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Heavy metal pollution has become a serious global problem in soil contamination. Cadmium (Cd) is a ubiquitous environmental toxic pollutant to plants, which can greatly affect their growth and development, or even lead to their death. In the present study, we performed a comparative transcriptome analysis to identify the regulators and pathways that were involved in Cd absorption and transportation in the roots, leaves and fruits of wild-type and mutant pepper plants. A total of 883,591,943 million reads were mapped to the reference genome, and 33,927 genes were identified. Sample reads and the reference genome alignment efficiency were between 78.99% to 84.57%. Finally, 798 differentially expressed genes (DEGs) were identified in the roots, leaves and fruits of the mutant and wild-type plants, including 80 up-regulated DEGs and 718 down-regulated DEGs. The DGEs were mostly related to the phenylpropanoid biosynthesis pathway and glyoxylate and dicarboxylate metabolism. In addition, some vital DEGs were identified in absorption/transportation-related pathways, such as ABC transporter and P-type ATPase. The results revealed that several genes were related to the regulation of Cd absorption and transportation. Collectively, our findings provided valuable insights into the molecular mechanism of Cd absorption and transportation in pepper.

Long non-coding RNAs (lncRNAs) participate in many biological processes and play extremely important regulatory roles in animals and plants. However, little is known about lncRNAs and their functions in cytoplasmic male sterility (CMS) during the developmental process of floral buds in pepper. In the present study, we systematically identified lncRNAs in the pepper CMS line (9704A) and maintainer line (9704B) using high-throughput sequencing and bioinformatic analyses. A total of 10,655 putative lncRNAs were identified during the developmental process of floral buds, and 262, 298 and 905 differentially expressed lncRNAs (DELs) were obtained in three stages, respectively. Moreover, 1137 unique DELs were identified between 9704A and 9704B, and their cis and trans target protein-coding genes were predicted. We used Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses to decipher the potential functions of DELs. The cell and cellular process GO terms were significantly enriched, and KEGG pathway analysis showed enrichments in the pathways for starch and sucrose metabolism genes, pentose and glucuronate interconversions, all of which were highly associated with pollen development. Besides, a regulatory network about the lncRNAs and the protein-coding genes involved in the formation of CMS was constructed. Our findings laid a foundation for further studies on the function of lncRNAs in pollen development and provided valuable insights into the regulatory mechanism underlying CMS.

The male-sterile line has been largely used in the hybrid seed production of pepper, which can effectively improve the efficiency of hybrid seed production. However, the formation mechanism of male sterility in pepper remains unclear. In the present study, we compared the gene expression patterns between pepper cytoplasmic male sterile line 9704A and its maintainer 9704B during floral bud development using RNA sequencing technology. A total of 547 976 and 2 416 Differentially Expressed Genes (DEGs) were identified in the stage S1, S2 and S3, respectively, and more than 70% of the DEGs were down-regulated in the sterile line. Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to further understand the functions of these identified DEGs. The results showed that the DEGs were mainly enriched in pathways of starch and sucrose metabolism, pentose and glucuronate interconversions. A number of genes, such as MS1, PME5, ATPB, and lots of transcription factors were found down-regulated in the sterile line, and we also identified a series of genes with large differences in expression patterns between sterile line and maintainer line. Collectively, our findings laid a foundation for further molecular breeding in pepper and provided new insights into its mechanism underlying the male sterility. Keywords: pepper, capsicum annuum, cytoplasmic male sterility, comparative transcriptome analysis, DEGs

Cold stress has become one of the main abiotic stresses in pepper, which severely limits the growth and development of pepper. In this study, the physiological indicators and transcriptome of a cold-tolerance (CT) inbred line A188 and a cold-sensitive (CS) inbred line A122 under cold–rewarm treatments were studied, the aim of this study was to determine the potential of the key factors in pepper response to cold stress. Compared with CT, CS wilts more seriously after cold stress, with poor resilience, higher content of malondialdehyde, and lower content of soluble sugar and total chlorophyll. Moreover, during cold treatment, 7333 and 5953 differentially expressed genes (DEGs) were observed for CT and CS, respectively. These DEGs were significantly enriched in pathways related to photosynthesis, plant hormone signal transduction, and DNA damage repair. Interestingly, in addition to the widely studied transcription factors related to cold, it was also found that 13 NAC transcription factors increased significantly in the T4 group, meanwhile, the NAC8 (Capana02g003557) and NAC72 (Capana07g002219) in CT were significantly higher than those in CS under rewarming for 1 h after 72 h cold treatment. Notably, weighted gene coexpression network analysis identified four positively correlated modules and eight hub genes, including zinc finger proteins, heat shock 70 kda protein, and cytochrome P450 family, which are related to cold tolerance. All of these pathways and genes may be responsible for the response to cold and even the cold tolerance in pepper.

Dwarfing is the development trend of pepper breeding. It is of great practical and scientific value to generate new dwarf germplasms, and identify new genes or alleles conferring dwarf traits in pepper. In our previous study, a weakly BR-insensitive dwarf mutant, E29, was obtained by EMS mutagenesis of the pepper inbred line 6421. It can be used as a good parent material for breeding new dwarf varieties. Here, we found that this dwarf phenotype was controlled by a single recessive gene. Whole-genome resequencing, dCAPs analysis, and VIGs validation revealed that this mutation was caused by a nonsynonymous single-nucleotide mutation (C to T) in CaBRI1. An enzyme activity assay, transcriptome sequencing, and BL content determination further revealed that an amino-acid change (Pro1157Ser) in the serine/threonine protein kinase and catalytic (S_TKc) domain of CaBRI1 impaired its kinase activity and caused the transcript levels of two important genes (CaDWF4 and CaROT3) participating in BR biosynthesis to increase dramatically in the E29 mutant, accompanied by significantly increased accumulation of brassinolide (BL). Therefore, we concluded that the novel single-base mutation in CaBRI1 conferred the dwarf phenotype and resulted in brassinosteroid (BR) accumulation in pepper. This study provides a new allelic variant of the height-regulating gene CaBRI1 that has theoretical and practical values for the breeding of the plants suitable for the facility cultivation and mechanized harvesting of pepper varieties.

Limited knowledge is available for phosphorylation modifications in pepper (Capsicum annuum L.), especially in pepper fruit development. In this study, we conducted the first comprehensive phosphoproteomic analysis of pepper fruit at four development stage by Tandem Mass Tag proteomic approaches. A total of 2639 unique phosphopeptides spanning 1566 proteins with 4150 nonredundant sites of phosphorylation were identified, among which 2327 peptides in 1413 proteins were accurately quantified at four different stages. Mature Green (MG) to breaker stage showed the largest number of differentially expressed phosphoproteins and the number of downregulated phosphoproteins was significantly higher than that of upregulated after MG stage. Twenty seven phosphorylation motifs, including 22 pSer motifs and five pThr motifs and 85 kinase including 28 serine/threonine kinases, 14 receptor protein kinases, six mitogen-activated protein kinases, seven calcium-dependent protein kinases, two casein kinases, and some other kinases were quantified. Then the dynamic changes of phosphorylated proteins in ethylene and abscisic acid signaling transduction pathways during fruit development were analyzed. Our results provide a cascade of phosphoproteins and a regulatory network of phosphorylation signals, which help to further understand the mechanism of phosphorylation in pepper fruit development.

Long non-coding RNAs (lncRNAs) have been shown to play critical regulatory roles in diverse biological processes in plants. However, the presence of lncRNAs and their potential functions in hot pepper are still unknown. Using strand-specific RNA-sequencing, a total of 2505 putative lncRNAs were identified in the fruits of hot pepper. The lncRNAs were transcribed from all pepper chromosomes and 95.37% of them originated from intergenic regions. And 1066 lncRNAs were differentially expressed among the four samples during pepper fruit development. Many potential protein-coding (PC) genes targeted by lncRNAs with cis or trans-acting were also identified. Six of them and their target genes were further validated by quantitative RT-PCR. Some of these target genes were involved in plant hormone signal transduction, cell wall formation and carotenoid biosynthesis, indicating the roles of lncRNAs in the regulation of fruit development and quality. This study identified a large number of lncRNAs in hot pepper, thereby providing some insights into the fruit development of hot pepper.

As a key determinant affecting yield and quality in pepper, inflorescence architecture is a significant feature of the ornamental plants. In our present study, a new mutant with fasciculate inflorescence named CL74 was derived from our germplasm resources. Genetic analysis of CL74 indicated that it was different from the fasciculate (fa) mutant, and its fasciculate inflorescence trait was controlled by a single recessive nuclear gene. A large F2 population was constructed and used for bulked segregant analysis (BSA) and linkage analysis. These findings revealed that the candidate gene was mapped to a segment with a physical distance of approximately 998 kb between Indel-197064077 and Indel-198110136. There were two agamous-like MADS-box protein genes Capanan11g001832 and Capanan11g001834 in the candidate region. Besides, two single nucleotide polymorphisms (SNPs) existed on the coding region of Capanan11g001832 in the CL74 mutant compared with the single-flower plants. In addition, the two SNPs were verified and fully co-segregated with fasciculate inflorescence phenotype in the F2 individuals. Collectively, Capanan11g001832 was a strong candidate gene for the fasciculate inflorescence. Furthermore, transcriptome analysis indicated that three WUSCHEL-like genes (Capana00g000667, Capana06g000476, and Capana11g000671) were downregulated and CLV1 (Capana04g000175) was upregulated in CL74. Moreover, we speculated that there was a complex relationship among the genes Capanan11g001832, Capanan11g001834, the three WUSCHEL-like genes, and CLV1, and these genes might co-regulate the formation of fasciculate inflorescence in pepper. Our study laid the foundation for the selection of high-yield, mechanized harvesting, or ornamental varieties of pepper.

Heat stress (HS), caused by extremely high temperatures, is one of the most severe forms of abiotic stress in pepper. In the present study, we studied the transcriptome and metabolome of a heat-tolerant cultivar (17CL30) and a heat-sensitive cultivar (05S180) under HS. Briefly, we identified 5754 and 5756 differentially expressed genes (DEGs) in 17CL30 and 05S180, respectively. Moreover, we also identified 94 and 108 differentially accumulated metabolites (DAMs) in 17CL30 and 05S180, respectively. Interestingly, there were many common HS-responsive genes (approximately 30%) in both pepper cultivars, despite the expression patterns of these HS-responsive genes being different in both cultivars. Notably, the expression changes of the most common HS-responsive genes were typically much more significant in 17CL30, which might explain why 17CL30 was more heat tolerant. Similar results were also obtained from metabolome data, especially amino acids, organic acids, flavonoids, and sugars. The changes in numerous genes and metabolites emphasized the complex response mechanisms involved in HS in pepper. Collectively, our study suggested that the glutathione metabolic pathway played a critical role in pepper response to HS and the higher accumulation ability of related genes and metabolites might be one of the primary reasons contributing to the heat resistance.

Hot pepper (Capsicum annuum L.) is one of the most important vegetable crops in China, but floods bring substantial decreases in production over the past several decades. In order to investigate the mechanisms of waterlogging resistance in mutant hot pepper, we measured the agronomic traits of mutant and wild-type, as well as the activities of root antioxidant enzymes and the contents of osmotic regulation substance. At the same time, we did transcriptome sequencing on the plant roots, and screened for differentially expressed genes between mutant and wild-type. The results showed that, under waterlogging stress, the mutants could grow normally, and the activities of their superoxide dismutase, peroxidase, catalase, and glutathione reductase were significantly increased, as well as the contents of proline and soluble sugar. The accumulation of malondialdehyde and hydroxyl radical in mutants was significantly reduced. Among the 61 differentially expressed genes from transcriptome analysis, 24 genes were up-regulated and 37 genes were down-regulated in mutants. After functional analysis, we found 8 genes related to the metabolism of endogenous hormone and protective enzymes, among which, auxin-induced protein related gene cap.ARATH, ethylene response related gene Cap.RAP2, MYB family related gene Cap.MYB1R1, and the 4 genes related to peroxidase Cap.POD, were significantly up-regulated in mutants, while the Capana01g001329 gene was down-regulated. These results suggest that under waterlogging stress, the mutant could enhance its resistance to waterlogging by regulating the genes involved in metabolism of endogenous hormones and protective enzymes. This study provides the scientific basis for elucidating the gene regulation network of mutant pepper under waterlogging stress

To understand the mechanism of the color formation of pepper fruit, integrative analysis of the metabolome and transcriptome profiles was performed in pepper varieties with 4 different fruit colors. A total of 188 flavonoids were identified, and most of the anthocyanins, flavonols and flavones showed markedly higher abundances in purple variety than in other varieties, which was linked to the high expression of flavonoid synthesis and regulatory genes. Using weighted gene co-expression network analyses, modules related to flavonoid synthesis and candidate genes that regulate flavonoid synthesis and transport were identified. Furthermore, the analysis of 12 carotenoids showed that the content of xanthophylls at 50 days after anthesis was significantly different between the four pepper varieties, which was resulted from the differential expressions of genes downstream of the carotenoid pathway. Our results provide new insights into the understanding of the synthesis and accumulation of flavonoids and carotenoids in pepper fruit.

Pepper is an important vegetable with great economic value and unique biological features. In the past few years, significant development has been made toward understanding the huge complex pepper genome; however, pepper functional genomics has not been well studied. To better understand the pepper gene structure and pepper gene regulation, we conducted full-length mRNA sequencing by PacBio sequencing and obtained 57 862 high-quality full-length mRNA sequences derived from 18 362 previously annotated and 5769 newly detected genes. New gene models were built that combined the full-length mRNA sequences and corrected approximately 500 fragmented gene models from previous annotations. Based on the full-length mRNA, we identified 4114 and 5880 pepper genes forming natural antisense transcript (NAT) genes in-cis and in-trans, respectively. Most of these genes accumulate small RNAs in their overlapping regions. By analyzing these NAT gene expression patterns in our transcriptome data, we identified many NAT pairs responsive to a variety of biological processes in pepper. Pepper formate dehydrogenase 1 (FDH1), which is required for R-gene-mediated disease resistance, may be regulated by nat-siRNAs and participate in a positive feedback loop in salicylic acid biosynthesis during resistance responses. Several cis-NAT pairs and subgroups of trans-NAT genes were responsive to pepper pericarp and placenta development, which may play roles in capsanthin and capsaicin biosynthesis. Using a comparative genomics approach, the evolutionary mechanisms of cis-NATs were investigated, and we found that an increase in intergenic sequences accounted for the loss of most cis-NATs, while transposon insertion contributed to the formation of most new cis-NATs. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at http://bigd.big.ac.cn/gsa Accession number, CRA001412.

MicroRNAs (miRNAs) are non-coding small RNAs which play an important regulatory role in various biological processes. Previous studies have reported that miRNAs are involved in fruit development in model plants. However, the miRNAs related to fruit development and quality in hot pepper (Capsicum annuum L.) remains unknown. In this study, small RNA populations from different fruit ripening stages and different varieties were compared using next-generation sequencing technology. Totally, 59 known miRNAs and 310 novel miRNAs were identified from four libraries using miRDeep2 software. For these novel miRNAs, 656 targets were predicted and 402 of them were annotated. GO analysis and KEGG pathways suggested that some of the predicted miRNAs targeted genes involved in starch sucrose metabolism and amino sugar as well as nucleotide sugar metabolism. Quantitative RT-PCR validated the contrasting expression patterns between several miRNAs and their target genes. These results will provide an important foundation for future studies on the regulation of miRNAs involved in fruit development and quality.