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

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

Metabolite biodiversity in pepper (Capsicum) fruits of thirty-two diverse accessions: variation in health-related compounds and implications for breeding

A comprehensive study on morphology and biochemical compounds of 32 Capsicum spp. accessions has been performed. Accessions represented four pepper species, Capsicum annuum, Capsicum frutescens, Capsicum chinense and Capsicum baccatum which were selected by their variation in morphological characters such as fruit color, pungency and origin. Major metabolites in fruits of pepper, carotenoids, capsaicinoids (pungency), flavonoid glycosides, and vitamins C and E were analyzed and quantified by high performance liquid chromatography. The results showed that composition and level of metabolites in fruits varied greatly between accessions and was independent of species and geographical location. Fruit color was determined by the accumulation of specific carotenoids leading to salmon, yellow, orange, red and brown colored fruits. Levels of both O- and C-glycosides of quercetin, luteolin and apigenin varied strongly between accessions. All non-pungent accessions were devoid of capsaicins, whereas capsaicinoid levels ranged from 0.07 up to 80 mg/100g fr. wt. in fruit pericarp. In general, pungent accessions accumulated the highest capsaicinoid levels in placenta plus seed tissue compared to pericarp. The non-pungent capsaicinoid analogs, capsiates, could be detected at low levels in some pungent accessions. All accessions accumulated high levels of vitamin C, up to 200 mg/100g fr. wt. The highest vitamin E concentration found was 16 mg/100g fr. wt. Based on these metabolic data, five accessions were selected for further metabolic and molecular analysis, in order to isolate key genes involved in the production of these compounds and to assist future breeding programs aimed at optimizing the levels of health-related compounds in pepper fruit.     

Biosynthesis of capsinoid is controlled by the Pun1 locus in pepper

Pungency in pepper (Capsicum annuum L.) has unique characteristics due to the alkaloid compound group, capsaicinoids, which includes capsaicin. Although capsaicinoids have been proved to have pharmacological and physiological effects on human health, the application of capsaicinoids has been limited because of their pungency. Capsinoids found in non-pungent peppers share closely related structures with capsaicinoids and show similar biological effects. Previous studies demonstrated that mutations in the p-AMT gene were related to the production of capsinoids; however, the pathway of capsinoid synthesis has not yet been fully elucidated. In this study, we performed genetic analysis to determine the mechanism of capsinoid synthesis using a F6 recombinant inbred line population. In this population, the presence/absence of capsinoids co-segregated with the genotype of the Pun1 locus, without exception. In addition, we screened the patterns of capsinoid synthesis and the correlation between the Pun1 locus and capsinoid synthesis in p-AMT mutant accessions. In Capsicum germplasms, we selected amino-acid-substituted mutants in the PLP binding domain of the p-AMT gene. Capsinoids were not synthesized with the recessive pun1 gene, regardless of the p-AMT genotype, and no relationship was found between p-AMT mutant type and capsinoid content. We concluded that the Pun1 gene, which is responsible for capsaicinoid synthesis, also controls capsinoid synthesis.     

Molecular mapping of the C locus for presence of pungency in Capsicum.

Pungency owing to the presence of capsaicinoids is a unique character of pepper (Capsicum spp.). Capsaicinoids are produced in the placenta and it has long been known that a single dominant gene, C, is required for pungent genotypes to produce capsaicinoids. We mapped C to pepper chromosome 2 in a cross between a pungent Capsicum frutescens wild accession and a non-pungent Capsicum annuum bell pepper. This position confirmed results from earlier studies. The RFLP marker TG 205 cosegregated with C and two additional RFLP markers were also located within 1 cM. The recessive allele at the C locus is used in breeding programs around the world focused on very diverse germplasm, hence any of these tightly linked markers may be of value as potential sources of useful markers for marker-assisted selection. To demonstrate this point, we developed a PCR-based CAPS (cleaved amplified polymorphic sequence) marker linked to C using the sequence of the Capsicum fibrillin gene located 0.4 cM from C. The use of molecular markers for high-throughput screening for the c allele in pepper breeding programs is discussed.     

Identification of Gene-Specific Polymorphisms and Association with Capsaicin Pathway Metabolites in Capsicum annuum L. Collections

Pepper (Capsicum annuum L.) is an economically important crop with added nutritional value. Production of capsaicin is an important quantitative trait with high environmental variance, so the development of markers regulating capsaicinoid accumulation is important for pepper breeding programs. In this study, we performed association mapping at the gene level to identify single nucleotide polymorphisms (SNPs) associated with capsaicin pathway metabolites in a diverse Capsicum annuum collection during two seasons. The genes Pun1, CCR, KAS and HCT were sequenced and matched with the whole-genome sequence draft of pepper to identify SNP locations and for further characterization. The identified SNPs for each gene underwent candidate gene association mapping. Association mapping results revealed Pun1 as a key regulator of major metabolites in the capsaicin pathway mainly affecting capsaicinoids and precursors for acyl moieties of capsaicinoids. Six different SNPs in the promoter sequence of Pun1 were found associated with capsaicin in plants from both seasons. Our results support that CCR is an important control point for the flux of p-coumaric acid to specific biosynthesis pathways. KAS was found to regulate the major precursors for acyl moieties of capsaicinoids and may play a key role in capsaicinoid production. Candidate gene association mapping of Pun1 suggested that the accumulation of capsaicinoids depends on the expression of Pun1, as revealed by the most important associated SNPs found in the promoter region of Pun1.     

Analysis of capsaicinoid biosynthesis pathway genes expression in callus cultures of Capsicum chinense Jacq. cv. ‘Umorok’

The placental tissue of the highly pungent chilli cultivar, Capsicum chinense Jacq. cv. ‘Umorok’, is used as explants for callus induction. Callus cultures were subcultured after every 32 days and growth curves for a period of six consecutive growth cycles were studied till a stable capsaicinoids producing callus cultures were obtained. The capsaicinoids content in placental tissue explants decreased gradually during the first 2 months of culture as the explants dedifferentiated to form friable callus while the biomass and capsaicinoid content did not show much change in the subsequent growth cycles. The maximum callus biomass of 7.8 g freshweight (FW) or 0.56 g dry weight (DW) per culture were obtained on the 24th day of every growth cycle and the maximum average capsaicinoids content (1.6 mg g^?1 FW capsaicin and 0.78 mg g^?1 FW dihydrocapsaicin) were obtained on the 20th day of every growth cycle. To investigate the underlying dynamics for capsaicinoid biosynthesis during callus formation, comparative gene expression analysis of the genes involved in capsaicinoid biosynthesis pathway were also studied by qRT-PCR analysis. When compared with placental tissue, all the studied genes showed reduced expression during callus formation, especially putative aminotransferase (pAMT) and pungent gene 1 (Pun1), which were extensively down regulated from the 3rd month onwards in the callus cultures. Therefore, the present study revealed that the down-regulated expression of mainly two putative genes in capsaicinoid biosynthetic pathway (pAMT and Pun1) resulted in lower accumulation of capsaicinoids in callus cultures compared to placental tissues of fruits.

     

Virus-induced silencing of <Emphasis Type="Italic">Comt</Emphasis>, p<Emphasis Type="Italic">Amt</Emphasis> and <Emphasis Type="Italic">Kas</Emphasis> genes results in a reduction of capsaicinoid accumulation in chili pepper fruits

Capsaicinoids are responsible for the pungent taste of chili pepper fruits of Capsicum species. Capsaicinoids are biosynthesized through both the phenylpropanoid and the branched-fatty acids pathways. Fragments of Comt (encoding a caffeic acid O-methyltransferase), pAmt (a putative aminotransferase), and Kas (a β-keto-acyl-[acyl-carrier-protein] synthase) genes, that are differentially expressed in placenta tissue of pungent chili pepper, were individually inserted into a Pepper huasteco yellow veins virus (PHYVV)-derived vector to determine, by virus-induced gene silencing, irrespective of whether these genes are involved in the biosynthesis of capsaicinoids. Reduction of the respective mRNA levels as well as the presence of related siRNAs confirmed the silencing of these three genes. Morphological alterations were evident in plants inoculated with PHYVV::Comt and PHYVV::Kas constructs; however, plants inoculated with PHYVV::pAmt showed no evident alterations. On the other hand, fruit setting was normal in all cases. Biochemical analysis of placenta tissues showed that, indeed, independent silencing of all three genes led to a dramatic reduction in capsaicinoid content in the fruits demonstrating the participation of these genes in capsaicinoid biosynthesis. Using this approach it was possible to generate non-pungent chili peppers at high efficiency.     

A versatile PCR marker for pungency in Capsicum spp.

Pungency in Capsicum spp. is an important quality trait for pepper breeding. The perception of pungency in pepper is due to the presence of a group of compounds named capsaicinoids, only found within the Capsicum genus. How pungency is controlled at genetic and molecular levels has not been completely elucidated. The use of molecular markers to assess pungency trait is required for molecular breeding, despite the difficulty of development of universal markers for this trait. In this work, a DNA sequence possibly related to pungency with a high similarity to Pun1 locus was studied, and sequence analysis of this homolog revealed a 15?bp deletion in non-pungent pepper accessions. An allele-specific pair of primers was designed and specific fragments of 479?bp from non-pungent and 494?bp from pungent accessions were obtained. Polymorphism of this marker, named MAP1, was tested in a wide range of accessions, belonging to several Capsicum species, including pungent and non-pungent accessions of C. annuum L., and pungent accessions of C. chinense, C. baccatum, C. frutescens, C. pubescens, C. galapagoense, C. eximium, C. tovarii, C. cardenasii, and C. chacoense. All these Capsicum accessions were correctly discriminated. The marker suitability to assess pungency in domesticated and wild Capsicum species was demonstrated, and therefore it will be very useful in marker assisted selection (MAS). Moreover, MAP1 was located in a saturated pepper linkage map and its possible relationship with the Pun1 locus has been discussed. Among the available markers for this complex quality trait, the marker developed in this study is the most universal so far.     

Development and application of a suite of non-pungency markers for the Pun1 gene in pepper (Capsicum spp.)

Pungency in peppers is due to the presence of capsaicinoid molecules, which are only produced in Capsicum species. The major gene Pun1 is required for the production of capsaicinoids. Three distinct mutant alleles of Pun1 have been found in three cultivated Capsicum species, one of which has been widely utilized by breeders. Although these mutations have been previously identified, a robust collection of molecular markers for the set of alleles is not available. This has been hindered by the existence of at least one paralogous locus that tends to amplify with Pun1. We present a suite of markers that can differentiate the four Pun1 alleles and test them on a diverse panel of pepper lines and in an F2 population segregating for pungency. These markers will be useful for pepper breeding, germplasm characterization, and seed purity testing.     

QTL analysis for capsaicinoid content in Capsicum

Pungency or “heat” found in Capsicum fruit results from the biosynthesis and accumulation of alkaloid compounds known as capsaicinoids in the dissepiment, placental tissue adjacent to the seeds. Pepper cultivars differ with respect to their level of pungency because of quantitative and qualitative variation in capsaicinoid content. We analyzed the segregation of three capsaicinoids: capsaicin, dihydrocapsaicin and nordihydrocapsaicin in an inter-specific cross between a mildly pungent Capsicum annuum ‘NuMex RNaky’ and the wild, highly pungent C. frutescens accession BG2814-6. F₃ families were analyzed in three trials in California and in Israel and a dense molecular map was constructed comprised mostly of loci defined by simple sequence repeat (SSR) markers. Six QTL controlling capsaicinoid content were detected on three chromosomes. One gene from the capsaicinoid biosynthetic pathway, BCAT, and one random fruit EST, 3A2, co-localized with QTL detected in this study on chromosomes 3 and 4. Because one confounding factor in quantitative determination of capsaicinoid is fruit size, fruit weight measurements were taken in two trials. Two QTL controlling fruit weight were detected, however, they did not co-localize with QTL detected for capsaicinoid content. The major contribution to the phenotypic variation of capsaicinoid content (24–42% of the total variation) was attributed to a digenic interaction between a main-effect QTL, cap7.1, and a marker located on chromosome 2 that did not have a main effect on the trait. A second QTL, cap7.2 is likely to correspond to the QTL, cap, identified in a previous study as having pronounced influence on capsaicinoid content.     

Why are not all chilies hot? A trade-off limits pungency

Evolutionary biologists increasingly recognize that evolution can be constrained by trade-offs, yet our understanding of how and when such constraints are manifested and whether they restrict adaptive divergence in populations remains limited. Here, we show that spatial heterogeneity in moisture maintains a polymorphism for pungency (heat) among natural populations of wild chilies (Capsicum chacoense) because traits influencing water-use efficiency are functionally integrated with traits controlling pungency (the production of capsaicinoids). Pungent and non-pungent chilies occur along a cline in moisture that spans their native range in Bolivia, and the proportion of pungent plants in populations increases with greater moisture availability. In high moisture environments, pungency is beneficial because capsaicinoids protect the fruit from pathogenic fungi, and is not costly because pungent and non-pungent chilies grown in well-watered conditions produce equal numbers of seeds. In low moisture environments, pungency is less beneficial as the risk of fungal infection is lower, and carries a significant cost because, under drought stress, seed production in pungent chilies is reduced by 50 per cent relative to non-pungent plants grown in identical conditions. This large difference in seed production under water-stressed (WS) conditions explains the existence of populations dominated by non-pungent plants, and appears to result from a genetic correlation between pungency and stomatal density: non-pungent plants, segregating from intra-population crosses, exhibit significantly lower stomatal density (p = 0.003), thereby reducing gas exchange under WS conditions. These results demonstrate the importance of trait integration in constraining adaptive divergence among populations.     

In Vivo and In Vitro Formation of Dihydrocapsaicin in Sweet Pepper Fruits,Capsicum annuum L. var. grossum

Dihydrocapsaicin, one of pungent principles in Capsicum fruits, was formed and accumulated in sweet pepper fruits after 6 days’ post-harvest ripening under continuous light in a medium containing vanillylamine and isocapric acid. No capsaicinoids were formed in sweet pepper fruits ripened in the dark even in the presence of both vanillylamine and isocapric acid. The capsaicinoid newly formed during the ripening was almost exclusively dihydrocapsaicin, as much as 92.8% of the total capsaicinoids. Dihydrocapsaicin was also formed by cell-free extracts prepared from the sweet pepper fruits in a reaction mixture containing vanillylamine and isocapric acid. Dihydrocapsaicin formed was quantified by TLC, GLC, GC-MS and MF.     

Non-pungent Capsicum contains a deletion in the capsaicinoid synthetase gene, which allows early detection of pungency with SCAR markers

The capsaicinoid synthetase (CS) gene cosegregated perfectly with the C locus, which controls the presence of pungency, in 121 F2 individuals from a cross between 'ECW123R' and 'CM334', both of Capsicum annuum. We concluded that CS and C are tightly linked. Sequence analysis of the genes of four pungent and four non-pungent pepper lines showed that the non-pungent peppers had a 2,529 bp-deletion in the 5' upstream region of CS. We have developed molecular markers of the C locus to detect pungency at the seedling stage. Based on the deleted sequence, we developed five SCAR markers, two of them being codominant. These SCAR markers will be useful for easy, accurate, and early detection of non-pungent individuals in breeding programs.     

Isolation of cDNA clones differentially accumulated in the placenta of pungent pepper by suppression subtractive hybridization

Capsaicinoids responsible for pungency of chili pepper are synthesized exclusively in the placenta tissue of the fruit. As an elementary step in the molecular genetics study of capsaicinoid biosynthesis, a cDNA library was constructed from the placenta of a highly pungent pepper, Capsicum chinense cv. Habanero using the suppression subtractive hybridization (SSH). Thirty-nine cDNA clones from about 400 subtracted clones were selected through dot blot analysis and according to their nucleotides sequence. Sequence information of the chosen clones was evaluated by comparing it with DNA and protein databases. Results showed that the cDNA clones could be divided into 4 groups; cDNAs with similarities in genes encoding metabolic enzymes including acyl transferase and fatty acid alcohol oxidase (Group I), putative cell wall proteins (Group II), biotic and abiotic stress-inducible proteins (Group III), and lastly, cDNAs with no similarity (Group IV). Northern blot analysis was performed to confirm that these clones are differentially expressed in pungent pepper. The results revealed that all cDNA clones were differentially expressed in pungent pepper. In addition, the cDNA clones of Groups I and IV were differentially or preferentially expressed in the placenta of pungent pepper.     

Peroxidases and the metabolism of capsaicin in Capsicum annuum L.

Capsaicinoids are acid amides of C₉ - C₁₁ branched-chain fatty acids and vanillylamine. These compounds are responsible for the pungency of the Capsicum species and of cultivars regarded as hot peppers. Moreover, it has been suggested that these compounds play an ecological role in seed dispersal. Because they are used in the pharmacological, food and pesticide industries, much attention has been paid on knowing how their accumulation is controlled, both in the fruit and in cell cultures. Such control involves the processes of biosynthesis, conjugation and catabolism. Recent progress has been made on the biosynthetic pathway, and several of the genes coding for biosynthetic enzymes have been cloned and expression studies performed. With regard to catabolism, cumulative evidence supports that capsaicinoids are oxidized in the pepper by peroxidases. Peroxidases are efficient in catalyzing in vitro oxidation of both capsaicin and dihydrocapsaicin. These enzymes are mainly located in placental and the outermost epidermal cell layers of pepper fruits, as occurs with capsaicinoids, and some peroxidases are present in the organelle of capsaicinoid accumulation, that is, the vacuole. Hence, peroxidases are in the right place for this function. The products of capsaicin oxidation by peroxidases have been characterized in vitro, and some of them have been found to appear in vivo in the Capsicum fruit. Details on the kinetics and catalytic cycle for capsaicin oxidation by peroxidases are also discussed.     

Evolution of Capsaicinoids in Peter Pepper (Capsicum annuum var. annuum) During Fruit Ripening

The evolution of individual and total contents of capsaicinoids present in Peter peppers (Capsicum annuum var. annuum) at different ripening stages has been studied. Plants were grown in a glasshouse and the new peppers were marked in a temporal space of ten days. The extraction of capsaicinoids was performed by ultrasound‐assisted extraction with MeOH. The capsaicinoids nordihydrocapsaicin (n‐DHC), capsaicin, dihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin were analyzed by ultraperformance liquid chromatography (UHPLC)‐fluorescence and identified by UHPLC‐Q‐ToF‐MS. The results indicate that the total capsaicinoids increase in a linear manner from the first point of harvest at ten days (0.283 mg/g FW) up to 90 days, at which point they reach a concentration of 1.301 mg/g FW. The evolution as a percentage of the individual capsaicinoids showed the initial predominance of capsaicin, dihydrocapsaicin, and n‐DHC. Dihydrocapsaicin was the major capsaicinoid up to day 50 of maturation. After 50 days, capsaicin became the major capsaicinoid as the concentration of dihydrocapsaicin fell slightly. The time of harvest of Peter pepper based on the total capsaicinoids content should be performed as late as possible. In any case, harvesting should be performed before overripening of the fruit is observed.