Lysine 2-hydroxyisobutyrylation proteomics analyses reveal the regulatory mechanism of CaMYB61-CaAFR1 module in regulating stem development in Capsicum annuum L.

Plant stems constitute the most abundant renewable resource on earth. The function of lysine (K)-2-hydroxyisobutyrylation (Khib), a novel post-translational modification (PTM), has not yet been elucidated in plant stem development. Here, by assessing typical pepper genotypes with straight stem (SS) and prostrate stem (PS), we report the first large-scale proteomics analysis for protein Khib to date. Khib-modifications influenced central metabolic processes involved in stem development, such as glycolysis/gluconeogenesis and protein translation. The high Khib level regulated gene expression and protein accumulation associated with cell wall formation in the pepper stem. Specially, we found that CaMYB61 knockdown lines that exhibited prostrate stem phenotypes had high Khib levels. Most histone deacetylases (HDACs, e.g., switch-independent 3 associated polypeptide function related 1, AFR1) potentially function as the "erasing enzymes" involved in reversing Khib level. CaMYB61 positively regulated CaAFR1 expression to erase Khib and promote cellulose and hemicellulose accumulation in the stem. Therefore, we propose a bidirectional regulation hypothesis of "Khib modifications" and "Khib erasing" in stem development, and reveal a novel epigenetic regulatory network in which the CaMYB61-CaAFR1 molecular module participating in the regulation of Khib levels and biosynthesis of cellulose and hemicellulose for the first time.

Genome-Wide Analysis of the MYB-Related Transcription Factor Family in Pepper and Functional Studies of CaMYB37 Involvement in Capsaicin Biosynthesis.

Chili pepper is an important economic vegetable worldwide. MYB family gene members play an important role in the metabolic processes in plant growth and development. In this study, 103 pepper MYB-related members were identified and grouped into nine subfamilies according to phylogenetic relationships. Additionally, a total of 80, 20, and 37 collinear gene pairs were identified between pepper and tomato, pepper and Arabidopsis, and tomato and Arabidopsis, respectively. We performed promoter cis-element analysis and showed that CaMYB-related members may be involved in multiple biological processes such as growth and development, secondary metabolism, and circadian rhythm regulation. Expression pattern analysis indicated that CaMYB37 is significantly more enriched in fruit placenta, suggesting that this gene may be involved in capsaicin biosynthesis. Through VIGS, we confirmed that CaMYB37 is critical for the biosynthesis of capsaicin in placenta. Our subcellular localization studies revealed that CaMYB37 localized in the nucleus. On the basis of yeast one-hybrid and dual-luciferase reporter assays, we found that CaMYB37 directly binds to the promoter of capsaicin biosynthesis gene AT3 and activates its transcription, thereby regulating capsaicin biosynthesis. In summary, we systematically identified members of the CaMYB-related family, predicted their possible biological functions, and revealed that CaMYB37 is critical for the transcriptional regulation of capsaicin biosynthesis. This work provides a foundation for further studies of the CaMYB-related family in pepper growth and development.

Full-length mRNA sequencing and gene expression profiling reveal broad involvement of natural antisense transcript gene pairs in pepper development and response to stresses.

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.

Integrative Transcriptome and Proteome Analysis Identifies Major Metabolic Pathways Involved in Pepper Fruit Development.

Pepper ( Capsicum annuum L.) fruit development is a complex and genetically programmed process. In this study, we conducted integrative analysis of transcriptome and proteome profiles during pepper fruit development. A total of 23 349 transcripts and 5455 protein groups were identified in four fruit developmental stages of two pepper varieties. The numbers of transcripts and proteins identified were decreased gradually across fruit development, and the most significant changes in transcript and protein levels happened from the mature green (MG) to breaker (Br) stages. Poor correlation between differentially expressed transcript and differentially expressed protein profiles was observed during pepper fruit development. We then analyzed expression profiles of transcripts and proteins involved in cell wall metabolism, and capsanthin, tocopherol, and ascorbate biosynthetic pathways during fruit development, and identified key regulatory proteins in these pathways. We presented a dynamic picture of pepper fruit development via comprehensive analysis of transcriptome and proteome profiles at different fruit developmental stages and in different varieties, revealing the temporal specificity of key protein expression. Our report provides insight into the transcription and translation dynamics of pepper fruit development and a reference for other nonclimacteric species.

Evaluation of the uptake capacities of heavy metals in Chinese cabbage.

Heavy metal (HM) contamination in soil result in second pollution damage to Chinese cabbage, leading to deleterious health impacts. To elucidate the common transfer and accumulation characteristics of HMs in Chinese cabbage cultivar is important for safety breeding and consumption. In this case, concentrations and transfer capacities of HMs (Cd, Cr, Hg, Pb and As) in 35 common Chinese cabbage genotypes and their genotypic difference were investigated in this work. Results indicate that Chinese cabbage cultivar was more susceptible to Cd pollution, Hg was easily sifted from underground part to aerial part, Cr and Pb have similar enrichment and translocation characteristics, and Chinese cabbage cultivar had tolerance to As toxicity to some extent. Moreover, genotypic difference in HM accumulation in different parts of Chinese cabbage cultivar was also observed, and for edible part, followed by Hg>As>Cd>Pb>Cr. Referring to overall HM pollution level and biomass yield of edible part of 35 tested Chinese cabbages, B18, B6, B2 and B3 could be considered as the potential HMs pollution-safe Chinese cabbage cultivars. Information founded in this work may be used to provide referential strategies and methods to minimize the influx of HMs pollutants to human being through consumption and cultivation of Chinese cabbages.

Full-length transcriptome sequencing of pepper fruit during development and construction of a transcript variation database.

Chili pepper is an important spice and a model plant for fruit development studies. Large-scale omics information on chili pepper plant development continues to be gathered for understanding development as well as capsaicin biosynthesis. In this study, a full-spectrum transcriptome data of eight chili pepper tissues at five growth stages using the Oxford Nanopore long-read sequencing approach was generated. Of the 485 351 transcripts, 35 336 were recorded as reference transcripts (genes), while 450 015 were novel including coding, lnc, and other non-coding RNAs. These novel transcripts belonged to unknown/intergenic (347703), those retained introns (26336), and had multi-exons with at least one junction match (20333). In terms of alternative splicing, retained intron had the highest proportion (14795). The number of tissue-specific expressed transcripts ranged from 22 925 (stem) to 40 289 (flower). The expression changes during fruit and placenta development are discussed in detail. Integration of gene expression and capsaicin content quantification throughout the placental development clarifies that capsaicin biosynthesis in pepper is mainly derived from valine, leucin, and isoleucine degradation as well as citrate cycle and/or pyrimidine metabolism pathways. Most importantly, a user-friendly Pepper Full-Length Transcriptome Variation Database (PFTVD 1.0) (http://pepper-database.cn/) has been developed. PFTVD 1.0 provides transcriptomics and genomics information and allows users to analyse the data using various tools implemented. This work highlights the potential of long-read sequencing to discover novel genes and transcripts and their diversity in plant developmental biology.

Fertility restorer gene CaRf and PepperSNP50K provide a promising breeding system for hybrid pepper.

Cytoplasmic male sterility (CMS) is pivotal in plant breeding and widely employed in various crop hybrids, including pepper. However, the functional validation of the restorer of fertility (Rf) gene in pepper has been lacking until now. This study identifies and characterizes CaRf, a single dominant locus crucial for restoring CMS in the pepper strong recovery inbred line Zhangshugang. The CaRf gene encodes a mitochondria-targeted pentatricopeptide repeat protein, validated through the induction of male sterility upon its silencing in hybrid F1 plants. To enhance pepper breeding efficiency, 176 important pepper breeding parent materials were resequenced, and a PepperSNP50K liquid-phase breeding chip was developed, comprising 51 172 markers. Integration of CaRf functional characterization and PepperSNP50K facilitated the development of a high-quality red pepper hybrid. These findings provide significant insights and practical strategies for advancing molecular-designed breeding in peppers.

Corrigendum: Genome-wide identification and capsaicinoid biosynthesis-related expression analysis of the R2R3-MYB gene family in Capsicum annuum L.

[This corrects the article DOI: 10.3389/fgene.2020.598183.].

A multi-omics approach identifies bHLH71-like as a positive regulator of yellowing leaf pepper mutants exposed to high-intensity light.

Light quality and intensity can have a significant impact on plant health and crop productivity. Chlorophylls and carotenoids are classes of plant pigments that are responsible for harvesting light energy and protecting plants from the damaging effects of intense light. Our understanding of the role played by plant pigments in light sensitivity has been aided by light-sensitive mutants that change colors upon exposure to light of variable intensity. In this study, we conducted transcriptomic, metabolomic, and hormone analyses on a novel yellowing mutant of pepper (yl1) to shed light on the molecular mechanism that regulates the transition from green to yellow leaves in this mutant upon exposure to high-intensity light. Our results revealed greater accumulation of the carotenoid precursor phytoene and the carotenoids phytofluene, antheraxanthin, and zeaxanthin in yl1 compared with wild-type plants under high light intensity. A transcriptomic analysis confirmed that enzymes involved in zeaxanthin and antheraxanthin biosynthesis were upregulated in yl1 upon exposure to high-intensity light. We also identified a single basic helix-loop-helix (bHLH) transcription factor, bHLH71-like, that was differentially expressed and positively correlated with light intensity in yl1. Silencing of bHLH71-like in pepper plants suppressed the yellowing phenotype and led to reduced accumulation of zeaxanthin and antheraxanthin. We propose that the yellow phenotype of yl1 induced by high light intensity could be caused by an increase in yellow carotenoid pigments, concurrent with a decrease in chlorophyll accumulation. Our results also suggest that bHLH71-like functions as a positive regulator of carotenoid biosynthesis in pepper.

Genomes of cultivated and wild Capsicum species provide insights into pepper domestication and population differentiation.

Pepper (Capsicum spp.) is one of the earliest cultivated crops and includes five domesticated species, C. annuum var. annuum, C. chinense, C. frutescens, C. baccatum var. pendulum and C. pubescens. Here, we report a pepper graph pan-genome and a genome variation map of 500 accessions from the five domesticated Capsicum species and close wild relatives. We identify highly differentiated genomic regions among the domesticated peppers that underlie their natural variations in flowering time, characteristic flavors, and unique resistances to biotic and abiotic stresses. Domestication sweeps detected in C. annuum var. annuum and C. baccatum var. pendulum are mostly different, and the common domestication traits, including fruit size, shape and pungency, are achieved mainly through the selection of distinct genomic regions between these two cultivated species. Introgressions from C. baccatum into C. chinense and C. frutescens are detected, including those providing genetic sources for various biotic and abiotic stress tolerances.

Transcriptome data reveal gene clusters and key genes in pepper response to heat shock.

Climate change and global warming pose a great threat to plant growth and development as well as crop productivity. To better study the genome-wide gene expression under heat, we performed a time-course (0.5 to 24 h) transcriptome analysis in the leaf and root of 40-day-old pepper plants under 40°C as well as in control plants. Clustering analysis (K-means) showed that the expression of 29,249 genes can be grouped into 12 clusters with distinct expression dynamics under stress. Gene ontology (GO) enrichment analysis and transcription factor (TF) identification were performed on the clusters with certain expression patterns. Comparative analysis between the heat-treated and control plants also identified differentially expressed genes (DEGs), which showed the largest degree of change at 24 h. Interestingly, more DEGs were identified in the root than in the leaf. Moreover, we analyzed the gene expression of 25 heat shock factor genes (HSFs) in pepper after heat stress, identified five of these HSFs that responded to heat stress, and characterized the role of these genes in heat-tolerant (17CL30) and heat-susceptible (05S180) pepper lines. The findings of this study improve our understanding of the genome-wide heat stress response in pepper.

Analysis of the Expression and Function of Key Genes in Pepper Under Low-Temperature Stress.

The mechanism of resistance of plants to cold temperatures is very complicated, and the molecular mechanism and related gene network in pepper are largely unknown. Here, during cold treatment, we used cluster analysis (k-means) to classify all expressed genes into 15 clusters, 3,680 and 2,405 differentially expressed genes (DEGs) were observed in the leaf and root, respectively. The DEGs associated with certain important basic metabolic processes, oxidoreductase activity, and overall membrane compositions were most significantly enriched. In addition, based on the homologous sequence alignment of Arabidopsis genes, we identified 14 positive and negative regulators of the ICE-CBF-COR module in pepper, including CBF and ICE, and compared their levels in different data sets. The correlation matrix constructed based on the expression patterns of whole pepper genes in leaves and roots after exposure to cold stress showed the correlation between 14 ICE-CBF-COR signaling module genes, and provided insight into the relationship between these genes in pepper. These findings not only provide valuable resources for research on cold tolerance, but also lay the foundation for the genetic modification of cold stress regulators, which would help us achieve improved crop tolerance. To our knowledge, this is the first study to demonstrate the relationship between positive and negative regulators related to the ICE-CBF-COR module, which is of great significance to the study of low-temperature adaptive mechanisms in plants.

The bHLH1-DTX35/DFR module regulates pollen fertility by promoting flavonoid biosynthesis in Capsicum annuum L.

High pollen fertility can ensure the yield and efficiency of breeding work, but factors that affect the fertility of pepper pollen have not been studied extensively. In this work, we screened the reduced pollen fertility 1 (rpf1) mutant of Capsicum annuum with reduced pollen fertility and yellow anthers from an EMS (ethyl methanesulfonate)-mutagenized pepper population. Through construction of an F 2 population followed by BSA (bulked segregant analysis) mapping and KASP genotyping, we identified CabHLH1 as a candidate gene for control of this trait. A G â†’ A mutation at a splice acceptor site in CabHLH1 causes a frameshift mutation in the mutant, and the translated protein is terminated prematurely. Previous studies on CabHLH1 have focused on the regulation of flavonoid synthesis. Here, we found that CabHLH1 also has an important effect on pollen fertility. Pollen vigor, anther flavonoid content, and seed number were lower in CabHLH1-silenced pepper plants, whereas anther H2O2 and MDA (malondialdehyde) contents were higher. RNA-seq analyses showed that expression of the flavonoid synthesis genes DFR, ANS, and RT was significantly reduced in anthers of CabHLH1-silenced plants and rpf1 plants, as was the expression of DTX35, a gene related to pollen fertility and flavonoid transport. Yeast one-hybrid and dual-luciferase reporter assays showed that CabHLH1 can directly bind to the promoters of DTX35 and DFR and activate their expression. These results indicate that CabHLH1 regulates reactive oxygen species homeostasis by promoting the synthesis of anther flavonoids and acts as a positive regulator of pepper pollen fertility.

Comprehensive Proteome and Lysine Acetylome Analysis Reveals the Widespread Involvement of Acetylation in Cold Resistance of Pepper (Capsicum annuum L.).

Pepper is a typical warmth-loving vegetable that lacks a cold acclimation mechanism and is sensitive to cold stress. Lysine acetylation plays an important role in diverse cellular processes, but limited knowledge is available regarding acetylation modifications in the resistance of pepper plants to cold stress. In this study, the proteome and acetylome of two pepper varieties with different levels of cold resistance were investigated by subjecting them to cold treatments of varying durations followed by recovery periods. In total, 6,213 proteins and 4,574 lysine acetylation sites were identified, and this resulted in the discovery of 3,008 differentially expressed proteins and 768 differentially expressed acetylated proteins. A total of 1,988 proteins were identified in both the proteome and acetylome, and the functional differences in these co-identified proteins were elucidated through GO enrichment. KEGG analysis showed that 397 identified acetylated proteins were involved in 93 different metabolic pathways. The dynamic changes in the acetylated proteins in photosynthesis and the "carbon fixation in the photosynthetic organisms" pathway in pepper under low-temperature stress were further analyzed. It was found that acetylation of the PsbO and PsbR proteins in photosystem II and the PsaN protein in photosystem I could regulate the response of pepper leaves to cold stress. The acetylation levels of key carbon assimilation enzymes, such as ribulose bisphosphate carboxylase, fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, glyceraldehyde 3-phosphate dehydrogenase, phosphoribulokinase, and triosephosphate isomerase decreased, leading to decreases in carbon assimilation capacity and photosynthetic efficiency, reducing the cold tolerance of pepper leaves. This study is the first to identify the acetylome in pepper, and it greatly expands the catalog of lysine acetylation substrates and sites in Solanaceae crops, providing new insights for posttranslational modification studies.

Improved immobilization of soil cadmium by regulating soil characteristics and microbial community through reductive soil disinfestation.

Cadmium (Cd) contamination arising from industrialization has attracted increasing attention in recent years. Reductive soil disinfestation (RSD) as an effective agricultural practice has been widely applied for soil sterilization. However, there is little research regarding RSD affecting Cd immobilization. Here, five treatments, namely untreated soil (CK), flooding-treated soil (FL), RSD with 2% ethyl alcohol (EA), 2% sugarcane bagasse (SB), and 2% bean dregs (BD) were designed to detect their performance for Cd immobilization in contaminated soils, and the change of soil properties and microbial communities were monitored. The results revealed that pH significantly increased in FL and RSD-treated soils, but was negatively correlated with the exchangeable fraction of Cd (EX-Cd), while Oxidation-Reduction Potential (Eh) significantly decreased in FL and RSD-treated soils, and was positively correlated with EX-Cd. BD treatment might contribute to the increase of CaCO3 as shown by X-Ray Diffractomer analysis and strongly decreased the EX-Cd in the soil, but increased the relative abundances of Firmicutes, Planctomycetes, Acidobacteria, and Gemmatimonadetes, which may promote Fe (III) reduction or induce resistance to Cd. Bacterial communities at the phylum and genus levels were closely related to Cd fraction. The FL and RSD treatments moderately altered bacterial specific functions, including iron respiration, which may contribute to remediation of Cd-polluted soil by Fe (III) reduction. Field experiments were conducted to confirm that BD treatment resulted in a significant increase in pH whereas the content of total available Cd was reduced in soils. Compared to the control, concentration of total available Cd of red amaranth, sweet potato, towel gourd, and cowpeas were reduced by approximately 46%, 74%, 72%, and 76% in a BD-treated field, respectively. Our study highlights the potential of RSD as an effective method for Cd immobilization in contaminated soils by improving soil characteristics and altering the composition of the microbial community.

iTRAQ-based quantitative proteomic analysis of heat stress-induced mechanisms in pepper seedlings.

BACKGROUND: As one of the most important vegetable crops, pepper has rich nutritional value and high economic value. Increasing heat stress due to the global warming has a negative impact on the growth and yield of pepper. METHODS: To understand the heat stress response mechanism of pepper, an iTRAQ-based quantitative proteomic analysis was employed to identify possible heat-responsive proteins and metabolic pathways in 17CL30 and 05S180 pepper seedlings under heat stress. RESULT: In the present study, we investigated the changes of phenotype, physiology, and proteome in heat-tolerant (17CL30) and heat-sensitive (05S180) pepper cultivars in response to heat stress. Phenotypic and physiological changes showed that 17CL30 had a stronger ability to resist heat stress compared with 05S180. In proteomic analysis, a total of 3,874 proteins were identified, and 1,591 proteins were considered to participate in the process of heat stress response. According to bioinformatic analysis of heat-responsive proteins, the heat tolerance of 17CL30 might be related to a higher ROS scavenging, photosynthesis, signal transduction, carbohydrate metabolism, and stress defense, compared with 05S180.

Genome-Wide Identification and Capsaicinoid Biosynthesis-Related Expression Analysis of the R2R3-MYB Gene Family in Capsicum annuum L.

Capsaicinoids are naturally specialized metabolites in pepper and are the main reason that Capsicum fruits have a pungent smell. During the synthesis of capsaicin, MYB transcription factors play key regulatory roles. In particular, R2R3-MYB subfamily genes are the most important members of the MYB family and are critical candidate factors in capsaicinoid biosynthesis. The 108 R2R3-MYB genes in pepper were identified in this study and all are shown to have two highly conserved MYB binding domains. Phylogenetic and structural analyses clustered CaR2R3-MYB genes into seven groups. Interspecies collinearity analysis found that the R2R3-MYB family contains 16 duplicated gene pairs and the highest gene density is on chromosome 00 and 03. The expression levels of CaR2R3-MYB differentially expressed genes (DEGs) and capsaicinoid-biosynthetic genes (CBGs) in fruit development stages were obtained via RNA-seq and quantitative polymerase chain reaction (qRT-PCR). Co-expression analyses reveal that highly expressed CaR2R3-MYB genes are co-expressed with CBGs during early stages of pericarp and placenta development processes. It is speculated that six candidate CaR2R3-MYB genes are involved in regulating the synthesis of capsaicin and dihydrocapsaicin. This study is the first systematic analysis of the CaR2R3-MYB gene family and provided references for studying their molecular functions. At the same time, these results also laid the foundation for further research on the capsaicin characteristics of CaR2R3-MYB genes in pepper.

Integrative analysis of metabolome and transcriptome reveals the mechanism of color formation in pepper fruit (Capsicum annuum L.).

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.

Identification, expression, alternative splicing and functional analysis of pepper WRKY gene family in response to biotic and abiotic stresses.

WRKY proteins are a large group of plant transcription factors that are involved in various biological processes, including biotic and abiotic stress responses, hormone response, plant development, and metabolism. WRKY proteins have been identified in several plants, but only a few have been identified in Capsicum annuum. Here, we identified a total of 62 WRKY genes in the latest pepper genome. These genes were classified into three groups (Groups 1-3) based on the structural features of their proteins. The structures of the encoded proteins, evolution, and expression under normal growth conditions were analyzed and 35 putative miRNA target sites were predicted in 20 CaWRKY genes. Moreover, the response to cold or CMV treatments of selected WRKY genes were examined to validate the roles under stresses. And alternative splicing (AS) events of some CaWRKYs were also identified under CMV infection. Promoter analysis confirmed that CaWRKY genes are involved in growth, development, and biotic or abiotic stress responses in hot pepper. The comprehensive analysis provides fundamental information for better understanding of the signaling pathways involved in the WRKY-mediated regulation of developmental processes, as well as biotic and abiotic stress responses.