Metabolome profiling and transcriptome analysis filling the early crucial missing steps of piperine biosynthesis in Piper nigrum L.

Black pepper (Piper nigrum L.), the world renown as the King of Spices, is not only a flavorsome spice but also a traditional herb. Piperine, a species-specific piper amide, is responsible for the major bioactivity and pungent flavor of black pepper. However, several key steps for the biosynthesis of piperoyl-CoA (acyl-donor) and piperidine (acyl-acceptor), two direct precursors for piperine, remain unknown. In this study, we used guilt-by-association analysis of the combined metabolome and transcriptome, to identify two feruloyldiketide-CoA synthases responsible for the production of the C5 side chain scaffold feruloyldiketide-CoA intermediate, which is considered the first and important step to branch metabolic fluxes from phenylpropanoid pathway to piperine biosynthesis. In addition, we also identified the first two key enzymes for piperidine biosynthesis derived from lysine in P. nigrum, namely a lysine decarboxylase and a copper amine oxidase. These enzymes catalyze the production of cadaverine and 1-piperideine, the precursors of piperidine. In vivo and in vitro experiments verified the catalytic capability of them. In conclusion, our findings revealed enigmatic key steps of piperine biosynthetic pathway and thus provide a powerful reference for dissecting the biosynthetic logic of other piper amides.

Transcriptome and metabolome analyses revealed the response mechanism of pepper roots to Phytophthora capsici infection.

BACKGROUND: Phytophthora root rot caused by the oomycete Phytophthora capsici is the most devastating disease in pepper production worldwide, and current management strategies have not been effective in preventing this disease. Therefore, the use of resistant varieties was regarded as an important part of disease management of P. capsici. However, our knowledge of the molecular mechanisms underlying the defense response of pepper roots to P. capsici infection is limited. METHODS: A comprehensive transcriptome and metabolome approaches were used to dissect the molecular response of pepper to P. capsici infection in the resistant genotype A204 and the susceptible genotype A198 at 0, 24 and 48 hours post-inoculation (hpi). RESULTS: More genes and metabolites were induced at 24 hpi in A204 than A198, suggesting the prompt activation of defense responses in the resistant genotype, which can attribute two proteases, subtilisin-like protease and xylem cysteine proteinase 1, involved in pathogen recognition and signal transduction in A204. Further analysis indicated that the resistant genotype responded to P. capsici with fine regulation by the Ca2+- and salicylic acid-mediated signaling pathways, and then activation of downstream defense responses, including cell wall reinforcement and defense-related genes expression and metabolites accumulation. Among them, differentially expressed genes and differentially accumulated metabolites involved in the flavonoid biosynthesis pathways were uniquely activated in the resistant genotype A204 at 24 hpi, indicating a significant role of the flavonoid biosynthesis pathways in pepper resistance to P. capsici. CONCLUSION: The candidate transcripts may provide genetic resources that may be useful in the improvement of Phytophthora root rot-resistant characters of pepper. In addition, the model proposed in this study provides new insight into the defense response against P. capsici in pepper, and enhance our current understanding of the interaction of pepper-P. capsici.

Rapid onsite detection of piper yellow mottle virus infecting black pepper by recombinase polymerase amplification-lateral flow assay (RPA-LFA).

Piper yellow mottle virus (PYMoV) is a pararetrovirus associated with stunt disease in black pepper. As the primary spread of the virus occurs through vegetative propagation, effective diagnostics are required for the production of virus-free plants. Currently available assays are time-consuming, require expensive equipment, and are not suitable for on-site detection. In the present study, two rapid assays based on the recombinase polymerase amplification (RPA) coupled with lateral flow assay (LFA) using (i) 6-carboxyfluorescein (FAM) labeled nfo probe and biotin-labeled reverse primer and (ii) FAM labeled forward and biotin-labeled reverse primer was developed for the detection of PYMoV. The assays were performed using TwistAmp DNA amplification reagents and crude extract from the infected plant and mealybug as templates. Both assays were optimized for parameters like concentration of magnesium acetate, temperature, and time. The RPA product was then diluted and applied to the sample pad of a lateral flow device for visualizing the results. The formation of a colored line at the test line was considered positive for PYMoV. The entire process from sample preparation to visualization of results could be completed in about 30 min. The developed assays were specific and 10 times more sensitive than PCR. The assays were validated using field samples of black pepper and mealybug vectors.

De novo transcriptome profiling unveils the regulation of phenylpropanoid biosynthesis in unripe Piper nigrum berries.

BACKGROUND: Black pepper (Piper nigrum L.) is rich in bioactive compounds that make it an imperative constituent in traditional medicines. Although the unripe fruits have long been used in different Ayurvedic formulations, the mechanism of gene regulation resulting in the production of the bioactive compounds in black pepper is not much investigated. Exploring the regulatory factors favouring the production of bioactive compounds ultimately help to accumulate the medicinally important content of black pepper. The factors that enhance the biosynthesis of these compounds could be potential candidates for metabolic engineering strategies to obtain a high level production of significant biomolecules. RESULTS: Being a non-model plant, de novo sequencing technology was used to unravel comprehensive information about the genes and transcription factors that are expressed in mature unripe green berries of P. nigrum from which commercially available black pepper is prepared. In this study, the key gene regulations involved in the synthesis of bioactive principles in black pepper was brought out with a focus on the highly expressed phenylpropanoid pathway genes. Quantitative real-time PCR analysis of critical genes and transcription factors in the different developmental stages from bud to the mature green berries provides important information useful for choosing the developmental stage that would be best for the production of a particular bioactive compound. Comparison with a previous study has also been included to understand the relative position of the results obtained from this study. CONCLUSIONS: The current study uncovered significant information regarding the gene expression and regulation responsible for the bioactivity of black pepper. The key transcription factors and enzymes analyzed in this study are promising targets for achieving a high level production of significant biomolecules through metabolic engineering.

Viromes of 15 Pepper (Capsicum annuum L.) Cultivars.

Pepper (Capsicum annuum L.) plants produce berry fruits that are used as spices. Here, we examined the viromes of 15 pepper cultivars through RNA sequencing. We obtained 1,325 virus-associated contigs derived from 8 virus species. Bean broad wilt virus 2 (BBWV2) and cucumber mosaic virus (CMV) were identified as the major viruses infecting pepper plants, followed by potato virus Y, bell pepper endornavirus, and hot pepper endornavirus. The proportion of viral reads in each transcriptome ranged from 0.04% to 24.5%. BBWV2 was the dominant virus in seven cultivars, whereas CMV was dominant in five cultivars. All the bell pepper cultivars showed severe viral disease symptoms, whereas the commercially developed hot pepper cultivars were asymptomatic or had mild symptoms. In addition, 111 complete viral segments were obtained from 7 viruses. Based on the obtained viral genomes, the genetic relationship between the identified viruses and quasispecies of BBWV2 and CMV in each pepper plant was determined. Newly designed primers for nine viruses confirmed the results of RNA sequencing. Taken together, this study, for the first time, provides a comprehensive overview of viromes in 15 major pepper cultivars through RNA sequencing.

Peppers in Diet: Genome-Wide Transcriptome and Metabolome Changes in Drosophila melanogaster.

The habanero pepper (Capsicum chinense) is an increasingly important spice and vegetable crop worldwide because of its high capsaicin content and pungent flavor. Diets supplemented with the phytochemicals found in habanero peppers might cause shifts in an organism's metabolism and gene expression. Thus, understanding how these interactions occur can reveal the potential health effects associated with such changes. We performed transcriptomic and metabolomic analyses of Drosophila melanogaster adult flies reared on a habanero pepper diet. We found 539 genes/59 metabolites that were differentially expressed/accumulated in flies fed a pepper versus control diet. Transcriptome results indicated that olfactory sensitivity and behavioral responses to the pepper diet were mediated by olfactory and nutrient-related genes including gustatory receptors (Gr63a, Gr66a, and Gr89a), odorant receptors (Or23a, Or59a, Or82a, and Orco), and odorant-binding proteins (Obp28a, Obp83a, Obp83b, Obp93a, and Obp99a). Metabolome analysis revealed that campesterol, sitosterol, and sucrose were highly upregulated and azelaic acid, ethyl phosphoric acid, and citric acid were the major metabolites downregulated in response to the habanero pepper diet. Further investigation by integration analysis between transcriptome and metabolome data at gene pathway levels revealed six unique enriched pathways, including phenylalanine metabolism; insect hormone biosynthesis; pyrimidine metabolism; glyoxylate, and dicarboxylate metabolism; glycine, serine, threonine metabolism; and glycerolipid metabolism. In view of the transcriptome and metabolome findings, our comprehensive analysis of the response to a pepper diet in Drosophila have implications for exploring the molecular mechanism of pepper consumption.

Fine Mapping and Gene Analysis of restorer-of-fertility Gene CaRfHZ in Pepper (Capsicum annuum L.).

Cytoplasmic male sterility (CMS) is a common biological phenomenon used in hybrid production of peppers (Capsicum annuum L.). Although several restorer-of-fertility (Rf) genes of pepper CMS lines have been mapped, there is no report that the Rf gene with clear gene function has been isolated. Here, pepper CMS line HZ1A and its restorer line HZ1C were used to construct (HZ1A × HZ1C) F2 populations and map the Rf gene. A single dominant gene CaRfHZ conferred male fertility according to inheritance analysis. Using sterile plants from (HZ1A × HZ1C) F2 populations and bulked segregant analysis (BSA), the CaRfHZ gene was mapped between P06gInDel-66 and P06gInDel-89 on chromosome 6. This region spans 533.81 kb, where four genes are annotated according to Zunla-1 V2.0 gene models. Based on the analysis of genomic DNA sequences, gene expressions, and protein structures, Capana06g002968 was proposed as the strongest candidate for the CaRfHZ gene. Our results may help with hybrid pepper breeding and to elucidate the mechanism of male fertility restoration in peppers.

In silico assisted identification of peppery aroma compound 'rotundone' backbone genes from black pepper.

Rotundone, an oxygenated sesquiterpene compound, responsible for the peppery aroma. The importance of the rotundone in the flavor industry warrants search for the precursor genes in plants. We report in this study, the first on the identification of rotundone backbone genes viz., α-guaiene synthase & α-guaiene oxidase in black pepper. We identified the precursor genes of rotundone using berry transcriptome profiling. The metabolite profiling using head space mass spectrometry showed the presence of the direct precursor compounds for rotundone biosynthesis in black pepper berries. The identification of the genes & compounds of the guaiene skeleton is expected to help in bioprospecting of black pepper varieties & also in recombinant production of the aroma compound.Communicated by Ramaswamy H. Sarma [Formula: see text]Identification of rotundone backbone genes & precursor compounds from Piper nigrum.

Genome-Wide Identification and Functional Exploration of SBP-Box Gene Family in Black Pepper (Piper nigrum L.).

Black pepper (Piper nigrum L.), is dubbed "the King of Spices". However, the lack of genic knowledge has limited the understanding of its physiological processes and hindered the development of its molecular breeding. The SBP-box gene family is an important family in plant development and integrates multiple physiological processes. Here, we made a genome-wide identification of the pepper SBP-box gene family to provide evolutionary and functional information about this conserved transcription factor. In total, 34 SBP genes were identified in pepper. All these pepper SBP genes were clustered into eight groups, and one pepper group was not found in Arabidopsis thaliana. Segment duplications played the most important role in the expansion process of pepper SBP genes, and all these duplications were subjected to purifying selection. Half of pepper SBP genes were found miR156 target sites, and 17 miR156s were predicted. The tissue expression analysis revealed the differential expression of pepper SBP genes. Eleven SBP genes were found in four co-expression networks, and the GO enrichment further provides a functional prediction for pepper SBP genes. This study lays a foundation for further studies of pepper and provides a valuable reference for functional mining of pepper SBP genes.

Transcriptome profiling of pepper leaves by RNA-Seq during an incompatible and a compatible pepper-tobamovirus interaction.

Upon virus infections, the rapid and comprehensive transcriptional reprogramming in host plant cells is critical to ward off virus attack. To uncover genes and defense pathways that are associated with virus resistance, we carried out the transcriptome-wide Illumina RNA-Seq analysis of pepper leaves harboring the L3 resistance gene at 4, 8, 24 and 48 h post-inoculation (hpi) with two tobamoviruses. Obuda pepper virus (ObPV) inoculation led to hypersensitive reaction (incompatible interaction), while Pepper mild mottle virus (PMMoV) inoculation resulted in a systemic infection without visible symptoms (compatible interaction). ObPV induced robust changes in the pepper transcriptome, whereas PMMoV showed much weaker effects. ObPV markedly suppressed genes related to photosynthesis, carbon fixation and photorespiration. On the other hand, genes associated with energy producing pathways, immune receptors, signaling cascades, transcription factors, pathogenesis-related proteins, enzymes of terpenoid biosynthesis and ethylene metabolism as well as glutathione S-transferases were markedly activated by ObPV. Genes related to photosynthesis and carbon fixation were slightly suppressed also by PMMoV. However, PMMoV did not influence significantly the disease signaling and defense pathways. RNA-Seq results were validated by real-time qPCR for ten pepper genes. Our findings provide a deeper insight into defense mechanisms underlying tobamovirus resistance in pepper.

A Genome-Wide Analysis of Pathogenesis-Related Protein-1 (PR-1) Genes from Piper nigrum Reveals Its Critical Role during Phytophthora capsici Infection.

Black pepper (Piper nigrum L.) is a prominent spice that is an indispensable ingredient in cuisine and traditional medicine. Phytophthora capsici, the causative agent of footrot disease, causes a drastic constraint in P. nigrum cultivation and productivity. To counterattack various biotic and abiotic stresses, plants employ a broad array of mechanisms that includes the accumulation of pathogenesis-related (PR) proteins. Through a genome-wide survey, eleven PR-1 genes that belong to a CAP superfamily protein with a caveolin-binding motif (CBM) and a CAP-derived peptide (CAPE) were identified from P. nigrum. Despite the critical functional domains, PnPR-1 homologs differ in their signal peptide motifs and core amino acid composition in the functional protein domains. The conserved motifs of PnPR-1 proteins were identified using MEME. Most of the PnPR-1 proteins were basic in nature. Secondary and 3D structure analyses of the PnPR-1 proteins were also predicted, which may be linked to a functional role in P. nigrum. The GO and KEGG functional annotations predicted their function in the defense responses of plant-pathogen interactions. Furthermore, a transcriptome-assisted FPKM analysis revealed PnPR-1 genes mapped to the P. nigrum-P. capsici interaction pathway. An altered expression pattern was detected for PnPR-1 transcripts among which a significant upregulation was noted for basic PnPR-1 genes such as CL10113.C1 and Unigene17664. The drastic variation in the transcript levels of CL10113.C1 was further validated through qRT-PCR and it showed a significant upregulation in infected leaf samples compared with the control. A subsequent analysis revealed the structural details, phylogenetic relationships, conserved sequence motifs and critical cis-regulatory elements of PnPR-1 genes. This is the first genome-wide study that identified the role of PR-1 genes during P. nigrum-P. capsici interactions. The detailed in silico experimental analysis revealed the vital role of PnPR-1 genes in regulating the first layer of defense towards a P. capsici infection in Panniyur-1 plants.

Secondary Metabolic Profile as a Tool for Distinction and Characterization of Cultivars of Black Pepper (Piper nigrum L.) Cultivated in Pará State, Brazil.

This study evaluated the chemical compositions of the leaves and fruits of eight black pepper cultivars cultivated in Pará State (Amazon, Brazil). Hydrodistillation and gas chromatography-mass spectrometry were employed to extract and analyze the volatile compounds, respectively. Sesquiterpene hydrocarbons were predominant (58.5-90.9%) in the cultivars "Cingapura", "Equador", "Guajarina", "Iaçará", and "Kottanadan", and "Bragantina", "Clonada", and "Uthirankota" displayed oxygenated sesquiterpenoids (50.6-75.0%). The multivariate statistical analysis applied using volatile composition grouped the samples into four groups: γ-Elemene, curzerene, and δ-elemene ("Equador"/"Guajarina", I); δ-elemene ("Iaçará"/"Kottanadan"/"Cingapura", II); elemol ("Clonada"/"Uthirankota", III) and α-muurolol, bicyclogermacrene, and cubebol ("Bragantina", IV). The major compounds in all fruit samples were monoterpene hydrocarbons such as α-pinene, ß-pinene, and limonene. Among the cultivar leaves, phenolics content (44.75-140.53 mg GAE·g-1 FW), the enzymatic activity of phenylalanine-ammonia lyase (20.19-57.22 µU·mL-1), and carotenoids (0.21-2.31 µg·mL-1) displayed significant variations. Due to black pepper's susceptibility to Fusarium infection, a molecular docking analysis was carried out on Fusarium protein targets using each cultivar's volatile components. F. oxysporum endoglucanase was identified as the preferential protein target of the compounds. These results can be used to identify chemical markers related to the susceptibility degree of black pepper cultivars to plant diseases prevalent in Pará State.

Drought responsiveness in black pepper (Piper nigrum L.): Genes associated and development of a web-genomic resource.

Black pepper (Piper nigrum L.; 2n = 52; Piperaceae), the king of spices, is a perennial, trailing woody flowering vine and has global importance with widespread dietary, medicinal, and preservative uses. It is an economically important germplasm cultivated for its fruit and the major cash crop in >30 tropical countries. Crop production is mainly affected by drought stress. The present study deals with the candidate gene identification from drought-affected black pepper leaf transcriptome generated by Illumina Hiseq2000. It also aims to mine putative molecular markers (namely SSRs, SNPs, and InDels) and generate primers for them. The identification of transcription factors and pathways involved in drought tolerance is also reported here. De novo transcriptome assembly was performed with trinity assembler. In total, 4914 differential expressed genes, 2110 transcriptional factors, 786 domains and 1137 families, 20,124 putative SSR markers, and 259,236 variants were identified. At2g30105 (unidentified gene containing leucine-rich repeats and ubiquitin-like domain), serine threonine protein kinase, Mitogen-activated protein kinase, Nucleotide Binding Site-Leucine Rich Repeat, Myeloblastosis-related proteins, basic helix-loop-helix are all found upregulated and are reported to be associated with plant tolerance against drought condition. All these information are catalogued in the Black Pepper Drought Transcriptome Database (BPDRTDb), freely accessible for academic use at http://webtom.cabgrid.res.in/bpdrtdb/. This database is a good foundation for the genetic improvement of pepper plants, breeding programmes, and mapping population of this crop. Putative markers can also be a reliable genomic resource to develop drought-tolerant variety for better black pepper productivity.

A new Piper nigrum cysteine proteinase inhibitor, PnCPI, with antifungal activity: molecular cloning, recombinant expression, functional analyses and molecular modeling.

MAIN CONCLUSION: A new Piper nigrum cysteine proteinase inhibitor, PnCPI, belonging to group I of phytocystatins, with inhibitory activity against papain and growth of Fusarium solani f. sp. piperis, was isolated and characterized. Previous studies (de Souza et al. 2011) have identified a partial cDNA sequence of putative cysteine proteinase inhibitor differentially expressed in roots of black pepper (P. nigrum L.) infected by F. solani f. sp. piperis. Here, we aimed to isolate the full-length cDNA and genomic sequences of the P. nigrum cysteine proteinase inhibitor gene, named PnCPI. Sequence analyses showed that the PnCPI gene encodes a deduced protein of 108 amino acid residues with a predicted molecular mass of 12.3 kDa and isoelectric point of 6.51. Besides the LARFAV-like sequence, common to all phytocystatins, PnCPI contains three conserved motifs of the superfamily cystatin: a glycine residue at the N-terminal region, the QxVxG reactive site more centrally positioned, and one tryptophan in the C-terminal region. PnCPI, belonging to group I of phytocystatins, showed high identity with cystatins isolated from several plant species. Sequence analyses also revealed no putative signal peptide at the N-terminal of PnCPI, as well as no introns within the genomic sequence corresponding to the PnCPI coding region. Molecular modeling showed the ability of PnCPI to interact with papain, while its inhibitory activity against this protease was confirmed after heterologous expression in Escherichia coli. The effects of heat treatments on the inhibitory activity of recombinant PnCPI, rPnCPI, were evaluated. In addition, rPnCPI exhibited in vitro activity against F. solani f. sp. piperis, revealing a new cystatin with the potential antifungal application. The identification of PnCPI as a functional cystatin able to inhibit the in vitro growth of F. solani f. sp. piperis indicates other factors contributing to in vivo susceptibility of black pepper to root rot disease.

Transcriptomic analysis of pepper plants provides insights into host responses to Fusarium solani infestation.

Black pepper is an important commodity crop in Malaysia that generates millions of annual revenue for the country. However, black pepper yield is affected by slow decline disease caused by a soil-borne fungus Fusarium solani. RNA sequencing transcriptomics approach has been employed in this study to explore the differential gene expression in susceptible Piper nigrum L. and resistant Piper colubrinum Link. Gene expression comparative analysis of the two pepper species has yielded 2,361 differentially expressed genes (DEGs). Among them, higher expression of 1,426 DEGs was detected in resistant plant. These DEGs practically demonstrated the major branches of plant-pathogen interaction pathway (Path: ko04626). We selected five groups of defence-related DEGs for downstream qRT-PCR analysis. Cf-9, the gene responsible for recognizing fungal avirulence protein activity was found inexpressible in susceptible plant. However, this gene exhibited promising expression in resistant plant. Inactivation of Cf-9 could be the factor that causes susceptible plant fail in recognition of F. solani and subsequently delay activation of adaptive response to fungal invasion. This vital study advance the understanding of pepper plant defence in response to F. solani and aid in identifying potential solution to manage slow decline disease in black pepper cultivation.

A piperic acid CoA ligase produces a putative precursor of piperine, the pungent principle from black pepper fruits.

Black pepper (Piper nigrum L.) is known for its high content of piperine, a cinnamoyl amide derivative regarded as largely responsible for the pungent taste of this widely used spice. Despite its long history and worldwide use, the biosynthesis of piperine and related amides has been enigmatic up to now. In this report we describe a specific piperic acid CoA ligase from immature green fruits of P. nigrum. The corresponding enzyme was cloned and functionally expressed in E. coli. The recombinant enzyme displays a high specificity for piperic acid and does not accept the structurally related feruperic acid characterized by a similar C-2 extension of the general C6-C3 phenylpropanoid structure. The enzyme is also inactive with the standard set of hydroxycinnamic acids tested including caffeic acid, 4-coumaric acid, ferulic acid, and sinapic acid. Substrate specificity is corroborated by in silico modelling that suggests a perfect fit for the substrate piperic acid to the active site of the piperic acid CoA ligase. The CoA ligase gene shows its highest expression levels in immature green fruits, is also expressed in leaves and flowers, but not in roots. Virus-induced gene silencing provided some preliminary indications that the production of piperoyl-CoA is required for the biosynthesis of piperine in black pepper fruits.

Genome wide identification and characterization of microsatellite markers in black pepper (Piper nigrum): A valuable resource for boosting genomics applications.

Black pepper is one of the most valued and widely used spices in the world and dominates multi-billion dollar global spices trade. India is amongst the major producers, consumers and exporters of black pepper. In spite of its commercial and cultural importance, black pepper has received meagre attention in terms of generation of genomic resources. Availability of markers distributed throughout the genome would facilitate and accelerate genetic studies, QTL identification, genetic enhancement and crop improvement in black pepper. In this perspective, the sequence information from the recently sequenced black pepper (Piper nigrum) genome has been used for identification and characterisation of Simple Sequence Repeats (SSRs). Total 69,126 SSRs were identified from assembled genomic sequence of P. nigrum. The SSR frequency was 158 per MB making it, one SSR for every 6.3 kb in the assembled genome. Among the different types of microsatellite repeat motifs, dinucleotides were the most abundant (48.6%), followed by trinucleotide (23.7%) and compound repeats (20.62%). A set of 85 SSRs were used for validation, of which 74 produced amplification products of expected size. Genetic diversity of 30 black pepper accessions using 50 SSRs revealed four distinct clusters. Further, the cross species transferability of the SSRs was checked in nine other Piper species. Out of 50 SSRs used, 19 and 31 SSRs were amplified in nine and seven species, respectively. Thus the identified SSRs may have application in other species of the genus Piper where genome sequence is not available yet. Present study reports the first NGS based genomic SSRs in black pepper and thus constitute a valuable resource for a whole fleet of applications in genetics and plant breeding studies such as genetic map construction, QTL identification, map-based gene cloning, marker-assisted selection and evolutionary studies in Piper nigrum and related species.

The chromosome-scale reference genome of black pepper provides insight into piperine biosynthesis.

Black pepper (Piper nigrum), dubbed the 'King of Spices' and 'Black Gold', is one of the most widely used spices. Here, we present its reference genome assembly by integrating PacBio, 10x Chromium, BioNano DLS optical mapping, and Hi-C mapping technologies. The 761.2 Mb sequences (45 scaffolds with an N50 of 29.8 Mb) are assembled into 26 pseudochromosomes. A phylogenomic analysis of representative plant genomes places magnoliids as sister to the monocots-eudicots clade and indicates that black pepper has diverged from the shared Laurales-Magnoliales lineage approximately 180 million years ago. Comparative genomic analyses reveal specific gene expansions in the glycosyltransferase, cytochrome P450, shikimate hydroxycinnamoyl transferase, lysine decarboxylase, and acyltransferase gene families. Comparative transcriptomic analyses disclose berry-specific upregulated expression in representative genes in each of these gene families. These data provide an evolutionary perspective and shed light on the metabolic processes relevant to the molecular basis of species-specific piperine biosynthesis.

The CaAP2/ERF064 Regulates Dual Functions in Pepper: Plant Cell Death and Resistance to Phytophthora capsici.

Phytophthora blight is one of the most destructive diseases of pepper (Capsicum annuum L.) globally. The APETALA2/Ethylene Responsive Factors (AP2/ERF) genes play a crucial role in plant response to biotic stresses but, to date, have not been studied in the context of Phytophthora resistance in pepper. Here, we documented potential roles for the pepper CaAP2/ERF064 gene in inducing cell death and conferring resistance to Phytophthora capsici (P. capsici) infection. Results revealed that the N-terminal, AP2 domain, and C-terminal of CaAP2/ERF064 protein is responsible for triggering cell death in Nicotiana benthamiana (N. benthamiana). Moreover, the transcription of CaAP2/ERF064 in plant is synergistically regulated by the Methyl-Jasmonate (MeJA) and ethephon (ET) signaling pathway. CaAP2/ERF064 was found to regulate the expression of CaBPR1, which is a pathogenesis-related (PR) gene of pepper. Furthermore, the silencing of CaAP2/ERF064 compromised the pepper plant resistance to P.capsici by reducing the transcript level of defense-related genes CaBPR1, CaPO2, and CaSAR82, while the ectopic expression of CaAP2/ERF064 in N. benthamiana plant elevated the expression level of NbPR1b and enhanced resistance to P.capsici. These results suggest that CaAP2/ERF064 could positively regulate the defense response against P. capsici by modulating the transcription of PR genes in the plant.

Sensitive detection of Piper nigrum L. adulterants by a novel screening approach based on qPCR.

The spice made from the fruits of Piper nigrum L. (Piperaceae) has high economic value since the beginnings of international trade. Because its price has been increasing, adulterations with papaya seeds, cayenne pepper and maize flour were reported. These have been screened by methodologies dedicated to the detection of single adulterants lacking sensitivity and specificity. Herein we propose a specific, highly-sensitive, high-throughput and affordable qPCR-based methodology for the detection of P. nigrum contaminants (Carica papaya, Zea mays and Capsicum annuum) using plant DNA barcodes trnL and psbA-trnH. The method enables the specific detection of contaminants in a short time with low limits of detection (LOD6 values of 1, 2 and 10 Haploid Genome Equivalents). A market survey (29 samples) revealed 41% of samples contaminated, though about ¾ at very low levels indicating accidental contamination. The proposed tool will contribute to the improvement of quality of this much traded spice.