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.

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.

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.

Identifying pathological slices of gastric cancer via deep learning.

BACKGROUND: The accuracy of histopathology diagnosis largely depends on the pathologist's experience. It usually takes over 10 years to cultivate a senior pathologist, and small numbers of them lead to a high workload for those available. Meanwhile, inconsistent diagnostic results may arise among different pathologists, especially in complex cases, because diagnosis based on morphology is subjective. Computerized analysis based on deep learning has shown potential benefits as a diagnostic strategy. METHODS: This research aims to automatically determine the location of gastric cancer (GC) in the images of GC slices through artificial intelligence. We use image data from a regional teaching hospital in Taiwan for training. We collect images of patients diagnosed with GC from January 1, 2019 to December 31, 2020. In this study, scanned images are used to dissect 13,600 images from 50 different patients with GC sections whose GC sections are stained with hematoxylin and eosin (H&E stained) through a whole slide scanner, the scanned images from 50 different GC slice patients are divided into 80% training combinations, 2200 images of 40 patients are trained. The remaining 20%, totaling 10 people, are validated from a test set of 550 images. RESULTS: The validation results show that 91% of the correct rates are interpreted as GC images through deep learning. The sensitivity, specificity, PPV, and NPV were 84.9%, 94%, 87.7%, and 92.5%, respectively. After creating a 3D model through the grayscale value, the position of the GC is completely marked by the 3D model. The purpose of this research is to use artificial intelligence (AI) to determine the location of the GC in the image of GC slice. CONCLUSION: In patients undergoing pancreatectomy for pancreatic cancer, intraoperative infusion of lidocaine did not improve overall or disease-free survival. Reduced formation of circulating NETs was absent in pancreatic tumour tissue. CONCLUSION: For AI to assist pathologists in daily practice, to help a pathologist making a definite diagnosis is not the prime purpose at present time. The benefits could come from cancer screening and double-check quality control in a heavy workload which could distract the attention of pathologist during the time constraint examination process. We propose a two-steps method to identify cancerous areas in endoscopic gastric biopsy slices via deep learning. Then a 3D model is used to further mark all the positions of GC in the picture, and the model overcomes the problem that deep learning can't catch all GC.

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.

Integration of mRNA and miRNA profiling reveals the heterosis of three hybrid combinations of Capsicum annuum varieties.

Capsicum annuum is also known as chili which is one of the most important vegetable crops grown in the world. Breeding new varieties with heterosis could improve the quality of pepper, increase yield, growth potential, disease resistance, adaptability, and seed viability. To investigate the heterosis among three cross combinations of different parents, the mRNA-miRNA integrated analysis was performed. A total number of 22,659,009 to 36,423,818 clean data were generated from mRNA-seq with 81 libraries, and the unique mapped reads were from 35,495,567 (86.81%) to 46,466,622 (88.95%). The plant-hormone signal transduction pathway (40 genes) was detected with a higher DEG number. The SAUR32L, GID1, PYR1, EIN2. ERF1, PR1, JAR1-like, IAA from this pathway play a key role in plant development. From the miRNA-seq, the number of clean reads was ranging from 12,132,221 to 25,632,680. A total of 220 miRNAs were predicted in this study, and all of them were identified as novel miRNA. The top three candidate KEGG pathways of miRNA were ribosome signaling pathway (13 miRNAs), spliceosome pathway (13 miRNAs), and plant hormone signal transduction pathways (10 miRNAs). With the mRNA and miRNA integrated analysis, we found some key genes were regulated by some miRNAs. Among them, the scarecrow-like 6 protein can be up or down regulated by mir8, mir120, mir184, mir_214, mir125, and mir130. The function of Della protein was regulated by mir24, mir74, mir94, mir139, and mir190. This study contributes to understanding how heterosis regulates the traits, such as crop production, fruit weight, and fruit length.

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.

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.

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.

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.

Surface micromachined electrostatically actuated micro peristaltic pump.

An electrostatically actuated micro peristaltic pump is reported. The micro pump is entirely surface micromachined using a multilayer parylene technology. Taking advantage of the multilayer technology, the micro pump design enables the pumped fluid to be isolated from the electric field. Electrostatic actuation of the parylene membrane using both DC and AC voltages was demonstrated and applied to fluid pumping based on a 3-phase peristaltic sequence. A maximum flow rate of 1.7 nL min(-1) and an estimated pumping pressure of 1.6 kPa were achieved at 20 Hz phase frequency. A dynamic analysis was also performed with a lumped-parameter model for the peristaltic pump. The analysis results allow a quantitative understanding of the peristaltic pumping operation, and correctly predict the trends exhibited by the experimental data. The small footprint of the micro pump is well suited for large-scale integration of microfluidics. Moreover, because the same platform technology has also been used to fabricate other devices (e.g. valves, electrospray ionization nozzles, filters and flow sensors), the integration of these different devices can potentially lead to versatile and functional micro total analysis systems (microTAS).