Pepper Fruit Development Research Articles

Morphological, histological and transcriptomic mechanisms underlying different fruit shapes in Capsicum spp.

Pepper (Capsicum spp.) has a long domestication history and has accumulated diverse fruit shape variations. The illustration of the mechanisms underlying different fruit shape is not only important for clarifying the regulation of pepper fruit development but also critical for fully understanding the plant organ morphogenesis. Thus, in this study, morphological, histological and transcriptional investigations have been performed on pepper accessions bearing fruits with five types of shapes. From the results it can be presumed that pepper fruit shape was determined during the developmental processes before and after anthesis, and the anthesis was a critical developmental stage for fruit shape determination. Ovary shape index variations of the studied accessions were mainly due to cell number alterations, while, fruit shape index variations were mainly attributed to the cell division and cell expansion variations. As to the ovary wall thickness and pericarp thickness, they were regulated by both cell division in the abaxial-adaxial direction and cell expansion in the proximal-distal and medio-lateral directions. Transcriptional analysis discovered that the OFP-TRM and IQD-CaM pathways may be involved in the regulation of the slender fruit shape and the largest ovary wall cell number in the blocky-shaped accession can be attributed to the higher expression of CYP735A1, which may lead to an increased cytokinin level. Genes related to development, cell proliferation/division, cytoskeleton, and cell wall may also contribute to the regulation of helical growth in pepper. The insights gained from this study are valuable for further investigations into pepper fruit shape development.

The SEPALLATA-like CaSEP5 gene regulates flower sepal, pedicel, and fruit development in pepper (Capsicum annuum L.)

Flowers are reproductive plant organs that determine final fruit or seed yield. SEPALLATA-like MADS-box genes participate in floral determination and/or flower organ specification. Nevertheless, their molecular mechanisms in bell pepper have not yet been elucidated. In this study, we identified CaSEP5, which encodes a duplicated SEPALLATA-like MADS-box protein grouped in the FBP9/23-subclade. CaSEP5 genes were detected in all whorls of floral organs, and were relatively highly expressed in the sepals and petals. Subcellular localization revealed that CaSEP5 was in the nucleus. This finding aligns with transcription factor characteristics. The physical interactions of CaSEP5 with B-, C-, and other CaSEPs indicated that CaSEP5 act as glue to form protein complexes to regulate flower development by altering the hormones level and distribution. CaSEP5-silenced plants had elongated, leaf-like sepals, rigid, straight pedicels, reduced fruit size, altered expression patterns of other floral homeotic genes, and varied hormone levels. We proposed that CaSEP5 plays an important role in pepper flower and fruit development by means of a complex hormone and genetic network.

MYB24 Negatively Regulates the Biosynthesis of Lignin and Capsaicin by Affecting the Expression of Key Genes in the Phenylpropanoid Metabolism Pathway in Capsicum chinense.

The wide application of pepper is mostly related to the content of capsaicin, and phenylpropanoid metabolism and its branch pathways may play an important role in the biosynthesis of capsaicin. The expression level of MYB24, a transcription factor screened from the transcriptome data of the pepper fruit development stage, was closely related to the spicy taste. In this experiment, CcMYB24 was cloned from Hainan Huangdenglong pepper, a hot aromatic pepper variety popular in the world for processing, and its function was confirmed by tissue expression characteristics, heterologous transformation in Arabidopsis thaliana, and VIGS technology. The results showed that the relative expression level of CcMYB24 was stable in the early stage of pepper fruit development, and increased significantly from 30 to 50 days after flowering. Heterologous expression led to a significant increase in the expression of CcMYB24 and decrease in lignin content in transgenic Arabidopsis thaliana plants. CcMYB24 silencing led to a significant increase in the expression of phenylpropanoid metabolism pathway genes PAL, 4CL, and pAMT; lignin branch CCR1 and CAD; and capsaicin pathway CS, AT3, and COMT genes in the placenta of pepper, with capsaicin content increased by more than 31.72% and lignin content increased by 20.78%. However, the expression of PAL, pAMT, AT3, COMT, etc., in the corresponding pericarps did not change significantly. Although CS, CCR1, and CAD increased significantly, the relative expression amount was smaller than that in placental tissue, and the lignin content did not change significantly. As indicated above, CcMYB24 may negatively regulate the formation of capsaicin and lignin by regulating the expression of genes from phenylpropanoid metabolism and its branch pathways.

Multiomics analyses reveal high temperature-induced molecular regulation of ascorbic acid and capsaicin biosynthesis in pepper fruits

Heat stress (HS) is one of the most severe abiotic stressors that affects pepper fruit development, coloration, nutritional quality and yield. However, the molecular basis of pepper fruit response to HS has not been extensively studied. In this study, we performed integrated transcriptomic, proteomic and metabolomic analyses in pepper fruits subjected to HS (36 ℃) for 6 h, 12 h and 24 h. A total of 14513 differentially expressed genes (DEGs), 1999 differentially abundant proteins (DAPs), and 39 differentially accumulated metabolites (DAMs) were identified in response to heat treatments. Molecular function, regulatory network, and metabolic pathway analyses revealed that the DEGs and DAPs in heat-stressed fruits were mainly attributed to photosynthesis, response to stimulus, carbohydrate metabolic process, and protein folding process. Interestingly, among the DAMs, the content of capsaicin and ascorbic acid (Vitamin C), which are important phytochemical substances of pepper as food, was substantially impaired by HS. By integrated analysis of the genes, proteins and intermediate metabolites related to the biosynthesis pathway of capsaicin and ascorbic acid, we found that transcripts of most of the capsaicin biosynthesis genes are positively correlated to capsaicin content, while there is no obvious correlation between the biosynthesis genes and the content of ascorbic acid. Furthermore, by analyzing the promoter sequence, we found 3 transcription factors that could potentially regulate the expression of key genes in the capsaicin biosynthesis pathway in response to HS. Our research here provides a better understanding of the molecular mechanism of pepper fruit response to HS, and also provides a theoretical basis for the pre-harvest temperature management to harvest high-quality pepper fruits.

Identification of the 12-oxophytoeienoic acid reductase (OPR) gene family in pepper (Capsicum annuum L.) and functional characterization of CaOPR6 in pepper fruit development and stress response.

The 12-oxophytoeienoic acid reductase (OPR) is a kind of enzyme in the octadecanoid biosynthesis pathway that determines the biosynthesis of jasmonic acid. Although the roles of OPRs have been extensively studied in several crop plants, little is known about the biological functions of OPR-encoding genes in Capsicum annuum plants. In this study, seven OPR family genes (CaOPR1-7) were identified from the C. annuum genome. The physical and chemical properties of CaOPR1-7 were further analyzed, including gene expression patterns, promoter elements, and chromosomal locations. The results showed that the seven CaOPR homologues could be divided into two subgroups, and CaOPR6 was highly similar to AtOPR3 in Arabidopsis. The expression of CaOPR6 was significantly induced by various stresses such as cold, salt, and pathogen infection, indicating that CaOPR6 plays important roles in response to abiotic and biotic stresses. Overall, these findings improve the understanding of the biological functions of CaOPR6 in the development of pepper fruit and stress response of pepper plants, and facilitate further studies on the molecular biology of OPR proteins in Solanaceae vegetables.

Metabolomic Analysis Identifies Differences Between Wild and Domesticated Chili Pepper Fruits During Development (Capsicum annuum L.).

Capsicum spp. members are a rich source of specialized compounds due to their secondary metabolism. Some metabolic pathways have suffered modifications during the domestication process and improvement of agricultural traits. Here, we compared non-targeted LC–MS profiles from several areas: wild accessions (C. annuum L. var. glabriusculum), domesticated cultivars (C. annuum L.), and the F1 progeny of a domesticated, and a wild accession cross (in both directions) throughout seven stages of fruit development of chili pepper fruits. The main detected differences were in glycerophospholipid metabolism, flavone and flavonol biosynthesis, sphingolipid metabolism, and cutin biosynthesis. The domesticated group exhibited a higher abundance in 12′-apo-β-carotenal, among others capsorubin, and β-tocopherol. Palmitic acid and derivates, terpenoids, and quercitrin were prevalent in the wild accessions. F1 progeny showed a higher abundance of capsaicin, glycol stearate, and soyacerebroside I. This work supports evidence of the side-affectation of trait selection over the metabolism of chili pepper fruit development. Furthermore, it was also observed that there was a possible heterosis effect over the secondary metabolism in the F1 progeny.

Fine Mapping and Candidate Gene Identification for the CapUp Locus Controlling Fruit Orientation in Pepper (Capsicum spp.).

The orientation of fruits is a distinguishing morphological feature of pepper (Capsicum spp.) varieties. The pendent (downward curved) growth of the fruit stalks, known as pedicels, is highly correlated with fruit weight and pedicel length. A previous genetic analysis revealed that the pendent fruit orientation is governed by a dominant gene, and incomplete inheritance is also observed in some Capsicum accessions. To identify and localize this gene, a single quantitative trait locus (QTL) analysis was performed on one F2 and two recombinant inbred line (RIL) populations, and a genome-wide association study (GWAS) was performed using a core collection. Common QTL regions associated with fruit orientation were detected on chromosome 12. A total of 187,966 SNPs were identified in a genotyping-by-sequencing (GBS) for GWAS analysis of 196 Capsicum annuum, 25 Capsicum baccatum, 21 Capsicum chinense, and 14 Capsicum frutescens accessions, representing the germplasm collection of South Korea. The results of these analyses enabled us to narrow down the CapUp region of interest to 200–250 Mbp on chromosome 12. Seven candidate genes were found to be located between two markers that were completely cosegregated with the fruit orientation phenotype. The findings and markers developed in this study will be helpful for additional understanding of pepper fruit development and breeding for fruit orientation.

Novel structural annotation and functional expression analysis of GTP_EFTU conserved genes in pepper based on the PacBio sequencing data

Genes containing GTP_EFTU domain mainly express elongation factors (EF), Small GTPases, and GTP-binding proteins, which are closely related to protein synthesis, extension and ATP synthesis. In this study, we identified 39 genes containing GTP_EFTU domains from peppers. The evolutionary trees constructed from capsicum, Arabidopsis, rice, and tomato are mainly divided into 7 subfamilies. Using PacBio (Pacific Biosciences) sequencing and assembly data, we extracted these 39 gene sequences, from which 25 genes had alternative splicing. Particularly, the Capana08g000545 had 16 alternative splicing processes. Accordingly, we performed promoter sequence analysis, subcellular location prediction, the expression analysis of different tissues and periods, and also the GO (Gene ontology) analysis of co-expressed genes. Lastly we did the qRT-PCR analysis in 5 stages of pepper fruit development. These analyses revealed important structural and functional information for the identified 39 genes that contain GTP_EFTU domains, providing important references for further follow-up experiments to verify the genes function on plants or their unique roles in peppers.

A method to analyze time expression profiles demonstrated in a database of chili pepper fruit development

RNA-Seq experiments allow genome-wide estimation of relative gene expression. Estimation of gene expression at different time points generates time expression profiles of phenomena of interest, as for example fruit development. However, such profiles can be complex to analyze and interpret. We developed a methodology that transforms original RNA-Seq data from time course experiments into standardized expression profiles, which can be easily interpreted and analyzed. To exemplify this methodology we used RNA-Seq data obtained from 12 accessions of chili pepper (Capsicum annuum L.) during fruit development. All relevant data, as well as functions to perform analyses and interpretations from this experiment, were gathered into a publicly available R package: “Salsa”. Here we explain the rational of the methodology and exemplify the use of the package to obtain valuable insights into the multidimensional time expression changes that occur during chili pepper fruit development. We hope that this tool will be of interest for researchers studying fruit development in chili pepper as well as in other angiosperms.

Transcriptome Analyses Throughout Chili Pepper Fruit Development Reveal Novel Insights into the Domestication Process.

Chili pepper (Capsicum spp.) is an important crop, as well as a model for fruit development studies and domestication. Here, we performed a time-course experiment to estimate standardized gene expression profiles with respect to fruit development for six domesticated and four wild chili pepper ancestors. We sampled the transcriptomes every 10 days from flowering to fruit maturity, and found that the mean standardized expression profiles for domesticated and wild accessions significantly differed. The mean standardized expression was higher and peaked earlier for domesticated vs. wild genotypes, particularly for genes involved in the cell cycle that ultimately control fruit size. We postulate that these gene expression changes are driven by selection pressures during domestication and show a robust network of cell cycle genes with a time shift in expression, which explains some of the differences between domesticated and wild phenotypes.

Comprehensive Phosphoproteomic Analysis of Pepper Fruit Development Provides Insight into Plant Signaling Transduction.

Limited knowledge is available for phosphorylation modifications in pepper (Capsicum annuum L.), especially in pepper fruit development. In this study, we conducted the first comprehensive phosphoproteomic analysis of pepper fruit at four development stage by Tandem Mass Tag proteomic approaches. A total of 2639 unique phosphopeptides spanning 1566 proteins with 4150 nonredundant sites of phosphorylation were identified, among which 2327 peptides in 1413 proteins were accurately quantified at four different stages. Mature Green (MG) to breaker stage showed the largest number of differentially expressed phosphoproteins and the number of downregulated phosphoproteins was significantly higher than that of upregulated after MG stage. Twenty seven phosphorylation motifs, including 22 pSer motifs and five pThr motifs and 85 kinase including 28 serine/threonine kinases, 14 receptor protein kinases, six mitogen-activated protein kinases, seven calcium-dependent protein kinases, two casein kinases, and some other kinases were quantified. Then the dynamic changes of phosphorylated proteins in ethylene and abscisic acid signaling transduction pathways during fruit development were analyzed. Our results provide a cascade of phosphoproteins and a regulatory network of phosphorylation signals, which help to further understand the mechanism of phosphorylation in pepper fruit development.

Genome-wide identification and expression analysis of the carotenoid metabolic pathway genes in pepper (Capsicum annuum L.)

Carotenoids belong to a major family of isoprenoids which play vital roles in plant growth and development. Pepper (Capsicum annuum L.) is not only a vegetable and condiment grown worldwide, but also can be used as a model organism in fruit color research. In this study, 61 genes known to be involved in mevalonate (MVA), plastidial 2-C-methyl-d-erythritol-4-phosphate (MEP) and carotenoid metabolism (biosynthesis and catabolism) pathways were identified and characterized based on the genome of pepper plants. In the present study, it was found that 56 of the 61 carotenoid metabolism genes in pepper were unevenly distributed throughout 12 chromosomes. CaGGPPS2, CaGGPPS4a and CaGGPPS4b were generally clustered into one region. This study identified CaHMGR1/CaHMGR3 and CaHMGR1/CaHMGR2 as segregation duplication events. In addition, the pepper carotenoid metabolism genes had high variations in gene structure. The key carotenoid metabolism genes showed different expression patterns during the pepper fruit development and under environmental stress conditions. The results obtained in this research investigation will contribute insight to the evolution and function of carotenoid metabolic pathways in pepper fruit.

Hormonal and transcriptional analyses of fruit development and ripening in different varieties of black pepper (Piper nigrum).

Black pepper (Piper nigrum L.) is one of the most popular and oldest spices in the world with culinary uses and various pharmacological properties. In order to satisfy the growing worldwide demand for black pepper, improved productivity of pepper is highly desirable. A primary constraint in black pepper production is the non-synchronous nature of flower development and non-uniform fruit ripening within a spike. The uneven ripening of pepper berries results in a high labour requirement for selective harvesting contributes to low productivity and affects the quality of the pepper products. In Malaysia, there are a few recommended varieties for black pepper planting, each having some limitations in addition to the useful characteristics. Therefore, a comparative study of different black pepper varieties will provide a better understanding of the mechanisms regulates fruit development and ripening. Plant hormones are known to influence the fruit development process and their roles in black pepper flower and fruit development were inferred based on the probe-based gene expression analysis and the quantification of the multiple plant hormones using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). In this study, jasmonic acid and salicylic acid were found to play roles in flowering and fruit setting, whereas auxin, gibberellin and cytokinins are important for fruit growth. Abscisic acid has positive role in fruit maturation and ripening in the development process. Distinct pattern of plant hormones related gene expression profiles with the hormones accumulation profiles suggested a complex network of regulation is involved in the signaling process and crosstalk between plant hormones was another layer of regulation in the black pepper fruit development mechanisms. The current study provides clues to help in elucidating the timing of the action of each specific plant hormone during fruit development and ripening which could be applied to enhance our ability to control the ripening process, leading to improving procedures for the production and post-harvest handling of pepper fruits.

Integrative Transcriptome and Proteome Analysis Identifies Major Metabolic Pathways Involved in Pepper Fruit Development

Pepper ( Capsicum annuum L.) fruit development is a complex and genetically programmed process. In this study, we conducted integrative analysis of transcriptome and proteome profiles during pepper fruit development. A total of 23 349 transcripts and 5455 protein groups were identified in four fruit developmental stages of two pepper varieties. The numbers of transcripts and proteins identified were decreased gradually across fruit development, and the most significant changes in transcript and protein levels happened from the mature green (MG) to breaker (Br) stages. Poor correlation between differentially expressed transcript and differentially expressed protein profiles was observed during pepper fruit development. We then analyzed expression profiles of transcripts and proteins involved in cell wall metabolism, and capsanthin, tocopherol, and ascorbate biosynthetic pathways during fruit development, and identified key regulatory proteins in these pathways. We presented a dynamic picture of pepper fruit development via comprehensive analysis of transcriptome and proteome profiles at different fruit developmental stages and in different varieties, revealing the temporal specificity of key protein expression. Our report provides insight into the transcription and translation dynamics of pepper fruit development and a reference for other nonclimacteric species.

Genome-Wide Identification, Expression, and Functional Analysis of the Alkaline/Neutral Invertase Gene Family in Pepper.

Alkaline/neutral invertase (NINV) proteins irreversibly cleave sucrose into fructose and glucose, and play important roles in carbohydrate metabolism and plant development. To investigate the role of NINVs in the development of pepper fruits, seven NINV genes (CaNINV1–7) were identified. Phylogenetic analysis revealed that the CaNINV family could be divided into α and β groups. CaNINV1–6 had typical conserved regions and similar protein structures to the NINVs of other plants, while CaNINV7 lacked amino acid sequences at the C-terminus and N-terminus ends. An expression analysis of the CaNINV genes in different tissues demonstrated that CaNINV5 is the dominant NINV in all the examined tissues (root, stem, leaf, bud, flower, and developmental pepper fruits stage). Notably, the expression of CaNINV5 was found to gradually increase at the pre-breaker stages, followed by a decrease at the breaker stages, while it maintained a low level at the post-breaker stages. Furthermore, the invertase activity of CaNINV5 was identified by functional complementation of the invertase-deficient yeast strain SEY2102, and the optimum pH of CaNINV5 was found to be ~7.5. The gene expression and enzymatic activity of CaNINV5 suggest that it might be the main NINV enzyme for hydrolysis of sucrose during pepper fruit development.

Noncoding and coding transcriptome analysis reveals the regulation roles of long noncoding RNAs in fruit development of hot pepper (Capsicum annuum L.)

Long non-coding RNAs (lncRNAs) have been shown to play critical regulatory roles in diverse biological processes in plants. However, the presence of lncRNAs and their potential functions in hot pepper are still unknown. Using strand-specific RNA-sequencing, a total of 2505 putative lncRNAs were identified in the fruits of hot pepper. The lncRNAs were transcribed from all pepper chromosomes and 95.37% of them originated from intergenic regions. And 1066 lncRNAs were differentially expressed among the four samples during pepper fruit development. Many potential protein-coding (PC) genes targeted by lncRNAs with cis or trans-acting were also identified. Six of them and their target genes were further validated by quantitative RT-PCR. Some of these target genes were involved in plant hormone signal transduction, cell wall formation and carotenoid biosynthesis, indicating the roles of lncRNAs in the regulation of fruit development and quality. This study identified a large number of lncRNAs in hot pepper, thereby providing some insights into the fruit development of hot pepper.

Identification of Optimal Reference Genes for Normalization of qPCR Analysis during Pepper Fruit Development.

Due to its high sensitivity and reproducibility, quantitative real-time PCR (qPCR) is practiced as a useful research tool for targeted gene expression analysis. For qPCR operations, the normalization with suitable reference genes (RGs) is a crucial step that eventually determines the reliability of the obtained results. Although pepper is considered an ideal model plant for the study of non-climacteric fruit development, at present no specific RG have been developed or validated for the qPCR analyses of pepper fruit. Therefore, this study aimed to identify stably expressed genes for their potential use as RGs in pepper fruit studies. Initially, a total of 35 putative RGs were selected by mining the pepper transcriptome data sets derived from the PGP (Pepper Genome Platform) and PGD (Pepper Genome Database). Their expression stabilities were further measured in a set of pepper (Capsicum annuum L. var. 007e) fruit samples, which represented four different fruit developmental stages (IM: Immature; MG: Mature green; B: Break; MR: Mature red) using the qPCR analysis. Then, based on the qPCR results, three different statistical algorithms, namely geNorm, Normfinder, and boxplot, were chosen to evaluate the expression stabilities of these putative RGs. It should be noted that nine genes were proven to be qualified as RGs during pepper fruit development, namely CaREV05 (CA00g79660); CaREV08 (CA06g02180); CaREV09 (CA06g05650); CaREV16 (Capana12g002666); CaREV21 (Capana10g001439); CaREV23 (Capana05g000680); CaREV26 (Capana01g002973); CaREV27 (Capana11g000123); CaREV31 (Capana04g002411); and CaREV33 (Capana08g001826). Further analysis based on geNorm suggested that the application of the two most stably expressed genes (CaREV05 and CaREV08) would provide optimal transcript normalization in the qPCR experiments. Therefore, a new and comprehensive strategy for the identification of optimal RGs was developed. This strategy allowed for the effective normalization of the qPCR analysis of the pepper fruit development at the whole pepper genome level. This study not only explored the optimal RGs specific for studying pepper fruit development, but also introduced a referable strategy of RG mining which could potentially be implicated in other plant species.

Predominant role of the l-galactose pathway in l-ascorbic acid biosynthesis in fruits and leaves of the Capsicum annuum L. chili pepper

l-ascorbic acid (vitamin C) is critical for animals and plants since it is a powerful antioxidant and cofactor involved in a wide number of biochemical reactions. Because humans cannot synthesize vitamin C, they must acquire it through the daily diet. Chili pepper fruits are an excellent source of ascorbic acid (AsA). However, how AsA is synthesized in the chili pepper remains poorly understood. Four pathways for AsA synthesis in plants have been reported namely the l-galactose, l-gulose, d-galacturonate, and myo-inositol pathways. In the present study, we investigated the pathways involved in vitamin C synthesis in the Serrano Tampiqueno 74 chili pepper. Our results showed a higher incorporation of labeled l-galactose into AsA compared to the incorporation of labeled myo-inositol and d-galacturonic acid. Based on these results, the l-galactose pathway is the predominant mechanism for AsA synthesis in the fruits and leaves of this chili pepper. In addition, qPCR analysis of the expression of genes involved in the l-galactose pathway revealed that, in general, GMP, GME, and GGP/VTC2 were most highly expressed. A positive correlation between gene expression and AsA levels in pericarp tissues during chili pepper fruit development and ripening was not observed. Expression analysis of GalUR and MIOX, which are involved in the d-galacturonic and myo-inositol pathways of AsA biosynthesis, respectively, also did not exhibit a positive correlation with ascorbic acid content in chili pepper fruits.

Mining secreted proteins that function in pepper fruit development and ripening using a yeast secretion trap (YST)

Plant cells secrete diverse sets of constitutively- and conditionally-expressed proteins under various environmental and developmental states. Secreted protein populations, or secretomes have multiple functions, including defense responses, signaling, metabolic processes, and developmental regulation. To identify genes encoding secreted proteins that function in fruit development and ripening, a yeast secretion trap (YST) screen was employed using pepper (Capsicum annuum) fruit cDNAs. The YST screen revealed 80 pepper fruit-related genes (CaPFRs) encoding secreted proteins including cell wall proteins, several of which have not been previously described. Transient GFP-fusion assay and an in planta secretion trap were used to validate the secretion of proteins encoded by selected YST clones. In addition, RNA gel blot analyses provided further insights into their expression and regulation during fruit development and ripening. Integrating our data, we conclude that the YST provides a valuable functional genomics tool for the identification of substantial numbers of novel secreted plant proteins that are associated with biological processes, including fruit development and ripening.

Anthocyanin accumulation and expression analysis of biosynthesis-related genes during chili pepper fruit development

Chili pepper (Capsicum annuum L.) cv. Arbol and Uvilla fruits differing in anthocyanin contents were analyzed to characterize the accumulation patterns. The maximum accumulation of the aglycon delphinidin occurred 20 days postanthesis (DPA) with higher content in Uvilla than in Arbol fruits. Regarding the cDNA library, 9 186 cDNA clones were selected. The clones with high homology to genes concerning anthocyanin biosynthesis, such as encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′,5′-hydroxylase (F3′5′H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), UDP Glc-flavonoid 3-O-gluco-syl transferase (UFGT), and also those possibly involved in anthocyanin transport into the vacuoles, an anthocyanin permease (ANP) and a glutathione S-transferase (GST) were used for gene expression analysis. In general, the expression of all investigated genes was developmentally regulated in both Arbol and Uvilla. CHS and CHI transcripts were expressed at the maximal level at 10 DPA, and then consistently declined throughout fruit development. F3′5′H, DFR, UFGT and GST expression exhibited a positive correlation with anthocyanin accumulation, and the highest transcript levels were detected prior to or by the time of maximum anthocyanin accumulation, depending on the chili pepper type. Pericarp fruit tissues from cv. Tampiqueno 74, an anthocyanin non-accumulator, also showed CHS, CHI, F3H, ANS and ANP expression at some developmental stages.