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R E V I E W

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 capsa-icinoids 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 path-

ways, 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 identified candidate genes possibly involved in

capsaicinoid biosynthesis. Genetic and molecular approa-

ches have also contributed to the knowledge of this

biosynthetic pathway; however, more studies are necessary

for a better understanding of the regulatory process thataccounts for different accumulation levels of capsaicinoids

in chili pepper fruits.

Keywords Capsaicinoid biosynthesis Capsicum Chili

pepper

Abbreviations

DPA Days post-anthesis

ROS Reactive oxygen species

pAMT Putative aminotransferase

Introduction

Capsaicinoids are the substances responsible for the pun-

gent 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) syn-

thesized 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: [email protected]

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

123

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DOI 10.1007/s00299-010-0968-8


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fruits approximately 20 days post-anthesis (DPA) (Iwai

et al. 1979). Capsaicinoid biosynthesis occurs in the pla-

cental epidermis cells, where they are secreted towards the

outer cell wall, and finally 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 growthconditions. 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 bio-

synthesis and some possibilities for genetic manipulations

of this trait in the Capsicum genus.

Capsaicinoid uses

Chili pepper fruits are consumed fresh in salads (pimien-

tos) and salsas as ingredients of different dishes around the

world or even processed as pickles and salsas. Capsaici-

noids 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 ofdishes. The food industry widely uses capsaicinoids for

multiple purposes because they are the basic ingredi-

ents for salsas, curries and dressings, among other

foods (Perkins et al. 2002).

2. Pharmaceutical and medical. Capsaicinoids have been

found to exert a series of physiological and pharmaco-

logical effects, including analgesia, anticancer, anti-

inflammatory, antioxidative and anti-obesity activities

(Negulesco et al. 1987; Govindarajan and Sathyanara-

yana 1991; Luo et al. 2010; Liu and Nair 2010).

Capsaicinoids demonstrate anti-inflammatory activities

(Spiller et al. 2008); therefore, they are used as the main

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-O-

methyltransferase, COMT

caffeic acid O-methyl

transferase, HCHLhydroxycinnamoyl-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

[modified from Stewart et al.

(2005), and Mazourek et al.

(2009)]

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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, hyperreflexia 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

field 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).

Fig. 2 Structures of common

capsaicinoids (a) and capsinoids

(b)

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)

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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 capsaicininduces apoptosis in cancer cells is associated with

production of reactive oxygen species (ROS) and

disruption of the mitochondrial transmembrane poten-

tial 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). Addi-

tionally, in colon cancer cells treated with capsaicin,

the activity of caspase-3-activity, the major apoptosis-

executing enzyme, was substantially increased (Yang

et al. 2009).

3. Cosmetic and dietary. Capsaicinoids are also used asadditives in a series of hair-loss-prevention shampoos

currently found in the market.

4. 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 ofFusarium fungus, which is a major problemin

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 inheri-

tance studies have suggested that one single dominant

gene, named locus C, is responsible for the pungent char-

acteristic (Deshpande 1935).

The general capsaicinoid biosynthetic pathway was

established at the end of the 1960s, when radiotracer

studies were used to investigate capsaicinoid precursors,

finding 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 phenylal-

anine ammonia lyase (PAL), cinnamate 4-hydroxylase

(C4H), coumarate 3-hydroxylase (C3H) and caffeic acid

O-methyltransferase (COMT) in phenylpropanoid-medi-

ated capsaicinoid biosynthesis was established by several

authors (Fujiwake et al. 1982a, b; Sukrasno and Yeoman1993). 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), caffeoyl-

CoA 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 approa-

ches 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 tran-

scripts 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 con-

version 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 first

developmental stages in the chili pepper fruits with the

highest pungency. By using tissue-specific expression

analysis in fruits, it was also found that both the 3-keto-

acyl-ACP synthase (Kas) and the putative aminotransferase

(pAmt) sequences were only expressed at significant levels

in placental tissues, where the capsaicinoids weresynthesized.

With the aim of detecting genes involved in the bio-

synthesis of the fatty acid component, Aluru et al. (2003)

carried out a differential screen of a Habanero ( C. chin-

ense) 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 accumu-

lation occurred in the immature green placenta. Interest-ingly, 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, flowers 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

significantly increased fatty acid formation ([C8). Fur-

thermore, 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 sub-

epidermal 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 com-

pared 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 pla-

centa tissues. The cloned sequences were analyzed by

northern blot analysis, and some of them were specifically

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 suggestedthat 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 partic-

ipate 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 inclose proximity to Pun 1 (locus C), which modifies 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 deficient in a specific 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 non-

pungent chili peppers, showing that sequences from non-

pungent fruits have a 2.5-kb deletion, encompassing part of

the putative promoter and the first exon. That allele was

named pun1, and because the SB2-66 clone has acyl-

transferase 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

specifically 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 significantly reduced in non-pungent peppers. These

results suggested that At3 might participate in the regula-

tion of other capsaicinoid-related genes.

In order to demonstrate that At3 was related to capsa-

icinoid 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 con-

tradictory, 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 infiltration process. This behavior ofcapsaicinoid production in chili pepper plants was previ-

ously 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 specifically 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 approxi-

mately 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 pro-

posed that the SB2-66 clone was gene C, which is thought

to be responsible for chili pepper fruit pungency. More-

over, 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 syn-

thase 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 first

exon, and this allele was named pun12. Due to that dele-

tion, 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

specific enzymes and genes was proposed, and some tran-

scripts for those genes specifically 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 (phenyl-

propanoid 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 (Abraham-

Juarez 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-PCRand 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, specific-

siRNAs 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 accu-mulation 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 efficiently converted to vanillyl-

amine (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 vanillyl-

amine. 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 ofpAmtfrom 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 bio-

synthesis by regulating the phenylpropanoid precursors

channeled into this pathway.

As previously stated, the participation ofPal, C4h, Comt

and Amt (from the phenylpropanoid pathway; Fig. 1) in

capsaicinoid production, as well as the roles ofBCAT, 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 pro-

posed. Although the exact process by which the branched-

chain fatty acids are synthesized is not known, some

authors (Blum et al. 2003; Stewart et al. 2005) have sug-

gested that a desaturase converts 8-methylnonanoic acid

into 8-methyl-6-nonenoic acid. Nonetheless, 8-methyl-trans-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

modification 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 aninnovative branched-chain fatty acid biosynthesis pathway

where, in addition to isobutyryl-CoA, some other inter-

mediaries like acetyl-CoA, isovaleryl-CoA, anteisovaleryl-

CoA 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 Bos-

land 2000) (Table 1). For instance, Estrada et al. (1999)

observed that flooding-stressed chili pepper plants accu-

mulated more capsaicinoids than control plants, and that

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 field conditions (Jurenitsch et al. 1979). The

effects of light and temperature on capsaicinoid accumu-

lation have also been studied by Murakami et al. (2006).

These authors showed that chili pepper plants accumulatedmore capsaicinoids under continuous fluorescent 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 scientific evidence has

been obtained about the genes that regulate the biosyn-

thesis 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 clearevidence 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

BCATand 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 met-

abolic flux of precursors, or by being an important com-

ponent of a regulatory complex (Stewart et al. 2005).

Recently, it has been proposed that capsaicin might

function as a feedback inhibitor in the capsaicinoid bio-

synthetic 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 con-

centrations (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 identified 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

Table 1 Capsaicinoid content in several chili peppers [from Koz-

ukue et al. (2005), and Bosland and Baral (2007)]

Capsicum species Capsaicinoidsb

(lg/g fresh weight; f.wt.)

C. frutescens Bhut jolokiaa 6,2581

C. chinense cv. Habanero 2,260

C. frutescens Thai 1,332

C. annuum L. Serrano type 76

C. annuum L. Jalapeno type 75

C. annuum L. Green bell type 0

a In Bhut joloquia, a factor of 16 was used to convert SHU to lg/gb Since some data were reported as dry weight, a 90% of humidity

was considered to carry out the conversions

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variation in capsaicinoid accumulation; therefore, it was

proposed that the cap QTL could be a regulator of capsa-

icinoid biosynthesis or perhaps could correspond to an

unidentified structural gene. In a similar study, six QTLs

related to capsaicinoid accumulation were identified 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 observedvariation in pungency. Four out of those five 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 identified in this study might be an ortholog of the cap

QTL that has been previously mapped (Blum et al. 2003).

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 first 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 four-

base 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 base-

pair 1,291 in the pAMT gene that is responsible for a non-

sense recessive mutation that causes a loss of pungency and

an accumulation of capsinoids instead of capsaicinoids. A

derived cleaved amplified polymorphic sequence (dCAPS)

DNA marker was developed to detect homozygous reces-

sive 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.

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 capsaici-

noids, capsinoids are non-pungent and do not cause a

burning sensation, facilitating their application in human

medicine (Sasahara et al. 2010). Recent advances in thestudy 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 non-

pungent pepper, Capsicum annuum cv. CH-19, which

possesses a functional loss of pAMT (Lang et al. 2009;

Tanaka et al. 2010). Capsiate, dihydrocapsiate and nordi-

hydrocapsiate 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 sub-sequent loss of mitochondrial transmembrane potential

(Macho et al. 2003). Inhibition of angiogenesis and vas-

cular permeability by capsiate has been demonstrated by

Pyon et al. (2008). Moreover, capsinoids show antioxida-

tive (Rosa et al. 2002) and anti-inflammatory 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 findings create an opportunity to manipulate the

capsinoid metabolic pathway to over-accumulate these

secondary compounds and use them medicinally.

Future research

The phenylpropanoid and branched-chain fatty acid bio-

synthesis 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 bio-

synthesis 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 bio-

synthetic pathway (Stewart et al. 2007) several genes have

neither been isolated nor identified. 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 puta-

tive genes encoding 4CL, HCT, C3H and ACS have been

recently isolated (Mazourek et al. 2009), it is necessary to

demonstrate their specific roles in the capsaicinoid bio-

synthetic 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 Pun1encodes 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 significantly

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 andKas 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 gener-

ated (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 anthocya-

nins, transcription factor expression is highly correlated

with structural gene expression (Spelt et al. 2000; Borov-

sky et al. 2004; Espley et al. 2007); thus, this approach

might be used as an initial criterion for the identification of

capsaicinoid biosynthesis-related transcription factors.

Furthermore, Mazourek et al. (2009) have recently pro-

posed 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 phenyl-

propanoid-related and branched-chain fatty acid-related

pathways but had not been previously considered as par-

ticipants in those pathways. The predicted cellular locali-

zation 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 locali-

zation. The 42 sequences were genetically mapped in chili

pepper plants. This information opens the possibility of

considering the influence 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 poten-

tally involved in capsaicinoid biosynthesis and regulation,

it is necessary to expand the molecular analysis of non-

pungent 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 biosyn-

thesis. Furthermore, perhaps some Capsicum plants with

mutations at different capsaicinoid biosynthetic steps could

be generated by chemical mutagenesis and analyzed byTILLING (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 partici-

pation of some putative genes in capsaicinoid biosynthe-

sis have already been published (Stewart et al. 2005;

Abraham-Juarez et al. 2008). Silencing the HCT gene in Nicotiana benthamiana and Arabidopsis plants has dem-

onstrated 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 func-

tional 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 specific functions. Furthermore, complementing

mutants with known genes can demonstrate gene function in

plants. Chili pepper tissue culture and Agrobacterium-

mediated 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 efficiency for in vitro plant regeneration and trans-

formation 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 accumula-

tion. 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

Plant Cell Rep (2011) 30:695706 703

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engineering, including the capsaicinoid biosynthesis path-

way. Chili pepper tissue culture and genetic transformation

protocols have been used for engineering some agricul-

turally 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 biosyn-thesis and regulation should certainly ease the task of

manipulating this metabolic pathway to produce chili

pepper with specific levels of pungency.

Acknowledgments This work was supported by Conacyt (Mexico),

project 55264. Aza-Gonzalez C. is a Conacyt (Mexico) graduate

fellowship recipient.

References

Abraham-Juarez MD, Rocha-Granados MD, Lopez MG, Rivera-

Bustamante RF, Ochoa-Alejo N (2008) Virus-induced silencing

of Comt, pAmt and Kas genes results in a reduction of

capsaicinoid accumulation in chili pepper fruits. Planta 227:

681695

Aluru MR, Mazourek M, Landry LG, Curry J, Jahn M, OConnell

MA (2003) Differential expression of fatty acid synthase genes,

Acl, Fat and Kas, in Capsicum fruit. J Exp Bot 54:16551664

Andrews J (1995) Peppers: the domesticated capsicums, New edition.

University of Texas Press, Austin

Ben-Chaim A, Borovsky Y, Falise M, Mazourek M, Kang BC, Paran

I, Jahn M (2006) QTL analysis for capsaicinoid content in

Capsicum. Theor Appl Genet 113:14811490

Bennett DJ, Kirby GW (1968) Constitution and biosynthesis of

capsaicin. J Chem Soc C 4:442446

Blum E, Liu K, Mazourek M, Yoo EY, Jahn M, Paran I (2002)

Molecular mapping of the C locus for presence of pungency in

Capsicum. Genome 45:702705

Blum E, Mazourek M, OConnell M, Curry J, Thorup T, Liu KD,

Jahn M, Paran I (2003) Molecular mapping of capsaicinoid

biosynthesis genes and quantitative trait loci analysis for

capsaicinoid content in Capsicum. Theor Appl Genet 108:7986

Borovsky Y, Oren-Shamir M, Ovadia R, De Jong W, Paran I (2004)

The A locus that controls anthocyanin accumulation in pepper

encodes a MYB transcription factor homologous to Anthocya-

nin2 of Petunia. Theor Appl Genet 109:2329

Bosland PW (1996) Capsicums: innovative uses of an ancient crop.

In: Janick J (ed) Progress in new crops. ASHS Press, Arlington,

pp 479487

Bosland PW, Baral JB (2007) Bhut Jolokiathe worlds hottest

known chile pepper is a putative naturally occurring interspecifichybrid. Hortscience 42:222224

Choi SH, Suh BS, Kozukue E, Kozukue N, Levin CE, Friedman M

(2006) Analysis of the contents of pungent compounds in fresh

Korean red peppers and in pepper-containing foods. J Agric

Food Chem 54:90249031

Cordell GA, Araujo OE (1993) Capsaicin: identification, nomencla-

ture, and pharmacotherapy. Ann Pharmacother 27:330336

Curry J, Aluru M, Mendoza M, Nevarez J, Melendrez M, OConnell

MA (1999) Transcripts for possible capsaicinoid biosynthetic

genes are differentially accumulated in pungent and non-pungent

Capsicum spp. Plant Sci 148:4757

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 elicitor-

inducible 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 identification 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 immuno-

reactive 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. Molecular-

cloning 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 fruitdoes not prevent depolymerization of cell wall polysaccharides

during ripening. Plant Mol Biol 50:345355

Harvell KP, Bosland PW (1997) The environment produces a

significant 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)

Purification, cloning, and properties of an acyltransferase

704 Plant Cell Rep (2011) 30:695706

123


11/12

controlling shikimate and quinate ester intermediates in phenyl-

propanoid metabolism. J Biol Chem 278:95103

Horffmann L, Besseau S, Geoffroy P, Ritzenthaler C, Meyer D,

Lapierre C, Pollet B, Legrand M (2004) Silencing of hydro-

xycinnamoyl-Coenzyme A shikimate/quinate hydroxycinna-

moyltransferase affects phenylpropanoid biosynthesis. Plant

Cell 16:14461465

Inoue N, Matsunaga Y, Sato H, Takahashi M (2007) Enhanced energy

expenditure and fat oxidation in humans with high BMI scores

by the ingestion of novel and non-pungent capsaicin analogues

(capsinoids). Biosci Biotechnol Biochem 71:380389

Ito K, Nakazato T, Yamato K, Miyakawa Y, Yamada T, Hozumi N,

Segawa K, Ikeda Y, Kizaki M (2004) Induction of apoptosis in

leukemic cells by homovanillic acid derivative, capsaicin,

through oxidative stress: implication of phosphorylation of p53

at Ser-15 residue by reactive oxygen species. Cancer Res

64:10711078

Iwai K, Suzuki T, Fujiwake H (1979) Formation and accumulation of

pungent principle of hot pepper fruits, capsaicin and its analogs,

in Capsicum annuun var. annuum cv. Karayatsubusa at different

growth-stages after flowering. Agric Biol Chem 43:24932498

Jurenitsch J, Kubelka W, Jentzsch K (1979) Identification of

cultivated taxa of Capsicum taxonomy, anatomy and composi-

tion of pungent principle. Planta Med 35:174183

Kim JD, Kim JM, Pyo JO, Kim SY, Kim BS, Yu R, Han IS (1997)

Capsaicin can alter the expression of tumor forming-related

genes which might be followed by induction of apoptosis of a

Korean stomach cancer cell line, SNU-1. Cancer Lett

120:235241

Kim M, Kim S, Kim S, Kim BD (2001) Isolation of cDNA clones

differentially accumulated in the placenta of pungent pepper by

suppression subtractive hybridization. Mol Cells 11:213219

Kim JS, Park M, Lee DJ, Kim BD (2009) Characterization of putative

capsaicin synthase promoter activity. Mol Cells 28:331339

Kobata K, Todo T, Yazawa S, Iwai K, Watanabe T (1998) Novel

capsaicinoid-like substances, capsiate and dihydrocapsiate, from

the fruits of a nonpungent cultivar, CH-19 sweet, of pepper

(Capsicum annuum L.). J Agric Food Chem 46:16951697

Kobata K, Sutoh K, Todo T, Yazawa S, Iway K, Watanabe T (1999)

Nordihydrocapsiate, a new capsinoid from the fruits of a non-

pungent pepper, Capsicum annuum. J Nat Prod 62:335336

Kobata K, Kawaguchi M, Watanabe T (2002) Enzymatic synthesis of

a capsinoid by the acylation of vanillyl alcohol with fatty acid

derivatives catalyzed by lipases. Biosci Biotechnol Biochem

66:319327

Kothari SL, Joshi A, Kachhwaha S, Ochoa-Alejo N (2010) Chilli

peppersa review on tissue culture and transgenesis. Biotechnol

Rev 28:3548

Kozukue N, Han JS, Kozukue E, Lee SJ, Kim JA, Lee KR, Levin CE,

Friedman M (2005) Analysis of eight capsaicinoids in peppers

and pepper-containing foods by high-performance liquid chro-

matography and liquid chromatography-mass spectrometry.

J Agric Food Chem 53:91729181

Lang YQ, Kisaka H, Sugiyama R, Nomura K, Morita A, Watanabe T,Tanaka Y, Yazawa S, Miwa T (2009) Functional loss of pAMT

results in biosynthesis of capsinoids, capsaicinoid analogs, in

Capsicum annuum cv. CH-19 Sweet. Plant J 59:953961

Lee YH, Kim HS, Kim JY, Jung M, Park YS, Lee JS, Choi SH, Her

NH, Lee JH, Hyung NI, Lee CH, Yang SG, Harn CH (2004) A

new selection method for pepper transformation: callus-mediated

shoot formation. Plant Cell Rep 23:5058

Lee CJ, Yoo EY, Shin J, Lee J, Hwang HS, Kim BD (2005) Non-

pungent Capsicum contains a deletion in the capsaicinoid

synthetase gene, which allows early detection of pungency with

SCAR markers. Mol Cells 19:262267

Lee YH, Jung M, Shin SH, Lee JH, Choi SH, Her NH, Lee JH, Ryu

KH, Paek KY, Harn CH (2009) Transgenic peppers that are

highly tolerant to a new CMV pathotype. Plant Cell Rep

28:223232

Leete E, Louden MCL (1968) Biosynthesis of capsaicin and

dihydrocapsaicin in Capsicum frutescens. J Am Chem Soc

90:68376841

Liu Y, Nair MG (2010) Capsaicinoids in the hottest pepper Bhut

Jolokia and its antioxidant and antiinflammatory activities. Nat

Prod Commun 5:9194

Luo X-J, Peng J, Li Y-J (2010) Recent advances in the study of

capsaicinoids and capsinoids. Eur J Pharmacol. doi:

10.1016/j.ejphar.2010.09.074

Macho A, Lucena C, Sancho R, Daddario N, Minassi A, Munoz E,

Appendino G (2003) Non-pungent capsaicinoids from sweet

peppersynthesis and evaluation of the chemopreventive and

anticancer potential. Eur J Nutr 42:29

Marabini S, Ciabatti PG, Polli G, Fusco BM, Geppetti P (1991)

Beneficial-effects of intranasal applications of capsaicin in

patients with vasomotor rhinitis. Eur Arch Oto Rhino Laryngol

248:191194

Mazourek M, Pujar A, Borovsky Y, Paran I, Mueller L, Jahn MM

(2009) A dynamic interface for capsaicinoid systems biology.

Plant Physiol 150:18061821

Merali Z, Mayer MJ, Parker ML, Michael AJ, Smith AC, Waldron

KW (2007) Metabolic diversion of the phenylpropanoid pathway

causes cell wall and morphological changes in transgenic

tobacco stems. Planta 225:11651178

Minoia S, Petrozza A, DOnofrio O, Piron F, Mosca G, Sozio G,

Cellini F, Bendahmane A, Carriero F (2010) A new mutant

genetic resource for tomato crop improvement by TILLING

technology. BMC Res Notes 3:69

Mori A, Lehmann S, OKelly J, Kumagai T, Desmond JC, Pervan M,

McBride WH, Kizaki M, Koeffler HP (2006) Capsaicin, a

component of red peppers, inhibits the growth of androgen-

independent, p53 mutant prostate cancer cells. Cancer Res

66:32223229

Murakami K, Ido M, Masuda M (2006) Fruit pungency of Shishito

pepper as affected by a dark interval in continuous fluorescent

illumination with temperature alteration. J Soc High Tech Agric

18:284289

Negulesco JA, Noel SA, Newman HA, Naber EC, Bhat HB, Witial

DT (1987) Effects of pure capsaicinoids (capsaicin and dihy-

drocapsaicin) on plasma lipid and lipoprotein concentrations of

turkey. Atherosclerosis 64:8590

Nelson EK (1919) The constitution of capsaicin, the pungent principle

of Capsicum. J Am Chem Soc 41:11151121

Ochoa-Alejo N, Ramrez-Malagon R (2001) In vitro chili pepper

biotechnology. In Vitro Cell Dev Biol Plant 37:701729

Ohnuki K, Haramizu S, Oki K, Watanabe T, Yazawa S, Fushiki T

(2001) Administration of capsiate, a non-pungent capsaicin

analog, promotes energy metabolism and suppresses body fat

accumulation in mice. Biosci Biotechnol Biochem 65:2735

2740Perkins B, Bushway R, Guthrie K, Fan T, Stewart B, Prince A,

Williams M (2002) Determination of capsaicinoids in salsa by

liquid chromatography and enzyme immunoassay. J AOAC Int

85:8285

Pyon B-J, Choi S, Lee Y, Kim T-W, Min J-K, Kim Y, Kim B-D, Kim

J-H, Kim T-Y, Kim Y-M, Kwon Y-G (2008) Capsiate, a non-

pungent capsaicin-like compound, inhibits angiogenesis and

vascular permeability via direct inhibition of Src kinase activity.

Cancer Res 68:227235

Rains C, Bryson HM (1995) Topical capsaicin- a review of its

pharmacological properties and therapeutic potential in

Plant Cell Rep (2011) 30:695706 705

123
http://dx.doi.org/10.1016/j.ejphar.2010.09.074http://dx.doi.org/10.1016/j.ejphar.2010.09.074


12/12

post-herpetic neuralgia, diabetic neuropathy and osteoarthritis.

Drug Aging 7:317328

Reilly CA, Crouc DJ, Yost GS, Fatah AA (2001) Determination of

capsaicin, dihydrocapsaicin, and nonivamide in self-defense

weapons by liquid chromatography-mass spectrometry and

liquid chromatography-tandem mass spectrometry. J Chromatogr

A 912:259267

Robbins W (2000) Clinical applications of capsaicinoids. Clin J Pain

16:S86S89

Rosa A, Deiana M, Casu V, Paccagnini S, Appendino G, Ballero M,

Dessi MA (2002) Antioxidant activity of capsinoids. J Agric

Food Chem 50:73967401

Sanchez AM, Sanchez MG, Malagarie-Cazenave S, Olea N, Daz-

Laviada I (2006) Induction of apoptosis in prostate tumor PC-3

cells and inhibition of xenograft prostate tumor growth by the

vanilloid capsaicin. Apoptosis 11:8999

Sancho R, Lucena C, Macho A, Calzado MA, Blanco-Molina M,

Minassi A, Appendino G, Munoz E (2002) Immunosuppressive

activity of capsaicinoids: capsiate derived from sweet peppers

inhibits NF-kappaB activation and is a potent anti-inflammatory

compound in vivo. Eur J Immunol 32:17531763

Sasahara I, Furuhata Y, Iwasaki Y, Inoue N, Sato H, Watanabe T,

Takahashi M (2010) Assessment of the biological similarity of

three capsaicin analogs (capsinoids) found in non-pungent chili

pepper (CH-19 Sweet) fruits. Biosci Biotechnol Biochem

74:274278

Singh S, Jarret R, Russo V, Majetich G, Shimkus J, Bushway R,

Perkins B (2009) Determination of capsinoids by HPLC-DAD in

Capsicum species. J Agric Food Chem 57:34523457

Snitker S, Fujishima Y, Shen H, Ott S, Pi-Sunyer X, Furuhata Y, Sato

H, Takahasshi M (2009) Effects of novel capsinoid treatment on

fatness and energy metabolism in humans: possible pharmaco-

genetic implications. Am J Clin Nutr 89:4550

Spelt C, Quattrocchio F, Mol JNM, Koes R (2000) Anthocyanin1 of

petunia encodes a basic helix-loop-helix protein that directly

activates transcription of structural anthocyanin genes. Plant Cell

12:16191631

Spiller F, Alves MK, Vieira SM, Carvalho TA, Leite CE, Lunardelli

A, Poloni JA, Cunha FQ, de Oliveira JR (2008) Anti-inflamma-

tory effects of red pepper (Capsicum baccatum) on carrageenan-

and antigen-induced inflammation. J Pharm Pharmacol

60:473478

Stewart C, Kang BC, Liu K, Mazourek M, Moore SL, Yoo EY, Kim

BD, Paran I, Jahn MM (2005) The Pun1 gene for pungency in

pepper encodes a putative acyltransferase. Plant J 42:675688

Stewart C, Mazourek M, Stellari GM, OConnell M, Jahn M (2007)

Genetic control of pungency in C. chinense via the Pun1 locus.

J Exp Bot 58:979991

Sukrasno N, Yeoman MM (1993) Phenylpropanoid metabolism

during growth and development of Capsicum frutescens fruits.

Phytochemistry 32:839844

Surh Y-J (2002) More than spice: capsaicin in hot chili peppers makes

tumor cells commit suicide. J Natl Cancer Inst 94:12631265

Sutoh K, Kobata K, Yazawa S, Watanabe T (2006) Capsinoid is

biosynthesized from phenylalanine and valine in a non-pungent

pepper, Capsicum annuum L. cv. CH-19 Sweet. Biosci Biotech-

nol Biochem 70:15131516

Suzuki T, Fujiwake H, Iwai K (1980) Formation and metabolism of

pungent principle of Capsicum fruits. 5. Intracellular-localiza-

tion of capsaicin and its analogs, capsaicinoid, in Capsicum fruit.

1. Microscopic investigation of the structure of the placenta of

Capsicum annuum var. annuum cv. Karayatsubusa. Plant Cell

Physiol 21:839853

Suzuki T, Kawada T, Iwai K (1981) Formation and metabolism of

pungent principle of Capsicum fruits. 9. Biosynthesis of acyl

moieties of capsaicin and its analogs from valine and leucine in

Capsicum fruits. Plant Cell Physiol 22:2332

Tanaka Y, Hosokawa M, Miwa T, Watanabe T, Yazawa S (2010)

Newly mutated putative-aminotransferase in nonpungent pepper

(Capsicum annuum) results in biosynthesis of capsinoids,

capsaicinoid analogues. J Agric Food Chem 58:17611767

Tewksbury JJ, Reagan KM, Machnicki NJ, Carlo TA, Haak DC,

Penaloza ALC, Levey DJ (2008) Evolutionary ecology of

pungency in wild chilies. Proc Natl Acad Sci USA 105:

1180811811

Thiele R, Mueller-Seitz E, Petz M (2008) Chili pepper fruits:

presumed precursors of fatty acids characteristic for capsaici-

noids. J Agric Food Chem 56:42194224

Xing FB, Cheng GX, Yi KK (2006) Study on the antimicrobial

activities of the capsaicin microcapsules. J Appl Polym Sci

102:13181321

Yang KM, Pyo JO, Kim GY, Yu R, Han IS, Ju SA, Kim WH, Kim BS

(2009) Capsaicin induces apoptosis by generating reactive

oxygen species and disrupting mitochondrial transmembrane

potential in human colon cancer cell lines. Cell Mol Biol Lett

14:497510

Zewdie Y, Bosland PW (2000) Evaluation of genotype, environment,

and genotype-by-environment interaction for capsaicinoids in

Capsicum annuum L. Euphytica 111:185190

706 Plant Cell Rep (2011) 30:695706

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