7 positions in its structure (Fig. 2). R. microsporus (Huffaker et al., 2011). Notably, zealexin A1 has signifi- cant broad-based inhibitory capacity on fungal growth at physiological- 5.1.1.4. Kauralexins. Kauralexins are acidic ent-kaurane-related ly relevant concentrations. The reduced or even abrogated antifungal diterpenoid phytoalexins that are thought to be downstream end- activity of zealexin A2 and A3 is thought to be due to the hydroxylation products of the activity of ent-copalyl diphosphate synthase, an of C-1 and C-8 in their respective structures (Huffaker et al., 2011). C.E.C.C. Ejike et al. / Food Research International 52 (2013) 167–177 173

5.2. Flavonoid phytoalexins system (Lo & Nicholson, 1998; Lo et al., 1999). The amount of 3-deoxyanthocyanidin in black sorghum bran is about 3 times Plants synthesize a spectrum of flavonoids with a wide array of func- that in brown sorghum, and apigeninidin and luteolinidin are the tions. Flavonoid phytoalexins are made strictly for defense purposes in re- two major 3-deoxyanthocyanidins representing up to 50% of total sponse to stressors. In cereals, sakuranetin and 3-deoxyanthocyanidins amounts in black and brown sorghum (Awika et al., 2004). The are the most important flavonoid phytoalexins. 3-deoxyanthocyanidins show similar antioxidant ability to anthocyanin, and very easily present overlapping peaks and same retention 5.2.1. Sakuranetin time in traditional adsorption analysis. Luetolinidin and its 5-methoxy Sakuranetin (5,4′-dihydroxy-7-methyoxyflavone or 7-O-methyl- derivative, at concentration of 3, 7.5 and 15 μM, were reported to naringenin) is a flavone phytoalexin that was first identified as a phy- inhibit the germination and viability of the conidia of Colletotrichum toalexin produced in rice leaves. Sakuranetin accumulates in response sublineolum, a fungus that causes anthracnose in some members of to UV light treatment (Kodama et al., 1992) or other elicitors such the Poaceae (Lo et al., 1996). as attack by pathogens and chemical (copper chloride and jasmonic acid) treatment (Kodama et al., 1992; Rakwal, Hasegawa, & Kodama, 6. Biosynthesis of phytoalexins produced by the Poaceae 1996). Other known elicitors are methionine, coronatine and chisotan oligomers (Nakazato, Tamogami, Kawai, Hasegawa, & Kodama, 2000; 6.1. Terpenoid phytoalexins Obara, Hasegawa, & Kodama, 2002; Tamogami & Kodama, 2000). The ability of sakuranetin to inhibit Magnaporthe grisea spore germination Isoprenoids are derived from a basic 5-carbon unit, isopentenyl

(EC50 value, 15 μg/mL) is thought to be due to the methyl group in diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate the C-7 position (Koga, 2003). Moreover, the ﬂavonoid phytoalexin (DMAPP). IPP is by a series of sequences, condensed to DMAPP to has been recently pursued as a potential antibacterial agent due to its yield short chain isoprenoid precursors, geranyl diphosphate, farnesyl inhibition of Helicobacter pylori β-hydroxyacyl–acyl carrier protein diphosphate, and geranylgeranyl diphosphate. These are usually further dehydratase, an enzyme required for cellular fatty acid synthesis, by metabolized sequentially by terpene cyclases and oxidases to the preventing the binding of substrate to the enzyme's active site (Zhang 10-carbon monoterpenes, 15-carbon sesquiterpenes, and 20-carbon et al., 2008). diterpenes (Okada, 2011). The methylerythritol phosphate (MEP) pathway leads to the formation of geranylgeranyl diphosphate (GGDP) in 5.2.2. 3-Deoxyanthocyanidins rice. GGDP, in a protonation-initiated reaction, is cyclized to ent- or 3-Deoxyanthocyanidins are a group of colored ﬂavonoid phyto- syn-copalyl diphosphate (ent-CDP and syn-CDP) by the class II alexins found mainly in sorghum bran. The production of these diterpene cyclases, ent-CDP and syn-CDP cyclases (Cho et al., 2004). secondary metabolites is induced in response to infection by the sor- As shown in Fig. 5, further conversions initiated by a diphosphate ghum leaf blight fungi Magnaporthe grisea and Colletotrichum spp. ester ionization are catalyzed by four class I diterpene synthases, (Snyder & Nicholson, 1990). The major 3-deoxyanthocyanidin phyto- ent-cassa-12,15-diene synthase, ent-sandaracopimaradien synthase, alexins, shown in Fig. 4, include apigeninidin (yellow color), columnidin 9β-H-pimara-7,15-diene synthase, and stemar-13-ene synthase to (orange red color), diosmetinidin, luteolinidin (orange color) and form ent-cassa-12,15-diene, ent-sandaracopimaradien, 9β-H-pimara-7, tricetinidin (intense red color) (Nielsen et al., 2004; Ozawa, 1982; 15-diene and stemar-13-ene, respectively. These hydrocarbons are Wolniak & Wawer, 2008). The 3-deoxyanthocyanidin phytoalexins ac- thereafter cyclized, forming phytocassanes A to E, oryzalexins A to F, cumulate, when pathogens attack the plant, in the form of inclusions momilactones A to B and oryzalexin S, respectively as end-products in epidermal cells, which are similar to transport vesicles in morphology (Cho et al., 2004; Mohan et al., 1996; Nemoto et al., 2007; Otomo but lack a membrane (Snyder & Nicholson, 1990). The development et al., 2004; Wickham & West, 1992). of inclusions is a process that is characterized by color and size trans- To date, little is known about the enzymes catalyzing the above down- formation. This seeming maturation process is followed by cell stream cyclization reactions, although speculative biosynthetic pathways rupture which releases 3-deoxyanthocyanidins into the apoplastic have been assembled (Peters, 2006). The fact that a 3-oxy group is

Fig. 5. Proposed cyclization reactions in the biosynthetic pathway for the labdane-related diterpenoid phytoalexins in rice. Reprinted from Nemoto et al. Promoter analysis of thericestemar-13-enesynthasegeneOsDTC2,whichisinvolvedinthebiosynthesisofthephytoalexin oryzalexin S, Biochimica et Biophysica Acta (BBA) — Gene Structure and Expression, 1769,678–683, Copyright (2007), with permission from Elsevier. 174 C.E.C.C. Ejike et al. / Food Research International 52 (2013) 167–177 present in oryzalexins A–F indicates that ent-sandaracopimaradien-3α-ol 6.2.2. 3-Deoxyanthocyanidins is a common precursor for their biosynthesis (Kato, Kodama, & Akatsuka, Naringenin is also the precursor of 3-deoxyanthocyanidins. In 1995). The C-3 hydroxyl group of 3-hydroxy-syn-pimara-7,15-dien-19, the biosynthesis of the flavonoid phytoalexins, as shown in Fig. 6, 6β-olide is oxidized by an inducible soluble dehydrogenase to produce flavanone-3′-hydroxylase catalyzes the C-3′ hydroxylation of flava- momilactones A (Atawong, Hasegawa, & Kodama, 2002). Momilactone nones, which is succeeded by a dihydroflavonol-4-reductase catalyzed A is potentially an intermediate in the path to momilactone B production. NADPH-dependent reduction of the carbonyl group at position C-4. The ubiquitous C-11 keto group and common C-2 oxy moiety in the Anthocyanidin synthase then catalyzes the removal of the resulting hy- phytocassanes suggest early incorporation of oxygen at these positions. droxyl group (Ibraheem, Gaffoor, & Chopra, 2010). These pathways re- It is important to note that none of the enzymes involved in elaboration sult in the synthesis of luteolinidin and apigeninidin. First, naringenin is of the rice diterpenoid phytoalexins has yet been identified (Peters, transformed by hydroxylation of C-3′ to produce eriodictyol in a reac- 2010). The Oryza sativa TGA factor for phytoalexin production 1 gene is tion catalyzed by flavonoid-3′-hydroxylase. Eriodictyol then undergoes thought to regulate the biosynthesis of diterpenoid phytoalexins through reduction of its C-4 keto group to produce luteoferol in a reaction cata- positive control of 9β-H-pimara-7,15-diene synthase expression by bind- lyzed by dihydroflavonol reductase. Naringenin also directly undergoes ing to the cis-element in the gene promoter region; it also influences the similar reaction to form apiferol, which can be hydroxylated at C-3′ upstream MEP pathway at the transcriptional level (Okada et al., 2009). to yield luteoferol. Apiferol and luteoferol can finally be transformed to apigeninidin and luteolinidin, respectively by the action of 6.2. Flavonoid phytoalexins anthocyanidin synthase; the biosynthesis of 3-deoxyanthocyanidins is thought to be regulated by a gene sequence designated “yellow seed 1” Flavonoid biosynthesis starts with a condensation reaction catalyzed (Ibraheem et al., 2010). Detailed molecular basis of the biosynthesis by chalcone synthase, which converts phenylpropanoid-CoA and of 3-deoxyanthocyanidin phytoalexins in plants, however, is still not malonyl-CoA into chalcones. Thereafter, the chalcones are isomerized very clearly elucidated. into flavanones by chalcone isomerase. The biosynthetic pathway for anthocyanidin and 3-deoxyanthocyanidin are thought to be similar up 7. Prospects of phytoalexins from the Poaceae in food preservation to naringenin, where they are diverged into distinct pathways. Since the advent of phytoalexins, a lot of attention has been devoted 6.2.1. Sakuranetin to their use in providing resistance to plant diseases and pests in plants. Naringenin (4′,5,7-trihydroxyflavanone), an antioxidant flavo- Progress has been made in transferring biosynthetic genes of known noid, is considered the precursor of sakuranetin. Naringenin-7-O- phytoalexins to plants that do not produce such defense compounds, methyltransferase (NOMT) catalyzes the biosynthesis of sakuranetin in thereby conferring resistance to the new hosts against pathogenic rice (Rakwal, Agrawal, Yonekura, & Kodama, 2000). 4-Coumaryol-CoA fungi. Moreover, some phytoalexins have attracted particular interest and malonyl-CoA react to produce chalcone by the action of chalcone as modulators of abnormal physiological processes relevant in human synthase and, through the action of chalcone isomerase, chalcone health promotion (Boue et al., 2009; Holland & O'Keefe, 2010; is converted to naringenin (Rakwal et al., 1996). The action of Jahangir et al., 2009; Nwachukwu et al., in press). However, despite NOMT, which catalyzes the transfer of the methyl group of the fact that phytoalexins are synthesized ab initio in plants to combat S-adenosyl-L-methionine to the C-7 hydroxyl group of naringenin, microbial infection, there is a dearth of literature information on the produces sakuranetin (Rakwal et al., 2000, 1996). Despite the pres- use of the inducible plant compounds as antimicrobial food preserva- enceofsakuranetininotherplantssuchasArtemisia campestris, tives for reducing food losses and wastages due to spoilage microbes, Prunus spp., Baccharis spp., Betula spp. and Juglans spp., there is a dearth or against food-borne pathogens. The prospects are particularly strong of evidence that demonstrates NOMT activities in these plants; there- with the abundance of natural structurally diverse phytoalexins in the fore, there may be plant-specific pathways for sakuranetin biosynthesis cereal food Poaceae and other sources, which will ensure sustainability (Harborne, Baxter, & Moss, 1999; Shimizu et al., 2012). and possible safety for use in the food system when compared to

Fig. 6. Proposed biosynthesis of the 3-deoxyanthocyanidins. CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; F3′H, flavonoid-3′-hydroxylase. C.E.C.C. Ejike et al. / Food Research International 52 (2013) 167–177 175 synthetic antimicrobial agents. Moreover, pigmented phytoalexins such References as the 3-deoxyanthocyanidins can potentially play dual roles in food product development as new natural food colorant and antimicrobial Agrawal, G. K., Rakwal, R., Tamogami, S., Yonekura, M., Kubo, A., & Saji, H. (2002). Chi- tosan activates defense/stress response(s) in the leaves of Oryza sativa seedlings. agents. The stability of 3-deoxyanthocyanidins relative to anthocyanin Plant Physiology and Biochemistry, 40, 1061–1069. also means that their use may forestall the degradation of the latter in Akar, T., Avci, M., & Dusunceli, F. (2004). Barley: Post harvest operations. Ankara, Turkey: the presence of ascorbic acid, a traditional food additive, a phenomenon The Central Research Institute for Field Crops (http://www.fao.org/fileadmin/user_ upload/inpho/docs/Post_Harvest_Compendium_-_BARLEY.pdf (Accessed December that causes fading in fruit juices (Iacobucci & Sweeny, 1983). This stabil- 17, 2012)) ity of 3-deoxyanthocyanidins has been demonstrated in extracts from Akatsuka, T., Takahashi, N., Kodama, O., Sekido, H., Kono, Y., & Takeuchi, S. (1985). sorghum bran where they retained 31% of their color even at low acidic Novel phytoalexins (oryzalexins A, B, and C) isolated from rice blast leaves infected pH values (Ojwang & Awika, 2008). with Pyricularia oryzae. I. Isolation, characterization and biological activities of oryzalexins. Agricultural and Biological Chemistry, 49, 1689–1694. The prospective food application of phytoalexins at the commer- Atawong, A., Hasegawa, M., & Kodama, O. (2002). Biosynthesis of rice phytoalexin: Enzy- cial scale depends on their accumulated amounts in the plants after matic conversion of 3ß-hydroxy-9ß-pimara-7,15-dien-19,6ß-olide to momilactone A. – elicitation, which varies depending on the Poacea food, phytoalexin Bioscience, Biotechnology, and Biochemistry, 66,566 570. Awika, J. M., Rooney, L. W., & Waniska, R. D. (2004). Properties of 3-deoxyanthocyanins type and duration of exposure to elicitors. In maize, kauralexins from sorghum. Journal of Agricultural and Food Chemistry, 52, 4388–4394. − have been reported to exceed 100 μgg 1 fresh weight in 48 h and Basse, C. W. (2005). Dissecting defense-related and developmental transcriptional up to 167 μgg−1 fresh weight in 10 days (Schmelz et al., 2011). responses of maize during Ustilago maydis infection and subsequent tumor formation. Plant Physiology, 138, 1774–1784. Moreover, a recent study reported that zealexins accumulate to levels Boue, S. M., Cleveland, T. E., Carter-Wientjes, C., Shih, B. Y., Bhatnagar, D., McLachlan, J. M., −1 greater than 800 μgg fresh weight in maize (Huffaker et al., 2011). et al. (2009). Phytoalexin-enriched functional foods. Journal of Agricultural and Food The production of rice phytoalein sakuranetin was reported to exceed Chemistry, 57,2614–2622. Carpita, N. C., & McCann, M. C. (2008). Maize and sorghum: genetic resources for 500 ng/wound (Tamogami & Kodama, 2000) while momilactone A bioenergy grasses. Trends in Plant Science, 13, 415–420. −1 was found to accumulate to levels up to 10.4 μgg fresh weight Cartwright,D.,Langcake,P.,Pryce,R.J.,Leworthy,D.P.,&Ride,J.P.(1977).Chemicalactiva- in rice (Nojiri et al., 1996). Likewise, the 3-deoxyanthocyanidin, tion of host defense mechanisms as a basis for crop protection. Nature, 267,511–513. Cartwright, D. W., Langcake, P., Pryce, R. J., Leworthy, D. P., & Ride, J. P. (1981). Isolation luteolinidin was produced in sorghum at concentrations exceeding and characterization of two phytoalexins from rice as momilactones A and B. Phyto- 150 ng/mg tissues after 120 h of elicitation (Weiergang et al., 1996). chemistry, 20,535–537. Given the advances in identifying eliciting factors and understanding Cho, E., Okada, A., Kenmoku, H., Otomo, K., Toyomasu, T., Mitsuhashi, W., et al. (2004). Molec- phytoalexin biosynthesis, increased accumulation of desired phyto- ular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured alexins can be readily achieved, thus making available sufficient rice cells treated with a chitin elicitor. The Plant Journal, 37,1–8. quantities of the compounds for diverse food applications. The abun- Corbo, M. R., Bevilacqua, A., Campaniello, D., D'Amato, D., Speranza, B., & Sinigaglia, M. dance of these phytoalexins in the Poaceae, the large scale production (2009). Prolonging microbial shelf life of foods through the use of natural compounds and non-thermal approaches — A review. International Journal of Food Sci- of their cereal crop sources, and the large data available on their ence and Technology, 44, 223–241. chemistry and genetics provide a promising platform for the develop- Cruickshank, I. A. M., & Perrin, D. R. (1960). Isolation of a phytoalexin from Pisum ment of novel natural food preservatives. sativum L. Nature, 197, 799–800. Deb, U. K., Bantilan, M. C. S., Roy, A. D., & Rao, P. (2004). Global sorghum production scenario. In M. C. S. Bantilan, U. K. Deb, C. L. L. Gowda, B. V. S. Reddy, A. B. Obilana, & R. E. Evenson (Eds.), Sorghum genetic enhancement: research process, 8. Conclusion and future direction dissemination and impacts (pp. 21–38). Andhra Pradesh, India: International Crops Research Institute for the Semi-Arid Tropics. Delannoy, E., Lyon, B. R., Marmey, P., Jalloul, A., Daniel, J. F., Montillet, J. L., et al. (2005). Cereal food crops belonging to the Poaceae are sources of struc- Resistance of cotton towards Xanthomonas campestris pv. malvacearum. Annual turally diverse phytoalexins that exhibit antimicrobial activities Review of Phytopathology, 43,63–82. Desjardins, A. E., Gardner, H. W., & Weltring, K. M. (1992). Detoxification of sesquiterpene against various plant pathogens. Moreover, phytoalexins, in general, phytoalexins by Gibberella pulicaris (Fusarium sambucinum)anditsimportancefor have recently gained particular attention as functional agents with virulence on potato tubers. Journal of Industrial Microbiology and Biotechnology, 9, prospective human health applications. Despite their primary roles 201–221. as antimicrobial agents, there is a knowledge gap on the use of phy- Doehlemann, G., Wahl, R., Horst, R. J., Voll, L. M., Usadel, B., Poree, F., et al. (2008). Reprogramming a maize plant: transcriptional and metabolic changes induced by toalexins in food preservation, and against food-borne pathogens. the fungal biotroph Ustilago maydis. The Plant Journal, 56, 181–195. Since the cereal food Poaceae are widely cultivated globally, phyto- Du, Y., Chu, H., Wang, M., Chu, I. K., & Lo, C. (2010). Identification of flavone phyto- fl alexins produced in various parts of these crops can be exploited alexins and a pathogeninducible avone synthase II gene (SbFNSII) in sorghum. Journal of Experimental Botany, 61, 983–994. within the food system especially as natural antimicrobial food pre- Dyson, T. (1996). Population and food: global trends and future prospects. London: servatives, a strategy towards the promotion of global food security. Routledge, 227. We propose that future research should evaluate the antimicrobial Evans, A. (2009). The feeding of the nine billion: Global food security for the 21st century. London: Chatham House, 59. activities of these phytoalexins against particular detrimental food FAO (2002b). Sweet sorghum in China. Spotlight, Agriculture and Consumer Protection microorganisms, and perhaps explore the use of isolated elicitors Department. Rome: FAO. from these organisms for inducing phytoalexin synthesis in the FAO (2004). International year of rice 2004 — rice and human nutrition, vol. 2006,Rome:FAO. FAO (2005). Summary of world food and agricultural statistics 2005. Rome: FAO, 64. plants. The latter strategy will particularly ensure the production of FAO (2006). “Agriculture: Towards 2015/30”, technical interim report. Prospects for food, specific array of responses against the growth, pathogenicity and nutrition, agriculture and major commodity groups. Rome: FAO. other negative activities induced by a particular microorganism, FAO (2012a). FAO statistical year book 2012. Rome: FAO, 216. FAO (2012b). FAO cereal supply and demand brief. http://www.fao.org/worldfood and will prospectively become a unique natural strategy for produc- situation/wfs-home/csdb/en/ (Accessed December 14, 2012) ing antimicrobial compounds for food applications. Furthermore, the FAOSTAT (2010). Food and Agriculture Organization, Rome. http://faostat.fao.org/site/ safety of these phytoalexins needs to be substantiated at applicable 567/default.aspx (Accessed December, 18, 2012) doses in order to evaluate their possible uses in the human food Food and Agriculture Organization (FAO) (2002a). The state of food insecurity in the world 2001. Rome: FAO. system. Fukuta, M., Xuan, T. D., Deba, F., Tawata, S., Khanh, T. D., & Chung, I. M. (2007). Compar- ative efficacies in vitro of antibacterial, fungicidal, antioxidant, and herbicidal activities of momilatones A and B. Journal of Plant Interactions, 2, 245–251. Godfray, H. C. J., Beddington, J. R., Crute, I. R., Haddad, L., Lawrence, D., Muir, J. F., et al. (2010). Acknowledgment Food security: The challenge of feeding 9 billion people. Science, 327, 812–818. Government Office for Science, London (2011). Foresight. The future of food and farm- CECCE is supported by a TETFund Nigeria research grant. A ing: Challenges and choices for global sustainability, executive summary. London: The Government Office for Science. start-up grant from the Faculty of Agriculture, Dalhousie University, Grayer, R. J., & Kokubun, T. (2001). Plant-fungal interactions: The search for phytoalexins Canada to CCU is also acknowledged. and other antifungal compounds from higher plants. Phytochemistry, 56,253–263. 176 C.E.C.C. Ejike et al. / Food Research International 52 (2013) 167–177

Großkinsky, D. K., van der Graff, E., & Roitsch, T. (2012). Phytoalexin transgenics in crop Müller, K. O., & Börger, H. (1940). Experimentelle untersuchungen über die protection — Fairy tale with a happy end? Plant Science, 195,54–70. phytophthora-resistenz der kartoffel, arbeiten aus der biologischen reichsanstalt Gustavsson, J., Cederberg, C., Sonesson, U., van Otterdijk, R., & Meybeck, A. (2011). für land-und forstwirtschaft. Berlin-Dahlem, 23, 189–231. Global food losses and food waste. Extent, causes and prevention. Rome: FAO. Nakazato, Y., Tamogami, S., Kawai, H., Hasegawa, M., & Kodama, O. (2000). Harborne, J. B., Baxter, H., & Moss, G. P. (1999). Phytochemical dictionary: A handbook of Methionine-induced phytoalexin production in rice leaves. Bioscience, Biotechnology, bioactive compounds from plants (2nd ed.). London: Taylor & Francis, 976. and Biochemistry, 64,577–583. Harris, L. J., Saparno, A., Johnston, A., Prisic, S., Xu, M., Allard, S., et al. (2005). The maize Nemoto, T., Okada, A., Okada, K., Shibuya, N., Toyomasu, T., Nojiri, H., et al. (2007). Pro- An2 gene is induced by Fusarium attack and encodes an ent-copalyl diphosphate moter analysis of the rice stemar-13-ene synthase gene OsDTC2, which is involved synthase. Plant Molecular Biology, 59, 881–894. in the biosynthesis of the phytoalexin oryzalexin S. Biochimica et Biophysica Acta, Holland, K. W., & O'Keefe, S. F. (2010). Recent applications of peanut phytoalexins. 1769, 678–683. Recent Patents on Food, Nutrition & Agriculture, 2, 221–232. Nevo, E. (1992). Origin, evolution, population genetics and resources for breeding Huffaker, A., Kaplan, F., Vaughan, M. M., Dafoe, N. J., Ni, X., Rocca, J. R., et al. (2011). of wild barley, Hordeum pontaneum in the fertile crescent. Chapter 2. In P. R. Novel acidic sesquiterpenoids constitute a dominant class of pathogen-induced Shewry (Ed.), Barley: Genetics, biochemistry, molecular biology and biotechnology phytoalexins in maize. Plant Physiology, 156, 2082–2097. (pp. 19–43). Wallingford, Oxon: CAB International. Iacobucci, G. A., & Sweeny, J. G. (1983). Effects of substitution on the stability of Nielsen, K. A., Gotfredsen, C. H., Buch-Pedersen, M. J., Ammitzbøll, H., Mattsson, O., 3-deoxyanthocyanidins in aqueous solutions. Journal of Agricultural and Food Duus, J.Ø., et al. (2004). Inclusions of flavonoid 3-deoxyanthocyanidins in sorghum Chemistry, 31, 531–533. bicolor self-organize into spherical structures. Physiological and Molecular Plant Pa- Ibraheem, F., Gaffoor, I., & Chopra, S. (2010). Flavonoid phytoalexin-dependent thology, 65, 187–196. resistance to anthracnose leaf blight requires a functional yellow seed1 in Sorghum Nojiri, H., Sugimori, M., Yamane, H., Nishimura, Y., Yamada, A., Shibuya, N., et al. bicolor. Genetics, 184, 915–926. (1996). lnvolvement of jasmonic acid in elicitor-induced phytoalexin production International Food Biotechnology Council (IFBC) (1990). Biotechnologies and food: in suspension-cultured rice cells. Plant Physiology, 110, 387–392. assuring the safety of foods produced by genetic modification. Regulatory Toxicology Nwachukwu, I. D., Luciano, F. B., & Udenigwe, C. C. (in press). The inducible soybean and Pharmacology, 12,S1–S196. glyceollin phytoalexins with multifunctional health-promoting properties. Food Iriti, M., & Faoro, F. (2009). Chemical diversity and defence metabolism: How plants cope Research International. http://dx.doi.org/10.1016/j.foodres.2013.01.024. with pathogens and ozone pollution. International Journal of Molecular Sciences, 10, Obara, N., Hasegawa, M., & Kodama, O. (2002). Induced volatiles in elicitor-treated and 3371–3399. rice blast fungus-inoculated rice leaves. Bioscience, Biotechnology, and Biochemistry, Jahangir, M., Kim, H. K., Choi, Y. H., & Verpoorte, R. (2009). Health-affecting compounds 66, 2549–2559. in Brassicaceae. Comprehensive Reviews in Food Science and Food Safety, 8,31–43. Ojwang, L., & Awika, J. M. (2008). Effect of pyruvic acid and ascorbic acid on stability of Julian, P., & Barthel, M. (2010). Foresight. Global food waste reduction: Priorities for a 3-deoxyanthocyanidins. Journal of the Science of Food and Agriculture, 88,1987–1996. world in transition, science review SR56. London: Government Office for Science. Okada, K. (2011). The biosynthesis of isoprenoids and the mechanisms regulating it in Kato, T., Kabuto, C., Sasaki, N., Tsunagawa, M., Aizawa, H., Fujita, K., et al. (1973). plants. Bioscience, Biotechnology, and Biochemistry, 75, 1219–1225. Momilactones, growth inhibitors from rice, Oryza sativa L. Tetrahedron Letters, 39, Okada, A., Okada, K., Miyamoto, K., Koga, J., Shibuya, N., Nojiri, H., et al. (2009). 3861–3864. OsTGAP1, a bZIP transcription factor, coordinately regulates the inductive production Kato, T., Kodama, O., & Akatsuka, T. (1994). Oryzalexin F, a diterpene phytoalexin from of diterpenoid phytoalexins in rice. Journal of Biological Chemistry, 284, 26510–26518. UV-irradiated rice leaves. Phytochemistry, 36, 299–301. Organization for Economic Co-operation and Development (OECD) (2004). Consensus Kato, T., Kodama, O., & Akatsuka, T. (1995). Characterization of an inducible P450 document on compositional considerations for new varieties of barley (Hordeum hydroxylase involved in the rice diterpene phytoalexin biosynthetic pathway. vulgare L.): key food and feed nutrients and anti-nutrients. Report no 12. Paris: Archives of Biochemistry and Biophysics, 316, 707–712. Environment Directorate, OECD. Kato-Noguchi, H. (2011). Convergent or parallel molecular evolution of momilactone A Otomo, K., Kanno, Y., Motegi, A., Kenmoku, H., Yamane, H., Mitsuhashi, W., et al. (2004). and B: Potent allelochemicals, momilactones have been found only in rice and the Diterpene cyclases responsible for the biosynthesis of phytoalexins, momilactones moss Hypnum plumaeforme. Journal of Plant Physiology, 168, 1511–1516. A, B, and oryzalexins A–F in rice. Bioscience, Biotechnology, and Biochemistry, 68, Kikuchi, S., Satoh, K., Nagata, T., Kawagashira, N., Doi, K., Kishimoto, N., et al. (2003). 2001–2006. Collection, mapping, and annotation of over 28,000 cDNA clones from japonica Ozawa, T. (1982). Separation of the components in black tea infusion by chromatography rice. Science, 301, 376–379. on toyopearl. Agricultural and Biological Chemistry, 46,1079–1081. Kodama, O., Miyakawa, J., Akatsuka, T., & Kiyosawa, S. (1992). Sakuranetin, a flavanone Paxton, J. D. (1980). A new working definition of the term “phytoalexin”. Plant Disease, phytoalexin from ultraviolet-irradiated rice leaves. Phytochemistry, 31, 3807–3809. 64, 734. Kodama, O., Suzuki, T., Miyakawa, J., & Akatsuka, T. (1988). Ultraviolet-induced accu- Pedras, M. S. C., Yaya, E. E., & Glawischnig, E. (2011). The phytoalexins from cultivated mulation of phytoalexins in rice leaves. Agricultural and Biological Chemistry, 52, and wild crucifers: chemistry and biology. Natural Product Reports, 28, 1381–1405. 2469–2473. Peters, R. J. (2006). Uncovering the complex metabolic network underlying diterpenoid Koga, J. (2003). Structure, function, and biological activity of rice phytoalexins and phytoalexin biosynthesis in rice and other cereal crop plants. Phytochemistry, 67, elicitors. In M. Rai, & D. Mares (Eds.), Plant-derived antimycotics (pp. 497–524). 2307–2317. New York: Haworth Press. Peters, R. J. (2010). Two rings in them all: the labdane-related diterpenoids. Natural Koga, J., Kubota, H., Gomi, S., Umemura, K., Ohnishi, M., & Kono, T. (2006). Cholic acid, a Product Reports, 27, 1521–1530. bile acid elicitor of hypersensitive cell death, pathogenesis-related protein synthe- Pinstrup-Andersen, P., Pandya-Lorch, R., & Rosegrant, M. W. (2001). Global food security: sis, and phytoalexin accumulation in rice. Plant Physiology, 140, 1475–1483. a review of the challenges. In P. Pinstrup-Andersen, & R. Pandya-Lorch (Eds.), The Koga, J., Ogawa, N., Yamauchi, T., Klkuchi, M., Ogasawara, N., & Shimura, M. (1997). unfinished agenda: Perspectives on overcoming hunger, poverty, and environmental Functional moiety for the antifungal activity of phytocassane E, a diterpene phyto- degradation. Washington, D.C.: International Food Policy Research Institute. alexin from rice. Phytochemistry, 44, 249–253. Puttalingaiah, B., Shetty, N. P., Shetty, H. S., Neergaard, E., & Jørgensen, H. J. L. (2009). Koga, J., Shimura, M., Oshima, K., Ogawa, N., Yamauchi, T., & Ogasawara, N. (1995). Infection biology and defence responses in sorghum against Colletotrichum Phytocassanes A, B, C and D, novel diterpene phytoalexins from rice, Oryza sativa sublineolum. Journal of Applied Microbiology, 107, 404–415. L. Tetrahedron, 51, 7907–7918. Rakwal, R., Agrawal, G. K., Yonekura, M., & Kodama, O. (2000). Naringenin Kollner, T. G., Schnee, C., Li, S., Svatos, A., Schneider, B., Gershenzon, J., et al. (2008). Proton- 7-O-methyltransferase involved in the biosynthesis of the flavanone phytoalexin ation of a neutral (S)-β-bisabolene intermediate is involved in (S)-β-macrocarpene sakuranetin from rice (Oryza sativa L.). Plant Science, 155, 213–221. formation by the maize sesquiterpene synthases TPS6 and TPS11. Journal of Biological Rakwal, R., Hasegawa, M., & Kodama, O. (1996). A methyltransferase for synthesis of Chemistry, 283, 20779–20788. the flavanone phytoalexin sakuranetin in rice leaves. Biochemical and Biophysical Kurusu, T., Hamada, J., Nokajima, H., Kitagawa, Y., Kiyoduka, M., Takahashi, A., et al. Research Communications, 222, 732–735. (2010). Regulation of microbeassociated molecular pattern-induced hypersensitive Schmelz, E. A., Kaplan, F., Huffaker, A., Dafoe, N. J., Vaughan, M. M., Ni, X., et al. (2011). cell death, phytoalexin production, and defense gene expression by calcineurin Identity, regulation, and activity of inducible diterpenoid phytoalexins in maize. B-like protein-interacting protein kinases, OsCIPK14/15, in rice-cultured cells. Plant Proceedings of the National Academy of Sciences of the United States of America, Physiology, 153,678–692. 108, 5455–5460. Lee, C. W., Yoneyama, K., Takeuchi, Y., Konnai, M., Tamogami, S., & Kodama, O. (1999). Shimizu, T., Jikumaru, Y., Okada, A., Okada, K., Koga, J., Umemura, K., et al. (2008). Momilactones A and B in rice straw harvested at different growth stages. Bioscience, Effects of a bile acid elicitor, cholic acid, on the biosynthesis of diterpenoid phyto- Biotechnology, and Biochemistry, 63,1318–1320. alexins in suspension-cultured rice cells. Phytochemistry, 69, 973–981. Lo, S. C., de Verdier, K., & Nicholson, R. L. (1999). Accumulation of 3-deoxyanthocyanidins Shimizu, T., Lin, F., Hasegawa, M., Okada, K., Nojiri, H., & Yamane, H. (2012). Purification phytoalexins and resistance to Colletotrichum sublineolum in sorghum. Physiological and identification of naringenin 7-O-methyltransferase, a key enzyme in biosyn- and Molecular Plant Pathology, 55,263–273. thesis of flavonoid phytoalexin sakuranetin in rice. Journal of Biological Chemistry, Lo, S. C., & Nicholson, R. L. (1998). Reduction of light-induced anthocyanin accumula- 287, 19315–19325. tion in inoculated sorghum mesocotyls: implications for a compensatory role in Snyder, B. A., & Nicholson, R. L. (1990). Synthesis of phytoalexins in sorghum as a the defense response. Plant Physiology, 116, 979–989. site-specific response to fungal ingress. Science, 248, 1637–1639. Lo, S. C., Weiergang, I., Bonham, C., Hipskind, J., Wood, K., & Nicholson, R. L. (1996). Spielmeyer, W., Ellis, M., Robertson, M., Ali, S., Lenton, J. R., & Chandler, P. M. (2004). Phytoalexin accumulation in sorghum: identification of a methyl ether of Isolation of gibberellin metabolic pathway genes from barley and comparative luteolinidin. Physiological and Molecular Plant Pathology, 49,21–31. mapping in barley, wheat and rice. Theoritical and Applied Genetics, 109, 847–855. Mohan, R. S., Yee, N. K. N., Coates, R. M., Ren, Y. Y., Stamenkovic, P., Mendez, I., et al. Stoessl, A., & Arditti, J. (1984). Orchid phytoalexins. In J. Arditti (Ed.), Orchid biology. (1996). Biosynthesis of cyclic diterpene hydrocarbons in rice cell suspensions: Reviews and perspectives, Vol. III. (pp. 151–175). Ithaca: Cornell University Press. conversion of 9,10-syn-labda-8(17),13-dienyl diphosphate to 9ß-pimara-7,15-diene Stoessla, A., Stothersb, J. B., & Ward, E. W. B. (1976). Sesquiterpenoid stress compounds and stemar-13-ene. Archives of Biochemistry and Biophysics, 330,33–47. of the solanaceae. Phytochemistry, 15, 855–872. C.E.C.C. Ejike et al. / Food Research International 52 (2013) 167–177 177

Tajkarimi, M. M., Ibrahim, S. A., & Cliver, D. O. (2010). Antimicrobial herb and spice Weiergang, I., Hipskind, J. D., & Nicholson, R. L. (1996). Synthesis of 3-deoxyanthocyanidin compounds in food. Food Control, 21, 1199–1218. phytoalexins in sorghum occurs independent of light. Physiological and Molecular Takahashi, N., Kato, T., Tsunagawa, M., Sasaki, N., & Kitahara, Y. (1976). Mechanisms of Plant Pathology, 49,377–388. dormancy in rice seeds. II. New growth inhibitors, momilactone-A and -B isolated Wickham, K. A., & West, C. A. (1992). Biosynthesis of rice phytoalexins: Identification of from the hulls of rice seeds. Japanese Journal of Breeding, 26,91–98. putative diterpene hydrocarbon precursors. Archives of Biochemistry and Biophysics, Tamogami, S., & Kodama, O. (2000). Coronatine elicits phytoalexin production in rice 293,320–332. leaves (Oryza sativa L.) in the same manner as jasmonic acid. Phytochemistry, 54, Wolniak, M., & Wawer, I. (2008). 13C CPMAS NMR and DFT calculations of 689–694. anthocyanidins. Solid State Nuclear Magnetic Resonance, 34,44–51. Tamogami, S., Mitani, M., Kodama, O., & Akatsuka, T. (1993). Oryzalexin S structure: A Wu, Y., Zhou, K., Toyomasu, T., Sugawara, C., Oku, M., & Abe, S. (2012). Functional new stemarane-type rice plant phytoalexin and its biogenesis. Tetrahedron, 49, characterization of wheat copalyl diphosphate synthases sheds light on the early 2025–2032. evolution of labdane-related diterpenoid metabolism in the cereals. Phytochemistry, Tamogami, S., Rakwal, R., & Kodama, O. (1997). Phytoalexin production elicited by 84,40–46. exogenously applied jasmonic acid in rice leaves (Oryza sativa L.) is under the Yamaguchi, T., Yamada, A., Hong, N., Ogawa, T., Ishii, T., & Shibuya, N. (2000). control of cytokinins and ascorbic acid. FEBS Letters, 412,61–67. Differences in the recognition of glucan elicitor signals between rice and soybean: Toyomasu, T., Kagahara, T., Okada, K., Koga, J., Hasegawa, M., Mitsuhashi, W., et al. β-Glucan fragments from the rice blast disease fungus Pyricularia oryzae that (2008). Diterpene phytoalexins are biosynthesized in and exuded from the roots elicit phytoalexin biosynthesis in suspension-cultured rice cells. The Plant Cell, 12, of rice seedlings. Bioscience, Biotechnology, and Biochemistry, 72, 562–567. 817–826. Turgis, M., Vu, K. D., Dupont, C., & Lacroix, M. (2012). Combined antimicrobial effect Zhang, L., Kong, Y., Wu, D., Zhang, H., Wu, J., Chen, J., et al. (2008). Three flavonoids of essential oils and bacteriocins against foodborne pathogens and food spoilage targeting the beta-hydroxyacyl-acyl carrier protein dehydratase from Helicobacter bacteria. Food Research International, 48, 696–702. pylori: Crystal structure characterization with enzymatic inhibition assay. Protein Udenigwe, C. C., & Aluko, R. E. (2012). Chemistry and bioactivity of food phytoalexins. Science, 17, 1971–1978. In F. O. Uruakpa (Ed.), Bioactive molecules in plant foods (pp. 13–130). New York: Zheng, L. Y., Guo, X. S., He, B., Sun, L. J., Peng, Y., Dong, S. S., et al. (2011). Genome-wide Nova Science Publishers. patterns of genetic variation in sweet and grain sorghum (Sorghum bicolor). Genome Umemura, K., Ogawa, N., Koga, J., Iwata, M., & Usami, H. (2002). Elicitor activity of Biology, 12,R114. cerebroside, a sphingolipid elicitor, in cell suspension cultures of rice. Plant & Cell Zhou, K., Xu, M., Tiernan, M., Xie, Q., Toyomasu, T., Sugawara, C., et al. (2012). Functional Physiology, 43, 778–784. characterization of wheat ent-kaurene(-like) synthases indicates continuing evolu- Van Etten, H., Mansfield, J. W., Bailey, J. A., & Farmer, E. E. (1994). Two classes of plant tion of labdane-related diterpenoid metabolism in the cereals. Phytochemistry, 84, antibiotics: Phytoalexins versus “phytoanticipins”. The Plant Cell, 6, 1191–1192. 47–55.