An R2R3-MYB Transcription Factor Regulates Capsaicinoid ... · Comt, Ca4H, and CaMYB31 in placental tissue from fruits of cv Tampiquen˜o 74 and cv California Wonder at different

An R2R3-MYB Transcription Factor RegulatesCapsaicinoid Biosynthesis1[OPEN]

Magda L. Arce-Rodríguez,a and Neftalí Ochoa-Alejoa,b,2

aDepartamento de Ingeniería Genética de Plantas, Centro de Investigación y de Estudios Avanzados delInstituto Politécnico Nacional, Unidad Irapuato, 36821 Irapuato, Guanajuato, MexicobDepartamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados del InstitutoPolitécnico Nacional, Unidad Irapuato, 36821 Irapuato, Guanajuato, Mexico

ORCID ID: 0000-0001-9940-5945 (N.O.-A.).

Capsaicinoids are responsible for the hot taste of chili peppers. They are restricted to the genus Capsicum and are synthesized bythe acylation of the aromatic compound vanillylamine (derived from the phenylpropanoid pathway) with a branched-chain fattyacid by the catalysis of the putative enzyme capsaicinoid synthase. R2R3-MYB transcription factors have been reported indifferent species of plants as regulators of structural genes of the phenylpropanoid pathway; therefore, we hypothesized thatMYB genes might be involved in the regulation of the biosynthesis of pungent compounds. In this study, an R2R3-MYBtranscription factor gene, designated CaMYB31, was isolated and characterized in Capsicum annuum ‘Tampiqueño 74’.Bioinformatic analysis suggested that CaMYB31 could be involved in secondary metabolism, stress and plant hormoneresponses, and development. CaMYB31 expression analysis from placental tissue of pungent and nonpungent chili pepperfruits showed a positive correlation with the structural genes Ca4H, Comt, Kas, pAmt, and AT3 expression and also with thecontent of capsaicin and dihydrocapsacin during fruit development. However, CaMYB31 also was expressed in vegetativetissues (leaves, roots, and stems). Moreover, CaMYB31 silencing significantly reduced the expression of capsaicinoidbiosynthetic genes and the capsaicinoid content. Additionally, CaMYB31 expression was affected by the plant hormonesindoleacetic acid, jasmonic acid, salicylic acid, and gibberellic acid or by wounding, temperature, and light, factors known toaffect the production of capsaicinoids. These findings indicate that CaMYB31 is indeed involved in the regulation of structuralgenes of the capsaicinoid biosynthetic pathway.

Chili peppers are members of the Capsicum genus,which belongs to the Solanaceae family. Capsicumannuum is the most cultivated species in the world. Theimportance of this crop is based on a great variety ofindustrial applications, such as food, pharmaceuti-cal, cosmetic, and agronomic uses (Ochoa-Alejo andRamírez-Malagón, 2001). One of the most valuedcompounds of chili pepper fruits are the capsaicinoids,which are synthesized and accumulated in the placen-tal tissue through the convergence of phenylpropanoidand the branched-chain fatty acid pathways (Aza-González et al., 2011; Fig. 1). Capsaicin and dihy-drocapsaicin are responsible for 90% of the pungencyin the fruit, and their accumulation depends on the

genotype, stage of fruit development, and environ-mental conditions (Lindsey and Bosland, 1996; Kimet al., 2009; Arce-Rodríguez and Ochoa-Alejo, 2015).

Research on the capsaicinoid biosynthesis pathwayhas uncovered key aspects of its biochemistry and mo-lecular biology (Aza-González et al., 2011). The genera-tion of chili pepper cDNA libraries and comparative geneexpression analysis of pungent and nonpungent fruits hasallowed the identification of several structural genes ofthe capsaicinoid biosynthesis pathway, such as Phe am-monia lyase (Pal), cinnamic acid 4-hydroxylase (Ca4H),4-coumarate-CoA ligase (4CL), hydroxycinnamoyl trans-ferase (HCT), caffeic acid O-methyltransferase (Comt),hydroxycinnamoyl-CoAhydratase/ligase, and aputativeacyltransferase (AT3) proposed to be the possible cap-saicinoid synthase (Curry et al., 1999; Kim et al., 2001;Stewart et al., 2005;Mazourek et al., 2009; Liu et al., 2013).

The role of transcription factors in the regulation ofcapsaicinoid biosynthesis is underexplored. Stewartet al. (2005) reported a differential expression analysisof two basic Leu zipper transcription factors in pungentand nonpungent fruits at different developmentalstages, but their expression pattern did not correlatepositively with the expression of the structural genesPal, Ca4H, Comt, pAMT, BCAT, Kas, Acl, FatA, and AT3.Recently, Keyhaninejad et al. (2014) reported two ethyleneresponse factor transcription factors whose expression

1 This work was supported by the National Council of Science andTechnology (grant no. 177063 and a scholarship to M.L.A.-R.).

2 Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Neftalí Ochoa-Alejo ([email protected]).

M.L.A.-R. designed and carried out the experimental work, ana-lyzed the data, and wrote the article; N.O.-A. designed, directed, andsupervised the experiments and complemented and corrected thearticle.

[OPEN] Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.17.00506

Plant Physiology�, July 2017, Vol. 174, pp. 1359–1370, www.plantphysiol.org � 2017 American Society of Plant Biologists. All Rights Reserved. 1359 www.plantphysiol.orgon August 6, 2020 - Published by Downloaded from

Copyright © 2017 American Society of Plant Biologists. All rights reserved.

http://orcid.org/0000-0001-9940-5945

http://orcid.org/0000-0001-9940-5945

http://orcid.org/0000-0001-9940-5945

http://orcid.org/0000-0001-9940-5945

http://orcid.org/0000-0001-9940-5945

http://orcid.org/0000-0001-9940-5945

http://orcid.org/0000-0001-9940-5945

http://orcid.org/0000-0001-9940-5945

http://crossmark.crossref.org/dialog/?doi=10.1104/pp.17.00506&domain=pdf&date_stamp=2017-06-20

mailto:[email protected]

http://www.plantphysiol.org

mailto:[email protected]

http://www.plantphysiol.org/cgi/doi/10.1104/pp.17.00506


correlated positively with the pungency level in nine chilipepper cultivars, and they were proposed as possibleregulators of capsaicinoid biosynthesis.

Previous studies have shown that the phenyl-propanoid pathway is regulated by R2R3-MYB tran-scription factors in different plant species, such asArabidopsis (Arabidopsis thaliana), Antirrhinum majus,and Pinus taeda (Tamagnone et al., 1998; Patzlaff et al.,2003; Zhou et al., 2009). R2R3-MYB transcription factorsrepresent one of the largest families in plants and regu-late different biological processes, such as primary andsecondary metabolism, responses to biotic and abioticstresses, developmental processes, and hormonal re-sponses (Dubos et al., 2010; Ambawat et al., 2013; Liu et al.,2015). Since the phenylpropanoid pathway is involved inthe biosynthesis of precursors of the capsaicinoid biosyn-thetic pathway, we hypothesized that the R2R3-MYBfamily transcription factors participate in the biosynthesisof capsaicinoids. Here, we report on the isolation of a

CaMYB31 cDNA, which encodes a putative R2R3-MYBprotein, and its role in the regulation of capsaicinoidbiosynthetic genes and in the accumulation of cap-saicinoids in chili pepper fruits. Furthermore, we charac-terized CaMYB31 with regard to its gene organization,phylogeny, function, and responses to hormones, light,temperature, and wounding.

RESULTS

Identification of CaMYB31 by Differential GeneExpression in Placental Tissue from Pungent andNonpungent Fruits

The accumulation of capsaicin and dihydrocapsaicinin placental tissue of chili pepper fruits of C. annuum‘Tampiqueño 74’ (pungent) and ‘California Wonder’(nonpungent) during fruit development is presentedin Table I. Capsaicin content was twice as high as

Figure 1. Capsaicinoid biosynthetic pathway.PAL, Phe ammonia lyase; Ca4H, cinnamate4-hydroxylase; 4CL, 4-coumaroyl-CoA ligase;HCT, hydroxycinnamoyl transferase; C3H,coumaroyl shikimate/quinate 3-hydroxylase;COMT, caffeic acid O-methyltransferase; pAMT,aminotransferase; BCAT, branched-chain aminoacid transferase; KAS, ketoacyl-ACP synthase;ACL, acyl-CoA synthetase; FAT, acyl-ACP thioes-terase; CS, capsaicinoid synthase; AT3, acyl-transferase. The potential target genes of theCaMYB31 transcription factor are marked in red.

Table I. Accumulation of capsaicin and dihydrocapsaicin in fruits of cv Tampiqueno 74 and cv CaliforniaWonder at different developmental stages

Separation and determination of capsaicin and dihydrocapsaicin was performed by HPLC. Each value isthe mean of six biological replicates 6 SD. Lpt, Lyophilized placental tissue; N.D., not detected.

Cultivar Stages of Fruit Development Capsaicin Dihydrocapsaicin

DPA mg g21 LptTampiqueno 74 10 N.D. N.D.

20 6.73 6 0.55 3.43 6 0.3130 9.91 6 1.42 5.44 6 1.5940 11.21 6 1.19 5.85 6 0.9150 7.94 6 0.61 4.98 6 0.8760 5.44 6 0.46 3.93 6 0.49

California Wonder 10 N.D. N.D.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.

1360 Plant Physiol. Vol. 174, 2017

Arce-Rodríguez and Ochoa-Alejo

www.plantphysiol.orgon August 6, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.


dihydrocapsaicin in cv Tampiqueño 74 fruits, but thesetwo capsaicinoids were not detected in cv CaliforniaWonder. In cv Tampiqueño 74, capsaicinoids were notrecorded at 10 DPA, began accumulating at 20 DPA,peaked at 30 to 40 DPA, and decreased at 50 to 60 DPA.The expression of Ca4H, Comt, pAmt, AT3, and Kas

genes was positively correlated with capsaicinoid accu-mulation in cv Tampiqueño 74 placental tissue. The ex-pression of pAmt, Kas, AT3, and Comt was very low oreven undetectable at 10 DPA, increased to a maximumbetween 30 and 40 DPA, decreased at 50 DPA, and wasvery low or undetectable at 60DPA.Ca4Hwas expressedmoderately at 10 DPA, increased to amaximum between20 and 40 DPA, and then diminished slightly between50 and 60 DPA. In cv California Wonder, the expressionof AT3 was undetectable at all stages of fruit develop-ment, while that of Kas and pAmt was very low at 30 to

50 DPA. However, Comt and Ca4Hwere expressed at allstages of the nonpungent fruits (Fig. 2).

Partial MYB sequences were identified from a cDNAlibrary of cv Tampiqueño 74 (Supplemental Table S1).Only sequences that showed greater than 40% homologyto MYB transcription factors and that were expressed inpungent placental tissue were selected for qRT-PCRanalysis (Supplemental Fig. S1).CaMYB31 showed a clearexpression pattern that correlated positivelywith both theexpression levels of the capsaicinoid biosynthetic struc-tural genes and capsaicinoid content during fruit devel-opment (Fig. 2).

CaMYB31 Structure and Phylogenetic Sequence Analyses

CaMYB31 contains a 747-bp reading frame encodinga putative R2R3-MYB protein of 249 amino acids that is

Figure 2. Quantitative reverse transcription (qRT)-PCR expression assays of the capsaicinoid biosynthetic genes Kas, pAmt, AT3,Comt, Ca4H, and CaMYB31 in placental tissue from fruits of cv Tampiqueno 74 and cv California Wonder at different devel-opmental stages. The data points represent means of three biological replicates 6 SD.

Plant Physiol. Vol. 174, 2017 1361

An R2R3-MYB Gene Regulates Pungency in Chili Pepper


http://www.plantphysiol.org/cgi/content/full/pp.17.00506/DC1



preceded by a 59 untranslated region (UTR) of 148 bpand followed by a 39 UTR of 189 bp. The CaMYB31cDNA sequence was compared with the chili peppergenome database (Qin et al., 2014), and three exons, of133, 130, and 484 bp, and two introns were identified.The first intron of 724 bp was localized between aminoacids 44 and 45 of the R2 domain, and the second one of425 bp was between amino acids 88 and 89 of the R3domain (Fig. 3A).

A phylogenetic tree of 167 plant MYB proteins wasconstructed using the neighbor-joining method, basedon alignment of the MYB domain, with a bootstrap test(n = 1,000; Fig. 3B). The full-length amino acid sequenceof CaMYB31 was used to look for MYB proteins fromRefSeq and the Swiss-Prot National Center for Biotech-nology Information protein database, and the MYBArabidopsis proteins also were included (SupplementalTable S2). The phylogenetic analysis was congruent with

Figure 3. Structural organization of the CaMYB31 gene and phylogenetic relationship of CaMYB31 with other plant MYBtranscription factors. A, Schematic diagram of CaMYB31 showing the coding regions (black boxes), 59 and 39UTRs (gray boxes),introns (solid lines), R2R3-MYB domains (dotted lines), and the fragment sequence of CaMYB31 used for virus-induced genesilencing (VIGS). B, Phylogenetic tree constructed using the neighbor-joining method based on the MYB domain alignment usingMEGA6 software. The bootstrap values are shown as percentages (1,000 replicates) when greater than 50%. The clades weregrouped as reported previously for Arabidopsis (Stracke et al., 2001; highlightedwith colored diamonds for each subgroup), whileCaMYB31 (highlighted with blue letters) grouped with solanaceous sequences (marked with red circles). Accession numbers forall protein sequences are listed in Supplemental Table S2.








the subgroups defined for Arabidopsis MYB proteins(Kranz et al., 1998; Stracke et al., 2001).According to our phylogenetic tree, CaMYB31 belongs

to a cluster of MYB Solanaceae proteins with no assignedbiological functions at this time (XP_016580728.1 with98% identity to XP_015081732.1 with 67% identity; Fig.3B). The nearest neighbor clade to CaMYB31 was sub-group 14 of Arabidopsis proteins (AtMYB36 with 63%identity to AtMYB68 with 46% identity), which are re-lated mainly to development, while some of them areimplicated in hormone response, stress response, and theregulation of lignin biosynthesis (Fig. 3B). We also con-structed the phylogenetic tree using the IT3F softwareonline (Bailey et al., 2008; Supplemental Fig. S2), and theresults were consistent with our phylogenetic analysis.CaMYB31 alignment with other MYB plant regula-

tory proteins showed a highly conserved R2R3 MYBdomain in the N-terminal region and a highly variableC-terminal region (Supplemental Fig. S3A). As a com-plement to the phylogenetic analysis, we analyzed theC-terminal region of CaMYB31 by comparison with thenearest neighbor clade to search for conserved motifsusingMEMEmotif-detection software, thinking that sucha motif might be important for the function of CaMYB31.The C-terminal region of CaMYB31 shares onemotif withthese proteins related to development (Supplemental Fig.S3B). Moreover, PlantPAN 2.0 (Chow et al., 2016) wasused as a database for signal scanning of MYB-bindingsites in the putative promoter sequences of the structuralcapsaicinoid genes, and we found MYB cis-elements onall predictive promoters of the capsaicinoid structuralgenes (Supplemental Fig. S4).

Differential CaMYB31 Expression Analyses in DifferentOrgan Tissues of Capsicum spp.

The expression of CaMYB31 and that of the structuralgenes Kas, pAmt, Comt, and Ca4H were analyzed byqRT-PCR in different tissues of the fruit (seeds, peri-carp, and placenta) and plant (roots, stems, flowers, andleaves) from pungent and nonpungent cultivars. Kasand pAmt gene expression was detected exclusively inplacental tissue (Fig. 4), although transcript levels weremuch higher in placental tissue from the immaturefruits of Capsicum chinense ‘Habanero’ than from cvTampiqueño 74, which correlated with the pungencylevel, whereas Comt and Ca4H also were expressed inseeds, pericarp, and vegetative tissues (Fig. 4).CaMYB31 showed the highest expression in placental

tissue of cv Habanero immature fruits, which are con-sidered highly hot (Fig. 4). Moreover, the characteristicexpression pattern of CaMYB31 during the developmentof cv Tampiqueño 74 fruitswas confirmed. TheCaMYB31expression observed in placental tissue of cv CaliforniaWonder fruitswasmuch lower.CaMYB31presented verylow expression in the seeds and pericarp.In the case of vegetative tissues,CaMYB31was expressed

at very low levels inflower tissues of cvTampiqueño 74 andwasundetectable inflowersof cvCaliforniaWonder (Fig. 4).

However, CaMYB31 was expressed moderately in rootsand highly in leaf and stem tissues when compared withthe placenta of cv Tampiqueño 74 chili pepper fruits.

Figure 4. Differential expression assays of CaMYB31 and of the cap-saicinoid biosynthetic genes pAmt, Kas, Comt, and Ca4H in differenttissues of cv Tampiqueno 74 and cv CaliforniaWonder and in fruits of cvHabanero BG-3821. M, Mature; I, immature; Pe, pericarp; Se, seed; Pl,placenta; Fl, flower; Le, leaf; St, stem; Ro, root. The data points aremeans of three biological replicates 6 SD.










Effects of CaMYB31 Silencing on the Expression ofCapsaicinoid Biosynthetic Genes and onCapsaicinoid Content

CaMYB31-silencing experiments were carried outusing a tobacco rattle virus (TRV)-derived vector togenerate the construct pTRV2-CaMYB31, which con-tained a 234-bp region from 90 amino acids of the R3domain toward the C-terminal region (Fig. 3A). Fruitsof 40 DPA were collected to investigate the effect ofCaMYB31 silencing.

Fruits from plants infected with the pTRV2-CaMYB31construct exhibited a significant diminution of CaMYB31gene expression (82%) compared with uninfected plants(Fig. 5A). CaMYB31 silencing caused a significant re-duction in the expression of the capsaicinoid biosyn-thetic genes Ca4H (81.8%), 4CL (80.6%), C3H (76.4%),HCT (49%), Comt (42.7%), pAmt (71%), BCAT (88.6%),BCKDH (48.5%), Kas (70%), and Acl (30.3%) comparedwith fruits from uninfected plants (Fig. 5A). It hasbeen shown previously that agroinfection with theempty pTRV2 vector did not cause statistically signifi-cant changes in the expression patterns of tested struc-tural genes in chili pepper fruits (Arce-Rodríguez andOchoa-Alejo, 2015).

Capsaicin and dihydrocapsaicin contents in fruits ofplants agroinfected with pTRV2-CaMYB31 showedsignificant reductions of 74.2% and 73%, respec-tively, compared with fruits from uninfected plants(Fig. 5B). Fruits from plants agroinfected with theempty pTRV2 vector exhibited capsaicin contentstatistically similar to that of uninfected plants;however, dihydrocapsaicin content was significantlyhigher. Therefore, fruits from plants agroinfectedwith pTRV2-CaMYB31 had an 82.4% decrease incapsaicin and dihydrocapsaicin compared with fruitsfrom plants infected with the empty pTRV2 vector(Fig. 5B).

Effects of Plant Hormones, Light, Temperature, andWounding on the Expression of CaMYB31 andCapsaicinoid Biosynthesis Structural Marker Genes

Environmental stimuli and some plant hormonesaffect capsaicinoid accumulation in chili pepper fruits(Kim et al., 2009; Gutiérrez-Carbajal et al., 2010). Inorder to test whether these factors regulate CaMYB31expression, several physical stimuli and plant hor-moneswere applied to 30-DPA fruits of cv Tampiqueño74. The expression of CaMYB31 and the capsaicinoidbiosynthetic genemarkers Kas and pAmtwere analyzedin placental tissue by qRT-PCR.

Fruits were treated with light or dark conditions withvarying exposure times, and the transcript levels wereexamined. Dark exposure caused a significant dimi-nution on CaMYB31 expression similar to the expres-sion pattern for Kas and pAmt (Fig. 6). Temperature wasanother tested stimulus; we monitored gene expressionat high temperature (37°C), low temperature (4°C), andstandard growth temperature (25°C) at different expo-sure times. CaMYB31,Kas, and pAmt expression at 37°Cwas significantly lower, while that in fruits at 4°C washigher, compared with standard growth conditions(Fig. 7). The expression of CaMYB31, Kas, and pAmt inwounded fruits had a significant decrease when com-pared with nonwounded fruits (Fig. 8).

In addition, the transcript levels of CaMYB31 in fruitstreated with 100 mM jasmonic acid (JA), salicylic acid(SA), gibberellic acid GA3, or indoleacetic acid (IAA)compared with control fruits (treated with Murashigeand Skoog [MS] medium) were determined. CaMYB31,Kas, and pAmt expression increased significantly byIAA, SA, and GA3 treatment, whereas a significantdiminution by JA treatment was detected, except at3 h of exposure (Fig. 9). The effect of these treatmentswas dependent on the time of exposure. In general,CaMYB31 expression correlated with the expression of

Figure 5. Effects of CaMYB31 gene silencing on biosynthetic structural gene expression and on capsaicinoid content in fruits ofchili pepper. A, qRT-PCR analysis of capsaicinoid biosynthetic genes and CaMYB31 in fruits of chili pepper plants infected withthe viral construct pTRV2-CaMYB31 compared with uninfected control plants. B, HPLC analysis of capsaicin and dihy-drocapsaicin in fruits of uninfected plants and pTRV2- and pTRV2-CaMYB31-infected plants. The data points are means of threebiological replicates 6 SD. Asterisks indicate significant differences between the control (uninfected) and infected plants (P #

0.05; Tukey’s test).





Kas and pAmt under exposure to different environ-mental stimuli.

DISCUSSION

The generation of chili pepper cDNA libraries andcomparative gene expression analysis of pungent(cv Tampiqueño 74) and nonpungent (cv CaliforniaWonder) fruits allowed us to identify CaMYB31, anR2R3-MYB transcription factor gene, as a strong can-didate to regulate the capsaicinoid pathway.In previous studies, the positive correlation between

the expression pattern of structural genes (Pal, Ca4H,Comt, Acl, Fat, Kas, and AT3) of the capsaicinoid biosyn-thetic pathway and the level of pungency of chili pepperfruits has been reported (Curry et al., 1999; Aluru et al.,2003; Arce-Rodríguez and Ochoa-Alejo, 2015). It has beenshown that capsaicinoid accumulation is dependent onthe developmental stage of chili pepper fruits, and thissynthesis and accumulation are highly likely to be regu-lated and coordinated at the transcriptional level.

Capsaicinoid Content and Expression of Genes Involved inthe Capsaicinoid Pathway in Chili Pepper Fruits

A positive correlation was observed between the ex-pression pattern of the structural genes Kas, pAmt, andAT3 and the pungency level during the development ofSerrano and bell pepper fruits, indicating that these arespecific genes of the capsaicinoid pathway. On the otherhand, Comt and Ca4H genes also were expressed innonpungent fruit and in vegetative tissues, suggestingthat they participate in other biological processes, such aslignin biosynthesis (Humphreys and Chapple, 2002).CaMYB31 showed an expression pattern similar to that ofthe Kas, pAmt, and AT3 genes in the placental tissue offruits that correlated with pungency, which stronglysuggested it as a candidate to regulate the struc-tural genes of the capsaicinoid pathway. However,CaMYB31 also was expressed in vegetative tissuessuch as roots, leaves, and stems, indicating a possiblerole in more than one biological process, such as ligninbiosynthesis, or other metabolites derived from thephenylpropanoid pathway.

Figure 6. Effects of light onCaMYB31,Kas, andpAmt expression in placental tissue from chilipepper fruits at 30 DPA. Gene expression wasmeasured at different incubation times (3, 6, 12,and 16 h). The data points are means of threebiological replicates 6 SD. Asterisks show sig-nificant differences between fruits incubatedunder dark and light conditions (P # 0.05;Tukey’s test).





Bioinformatic Analysis of CaMYB31

Bioinformatic analysis of R2R3-MYB proteins showedthat the putative CaMYB31 protein was closer to un-known function proteins of chili pepper, tomato (Solanumlycopersicum), potato (Solanum tuberosum), and to-bacco (Nicotiana tabacum). Moreover, subgroup 14 ofMYB Arabidopsis proteins was the nearest clade toCaMYB31, which shared one motif in the C-terminalregion and, therefore, might have some similarfunctions (lignin biosynthesis, stress and plant hor-mone response, or development). The lignin andcapsaicinoid biosynthetic pathways have commonsteps at early stages of the phenylpropanoid bio-synthesis, suggesting that CaMYB31 is probablyimplicated in the early regulation of the capsaicinoidpathway. Additionally, we found putative MYB-binding sites on all the predicted promoters of thestructural genes of the capsaicinoid pathway, sug-gesting that they are potential target genes ofCaMYB31.

Effects of CaMYB31 Silencing on the Expression ofCapsaicinoid Biosynthesis-Related Genes andCapsaicinoid Content

Virus-induced gene silencing was used to investigatethe function of CaMYB31 in the capsaicinoid pathway.CaMYB31 silencing caused a significant decrease incapsaicinoid accumulation and on the expression of allstructural genes of the phenylpropanoid pathway, ex-cept that of Pal, and also on the early genes of thebranched-chain fatty acid pathway (BCAT, BCKDH,Kas, and Acl) of capsaicinoid biosynthesis, showingstrong evidence of its participation in the regulation ofthis metabolic process. Based on these results, we pro-pose that the structural genes that showed a significantdecrease in expression when CaMYB31 was silencedare potential target genes of CaMYB31. Future studiesof the CaMYB31 interaction with promoters of thestructural geneswhose expressionwas affected shouldprovide more evidence of the direct transcriptionaleffect.

Figure 7. Effects of temperature on CaMYB31,Kas, and pAmt expression in placental tissuefrom chili pepper fruits at 30 DPA. Gene ex-pression was quantified at different incubationtimes (3, 6, 12, and 16 h) and different tem-peratures (4°C, 25°C, and 37°C). The datapoints are means of three biological repli-cates 6 SD. Asterisks show significant differ-ences between fruits incubated at standardtemperature (25°C) and high temperature(37°C) or low temperature (4°C; P # 0.05;Tukey’s test).





Effects of Hormones, Light, Temperature, and Woundingon CaMYB31 Expression

The biosynthesis and accumulation of capsaicinoidsin chili pepper fruits is sensitive to environmental fac-tors such as temperature and light, to stress conditions(wounding and drought), and also to plant hormones(Lindsey and Bosland, 1996; Suresh and Ravishankar,2005; Kim et al., 2009; Gutiérrez-Carbajal et al., 2010).For this reason, we analyzed the effects of plant hor-mones and stress on CaMYB31 expression and two ofthe possible target genes: Kas and pAmt.In previous studies, it was reported that SA induced

the production of capsaicinoids and intermediarycompounds (Gutiérrez-Carbajal et al., 2010; Altúzar-Molina et al., 2011; Rodas-Junco et al., 2013) while JAinduced or repressed the accumulation of capsaicinoidsand intermediary metabolites depending on the con-centration and exposure time (Suresh and Ravishankar,2005; Gutiérrez-Carbajal et al., 2010; Altúzar-Molinaet al., 2011). CaMYB31, Kas, and pAmt expression wasincreased significantly with SA treatment, while JA

treatment caused a significant decrease in its expres-sion, except at 3 h of exposure. In general, a typicalantagonistic effect between SA and JA treatment wasrevealed, similar to that observed in genes related todefense against pathogens.

Wounding stress has been reported to promote theformation of JA while repressing SA production (Leeet al., 2004). A significant decrease in CaMYB31, Kas, andpAmt expression in wounded chili pepper fruits was ob-served consistently with JA treatment, except at 3 h.

Other hormones, such as GA3 and auxin, interactwith the SA and JA pathways, increasing the com-plexity of the signaling pathway in the mechanism ofdefense in plants (Robert-Seilaniantz et al., 2011; Pieterseet al., 2012). CaMYB31, Kas, and pAmt expression wasincremented significantly under IAA and GA3 treatment.It has been reported that GAs suppress JA signaling andinduce SA signaling (Pieterse et al., 2012), consistent withour results. Conversely, it has been reported that the auxinresponse was antagonistic to the SA response (Pieterseet al., 2012); however, we did not observe an antagonistic

Figure 8. Effects of wounding on CaMYB31,Kas, and pAmt expression in placental tissuefrom chili pepper fruits at 30 DPA. Gene ex-pression was quantified at different incubationtimes (3, 6, 12, and 16 h). The data points aremeans of three biological replicates 6 SD. As-terisks show significant differences betweencontrol (no injuries) and wounded fruits (P #

0.05; Tukey’s test).





effect on the expression of CaMYB31, Kas, and pAmt. Theconcentration of the plant hormone and exposure timehighly influence gene expression.

Temperature is another factor that positively ornegatively affects the level of pungency in chili pepperfruits and the expression of capsaicinoid structuralgenes (Kim et al., 2009; Rahman and Inden, 2012; González-Zamora et al., 2013). The expression of CaMYB31, Kas, andpAmtwas increased significantly at 4°C and was reducedat 37°C compared with 25°C; thus, cv Tampiqueño74 perhaps is a variety in which the accumulation ofcapsaicinoids is negatively affected by high temperature.

Light intensity has been found to exert a significanteffect on capsaicinoid content in chili pepper fruits(Iwai et al., 1979; Gangadhar et al., 2012). Kim et al.(2009) reported that the enzymatic activity of AT3 wasinduced by exposure to light and repressed in the dark.Similarly, the expression of CaMYB31, Kas, and pAmtwas significantly higher under light compared withdark conditions in our investigation.

CONCLUSION

Our work showed strong evidence of the involve-ment of a MYB transcription factor in the regulation ofcapsaicinoid biosynthesis and its stress response in chilipepper fruits. Future studies are necessary to investigatewhether CaMYB31 acts directly on the promoter ofstructural genes of the capsaicinoid pathway or throughthe formation of a protein complex with other transcrip-tion factors.

MATERIALS AND METHODS

Plant Growth Conditions

Chili pepper (Capsicum annuum) ‘Tampiqueño 74’ (Serrano type) and ‘Cal-iforniaWonder’ (bell pepper type) plants were grown in greenhouse conditionsand fertilized every 2 weeks with FerviaFol (Agroquímicos Rivas) solution(N:P:K, 30:20:10). Chili pepper fruits were harvested at 10, 20, 30, 40, 50, and60 DPA from at least six plants. Only placental tissue was collected and wasimmediately frozen in liquid nitrogen, stored at280°C, and used for qRT-PCRexpression analysis to select MYB gene candidates.

Figure 9. Effects of plant hormones onCaMYB31, Kas, and pAmt expression in placental tissue from chili pepper fruits at 30DPA.Gene expression was quantified at different incubation times (3, 6, 12, and 16 h) in fruits treatedwith 100 mM IAA (A), GA3 (B), SA(C), or JA (D). The data points are means of three biological replicates6 SD. Asterisks show significant differences between control(MS medium) and treated fruits (P # 0.05; Tukey’s test).





The cv Tampiqueño 74 and cv California Wonder and Capsicum chinense‘Habanero’ BG-3821 plants were grown for differential expression analysis indifferent organs. Chili pepper fruits were harvested at 10, 20, 40, and 60 DPAfrom C. annuum, and immature and mature stages from C. chinense were dis-sected to separate the seeds, placenta, and pericarp, which were storedseparately. Roots, leaves, stems, and flowers of adult plants from the dif-ferent genotypes of C. annuum were collected. All tissues were frozen andstored at 280°C.

Seeds of cv Tampiqueño 74 were germinated in a growth chamber at atemperature of 28°C under a 16-h photoperiod, a photon flux of 70 mmol m22 s21

(T8W/Starcoat GE fluorescent lamps), and a relative humidity of 66% for si-lencing experiments. Three-week-old chili pepper seedlings were agroinfectedand, after 6 weeks, were transferred to greenhouse conditions until 40-DPA chilipepper fruits were collected for each treatment.

Seeds of cv Tampiqueño 74 were germinated and grown in a greenhouse forthe studies of environmental stimuli in entire fruits. Placental tissue was col-lected from 30-DPA chili pepper fruits for each treatment.

Identification and Isolation of the CaMYB31 Gene

Weselected24MYBpartial sequences fromacDNAlibraryof cvTampiqueño74 with more than 40% identity to MYB proteins (Supplemental Table S1). Theexpression of these MYB genes was analyzed by qRT-PCR, comparatively withthe expression of the capsaicinoid biosynthetic genesAT3,Kas, pAmt,Ca4H, andComt, in placental tissue of fruits at different developmental stages (10, 20, 30,40, 50, and 60 DPA) of cv Tampiqueño 74 (pungent) and cv California Wonder(nonpungent). Moreover, the levels of capsaicin and dihydrocapsaicin werequantified at the same stages of development in pungent and nonpungentfruits. The CaMYB31 gene was selected as the candidate based on the positivecorrelation of its expression pattern with capsaicinoid content and the expres-sion levels of the structural genes.

The full-length CaMYB31 gene was predicted using the GeneScan Webserver from the pepper genome (Qin et al., 2014), and primers were designed(forward, 59-TACACGTGATGGTGAGAACAACACCTTGCT-39, and reverse,59-CGCACGTGTTAATAATTAAAATGATCAAA-39) for amplification by re-verse transcription-PCR from placental tissue of 40-DPA chili pepper fruits ofcv Tampiqueño 74. The PCR conditions for amplification were 94°C for 3 min,followed by 30 cycles (94°C for 30 s, 57.5°C for 30 s, and 72°C for 1 min), and72°C for 7 min. The PCR product was diluted (1 mL of PCR product with 19 mLof sterile water) and used as a template for a second PCR under the sameconditions. The PCR product was cloned into the pCR 8 vector (Invitrogen)according to the manufacturer’s instructions and sequenced.

Phylogenetic and Sequence Analysis

BLASTpwas used to searchMYBprotein sequences from theRefSeq_proteinand Swiss-Prot National Center for Biotechnology Information databases usingthe full-length amino acid sequence of CaMYB31. The characterized Solanaceaeproteins (SlMYB12, SlBLIND,CaBLIND,CaMYB,CaMYB1,CaA, andCaMYB3)and all the Arabidopsis (Arabidopsis thaliana) MYB proteins reported by Strackeet al. (2001) and at the IT3F Web site (Bailey et al., 2008) also were included(Supplemental Table S2). The sequences were aligned with ClustalW usingdefault parameters, and it was manually adjusted. Based on the MYB domainalignment, a phylogenetic tree was constructed with the neighbor-joiningmethod, model JTT+G, and a bootstrap test (1,000 replicates) using MEGA6software. In addition, we constructed a phylogenetic tree using the IT3F Website with CaMYB31 and the MYB proteins of Arabidopsis. The clades weregrouped as reported previously (Kranz et al., 1998; Stracke et al., 2001). MEMEversion 4.11.3 (http://meme-suite.org/tools/meme) was used to discoverputative motifs in the C-terminal region of CaMYB31. PlantPAN 2.0 (Chowet al., 2016) was used to searchMYB-binding sites in the predictive promoters ofthe structural genes of the capsaicinoid biosynthesis pathway.

Virus-Induced Gene Silencing of the CaMYB31 Gene

A 234-bp fragment of CaMYB31 was amplified (bases 269–503) using theprimers forward (59-AGTCTAGATCCTTTGGTGACCATTCTGA-39) and re-verse (59-AGTCTAGAGGCGATTGCTGCTCACTT-39). Amplification condi-tions were 94°C for 5min, followed by 30 cycles (94°C for 30 s, 55°C for 30 s, and72°C for 1 min), and extension at 72°C for 7 min. CaMYB31 viral silencingplasmid construction and agroinfiltration were carried out as reported for

tomato (Solanum lycopersicum; Liu et al., 2002), with some modifications (Arce-Rodríguez and Ochoa Alejo, 2015).

Quantification of Capsaicinoids

Capsaicin and dihydrocapsaicin were quantified in placental tissue from cvTampiqueño 74 (pungent) and cv CaliforniaWonder (nonpungent) chili pepperfruits in three biological samples composed of 10 placentas each in the case of10-DPA fruits and three placentas for the 20-, 30-, 40-, 50-, and 60-DPA fruits.The extraction and quantification of capsaicin and dihydrocapsaicin were car-ried out as reported by Arce-Rodríguez and Ochoa-Alejo (2015).

Plant Hormones, Light, Temperature, and Wounding ChiliPepper Fruit Treatments

Chili pepper fruits of cv Tampiqueño 74 at 30DPAwere collected to evaluatethe effect of different factors on the expression ofCaMYB31 and the capsaicinoidbiosynthetic genes Kas and pAmt for 3, 6, 12, and 16 h. For light/dark responseassays, complete fruits were first wrapped with plastic film and foil for 24 h andthenwereuncovered and exposed tofluorescent light conditions (50mmolm22 s21).For temperature treatments, entire fruits were wrapped with plastic film andfoil and incubated at 4°C, 25°C, and 37°C. For the wounding responses ex-periment, a scalpel blade was introduced approximately 6 mm deep severaltimes into whole fruits, which were then covered with plastic film and incu-bated at 25°C under standard light conditions. To apply plant hormone treat-ments, placental tissue was separated from 30-DPA fruits and submerged intoMS (Murashige and Skoog, 1962) liquid medium containing 0 or 100 mM JA, SA,GA3, or IAA (Sigma) and incubated at 25°C under standard light conditions.After incubation, the placental tissues from all treatments were immediatelyfrozen in liquid nitrogen and stored at 280°C for gene expression analysis.

Total RNA Extraction and cDNA Synthesis

Total RNA was extracted from placenta, pericarp, seeds, flowers, stems,leaves, and roots with Trizol (Invitrogen) according to the protocol provided bythe manufacturer. The extraction was performed in triplicate for each sample.RNA was purified with the PureLink Micro-To-Midi kit (Invitrogen) accordingto the instructions. cDNAwas synthesized from purified RNAwith SuperScriptII or SuperScript III reverse transcriptase (Invitrogen) following the protocolprovided by the manufacturer.

Real-Time Quantitative PCR Assays

Total RNA was extracted, purified, and treated with DNase I (Invitrogen)following the manufacturer’s instructions. cDNA was synthesized withSuperScript III reverse transcriptase (Invitrogen) and adjusted to 100 ng mL21.qRT-PCR was performed as reported by Arce-Rodríguez and Ochoa-Alejo(2015) using the primer pairs described in Supplemental Table S3.

Statistical Analysis

Thedata generated in silencing experiments and the effect of different factorson the expression ofCaMYB31were subjected toANOVAwith Tukey’s test (P#

0.05) to find statistically significant differences between the mean values.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. qRT-PCR expression assays of the putative MYBtranscription factor genes in placental tissue from chili pepper fruits of cvTampiqueño 74 and cv California Wonder at different developmental stages.

Supplemental Figure S2. Phylogenetic relationship of CaMYB31 withMYB transcription factors of Arabidopsis.






http://meme-suite.org/tools/meme





Supplemental Figure S3. Alignment of amino acid sequences of CaMYB31with others plant R2R3-MYB proteins.

Supplemental Figure S4. Schematic diagram of the putative MYB-bindingsite localizations in the predicted promoter of capsaicinoid structuralgenes.

Supplemental Table S1. Sequences from the cDNA library of cv Tampi-queño 74 (Serrano type) for qRT-PCR analysis.

Supplemental Table S2. Accession numbers of MYB proteins listed inFigure 3.

Supplemental Table S3. Primers used for qRT-PCR analysis.

ACKNOWLEDGMENTS

We thank Yolanda Rodríguez for technical assistance in the HPLC analysis;Dr. Cesar Aza-González, Dr. María del Rocío Gomez-García, and José LuisPablo-Rodríguez for technical assistance in the agroinfiltration experiments;and Dr. Luis Delaye, Dr. Octavio Martínez de la Vega, and Magdalena Riverafor their important insights in bioinformatic analysis.

Received April 12, 2017; accepted May 4, 2017; published May 8, 2017.

LITERATURE CITED

Altúzar-Molina AR, Muñoz-Sánchez JA, Vázquez-Flota F, Monforte-González M, Racagni-Di Palma G, Hernández-Sotomayor SMT (2011)Phospholipidic signaling and vanillin production in response to salicylicacid and methyl jasmonate in Capsicum chinense J. cells. Plant PhysiolBiochem 49: 151–158

Aluru MR, Mazourek M, Landry LG, Curry J, Jahn M, O’Connell MA(2003) Differential expression of fatty acid synthase genes, Acl, Fat andKas, in Capsicum fruit. J Exp Bot 54: 1655–1664

Ambawat S, Sharma P, Yadav NR, Yadav RC (2013) MYB transcriptionfactor genes as regulators for plant responses: an overview. Physiol MolBiol Plants 19: 307–321

Arce-Rodríguez ML, Ochoa-Alejo N (2015) Silencing AT3 gene reduces theexpression of pAmt, BCAT, Kas, and Acl genes involved in capsaicinoidbiosynthesis in chili pepper fruits. Biol Plant 59: 477–484

Aza-González C, Núñez-Palenius HG, Ochoa-Alejo N (2011) Molecularbiology of capsaicinoid biosynthesis in chili pepper (Capsicum spp.).Plant Cell Rep 30: 695–706

Bailey PC, Dicks J, Wang TL, Martin C (2008) IT3F: a web-based tool forfunctional analysis of transcription factors in plants. Phytochemistry 69:2417–2425

Chow CN, Zheng HQ, Wu NY, Chien CH, Huang HD, Lee TY, Chiang-Hsieh YF, Hou PF, Yang TY, Chang WC (2016) PlantPAN 2.0: an updateof plant promoter analysis navigator for reconstructing transcriptionalregulatory networks in plants. Nucleic Acids Res 44: D1154–D1160

Curry J, Aluru M, Mendoza M, Nevarez J, Melendrez M, O’Connell MA(1999) Transcripts for capsaicinoids biosynthetic genes are differentiallyaccumulated in pungent and non-pungent Capsicum spp. Plant Sci 148:47–57

Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L(2010) MYB transcription factors in Arabidopsis. Trends Plant Sci 15: 573–581

Gangadhar BH, Mishra RK, Pandian G, Park SW (2012) Comparativestudy of color, pungency, and biochemical composition in chili pepper(Capsicum annuum) under different light-emitting diode treatments.HortScience 47: 1729–1735

González-Zamora A, Sierra-Campos E, Luna-Ortega JG, Pérez-Morales R,Rodríguez Ortiz JC, García-Hernández JL (2013) Characterization ofdifferent Capsicum varieties by evaluation of their capsaicinoids contentby high performance liquid chromatography, determination of pun-gency and effect of high temperature. Molecules 18: 13471–13486

Gutiérrez-Carbajal MG, Monforte-González M, Miranda-Ham ML,Godoy-Hernández G, Vázquez-Flota F (2010) Induction of capsaicinoidsynthesis in Capsicum chinense cell cultures by salicylic acid or methyljasmonate. Biol Plant 54: 430–434

Humphreys JM, Chapple C (2002) Rewriting the lignin roadmap. CurrOpin Plant Biol 5: 224–229

Iwai K, Suzuki T, Fujiwake H (1979) Formation and accumulation ofpungent principle of hot pepper fruits, capsaicin and its analogues, inCapsicum annuum var. annuum cv. Karayatsubusa at different growthstages after flowering. Agric Biol Chem 43: 2493–2498

Keyhaninejad N, Curry J, Romero J, O’Connell MA (2014) Fruit specificvariability in capsaicinoid accumulation and transcription of structuraland regulatory genes in Capsicum fruit. Plant Sci 215-216: 59–68

Kim M, Kim S, Kim S, Ki BD (2001) Isolation of cDNA clones differentiallyaccumulated in the placenta of pungent pepper by suppression sub-tractive hybridization. Mol Cells 11: 213–219

Kim JS, Park M, Lee DJ, Kim BD (2009) Characterization of putativecapsaicin synthase promoter activity. Mol Cells 28: 331–339

Kranz HD, Denekamp M, Greco R, Jin H, Leyva A, Meissner RC, PetroniK, Urzainqui A, Bevan M, Martin C, et al (1998) Towards functionalcharacterisation of the members of the R2R3-MYB gene family fromArabidopsis thaliana. Plant J 16: 263–276

Lee A, Cho K, Jang S, Rakwal R, Iwahashi H, Agrawal GK, Shim J, Han O(2004) Inverse correlation between jasmonic acid and salicylic acidduring early wound response in rice. Biochem Biophys Res Commun318: 734–738

Lindsey K, Bosland PW (1996) A field study of environmental interactionon pungency. Capsicum Eggplant Newslett 14: 36–38

Liu J, Osbourn A, Ma P (2015) MYB transcription factors as regulators ofphenylpropanoid metabolism in plants. Mol Plant 8: 689–708

Liu S, Li W, Wu Y, Chen C, Lei J (2013) De novo transcriptome assembly inchili pepper (Capsicum frutescens) to identify genes involved in the bio-synthesis of capsaicinoids. PLoS ONE 8: e48156

Liu Y, Schiff M, Dinesh-Kumar SP (2002) Virus-induced gene silencing intomato. Plant J 31: 777–786

Mazourek M, Pujar A, Borovsky Y, Paran I, Mueller L, Jahn MM (2009) Adynamic interface for capsaicinoid systems biology. Plant Physiol 150:1806–1821

Murashige T, Skoog F (1962) A revised medium for rapid growth andbioassays with tobacco tissue cultures. Physiol Plant 15: 473–497

Ochoa-Alejo N, Ramírez-Malagón R (2001) In vitro chili pepper biotech-nology. In Vitro Cell Dev Biol Plant 37: 730–741

Patzlaff A, McInnis S, Courtenay A, Surman C, Newman LJ, Smith C,Bevan MW, Mansfield S, Whetten RW, Sederoff RR, et al (2003)Characterisation of a pine MYB that regulates lignification. Plant J 36:743–754

Pieterse CMJ, Van der Does D, Zamioudis C, Leon-Reyes A, Van WeesSCM (2012) Hormonal modulation of plant immunity. Annu Rev CellDev Biol 28: 489–521

Qin C, Yu C, Shen Y, Fang X, Chen L, Min J, Cheng J, Zhao S, Xu M, LuoY, et al (2014) Whole-genome sequencing of cultivated and wild peppersprovides insights into Capsicum domestication and specialization. ProcNatl Acad Sci USA 111: 5135–5140

Rahman MJ, Inden H (2012) Effect of nutrient solution and temperature oncapsaicin content and yield contributing characteristics in six sweetpepper (Capsicum annuum L.) cultivars. J Food Agric Environ 10: 524–529

Robert-Seilaniantz A, Grant M, Jones JDG (2011) Hormone crosstalk inplant disease and defense: more than just jasmonate-salicylate antago-nism. Annu Rev Phytopathol 49: 317–343

Rodas-Junco BA, Cab-Guillén Y, Muñoz-Sánchez JA, Vázquez-Flota F,Monforte-González M, Hernández-Sotomayor SMT (2013) Salicylicacid induces vanillin synthesis through the phospholipid signalingpathway in Capsicum chinense cell cultures. Plant Signal Behav 8: doi/10.4161/psb.26752

Stewart C Jr, Kang BC, Liu K, Mazourek M, Moore SL, Yoo EY, Kim BD,Paran I, Jahn MM (2005) The Pun1 gene for pungency in pepper encodesa putative acyltransferase. Plant J 42: 675–688

Stracke R, Werber M, Weisshaar B (2001) The R2R3-MYB gene family inArabidopsis thaliana. Curr Opin Plant Biol 4: 447–456

Suresh B, Ravishankar GA (2005) Methyl jasmonate modulated biotrans-formation of phenylpropanoids to vanillin related metabolites usingCapsicum frutescens root cultures. Plant Physiol Biochem 43: 125–131

Tamagnone L, Merida A, Parr A, Mackay S, Culianez-Macia FA, RobertsK, Martin C (1998) The AmMYB308 and AmMYB330 transcription fac-tors from Antirrhinum regulate phenylpropanoid and lignin biosynthesisin transgenic tobacco. Plant Cell 10: 135–154

Zhou J, Lee C, Zhong R, Ye ZH (2009) MYB58 and MYB63 are transcrip-tional activators of the lignin biosynthetic pathway during secondarycell wall formation in Arabidopsis. Plant Cell 21: 248–266










Documents

An R2R3-MYB Transcription Factor Regulates Capsaicinoid ... · Comt, Ca4H, and CaMYB31 in placental tissue from fruits of cv Tampiquen˜o 74 and cv California Wonder at different