Plant Cell Rep (2011) 30:695706 DOI 10.

1007/s00299-010-0968-8

REVIEW

Molecular biology of capsaicinoid biosynthesis in chili pepper (Capsicum spp.)

Cesar Aza-Gonzalez Hector G. Nunez-Palenius Neftal Ochoa-Alejo

Received: 23 September 2010 / Revised: 29 November 2010 / Accepted: 30 November 2010 / Published online: 14 December 2010 Springer-Verlag 2010

Abstract Capsicum species produce fruits that synthesize and accumulate unique hot compounds known as capsaicinoids in placental tissues. The capsaicinoid biosynthetic pathway has been established, but the enzymes and genes participating in this process have not been extensively studied or characterized. Capsaicinoids are synthesized through the convergence of two biosynthetic pathways: the phenylpropanoid and the branched-chain fatty acid pathways, which provide the precursors phenylalanine, and valine or leucine, respectively. Capsaicinoid biosynthesis and accumulation is a genetically determined trait in chili pepper fruits as different cultivars or genotypes exhibit differences in pungency; furthermore, this characteristic is also developmentally and environmentally regulated. The establishment of cDNA libraries and comparative gene expression studies in pungent and non-pungent chili pepper fruits has identied candidate genes possibly involved in capsaicinoid biosynthesis. Genetic and molecular approaches have also contributed to the knowledge of this

biosynthetic pathway; however, more studies are necessary for a better understanding of the regulatory process that accounts for different accumulation levels of capsaicinoids in chili pepper fruits. Keywords pepper Capsaicinoid biosynthesis Capsicum Chili

Abbreviations DPA Days post-anthesis ROS Reactive oxygen species pAMT Putative aminotransferase

Introduction Capsaicinoids are the substances responsible for the pungent sensation that occurs when mammals bite Capsicum fruits. Only chili pepper fruits synthesize these compounds in nature. It has been suggested that capsaicinoids might provide protection against some pathogens (Tewksbury et al. 2008). Capsaicinoids are synthesized by condensing a molecule of vanillylamine, derived from phenylalanine, to a branched fatty acid (from 9 to 11 carbon atoms) synthesized from either valine or leucine (Curry et al. 1999) (Fig. 1). Although more than ten different capsaicinoid structures exist (Mazourek et al. 2009), capsaicin (CAP) and dihydrocapsaicin (DHCAP) are the most predominant, accounting for almost 90% of all capsaicinoids (Kozukue et al. 2005; Choi et al. 2006) (Fig. 2). CAP differs from DHCAP by an unsaturated double bond at carbon 9 of the branched-chain fatty acid. It is known, for the majority of Capsicum species, that capsaicinoids start accumulating in

Communicated by R. Reski. A contribution to the Special Issue: Plant Biotechnology in Support of the Millennium Development Goals. C. Aza-Gonzalez H. G. Nunez-Palenius N. Ochoa-Alejo (&) Departamento de Ingeniera Genetica de Plantas, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional (Cinvestav)-Unidad Irapuato, Km 9.6 libramiento norte carretera Irapuato-Leon, 36821 Irapuato, Guanajuato, Mexico e-mail: nochoa@ira.cinvestav.mx N. Ochoa-Alejo Departamento de Biotecnologa y Bioqumica, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional (Cinvestav)-Unidad Irapuato, Km 9.6 libramiento norte carretera Irapuato-Leon, 36821 Irapuato, Guanajuato, Mexico

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fruits approximately 20 days post-anthesis (DPA) (Iwai et al. 1979). Capsaicinoid biosynthesis occurs in the placental epidermis cells, where they are secreted towards the outer cell wall, and nally accumulate within structures named blisters located on the placenta surface (Suzuki et al. 1980; Stewart et al. 2007) (Fig. 3). Capsaicinoids are found at different amounts in Capsicum fruits, depending mostly on the genotype, developmental stage and growth conditions. The mechanisms by which the capsaicinoid amounts are regulated in chili pepper fruits are still unknown. This article deals with the current state of knowledge of the molecular biology of capsaicinoid biosynthesis and some possibilities for genetic manipulations of this trait in the Capsicum genus. Capsaicinoid uses Chili pepper fruits are consumed fresh in salads (pimientos) and salsas as ingredients of different dishes around the

world or even processed as pickles and salsas. Capsaicinoids are one of the groups of compounds produced by chili pepper fruits that are used for industrial and medical purposes. 1. Food. Capsaicinoids are of great importance and are principally used by humans as food additives because chili peppers are widely used to season a variety of dishes. The food industry widely uses capsaicinoids for multiple purposes because they are the basic ingredients for salsas, curries and dressings, among other foods (Perkins et al. 2002). Pharmaceutical and medical. Capsaicinoids have been found to exert a series of physiological and pharmacological effects, including analgesia, anticancer, antiinammatory, antioxidative and anti-obesity activities (Negulesco et al. 1987; Govindarajan and Sathyanarayana 1991; Luo et al. 2010; Liu and Nair 2010). Capsaicinoids demonstrate anti-inammatory activities (Spiller et al. 2008); therefore, they are used as the main

2.

Fig. 1 Capsaicinoid biosynthetic pathway. PAL phenylalanine ammonia lyase, C4H cinnamate 4-hydroxylase, 4CL 4-coumaroyl-CoA ligase, HCT hydroxycinnamoyl transferase, C3H coumaroyl shikimate/quinate 3-hydroxylase, CCoAOMT caffeoyl-CoA 3-Omethyltransferase, COMT caffeic acid O-methyl transferase, HCHL hydroxycinnamoyl-CoA hydratase/lyase, pAMT putative aminotransferase, BCAT branched-chain amino acid transferase, KAS ketoacyl-ACP synthase, ACL acyl carrier protein, FAT acyl-ACP thioesterase, ACS acyl-CoA synthetase, CS capsaicin or capsaicinoid synthase. COMT is indicated in parentheses because it was the enzyme proposed early on to participate in the phenylpropanoid pathway [modied from Stewart et al. (2005), and Mazourek et al. (2009)]

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Plant Cell Rep (2011) 30:695706 Fig. 2 Structures of common capsaicinoids (a) and capsinoids (b)

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Fig. 3 Tissues of chili pepper fruits. C. chinense Habanero dissected and without seeds (a) and the interlocular septum showing the blisters where capsaicinoids accumulate (b)

components of ointments, patches, oils and creams designed to relieve the pain caused by several diseases (Rains and Bryson 1995). Nonetheless, these alkaloids

also have other applications of medical relevance. For instance, capsaicinoids are able to reduce the painful discomforts caused by vasomotor rhinitis, osteoarthritis and rheumatoid arthritis (Deal et al. 1991; Marabini et al. 1991; Cordell and Araujo 1993; Robbins 2000). What is more, it has been observed that capsaicinoids can participate as pain relievers for cluster headaches, neck pain, oral mucositis, rhinopathy, hyperreexia and cutaneous pain caused by skin tumors (Hautkappe et al. 1998). These pharmacological properties are due to the release of Substance P (a neuropeptide implicated in pain transmission) from terminals of primary sensory neurons by the action of capsaicinoids (Gamse et al. 1981). Currently, the capsaicin studies in the medical eld are focused on the ability of capsaicin to inhibit the growth of cancerous cells. It has been reported that capsaicin induces apoptosis cell death in in vitro human-gastric cancer cells (SNU-1) (Kim et al. 1997). In another study, it was described that capsaicin was able to reduce the growth of numerous lines of leukemic cells through G0G1 phase cell cycle arrest and apoptosis. It was observed in mice that tumor weight was reduced by 50% when capsaicin (50 mg/kg) was injected daily for 6 days (Ito et al. 2004).

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3.

4.

Similarly, Mori et al. (2006) reported that capsaicin is able to inhibit the growth of prostate cancer cells in mice, without producing any toxicity; tumor weight was reduced to *50% when 5 mg/kg/d capsaicin was administered to mice 3 days per week for 4 weeks. Sanchez et al. (2006) have also reported the effect of capsaicin on the apoptosis of prostate tumor PC-3 cells. It seems that the mechanism by which capsaicin induces apoptosis in cancer cells is associated with production of reactive oxygen species (ROS) and disruption of the mitochondrial transmembrane potential by the suppression of a NADH-oxidoreductase, an enzyme that transfers electrons from cytoplasmic NADH via coenzyme Q (ubiquinone) to the external electron acceptors such as oxygen (Surh 2002). Additionally, in colon cancer cells treated with capsaicin, the activity of caspase-3-activity, the major apoptosisexecuting enzyme, was substantially increased (Yang et al. 2009). Cosmetic and dietary. Capsaicinoids are also used as additives in a series of hair-loss-prevention shampoos currently found in the market. Miscellaneous uses. Self-protection aerosol sprays using capsaicinoids as the main active ingredient are currently on the market (Fung et al. 1982; Andrews 1995; Reilly et al. 2001). Capsaicinoids have been tried as a repellent to prevent mice from gnawing on underground electrical cables (Bosland 1996). Further, capsaicinoids have antimicrobial properties (Xing et al. 2006); for example, Tewksbury et al. (2008) described that these substances are capable of inhibiting the growth of Fusarium fungus, which is a major problem in post-harvest fruits and vegetables. Consequently, capsaicinoids might be useful as biopesticides.

The capsaicinoid biosynthetic pathway Capsaicinoids have been studied since the beginning of 1800s; nonetheless, their chemical structure was not fully established until 1919 (Nelson 1919). Chili pepper inheritance studies have suggested that one single dominant gene, named locus C, is responsible for the pungent characteristic (Deshpande 1935). The general capsaicinoid biosynthetic pathway was established at the end of the 1960s, when radiotracer studies were used to investigate capsaicinoid precursors, nding that the vanillyamine moiety was synthesized from phenylalanine, and that the branched-chain fatty acid was derived from valine (Bennett and Kirby 1968; Leete and Louden 1968). The biosynthetic phenylpropanoid pathway proposed at that time involved the sequential synthesis of

phenylalanine, cinnamic, p-coumaric, caffeic and ferulic acids, and then the formation of vanillin and vanillylamine (Bennett and Kirby 1968). The participation of phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), coumarate 3-hydroxylase (C3H) and caffeic acid O-methyltransferase (COMT) in phenylpropanoid-mediated capsaicinoid biosynthesis was established by several authors (Fujiwake et al. 1982a, b; Sukrasno and Yeoman 1993). At the beginning of the 1980s, it was discovered that acyl moieties were derived from either valine or leucine (Suzuki et al. 1981). More recently, Stewart et al. (2005) and Mazourek et al. (2009) have proposed the participation of some other enzymes, such as 4-coumaroyl-CoA ligase (4CL), hydroxycinnamoyl transferase (HCT), caffeoylCoA O-methyltransferase (CCoAOMT; instead of COMT) and hydroxycinnamoyl-CoA hydratase/lyase (HCHL), in the phenylpropanoid pathway that lead to capsaicinoid formation based on different experimental sources (see for example Gasson et al. 1998; Hoffmann et al. 2003; Merali et al. 2007) (Fig. 1). One of the most important molecular biology approaches to understand the capsaicinoid biosynthesis pathway started with Curry et al. (1999). Because it was previously known that the phenylpropanoid pathway was involved in supplying precursors for capsaicinoid biosynthesis, and considering that the PAL-, C4H- and COMT-encoding genes were already cloned in other plants (Estabrook and Senguptagopalan 1991; Gowri et al. 1991; Fahrendorf and Dixon 1993), Curry et al. (1999) decided to isolate some of the phenylpropanoid-pathway genes from chili peppers. They generated cDNAs for the Pal, C4h and Comt genes from a Capsicum chinense cv. Habanero cDNA library, using heterologous sequences from alfalfa and soybean cDNAs. It was observed by northern blot analysis in Habanero chili pepper fruit placentas that Pal, C4h and Comt transcripts accumulated the most in immature fruits, but their concentration started to diminish as fruits ripen. Transcript levels of these three genes correlated with pungency levels because a higher transcript level was observed in more pungent chili pepper fruits. Afterwards, a differential screen was performed to detect abundant transcripts of additional genes in Habanero samples that were undetectable in non-pungent peppers (C. chinense PI 1721) as an approach to gain information on some other genes involved in capsaicinoid-biosynthesis. Two transcripts were characterized: one showing high homology to a 3-keto-acyl-ACP synthase (Kas gene), which might be involved in the biosynthesis of the branched-chain fatty acid, and the other with high homology to a putative aminotransferase (pAmt gene). It was previously predicted that an aminotransferase should be involved in the conversion of vanillin to vanillylamine. Northern blot expression analyses were carried out for the two newly

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found sequences and, much like Pal, C4h and Comt, the transcripts showed maximal accumulation during the rst developmental stages in the chili pepper fruits with the highest pungency. By using tissue-specic expression analysis in fruits, it was also found that both the 3-ketoacyl-ACP synthase (Kas) and the putative aminotransferase (pAmt) sequences were only expressed at signicant levels in placental tissues, where the capsaicinoids were synthesized. With the aim of detecting genes involved in the biosynthesis of the fatty acid component, Aluru et al. (2003) carried out a differential screen of a Habanero (C. chinense) placenta cDNA library. They recovered three cDNA sequences with high similarity to branched-chain fatty acid biosynthesis enzyme genes: an acyl carrier protein (Acl), a thioesterase (Fat) and a b-keto-acyl-ACP synthase (Kas). Through RNA blots the expression analysis of those three genes was accomplished for several Habanero chili pepper tissues, showing that their greatest accumulation occurred in the immature green placenta. Interestingly, as Habanero chili pepper fruits ripen, transcript accumulation diminishes. Moreover, it was found that the transcript accumulation of those three sequences was positively correlated with pungency levels in several Capsicum varieties. The Kas transcripts only accumulated in the placental tissues (Curry et al. 1999), whereas Acl and Fat transcripts were detected in other tissues, such as that of roots, stems, leaves, owers and seeds. In order to test whether Kas encoded for a protein with KAS activity, the protein was over-expressed in E. coli, and the protein encoded by Kas signicantly increased fatty acid formation ([C8). Furthermore, KAS activity was inhibited by *50% by 20 mM cerulenin, which is a basic characteristic of class 1 KAS enzymes. Using antibodies against KAS, the protein was found to accumulate in the placental epidermal and subepidermal layers of chili pepper fruits. Knowing that capsaicinoid biosynthesis genes are highly expressed in placenta tissues from chili pepper fruits with high pungency levels, Kim et al. (2001) generated a cDNA subtractive library from the highly pungent chili pepper placenta tissues of C. chinense cv. Habanero. The authors utilized placenta tissues from 30 DPA chili pepper Habanero fruits as the tester (highly pungent) and compared these with either 10 DPA Habanero or C. annuum cv. Haehwa III placental tissues (non-pungents). The results showed that 39 cDNA sequences were highly expressed in placenta tissues from 30 DPA Habanero chili pepper fruits, but not in either 10 DPA Habanero and Haehwa III placenta tissues. The cloned sequences were analyzed by northern blot analysis, and some of them were specically expressed in placenta tissues. SB2-149 and SB1-158 clones showed a high similarity to the pAmt and Kas genes,

respectively, which had formerly been reported as putative genes involved in the capsaicinoid biosynthetic pathway (Curry et al. 1999). Similarly, the SB2-66 and SB2-115 clones might be related to capsaicinoid biosynthesis as well. Mainly, the SB2-66 clone showed homology to a group of coenzyme A-dependent acyl transferases, which are involved in the transfer of acyl groups in a coenzyme A-dependent manner. Therefore, these authors suggested that the SB2-66 clone might be the capsaicinoid synthase (CS), the last enzyme responsible for the condensation of vanillylamine to a branched-chain fatty acid moiety in the capsaicinoid biosynthetic pathway. Another remarkable clone was SB2-115 because it showed high homology to long chain fatty-acid alcohol oxidases from Arabidopsis and Candida tropicalis. Therefore, SB2-115 could participate in fatty acid biosynthesis, and the products might be used for capsaicinoid production. Soon after, Stewart et al. (2005) found that the SB2-66 clone, previously isolated by Kims group, co-segregated with the pungency trait, and it was mapped to a locus in close proximity to Pun 1 (locus C), which modies the pungency level (Blum et al. 2002). The full SB2-66 cDNA was used as a probe for a DNA blot of genomic DNA isolated from numerous pungent and non-pungent chili pepper fruits. It was observed that DNA from non-pungent peppers was decient in a specic hybridization band that appeared in DNA from pungent fruits. Using genome walking, the SB2-66 genomic DNA was isolated and compared with certain sequences from pungent and nonpungent chili peppers, showing that sequences from nonpungent fruits have a 2.5-kb deletion, encompassing part of the putative promoter and the rst exon. That allele was named pun1, and because the SB2-66 clone has acyltransferase domains it was labeled At3. The expression pattern for At3 was determined by northern blot assays in Habanero (Capsicum chinense-hot) and Bell (Capsicum annuum-sweet) peppers, showing that At3 expression was specically located in the placental tissues from pungent peppers and that its maximal accumulation was observed at *20 DPA. A series of northern blot analyses in Habanero and Bell pepper placenta tissues were carried out for capsaicinoid biosynthesis-related genes, such as Pal, C4H, Comt, pAmt, BCAT, Kas, Acl and FatA (Fig. 1). The results showed that, with the exception of BCAT and Acl, the candidate genes were either undetectable or their levels were signicantly reduced in non-pungent peppers. These results suggested that At3 might participate in the regulation of other capsaicinoid-related genes. In order to demonstrate that At3 was related to capsaicinoid production, virus-induced gene silencing (VIGS) with Tobacco rattle virus (TRV) as the vector was used to silence the At3 gene (Stewart et al. 2005). Capsaicinoid production was reduced by 50% compared with a control

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plant when the At3 gene was silenced. Interestingly, the observed capsaicinoid reduction caused by At3 silencing reached 70% when the comparison was made with the empty vector. Although these results seemed to be contradictory, the plant inoculated with the empty vector accumulated more capsaicinoids than the non-inoculated plant, probably because of wounding and infection that occurred during the inltration process. This behavior of capsaicinoid production in chili pepper plants was previously observed when pepper plants were subjected to environmental stresses (Estrada et al. 1999). The At3 gene was proposed (Stewart et al. 2005) to encode the capsaicinoid synthase for several reasons: (1) the transcript accumulated specically in pungent-placenta tissues, (2) non-pungent pepper fruits showed an explicit deletion in that gene, (3) its expression pattern was similar to other capsaicinoid-related genes, and (4) silencing it by VIGS reduced capsaicinoid accumulation by approximately 70%. However, some results did not support the view that the At3 gene encodes the capsaicinoid synthase; for instance, it was proposed that the capsaicinoid synthase should be a coenzyme-A dependent acyltransferase, and this was not the case for AT3. Therefore, more research is necessary to fully establish the function of AT3 and its role as putative regulator of the capsaicinoid biosynthetic pathway. On the other hand, Lee et al. (2005) also proposed that the gene corresponding to the SB2-66 clone might be the capsaicinoid synthase. They analyzed the F2 population from a cross between a non-pungent C. annuum and a mildly pungent C. annuum. According to their results, the capsaicinoid synthase (1) co-segregated with the pungency trait, (2) was only expressed in the fruit placenta, and (3) co-segregated with locus C. Therefore, these authors proposed that the SB2-66 clone was gene C, which is thought to be responsible for chili pepper fruit pungency. Moreover, similarly to Stewart et al. (2005), Lee et al. (2005) found that non-pungent Capsicum peppers had a 2,529-bp deletion in the 50 -region of the putative capsaicinoid synthase gene. Later, Stewart et al. (2007) analyzed the At3 gene in a non-pungent C. chinense NMCA 30036 chili pepper. The At3 gene sequence revealed a 4-bp deletion in the rst exon, and this allele was named pun12. Due to that deletion, the AT3 protein was not detected in NMCA 30036 fruits, but low levels of the transcript were detected in 20 and 50 DPA chili pepper fruits. Although a capsaicinoid biosynthetic pathway involving specic enzymes and genes was proposed, and some transcripts for those genes specically accumulated in placenta tissues, except for At3 (Pun1), no direct evidence of their participation in capsaicinoid production had been reported. Therefore, 30 UTR sequences for Comt, pAmt (phenylpropanoid pathway) and Kas (fatty acid biosynthesis

pathway), as reported by Curry et al. (1999), were inserted into a viral vector derived from Pepper huasteco yellow veins virus (PHYVV), in order to investigate whether the genes were involved in capsaicinoid production (AbrahamJuarez et al. 2008). Four-week-old Serrano pepper plants (C. annuum L. cv. Tampiqueno 74) were infected with the PHYVV-vector bearing Comt, pAmt and Kas constructs. Comt, pAmt and Kas transcripts were analyzed by RT-PCR and northern blot in the placenta tissue of 40 DPA chili pepper fruits. The results showed that Comt, pAmt and Kas transcripts were almost undetectable in infected plants but were detectable in wild-type plants. Furthermore, specicsiRNAs for Comt, pAmt and Kas were observed in the placenta tissues of silenced chili pepper plants. Although some infected plants did not show a homogenous decrease in Comt, pAmt and Kas transcript levels, it was possible to observe that the infected plants with undetectable transcript levels also had undetectable capsaicinoid levels. On the other hand, infected chili pepper plants with detectable Comt, pAmt and Kas transcripts depicted an average accumulation of 9.6, 7.1 and 11.7%, respectively, compared with non-infected plants. In this context, the participation of Comt, pAmt and Kas in capsaicinoid-biosynthesis was proven, supporting the previously proposed capsaicinoid biosynthetic pathway (Fig. 1). The participation of the pAmt gene in the capsaicinoid pathway was also ascertained (Sutoh et al. 2006). In vivo experiments carried out in a pungent chili pepper (C. annuum cv. Takanotsume) showed that [14C]-vanillin injected into fruits was efciently converted to vanillylamine (Sutoh et al. 2006). Additionally, it was found that [14C]-vanillin was transformed into vanillyl alcohol, a precursor of a non-pungent compound named capsinoid (Fig. 2). Nevertheless, Lang et al. (2009) reported that C. annuum cv. CH-19 Sweet pepper fruits, which do not accumulate capsaicinoids, were only capable of converting [14C]-vanillin into vanillyl alcohol, but not into vanillylamine. The pAMT activity was measured in cell-free extracts from C. annuum cv. CH-19 Sweet placenta, showing that conversion of vanillin into vanillylamine and capsaicinoid production were reduced to 60 and 9%, respectively, compared with pungent varieties. After comparing the sequences of pAmt from C. annuum cv. CH-19 Sweet and Habanero (C. chinense), it was discovered that a T nucleotide insertion in the pAmt sequence of CH-19 Sweet pepper had occurred, producing a stop codon, which affects the production of active pAMT. It was concluded that pAMT actively participates in capsaicinoid biosynthesis by regulating the phenylpropanoid precursors channeled into this pathway. As previously stated, the participation of Pal, C4h, Comt and Amt (from the phenylpropanoid pathway; Fig. 1) in capsaicinoid production, as well as the roles of BCAT, Kas,

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Acl and FatA (from the branched-chain fatty acid pathway) in capsaicinoid accumulation, is known. Moreover, a putative capsaicin or capsaicinoid synthase has been proposed. Although the exact process by which the branchedchain fatty acids are synthesized is not known, some authors (Blum et al. 2003; Stewart et al. 2005) have suggested that a desaturase converts 8-methylnonanoic acid into 8-methyl-6-nonenoic acid. Nonetheless, 8-methyltrans-6-nonenoic acid, the branched-chain fatty acid used for capsaicin synthesis, was detected in the thioester pool (acyl-ACP and acyl-CoA) isolated from two chili pepper placenta tissues (C. chinense var. Habanero orange and C. annuum var. Jalapeno). This result suggests that the desaturation reaction takes place before the thioesterase FAT removes the branched-chain fatty acids, and no modication occurs once the fatty acid is attached to the vanillylamine moiety (Thiele et al. 2008). Furthermore, the fatty acid moieties attached to ACP and CoA corresponded to those found in capsaicinoid molecules. On the other hand, Mazourek et al. (2009) very recently proposed an innovative branched-chain fatty acid biosynthesis pathway where, in addition to isobutyryl-CoA, some other intermediaries like acetyl-CoA, isovaleryl-CoA, anteisovalerylCoA and propinyl-CoA could be used as substrates for capsaicinoid biosynthesis.

Regulation of the biosynthetic pathway The pungency threshold found in any Capsicum fruit is an inherited characteristic, and the ability to accumulate capsaicinoids is a trait that depends on the chili pepper species, variety, genotype, and environmental growth conditions (Harvell and Bosland 1997; Zewdie and Bosland 2000) (Table 1). For instance, Estrada et al. (1999) observed that ooding-stressed chili pepper plants accumulated more capsaicinoids than control plants, and that

Table 1 Capsaicinoid content in several chili peppers [from Kozukue et al. (2005), and Bosland and Baral (2007)] Capsicum species C. frutescens Bhut jolokiaa C. chinense cv. Habanero C. frutescens Thai C. annuum L. Serrano type C. annuum L. Jalapeno type C. annuum L. Green bell type
a b

Capsaicinoidsb (lg/g fresh weight; f.wt.) 6,2581 2,260 1,332 76 75 0

In Bhut joloquia, a factor of 16 was used to convert SHU to lg/g

Since some data were reported as dry weight, a 90% of humidity was considered to carry out the conversions

capsaicinoid accumulation was more noticeable when the pepper plants were drought-stressed. In a similar study, it was found that the effect on capsaicinoid accumulation depended on whether chili pepper plants were grown under greenhouse or eld conditions (Jurenitsch et al. 1979). The effects of light and temperature on capsaicinoid accumulation have also been studied by Murakami et al. (2006). These authors showed that chili pepper plants accumulated more capsaicinoids under continuous uorescent light and temperature (150350 lmols m-2, 28C) than pepper plants kept 18 h at 28C/6 h at 16C (light/dark) cycles. To our knowledge, no conclusive scientic evidence has been obtained about the genes that regulate the biosynthesis and accumulation patterns of different capsaicinoids. However, several papers have been published regarding this matter. It has been suggested from comparative expression studies in pungent and non-pungent chili pepper fruits that two bZIP transcription factors might be involved in regulating the capsaicinoid biosynthetic pathway (Blum et al. 2003; Stewart et al. 2005); nonetheless, no clear evidence exists to verify their participation. The Pun1 gene, which encodes an acyltransferase and has been found to be involved in capsaicinoid production, was analyzed in a non-pungent pepper variety (C. annuum Bell) with a mutation in that gene. With the exception of BCAT and Acl, which are constitutively expressed in fruits, no transcripts of any of the structural genes involved in capsaicinoid biosynthesis were detected in leaves or during fruit development (Stewart et al. 2005). These results suggest the possibility that the Pun1 gene might participate in regulating the capsaicinoid biosynthetic pathway by controlling some structural genes, by controlling the metabolic ux of precursors, or by being an important component of a regulatory complex (Stewart et al. 2005). Recently, it has been proposed that capsaicin might function as a feedback inhibitor in the capsaicinoid biosynthetic pathway because the immersion of immature green pepper fruit placenta tissues in several capsaicin solutions (0, 0.15, 0.3 or 0.6 mg ml-1) caused a drastic reduction (*50% compared with control) in CS, Kas, Pal and pAmt transcript accumulation (Kim et al. 2009). This result might explain the lack of a correlation between maximal transcript accumulation and capsaicinoid concentrations (Kim et al. 2009). Another approach that has been utilized to investigate the regulation of capsaicinoid biosynthesis is to search for a quantitative trait locus (QTL) that affects capsaicinoid production. A QTL named cap was identied in chili pepper chromosome 7 by analyzing the F2 population from a cross between pungent (C. frutescens parent, accession BG 2816) and non-pungent (C. annuum parent cv. Maor) pepper plants (Blum et al. 2003). From this analysis, it was observed that cap contributed 3438% of the observed

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variation in capsaicinoid accumulation; therefore, it was proposed that the cap QTL could be a regulator of capsaicinoid biosynthesis or perhaps could correspond to an unidentied structural gene. In a similar study, six QTLs related to capsaicinoid accumulation were identied and localized in chromosomes 3, 4 and 7 (Ben-Chaim et al. 2006). Five of these QTLs contributed to capsaicin increase and accumulation and explained 37% of the observed variation in pungency. Four out of those ve QTLs seemed to be involved in dihydrocapsacin accumulation and explained 25% of the phenotypic variation. Only one QTL was associated with nordihydrocapsaicin levels, and this QTL did not co-localize with other QTLs that control the accumulation of other capsaicinoids. These six QTLs explained 31% of the phenotypic variation. In addition, an interaction between the cap7.1 QTL and a marker located in chromosome 2 was observed, which explained 42% of the variation in capsaicinoid content. Likewise, the cap7.2 QTL identied in this study might be an ortholog of the cap QTL that has been previously mapped (Blum et al. 2003).

Capsinoids, non-pungent analogs of capsaicinoids In addition to capsaicinoids, other secondary metabolites named capsinoids are produced in chili pepper fruits (Kobata et al. 1998; Singh et al. 2009). Unlike capsaicinoids, capsinoids are non-pungent and do not cause a burning sensation, facilitating their application in human medicine (Sasahara et al. 2010). Recent advances in the study of capsinoids have been summarized by Luo et al. (2010). Capsinoids are synthesized by the condensation of branched-chain fatty acid moieties and vanillyl alcohol, instead of the vanillylamine used for capsaicinoids (Kobata et al. 2002) (Fig. 2). Capsinoids over-accumulate in a nonpungent pepper, Capsicum annuum cv. CH-19, which possesses a functional loss of pAMT (Lang et al. 2009; Tanaka et al. 2010). Capsiate, dihydrocapsiate and nordihydrocapsiate are the three capsinoids found in these chili pepper fruits (Kobata et al. 1998; Kobata et al. 1999). Like capsaicinoids, these capsinoids induce apoptosis, which is preceded by an increase production of ROS and a subsequent loss of mitochondrial transmembrane potential (Macho et al. 2003). Inhibition of angiogenesis and vascular permeability by capsiate has been demonstrated by Pyon et al. (2008). Moreover, capsinoids show antioxidative (Rosa et al. 2002) and anti-inammatory properties (Sancho et al. 2002). Other researchers have shown that capsinoids induce energy expenditure and body fat loss in rats and humans (anti-obesity effects) (Ohnuki et al. 2001; Iwai et al. 1979; Inoue et al. 2007; Snitker et al. 2009). These ndings create an opportunity to manipulate the capsinoid metabolic pathway to over-accumulate these secondary compounds and use them medicinally.

Molecular markers for non-pungency Detecting the non-pungency trait in chili peppers during the early stages of development can certainly reduce the selection time for breeding programs that cater to consumer and industrial requirements. As previously mentioned, Stewart et al. (2005) reported that a large deletion (ca. 2.5 kb spanning 1.8 kb of the putative promoter and 0.7 kb of the rst exon) at the Pun1 locus (pun1 allele), encoding a putative acyltransferase, was positively correlated with non-pungency in chili pepper fruits. Based on this deletion, Lee et al. (2005) developed SCAR markers for easy, accurate and early detection of non-pungent chili peppers. Stewart et al. (2007) detected a novel allele named pun12, a recessive allele of Pun1 associated with the absence of blisters in non-pungent chili pepper fruits. A PCR-based co-dominant analysis of this pun12 revealed that a fourbase pair deletion led to a frameshift mutation. More recently, Lang et al. (2009) applied SNP analysis to the CH-19 Sweet pepper and found a T insertion at basepair 1,291 in the pAMT gene that is responsible for a nonsense recessive mutation that causes a loss of pungency and an accumulation of capsinoids instead of capsaicinoids. A derived cleaved amplied polymorphic sequence (dCAPS) DNA marker was developed to detect homozygous recessive mutants for this condition. A similar approach was used by Tanaka et al. (2010) to detect a nonsense mutation in the pAMT gene of the non-pungent cultivar Himo (C. annuum L.), in which a single-nucleotide substitution results in a single amino acid change from a cysteine to an arginine in the pyridoxal 5-phosphate binding domain.

Future research The phenylpropanoid and branched-chain fatty acid biosynthesis pathways are used to synthesize capsaicinoids through the action of a putative capsaicinoid synthase. As previously stated, there have been some advances towards our understanding of the genes in the capsaicinoid biosynthesis pathway, such as identifying the Pal, C4h, Comt, Amt, BCAT, Kas, Acl and Fat genes. Additionally, it has been suggested that the AT3 acyltransferase might be the capsaicinoid synthase. Nonetheless, some further research is needed, given that in the proposed capsaicinoid biosynthetic pathway (Stewart et al. 2007) several genes have neither been isolated nor identied. More importantly, how those genes are involved in the capsaicinoid pathway, whether by controlling it or as structural genes, should be precisely discerned. Another challenge is to identify the genes and enzymes involved in producing vanillin from feruloyl CoA, as well as the regulatory steps in this

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conversion. On the other hand, even though several putative genes encoding 4CL, HCT, C3H and ACS have been recently isolated (Mazourek et al. 2009), it is necessary to demonstrate their specic roles in the capsaicinoid biosynthetic pathway. To our knowledge, the Pun1 gene is the only gene that determines whether capsaicinoid is present or absent in Capsicum fruits (Stewart et al. 2005, 2007). Albeit Pun1 encodes an acyltransferase, it is not yet known which reaction it catalyzes or what role it plays in capsaicinoid biosynthesis. Another interesting question to address in the near future is whether Pun1 functions as a transcription factor because it was observed that Pal, C4H, Comt, pAmt, BCAT, Kas, Acl and FatA transcripts were signicantly diminished in a non-pungent pepper variety bearing the pun1 mutation (Stewart et al. 2005). However, the same authors (Stewart et al. 2007) later found a novel mutation named pun12 in other non-pungent chili pepper fruits harboring a four-base pair deletion in Pun1, causing a frameshift mutation. In this non-pungent pepper Pal and Kas gene expression was similar or even higher in Habanero chili pepper fruits, contradicting the idea that Pun1 functions as a transcription factor. Several chili pepper cDNA libraries have been generated (Curry et al. 1999; Kim et al. 2001; Mazourek et al. 2009) and could be used to search for transcription factors involved in the capsaicinoid biosynthetic pathway using blot analysis or microarrays. In other plant metabolic pathways, such as the biosynthetic pathway of anthocyanins, transcription factor expression is highly correlated with structural gene expression (Spelt et al. 2000; Borovsky et al. 2004; Espley et al. 2007); thus, this approach might be used as an initial criterion for the identication of capsaicinoid biosynthesis-related transcription factors. Furthermore, Mazourek et al. (2009) have recently proposed that phenylpropanoid and branched-chain fatty acid pathways are interconnected with other metabolic systems, such as amino acids, that can greatly affect the capsaicinoid accumulation. These authors cloned 42 sequences from chili pepper plants, 29 of which corresponded to phenylpropanoid-related and branched-chain fatty acid-related pathways but had not been previously considered as participants in those pathways. The predicted cellular localization of those proteins indicates that, with the exception of BCAT, pAMT and acyl-CoA synthetases, the enzymes did not show any discrepancies regarding their cellular localization. The 42 sequences were genetically mapped in chili pepper plants. This information opens the possibility of considering the inuence of other metabolic pathways, in addition to phenylpropanoids and branched-chain fatty acids, on capsaicinoid accumulation. In order to do a functional analysis of the genes potentally involved in capsaicinoid biosynthesis and regulation,

it is necessary to expand the molecular analysis of nonpungent versus pungent chili peppers. Detection of mutants by comparative SNP analysis in non-pungent and pungent chili peppers might render new information on structural or regulatory genes that participate in capsaicinoid biosynthesis. Furthermore, perhaps some Capsicum plants with mutations at different capsaicinoid biosynthetic steps could be generated by chemical mutagenesis and analyzed by TILLING (Targeting-Induced Local Lesions in Genomes) as a reverse-genetics tool for functional analysis. This approach has been applied to tomato plants (Gady et al. 2009; Minoia et al. 2010); however, its application to chili pepper genetic analysis may depend on the establishment of an associated database and a TILLING platform for this crop. Although VIGS is a limited approach for studying gene functions, successful examples demonstrating the participation of some putative genes in capsaicinoid biosynthesis have already been published (Stewart et al. 2005; Abraham-Juarez et al. 2008). Silencing the HCT gene in Nicotiana benthamiana and Arabidopsis plants has demonstrated its participation in the phenylpropanoid pathway (Horffmann et al. 2004) and could surely be very useful in studying its role in capsaicinoid biosynthesis in chili pepper fruits. Genetic transformation has been used as a tool for functional analysis in different plant species. Over-expression or suppression of candidate genes by sense and anti-sense technology can demonstrate the participation of certain genes in specic functions. Furthermore, complementing mutants with known genes can demonstrate gene function in plants. Chili pepper tissue culture and Agrobacteriummediated genetic transformation protocols have been developed by different authors (Kothari et al. 2010), but the main problem for their application in gene function studies is the low efciency for in vitro plant regeneration and transformation due to the recalcitrance of Capsicum species (Ochoa-Alejo and Ramrez-Malagon 2001). However, a gene function study using genetic transformation in chili pepper plants was reported by Harpster et al. (2002), opening the possibility of applying this approach to functional studies of genes involved in capsaicinoid biosynthesis. The processes by which the different capsaicinoid types and analogs are produced, and how their production is regulated, remain unknown and must be a priority for future research studies. As previously mentioned, several environmental factors such as light, temperature and water availability, among others, can affect capsaicinoid production and accumulation. Despite this, the precise molecular events that occur during capsaicinoid accumulation are unknown. Finally, basic knowledge is of paramount importance in manipulating any metabolic pathway by genetic

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Plant Cell Rep (2011) 30:695706 Deal CL, Schnitzer TJ, Lipstein E, Seibold JR, Stevens RM, Levy MD, Albert D, Renold F (1991) Treatment of arthritis with topical capsaicina double-blind trial. Clin Ther 13:383395 Deshpande RB (1935) Studies in Indian chillies: 4. Inheritance of pungency in Capsicum annuum L. Indian J Agric Sci 5:513516 Espley RV, Hellens RP, Putterill J, Stevenson DE, Kutty-Amma S, Allan AC (2007) Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. Plant J 49:414427 Estabrook EM, Senguptagopalan C (1991) Differential expression of phenylalanine ammonia-lyase and chalcone synthase during soybean nodule development. Plant Cell 3:299308 Estrada B, Pomar F, Diaz J, Merino F, Bernal MA (1999) Pungency level in fruits of the Padron pepper with different water supply. Sci Hortic 81:385396 Fahrendorf T, Dixon RA (1993) Stress responses in alfalfa (Medicago sativa L).18. Molecular-cloning and expression of the elicitorinducible cinnamate 4-hydroxylase cytochrome-P450. Arch Biochem Biophys 305:509515 Fujiwake H, Suzuki T, Iwai K (1982a) Intracellular distribution of enzymes and intermediates involved in biosynthesis of capsaicin and its analogues in Capsicum fruits. Agric Biol Chem 46:26852689 Fujiwake H, Suzuki T, Iwai K (1982b) Capsaicinoid formation in the protoplast from placenta of Capsicum fruits. Agric Biol Chem 46:25912592 Fung T, Jeffery W, Beveridge AD (1982) The identication of capsaicinoids in tear-gas spray. J Forensic Sci 27:812821 Gady AL, Hermans FW, Van de Wal MH, van Loo EN, Visser RG, Bachem CW (2009) Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations. Plant Methods 5:13 Gamse R, Lackner D, Gamse G, Leeman SE (1981) Effect of capsaicin pretreatment on capsaicin-evoked release of immunoreactive somatostatin and substance-P from primary sensory neurons. Naunyn Schmiedebergs Arch Pharmacol 316:3841 Gasson MJ, Kitamura Y, McLauchlan WR, Narband A, Parr AJ, Parsons ELH, Payne J, Rhodes MJC, Walton NJ (1998) Metabolism of ferulic acid to vanillin: a bacterial gene of the enoyl-SCoA hydratase/isomerase superfamily encodes an enzyme for the hydration and cleavage of hydroxycinnamic acid SCoA thioester. J Biol Chem 273:41634170 Govindarajan VS, Sathyanarayana MN (1991) Capsicum-production, technology, chemistry, and quality. V. Impact on physiology, pharmacology, nutrition, and metabolism; structure, pungency, pain, and desensitization sequences. Crit Rev Food Sci Nutr 29:435474 Gowri G, Bugos RC, Campbell WH, Maxwell CA, Dixon RA (1991) Stress responses in alfalfa (Medicago sativa L). 10. Molecularcloning and expression of S-adenosyl-L-methionine-caffeic acid 3-O-methyltransferase, a key enzyme of lignin biosynthesis. Plant Physiol 97:714 Harpster MH, Brummel DA, Dunsmuir P (2002) Supression of a ripening-related endo-1,4-b-glucanase in transgenic pepper fruit does not prevent depolymerization of cell wall polysaccharides during ripening. Plant Mol Biol 50:345355 Harvell KP, Bosland PW (1997) The environment produces a signicant effect on pungency of chiles. Hortscience 32: 12921292 Hautkappe M, Roizen MF, Toledano A, Roth S, Jeffries JA, Ostermeier AM (1998) Review of the effectiveness of capsaicin for painful cutaneous disorders and neural dysfunction. Clin J Pain 14:97106 Hoffmann L, Maury S, Martz F, Geoffroy P, Legrand M (2003) Purication, cloning, and properties of an acyltransferase

engineering, including the capsaicinoid biosynthesis pathway. Chili pepper tissue culture and genetic transformation protocols have been used for engineering some agriculturally important traits, such as virus resistance (Lee et al. 2004, 2009), but until now, no biosynthetic or regulatory genes have been manipulated by genetic engineering in Capsicum species (Kothari et al. 2010). Therefore, a basic knowledge of the genes involved in capsaicinoid biosynthesis and regulation should certainly ease the task of manipulating this metabolic pathway to produce chili pepper with specic levels of pungency.
Acknowledgments This work was supported by Conacyt (Mexico), project 55264. Aza-Gonzalez C. is a Conacyt (Mexico) graduate fellowship recipient.

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