CaWRKY01-10 and CaWRKY08-4 Confer Pepper's Resistance to Phytophthora capsici Infection by Directly Activating a Cluster of Defense-Related Genes.

Phytophthora blight of pepper, which is caused by the notorious oomycete pathogen Phytophthora capsici, is a serious disease in global pepper production regions. Our previous study had identified two WRKY transcription factors (TFs), CaWRKY01-10 and CaWRKY08-4, which are prominent modulators in the resistant pepper line CM334 against P. capsici infection. However, their functional mechanisms and underlying signaling networks remain unknown. Herein, we determined that CaWRKY01-10 and CaWRKY08-4 are localized in plant nuclei. Transient overexpression assays indicated that both CaWRKY01-10 and CaWRKY08-4 act as positive regulators in pepper resistance to P. capsici. Besides, the stable overexpression of CaWRKY01-10 and CaWRKY08-4 in transgenic Nicotiana benthamiana plants also significantly enhanced the resistance to P. capsici. Using comprehensive approaches including RNA-seq, CUT&RUN-qPCR, and dual-luciferase reporter assays, we revealed that overexpression of CaWRKY01-10 and CaWRKY08-4 can activate the expressions of the same four Capsicum annuum defense-related genes (one PR1, two PR4, and one pathogen-related gene) by directly binding to their promoters. However, we did not observe protein-protein interactions and transcriptional amplification/inhibition effects of their shared target genes when coexpressing these two WRKY TFs. In conclusion, these data suggest that both of the resistant line specific upregulated WRKY TFs (CaWRKY01-10 and CaWRKY08-4) can confer pepper's resistance to P. capsici infection by directly activating a cluster of defense-related genes and are potentially useful for genetic improvement against Phytophthora blight of pepper and other crops.

Capsicum chinense Jacq.-derived glutaredoxin (CcGRXS12) alters redox status of the cells to confer resistance against pepper mild mottle virus (PMMoV-I).

KEY MESSAGE: The CcGRXS12 gene protects plants from cellular oxidative damage that are caused by both biotic and abiotic stresses. The protein possesses GSH-disulphide oxidoreductase property but lacks Fe-S cluster assembly mechanism. Glutaredoxins (Grxs) are small, ubiquitous and multi-functional proteins. They are present in different compartments of plant cells. A chloroplast targeted Class I GRX (CcGRXS12) gene was isolated from Capsicum chinense during the pepper mild mottle virus (PMMoV) infection. Functional characterization of the gene was performed in Nicotiana benthamiana transgenic plants transformed with native C. chinense GRX (Nb:GRX), GRX-fused with GFP (Nb:GRX-GFP) and GRX-truncated for chloroplast sequences fused with GFP (Nb:Δ2MGRX-GFP). Overexpression of CcGRXS12 inhibited the PMMoV-I accumulation at the later stage of infection, accompanied with the activation of salicylic acid (SA) pathway pathogenesis-related (PR) transcripts and suppression of JA/ET pathway transcripts. Further, the reduced accumulation of auxin-induced Glutathione-S-Transferase (pCNT103) in CcGRXS12 overexpressing lines indicated that the protein could protect the plants from the oxidative stress caused by the virus. PMMoV-I infection increased the accumulation of pyridine nucleotides (PNs) mainly due to the reduced form of PNs (NAD(P)H), and it was high in Nb:GRX-GFP lines compared to other transgenic lines. Apart from biotic stress, CcGRXS12 protects the plants from abiotic stress conditions caused by H2O2 and herbicide paraquat. CcGRXS12 exhibited GSH-disulphide oxidoreductase activity in vitro; however, it was devoid of complementary Fe-S cluster assembly mechanism found in yeast. Overall, this study proves that CcGRXS12 plays a crucial role during biotic and abiotic stress in plants.

Characterization of leucine aminopeptidase (LAP) activity in sweet pepper fruits during ripening and its inhibition by nitration and reducing events.

KEY MESSAGE: Pepper fruits contain two leucine aminopeptidase (LAP) genes which are differentially modulated during ripening and by nitric oxide. The LAP activity increases during ripening but is negatively modulated by nitration. Leucine aminopeptidase (LAP) is an essential metalloenzyme that cleaves N-terminal leucine residues from proteins but also metabolizes dipeptides and tripeptides. LAPs play a fundamental role in cell protein turnover and participate in physiological processes such as defense mechanisms against biotic and abiotic stresses, but little is known about their involvement in fruit physiology. This study aims to identify and characterize genes encoding LAP and evaluate their role during the ripening of pepper (Capsicum annuum L.) fruits and under a nitric oxide (NO)-enriched environment. Using a data-mining approach of the pepper plant genome and fruit transcriptome (RNA-seq), two LAP genes, designated CaLAP1 and CaLAP2, were identified. The time course expression analysis of these genes during different fruit ripening stages showed that whereas CaLAP1 decreased, CaLAP2 was upregulated. However, under an exogenous NO treatment of fruits, both genes were downregulated. On the contrary, it was shown that during fruit ripening LAP activity increased by 81%. An in vitro assay of the LAP activity in the presence of different modulating compounds including peroxynitrite (ONOO-), NO donors (S-nitrosoglutathione and nitrosocyteine), reducing agents such as reduced glutathione (GSH), L-cysteine (L-Cys), and cyanide triggered a differential response. Thus, peroxynitrite and reducing compounds provoked around 50% inhibition of the LAP activity in green immature fruits, whereas cyanide upregulated it 1.5 folds. To our knowledge, this is the first characterization of LAP in pepper fruits as well as of its regulation by diverse modulating compounds. Based on the capacity of LAP to metabolize dipeptides and tripeptides, it could be hypothesized that the LAP might be involved in the GSH recycling during the ripening process.

Chili pepper extracts, capsaicin, and dihydrocapsaicin as potential anticancer agents targeting topoisomerases.

DNA topoisomerases regulate conformational changes in DNA topology during normal cell growth, such as replication, transcription, recombination, and repair, and may be targeted for anticancer drugs. A DNA topology assay was used to investigate DNA-damaging/protective activities of extracts from Habanero Red (HR), Habanero Maya Red (HMR), Trinidad Moruga Scorpion (TMS), Jalapeno (J), Serrano pepper (SP), Habanero Red Savina (HRS), Bhut Jolokia (BJ), and Jamaica Rosso (JR) peppers, demonstrating their inhibitory effect on the relaxation of pBR by Topo I. DNA topoisomerase II (Topo II) is proven therapeutic target of anticancer drugs. Complete inhibition of Topo II was observed for samples TMS, HR, and HMR. Extracts J and SP had the lowest capsaicin and dihydrocapsaicin content compared to other peppers. HR, HMR, TMS, J, S, HRS, BJ, JR extracts showed the anticancer effect, examined by MTS and xCell assay on the in vitro culture of human colon carcinoma cell line HCT116.

Comparative transcriptome analysis and Arabidopsis thaliana overexpression reveal key genes associated with cadmium transport and distribution in root of two Capsicum annuum cultivars.

The molecular mechanisms underlying high and low cadmium (Cd) accumulation in hot pepper cultivars remain unclear. In this study, comparative transcriptome analysis of root between high-Cd (J) and low-Cd (Z) cultivars was conducted under hydroponic cultivation with 0 and 0.4 mg/L Cd, respectively. The results showed that J enhanced the root uptake of Cd by elevating the expression of Nramp5 and counteracting Cd toxicity by increasing the expression of genes, such as NIR1, GLN1, and IAA9. Z reduced Cd accumulation by enhancing the cell wall lignin synthesis genes PAL, COMT, 4CL, LAC, and POD and the Cd transporters ABC, MTP1, and DTX1. Elevated expression of genes related to sulfur metabolism was observed in Z, potentially contributing to its ability to detoxify Cd. To investigate the function of CaCOMT1, an Arabidopsis thaliana overexpression line (OE-CaCOMT1) was constructed. The results revealed that OE-CaCOMT1 drastically increased the lignin content by 38-42% and reduced the translocation of Cd to the aboveground parts by 32%. This study provides comprehensive insights into the mechanisms underlying Cd accumulation in hot pepper cultivars using transcriptome analysis. Moreover, this study elucidates the critical function of CaCOMT1, providing a theoretical foundation for the production of low-Cd vegetables for food safety.

Transcription factor CaHDZ15 promotes pepper basal thermotolerance by activating HEAT SHOCK FACTORA6a.

High temperature stress (HTS) is a serious threat to plant growth and development and to crop production in the context of global warming, and plant response to HTS is largely regulated at the transcriptional level by the actions of various transcription factors (TFs). However, whether and how homeodomain-leucine zipper (HD-Zip) TFs are involved in thermotolerance are unclear. Herein, we functionally characterized a pepper (Capsicum annuum) HD-Zip I TF CaHDZ15. CaHDZ15 expression was upregulated by HTS and abscisic acid in basal thermotolerance via loss- and gain-of-function assays by virus-induced gene silencing in pepper and overexpression in Nicotiana benthamiana plants. CaHDZ15 acted positively in pepper basal thermotolerance by directly targeting and activating HEAT SHOCK FACTORA6a (HSFA6a), which further activated CaHSFA2. In addition, CaHDZ15 interacted with HEAT SHOCK PROTEIN 70-2 (CaHsp70-2) and glyceraldehyde-3-phosphate dehydrogenase1 (CaGAPC1), both of which positively affected pepper thermotolerance. CaHsp70-2 and CaGAPC1 promoted CaHDZ15 binding to the promoter of CaHSFA6a, thus enhancing its transcription. Furthermore, CaHDZ15 and CaGAPC1 were protected from 26S proteasome-mediated degradation by CaHsp70-2 via physical interaction. These results collectively indicate that CaHDZ15, modulated by the interacting partners CaGAPC1 and CaHsp70-2, promotes basal thermotolerance by directly activating the transcript of CaHSFA6a. Thus, a molecular linkage is established among CaHsp70-2, CaGAPC1, and CaHDZ15 to transcriptionally modulate CaHSFA6a in pepper thermotolerance.

CaCP15 Gene Negatively Regulates Salt and Osmotic Stress Responses in Capsicum annuum L.

Salt and osmotic stress seriously restrict the growth, development, and productivity of horticultural crops in the greenhouse. The papain-like cysteine proteases (PLCPs) participate in multi-stress responses in plants. We previously demonstrated that salt and osmotic stress affect cysteine protease 15 of pepper (Capsicum annuum L.) (CaCP15); however, the role of CaCP15 in salt and osmotic stress responses is unknown. Here, the function of CaCP15 in regulating pepper salt and osmotic stress resistance was explored. Pepper plants were subjected to abiotic (sodium chloride, mannitol, salicylic acid, ethrel, methyl jasmonate, etc.) and biotic stress (Phytophthora capsici inoculation). The CaCP15 was silenced through the virus-induced gene silencing (VIGS) and transiently overexpressed in pepper plants. The full-length CaCP15 fragment is 1568 bp, with an open reading frame of 1032 bp, encoding a 343 amino acid protein. CaCP15 is a senescence-associated gene 12 (SAG12) subfamily member containing two highly conserved domains, Inhibitor 129 and Peptidase_C1. CaCP15 expression was the highest in the stems of pepper plants. The expression was induced by salicylic acid, ethrel, methyl jasmonate, and was infected by Phytophthora capsici inoculation. Furthermore, CaCP15 was upregulated under salt and osmotic stress, and CaCP15 silencing in pepper enhanced salt and mannitol stress resistance. Conversely, transient overexpression of CaCP15 increased the sensitivity to salt and osmotic stress by reducing the antioxidant enzyme activities and negatively regulating the stress-related genes. This study indicates that CaCP15 negatively regulates salt and osmotic stress resistance in pepper via the ROS-scavenging.

Genetic Regulation, Environmental Cues, and Extraction Methods for Higher Yield of Secondary Metabolites in Capsicum.

Capsicum (chili pepper) is a widely popular and highly consumed fruit crop with beneficial secondary metabolites such as capsaicinoids, carotenoids, flavonoids, and polyphenols, among others. Interestingly, the secondary metabolite profile is a dynamic function of biosynthetic enzymes, regulatory transcription factors, developmental stage, abiotic and biotic environment, and extraction methods. We propose active manipulable genetic, environmental, and extraction controls for the modulation of quality and quantity of desired secondary metabolites in Capsicum species. Specific biosynthetic genes such as Pun (AT3) and AMT in the capsaicinoids pathway and PSY, LCY, and CCS in the carotenoid pathway can be genetically engineered for enhanced production of capsaicinoids and carotenoids, respectively. Generally, secondary metabolites increase with the ripening of the fruit; however, transcriptional regulators such as MYB, bHLH, and ERF control the extent of accumulation in specific tissues. The precise tuning of biotic and abiotic factors such as light, temperature, and chemical elicitors can maximize the accumulation and retention of secondary metabolites in pre- and postharvest settings. Finally, optimized extraction methods such as ultrasonication and supercritical fluid method can lead to a higher yield of secondary metabolites. Together, the integrated understanding of the genetic regulation of biosynthesis, elicitation treatments, and optimization of extraction methods can maximize the industrial production of secondary metabolites in Capsicum.

New insights into short-term water stress tolerance through transcriptomic and metabolomic analyses on pepper roots.

In the current climate change scenario, water stress is a serious threat to limit crop growth and yields. It is necessary to develop tolerant plants that cope with water stress and, for this purpose, tolerance mechanisms should be studied. NIBER® is a proven water stress- and salt-tolerant pepper hybrid rootstock (Gisbert-Mullor et al., 2020; López-Serrano et al., 2020), but tolerance mechanisms remain unclear. In this experiment, NIBER® and A10 (a sensitive pepper accession (Penella et al., 2014)) response to short-term water stress at 5 h and 24 h was studied in terms of gene expression and metabolites content in roots. GO terms and gene expression analyses evidenced constitutive differences in the transcriptomic profile of NIBER® and A10, associated with detoxification systems of reactive oxygen species (ROS). Upon water stress, transcription factors like DREBs and MYC are upregulated and the levels of auxins, abscisic acid and jasmonic acid are increased in NIBER®. NIBER® tolerance mechanisms involve an increase in osmoprotectant sugars (i.e., trehalose, raffinose) and in antioxidants (spermidine), but lower contents of oxidized glutathione compared to A10, which indicates less oxidative damage. Moreover, the gene expression for aquaporins and chaperones is enhanced. These results show the main NIBER® strategies to overcome water stress.

The bs5 allele of the susceptibility gene Bs5 of pepper (Capsicum annuum L.) encoding a natural deletion variant of a CYSTM protein conditions resistance to bacterial spot disease caused by Xanthomonas species.

KEY MESSAGE: The bs5 resistance gene against bacterial spot was identified by map-based cloning. The recessive bs5 gene of pepper (Capsicum annuum L.) conditions a non-hypersensitive resistance trait, characterized by a slightly swollen, pale green, photosynthetically active leaf tissue, following Xanthomonas euvesicatoria infection. The isolation of the bs5 gene by map-based cloning revealed that the bs5 protein was shorter by 2 amino acids as compared to the wild type Bs5 protein. The natural 2 amino acid deletion occurred in the cysteine-rich transmembrane domain of the tail-anchored (TA) protein, Ca_CYSTM1. The protein products of the wild type Bs5 and mutant bs5 genes were shown to be located in the cell membrane, indicating an unknown function in this membrane compartment. Successful infection of the Bs5 pepper lines was abolished by the 6 bp deletion in the TM encoding domain of the Ca_CYSTM1 gene in bs5 homozygotes, suggesting, that the resulting resistance might be explained by the lack of entry of the Xanthomonas specific effector molecules into the plant cells.

Diversity and expression analysis of ZIP transporters and associated metabolites under zinc and iron stress in Capsicum.

The members of ZRT, IRT-like protein (ZIP) family are involved in the uptake and transportation of several metal ions. Here, we report a comprehensive identification of ZIP transporter genes from Capsicum annuum, C. chinense, and C. baccatum, and their expression analysis under Zn and Fe stress. Changes in root morphology and differential accumulation of several metabolites from sugars, amino acids, carboxylic acids, and fatty acids in root and leaf tissues of plants in the absence of Zn and Fe were observed. Further, metabolites such as L-aspartic acid, 2-ketoglutaric acids, ß-L-fucopyranose, quininic acid, chlorogenic acid, and aucubin were significantly upregulated in root and leaf tissues under Zn/Fe deprived conditions. qRT-PCR analysis of 17 CaZIPs in different tissues revealed tissue-specific expression of CaZIP1-2, CaZIP4-8, CaZIP13, and CaZIP16-17 under normal conditions. However, the absence of Zn and Fe significantly induced the expression of CaZIP4-5, CaZIP7-9, and CaZIP14 genes in root and leaf tissues. Additionally, in the absence of Fe, upregulation of CaZIP4-5 and CaZIP8 and increased uptake of mineral elements Cu, Zn, Mg, P, and S were observed in roots, suggesting their potential role in metal-ion uptake in Capsicum. The identified genes provide the basis for future studies of mineral uptake and their biofortification to increase the nutritional values in Capsicum.

Genome-wide identification of RUB activating enzyme and conjugating enzyme gene families and their expression analysis under abiotic stresses in Capsicum annuum.

NEDD8/RUB, as a ubiquitin-like protein, participates in the post-translational modification of protein and requires unique E1, E2, and E3 enzymes to bind to its substrate. The RUB E1 activating enzyme and E2 conjugating enzyme play a significant role in the neddylation. However, it is unknown whether RUB E1 and E2 exist in pepper and what its function is. In this study, a total of three putative RUB E1 and five RUB E2 genes have been identified in the pepper genome. Subsequently, their physical and chemical properties, gene structure, conserved domains and motifs, phylogenetic relationship, and cis-acting elements were analyzed. The structure and conserved domain of RUB E1 and E2 are similar to that of Arabidopsis and tomato. The RUB E1 and E2 genes were randomly distributed on seven chromosomes, and there were two pairs of collinearity between pepper and Arabidopsis and eight pairs of collinearity between pepper and tomato. Phylogenetic analysis reveals that RUB E1 and E2 genes of pepper have a closer relationship with that of tomato, potato, and Nicotiana attenuate. The cis-elements of RUB E1 and E2 genes contained hormone response and stress response. RUB E1 and E2 genes were expressed in at least one tissue and CaRCE1.3 and CaRCE2.1 were exclusively expressed in flowers and anthers. Moreover, the expression of RUB E1 genes (CaECR1, CaAXR1.1, and CaAXR1.2) and RUB E2 genes (CaRCE1.1, CaRCE1.2, and CaRCE2.1) was increased to varying degrees under low-temperature, drought, salt, ABA, and IAA treatments, while CaRCE1.3 and CaRCE2.2 were down-regulated under low-temperature treatment. In addition, these genes were hardly expressed under MeJA treatment. In summary, this study provides a theoretical foundation to explore the role of RUB E1 and E2 in the response of plants to stress.

Role of the cAMP signaling pathway in the dissemination and development on pepper fruit anthracnose disease caused by Colletotrichum scovillei.

The ascomycete fungus Colletotrichum scovillei causes severe anthracnose disease on the fruit of sweet pepper and chili pepper (Capsicum annuum L.) worldwide. Understanding the biology of C. scovillei would improve the management of fruit anthracnose diseases. The cyclic adenosine monophosphate (cAMP) signaling pathway regulates diverse cellular and physiological processes in several foliar fungal pathogens. We investigated the roles of the cAMP signaling pathway in C. scovillei using pharmaceutical and genetic approaches. Exogenous cAMP was found to increase conidiation, appressorium formation, and anthracnose disease development in C. scovillei. CsAc1, CsCap1, and CsPdeH, which regulate the intracellular cAMP level, were deleted by homology-dependent gene replacement. Expectedly, the intracellular cAMP level was significantly decreased in ΔCsac1 and ΔCscap1 but increased in ΔCspdeh. All three deletion mutants exhibited serious defects in multiple fungal developments and pathogenicity, suggesting regulation of the intracellular cAMP level is important for C. scovillei. Notably, exogenous cAMP recovered the defect of ΔCsac1 in appressorium development, but not penetration, which was further recovered by adding CaCl2. This result suggests that CsAc1 is associated with both the cAMP and Ca2+ signaling pathways in C. scovillei. ΔCscap1 produced morphologically abnormal conidia with reduced tolerance to thermal stress. ΔCspdeh was completely defective in conidiation in C. scovillei, unlike other foliar pathogens. Taken together, these results demonstrate the importance of cAMP signaling in anthracnose disease caused by C. scovillei.

Detection of indigenous gut bacteria related to red chilli pepper (Capsicum annuum) in murine caecum and human faecal cultures.

BACKGROUND: Red chilli pepper (Capsicum annuum; RP) is a popular spice containing the active compound capsaicin. Indigenous gut bacteria and metabolism can affect host health. The functions of capsaicin, including the regulation of metabolic health and anti-oxidant properties, may be correlated with the gut microbiota. METHODS: To identify indigenous gut bacteria that are responsive to RP, Institute of Cancer Research mice fed a diet with no fibre or with 5% (w/w) RP for 14 days. Additionally, human stool samples collected from four healthy volunteers were incubated without (control) or with 2% (w/v) RP at 37 °C for 24 h. Microbiota in murine caecal samples and human faecal cultures were analysed using 16S rRNA (V4) amplicon sequencing. RESULTS: Compared with the microbiota in mice fed no-fibre diets, Lachnospiraceae spp.-, Muribaculaceae spp.-, and Phacaeicola vulgatus-like bacteria were defined as murine RP-responsive indigenous gut bacteria (RP-RIB). In the human faecal cultures, acetate and propionate levels were higher in RP cultures than in the control cultures. Subdoligranulum spp.-, Blautia spp.-, Faecalibacterium prausnitzii-, P. vulgatus-, and Prevotella copri-like bacteria were defined as human RP-RIB. Compared with control culture Fe-reducing power was increased in the culture with RP. CONCLUSION: RP increases the amount of short-chain fatty acid-producing bacteria and beneficial gut bacteria in mouse and human faecal cultures. Overall, RP could have a positive effect on the host by altering the gut microbiota.

Integrated omics analysis identified genes and their splice variants involved in fruit development and metabolites production in Capsicum species.

To date, several transcriptomic studies during fruit development have been reported; however, no comprehensive integrated study on expression diversity, alternative splicing, and metabolomic profiling was reported in Capsicum. This study analyzed RNA-seq data and untargeted metabolomic profiling from early green (EG), mature green (MG), and breaker (Br) fruit stages from two Capsicum species, i.e., C. annuum (Cann) and C. frutescens (Cfrut) from Northeast India. A total of 117,416 and 96,802 alternatively spliced events (AltSpli-events) were identified from Cann and Cfrut, respectively. Among AltSpli-events, intron retention (IR; 32.2% Cann and 25.75% Cfrut) followed by alternative acceptor (AA; 15.4% Cann and 18.9% Cfrut) were the most abundant in Capsicum. Around 7600 genes expressed in at least one fruit stage of Cann and Cfrut were AltSpli. The study identified spliced variants of genes including transcription factors (TFs) potentially involved in fruit development/ripening (Aux/IAA 16-like, ETR, SGR1, ARF, CaGLK2, ETR, CaAGL1, MADS-RIN, FUL1, SEPALLATA1), carotenoid (PDS, CA1, CCD4, NCED3, xanthoxin dehydrogenase, CaERF82, CabHLH100, CaMYB3R-1, SGR1, CaWRKY28, CaWRKY48, CaWRKY54), and capsaicinoids or flavonoid biosynthesis (CaMYB48, CaWRKY51), which were significantly differentially spliced (DS) between consecutive Capsicum fruit stages. Also, this study observed that differentially expressed isoforms (DEiso) from 38 genes with differentially spliced events (DSE) were significantly enriched in various metabolic pathways such as starch and sucrose metabolism, amino acid metabolism, cysteine cutin suberin and wax biosynthesis, and carotenoid biosynthesis. Furthermore, the metabolomic profiling revealed that metabolites from aforementioned pathways such as carbohydrates (mainly sugars such as D-fructose, D-galactose, maltose, and sucrose), organic acids (carboxylic acids), and peptide groups significantly altered during fruit development. Taken together, our findings could help in alternative splicing-based targeted studies of candidate genes involved in fruit development and ripening in Capsicum crop.

Genome sequence for the blue-flowered Andean shrub Iochroma cyaneum reveals extensive discordance across the berry clade of Solanaceae.

The tomato (Solanum lycopersicum L.) family, Solanaceae, is a model clade for a wide range of applied and basic research questions. Currently, reference-quality genomes are available for over 30 species from seven genera, and these include numerous crops as well as wild species [e.g., Jaltomata sinuosa (Miers) Mione and Nicotiana attenuata Torr. ex S. Watson]. Here we present the genome of the showy-flowered Andean shrub Iochroma cyaneum (Lindl.) M. L. Green, a woody lineage from the tomatillo (Physalis philadelphica Lam.) subfamily Physalideae. The assembled size of the genome (2.7 Gb) is more similar in size to pepper (Capsicum annuum L.) (2.6 Gb) than to other sequenced diploid members of the berry clade of Solanaceae [e.g., potato (Solanum tuberosum L.), tomato, and Jaltomata]. Our assembly recovers 92% of the conserved orthologous set, suggesting a nearly complete genome for this species. Most of the genomic content is repetitive (69%), with Gypsy elements alone accounting for 52% of the genome. Despite the large amount of repetitive content, most of the 12 I. cyaneum chromosomes are highly syntenic with tomato. Bayesian concordance analysis provides strong support for the berry clade, including I. cyaneum, but reveals extensive discordance along the backbone, with placement of chili pepper and Jaltomata being highly variable across gene trees. The I. cyaneum genome contributes to a growing wealth of genomic resources in Solanaceae and underscores the need for expanded sampling of diverse berry genomes to dissect major morphological transitions.

Identification of CaAN3 as a fruit-specific regulator of anthocyanin biosynthesis in pepper (Capsicum annuum).

KEY MESSAGE: The novel gene CaAN3 encodes an R2R3 MYB transcription factor that regulates fruit-specific anthocyanin accumulation. The key regulatory gene CaAN2 encodes an R2R3 MYB transcription factor that regulates anthocyanin biosynthesis in various tissues in pepper (Capsicum annuum). However, CaAN2 is not expressed in certain pepper accessions showing fruit-specific anthocyanin accumulation. In this study, we identified the novel locus CaAN3 as a regulator of fruit-specific anthocyanin biosynthesis, using an F2 population derived from a hybrid cultivar with purple immature fruits and segregating for CaAN3. We extracted total RNA, assembled two RNA pools according to fruit color, and carried out bulked segregant RNA sequencing. We aligned the raw reads to the pepper reference genome Dempsey and identified 6,672 significant single nucleotide polymorphisms (SNPs) by calculating the Δ(SNP-index) between the two pools. We then conducted molecular mapping to delimit the target region of CaAN3 to the interval 184.6-186.4 Mbp on chromosome 10. We focused on Dem.v1.00043895, encoding an R2R3 MYB transcription factor, as the strongest candidate gene. Sequence analysis revealed four insertion/deletion polymorphisms in the promoter region of the green CaAN3 allele. We employed virus-induced gene silencing and transient overexpression assays to characterize the function of the candidate gene. When Dem.v1.00043895 was silenced in pepper, anthocyanin accumulation decreased in the pericarp, while the transient overexpression of Dem.v1.00043895 in Nicotiana benthamiana leaves resulted in the accumulation of anthocyanins around the infiltration sites. These results showed that Dem.v1.00043895 is CaAN3, an activator of anthocyanin biosynthesis in pepper fruits.

A lineage-specific arginine in POS1 is required for fruit size control in Physaleae (Solanaceae) via gene co-option.

Solanaceae have important economic value mainly due to their edible fruits. Physalis organ size 1/cytokinin response factor 3 (POS1/CRF3), a unique gene in Solanaceae, is involved in fruit size variation in Physalis but not in Solanum. However, the underlying mechanisms remain elusive. Here, we found that POS1/CRF3 was likely created via the fusion of CRF7 and CRF8 duplicates. Multiple genetic manipulations revealed that only POS1 and Capsicum POS1 (CaPOS1) functioned in fruit size control via the positive regulation of cell expansion. Comparative studies in a phylogenetic framework showed the directional enhancement of POS1-like expression in the flowers and fruits of Physaleae and the specific gain of certain interacting proteins associated with cell expansion by POS1 and CaPOS1. A lineage-specific single nucleotide polymorphism (SNP) caused the 68th amino acid histidine in the POS1 orthologs of non-Physaleae (Nicotiana and Solanum) to change to arginine in Physaleae (Physalis and Capsicum). Substituting the arginine in Physaleae POS1-like by histidine completely abolished their function in the fruits and the protein-protein interaction (PPI) with calreticulin-3. Transcriptomic comparison revealed the potential downstream pathways of POS1, including the brassinosteroid biosynthesis pathway. However, POS1-like may have functioned ancestrally in abiotic stress within Solanaceae. Our work demonstrated that heterometric expression and a SNP caused a single amino acid change to establish new PPIs, which contributed to the co-option of POS1 in multiple regulatory pathways to regulate cell expansion and thus fruit size in Physaleae. These results provide new insights into fruit morphological evolution and fruit yield control.

Jasmonate resistant 1 and ethylene responsive factor 11 are involved in chilling sensitivity in pepper fruit (Capsicum annuum L.).

Pepper fruit (Capsicum annuum L.) is sensitive to chilling stress with chilling injuries occurring below 7 °C; however, chilling injuries occur at different temperatures depending on the genotype. The present study aimed to identify the factors that affect chilling sensitivity in pepper fruits. A total of 112 F2 pepper fruits crossed between chilling-insensitive 'UZB-GJG-1999-51' and chilling-sensitive 'C00562' pepper were grouped according to the seed browning rate, which is a typical chilling symptom of pepper fruit under chilling conditions. Physiological traits, amino acids, fatty acids, as well as ethylene responsive factor (ERF) and jasmonate resistant 1 (JAR1) expression levels were analyzed, and their correlations with the seed browning rate were confirmed. The expression level of JAR1 showed a strong negative correlation with the seed browning rate (r = - 0.7996). The expression level of ERF11 and content of hydrogen peroxide showed strong positive correlation with the seed browning rate (r = 0.7622 and 0.6607, respectively). From these results, we inferred that JAR1 and ERF11 are important factors influencing the chilling sensitivity of pepper fruit.

Functional analysis of hot pepper ethylene responsive factor 1A in plant defense.

Ethylene-responsive factors play important roles in the biotic and abiotic stresses. Only some ERF genes from Capsicum annuum have been characterized. In the study, the CaERF1A gene is characterized in response to biotic stress. CaERF1A transcripts were induced by various plant defense-related hormone treatments. Knockdown of CaERF1A in hot pepper plants are negatively affected Tobacco mosaic virus-P0-mediated hypersensitive response cell death, resulting in reduced gene expression of pathogenesis-related genes and ethylene and jasmonic acid synthesis-related gene. Overexpressing CaERF1A transgenic plants show enhanced resistance to fungal pathogen via regulating ethylene and jasmonic acid synthesis-related gene expression. Thus, CaERF1A is a positive regulator of plant defense by modulating ethylene and jasmonic acid synthesis-related gene expressions.