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Plant Physiology Preview. Published on May 8, 2017, as DOI:10.1104/pp.17.00506
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Running head:
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An R2R3-MYB gene regulates pungency in chili pepper
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Corresponding author:
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Neftalí Ochoa-Alejo
Mailing address: Departamento de Ingeniería Genética de Plantas, Centro de
Investigación y de Estudios Avanzados del Instituto Politécnico Nacional
(Cinvestav-Unidad Irapuato). Km. 9.6 Libramiento Norte Carretera Irapuato-León;
Apartado Postal 629; 36821-Irapuato, Gto., México.
Phone: (+52) 462 623 9654
Fax: (+52) 462 624 5849
E-mail: [email protected]
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An R2R3-MYB Transcription Factor in Capsaicinoid Biosynthesis
Magda L. Arce-Rodríguez1 and Neftalí Ochoa-Alejo1, 2
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Departamento de Ingeniería Genética de Plantas1, and Departamento de
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Biotecnología y Bioquímica2, Centro de Investigación y de Estudios Avanzados del
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Instituto Politécnico Nacional, Unidad Irapuato, Km 9.6 Libramiento Norte
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Carretera Irapuato-León, 36821, Irapuato, Guanajuato, México.
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One sentence summary:
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Copyright 2017 by the American Society of Plant Biologists
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CaMYB31, an R2R3-MYB transcription factor, regulates the capsaicinoid
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biosynthetic pathway in chili pepper fruits, and is regulated by plant hormones,
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wounding, temperature and light.
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Financial source:
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The National Council of Science and Technology (Conacyt, Mexico) financially
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supported this research, through the project 177063, and an MLA-R scholarship.
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Corresponding author e-mail: [email protected]
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The author responsible for distribution of materials integral to the findings
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presented in this article in accordance with the policy described in the Instructions
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for
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([email protected]).
Authors
(www.plantphysiol.org)
is:
Neftalí
Ochoa-Alejo
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Author´s contributions:
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MLA-R. designed and carried out the experimental work, analyzed the data and
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wrote the manuscript. N.O-A. designed, directed and supervised the experiments,
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and complemented and corrected the article.
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Capsaicinoids are responsible for the hot taste of chili peppers. They are restricted
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to the genus Capsicum and are synthesized by the acylation of the aromatic
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compound vanillylamine (derived from the phenylpropanoid pathway) with a
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branched-chain fatty acid by the catalysis of the putative enzyme capsaicinoid
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synthase. R2R3-MYB transcription factors have been reported in different species
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of plants as regulators of structural genes of the phenylpropanoid pathway;
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therefore, we hypothesized that MYB genes might be involved in the regulation of
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the biosynthesis of pungent compounds. In this study, an R2R3-MYB transcription
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factor gene, designated CaMYB31, was isolated and characterized in Capsicum
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annuum L. cv. Tampiqueño 74. Bioinformatic analysis suggested that CaMYB31
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could be involved in secondary metabolism, stress and plant hormone responses,
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and development. CaMYB31 expression analysis from placental tissue of pungent
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and non-pungent chili pepper fruits showed a correlation positive with the structural
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genes Ca4H, Comt, Kas, pAmt and AT3 expression and also with the content of
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capsaicin and dihydrocapsacin during fruit development. However, CaMYB31 was
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also expressed in vegetative tissues (leaves, roots and stems). Moreover,
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CaMYB31
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biosynthetic genes and the capsaicinoid content. Additionally, CaMYB31
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expression was affected by the plant hormones indoleacetic acid, jasmonic acid,
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salicylic acid and gibberellic acid or by wounding, temperature and light, factors
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known to affect the production of capsaicinoids. These findings indicate that
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CaMYB31 is indeed involved in the regulation of structural genes of the
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capsaicinoid biosynthetic pathway.
silencing
significantly
reduced
the
expression
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of
capsaicinoid
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INTRODUCTION
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Chili peppers are members of the Capsicum genus, which belongs to the
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Solanaceae family. Capsicum annuum is the most cultivated species in the world.
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The importance of this crop is based on a great variety of industrial applications,
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such as food, pharmaceutical, cosmetic and agronomic uses (Ochoa-Alejo and
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Ramírez-Malagón, 2001). One of the most valued compounds of chili pepper fruits
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are the capsaicinoids, which are synthesized and accumulated in the placental
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tissue through the convergence of phenylpropanoid and the branched-chain fatty
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acid
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dihydrocapsaicin are responsible for 90% of the pungency in the fruit, and their
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accumulation depends on the genotype, stage of fruit development and
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environmental conditions (Lindsey and Bosland, 1996; Kim et al., 2009; Arce-
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Rodríguez and Ochoa-Alejo, 2015).
pathways
(Aza-González
et
al.,
2011)
(Fig.
1).
Capsaicin
and
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Research on the capsaicinoid biosynthesis pathway has uncovered key
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aspects of its biochemistry and molecular biology (Aza-González et al., 2011). The
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generation of chili pepper cDNA libraries and comparative gene expression
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analysis of pungent and non-pungent fruits has allowed the identification of several
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structural genes of the capsaicinoid biosynthesis pathway, such as phenylalanine
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ammonia-lyase (Pal), cinnamic acid 4-hydroxylase (Ca4H), 4-coumarate-CoA
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ligase
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methyltransferase (Comt), hydroxycinnamoyl-CoA hydratase/ligase (HCHL) and a
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putative acyltransferase (AT3) proposed to be the possible capsaicinoid synthase
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(CS) (Curry et al., 1999; Kim et al., 2001; Stewart et al., 2005; Mazourek et al.,
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2009; Liu et al, 2013).
(4CL),
hydroxycinnamoyl
transferase
(HCT),
caffeic
acid
O-
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The role of transcription factors in the capsaicinoid biosynthesis regulation is
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underexplored. Stewart et al. (2005) reported a differential expression analysis of
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two bZIP (Basic Leucine Zipper) transcription factors in pungent and non-pungent
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fruits at different developmental stages, but their expression pattern did not
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correlate positively with the expression of the structural genes Pal, Ca4H, Comt,
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pAMT, BCAT, Kas, Acl, FatA and AT3. Recently, Keyhaninejad et al. (2014)
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reported two ERF (Ethylene Response Factor) transcription factors whose
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expression correlated positively with the pungency level in nine chili pepper
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cultivars and they were proposed as possible regulators of capsaicinoid
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biosynthesis.
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Previous studies have shown that the phenylpropanoid pathway is regulated
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by R2R3-MYB transcription factors in different plant species such as Arabidopsis
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thaliana, Antirrhinum majus, and Pinus taeda (Tamagnone et al., 1998; Patzlaff et
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al., 2003; Zhou et al., 2009). R2R3-MYB transcription factors represent one of the
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largest family in plants and regulate different biological processes such as primary
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and
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developmental processes, and hormonal responses (Dubos et al., 2010; Ambawat
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et al., 2013; Liu et al., 2015). Since the phenylpropanoid pathway is involved in the
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biosynthesis of precursors of capsaicinoid biosynthetic pathway, we hypothesized
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that the R2R3-MYB family transcription factors participate in the biosynthesis of
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capsaicinoids. Here, we report on the isolation of a CaMYB31 cDNA, which
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encodes a putative R2R3-MYB protein, and its role in the regulation of capsaicinoid
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biosynthetic genes and in the accumulation of capsaicinoids in chili pepper fruits.
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Furthermore, we characterized CaMYB31 regarding its gene organization,
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phylogeny, function and its responses to hormones, light, temperature and
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wounding.
secondary
metabolism,
responses
to
biotic
and
abiotic
stresses,
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RESULTS
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Identification of CaMYB31 by Differential Gene Expression in Placental
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Tissue from Pungent and Non-Pungent Fruits
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The accumulation of capsaicin and dihydrocapsaicin in placental tissue of
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chili pepper fruits of C. annuum cv. Tampiqueño 74 (pungent) and cv. California
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Wonder (non-pungent) during fruit development is presented in Table I. Capsaicin
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content was twice as high as dihydrocapsaicin in ‘Tampiqueño 74’ fruits, but these
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two capsaicinoids were not detected in California Wonder. In ‘Tampiqueño 74’,
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capsaicinoids were not recorded at 10 days post-anthesis (DPA), began
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accumulating at 20 DPA, peaked at 30-40 DPA, and decreased at 50-60 DPA.
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The expression of Ca4H, Comt, pAmt, AT3 and Kas genes was positively
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correlated with capsaicinoid accumulation in ‘Tampiqueño 74’ placental tissue. The
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expression of pAmt, Kas, AT3 and Comt was very low or even undetectable at 10
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DPA, increased to a maximum between 30 and 40 DPA, decreased at 50 DPA,
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and was very low or undetectable at 60 DPA. Ca4H was expressed moderately at
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10 DPA, increased to a maximum between 20 and 40 DPA, and then diminished
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slightly between 50 and 60 DPA. In California Wonder, the expression of AT3 was
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undetectable at all stages of fruit development, while that of Kas and pAmt was
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very low at 30-50 DPA. However, Comt and Ca4H were expressed at all stages of
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the non-pungent fruits (Fig. 2).
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Partial MYB sequences were identified from a cDNA library of C. annuum
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cv. Tampiqueño 74. Only sequences that showed >40% homology and that
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expressed in pungent placental tissue were selected for qRT-PCR analysis (see
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Supplemental Figure 1). CaMYB31 showed a clear expression pattern that
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correlated positively with both the expression levels of the capsaicinoid
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biosynthetic
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development (Fig. 2).
structural
genes
and
with
capsaicinoid
content
during
fruit
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CaMYB31 Structure and Phylogenetic Sequence Analyses
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CaMYB31 contains a 747 bp reading frame encoding a putative R2R3-MYB
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protein of 249 amino acids that is preceded by a 5’ UTR of 148 bp and followed by
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a 3’ UTR of 189 bp. CaMYB31 cDNA sequence was compared to the chili pepper
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genome database (Qin et al., 2014) and three exons of 133, 130 and 484 bp, and
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two introns were identified. The first intron of 724 bp was localized between aa 44
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and 45 of the R2 domain, and the second one of 425 bp was between aa 88 and
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89 of the R3 domain (Fig. 3A).
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A phylogenetic tree of 167 plant MYB proteins was constructed using the
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neighbor joining method, based on alignment of the MYB domain, with a bootstrap
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test (n = 1000) (Fig. 3B). The full-length amino acid sequence of CaMYB31 was
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used to look for MYB proteins from RefSeq and Swiss-Prot NCBI protein database,
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and the MYB Arabidopsis proteins were also included (See Supplemental Table II).
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The phylogenetic analysis was congruent with the subgroups defined for
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Arabidopsis MYB proteins (Kranz 1998; Stracke et al., 2001).
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According to our phylogenetic tree, CaMYB31 belongs to a cluster of MYB
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Solanaceae proteins with no assigned biological functions at this time
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(XP_016580728.1 with 98% identity to XP_015081732.1 with 67% identity) (Fig.
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3B). The nearest neighbor clade to CaMYB31 was the subgroup 14 of Arabidopsis
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thaliana proteins (AtMYB36 with 63% identity to AtMYB68 with 46% identity), which
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are mainly related to development, while some of them are implicated in hormone
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response, stress response and in the regulation of the lignin biosynthesis (Fig. 3B).
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We also constructed the phylogenetic tree using the IT3F software online (Bailey et
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al., 2008) (see Supplemental Fig. 2) and the results were consistent with our
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phylogenetic analysis.
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CaMYB31 alignment with other MYB plant regulatory proteins showed a
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highly conserved R2R3 MYB domain in the N-terminal region, and a highly variable
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C-terminal region (see Supplemental Fig. 3A). As a complement to the
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phylogenetic analysis, we analyzed the C-terminal region of CaMYB31 by
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comparison with the nearest neighbor clade to search for conserved motifs using
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MEME motif-detection software, thinking that such motif might be important for the
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function of CaMYB31. The C-terminal region of CaMYB31 share one motif with
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these proteins related to development (see Supplemental Fig. 3B). Moreover,
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PlantPAN 2.0 (Chow et al., 2016) was used as a database for signal scanning of
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MYB binding sites in the putative promoter sequences of the structural
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capsaicinoid genes, and we found MYB cis-elements on all predictive promoters of
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the capsaicinoid structural genes (see Supplemental Fig. 4).
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Differential CaMYB31 Expression Analyses in Different Organ Tissues of
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Capsicum spp.
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The expression of CaMYB31 and that of the structural genes Kas, pAmt,
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Comt and Ca4H were analyzed by qRT-PCR in different tissues of the fruit (seeds,
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pericarp and placenta) and plant (roots, stems, flowers and leaves) from pungent
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and non-pungent cultivars. Kas and pAmt genes expression was detected
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exclusively in placental tissue (Fig. 4), although transcript levels were much higher
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in placental tissue from the immature fruits of Habanero than from ‘Tampiqueño
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74’, which correlated with the pungency level, whereas Comt and Ca4H were also
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expressed in seeds, pericarp and vegetative tissues (Fig 4).
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CaMYB31 showed the highest expression in placental tissue of Habanero
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immature fruits, which are considered highly hot (Fig. 4). Moreover, the
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characteristic expression pattern of CaMYB31 during the development of
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‘Tampiqueño 74’ fruits was confirmed. The CaMYB31 expression observed in
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placental tissue of California Wonder fruits was much lower. CaMYB31 presented
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very low expression in the seeds and pericarp.
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In the case of vegetative tissues, CaMYB31 expressed at very low levels in
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flower tissues of ‘Tampiqueño 74’ and was undetectable in flowers of California
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Wonder (Fig. 4). However, CaMYB31 was expressed moderately in roots and
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highly in leaf and stem tissues when compared to the placenta of ‘Tampiqueño 74’
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chili pepper fruits.
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Effect of CaMYB31 Silencing on the Expression of Capsaicinoid Biosynthetic
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Genes and on Capsaicinoid Content
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CaMYB31 silencing experiments were carried out using a Tobacco rattle
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virus (TRV)-derived vector to generate the construct pTRV2-CaMYB31, which
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contained a 234 bp region from 90 aa of the R3 domain towards the C-terminal
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region (Fig. 3A). Fruits of 40 DPA were collected to investigate the effect of
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CaMYB31 silencing.
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Fruits from plants infected with the pTRV2-CaMYB31 construct exhibited a
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significant diminution of the CaMYB31 gene expression (82%) compared to
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uninfected plants (Fig. 5A). CaMYB31 silencing caused a significant reduction in
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the expression of the capsaicinoid biosynthetic genes Ca4H (81.8%), 4CL (80.6%),
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C3H (76.4%), HCT (49%), Comt (42.7%), pAmt (71%), BCAT (88.6%), BCKDH
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(48.5%), Kas (70%) and Acl (30.3%), compared to fruits from uninfected plants
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(Fig. 5A). It has been previously shown that agroinfection with the empty pTRV2
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vector did not cause statistically significant changes in the expression patterns of
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tested structural genes in chili pepper fruits (Arce-Rodríguez and Ochoa-Alejo,
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2015).
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Capsaicin and dihydrocapsaicin contents in fruits of plants agroinfected with
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pTRV2-CaMYB31 showed a significant reduction of 74.2 and 73%, respectively,
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compared with fruits from uninfected plants (Fig. 5B). Fruits from plants
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agroinfected with the empty pTRV2 vector exhibited capsaicin content statistically
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similar to that of uninfected plants; however, dihydrocapsaicin content was
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significantly higher. Therefore, fruits from plants agroinfected with pTRV2-
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CaMYB31 had an 82.4% decrease in capsaicin and dihydrocapsaicin compared to
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fruits from plants infected with the empty pTRV2 vector (Fig. 5B).
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Effect of Plant Hormones, Light, Temperature and Wounding on the
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Expression of CaMYB31 and Capsaicinoid Biosynthesis Structural Marker
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Genes
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Environmental stimuli and some plant hormones affect capsaicinoid
254
accumulation in chili pepper fruits (Kim et al., 2009; Gutiérrez-Carbajal et al.,
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2010). In order to test whether these factors regulate the CaMYB31 expression,
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several physical stimuli and plant hormones were applied to 30 DPA fruits of
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Serrano ‘Tampiqueño 74’. The expression of CaMYB31 and the capsaicinoid
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biosynthetic gene markers Kas and pAmt were analyzed in placental tissue by
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qRT-PCR.
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Fruits were treated with light or dark conditions with varying exposure time,
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and the transcript levels were examined. Dark exposure caused a significant
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diminution on CaMYB31 expression similar to the expression pattern for Kas and
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pAmt (Fig. 6). Temperature was another tested stimulus; we monitored gene
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expression at high temperature (37°C), low temperature (4°C) and standard growth
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temperature (25°C) at different exposure times. CaMYB31, Kas and pAmt
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expression at 37°C was significantly lower, while that in fruits at 4°C was higher
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compared to standard growth conditions (Fig. 7). The expression of CaMYB31,
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Kas and pAmt in wounded fruits had a significant decrease when compared to non-
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wounded fruits (Fig. 8).
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In addition, the transcript levels of CaMYB31 in fruits treated with 100 µM of
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jasmonic acid (JA), salicylic acid (SA), gibberellic acid (GA3) or indoleacetic acid
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(IAA) compared to control fruits (treated with MS) were determined. CaMYB31,
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Kas and pAmt expression increased significantly by IAA, SA and GA3 treatment,
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whereas a significant diminution by JA treatment was detected, except at 3 h of
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exposure (Fig. 9). The effect of these treatments was dependent on the time of
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exposure. In general, CaMYB31 expression correlated with the expression of Kas
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and pAmt under exposure to different environmental stimulus.
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DISCUSSION
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The generation of chili pepper cDNA libraries and comparative gene
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expression analysis of pungent (Serrano cv. Tampiqueño 74) and non-pungent
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fruits (Bell pepper cv. California Wonder) allowed us to identify CaMYB31, an
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R2R3-MYB transcription factor gene, as a strong candidate to regulate the
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capsaicinoid pathway.
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In previous studies, the positive correlation between the expression pattern
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of structural genes (Pal, Ca4H, Comt, Acl, Fat, Kas and AT3) of the capsaicinoid
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biosynthetic pathway and the level of pungency of chili pepper fruits has been
289
reported (Curry et al., 1999; Aluru et al., 2003, Arce-Rodríguez and Ochoa-Alejo,
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2015). It has been shown that capsaicinoid accumulation is dependent on the
291
developmental stage of chili pepper fruits, and this synthesis and accumulation is
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highly likely to be regulated and coordinated at the transcriptional level.
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Capsaicinoid Content and Expression of Genes Involved in the Capsaicinoid
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Pathway in Chili Pepper Fruits
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A positive correlation was observed between the expression pattern of the
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structural genes Kas, pAmt and AT3 and the pungency level during the
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development of Serrano and Bell pepper fruits, indicating that these are specific
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genes of the capsaicinoid pathway. On the other hand, Comt and Ca4H genes
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were also expressed in non-pungent fruit and in vegetative tissues, suggesting that
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they participate in other biological process, such as lignin biosynthesis (Humphreys
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and Chapple, 2002). CaMYB31 showed an expression pattern similar to that of the
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Kas, pAmt and AT3 genes in the placental tissue of fruits that correlated with
305
pungency, which strongly suggested it as a candidate to regulate the structural
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genes of the capsaicinoid pathway. However, CaMYB31 was also expressed in
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vegetative tissues such as roots, leaves and stems, indicating a possible role in
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more than one biological process, such as lignin biosynthesis or other metabolites
309
derived from the phenylpropanoid pathway.
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Bioinformatic Analysis of CaMYB31
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Bioinformatic analysis of R2R3-MYB proteins showed that the putative
314
CaMYB31 protein was closer to unknown function proteins of chili pepper, tomato,
315
potato and tobacco. Moreover, the subgroup 14 of MYB Arabidopsis proteins was
316
the nearest clade to CaMYB31, which shared one motif in the C-terminal region,
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and therefore might have some similar functions (lignin biosynthesis, stress and
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plant hormone response or development). The lignin and capsaicinoid biosynthetic
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pathways have common steps at the early stages of the phenylpropanoid
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biosynthesis, suggesting that CaMYB31 is probably implicated in the early
321
regulation of the capsaicinoid pathway. Additionally, we found putative MYB
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binding sites on all the predicted promoters of the structural genes of the
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capsaicinoid pathway, suggesting that they are potential target genes of
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CaMYB31.
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Effect
of
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Biosynthesis-Related Genes and Capsaicinoid Content
CaMYB31
Silencing
on
the
Expression
of
Capsaicinoid
328
329
Virus induced gene silencing (VIGS) was used to investigate the function of
330
CaMYB31 in the capsaicinoid pathway. CaMYB31 silencing caused a significant
331
decrease in capsaicinoid accumulation and on the expression of all structural
332
genes of the phenylpropanoid pathway, except that of Pal, and also on the early
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genes of the branched-chain fatty acids pathway (BCAT, BCKDH, Kas and Acl) of
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capsaicinoid biosynthesis, showing strong evidence of its participation in the
335
regulation of this metabolic process. Based on these results, we propose that the
336
structural genes that showed a significant decrease in expression when CaMYB31
337
was silenced are potential target genes of CaMYB31. Future studies of CaMYB31
338
interaction with promoters of the structural genes whose expression was affected
339
should provide more evidence on the direct transcriptional effect.
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Effect of Hormones, Light, Temperature and Wounding on CaMYB31
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Expression
343
344
The biosynthesis and accumulation of capsaicinoids in chili pepper fruits is
345
sensitive to environmental factors such as temperature and light, by stress
346
conditions (wounding, drought), and also by plant hormones (Lindsey and Bosland,
347
1996; Suresh et al., 2005; Kim et al., 2009; Gutiérrez-Carbajal et al., 2010). For
348
this reason, we analyzed the effect of plant hormones and stress on CaMYB31
349
expression and two of the possible target genes: Kas and pAmt.
350
In previous studies, it was reported that SA induced the production of
351
capsaicinoids and intermediary compounds (Gutierrez-Carbajal et al., 2010;
352
Altuzar-Molina et al., 2011; Rodas-Junco et al., 2013), while JA induced or
353
repressed the accumulation of capsaicinoids and intermediary metabolites
354
depending on the concentration and exposure time (Suresh et al., 2005; Gutierrez-
355
Carbajal et al., 2010; Altuzar-Molina et al., 2011). CaMYB31, Kas and pAmt
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356
expression was significantly increased with SA treatment, while JA treatment
357
caused a significant decrease in its expression, except at 3 h of exposure. In
358
general, a typical antagonistic effect between SA and JA treatment was revealed,
359
similar to that observed in genes related to defense against pathogens.
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Wounding stress has been reported to promote the formation of JA while
361
repressing SA production (Lee et al., 2004). A significant decrease in CaMYB31,
362
Kas and pAmt expression in wounded chili pepper fruits was consistently observed
363
with JA treatment, except at 3 h.
364
Other hormones, such as gibberellic acid and auxin, interact with the SA and
365
JA pathways, increasing the complexity of the signaling pathway in the mechanism
366
of defense in plants (Robert-Seilaniantz et al., 2011; Pieterse et al., 2012).
367
CaMYB31, Kas and pAmt expression was significantly incremented under IAA and
368
GA3 treatment. It has been reported that gibberellins suppress JA signaling and
369
induce SA signaling (Pieterse et al., 2012), consistent with our results. Conversely,
370
it has been reported that the auxin response was antagonistic to the SA response
371
(Pieterse et al., 2012); however, we did not observe an antagonistic effect on the
372
expression of CaMYB31, Kas and pAmt. The concentration of the plant hormone
373
and exposure time highly influence gene expression.
374
Temperature is another factor that affects positively or negatively the level of
375
pungency in chili pepper fruits and the expression of capsaicinoid structural genes
376
(Rahman and Inden, 2012; Kim et al., 2009; González-Zamora et al., 2013). The
377
expression of CaMYB31, Kas and pAmt was significantly increased at 4°C and was
378
reduced at 37°C compared to 25°C; thus, Serrano ‘Tampiqueño 74’ perhaps is a
379
variety in which the accumulation of capsaicinoids is negatively affected by high
380
temperature.
381
Light intensity has been found to exert a significant effect on capsaicinoid
382
content in chili pepper fruits (Iwai et al., 1979; Gangadhar et al., 2012). Kim et al.
383
(2009) reported that the enzymatic activity of AT3 was induced by exposure to light
384
and repressed in the dark. Similarly, the expression of CaMYB31, Kas and pAmt
385
was significantly higher under light compared to dark conditions in the present
386
investigation.
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387
388
CONCLUSION
389
390
Our work showed strong evidence of the involvement of a MYB transcription factor
391
in the regulation of capsaicinoid biosynthesis and its stress response in chili pepper
392
fruits. Future studies are necessary to investigate whether CaMYB31 acts directly
393
on the promoter of structural genes of the capsaicinoid pathway or through the
394
formation of a protein complex with other transcription factors.
395
396
MATERIALS AND METHODS
397
398
Plant Growth Conditions
399
Chili pepper (Capsicum annuum L.) cv. Tampiqueño 74 (Serrano type) and
400
cv. California Wonder plants were grown in greenhouse conditions and fertilized
401
every two weeks with FerviaFol (Agroquímicos Rivas) solution (N:P:K 30:20:10).
402
Chili pepper fruits were harvested at 10, 20, 30, 40, 50 and 60 days post-anthesis
403
(DPA) from at least six plants. Only placental tissue was collected and was
404
immediately frozen in liquid nitrogen, stored at -80°C and used for qRT-PCR
405
expression analysis to select MYB gene candidates.
406
Capsicum annuum L. cv. Tampiqueño 74, cv. California Wonder and
407
Capsicum chinense Habanero BG-3821 plants were grown for differential
408
expression analysis in different organs. Chili pepper fruits were harvested at 10,
409
20, 40 and 60 DPA from Capsicum annuum and immature and mature stages from
410
Capsicum chinense were dissected to separate the seeds, placenta and pericarp,
411
which were stored separately. Roots, leaves, stem and flowers of adult plants from
412
the different genotypes of C. annuum were collected. All tissues were frozen and
413
stored at -80°C.
414
Seeds of cv. Tampiqueño 74 were germinated in a growth chamber at a
415
temperature of 28°C under a 16 h photoperiod, a photon flux of 70 µmol m-2 s-1
416
(T8W/starcoat GE fluorescent lamps) and a relative humidity of 66% for silencing
417
experiments. Three-week-old chili pepper seedlings were agroinfected and after 6
14
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
418
weeks were transferred to greenhouse conditions until 40 DPA chili pepper fruits
419
were collected for each treatment.
420
Seeds of Capsicum annuum L. cv. Tampiqueño 74 were germinated and
421
grown in a greenhouse for the studies of environment stimuli in entire fruits.
422
Placental tissue was collected from 30 DPA chili pepper fruits for each treatment.
423
424
Identification and Isolation of CaMYB31 Gene
425
426
We selected 24 MYB partial sequences from a cDNA library of Capsicum
427
annuum L. cv. Tampiqueño 74 with more than 40% identity to MYB proteins (see
428
Supplemental Table I). The expression of these MYB genes was analyzed by qRT-
429
PCR, comparatively with the expression of the capsaicinoid biosynthetic genes
430
AT3, Kas, pAmt, Ca4H and Comt in placental tissue of fruits at different
431
developmental stages (10, 20, 30, 40, 50 and 60 DPA) of C. annuum L. cv.
432
Tampiqueño 74 (pungent) and cv. California Wonder (non-pungent). Moreover, the
433
levels of capsaicin and dihydrocapsaicin were quantified at the same stages of
434
development in pungent and non-pungent fruits. CaMYB31 gene was selected as
435
candidate based on the positive correlation of its expression pattern with
436
capsaicinoid content and the expression levels of the structural genes.
437
The full-length CaMYB31 gene was predicted using GeneScan web server
438
from the pepper genome (Quin et al., 2014) and primers were designed (forward:
439
5’-TACACGTGATGGTGAGAACAACACCTTGCT-3´
440
CGCACGTGTTAATAATTAAAATGATCAAA-3’) for amplification by RT-PCR from
441
placental tissue of 40 DPA chili pepper fruits of cv. Tampiqueño 74. The PCR
442
conditions for amplification were 94°C for 3 min, followed by 30 cycles (94°C for 30
443
s; 57.5°C for 30 s; 72°C for 1 min), and 72°C for 7 min. The PCR product was
444
diluted (1 µL of PCR product with 19 µL of sterile water) and used as template for a
445
second PCR under the same conditions. PCR product was cloned into the pCR 8
446
vector (Invitrogen) according to the manufacturer’s instructions and sequenced.
and
447
448
Phylogenetic and Sequence Analysis
15
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
reverse:
5’-
449
450
BLASTp was used to search MYB protein sequences from RefSeq_protein
451
and Swiss-Prot NCBI database using the full-length amino acid sequence of
452
CaMYB31. The characterized Solanaceae proteins (SlMYB12, SlBLIND, CaBLIND,
453
CaMYB, CaMYB1, CaA and CaMYB3) and all the Arabidopsis thaliana MYB
454
proteins reported in Stracke et al., (2001) and in the IT3F Website (Bailey et al.,
455
2008) were also included (See Supplemental Table II). The sequences were
456
aligned with CLUSTAL W using default parameters, and it was manually adjusted.
457
Based on the MYB domain aligment, a phylogenetic tree was constructed with the
458
neighbor joining method, model JTT+G, and a bootstrap test (1000 replicates)
459
using MEGA 6 software. In addition, we constructed a phylogenetic tree using the
460
IT3F Website with CaMYB31 and the MYB proteins of Arabidopsis thaliana. The
461
clades were grouped as previously reported (Kranz et al., 1998; Stracke et al.,
462
2001). MEME (Multiple Em for Motif Elicitation) version 4.11.3 (http://meme-
463
suite.org/tools/meme) was used to discover putative motifs in the C-terminal region
464
of CaMYB31. PlantPAN 2.0 (Chow et al., 2016) was used to search MYB binding
465
sites in the predictive promoters of the structural genes of the capsaicinoid
466
biosynthesis pathway.
467
468
Virus-induced gene silencing of CaMYB31 gene
469
470
A 234 bp fragment of CaMYB31 was amplified (bases 269 to 503) using the
471
primers forward 5´-AGTCTAGATCCTTTGGTGACCATTCTGA-3’ and reverse 5´-
472
AGTCTAGAGGCGATTGCTGCTCACTT-3’. Amplification conditions were 94°C for
473
5 min, followed by 30 cycles (94°C for 30 s; 55°C for 30 s; 72°C for 1 min) and
474
extension at 72°C for 7 min. The CaMYB31-viral silencing plasmid construction
475
and agroinfiltration was carried out as reported for tomato (Liu et al., 2002), with
476
some modifications (Arce-Rodríguez and Ochoa Alejo, 2015).
477
478
Quantification of Capsaicinoids
479
16
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
480
Capsaicin and dihydrocapsaicin were quantified in placental tissue from C.
481
annuum cv. Tampiqueño 74 (pungent) and cv. Bell pepper California Wonder (non-
482
pungent) chili pepper fruits in three biological samples composed of 10 placentas
483
each in the case of 10 DPA fruits, and 3 placentas for the 20, 30, 40, 50 and 60
484
DPA fruits. The extraction and quantification of capsaicin and dihydrocapsaicin was
485
carried out as reported by Arce-Rodríguez and Ochoa-Alejo (2015).
486
487
Plant Hormones, Light, Temperature and Wounding Chili Pepper Fruit
488
Treatments
489
490
Chili pepper fruits of C. annuum cv. Tampiqueño 74 at 30 DPA were
491
collected to evaluate the effect of different factors on the expression of CaMYB31
492
and the capsaicinoid biosynthetic genes Kas and pAmt for 3, 6, 12 and 16 h. For
493
light/dark response assays, complete fruits were first wrapped with plastic film and
494
foil for 24 h and then were uncovered and exposed to fluorescent light conditions
495
(50 µM m-2 s-1). For temperature treatments, entire fruits were wrapped with plastic
496
film and foil and incubated at 4°C, 25°C and 37°C. For the wounding responses
497
experiment, a scalpel blade was introduced approximately 6 mm deep several
498
times into whole fruits, which were then covered with plastic film and incubated at
499
25°C under standard light conditions. To apply plant hormone treatments, placental
500
tissue was separated from 30 DPA fruits and submerged into MS (Murashige and
501
Skoog 1962) liquid medium containing 0 or either 100 µM jasmonic acid, salicylic
502
acid, gibberellic acid or indoleacetic acid (SIGMA) and incubated at 25°C under
503
standard light conditions. After incubation, the placental tissues from all treatments
504
were immediately frozen in liquid nitrogen and stored at -80°C for gene expression
505
analysis.
506
507
Total RNA Extraction and cDNA Synthesis
508
509
Total RNA was extracted from placenta, pericarp, seeds, flowers, stems,
510
leaves and roots with Trizol (Invitrogen) according to the protocol provided by the
17
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
511
manufacturer. The extraction was performed in triplicate for each sample. RNA
512
was purified with the PureLinkTM Micro-To-Midi kit (Invitrogen) according to the
513
instructions. cDNA was synthesized from purified RNA with the Super Script II or
514
Super Script III reverse transcriptase (Invitrogen) following the protocol provided by
515
the manufacturer.
516
517
Real-Time Quantitative PCR Assays
518
519
Total RNA was extracted, purified and treated with DNAse I (Invitrogen)
520
following the manufacturer’s instructions. cDNA was synthesized with Super Script
521
III reverse transcriptase (Invitrogen) and adjusted to 100 ng mL-1. qRT-PCR was
522
performed as reported by Arce-Rodríguez and Ochoa-Alejo (2015) using the
523
primer pairs described in Supplemental Table III.
524
525
Statistical Analysis
526
The data generated in silencing experiments and the effect of different
527
factors on the expression of CaMYB31 were subjected to Analysis of Variance
528
(ANOVA) with the Tukey test (P ≤ 0.05) to find statistically significant differences
529
between the mean values.
530
531
532
The coding sequence of CaMYB31 can be found in GenBank under
accession number XXX.
533
534
Supplemental Data
535
Supplemental Table I. Sequences from the cDNA library of Capsicum annuum L.
536
cv. Tampiqueño 74 (Serrano type) for qRT-PCR analysis.
537
Supplemental Table II. Accession numbers of MYB proteins listed in Figure 3.
538
Supplemental Table III. Primers used for qRT-PCR analysis.
539
Supplemental Figure 1. qRT-PCR expression assays of the putative MYB
540
transcription factor genes in placental tissue from chili pepper fruits of Capsicum
541
annuum cv.
18
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
542
Supplemental Figure 2. Phylogenetic relationship of CaMYB31 with MYB
543
transcription factors of Arabidopsis thaliana.
544
Supplemental Figure 3. Alignment of amino acid sequences of CaMYB31 with
545
others plant R2R3-MYB proteins.
546
Supplemental Figure 4. Schematic diagram of the putative MYB binding sites
547
localization in the predicted promoter of capsaicinoid structural genes.
548
Supplemental Table I. Sequences from the cDNA library of Capsicum annuum L.
549
cv. Tampiqueño 74 (Serrano type) for qRT-PCR analysis.
550
Supplemental Table II. Accession numbers of MYB proteins listed in Figure 3.
551
Supplemental Table III. Primers used for qRT-PCR analysis.
552
553
Supplemental Figure 1. qRT-PCR expression assays of the putative MYB
554
transcription factor genes in placental tissue from chili pepper fruits of Capsicum
555
annuum cv. Tampiqueño 74 and cv. California Wonder at different developmental
556
stages. The data points represent the means of three biological replicates ± SD.
557
558
Supplemental Figure 2. Phylogenetic relationship of CaMYB31 with MYB
559
transcription factors of Arabidopsis thaliana. Phylogenetic tree was constructed
560
online using the IT3F website. The clades were grouped as previously reported for
561
Arabidopsis thaliana (Kranz et al., 1998; Stracke et al., 2001) (highlighted with
562
colored diamonds for each subgroup). The nearest neighbor clade to CaMYB31
563
(highlighted with blue letters) was the subgroup 14. Accession numbers for all
564
protein sequences are listed in Supplemental Table II.
565
566
Supplemental Figure 3. Alignment of amino acid sequences of CaMYB31 with
567
others plant R2R3-MYB proteins. A, Comparison of R2R3-MYB proteins.
568
CaMYB31 (XXX), AtMYB36 (AT5G57620), AtMYB37 (AT5G23000), AtMYB35
569
(AT3G28470), AtMYB80 (AT5G56110) OsMYB4 (Q7XBH4), CaBLIND (F5C7S0),
570
and SlMYB12 (B4YAJ8). Dark gray indicates identical amino acids and gray shows
571
similar amino acids. Lines above the sequence highlight the R2 and R3 domains.
572
B, Schematic diagram of the comparison of CaMYB31 with proteins of the nearest
19
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
573
clade in the phylogenetic tree showing the putative motifs. Each box indicates a
574
putative motif, and specifically the red box indicates the motif that CaMYB31
575
shared with these proteins.
576
577
Supplemental Figure 4. Schematic diagram of the putative MYB binding sites
578
localization in the predicted promoter of capsaicinoid structural genes. The
579
horizontal black line indicates the length of promoter sequence from ATG codon to
580
-1500 pb. The vertical red lines indicate the putative TATA box, and the vertical
581
green and black lines indicate the putative MYB binding sites TAACAAA and
582
[AG]GATT, respectively; and the orientation towards up or down indicates the
583
position in the positive or negative strand, respectively.
584
585
586
ACKNOWLEDGMENTS
587
We thank Yolanda Rodríguez for technical assistance in the HPLC analysis;
588
Dr. Cesar Aza-González, Dr. María del Rocío Gomez-García, and José Luis Pablo-
589
Rodríguez for technical assistance in the agroinfiltration experiments; and Dr. Luis
590
Delaye, Dr. Octavio Martínez de la Vega and Magdalena Rivera for their important
591
insights in bioinformatic analysis.
592
593
Figure legends:
594
595
Figure 1. Capsaicinoid biosynthetic pathway. PAL, phenylalanine ammonia lyase;
596
Ca4H,
597
hydroxycinnamoyl transferase; C3H, coumaroyl shikimate/quinate 3-hydroxylase;
598
COMT, caffeic acid O-methyltransferase; pAMT, aminotransferase; BCAT,
599
branched-chain amino acid transferase; KAS, ketoacyl-ACP synthase; ACL, acyl-
600
CoA synthetase; FAT, acyl-ACP thiosterase; CS, capsaicinoid synthase; AT3,
601
acyltransferase. The potential target genes of CaMYB31 transcription factor are
602
marked in red.
cinnamate
4-hydroxylase;
4CL,
4-coumaroyl-CoA
603
20
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
ligase;
HCT,
604
Figure 2. qRT-PCR expression assays of the capsaicinoid biosynthetic genes Kas,
605
pAmt, AT3, Comt and Ca4H and CaMYB31 in placental tissue from chili pepper
606
fruits of Capsicum annuum cv. Tampiqueño 74 and cv. California Wonder at
607
different developmental stages. The data points represent the means of three
608
biological replicates ± SD.
609
610
Figure 3. Structural organization of the CaMYB31 gene and phylogenetic
611
relationship of CaMYB31 with other plant MYB transcription factors. A, Schematic
612
diagram of CaMYB31 showing the coding regions (black boxes), 5’ and 3’ UTR
613
regions (gray boxes), introns (solid lines), R2R3-MYB domains (dotted lines), and
614
the fragment sequence of CaMYB31 used for VIGS. B, Phylogenetic tree was
615
constructed using the neighbor joining method based on the MYB domain
616
alignment using MEGA 6 software. The bootstrap values are shown as
617
percentages (1000 replicates) when greater than 50%. The clades were grouped
618
as previously reported for Arabidopsis thaliana (Stracke et al., 2001) (highlighted
619
with colored diamonds for each subgroup), while CaMYB31 (highlighted with blue
620
letters) grouped with solanaceous sequences (marked with red circles). Accession
621
numbers for all protein sequences are listed in Supplemental Table II.
622
623
Figure 4. Differential expression assays of CaMYB31 and of the capsaicinoid
624
biosynthetic genes pAmt, Kas, Comt and Ca4H in different tissues of Capsicum
625
annuum cv. Tampiqueño 74 and cv. California Wonder, and in fruits of Capsicum
626
chinense Habanero BG-3821. M, mature; I, immature; Pe, pericarp; Se, seed; Pl,
627
placenta; Fl, flower; Le, leaf; St, stem; Ro, root. The data points are the means of
628
three biological replicates ± SD.
629
630
Figure 5. Effect of CaMYB31 gene silencing on biosynthetic structural gene
631
expression and on capsaicinoid content in fruits of chili pepper. A, qRT-PCR
632
analysis of capsaicinoid biosynthetic genes and CaMYB31 in fruits of chili pepper
633
plants infected with the viral construct pTRV2-CaMYB31 compared to the
634
uninfected control plants. B, HPLC analysis of capsaicin and dihydrocapsaicin in
21
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
635
fruits of uninfected plants and pTRV2 and pTRV2-CaMYB31 infected plants. The
636
data points are the means of three biological replicates ± SD. Asterisks indicate
637
significant differences between the control (uninfected) and infected plants (P ≤
638
0.05; Tukey test).
639
640
Figure 6. Effect of light on CaMYB31, Kas and pAmt expression in placental tissue
641
from chili pepper fruits at 30 DPA. Gene expression was measured at different
642
incubation times (3, 6, 12 and 16 h). The data points are the means of three
643
biological replicates ± SD. Asterisks show significant differences between fruits
644
incubated under dark and light conditions (P ≤ 0.05; Tukey test).
645
646
Figure 7. Effect of temperature on CaMYB31, Kas and pAmt expression in
647
placental tissue from chili pepper fruits at 30 DPA. Gene expression was quantified
648
at different incubation times (3, 6, 12 and 16 h) and different temperatures (4, 25
649
and 37°C). The data points are the means of three biological replicates ± SD.
650
Asterisks show significant differences between fruits incubated at standard
651
temperature (25°C), and high temperature (37°C) or low temperature (4°C) (P ≤
652
0.05; Tukey test).
653
654
Figure 8. Effect of wounding on CaMYB31, Kas and pAmt expression in placental
655
tissue from chili pepper fruits at 30 DPA. Gene expression was quantified at
656
different incubation times (3, 6, 12 and 16 h). The data points are the means of
657
three biological replicates ± SD. Asterisks show significant differences between
658
control (no injuries) and wounded fruits (P ≤ 0.05; Tukey test).
659
660
Figure 9. Effect of plant hormones on CaMYB31, Kas and pAmt expression in
661
placental tissue from chili pepper fruits at 30 DPA. Gene expression was quantified
662
at different incubation times (3, 6, 12 and 16 h) in fruits treated with 100 µM IAA
663
(A), GA3 (B), SA (C) or JA (D). The data points are the means of three biological
664
replicates ± SD. Asterisks show significant differences between control (MS
665
medium) and treated fruits (P ≤ 0.05; Tukey test).
22
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
666
667
668
669
670
671
672
673
674
675
Table I. Accumulation of capsaicin and dihydrocapsaicin in chili pepper fruits of
C. annumm cv. Tampiqueño 74 and cv. California Wonder at different
developmental stages
Cultivar
Stages of fruit
Capsaicin
Dihydrocapsaicin
(mg g-1Lpt)
(mg g-1Lpt)
10
N.D.
N.D.
20
6.73 ± 0.55
3.43 ± 0.31
30
9.91 ± 1.42
5.44 ± 1.59
40
11.21 ± 1.19
5.85 ± 0.91
50
7.94 ± 0.61
4.98 ± 0.87
60
5.44 ± 0.46
3.93 ± 0.49
N.D.
N.D.
development (DPA,
Days post-anthesis)
‘Tampiqueño 74’
‘California Wonder’ 10
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
20
N.D.
N.D.
30
N.D.
N.D.
40
N.D.
N.D.
50
N.D.
N.D.
60
N.D.
N.D.
676
Separation and determination of capsaicin and dihydrocapsaicin was performed by
677
HPLC. Lpt, lyophilized placental tissue. Each value is the mean of six biological
678
replicates ± SD. N.D., Not detected.
679
680
681
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751
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752
capsaicinoids. Plos One 8 (1): e48156 doi:10.1371/journal.pone.0048156
753
Liu Y, Schiff M, Dinesh-Kumar S (2002) Virus-induced gene silencing in tomato.
754
Plant J 31: 777-786
755
Mazourek M, Pujar A, Borovsky Y, Paran I, Mueller L, Jahn MM (2009) A
756
dynamic interface for capsaicinoid systems biology. Plant Physiol 150: 1806-1821
26
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
757
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays
758
with tobacco tissue cultures. Physiol Plant 15: 473-497
759
Ochoa-Alejo N, Ramírez-Malagón R (2001) In vitro chili pepper biotechnology. In
760
Vitro Cell Dev Biol-Plant 37: 730 -741
761
Patzlaff A, McInnis S, Courtenay A, Surman C, Newman LJ, Smith C, Bevan
762
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763
pine MYB that regulates lignification. Plant J 36: 743–754
764
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765
(2012) Hormonal modulation of plant immunity. Annu Rev Cell Dev Biol 28: 489–
766
521
767
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768
al (2014) Whole-genome sequencing of cultivated and wild peppers provides
769
insights into Capsicum domestication and specialization. Proc Natl Acad Sci USA
770
111: 5135-5140
771
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772
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773
(Capsicum annuum L.) cultivars. J Food Agric Environ 10: 524-529
774
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775
disease and defense: more than just jasmonate-salicylate antagonism. Annu Rev
776
Phytopathol 49: 317-343
777
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778
Monforte-Gonzalez M, Hérnandez-Sotomayor SMT (2013) Salicylic acid induces
779
vanillin synthesis through the phospholipid signaling pathway in Capsicum
780
chinense cell cultures. Plant Signal Behav 8(10): e26752 doi: 10.4161/psb.26752
781
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782
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783
acyltransferase. Plant J 42: 675-688
784
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785
Arabidopsis thaliana. Curr Opin Plant Biol 4: 447–456
27
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
786
Suresh
B,
Ravishankar
GA
(2005)
Methyl
jasmonate
787
biotransformation of phenylpropanoids to vanillin related metabolites using
788
Capsicum frutescens root cultures. Plant Physiol Biochem 43: 125-131
789
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792
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793
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794
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795
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796
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797
activators of the lignin biosynthetic pathway during secondary cell wall formation in
798
Arabidopsis. Plant Cell 21: 248–266
799
800
801
802
803
804
28
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
modulated
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Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.