Estimating Transcriptome Diversity and Specialization in Capsicum annuum L.

Chili pepper fruits of the genus Capsicum represent excellent experimental models to study the growth, development, and ripening processes in a non-climacteric species at the physiological, biochemical, and molecular levels. Fruit growth, development, and ripening involve a complex, harmonious, and finely controlled regulation of gene expression. The purpose of this study was to estimate the changes in transcriptome diversity and specialization, as well as gene specificities during fruit development in this crop, and to illustrate the advantages of estimating these parameters. To achieve these aims, we programmed and made publicly available an R package. In this study, we applied these methods to a set of 179 RNA-Seq libraries from a factorial experiment that includes 12 different genotypes at various stages of fruit development. We found that the diversity of the transcriptome decreases linearly from the flower to the mature fruit, while its specialization follows a complex and non-linear behavior during this process. Additionally, by defining sets of genes with different degrees of specialization and applying Gene Ontology enrichment analysis, we identified processes, functions, and components that play a central role in particular fruit development stages. In conclusion, the estimation of diversity, specialization, and specificity summarizes the global properties of the transcriptomes, providing insights that are difficult to achieve by other means.

Anthocyanin metabolic engineering of Euphorbia pulcherrima: advances and perspectives.

The range of floral colors is determined by the type of plant pigment accumulated by the plant. Anthocyanins are the most common flavonoid pigments in angiosperms; they provide a wide range of visible colors from red-magenta to blue-purple, products of cyanidin and delphinidin biosynthesis, respectively. For the floriculture industry, floral color is one of the most important ornamental characteristics for the development of new commercial varieties; however, most plant species are restricted to a certain color spectrum, limited by their own genetics. In fact, many ornamental crops lack bluish varieties due to the lack of activity of essential biosynthetic enzymes for the accumulation of delphinidin. An example is the poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch), the ornamental plant symbol of Christmas and native to Mexico. Its popularity is the result of the variety of colors displayed by its bracts, a kind of modified leaves that accumulate reddish pigments based mainly on cyanidin and, to a lesser extent, on pelargonidin. The commercial success of this plant lies in the development of new varieties and, although consumers like the typical red color, they are also looking for poinsettias with new and innovative colors. Previous research has demonstrated the possibility of manipulating flower color through metabolic engineering of the anthocyanin biosynthesis pathway and plant tissue culture in different ornamental plant species. For example, transgenic cultivars of flowers such as roses, carnations or chrysanthemums owe their attractive bluish colors to a high and exclusive accumulation of delphinidin. Here, we discuss the possibilities of genetic engineering of the anthocyanin biosynthetic pathway in E. pulcherrima through the introduction of one or more foreign delphinidin biosynthetic genes under the transcriptional control of a pathway-specific promoter, and the genome editing possibilities as an alternative tool to modify the color of the bracts. In addition, some other approaches such as the appropriate selection of the cultivars that presented the most suitable intracellular conditions to accumulate delphinidin, as well as the incorporation of genes encoding anthocyanin-modifying enzymes or transcription factors to favor the bluish pigmentation of the flowers are also revised.

Gene Functional Networks from Time Expression Profiles: A Constructive Approach Demonstrated in Chili Pepper (Capsicum annuum L.).

Gene co-expression networks are powerful tools to understand functional interactions between genes. However, large co-expression networks are difficult to interpret and do not guarantee that the relations found will be true for different genotypes. Statistically verified time expression profiles give information about significant changes in expressions through time, and genes with highly correlated time expression profiles, which are annotated in the same biological process, are likely to be functionally connected. A method to obtain robust networks of functionally related genes will be useful to understand the complexity of the transcriptome, leading to biologically relevant insights. We present an algorithm to construct gene functional networks for genes annotated in a given biological process or other aspects of interest. We assume that there are genome-wide time expression profiles for a set of representative genotypes of the species of interest. The method is based on the correlation of time expression profiles, bound by a set of thresholds that assure both, a given false discovery rate, and the discard of correlation outliers. The novelty of the method consists in that a gene expression relation must be repeatedly found in a given set of independent genotypes to be considered valid. This automatically discards relations particular to specific genotypes, assuring a network robustness, which can be set a priori. Additionally, we present an algorithm to find transcription factors candidates for regulating hub genes within a network. The algorithms are demonstrated with data from a large experiment studying gene expression during the development of the fruit in a diverse set of chili pepper genotypes. The algorithm is implemented and demonstrated in a new version of the publicly available R package "Salsa" (version 1.0).

Putative Transcription Factor Genes Associated with Regulation of Carotenoid Biosynthesis in Chili Pepper Fruits Revealed by RNA-Seq Coexpression Analysis.

During the ripening process, the pericarp of chili pepper (Capsicum spp.) fruits accumulates large amounts of carotenoids. Although the carotenoid biosynthesis pathway in the Capsicum genus has been widely studied from different perspectives, the transcriptional regulation of genes encoding carotenoid biosynthetic enzymes has not been elucidated in this fruit. We analyzed RNA-Seq transcriptomic data from the fruits of 12 accessions of Capsicum annuum during the growth, development, and ripening processes using the R package named Salsa. We performed coexpression analyses between the standardized expression of genes encoding carotenoid biosynthetic enzymes (target genes (TGs)) and the genes of all expressed transcription factors (TFs). Additionally, we analyzed the promoter region of each biosynthetic gene to identify putative binding sequences for each selected TF candidate. We selected 83 TFs as putative regulators of the carotenogenic structural genes. From them, putative binding sites in the promoters of the carotenoid-biosynthesis-related structural genes were found for only 54 TFs. These results could guide the search for transcription factors involved in the regulation of the carotenogenic pathway in chili pepper fruits and might facilitate the collection of corresponding experimental evidence to corroborate their participation in the regulation of this biosynthetic pathway in Capsicum spp.

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-ß-carotenal, among others capsorubin, and ß-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.

Transcriptional Regulation of Ripening in Chili Pepper Fruits (Capsicum spp.).

Chili peppers represent a very important horticultural crop that is cultivated and commercialized worldwide. The ripening process makes the fruit palatable, desirable, and attractive, thus increasing its quality and nutritional value. This process includes visual changes, such as fruit coloration, flavor, aroma, and texture. Fruit ripening involves a sequence of physiological, biochemical, and molecular changes that must be finely regulated at the transcriptional level. In this review, we integrate current knowledge about the transcription factors involved in the regulation of different stages of the chili pepper ripening process.

Inheritance of gene expression throughout fruit development in chili pepper.

Gene expression is the primary molecular phenotype and can be estimated in specific organs or tissues at particular times. Here we analyzed genome-wide inheritance of gene expression in fruits of chili pepper (Capsicum annuum L.) in reciprocal crosses between a domesticated and a wild accession, estimating this parameter during fruit development. We defined a general hierarchical schema to classify gene expression inheritance which can be employed for any quantitative trait. We found that inheritance of gene expression is affected by both, the time of fruit development as well as the direction of the cross, and propose that such variations could be common in many developmental processes. We conclude that classification of inheritance patterns is important to have a better understanding of the mechanisms underlying gene expression regulation, and demonstrate that sets of genes with specific inheritance pattern at particular times of fruit development are enriched in different biological processes, molecular functions and cell components. All curated data and functions for analysis and visualization are publicly available as an R package.

Anther Culture of the Gametophytic Self-Incompatible Species Physalis ixocarpa Brot.

Here we present an optimized protocol for in vitro embryo formation and plant regeneration through anther culture of the Mexican husk tomato (Physalis ixocarpa Brot.). This protocol relies on the application of an anther thermal shock at a specific developmental stage prior to the in vitro culture, ensures embryo formation from anthers without callus formation, and allows spending less time to regenerate doubled haploid complete plants. This protocol has been used for different cultivars of Physalis ixocarpa (Chapingo, Rendidora, Puebla, Arandaz, Manzano, Tamazula, Salamanca, and Milpero), and also for two wild-type accessions, all of them cultivated in Mexico. Chapingo cultivar responded with the highest percentage of androgenesis on the embryo induction medium (EIM).

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.

Genome-Wide Identification and Analysis of the MYB Transcription Factor Gene Family in Chili Pepper (Capsicum spp.).

The MYB transcription factor family is very large and functionally diverse in plants, however, only a few members of this family have been reported and characterized in chili pepper (Capsicum spp.). In the present study, we performed genome-wide analyses of the MYB family in Capsicum annuum, including phylogenetic relationships, conserved domain, gene structure organization, motif protein arrangement, chromosome distribution, chemical properties predictions, RNA-seq expression, and RT-qPCR expression assays. A total of 235 non-redundant MYB proteins were identified from C. annuum, including R2R3-MYB, 3R-MYB, atypical MYB, and MYB-related subclasses. The sequence analysis of CaMYBs compared with other plant MYB proteins revealed gene conservation, but also potential specialized genes. Tissue-specific expression profiles showed that CaMYB genes were differentially expressed, suggesting that they are functionally divergent. Furthermore, the integration of our data allowed us to propose strong CaMYBs candidates to be regulating phenylpropanoid, lignin, capsaicinoid, carotenoid, and vitamin C biosynthesis, providing new insights into the role of MYB transcription factors in secondary metabolism. This study adds valuable knowledge about the functions of CaMYB genes in various processes in the Capsicum genus.

Chili Pepper Carotenoids: Nutraceutical Properties and Mechanisms of Action.

Chili pepper is a prominent cultivated horticultural crop that is traditionally used for food seasoning and is applied for the treatment and prevention of multiple diseases. Its beneficial health properties are due to its abundance and variety of bioactive components, such as carotenoids, capsaicinoids, and vitamins. In particular, carotenoids have important nutraceutical properties, and several studies have focused on their potential in the prevention and treatment of human diseases. In this article, we reviewed the state of knowledge of general aspects of chili pepper carotenoids (biosynthesis pathway, types and content in Capsicum spp., and the effects of processing on carotenoid content) and recent findings on the effects of carotenoid nutraceuticals, such as antioxidant, cancer preventive, anti-inflammatory, cardiovascular disorder preventive, and anti-obesity effects.

Virus-Induced Gene Silencing (VIGS) in Chili Pepper (Capsicum spp.).

Virus-induced gene silencing (VIGS) is a transcript suppression technique that enables the functional characterization of genes in recalcitrant transformation plants. This technique consists in cloning a short fragment of a gene of interest into a viral vector, such as TRV (Tobacco rattle virus), and this viral construction is used to agro-infiltrate the plant. VIGS induces posttranscriptional gene silencing (PTGS) that results in the specific sequence degradation of target RNAs. Here we describe a VIGS protocol using the Gateway-based TRV vector for the study of genes in chili pepper plants.

Assessment of Capsaicinoid and Capsinoid Accumulation Patterns during Fruit Development in Three Chili Pepper Genotypes (Capsicum spp.) Carrying Pun1 and pAMT Alleles Related to Pungency.

Quantification, using an accurate analytical approach, of capsinoids and capsaicinoids was performed on three chili pepper (Capsicum spp.) genotypes: "Chiltepín", "Tampiqueño 74", and "Bhut Jolokia" at various stages of fruit development. The accumulation of capsinoids, in all these peppers started between 10 to 20 days post-anthesis (dpa), increased and reached the highest capsinoid amount at 40 dpa, and then decreased until 60 dpa. Conversely, capsaicinoids could already be determined at 10 dpa in "Bhut Jolokia" and their accumulation pattern was different from that of the capsinoids in this genotype. The capsiate/dihydrocapsiate ratio presented a higher variation between genotypes and developmental stages than the capsaicin/dihydrocapsaicin ratio. Capsinoid ratios (4-24%) and Pun1/pAMT genotyping were determined. These results provide information on the progress of the accumulation of capsinoids in the aforementioned pungent and superhot cultivars and could support future breeding studies toward the understanding of the factors affecting their accumulation.

Generation of BSA-capsaicin Nanoparticles and Their Hormesis Effect on the Rhodotorula mucilaginosa Yeast.

Capsaicin is a chemical compound found in pungent chili peppers (Capsicum spp.). In biotechnology, capsaicin has been proposed as a pathogen control; however, its low solubility in water and high instability limits its uses. The aim of this work was to study the effect of high concentrations of capsaicin on the synthesis of nanoparticles and to evaluate their inhibitory effect on the growth of Rhodotorula mucilaginosa yeast. Bovine serum albumin (BSA)-capsaicin nanoparticles were formulated at 0, 16.2, 32.5, 48.7 and 65.0 µg of capsaicin per mg of BSA. Nanoparticle properties were evaluated and they were added to cultures of R. mucilaginosa to quantify their effect on cell viability. We found that increased capsaicin levels caused several changes to the physicochemical parameters, probably due to changes in the hydrophobicity sites of the albumin during the nanostructuration. The administration of nanoparticles to cultures of R. mucilaginosa produced a maximal viability with nanoparticles at 16.2 µg/mg; on the contrary, nanoparticles at 65.0 µg/mg caused maximal cell death. R. mucilaginosa cells displayed a hormesis effect in response to the nanoparticle dose concentration. The nanoparticles showed different responses during the uptake process, probably as a consequence of the nanostructural properties of capsaicin in the BSA molecules.

Biochemistry and molecular biology of capsaicinoid biosynthesis: recent advances and perspectives.

The most widely known characteristic of chili pepper fruits is their capacity to produce capsaicinoids, which are responsible for the pungent sensation. The capsaicinoids have several uses in different areas, such as the pharmaceutical, cosmetic and agronomic industries, among others. They are synthesized by the condensation of vanillylamine (derived from phenylalanine) with a branched-chain fatty acid (from valine or leucine precursors), and they generally accumulate in the placental tissue of the chili pepper fruits. The pungency grade depends on the genotype of the plant but is also affected by external stimuli. In recent years, new structural and regulatory genes have been hypothesized to participate in the capsaicinoid biosynthetic pathway. Moreover, the role of some of these genes has been investigated. Substantial progress has been made in discerning the molecular biology of this pathway; however, many questions remain unsolved. We previously reviewed some aspects of the biochemistry and molecular biology of capsaicinoid biosynthesis (Aza-González et al. Plant Cell Rep 30:695-706. Aza-González et al., Plant Cell Rep 30:695-706, 2011), and in this review, we describe advances made by different researchers since our previous review, including the contribution of omics to the knowledge of this pathway.

An Introduction to Plant Tissue Culture: Advances and Perspectives.

Plant tissue culture techniques are the most frequently used biotechnological tools for basic and applied purposes ranging from investigation on plant developmental processes, functional gene studies, commercial plant micropropagation, generation of transgenic plants with specific industrial and agronomical traits, plant breeding and crop improvement, virus elimination from infected materials to render high-quality healthy plant material, preservation and conservation of germplasm of vegetative propagated plant crops, and rescue of threatened or endangered plant species. Additionally, plant cell and organ cultures are of interest for the production of secondary metabolites of industrial and pharmaceutical interest. New technologies, such as the genome editing ones combined with tissue culture and Agrobacterium tumefaciens infection, are currently promising alternatives for the highly specific genetic manipulation of interesting agronomical or industrial traits in crop plants. Application of omics (genomics, transcriptomics, and proteomics) to plant tissue culture will certainly help to unravel complex developmental processes such as organogenesis and somatic embryogenesis, which will probably enable to improve the efficiency of regeneration protocols for recalcitrant species. Additionally, metabolomics applied to tissue culture will facilitate the extraction and characterization of complex mixtures of natural plant products of industrial interest. General and specific aspects and applications of plant tissue culture and the advances and perspectives are described in this edition.

An R2R3-MYB Transcription Factor Regulates Capsaicinoid Biosynthesis.

Capsaicinoids are responsible for the hot taste of chili peppers. They are restricted to the genus Capsicum and are synthesized by the acylation of the aromatic compound vanillylamine (derived from the phenylpropanoid pathway) with a branched-chain fatty acid by the catalysis of the putative enzyme capsaicinoid synthase. R2R3-MYB transcription factors have been reported in different species of plants as regulators of structural genes of the phenylpropanoid pathway; therefore, we hypothesized that MYB genes might be involved in the regulation of the biosynthesis of pungent compounds. In this study, an R2R3-MYB transcription 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 hormone responses, and development. CaMYB31 expression analysis from placental tissue of pungent and nonpungent chili pepper fruits showed a positive correlation with the structural genes Ca4H, Comt, Kas, pAmt, and AT3 expression and also with the content of capsaicin and dihydrocapsacin during fruit development. However, CaMYB31 also was expressed in vegetative tissues (leaves, roots, and stems). Moreover, CaMYB31 silencing significantly reduced the expression of capsaicinoid biosynthetic genes and the capsaicinoid content. Additionally, CaMYB31 expression was affected by the plant hormones indoleacetic acid, jasmonic acid, salicylic acid, and gibberellic acid or by wounding, temperature, and light, factors known to affect the production of capsaicinoids. These findings indicate that CaMYB31 is indeed involved in the regulation of structural genes of the capsaicinoid biosynthetic pathway.

Dynamics of the chili pepper transcriptome during fruit development.

BACKGROUND: The set of all mRNA molecules present in a cell constitute the transcriptome. The transcriptome varies depending on cell type as well as in response to internal and external stimuli during development. Here we present a study of the changes that occur in the transcriptome of chili pepper fruit during development and ripening. RESULTS: RNA-Seq was used to obtain transcriptomes of whole Serrano-type chili pepper fruits (Capsicum annuum L.; 'Tampiqueño 74') collected at 10, 20, 40 and 60 days after anthesis (DAA). 15,550,468 Illumina MiSeq reads were assembled de novo into 34,066 chili genes. We classified the expression patterns of individual genes as well as genes grouped into Biological Process ontologies and Metabolic Pathway categories using statistical criteria. For the analyses of gene groups we added the weighted expression of individual genes. This method was effective in interpreting general patterns of expression changes and increased the statistical power of the analyses. We also estimated the variation in diversity and specialization of the transcriptome during chili pepper development. Approximately 17% of genes exhibited a significant change of expression in at least one of the intervals sampled. In contrast, significant differences in approximately 63% of the Biological Processes and 80% of the Metabolic Pathways studied were detected in at least one interval. Confirming previous reports, genes related to capsaicinoid and ascorbic acid biosynthesis were significantly upregulated at 20 DAA while those related to carotenoid biosynthesis were highly expressed in the last period of fruit maturation (40-60 DAA). Our RNA-Seq data was validated by examining the expression of nine genes involved in carotenoid biosynthesis by qRT-PCR. CONCLUSIONS: In general, more profound changes in the chili fruit transcriptome were observed in the intervals between 10 to 20 and 40 to 60 DAA. The last interval, between 40 to 60 DAA, included 49% of all significant changes detected, and was characterized predominantly by a global decrease in gene expression. This period signals the end of maturation and the beginning of senescence of chili pepper fruit. The transcriptome at 60 DAA was the most specialized and least diverse of the four states sampled.