Osmosensor-mediated control of Ca2+ spiking in pollen germination.

Higher plants survive terrestrial water deficiency and fluctuation by arresting cellular activities (dehydration) and resuscitating processes (rehydration). However, how plants monitor water availability during rehydration is unknown. Although increases in hypo-osmolarity-induced cytosolic Ca2+ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration1,2, the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo3-5, these channels have been classified as a subtype of mechanosensors. Here we identify bona fide cell surface hypo-osmosensors in Arabidopsis and find that pollen Ca2+ spiking is controlled directly by water through these hypo-osmosensors-that is, Ca2+ spiking is the second messenger for water status. We developed a functional expression screen in Escherichia coli for hypo-osmosensitive channels and identified OSCA2.1, a member of the hyperosmolarity-gated calcium-permeable channel (OSCA) family of proteins6. We screened single and high-order OSCA mutants, and observed that the osca2.1/osca2.2 double-knockout mutant was impaired in pollen germination and HOSCA. OSCA2.1 and OSCA2.2 function as hypo-osmosensitive Ca2+-permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca2+ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca2+ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants.

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.

Fine mapping and candidate gene analysis of CaFCD1 affecting cuticle biosynthesis in Capsicum annuum L.

KEY MESSAGE: CaFCD1 gene regulates pepper cuticle biosynthesis. Pepper (Capsicum annuum L.) is an economically important vegetable crop that easily loses water after harvesting, which seriously affects the quality of its product. The cuticle is the lipid water-retaining layer on the outside of the fruit epidermis, which regulates the biological properties and reduces the rate of water-loss. However, the key genes involved in pepper fruit cuticle development are poorly understood. In this study, a pepper fruit cuticle development mutant fcd1 (fruit cuticle deficiency 1) was obtained by ethyl methanesulfonate mutagenesis. The mutant has great defects in fruit cuticle development, and the fruit water-loss rate of fcd1is significantly higher than that of the wild-type '8214' line. Genetic analysis suggested that the phenotype of the mutant fcd1 cuticle development defect was controlled by a recessive candidate gene CaFCD1 (Capsicum annuum fruit cuticle deficiency 1) on chromosome 12, which is mainly transcribed during fruit development. In fcd1, a base substitution within the CaFCD1 domain resulted in the premature termination of transcription, which affected cutin and wax biosynthesis in pepper fruit, as revealed by the GC-MS and RNA-seq analysis. Furthermore, the yeast one-hybrid and dual-luciferase reporter assays verified that the cutin synthesis protein CaCD2 was directly bound to the promoter of CaFCD1, suggesting that CaFCD1 may be a hub node in the cutin and wax biosynthetic regulatory network in pepper. This study provides a reference for candidate genes of cuticle synthesis and lays a foundation for breeding excellent pepper varieties.

Evolution and functional diversification of catalase genes in the green lineage.

BACKGROUND: Catalases (CATs) break down hydrogen peroxide into water and oxygen to prevent cellular oxidative damage, and play key roles in the development, biotic and abiotic stresses of plants. However, the evolutionary relationships of the plant CAT gene family have not been systematically reported. RESULTS: Here, we conducted genome-wide comparative, phylogenetic, and structural analyses of CAT orthologs from 29 out of 31 representative green lineage species to characterize the evolution and functional diversity of CATs. We found that CAT genes in land plants were derived from core chlorophytes and detected a lineage-specific loss of CAT genes in Fabaceae, suggesting that the CAT genes in this group possess divergent functions. All CAT genes were split into three major groups (group α, ß1, and ß2) based on the phylogeny. CAT genes were transferred from bacteria to core chlorophytes and charophytes by lateral gene transfer, and this led to the independent evolution of two types of CAT genes: α and ß types. Ten common motifs were detected in both α and ß groups, and ß CAT genes had five unique motifs, respectively. The findings of our study are inconsistent with two previous hypotheses proposing that (i) new CAT genes are acquired through intron loss and that (ii) the Cys-343 residue is highly conserved in plants. We found that new CAT genes in most higher plants were produced through intron acquisition and that the Cys-343 residue was only present in monocots, Brassicaceae and Pp_CatX7 in P. patens, which indicates the functional specificity of the CATs in these three lineages. Finally, our finding that CAT genes show high overall sequence identity but that individual CAT genes showed developmental stage and organ-specific expression patterns suggests that CAT genes have functionally diverged independently. CONCLUSIONS: Overall, our analyses of the CAT gene family provide new insights into their evolution and functional diversification in green lineage species.

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.

Orf165 is associated with cytoplasmic male sterility in pepper.

Cytoplasmic male sterility (CMS) is a maternally inherited trait that derives from the inability to produce functional pollen in higher plants. CMS results from recombination of the mitochondrial genome. However, understanding of the molecular mechanism of CMS in pepper is limited. In this study, comparative transcriptomic analyses were performed using a near-isogenic CMS line 14A (CMS-14A) and a maintainer line 14B (ML-14B) as experimental materials. A total of 17,349 differentially expressed genes were detected between CMS-14A and ML-14B at the PMC meiosis stage. Among them, six unigenes associated with CMS and 108 unigenes involved in energy metabolism were identified. The gene orf165 was found in CMS-14A. When orf165 was introduced into ML-14B, almost 30% of transgenic plants were CMS. In addition, orf165 expression in transgenic CMS plants resulted in abnormal function of some genes involved in energy metabolism. When orf165 in transgenic CMS plant was silenced, the resulted orf165-silenced plant was male fertile and the expression patterns of some genes associated with energy metabolism were similar to ML-14B. Thus, we confirmed that orf165 influenced CMS in pepper.

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.

A novel single-base mutation in CaBRI1 confers dwarf phenotype and brassinosteroid accumulation 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.

Comprehensive Phosphoproteomic Analysis of Pepper Fruit Development Provides Insight into Plant Signaling Transduction.

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.

Flower transcriptome dynamics during nectary development in pepper (Capsicum annuum L.).

The measurement of gene expression can provide important information about gene function and the molecular basis for developmental processes. We analyzed the transcriptomes at three different developmental stages of pepper flower [sporogenous cell division, stage (B1); pollen mother cell meiosis, stage (B2); and open flower (B3)]. In the cDNA libraries for B1, B2, and B3: 82718, 77061, and 91491 unigenes were assembled, respectively. A total of 34,445 unigene sequences and 128 pathways were annotated by KEGG pathway analysis. Several genes associated with nectar biosynthesis and nectary development were identified, and 8,955, 12,182, and 23,667 DEGs were identified in the B2 vs B1, B3 vs B1, and B3 vs. B2 comparisons. DEGs were involved in various metabolic processes, including flower development, nectar biosynthesis, and nectary development. According to the RNA-seq data, all 13 selected DEGs showed similar expression patterns after q-PCR analysis. Sucrose-phosphatase, galactinol-sucrose galactosyltransferase, and sucrose synthase played very important roles in nectar biosynthesis, and CRABS CLAW could potentially be involved in mediating nectary development. A significant number of simple sequence repeat and single nucleotide polymorphism markers were predicted in the Capsicum annuum sequences. The new results provide valuable genetic information about flower development in pepper.

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.

Genome-Wide Identification and Characterization of the Mitochondrial Transcription Termination Factors (mTERFs) in Capsicum annuum L.

Mitochondrial transcription termination factors (mTERFs) regulate the expression of mitochondrial genes and are closely related to the function of the mitochondrion and chloroplast. In this study, the mTERF gene family in capsicum (Capsicum annuum L.) was identified and characterized through genomic and bioinformatic analyses. Capsicum was found to possess at least 35 mTERF genes (CamTERFs), which were divided into eight major groups following phylogenetic analysis. Analysis of CamTERF promoters revealed the presence of many cis-elements related to the regulation of cellular respiration and photosynthesis. In addition, CamTERF promoters contained cis-elements related to phytohormone regulation and stress responses. Differentially expressed genes in different tissues and developmental phases were identified using RNA-seq data, which revealed that CamTERFs exhibit various expression and co-expression patterns. Gene ontology (GO) annotations associated CamTERFs primarily with mitochondrion and chloroplast function and composition. These results contribute towards understanding the role of mTERFs in capsicum growth, development, and stress responses. Moreover, our data assist in the identification of CamTERFs with important functions, which opens avenues for future studies.

Integration of Transcriptomics and Metabolomics for Pepper (Capsicum annuum L.) in Response to Heat Stress.

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.

The changes of rhizosphere microbial communities in pepper varieties with different capsaicinoids.

Capsaicinoids are produced uniquely in pepper fruits, and its level determines the commercial quality and health-promoting properties of pepper. So, it is particularly important to increase capsaicinoids content in pepper. Rhizosphere microbiota is critical to plant growth and performance, and affected by plant varieties. However, the impact of pepper varieties with different capsaicinoids yields on the rhizosphere microbiota is poorly understood. Using high-throughput sequencing of the 16S rRNA and internal transcribed spacer (ITS) region, we investigated the rhizosphere microbial community among five pepper varieties containing different capsaicinoids. Our results demonstrated that pepper variety significantly influenced the diversity and structure of rhizosphere microbial community. Bacterial diversity in varieties with high capsaicinoids content was significantly higher than in varieties with low capsaicinoids content, while fungal diversity was opposite to bacterial diversity. The correlation analysis revealed that 19 dominant bacterial genera (e.g., Chujaibacter, Rhodanobacter, and Gemmatimonas) were significantly correlated with capsaicinoids content, and nine of them were also significantly associated with soil nutrients, whereas only one fungal genus (Podospora) was significantly correlated with capsaicinoids content. Additionally, almost all genera which significantly correlated to capsaicinoids content were biomarkers of the five pepper varieties and the correlation was well corresponding to the capsaicinoids content. Overall, our results confirmed that the variety of pepper significantly affected the rhizosphere microbial community in the fields, and bacteria and fungi responded differently to capsaicinoids, which may affect the biosynthesis of capsaicinoids and contribute to further improvement of capsaicinoids production in pepper fruits.

Stem lodging Resistance-1 controls stem strength by positively regulating the biosynthesis of cell wall components in Capsicum annuum L.

Lodging presents a significant challenge in cultivating high-yield crops with extensive above-ground biomass, yet the molecular mechanisms underlying this phenomenon in the Solanaceae family remain largely unexplored. In this study, we identified a gene, CaSLR1 (Capsicum annuum Stem Lodging Resistance 1), which encodes a MYELOBLASTOSIS (MYB) family transcription factor, from a lodging-affected C. annuum EMS mutant. The suppression of CaSLR1 expression in pepper led to notable stem lodging, reduced thickness of the secondary cell wall, and decreased stem strength. A similar phenotype was observed in tomato with the knockdown of SlMYB61, the orthologous gene to CaSLR1. Further investigations demonstrated that CaNAC6, a gene involved in secondary cell wall (SCW) formation, is co-expressed with CaSLR1 and acts as a positive regulator of its expression, as confirmed through yeast one-hybrid, dual-luciferase reporter assays, and electrophoretic mobility shift assays. These findings elucidate the CaNAC6-CaSLR1 module that contributes to lodging resistance, emphasizing the critical role of CaSLR1 in the lodging resistance regulatory network.

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.

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.].

Studying the effect of light intensity on the photosynthetic mechanism of pepper leaf yellowing mutants by proteomics and phosphoproteomics.

The leaf is an important plant organ and is closely related to agricultural yield. Photosynthesis plays a critical role in promoting plant growth and development. Understanding the mechanism of leaf photosynthesis regulation will help improve crop yield. In this study, the pepper yellowing mutant was used as the experimental material, and the photosynthetic changes of pepper leaves (yl1 and 6421) under different light intensities were analyzed by chlorophyll fluorimeter and photosynthesis meter. Changes in proteins and enrichment of phosphopeptides in pepper leaves were determined. The results showed that different light intensities had significant effects on the chlorophyll fluorescence and photosynthetic parameters of pepper leaves. The differentially expressed proteins (DEPs) and differentially expressed phosphorylated proteins (DEPPs) were mainly involved in photosynthesis, photosynthesis-antenna proteins, and carbon fixation in photosynthetic organisms. In yl1 leaves, the phosphorylation levels of photosynthesis and photosynthesis-antenna proteins LHCA2, LHCA3, PsbC, PsbO, and PsbP were lower under low light treatment, but significantly higher under high light intensity compared with wild-type leaves. In addition, many proteins involved in the carbon assimilation pathway, including TKT, Rubisco, and PGK, were phosphorylated, and this modification level was significantly higher in yl1 than in the wild type under high light intensity. These results provide a new perspective for studying the photosynthesis mechanism of pepper under different light intensities.