Plant glutathione peroxidases (GPXs) are important enzymes for removing reactive oxygen species in plant cells and are closely related to the stress resistance of plants. This study identified the GPX gene family members of pepper (Capsicum annuum L.), \"CM333\", at the whole-genome level to clarify their expression patterns and enzyme activity changes under abiotic stress and ABA treatment. The results showed that eight CaGPX genes were unevenly distributed across four chromosomes and one scaffold of the pepper genome, and their protein sequences had Cys residues typical of the plant GPX domains. The analysis of collinearity, phylogenetic tree, gene structure, and conserved motifs indicated that the CaGPX gene sequence is conserved, structurally similar, and more closely related to the sequence structure of Arabidopsis. Meanwhile, many cis elements involved in stress, hormones, development, and light response were found in the promoter region of the CaGPX gene. In addition, CaGPX1/4 and CaGPX6 were basically expressed in all tissues, and their expression levels were significantly upregulated under abiotic stress and ABA treatment. Subcellular localization showed that CaGPX1 and CaGPX4 are localized in chloroplasts. Additionally, the variations in glutathione peroxidase activity (GSH-Px) mostly agreed with the variations in gene expression. In summary, the CaGPXs gene may play an important role in the development of peppers and their response to abiotic stress and hormones.

As important transcription factors, WRKYs play a vital role in the defense response of plants against the invasion of multiple pathogens. Though some WRKY members have been reported to participate in pepper immunity in response to Ralstonia solanacearum infection, the functions of the majority of WRKY members are still unknown. Herein, CaWRKY22b was cloned from the pepper genome and its function against R. solanacearum was analyzed. The transcript abundance of CaWRKY22b was significantly increased in response to the infection of R. solanacearum and the application of exogenous methyl jasmonate (MeJA). Subcellular localization assay in the leaves of Nicotiana benthamiana showed that CaWRKY22b protein was targeted to the nuclei. Agrobacterium-mediated transient expression in pepper leaves indicated that CaWRKY22b overexpression triggered intensive hypersensitive response-like cell death, H2O2 accumulation, and the up-regulation of defense- and JA-responsive genes, including CaHIR1, CaPO2, CaBPR1, and CaDEF1. Virus-induced gene silencing assay revealed that knock-down of CaWRKY22b attenuated pepper\'s resistance against R. solanacearum and the up-regulation of the tested defense- and jasmonic acid (JA)-responsive genes. We further assessed the role of CaWRKY22b in modulating the expression of JA-responsive CaDEF1, and the result demonstrated that CaWRKY22b trans-activated CaDEF1 expression by directly binding to its upstream promoter. Collectively, our results suggest that CaWRKY22b positively regulated pepper immunity against R. solanacearum in a manner associated with JA signaling, probably by modulating the expression of JA-responsive CaDEF1.

The SBP-box gene significantly influences plant growth, development, and stress responses, yet its function in pepper plants during drought stress remains unexplored. Using virus-induced gene silencing and overexpression strategies, we examined the role of CaSBP13 during drought stress in plants. The results revealed that the expression of CaSBP13 can be induced by drought stress. Silencing of CaSBP13 in pepper notably boosted drought resistance, as evident by decreased active oxygen levels. Furthermore, the water loss rate, relative electrical conductivity, malondialdehyde content, and stomatal density were reduced in CaSBP13-silenced plants compared to controls. In contrast, CaSBP13 overexpression in Nicotiana benthamiana decreased drought tolerance with elevated reactive oxygen levels and stomatal density. Additionally, ABA signaling pathway genes (CaPP2C, CaAREB) exhibited reduced expression levels in CaSBP13-silenced plants post drought stress, as compared to control plants. On the contrary, CaPYL9 and CaSNRK2.4 showed heightened expression in CaSBP13-sienced plants under the same conditions. However, a converse trend for NbAREB, NbSNRK2.4, and NbPYL9 was observed post-four day drought in CaSBP13-overexpression plants. These findings suggest that CaSBP13 negatively regulates drought tolerance in pepper, potentially via ROS and ABA signaling pathways.

Pepper is an economically important vegetable worldwide, containing various specialized metabolites crucial for its development and flavor. Capsaicinoids, especially, are genus-specialized metabolites that confer a spicy flavor to Capsicum fruits. In this work, two pepper cultivars, YB (Capsicum frutescens L.) and JC (Capsicum baccatum L.) pepper, showed distinct differences in the accumulation of capsaicin and flavonoid. However, the molecular mechanism underlying them was still unclear. Metabolome analysis showed that the JC pepper induced a more abundant accumulation of metabolites associated with alkaloids, flavonoids, and capsaicinoids in the red ripening stages, leading to a spicier flavor in the JC pepper. Transcriptome analysis confirmed that the increased expression of transcripts associated with phenylpropanoid and flavonoid metabolic pathways occurred in the JC pepper. Integrative analysis of metabolome and transcriptome suggested that four structural genes, 4CL7, 4CL6, CHS, and COMT, were responsible for the higher accumulation of metabolites relevant to capsaicin and flavonoids. Through weighted gene co-expression network analyses, modules related to flavonoid biosynthesis and potential regulators for candidate genes were identified. The promoter analysis of four candidate genes showed they contained several cis-elements that were bonded to MYB, bZIP, and WRKY transcription factors. Further RT-qPCR examination verified three transcription factors, MYB, bZIP53, and WRKY25, that exhibited increased expression in the red ripening stage of the JC pepper compared to YB, which potentially regulated their expression. Altogether, our findings provide comprehensive understanding and valuable information for pepper breeding programs in the future.

Cytoplasmic male sterility (CMS) is a very important factor to produce hybrid seeds, and the restoration of fertility involves the expression of many fertility-related genes. Our previous study showed that the expression of CaPIPLC5 was significantly up-regulated in pepper restorer accessions and minimally expressed in sterile accessions, speculating that CaPIPLC5 is related to the restoration of fertility. In this study, we further validated the function of CaPIPLC5 in the restoration of fertility. The results showed that CaPIPLC5 was specifically expressed in the anthers of the restorer accessions with the subcellular localization in the cytoplasm. Furthermore, the expression of CaPIPLC5 was significantly higher in restorer lines and restorer combinations than that in CMS lines and their maintainer lines. Silencing CaPIPLC5 led to the number of pollen decreased, pollen grains wrinkled, and the ratio of pollen germination reduced. In addition, the joint analysis of Yeast One-Hybrid (Y1H) and Dual-Luciferase (dual-LUC) assays suggested that transcription factors such as CaARF5, CabZIP24 and CaMYB-like1, interacted with the promoter regions of CaPIPLC5, which regulated the expression of CaPIPLC5. The present results provide new insights into the study of CaPIPLC5 involved in the restoration of fertility in pepper.

Despite the therapeutic properties of capsaicin for some diseases, it shows some side effects for human health. The goal of this study was to develop a precise and accurate analytical strategy for the trace determination of capsaicin in different food, biological and environmental samples including pepper, saliva and wastewater by gas chromatography-mass spectrometry (GC-MS) after spraying-based fine droplet formation-liquid phase microextraction (SFDF-LPME) and quadruple isotope dilution (ID4) method. Acetic anhydride was used as derivatizing agent, and the extraction method was used to enrich the analyte derivative to reach low detection limits. Under the optimum conditions, limit of detection (LOD) and limit of quantitation (LOQ) were determined to be 0.33 and 1.10 µg/kg, respectively. Percent recoveries calculated for SFDF-LPME-GC-MS method ranged between 84.1 and 131.7 %. After the application of ID4-SFDF-LPME-GC-MS method, percent recoveries were obtained in the range of 94.9 and 104.0 % (%RSD ≤ 2.8) for the selected samples. It is obvious that the isotope dilution-based method provided high accurate and precise results due to the elimination of errors during the derivatization, extraction and measurement steps.

The yield and quality of pepper are considerably influenced by the cold conditions. Herein, we performed morphological, physiological and transcriptomic analyses by using two pepper seedlings, \'2379\' (cold-resistant) and \'2380\' (cold-sensitive). Briefly, 60 samples from each cultivar were analyzed at four distinct time points (0, 6, 24 and 48 h) at 5 °C in darkness. The physiological indices and activities of enzymes exhibited marked differences between the two cultivars. Transcriptomic analysis indicated that, compared to the control group, 11,415 DEGs were identified in \'2379\' and \'2380\' at 24 h. In the early stage, the number of DEGs in \'2379\' was 5.68 times higher than that in \'2380\', potentially explaining the observed differences in tolerance to colds. Processes such as protein targeting to membranes, jasmonic acid (JA)-mediated signalling, cold response and abscisic acid-activated signalling were involved. Subsequently, we identified a hub gene, CaAOS, that is involved in JA biosynthesis, positively influences cold tolerance and is a target of CaMYC2. Variations in the GC-motif of the CaAOS\'s promoter may influence the expression levels of CaAOS under cold treatment. The result of this study may lead to the development of more effective strategies for enhancing cold tolerance, potentially benefitting pepper breeding in cold regions.

UNASSIGNED: Domestic production of pepper (Capsicum spp.) is shrinking while demand within the US is growing. Lack of availability and cost of labor often present an obstacle for domestic producers both practically and economically. As a result, switching to harvesting peppers mechanically is anticipated as a key strategy to help domestic producers compete in the international market. Mechanical harvest efficiency can be improved through breeding. One important trait that mechanical harvest compatible material should have is an easy destemming trait: low force separation of the pedicel and calyx from the fruit.
UNASSIGNED: To detect the genetic sources underlying a novel easy destemming trait for the purpose of future breeding efforts in New Mexico pod-type green chile, we performed QTL analysis on three F2:F3 populations, coming from three New Mexico pod-type varieties: \'NuMex Odyssey,\' \'NuMex Iliad,\' and \'NuMex Joe E. Parker,\' each crossed with a parent with an easy destemming trait: MUC14. Genotyping was done through genotyping by sequencing (GBS) and phenotyping was done for destemming and fruit trait measurements. Correlations between measurements were found through the R package hmisc and QTL analysis was done through R/qtl.
UNASSIGNED: A strong relationship was seen between destemming and aspects of fruit morphology, particularly, destemming force and fruit width (Pearson\'s correlation coefficient r=0.75). Major QTLs for destemming and fruit size were discovered. Of these, the largest destemming force QTLs for all populations (PVE=34.5-69.9%) were on chromosome 10, and in two populations QTLs for destemming force were found on chromosome 3 (Percent Variance Explained (PVE)=10.7-18.8%). Fruit size-related QTLs in all populations colocalized in these same areas on chromosomes 3 and 10.
UNASSIGNED: This suggests that fruit shape may be genetically linked to destemming, and breeders interested in selecting for easy destemming pepper will also have to pay attention to fruit size and shape.

Red pepper (Capsicum annuum var. conoides) is commonly used for dried pepper production in China, and the drying process, particularly the during duration, profoundly affects its quality. The findings indicate that prolonged exposure to high temperatures during thermal drying results in significant darkening, an evident decrease in red and yellow tones, and gradual transformation of the pepper\'s microscopic structure from granular to compact, along with 88% reduction in moisture content and 81% decrease in thickness. The capsaicinoid content increased, resulting in a 4.3-fold increase in spiciness after drying compared to that of fresh pepper. The pepper aroma shifts from fruity, choking, and grassy to herb, dry wood, and smoky. Compounds such as 2-Acetylfuran, furfural, 2-methylfuran, 1-methylpyrrole, 2-methylpyrazine, and 2,5-dimethylpyrazine exhibited positive correlations with drying time, whereas ethyl 2-methylpropanoate, ethyl butanoate, ethyl 2-methylbutanoate, ethyl hexanoate, and 3-methylbutyl butanoate showed negative correlations, indicating their potential as markers for monitoring thermal drying processes.

Drought stress caused by the global climate considerably disturbs plant yield and growth. Here, we explored the putative roles of silicon in repressing drought mechanisms in pepper and the prominent involvement of secondary metabolites, GA pathway, and photosystem II. Our research revealed that the transcript level of the flavonoid biosynthesis-associated genes, including the PAL, 4-CL, CHS, FLS-1, F3H and DFR, progressively induced in the pepper leaves treated with silicon during the drought stress duration. Moreover, the phenolic and flavonoid compounds extensively induced in the pepper plants. Furthermore, the pepper plants markedly inhibited chlorophyll catabolic-allied genes, senescence-related marker gene, and the Rbohs gene. Silicon application also sustained the membrane stability, supported via fewer electrolyte leakage processes and minor, O2- H2O2 and MDA levels during drought. Apart from this, the pepper plants significantly induced the expression level of the photosystem II-related genes, osmoprotectants pathway-associated genes, and antioxidant defense genes. Moreover, the GA biosynthesis genes were prompted, while the ABA signaling and biosynthesis genes were suppressed in the silicon-supplemented plants. These consequences infer that the role of Si supplementation on enhancing drought tolerance could be elucidated through the activation of secondary metabolites, flavonoid biosynthesis, osmoprotectants, GA pathway, the efficiency of PSII, and the suppression of chlorophyll degradation. Our research outcomes unveil new and remarkable characteristics of silicon supplementation and offer a series of candidate targets for improving the tolerance of pepper plants to drought stress.