Pepper Genome Research Articles

Characterization of GPX Gene Family in Pepper (Capsicum annuum L.) under Abiotic Stress and ABA Treatment.

Plant glutathione peroxidases (GPXs) are important enzymes for removing reactive oxygen species in plant cells and are closely related to the stress resistance of plants. This study identified the GPX gene family members of pepper (Capsicum annuum L.), "CM333", at the whole-genome level to clarify their expression patterns and enzyme activity changes under abiotic stress and ABA treatment. The results showed that eight CaGPX genes were unevenly distributed across four chromosomes and one scaffold of the pepper genome, and their protein sequences had Cys residues typical of the plant GPX domains. The analysis of collinearity, phylogenetic tree, gene structure, and conserved motifs indicated that the CaGPX gene sequence is conserved, structurally similar, and more closely related to the sequence structure of Arabidopsis. Meanwhile, many cis elements involved in stress, hormones, development, and light response were found in the promoter region of the CaGPX gene. In addition, CaGPX1/4 and CaGPX6 were basically expressed in all tissues, and their expression levels were significantly upregulated under abiotic stress and ABA treatment. Subcellular localization showed that CaGPX1 and CaGPX4 are localized in chloroplasts. Additionally, the variations in glutathione peroxidase activity (GSH-Px) mostly agreed with the variations in gene expression. In summary, the CaGPXs gene may play an important role in the development of peppers and their response to abiotic stress and hormones.

CaWRKY22b Plays a Positive Role in the Regulation of Pepper Resistance to Ralstonia solanacearum in a Manner Associated with Jasmonic Acid Signaling.

As important transcription factors, WRKYs play a vital role in the defense response of plants against the invasion of multiple pathogens. Though some WRKY members have been reported to participate in pepper immunity in response to Ralstonia solanacearum infection, the functions of the majority of WRKY members are still unknown. Herein, CaWRKY22b was cloned from the pepper genome and its function against R. solanacearum was analyzed. The transcript abundance of CaWRKY22b was significantly increased in response to the infection of R. solanacearum and the application of exogenous methyl jasmonate (MeJA). Subcellular localization assay in the leaves of Nicotiana benthamiana showed that CaWRKY22b protein was targeted to the nuclei. Agrobacterium-mediated transient expression in pepper leaves indicated that CaWRKY22b overexpression triggered intensive hypersensitive response-like cell death, H2O2 accumulation, and the up-regulation of defense- and JA-responsive genes, including CaHIR1, CaPO2, CaBPR1, and CaDEF1. Virus-induced gene silencing assay revealed that knock-down of CaWRKY22b attenuated pepper's resistance against R. solanacearum and the up-regulation of the tested defense- and jasmonic acid (JA)-responsive genes. We further assessed the role of CaWRKY22b in modulating the expression of JA-responsive CaDEF1, and the result demonstrated that CaWRKY22b trans-activated CaDEF1 expression by directly binding to its upstream promoter. Collectively, our results suggest that CaWRKY22b positively regulated pepper immunity against R. solanacearum in a manner associated with JA signaling, probably by modulating the expression of JA-responsive CaDEF1.

Characterization of the GGP gene family in potato (Solanum tuberosum L.) and Pepper (Capsicum annuum L.) and its expression analysis under hormonal and abiotic stresses

GDP-l-galactose phosphorylase (GGP) is a key rate-limiting enzyme in plant ascorbic acid synthesis, which plays an important role in plant growth and development as well as stress response. However, the presence of GGP and its function in potato and pepper are not known. In this study, we first identified two GGP genes in each potato and pepper genomes using a genome-wide search approach. We then analyzed their physicochemical properties, conserved domains, protein structures and phylogenetic relationships. Phylogenetic tree analysis revealed that members of the potato and pepper GGP gene families are related to eggplant (Solanum melongena L.), Arabidopsis (Arabidopsis thaliana L.), tobacco (Nicotiana tabacum L.) and tomato (Solanum lycopersicum L.), with tomato being the most closely related. The promoter sequences mainly contain homeopathic elements such as light-responsive, hormone-responsive and stress-responsive, with light-responsive elements being the most abundant. By analyzing the structure of the genes, it was found that there is no transmembrane structure or signal peptide in the GGP gene family of potatoes and peppers, and that all of its members are hydrophilic proteins. The expression profiles of different tissues show that StGGP1 has the highest expression levels in leaves, StGGP2 has the highest expression levels in stamens, and CaGGPs have the highest expression levels in the early stages of fruit development (Dev1). It was found that StGGPs and CaGGPs genes showed different response to phytohormones and abiotic stresses. Abscisic acid (ABA) treatment induced the most significant change in the expression of StGGPs, while the expression of CaGGPs showed the most pronounced change under methyl jasmonate (MeJA) treatment. StGGPs responded mainly to dark treatment, whereas CaGGPs responded mainly to NaCl stress. These results provide an important basis for a detailed study about the functions of GGP homologous genes in potato and pepper in response to abiotic stresses.

CaMAPK1 Plays a Vital Role in the Regulation of Resistance to Ralstonia solanacearum Infection and Tolerance to Heat Stress.

As an important member of mitogen-activated protein kinase (MAPK) cascades, MAPKs play an important role in plant defense response against biotic and abiotic stresses; however, the involvement of the majority of the MAPK family members against Ralstonia solanacearum and heat stress (HS) remains poorly understood. In the present study, CaMAPK1 was identified from the genome of pepper and its function against R. solanacearum and HS was analyzed. The transcript accumulations of CaMAPK1 and the activities of its native promoter were both significantly induced by R. solanacearum inoculation, HS, and the application of exogenous hormones, including SA, MeJA, and ABA. Transient expression of CaMAPK1 showed that CaMAPK1 can be targeted throughout the whole cells in Nicotiana benthamiana and triggered chlorosis and hypersensitive response-like cell death in pepper leaves, accompanied by the accumulation of H2O2, and the up-regulations of hormones- and H2O2-associated marker genes. The knock-down of CaMAPK1 enhanced the susceptibility to R. solanacearum partially by down-regulating the expression of hormones- and H2O2-related genes and impairing the thermotolerance of pepper probably by attenuating CaHSFA2 and CaHSP70-1 transcripts. Taken together, our results revealed that CaMAPK1 is regulated by SA, JA, and ABA signaling and coordinates responses to R. solanacearum infection and HS in pepper.

Development of a 45K pepper GBTS liquid-phase gene chip and its application in genome-wide association studies.

Pepper (Capsicum spp.) is a vegetable that is cultivated globally and has undergone extensive domestication, leading to a significant diversification in its agronomic traits. With the advancement of genomics in pepper and the reduction in sequencing costs, the high-throughput detection of single nucleotide polymorphisms (SNPs) and small insertions-deletions (indels) has become increasingly critical for analyzing pepper germplasms and improving breeding programs. As a result, there is a pressing need for a cost-effective, high-throughput, and versatile technique suitable for both foreground and background selection in pepper breeding. In the present study, Python-based web scraping scripts were utilized to systematically extract data from published literatures and relevant sequence databases focusing on pepper genomes. Subsequent to data extraction, SNPs and indels were meticulously identified and filtered. This process culminated in the delineation of core polymorphic sites, which were instrumental in the development of specific probes. Following this, comprehensive phenotypic and genotypic analyses were conducted on a diverse collection of 420 pepper germplasms. Concurrently, a genome-wide association study (GWAS) was conducted to elucidate the genetic determinants of helical fruit shape in peppers. In this study, a 45K pepper Genotyping-By-Target-Sequencing (GBTS) liquid-phase gene chip was developed on the GenoBaits platform. This chip is composed of 45,389 probes, of which 42,535 are derived from core polymorphic sites (CPS) in the background genetic landscape, while 2,854 are associated with foreground agronomic traits, spanning across 43 traits. The CPS probes are spaced at an average interval of 68 Kb. We have assessed the performance of this chip on 420 pepper germplasms, with successful capture of target DNA fragments by 45,387 probes. Furthermore, the probe capture ratio surpassed 70% in 410 of the 420 germplasms tested. Using this chip, we have efficiently genotyped 273 germplasms for spiciness levels and elucidated the genetic relationships among 410 pepper germplasms. Our results allowed for precise clustering of sister lines and C. chinense germplasms. In addition, through a GWAS for helical fruit shape, we identified three quantitative trait loci (QTLs): heli2.1, heli11.1, and heli11.2. Within the heli11.1 QTL, a gene encoding the tubulin alpha chain was identified, suggesting its potential role in the helical growth pattern of pepper fruits. In summary, the 45K pepper GBTS liquid-phase gene chip offers robust detection of polymorphic sites and is a promising tool for advancing research into pepper germplasm and the breeding of new pepper varieties.

Genome-Wide Analysis of the TCP Transcription Factor Gene Family in Pepper (Capsicum annuum L.).

TCP transcription factors play a key role in regulating various developmental processes, particularly in shoot branching, flower development, and leaf development, and these factors are exclusively found in plants. However, comprehensive studies investigating TCP transcription factors in pepper (Capsicum annuum L.) are lacking. In this study, we identified 27 CaTCP members in the pepper genome, which were classified into Class I and Class II through phylogenetic analysis. The motif analysis revealed that CaTCPs in the same class exhibit similar numbers and distributions of motifs. We predicted that 37 previously reported miRNAs target 19 CaTCPs. The expression levels of CaTCPs varied in various tissues and growth stages. Specifically, CaTCP16, a member of Class II (CIN), exhibited significantly high expression in flowers. Class I CaTCPs exhibited high expression levels in leaves, while Class II CaTCPs showed high expression in lateral branches, especially in the CYC/TB1 subclass. The expression profile suggests that CaTCPs play specific roles in the developmental processes of pepper. We provide a theoretical basis that will assist in further functional validation of the CaTCPs.

Conserved and unique features of pepper FLOWERING LOCUS T-like genes revealed by comparative analysis among solanaceous crops

FLOWERING LOCUS T (FT)-like genes play crucial and antagonistic roles in flowering induction in plants. We analyzed the functional and evolutionary characteristics of pepper FT-like genes. According to their similarity to an FT ortholog of tomato, 10 phosphatidylethanolamine-binding protein family genes were retrieved from the pepper genome. Phylogenetic analysis placed the proteins encoded by six of these genes (CaFT1–CaFT6) into an FT-like clade. Comparative genomic analysis showed that four of pepper FT-like genes (CaFT1, CaFT2, CaFT3, CaFT4) have orthologous counterparts in other three solanaceous crops (tomato, potato, and tobacco). CaFT5, which is unique to pepper, was inferred to have arisen by genomic rearrangement followed by duplication of CaFT2. Interestingly, CaFT6, which has been nominated as a pepper FT ortholog, was located in a genomic region not syntenic to the loci of FT orthologs in other crops and was clustered with two other FT-like genes. This result implies the involvement of a unique evolutionary process in the origin of CaFT6. Pepper FT-like genes were mostly expressed in leaves, and their protein products interacted with FLOWERING LOCUS D. When they were overexpressed in Arabidopsis, flowering was promoted by two genes (CaFT6 and CaFT4) and delayed by two others (CaFT1 and CaFT3). The expressions of CaFT6 and CaFT4 were also negatively related to flowering time in pepper accessions implying that they act as flowering promoter genes. The conserved and unique characteristics of pepper FT-like genes provide insights into the dynamic evolution of FT-like genes and useful information for manipulation of FT-like genes to improve the productivity.

Representing true plant genomes: haplotype-resolved hybrid pepper genome with trio-binning.

As sequencing costs decrease and availability of high fidelity long-read sequencing increases, generating experiment specific de novo genome assemblies becomes feasible. In many crop species, obtaining the genome of a hybrid or heterozygous individual is necessary for systems that do not tolerate inbreeding or for investigating important biological questions, such as hybrid vigor. However, most genome assembly methods that have been used in plants result in a merged single sequence representation that is not a true biologically accurate representation of either haplotype within a diploid individual. The resulting genome assembly is often fragmented and exhibits a mosaic of the two haplotypes, referred to as haplotype-switching. Important haplotype level information, such as causal mutations and structural variation is therefore lost causing difficulties in interpreting downstream analyses. To overcome this challenge, we have applied a method developed for animal genome assembly called trio-binning to an intra-specific hybrid of chili pepper (Capsicum annuum L. cv. HDA149 x Capsicum annuum L. cv. HDA330). We tested all currently available softwares for performing trio-binning, combined with multiple scaffolding technologies including Bionano to determine the optimal method of producing the best haplotype-resolved assembly. Ultimately, we produced highly contiguous biologically true haplotype-resolved genome assemblies for each parent, with scaffold N50s of 266.0 Mb and 281.3 Mb, with 99.6% and 99.8% positioned into chromosomes respectively. The assemblies captured 3.10 Gb and 3.12 Gb of the estimated 3.5 Gb chili pepper genome size. These assemblies represent the complete genome structure of the intraspecific hybrid, as well as the two parental genomes, and show measurable improvements over the currently available reference genomes. Our manuscript provides a valuable guide on how to apply trio-binning to other plant genomes.

In Silico RNAseq and Biochemical Analyses of Glucose-6-Phosphate Dehydrogenase (G6PDH) from Sweet Pepper Fruits: Involvement of Nitric Oxide (NO) in Ripening and Modulation.

Pepper (Capsicum annuum L.) fruit is a horticultural product consumed worldwide which has great nutritional and economic relevance. Besides the phenotypical changes that pepper fruit undergo during ripening, there are many associated modifications at transcriptomic, proteomic, biochemical, and metabolic levels. Nitric oxide (NO) is a recognized signal molecule that can exert regulatory functions in diverse plant processes including fruit ripening, but the relevance of NADPH as a fingerprinting of the crop physiology including ripening has also been proposed. Glucose-6-phosphate dehydrogenase (G6PDH) is the first and rate-limiting enzyme of the oxidative phase of the pentose phosphate pathway (oxiPPP) with the capacity to generate NADPH. Thus far, the available information on G6PDH and other NADPH-generating enzymatic systems in pepper plants, and their expression during the ripening of sweet pepper fruit, is very scarce. Therefore, an analysis at the transcriptomic, molecular and functional levels of the G6PDH system has been accomplished in this work for the first time. Based on a data-mining approach to the pepper genome and fruit transcriptome (RNA-seq), four G6PDH genes were identified in pepper plants and designated CaG6PDH1 to CaG6PDH4, with all of them also being expressed in fruits. While CaG6PDH1 encodes a cytosolic isozyme, the other genes code for plastid isozymes. The time-course expression analysis of these CaG6PDH genes during different fruit ripening stages, including green immature (G), breaking point (BP), and red ripe (R), showed that they were differentially modulated. Thus, while CaG6PDH2 and CaG6PDH4 were upregulated at ripening, CaG6PDH1 was downregulated, and CaG6PDH3 was slightly affected. Exogenous treatment of fruits with NO gas triggered the downregulation of CaG6PDH2, whereas the other genes were positively regulated. In-gel analysis using non-denaturing PAGE of a 50-75% ammonium-sulfate-enriched protein fraction from pepper fruits allowed for identifying two isozymes designated CaG6PDH I and CaG6PDH II, according to their electrophoretic mobility. In order to test the potential modulation of such pepper G6PDH isozymes, in vitro analyses of green pepper fruit samples in the presence of different compounds including NO donors (S-nitrosoglutathione and nitrosocysteine), peroxynitrite (ONOO-), a hydrogen sulfide (H2S) donor (NaHS, sodium hydrosulfide), and reducing agents such as reduced glutathione (GSH) and L-cysteine (L-Cys) were assayed. While peroxynitrite and the reducing compounds provoked a partial inhibition of one or both isoenzymes, NaHS exerted 100% inhibition of the two CaG6PDHs. Taken together these data provide the first data on the modulation of CaG6PDHs at gene and activity levels which occur in pepper fruit during ripening and after NO post-harvest treatment. As a consequence, this phenomenon may influence the NADPH availability for the redox homeostasis of the fruit and balance its active nitro-oxidative metabolism throughout the ripening process.

Tryptophan decarboxylase (TDC) in sweet pepper (Capsicum annuum L.): Gene expression analysis during fruit ripening and after nitric oxide exposure

Tryptophan decarboxylase (TDC) catalyzes the conversion of L-tryptophan (Trp) to tryptamine, a first step in the biosynthesis of serotonin and melatonin in plants. Pepper (Capsicum annuum L.) fruit, a globally popular horticultural product has great nutritional and economic values. In addition to that pepper fruit undergoes phenotypical changes during ripening, many other alterations also occur at the transcriptomic, proteomic, biochemical, and metabolic levels. However, little information is known on how many genes encoding for TDC in pepper plants and their expression levels during the ripening of sweet pepper fruit. In the current study, based on a data-mining approach on the pepper genome and transcriptome (RNA-seq), five putative CaTDC genes were identified. They are designated as 1 to 5 based on their localizations in chromosomes and also their previous biochemical data. Among them, CaTDC3 and CaTDC4 encode proteins with tryptophan decarboxylase activity; however, CaTDC1, CaTDC2 and CaTDC5 encode either tyrosine decarboxylase (TYDC) or aromatic aldehyde synthase (AAS), although CaTDC5 shares some degree TDC homology. Therefore, they are considered as the putative CaTDCs until their activity is corroborated. The CaTDC4 and putative CaTDC5 are expressed in pepper fruit. The time-course analysis of these genes during fruit ripening (green immature, breaking point, and red ripe) showed that they were differentially expressed, i.e., CaTDC4 was upregulated, and putative CaTDC5 was downregulated. CaTDC4 was positively modulated by two light-responsive elements, Box4 and TCT-motif, while CaTDC5 was influenced by GT1-motif and G-Box. The protein sequence analysis also allowed identifying the Trp-substrate-binding pocket which is a characteristic of the TDC proteins. Exogenous NO (a signaling molecule) treatment triggered the downregulation of CaTDC4 but not putative CaTDC5. These data provide a novel insight on the potential functions involved in the secondary metabolism of TDCs in fleshy fruits. In the identified three new CaTDC genes, two (CaTDC4 and CaTDC5) expressed in pepper fruits are modulated by exogenous NO treatment during ripening.

Expression of alcohol acyltransferase is a potential determinant of fruit volatile ester variations in Capsicum.

The transcript level of alcohol acyltransferase 1 (AAT1) may be the main factor influencing the variations in volatile esters that characterizing the fruity/exotic aroma of pepper fruit. Volatile esters are key components for characterizing the fruity/exotic aroma of pepper (Capsicum spp.) fruit. In general, the volatile ester content in the fruit is the consequence of a delicate balance between their synthesis by alcohol acyltransferases (AATs) and degradation by carboxylesterases (CXEs). However, the precise role of these families of enzymes with regard to volatile ester content remains unexplored in Capsicum. In this study, we found that the volatile ester content was relatively low in C. annuum and much higher in C. chinense, particularly in pungent varieties. Additionally, fruits collected from multiple non-pungent C. chinense varieties, which harbor loss-of-function mutations in capsaicinoid biosynthetic genes, acyltransferase (Pun1), putative aminotransferase (pAMT), or putative ketoacyl-ACP reductase (CaKR1) were analyzed. The volatile ester contents of non-pungent C. chinense varieties (pamt/pamt) were equivalent to those of pungent varieties, but their levels were significantly lower in non-pungent NMCA30036 (pun12/pun12) and C. chinense (Cakr1/Cakr1) varieties. Multiple AAT-like sequences were identified from the pepper genome sequences, whereas only one CXE-like sequence was identified. Among these, AAT1, AAT2, and CXE1 were isolated from fruits of C. chinense and C. annuum. Gene expression analysis revealed that the AAT1 transcript level is a potential determinant of fruit volatile ester variations in Capsicum. Furthermore, enzymatic assays demonstrated that AAT1 is responsible for the biosynthesis of volatile esters in pepper fruit. Identification of a key gene for aroma biosynthesis in pepper fruit will provide a theoretical basis for the development of molecular tools for flavor improvement.

Comprehensive identification of glutathione peroxidase (GPX) gene family in response to abiotic stress in pepper (Capsicum annuum L.)

Plant glutathione peroxidase (GPX) plays an important role in the maintenance of cell homeostasis and in the antioxidant response in plants. In this study, the peroxidase (GPX) gene family was identified in the whole genome of pepper using bioinformatic method. As a result, a total of 5 CaGPX genes were identified, which were unevenly distributed on 3 of the 12 chromosomes of pepper genome. Based on phylogenetic analysis, 90 GPX genes in 17 species from lower plants to higher plants can be divided into 4 groups (GroupⅠ, Group Ⅱ, Group Ⅲ, Group Ⅳ). The MEME Suite analysis of GPX proteins shows that all these proteins contain four highly conserved motifs, as well as other conserved sequences and amino acid residues. Gene structure analysis revealed the conservative exon-intron organization pattern of these genes. In the promoter region of CaGPX genes, many cis elements of plant hormone and abiotic stress response were identified in each of CaGPX proteins. In addition, expression patterns of CaGPX genes in different tissues, developmental stages and responses to abiotic stress were also performed. The results of qRT-PCR showed that the transcripts of CaGPX genes varied greatly under abiotic stress at different time points. There results suggest that the GPX gene family of pepper may play a role in plant development andstress response. In conclusion, our research provides new insights into the evolution of pepper GPX gene family, and understanding for functional of these genes in response to abiotic stresses.

Identification and expression characteristics of NLP (NIN-like protein) gene family in pepper (Capsicum annuum L.).

Pepper (Capsicum annum L.) is the main crop in the vegetable industry. The growth and development of peppers are regulated by nitrate, but there is limited research on the molecular mechanisms of nitrate absorption and assimilation in peppers. A plant specific transcription factor NLP plays an important role in nitrate signal transduction. In this study, a total of 7 NLP members were identified based on pepper genome data. Two nitrogen transport elements (GCN4) were found in the CaNLP5 promoter. In the phylogenetic tree, CaNLP members are divided into three branches, with pepper NLP and tomato NLP having the closest genetic relationship. The expression levels of CaNLP1, CaNLP3, and CaNLP4 are relatively high in the roots, stems, and leaves. The expression level of CaNLP7 gene is relatively high during the 5-7days of pepper fruit color transformation. After various non-Biotic stress and hormone treatments, the expression of CaNLP1 was at a high level. The expression of CaNLP3 and CaNLP4 was down regulated in leaves, but up regulated in roots. Under conditions of nitrogen deficiency and sufficient nitrate, the expression patterns of NLP genes in pepper leaves and roots were determined. These results provide important insights into the multiple functions of CaNLPs in regulating nitrate absorption and transport.

Genome-wide analysis and identification of stress-responsive genes of the CCCH zinc finger family in Capsicum annuum L.

The CCCH zinc finger gene family encodes a class of proteins that can bind to both DNA and RNA, and an increasing number of studies have demonstrated that the CCCH gene family plays a key role in growth and development and responses to environmental stress. Here, we identified 57 CCCH genes in the pepper (Capsicum annuum L.) genome and explored the evolution and function of the CCCH gene family in C. annuum. Substantial variation was observed in the structure of these CCCH genes, and the number of exons ranged from one to fourteen. Analysis of gene duplication events revealed that segmental duplication was the main driver of gene expansion in the CCCH gene family in pepper. We found that the expression of CCCH genes was significantly up-regulated during the response to biotic and abiotic stress, especially cold and heat stress, indicating that CCCH genes play key roles in stress responses. Our results provide new information on CCCH genes in pepper and will aid future studies of the evolution, inheritance, and function of CCCH zinc finger genes in pepper.

Mapping of the bs5 and bs6 non-race-specific recessive resistances against bacterial spot of pepper.

Bacterial spot caused by Xanthomonas euvesicatoria is a major disease of pepper (Capsicum annuum L.) in warm and humid production environments. Use of genetically resistant cultivars is an effective approach to manage bacterial spot. Two recessive resistance genes, bs5 and bs6, confer non-race-specific resistance against bacterial spot. The objective of our study was to map these two loci in the pepper genome. We used a genotyping-by-sequencing approach to initially map the position of the two resistances. Segregating populations for bs5 and bs6 were developed by crossing susceptible Early CalWonder (ECW) with near-isogenic lines ECW50R (bs5 introgression) or ECW60R (bs6 introgression). Following fine-mapping, bs5 was delimited to a ~535 Kbp interval on chromosome 3, and bs6 to a ~666 Kbp interval in chromosome 6. We identified 14 and 8 candidate resistance genes for bs5 and bs6, respectively, based on predicted protein coding polymorphisms between ECW and the corresponding resistant parent. This research enhances marker-assisted selection of bs5 and bs6 in breeding programs and is a crucial step towards elucidating the molecular mechanisms underlying the resistances.

Genome-wide characterization of ascorbate peroxidase gene family in pepper (Capsicum annuum L.) in response to multiple abiotic stresses.

Pepper is widely grown all over the world, so it faces many abiotic stresses, such as drought, high temperature, low temperature, salt damage, and so on. Stresses causing the accumulation of reactive oxidative species (ROS) in plants are removed by antioxidant defense systems, and ascorbate peroxidase (APX) is an important antioxidant enzyme. Therefore, the present study performed genome-wide identification of the APX gene family in pepper. We identified nine members of the APX gene family in the pepper genome according to the APX proteins' conserved domain in Arabidopsis thaliana. The physicochemical property analysis showed that CaAPX3 had the longest protein sequence and the largest molecular weight of all genes, while CaAPX9 had the shortest protein sequence and the smallest MW. The gene structure analysis showed that CaAPXs were composed of seven to 10 introns. The CaAPX genes were divided into four groups. The APX genes of groups I and IV were localized in the peroxisomes and chloroplasts, respectively; the group II genes were localized in the chloroplasts and mitochondria; and the group III genes were located in the cytoplasm and extracell. The conservative motif analysis showed that all APX genes in the pepper had motif 2, motif 3, and motif 5. The APX gene family members were distributed on five chromosomes (Chr. 2, 4, 6, 8, and 9). The cis-acting element analysis showed that most CaAPX genes contain a variety of cis-elements related to plant hormones and abiotic stress. RNA-seq expression analysis showed that the expression patterns of nine APXs were different in vegetative and reproductive organs at different growth and development stages. In addition, the qRT-PCR analysis of the CaAPX genes revealed significant differential expression in response to high temperature, low temperature, and salinity stresses in leaf tissue. In conclusion, our study identified the APX gene family members in the pepper and predicted the functions of this gene family, which would provide resources for further functional characterization of CaAPX genes.

Class III Peroxidases (POD) in Pepper (Capsicum annuum L.): Genome-Wide Identification and Regulation during Nitric Oxide (NO)-Influenced Fruit Ripening.

The class III peroxidases (PODs) catalyze the oxidation of several substrates coupled to the reduction of H2O2 to water, and play important roles in diverse plant processes. The POD family members have been well-studied in several plant species, but little information is available on sweet pepper fruit physiology. Based on the existing pepper genome, a total of 75 CaPOD genes have been identified, but only 10 genes were found in the fruit transcriptome (RNA-Seq). The time-course expression analysis of these genes showed that two were upregulated during fruit ripening, seven were downregulated, and one gene was unaffected. Furthermore, nitric oxide (NO) treatment triggered the upregulation of two CaPOD genes whereas the others were unaffected. Non-denaturing PAGE and in-gel activity staining allowed identifying four CaPOD isozymes (CaPOD I-CaPOD IV) which were differentially modulated during ripening and by NO. In vitro analyses of green fruit samples with peroxynitrite, NO donors, and reducing agents triggered about 100% inhibition of CaPOD IV. These data support the modulation of POD at gene and activity levels, which is in agreement with the nitro-oxidative metabolism of pepper fruit during ripening, and suggest that POD IV is a target for nitration and reducing events that lead to its inhibition.

CaREM1.4 interacts with CaRIN4 to regulate Ralstonia solanacearum tolerance by triggering cell death in pepper.

Remorins, plant-specific proteins, have a significant role in conferring on plants the ability to adapt to adverse environments. However, the precise function of remorins in resistance to biological stress remains largely unknown. Eighteen CaREM genes were identified in pepper genome sequences based on the C-terminal conserved domain that is specific to remorin proteins in this research. Phylogenetic relations, chromosomal localization, motif, gene structures, and promoter regions of these remorins were analyzed and a remorin gene, CaREM1.4, was cloned for further study. The transcription of CaREM1.4 in pepper was induced by infection with Ralstonia solanacearum. Knocking down CaREM1.4 in pepper using virus-induced gene silencing (VIGS) technologies reduced the resistance of pepper plants to R. solanacearum and downregulated the expression of immunity-associated genes. Conversely, transient overexpression of CaREM1.4 in pepper and Nicotiana benthamiana plants triggered hypersensitive response-mediated cell death and upregulated expression of defense-related genes. In addition, CaRIN4-12, which interacted with CaREM1.4 at the plasma membrane and cell nucleus, was knocked down with VIGS, decreasing the susceptibility of Capsicum annuum to R. solanacearum. Furthermore, CaREM1.4 reduced ROS production by interacting with CaRIN4-12 upon co-injection in pepper. Taken together, our findings suggest that CaREM1.4 may function as a positive regulator of the hypersensitive response, and it interacts with CaRIN4-12, which negatively regulates plant immune responses of pepper to R. solanacearum. Our study provides new evidence for comprehending the molecular regulatory network of plant cell death.

Characterization of the B-BOX gene family in pepper and the role of CaBBX14 in defense response against Phytophthora capsici infection

The B-box (BBX) transcription factors are widely implicated in plant growth, development, and response to various biotic and abiotic stresses. However, their roles in the response of pepper to Phytophthora capsici infection (PCI) remain largely unexplored. Here, we report a total of 25 CaBBX genes with an uneven distribution were identified in pepper genome, and their characteristics, phylogenetic relationships, gene structures, conserved domains, and expression profiles were validated. CaBBXs were classified into five major clades (I to V) based on their phylogenetic relationships and conserved domains (presence of one or two B­box domains and a CCT domain). Gene duplication analysis demonstrated that there are two segmental duplication events but no tandem duplication event within pepper genome. Conserved motif and gene structure analysis revealed that the CaBBXs in the same clade have relatively similar motif arrangements and exon-intron patterns. Expression analysis revealed that the CaBBX genes have different expression levels in various tissues, and some of which were significantly induced during PCI and exogenous salicylic acid (SA) treatment. Among them, CaBBX14 displayed remarkable changed expression during PCI and SA treatment. The silencing of CaBBX14 increases pepper susceptibility to PCI, and also decreases in SA content and expression of pathogenesis-related (PR) and SA-related genes compared with control plants. Together, these findings advance our knowledge base on biological functions of CaBBXs in pepper during PCI through the SA signaling pathway, and we provide an example demonstrating that the potential of CaBBX14 to improve pepper resistance to PCI.

CRS: An online database of Capsicum annuum RNA-seq libraries

RNA sequencing (RNA-seq) technique plays an important role in the analyses of gene expression profiling, transcript variation, non-coding sequence function, and so on. RNA-seq technique has been widely used in Capsicum annuum (chili pepper) research. The number of current pepper RNA-seq libraries is huge and is increasing every year. It is not only difficult for researchers to obtain the underlying information of these data, but also causes a waste of resources to some extent. To solve this problem, we developed an online database, namely CRS, to host 467 publicly available RNA-seq libraries of C. annuum obtained from the Sequence Read Archive database (as of November 2021), which integrates the information of gene expression, tissue specificity, Gene Ontology terms, and KEGG Orthology. We believe that CRS will enable more convenient and efficient exploration of pepper RNA-seq data and be conducive to a deeper understanding of the pepper genome.