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.     

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.

     

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.     

Biosynthesis of Acyl Moieties of Capsaicin and its Analogues from Valine and Leucine in Capsicum Fruits

Biosynthetic pathways of acyl moieties of capsaicinoid in intactCapsicum fruits and spheroplasts prepared from placentas ofCapsicum fruits were examined using a radioisotopic technique.In intact Capsicum fruits, L-[U-¹⁴C] valine was incorporatedinto capsaicin and dihydrocapsaicin, the acyl constituents ofwhich are even-number branched chain fatty acids, while L-[U-¹⁴C]leucine was incorporated into nordihydrocapsaicin and homodihydrocapsaicin,which have odd-number branched chain facty acids as the acylmoieties. The intermediates of the odd- and even-number branchedchain fatty acids were identified with GLC/GPC after the spheroplastshad been incubated with L-[U-¹⁴C] valine or L-[U-¹⁴C] leucine.After incubation with L-[U-¹⁴C] valine, isobutyric acid and8-methyl nonanoic acid were detected, while isopentanoic acidand 9-methyl decanoic acid were found after incubation withL-[U-¹⁴C] leucine. The involvement of a-ketoisovalerate or a-ketoisocaproatein the biosynthesis of acyl moieties of capsaicinoid was alsodemonstrated in vitro using cell-free extracts of the placentasof Capsicum fruits. These findings suggest that the acyl moietiesof individual capsaicinoids in Capsicum fruits are synthesizedby pathways similar to those proposed for adipose tissue andbacteria. ¹Formation and Metabolism of Pungent Principle of Capsicum Fruits.Part IX. (Received September 2, 1980; Accepted November 17, 1980)     

Capsicum spp in vitro liquid cell suspensions: A useful system for the production of capsaicinoids and polyphenols

Capsicum are among the most extensively cultivated and consumed plant species in the world, because of their unique pungency, aroma and colour. The typical burning sensation caused by chili peppers is due to the occurrence of a group of alkaloids named capsaicinoids. In the present study, the production of solid callus and cell suspensions from hypocotyl explants of three different chili pepper cultivars (Capsicum annuum L. cv. Mazzolino, Capsicum chinense Jacq. cv. Naga Morich and Pimenta de Neyde), was optimised. In addition, C. chinense cv. Naga Morich cell suspensions were supplemented with biotic elicitors (methyl-jasmonate and chitosan) and with precursors and intermediates of capsaicin biosynthesis (vanillin, phenylalanine and valine), and both cells and media were analysed for capsaicinoid, polyphenol, flavonoid contents and for antioxidant activity. This is the first report regarding capsaicinoid elicitation with pure chitosan and with a combination of precursors of both phenylpropanoid and valine pathways. Overall, the highest capsaicinoid levels were detected in cell extracts from cultures treated with 10 μM methyl-jasmonate and with a combination of phenylalanine and valine amino acids (100 μM each). The present results confirm the possibility of using hypocotyl chili pepper cell suspensions to produce high amounts of health beneficial metabolites.     

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.     

Effect of phenylalanine and phenylpropanoids on the accumulation of capsaicinoids and lignin in cell cultures of chili pepper (<Emphasis Type="Italic">capsicum annuum</Emphasis> L.)

Summary Chili pepper (Capsicum annuum L., cv. Tampique?o 74) cell suspensions were employed to study the influence of phenylalanine and phenylpropanoids on the total production of capsaicinoids, the hot taste compounds of chili pepper fruits. The effect of capsaicinoid precursors and intermediates on the accumulation of lignin as an indicator of metabolic diversion was also investigated. Addition of 100 μM of either phenylalanine, cinnamic or caffeic acids to chili pepper cell cultures did not cause significant increases in total capsaicinoids (expressed as capsaicin content, and calculated as averages of the measured values) during the growth cycle. The highest total capsaicinoid content was recorded in cultures grown in the presence of vanillin (142.61 μg g⁻¹ f.wt.), followed by cells treated with 100 μM vanillylamine (104.88 μg g⁻¹ f.wt.), p-coumaric acid (72.36 μg g⁻¹ f.wt.). and ferulic acid (34.67 μg g⁻¹ f.wt.). Capsaicinoid content for control cells was 13.97 μg g⁻¹ f.wt. Chili pepper cell suspensions cultured in the presence of 100 μM of either phenylalanine, or cinnamic, caffeic, or ferulic acids, or the same concentration, of vanillin and vanillylamine, did not exhibit statistically significant differences in the content of lignin as compared with control cells. However, addition of p-coumaric acid (100 μM) to the cultute medium significantly increased thelignin production (c. 10–15 times the contents of control cells).     

Induction of capsaicinoid synthesis in <Emphasis Type="Italic">Capsicum chinense</Emphasis> cell cultures by salicylic acid or methyl jasmonate

Suspension cultures of Habanero pepper (Capsicum chinense Jacq.) were exposed to salicylic acid or methyl jasmonate to change secondary metabolism. Both treatments led to the accumulation of capsaicinoids and their late biosynthetic intermediate, vanillin. Both elicitors had a positive effect on the activities of phenylalanine ammonia lyase and coumarate O-methyltransferase, but none of them represented the main limiting step for capsaicinoid accumulation since vanillin contents were two orders of magnitude higher than those of capsaicinoids.     

Capsaicinoid profiles are not good chemotaxonomic indicators for Capsicum species

Capsaicinoids have been suggested as an aid in identifying Capsicum species. The distribution of seven capsaicinoids and their chemotaxonomic significance were examined within nearly 200 accessions of six Capsicum species. The seven capsaicinoids were separated and quantified using high-performance liquid chromatography. The capsaicinoid profiles were not consistent when examined within a species, therefore they have limited use as a chemotaxonomic indicator. In addition, the generalization that capsaicin and dihydrocapsaicin are always the major capsaicinoids was not true, exceptions were found for some of the accessions studied.     

Effect of foliar application of salicylic acid,hydrogen peroxide and a xyloglucan oligosaccharide on capsiate content and gene expression associated with capsinoids synthesis in <Emphasis Type="Italic">Capsicum annuum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>

Capsinoids are non-pungent analogues of capsaicinoids in pepper (Capsicum spp). The absence of pungency, in addition to their biological activities similar to that of capsaicinoids such as anti-inflammatory, antimicrobial, and antioxidant properties, makes capsinoids an excellent option for increasing use in human and animal nutrition, as well as health and pharmaceutical industries. There are only few sources of pepper producing capsinoids, and one of them (accession 509–45-1), Capsicum annuum L., is a potential source for increasing capsinoids content using strategies as controlled elicitation during plant production in the greenhouse. In this research we evaluated the effect of weekly and one-day-before-harvest foliar applications of hydrogen peroxide, salicylic acid and a xyloglucan oligosaccharide on the concentration of capsiate in fruits of this pepper accession, as well as the gene expression of phenylalanine ammonia-lyase (pal), putative aminotransferase (pamt), capsaicin synthase (at3) and β-keto acyl synthase (kas). Results showed that the two tested concentrations of H₂O₂ significantly increased capsiate content and gene expression associated with capsaicinoids (pamt, at3 and kas) and the phenylpropanoids (pal) pathways. Plant yield was not affected using this induction strategy. Our results indicated that the pre-harvest and weekly application of hydrogen peroxide and xyloglucan oligosaccharide improved production of capsiate in C. annuum L.     

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.     

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.     

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.     

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.     

Peroxidase-catalyzed oxidation of capsaicinoids: steady-state and transient-state kinetic studies

Capsaicinoids are the pungent compounds in Capsicum fruits (i.e., "hot" peppers). Peroxidases catalyze capsaicinoid oxidation and may play a central role in their metabolism. However, key kinetic aspects of peroxidase-catalyzed capsaicinoid oxidation remain unresolved. Using transient-state methods, we evaluated horseradish peroxidase compound I and II reduction by two prominent capsaicinoids (25 degrees C, pH 7.0). We determined rate constants approaching 2 x 10(7) and 5 x 10(5)M(-1)s(-1) for compound I and compound II reduction, respectively. We also determined k(app) values for steady-state capsaicinoid oxidation approaching 8 x 10(5)M(-1)s(-1) (25 degrees C, pH 7.0). Accounting for stoichiometry, these are in excellent agreement with constants for compound II reduction, suggesting that this reaction governs capsaicinoid-dependent peroxidase turnover. Ascorbate rapidly reduced capsaicinoid radicals, assisting in the determination of the kinetic constants reported. Because ascorbate accumulates in Capsicum fruits, it may also be an important determinant for capsaicinoid content and preservation in Capsicum fruits and related products.