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.

Transcriptome Analysis Reveals Key Genes Involved in Trichome Formation in Pepper (Capsicum annuum L.).

Trichomes are specialized organs located in the plant epidermis that play important defense roles against biotic and abiotic stresses. However, the mechanisms regulating the development of pepper epidermal trichomes and the related regulatory genes at the molecular level are not clear. Therefore, we performed transcriptome analyses of A114 (less trichome) and A115 (more trichome) to dig deeper into the genes involved in the regulatory mechanisms of epidermal trichome development in peppers. In this study, the epidermal trichome density of A115 was found to be higher by phenotypic observation and was highest in the leaves at the flowering stage. A total of 39,261 genes were quantified by RNA-Seq, including 11,939 genes not annotated in the previous genome analysis and 18,833 differentially expressed genes. Based on KEGG functional enrichment, it was found that DEGs were mainly concentrated in three pathways: plant-pathogen interaction, MAPK signaling pathway-plant, and plant hormone signal transduction. We further screened the DEGs associated with the development of epidermal trichomes in peppers, and the expression of the plant signaling genes GID1B-like (Capana03g003488) and PR-6 (Capana09g001847), the transcription factors MYB108 (Capana05g002225) and ABR1-like (Capana04g001261), and the plant resistance genes PGIP-like (Capana09g002077) and At5g49770 (Capana08g001721) in the DEGs were higher at A115 compared to A114, and were highly expressed in leaves at the flowering stage. In addition, based on the WGCNA results and the establishment of co-expression networks showed that the above genes were highly positively correlated with each other. The transcriptomic data and analysis of this study provide a basis for the study of the regulatory mechanisms of pepper epidermal trichomes.

Integrated transcriptome and metabolome analysis reveals anthocyanin biosynthesis mechanisms in pepper (Capsicum annuum L.) leaves under continuous blue light irradiation.

BACKGROUND: Different metabolic compounds give pepper leaves and fruits their diverse colors. Anthocyanin accumulation is the main cause of the purple color of pepper leaves. The light environment is a critical factor affecting anthocyanin biosynthesis. It is essential that we understand how to use light to regulate anthocyanin biosynthesis in plants. RESULT: Pepper leaves were significantly blue-purple only in continuous blue light or white light (with a blue light component) irradiation treatments, and the anthocyanin content of pepper leaves increased significantly after continuous blue light irradiation. This green-to-purple phenotype change in pepper leaves was due to the expression of different genes. We found that the anthocyanin synthesis precursor-related genes PAL and 4CL, as well as the structural genes F3H, DFR, ANS, BZ1, and F3'5'H in the anthocyanin synthesis pathway, had high expression under continuous blue light irradiation. Similarly, the expression of transcription factors MYB1R1-like, MYB48, MYB4-like isoform X1, bHLH143-like, and bHLH92-like isoform X3, and circadian rhythm-related genes LHY and COP1, were significantly increased after continuous blue light irradiation. A correlation network analysis revealed that these transcription factors and circadian rhythm-related genes were positively correlated with structural genes in the anthocyanin synthesis pathway. Metabolomic analysis showed that delphinidin-3-O-glucoside and delphinidin-3-O-rutinoside were significantly higher under continuous blue light irradiation relative to other light treatments. We selected 12 genes involved in anthocyanin synthesis in pepper leaves for qRT-PCR analysis, and the accuracy of the RNA-seq results was confirmed. CONCLUSIONS: In this study, we found that blue light and 24-hour irradiation together induced the expression of key genes and the accumulation of metabolites in the anthocyanin synthesis pathway, thus promoting anthocyanin biosynthesis in pepper leaves. These results provide a basis for future study of the mechanisms of light quality and photoperiod in anthocyanin synthesis and metabolism, and our study may serve as a valuable reference for screening light ratios that regulate anthocyanin biosynthesis in plants.

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.

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.

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.

Effects of Soil Type on Trace Element Absorption and Fruit Quality of Pepper.

The inbred "SJ11-3" pepper was cultured in yellow brown soil, paddy soil, fluvo-aquic soil, and pastoral soil, and the factors affecting the absorption of trace elements and fruit quality were analyzed. The results showed that the physicochemical properties of the soils were significantly different, which led to differences in the nutritional quality of pepper fruits. The pH value had a significant effect on the absorption of trace elements in pepper. The increase of pH promoted the absorption of magnesium and molybdenum but inhibited the absorption of zinc, copper, manganese, and iron. The stepwise multivariable regression analysis showed that the amount of molybdenum in soil was the main factor affecting the total amino acid content of pepper. Total nitrogen, zinc, and copper were the main factors that contributed to the soluble sugar content of pepper, and the available potassium was the major determinant of the vitamin C content of pepper. This study provides new insight on the pepper fruit quality grown on different types of soil with varying levels of trace elements.

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.

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.

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.

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.

Identification and characterization of novel microRNAs for fruit development and quality in hot pepper (Capsicum annuum L.).

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.