capsaicin in peppers 200405_1 will use
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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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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
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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.
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