Annuum Genome Research Articles

Identification and expression analysis of LEA gene family members in pepper (Capsicum annuum L.).

Pepper (Capsicum annuum L.) is an economically important crop containing capsaicinoids in the seed and placenta, which has various culinary, medical, and industrial applications. Late embryogenesis abundant (LEA) proteins are a large group of hydrophilic proteins participating in the plant stress response and seed development. However, to date there have been no genome-wide analyses of the LEA gene family in pepper. In the present study, 82 LEA genes were identified in the C. annuum genome and classified into nine subfamilies. Most CaLEA genes contain few introns (≤ 2) and are unevenly distributed across 10 chromosomes. Eight pairs of tandem duplication genes and two pairs of segmental duplication genes were identified in the LEA gene family; these duplicated genes were highly conserved and may have performed similar functions during evolution. Expression profile analysis indicated that CaLEA genes exhibited different tissue expression patterns, especially during embryonic development and stress response, particularly in cold stress. Three out of five CaLEA genes showed induced expression upon cold treatment. In summary, we have comprehensively reviewed the LEA gene family in pepper, offering a new perspective on the evolution of this family.

The Capsicum terpenoid biosynthetic module is affected by spider-mite herbivory

In response to herbivory, Capsicum annuum leaves adapt their specialized metabolome that may protect the plant against herbivore feeding either directly or indirectly through volatile metabolites acting as cues for natural enemies of the herbivore. The volatile blend of spider-mite infested leaves differs from non-challenged leaves predominantly by a higher contribution of mono- and sesquiterpenes. In addition to these terpenoids released into the headspace, the terpenoid composition of the leaves alters upon herbivory. All this suggests an important role for terpenoids and their biosynthetic machinery in the defence against herbivory. Here, we show that the C. annuum genome contains a terpene synthase (TPS) gene family of 103 putative members of which structural analysis revealed that 27 encode functional enzymes. Transcriptome analysis showed that several TPS loci were differentially expressed upon herbivory in leaves of two C. annuum genotypes, that differ in susceptibility towards spider mites. The relative expression of upstream biosynthetic genes from the mevalonate and the methylerythritol phosphate pathway also altered upon herbivory, revealing a shift in the metabolic flux through the terpene biosynthetic module. The expression of multiple genes potentially acting downstream of the TPSs, including cytochrome P450 monooxygenases, UDP-glucosyl transferases, and transcription factors strongly correlated with the herbivory-induced TPS genes. A selection of herbivory-induced TPS genes was functionally characterized through heterologous expression and the products that these enzymes catalysed matched with the volatile and non-volatile terpenoids induced in response to herbivory.

Detection of tandem repeats in the Capsicum annuum genome

In this study, we modified the multiple alignment method based on the generation of random position weight matrices (RPWM) and used it to search for tandem repeats (TRs) in the Capsicum annuum genome. The application of the modified (m)RPWM method, which considers the correlation of adjusting nucleotides, resulted in the identification of 908,072 TR regions with repeat lengths from 2 to 200bp in the C. annuum genome, where they occupied ~29%. The most common TRs were 2 and 3bp long followed by those of 21, 4, and 15bp. We performed clustering analysis of TRs with repeat lengths of 2 and 21bp and created position-weight matrices (PWMs) for each group; these templates could be used to search for TRs of a given length in any nucleotide sequence. All detected TRs can be accessed through publicly available database (http : //victoria.biengi.ac.ru/capsicum_tr/). Comparison of mRPWM with other TR search methods such as Tandem Repeat Finder, T-REKS, and XSTREAM indicated that mRPWM could detect significantly more TRs at similar false discovery rates, indicating its superior performance. The developed mRPWM method can be successfully applied to the identification of highly divergent TRs, which is important for functional analysis of genomes and evolutionary studies.

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.

Database of Potential Promoter Sequences in the Capsicum annuum Genome

Simple SummaryIn this study, we searched for potential promoter sequences (PPS) in the pepper genome. We used a new mathematical method for the multiple alignment of highly divergent sequences. Hence, 20 statistically significant classes of sequences in the range from −499 to +100 nucleotides near the annotated genes were calculated. A profile was constructed for each class, which was then used as a position–weight matrix to build a two-dimensional alignment. We found 825,136 potential promoter sequences with a false positive rate of 0.13% in the pepper genome. They were subsequently merged into a database. Potential promoter sequences were analyzed by TSSFinder software, which detected transcription start sites in more than a half of our data. The results show that the pepper genome contains many PPSs. We assume that most of them could be associated with various transposons, dispersed repeats, or viruses.In this study, we used a mathematical method for the multiple alignment of highly divergent sequences (MAHDS) to create a database of potential promoter sequences (PPSs) in the Capsicum annuum genome. To search for PPSs, 20 statistically significant classes of sequences located in the range from −499 to +100 nucleotides near the annotated genes were calculated. For each class, a position–weight matrix (PWM) was computed and then used to identify PPSs in the C. annuum genome. In total, 825,136 PPSs were detected, with a false positive rate of 0.13%. The PPSs obtained with the MAHDS method were tested using TSSFinder, which detects transcription start sites. The databank of the found PPSs provides their coordinates in chromosomes, the alignment of each PPS with the PWM, and the level of statistical significance as a normal distribution argument, and can be used in genetic engineering and biotechnology.

Supplementary materials for article V.Rudenko, E.Korotkov "Database of potential promoter sequences in the Capsicum annuum genome"

Supplementary materials for article V.Rudenko, E.Korotkov "Database of potential promoter sequences in the Capsicum annuum genome"

Genome-wide identification of the Capsicum bHLH transcription factor family: discovery of a candidate regulator involved in the regulation of species-specific bioactive metabolites

BackgroundThe basic helix–loop–helix (bHLH) transcription factors (TFs) serve crucial roles in regulating plant growth and development and typically participate in biological processes by interacting with other TFs. Capsorubin and capsaicinoids are found only in Capsicum, which has high nutritional and economic value. However, whether bHLH family genes regulate capsorubin and capsaicinoid biosynthesis and participate in these processes by interacting with other TFs remains unknown.ResultsIn this study, a total of 107 CabHLHs were identified from the Capsicum annuum genome. Phylogenetic tree analysis revealed that these CabHLH proteins were classified into 15 groups by comparing the CabHLH proteins with Arabidopsis thaliana bHLH proteins. The analysis showed that the expression profiles of CabHLH009, CabHLH032, CabHLH048, CabHLH095 and CabHLH100 found in clusters C1, C2, and C3 were similar to the profile of carotenoid biosynthesis in pericarp, including zeaxanthin, lutein and capsorubin, whereas the expression profiles of CabHLH007, CabHLH009, CabHLH026, CabHLH063 and CabHLH086 found in clusters L5, L6 and L9 were consistent with the profile of capsaicinoid accumulation in the placenta. Moreover, CabHLH007, CabHLH009, CabHLH026 and CabHLH086 also might be involved in temperature-mediated capsaicinoid biosynthesis. Yeast two-hybrid (Y2H) assays demonstrated that CabHLH007, CabHLH009, CabHLH026, CabHLH063 and CabHLH086 could interact with MYB31, a master regulator of capsaicinoid biosynthesis.ConclusionsThe comprehensive and systematic analysis of CabHLH TFs provides useful information that contributes to further investigation of CabHLHs in carotenoid and capsaicinoid biosynthesis.

Full-length LTR retroelements in Capsicum annuum revealed a few species-specific family bursts with insertional preferences.

Capsicum annuum is a species that has undergone an expansion of the size of its genome caused mainly by the amplification of repetitive DNA sequences, including mobile genetic elements. Based on information obtained from sequencing the genome of pepper, the estimated fraction of retroelements is approximately 81%, and previous results revealed an important contribution of lineages derived from Gypsy superfamily. However, the dynamics of the retroelements in the C. annuum genome is poorly understood. In this way, the present work seeks to investigate the phylogenetic diversity and genomic abundance of the families of autonomous (complete and intact) LTR retroelements from C. annuum and inspect their distribution along its chromosomes. In total, we identified 1151 structurally full-length retroelements (340 Copia; 811 Gypsy) grouped in 124 phylogenetic families in the base of their retrotranscriptase. All the evolutive lineages of LTR retroelements identified in plants were present in pepper; however, three of them comprise 83% of the entire LTR retroelements population, the lineages Athila, Del/Tekay, and Ale/Retrofit. From them, only three families represent 70.8% of the total number of the identified retroelements. A massive family-specific wave of amplification of two of them occurred in the last 0.5 Mya (GypsyCa_16; CopiaCa_01), whereas the third is more ancient and occurred 3.0 Mya (GypsyCa_13). Fluorescent in situ hybridization performed with family and lineage-specific probes revealed contrasting patterns of chromosomal affinity. Our results provide a database of the populations LTR retroelements specific to C. annuum genome. The most abundant families were analyzed according to chromosome insertional preferences, suppling useful tools to the design of retroelement-based markers specific to the species.

Tissue-Specific RNA-Seq Analysis and Identification of Receptor-Like Proteins Related to Plant Growth in Capsicum annuum.

Receptor-like proteins (RLPs) are a gene family of cell surface receptors that are involved in plant growth, development, and disease resistance. In a recent study, 438 pepper RLP genes were identified in the Capsicum annuum genome (CaRLPs) and determined to be present in response to multiple biotic stresses. To further understand the role of CaRLPs in plant growth and development, we analyzed expression patterns of all CaRLPs from various pepper tissues and developmental stages using RNA-seq. Ten CaRLP genes were selected for further analysis according to transcript levels with hierarchical clustering. The selected CaRLP genes displayed similarity of motifs within the same groups and structures typical of RLPs. To examine RLP function in growth and development, we performed loss-of-function analysis using a virus-induced gene silencing system. Three of the ten tested CaRLPs (CaRLP238, 253, and 360) in silenced plants exhibited phenotypic alteration with growth retardation compared to controls. All three gene-silenced peppers showed significant differences in root dry weight. Only CaRLP238 had significant differences in both root and shoot dry weight. Our results suggest that CaRLPs may play important roles in regulation of plant growth and development as well as function in defense responses to biotic stresses in the RLP gene family.

Structural and functional features of phytoene synthase isoforms PSY1 and PSY2 in pepper Capsicum annuum L. cultivars.

The fruits of various pepper cultivars are characterized by a different color, which is determined by the pigment ratio; carotenoids dominate in ripe fruits, while chlorophylls, in immature fruits. A key regulator of carotenoid biosynthesis is the phytoene synthase encoded by the PSY gene. The Capsicum annuum genome contains two isoforms of this enzyme, localized in leaf (PSY2) and fruit (PSY1) plastids. In this work, the complete PSY1 and PSY2 genes were identified in nine C. annuum cultivars, which differ in ripe fruit color. PSY1 and PSY2 sequence variability was 2.43 % (69 SNPs) and 1.21 % (36 SNPs). The most variable were PSY1 proteins of the cultivars ‘Maria’ (red-fruited) and ‘Sladkij shokolad’ (red-brown-fruited). All identified PSY1 and PSY2 homologs contained the phytoene synthase domain HH-IPPS and the transit peptide. In the PSY1 and PSY2 HH-IPPS domains, functionally significant sites were determined. For all accessions studied, the active sites (YAKTF and RAYV), aspartate-rich substrate-Mg2+-binding sites (DELVD and DVGED), and other functional residues were shown to be conserved. Transit peptides were more variable, and their similarity in the PSY1 and PSY2 proteins did not exceed 78.68 %. According to the biochemical data obtained, the largest amounts of chlorophylls and carotenoids across the cultivars studied were detected in immature and ripe fruits of the cv. ‘Sladkij shokolad’ and ‘Shokoladnyj’. Also, ripe fruits of the cv. ‘Nesozrevayuschij’ (green-fruited) were marked by significant chlorophyll content, but a minimum of carotenoids. The PSY1 and PSY2 expression patterns were determined in the fruit pericarp at three ripening stages in ‘Zheltyj buket’, ‘Sladkij shokolad’, ‘Karmin’ and ‘Nesozrevayuschij’, which have different ripe fruit colors: yellow, red-brown, dark red and green, respectively. In the leaves of the cultivars studied, PSY1 expression levels varied significantly. All cultivars were characterized by increased PSY1 transcription as the fruit ripened; the maximum transcription level was found in the ripe fruit of ‘Sladkij shokolad’, and the lowest, in ‘Nesozrevayuschij’. PSY2 transcripts were detected not only in the leaves and immature fruits, but also in ripe fruits. Assessment of a possible correlation of PSY1 and PSY2 transcription with carotenoid and chlorophyll content revealed a direct relationship between PSY1 expression level and carotenoid pigmentation during fruit ripening. It has been suggested that the absence of a typical pericarp pigmentation pattern in ‘Nesozrevayuschij’ may be associated with impaired chromoplast formation.

Systematic analysis of the Capsicum ERF transcription factor family: identification of regulatory factors involved in the regulation of species-specific metabolites

BackgroundERF transcription factors (TFs) belong to the Apetala2/Ethylene responsive Factor (AP2/ERF) TF family and play a vital role in plant growth and development processes. Capsorubin and capsaicinoids have relatively high economic and nutritional value, and they are specifically found in Capsicum. However, there is little understanding of how ERFs participate in the regulatory networks of capsorubin and capsaicinoids biosynthesis.ResultsIn this study, a total of 142 ERFs were identified in the Capsicum annuum genome. Subsequent phylogenetic analysis allowed us to divide ERFs into DREB (dehydration responsive element binding proteins) and ERF subfamilies, and further classify them into 11 groups with several subgroups. Expression analysis of biosynthetic pathway genes and CaERFs facilitated the identification of candidate genes related to the regulation of capsorubin and capsaicinoids biosynthesis; the candidates were focused in cluster C9 and cluster C10, as well as cluster L3 and cluster L4, respectively. The expression patterns of CaERF82, CaERF97, CaERF66, CaERF107 and CaERF101, which were found in cluster C9 and cluster C10, were consistent with those of accumulating of carotenoids (β-carotene, zeaxanthin and capsorubin) in the pericarp. In cluster L3 and cluster L4, the expression patterns of CaERF102, CaERF53, CaERF111 and CaERF92 were similar to those of the accumulating capsaicinoids. Furthermore, CaERF92, CaERF102 and CaERF111 were found to be potentially involved in temperature-mediated capsaicinoids biosynthesis.ConclusionThis study will provide an extremely useful foundation for the study of candidate ERFs in the regulation of carotenoids and capsaicinoids biosynthesis in peppers.

In silico mining and FISH mapping of a chromosome-specific satellite DNA in Capsicum annuum L.

A large proportion of eukaryote nuclear genomes is composed of repetitive DNA. Tracing the dynamics of repetitive elements in the genomes of related taxa can reveal important information about their phylogenic relationships as well as traits that have become distinct to a lineage. Study the genomic abundance and chromosomal location of repetitive DNA in Capsicum annuum L. to understand the repeat dynamics. We quantified repeated DNA content in the C. annuum genome using the RepeatExplorer pipeline. About 42% of the C. annuum genome dataset comprised repetitive elements. Of these, 0.011, 0.98, 3.09, and 0.024% represented high and low confidence satellite repeats, putative long-terminal repeats (LTRs), and rDNA sequences, respectively. One novel high confidence 167-bp satellite repeat with a genomic proportion of 0.011%, Ca167TR, was identified. Furthermore, FISH with Ca167TR on metaphase chromosomes of C. annuum revealed signals in the subtelomeric regions of the short and long arms of chromosome 3 and 4, respectively. Further understanding of the origin and associated functions of Ca167TR and other repeats in Capsicum will give us insights into the genomic relationships and functions of the genome.

Capsaicinoids: Pungency beyond Capsicum.

Capsaicinoids are metabolites responsible for the appealing pungency of Capsicum (chili pepper) species. The completion of the Capsicum annuum genome has sparked new interest into the development of biotechnological applications involving the manipulation of pungency levels. Pungent dishes are already part of the traditional cuisine in many countries, and numerous health benefits and industrial applications are associated to capsaicinoids. This raises the question of how to successfully produce more capsaicinoids, whose biosynthesis is strongly influenced by genotype-environment interactions in fruits of Capsicum. In this Opinion article we propose that activating the capsaicinoid biosynthetic pathway in a more amenable species such as tomato could be the next step in the fascinating story of pungent crops.

Fine mapping of the root-knot nematode resistance gene Me1 in pepper (Capsicum annuum L.) and development of markers tightly linked to Me1

Root-knot nematodes (RKNs) can severely damage crops, including peppers, worldwide. The application of resistance genes identified in the Capsicum annuum genome may represent a safe and economically relevant strategy for controlling RKNs. Among the Me genes (Me1, Me3, Me7, and N) that have been mapped to a cluster on chromosome P9, Me1 confers a heat-stable and broad-spectrum resistance that is difficult for virulent RKNs to overcome. In this study, we developed several closely linked kompetitive allele-specific PCR (KASPar) markers, simple sequence repeat (SSR) markers, sequence characterized amplified region (SCAR) markers, and high-resolution melting (HRM) markers for the mapping of RKN-resistance genes. Analyses of 948 individuals (BC1 and F2 progenies) revealed that Me1 was located between SCAR marker 16880-1-V2 and HRM marker 16830-H-V2, with 13 and 0 recombination events with Me1, respectively. These markers were localized to a 132-kb interval, which included six genes. The development of several PCR-based markers closely linked to Me1 will be useful for the marker-assisted selection of RKN resistance in pepper cultivars. Among these markers, 16830-H-V2 and 16830-CAPS are present in the CA09g16830 gene, which is predicted to be a putative late blight resistance protein homolog R1A-3 gene. This gene appears to be a suitable Me1 candidate gene.

Combined use of bulked segregant analysis and microarrays reveals SNP markers pinpointing a major QTL for resistance to Phytophthora capsici in pepper.

Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed candidate genes underlying the major QTL for Phytophthora capsici resistance in Capsicum. Using the candidate genes, reliable markers for Phytophthora resistance were developed and validated. Phytophthora capsici L. is one of the most destructive pathogens of pepper (Capsicum spp.). Resistance of pepper against P. capsici is controlled by quantitative trait loci (QTL), including a major QTL on chromosome 5 that is the predominant contributor to resistance. Here, to maximize the effect of this QTL and study its underlying genes, an F2 population and recombinant inbred lines were inoculated with P. capsici strain JHAI1-7 zoospores at a low concentration (3 × 10(3)/mL). Resistance phenotype segregation ratios for the populations fit a 3:1 and 1:1 (resistant:susceptible) segregation model, respectively, consistent with a single dominant gene model. Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed a single position polymorphism (SPP) marker mapping to the major QTL. When this SPP marker (Phyto5SAR) together with other SNP markers located on chromosome 5 was used to confirm the position of the major QTL, Phyto5SAR showed the highest LOD value at the QTL. A scaffold sequence (scaffold194) containing Phyto5SAR was identified from the C. annuum genome database. The scaffold contained two putative NBS-LRR genes and one SAR 8.2A gene as candidates for contributing to P. capsici resistance. Markers linked to these genes were developed and validated by testing 100 F1 commercial cultivars. Among the markers, Phyto5NBS1 showed about 90% accuracy in predicting resistance phenotypes to a low-virulence P. capsici isolate. These results suggest that Phyto5NBS1 is a reliable marker for P. capsici resistance and can be used for identification of a gene(s) underlying the major QTL on chromosome 5.

A BAX inhibitor-1 gene in Capsicum annuum is induced under various abiotic stresses and endows multi-tolerance in transgenic tobacco

Programmed cell death (PCD) is a highly conserved cellular suicide process important in developmental processes and elimination of damaged cells upon environmental stresses. Among the important regulators of PCD, much interest has been centered on BCL2-associated x protein (BAX) as the pro-PCD factor. On the other hand, BAX inhibitor-1 (BI-1) has been implicated as an anti-PCD factor that balances out the activity of BAX in the developmental processes and responses to environment. A cDNA clone coding a BI-1 gene was isolated from a cDNA library of heat-stressed hot pepper (Capsicum annuum) and named as CaBI-1. This gene contains an open reading frame (ORF) of 248 amino acids encoding a BI-1 protein. Genomic DNA-blot analysis for CaBI-1 suggested one or two loci in the C. annuum genome. Transcription of CaBI-1 was induced in response to high or low temperatures, drought, high salinity, flooding and heavy metal stresses, and ABA. We introduced the ORF of CaBI-1 under the control of the CaMV 35S promoter (P(35S)) into tobacco (Nicotiana tabacum cv. Wisconsin 38) genome by Agrobacterium-mediated transformation. The P(35S):CaBI-1 transgenic plants displayed markedly improved tolerance to high temperature, water deficit, and high salinity in comparison to the control plants. The results indicate that CaBI-1 is a BI-1 gene of which expression induced under various abiotic stresses and endows tolerance to several types of environmental stresses.