Capsicum chinense Jacq.-derived glutaredoxin (CcGRXS12) alters redox status of the cells to confer resistance against pepper mild mottle virus (PMMoV-I).

KEY MESSAGE: The CcGRXS12 gene protects plants from cellular oxidative damage that are caused by both biotic and abiotic stresses. The protein possesses GSH-disulphide oxidoreductase property but lacks Fe-S cluster assembly mechanism. Glutaredoxins (Grxs) are small, ubiquitous and multi-functional proteins. They are present in different compartments of plant cells. A chloroplast targeted Class I GRX (CcGRXS12) gene was isolated from Capsicum chinense during the pepper mild mottle virus (PMMoV) infection. Functional characterization of the gene was performed in Nicotiana benthamiana transgenic plants transformed with native C. chinense GRX (Nb:GRX), GRX-fused with GFP (Nb:GRX-GFP) and GRX-truncated for chloroplast sequences fused with GFP (Nb:Δ2MGRX-GFP). Overexpression of CcGRXS12 inhibited the PMMoV-I accumulation at the later stage of infection, accompanied with the activation of salicylic acid (SA) pathway pathogenesis-related (PR) transcripts and suppression of JA/ET pathway transcripts. Further, the reduced accumulation of auxin-induced Glutathione-S-Transferase (pCNT103) in CcGRXS12 overexpressing lines indicated that the protein could protect the plants from the oxidative stress caused by the virus. PMMoV-I infection increased the accumulation of pyridine nucleotides (PNs) mainly due to the reduced form of PNs (NAD(P)H), and it was high in Nb:GRX-GFP lines compared to other transgenic lines. Apart from biotic stress, CcGRXS12 protects the plants from abiotic stress conditions caused by H2O2 and herbicide paraquat. CcGRXS12 exhibited GSH-disulphide oxidoreductase activity in vitro; however, it was devoid of complementary Fe-S cluster assembly mechanism found in yeast. Overall, this study proves that CcGRXS12 plays a crucial role during biotic and abiotic stress in plants.

Exogenous strigolactone enhanced the drought tolerance of pepper (Capsicum chinense) by mitigating oxidative damage and altering the antioxidant mechanism.

KEY MESSAGE: Exogenous SL positively regulates pepper DS by altering the root morphology, photosynthetic character, antioxidant enzyme activity, stomatal behavior, and SL-related gene expression. Drought stress (DS) has always been a problem for the growth and development of crops, causing significant negative impacts on crop productivity. Strigolactone (SL) is a newly discovered class of plant hormones that are involved in plants' growth and development and environmental stresses. However, the role of SL in response to DS in pepper remains unknown. DS considerably hindered photosynthetic pigments content, damaged root architecture system, and altered antioxidant machinery. In contrast, SL application significantly restored pigment concentration modified root architecture system, and increased relative chlorophyll content (SPAD). Additionally, SL treatment reduced oxidative damage by reducing hydrogen peroxide (H2O2) (24-57%) and malondialdehyde (MDA) (79-89%) accumulation in pepper seedlings. SL-pretreated pepper seedlings showed significant improvement in antioxidant enzyme activity, proline accumulation, and soluble sugar content. Furthermore, SL-related genes (CcSMAX2, CcSMXL6, and CcSMXL3) were down-regulated under DS. These findings suggest that the foliar application of SL can alleviate the adverse effects of drought tolerance by up-regulating chlorophyll content and activating antioxidant defense mechanisms.

Identification and expression analyses of B3 genes reveal lineage-specific evolution and potential roles of REM genes in pepper.

BACKGROUND: The B3 gene family, one of the largest plant-specific transcription factors, plays important roles in plant growth, seed development, and hormones. However, the B3 gene family, especially the REM subfamily, has not been systematically and functionally studied. RESULTS: In this study, we performed genome-wide re-annotation of B3 genes in five Solanaceae plants, Arabidopsis thaliana, and Oryza sativa, and finally predicted 1,039 B3 genes, including 231 (22.2%) newly annotated genes. We found a striking abundance of REM genes in pepper species (Capsicum annuum, Capsicum baccatum, and Capsicum chinense). Comparative motif analysis revealed that REM and other subfamilies (ABI3/VP1, ARF, RAV, and HSI) consist of different amino acids. We verified that the large number of REM genes in pepper were included in the specific subgroup (G8) through the phylogenetic analysis. Chromosome location and evolutionary analyses suggested that the G8 subgroup genes evolved mainly via a pepper-specific recent tandem duplication on chromosomes 1 and 3 after speciation between pepper and other Solanaceae. RNA-seq analyses suggested the potential functions of REM genes under salt, heat, cold, and mannitol stress conditions in pepper (C. annuum). CONCLUSIONS: Our study provides evolutionary and functional insights into the REM gene family in pepper.

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.

Characterization of leucine aminopeptidase (LAP) activity in sweet pepper fruits during ripening and its inhibition by nitration and reducing events.

KEY MESSAGE: Pepper fruits contain two leucine aminopeptidase (LAP) genes which are differentially modulated during ripening and by nitric oxide. The LAP activity increases during ripening but is negatively modulated by nitration. Leucine aminopeptidase (LAP) is an essential metalloenzyme that cleaves N-terminal leucine residues from proteins but also metabolizes dipeptides and tripeptides. LAPs play a fundamental role in cell protein turnover and participate in physiological processes such as defense mechanisms against biotic and abiotic stresses, but little is known about their involvement in fruit physiology. This study aims to identify and characterize genes encoding LAP and evaluate their role during the ripening of pepper (Capsicum annuum L.) fruits and under a nitric oxide (NO)-enriched environment. Using a data-mining approach of the pepper plant genome and fruit transcriptome (RNA-seq), two LAP genes, designated CaLAP1 and CaLAP2, were identified. The time course expression analysis of these genes during different fruit ripening stages showed that whereas CaLAP1 decreased, CaLAP2 was upregulated. However, under an exogenous NO treatment of fruits, both genes were downregulated. On the contrary, it was shown that during fruit ripening LAP activity increased by 81%. An in vitro assay of the LAP activity in the presence of different modulating compounds including peroxynitrite (ONOO-), NO donors (S-nitrosoglutathione and nitrosocyteine), reducing agents such as reduced glutathione (GSH), L-cysteine (L-Cys), and cyanide triggered a differential response. Thus, peroxynitrite and reducing compounds provoked around 50% inhibition of the LAP activity in green immature fruits, whereas cyanide upregulated it 1.5 folds. To our knowledge, this is the first characterization of LAP in pepper fruits as well as of its regulation by diverse modulating compounds. Based on the capacity of LAP to metabolize dipeptides and tripeptides, it could be hypothesized that the LAP might be involved in the GSH recycling during the ripening process.

Controlling soft rot of green pepper by bacteriocin paracin wx3 and its effect on storage quality of green pepper.

A bacteriocin paracin wx3 was investigated as a candidate of natural preservative to control green pepper soft rot. Firstly, paracin wx3 was heterologously expressed in Pichia pastoris X33 with an improved yield of 0.537 g/L. Its size and amino acid sequence were confirmed by Tricine-SDS-PAGE and LC-MS/MS. Then, result of antibacterial activity showed that its MIC value against Pectobacterium carotovorum was 16 µg/mL. In vitro, paracin wx3 completely killed the pathogen at high concentrations ≥8 × MIC. In vivo, disease incidence of green pepper soft rot was decreased from 90% (control) to <2% (8 × MIC). Subsequently, results of action mode showed that paracin wx3 inhibited the growth of pathogen by pore-formation on cell membrane. Last, paracin wx3 treatment reduced losses of weight, firmness, total soluble solid, Vc of green pepper during storage. It also inhibited the production of soft rot volatile p-xylene, 1-butanol, 2-methyl-2-propanol, 3-hydroxybutan-2-one-D, 2-pentyl furan, butanal, etc.

Chili pepper extracts, capsaicin, and dihydrocapsaicin as potential anticancer agents targeting topoisomerases.

DNA topoisomerases regulate conformational changes in DNA topology during normal cell growth, such as replication, transcription, recombination, and repair, and may be targeted for anticancer drugs. A DNA topology assay was used to investigate DNA-damaging/protective activities of extracts from Habanero Red (HR), Habanero Maya Red (HMR), Trinidad Moruga Scorpion (TMS), Jalapeno (J), Serrano pepper (SP), Habanero Red Savina (HRS), Bhut Jolokia (BJ), and Jamaica Rosso (JR) peppers, demonstrating their inhibitory effect on the relaxation of pBR by Topo I. DNA topoisomerase II (Topo II) is proven therapeutic target of anticancer drugs. Complete inhibition of Topo II was observed for samples TMS, HR, and HMR. Extracts J and SP had the lowest capsaicin and dihydrocapsaicin content compared to other peppers. HR, HMR, TMS, J, S, HRS, BJ, JR extracts showed the anticancer effect, examined by MTS and xCell assay on the in vitro culture of human colon carcinoma cell line HCT116.

Members of the Capsicum annuum CaTrxh Family Respond to High Temperature and Exhibit Dynamic Hetero/Homo Interactions.

Climate change adversely affects the water and temperature conditions required for plant growth, leading to a decrease in yield. In high temperatures, oxidative stress causes cellular damage in plant cells, which is a negative factor for crop production. Thioredoxin (Trx) is a small redox protein containing a conserved WC(G/P)PC motif that catalyzes the exchange of disulfide bonds. It is known to play an important role in maintaining cellular redox homeostasis. Trx proteins are widely distributed across various subcellular locations, and they play a crucial role in responding to cellular stresses. In this study, seven CaTrxh-type genes present in pepper were identified and the CaTrxh-type family was classified into three subgroups. CaTrxh genes responded to heat stress. Moreover, subcellular locations of the CaTrxh family exhibited dynamic patterns in normal conditions, and we observed relocalizations in heat stress conditions. Each CaTrxh family protein member formed homo-/heteromeric protein complexes in BiFC assay. Unexpectedly, subgroup III CaTrxh9 and CaTrxh10 can recruit subgroup I and II CaTrxh proteins into the plasma membrane. Thus, the function of the CaTrxh-type family is expected to play a protective role in the cell in response to high-temperature stress via protein complex formations. CaTrxh may have potential applications in the development of crops with enhanced tolerance to oxidative stress.

Leaky mutations in the zeaxanthin epoxidase in Capsicum annuum result in bright-red fruit containing a high amount of zeaxanthin.

Fruit color is one of the most important traits in peppers due to its esthetic value and nutritional benefits and is determined by carotenoid composition, resulting from diverse mutations of carotenoid biosynthetic genes. The EMS204 line, derived from an EMS mutant population, presents bright-red color, compared with the wild type Yuwolcho cultivar. HPLC analysis indicates that EMS204 fruit contains more zeaxanthin and less capsanthin and capsorubin than Yuwolcho. MutMap was used to reveal the color variation of EMS204 using an F3 population derived from a cross of EMS204 and Yuwolcho, and the locus was mapped to a 2.5-Mbp region on chromosome 2. Among the genes in the region, a missense mutation was found in ZEP (zeaxanthin epoxidase) that results in an amino acid sequence alteration (V291 → I). A color complementation experiment with Escherichia coli and ZEP in vitro assay using thylakoid membranes revealed decreased enzymatic activity of EMS204 ZEP. Analysis of endogenous plant hormones revealed a significant reduction in abscisic acid content in EMS204. Germination assays and salinity stress experiments corroborated the lower ABA levels in the seeds. Virus-induced gene silencing showed that ZEP silencing also results in bright-red fruit containing less capsanthin but more zeaxanthin than control. A germplasm survey of red color accessions revealed no similar carotenoid profiles to EMS204. However, a breeding line containing a ZEP mutation showed a very similar carotenoid profile to EMS204. Our results provide a novel breeding strategy to develop red pepper cultivars containing high zeaxanthin contents using ZEP mutations.

Genome-wide identification of the B3 transcription factor family in pepper (Capsicum annuum) and expression patterns during fruit ripening.

In plants, B3 transcription factors play important roles in a variety of aspects of their growth and development. While the B3 transcription factor has been extensively identified and studied in numerous species, there is limited knowledge regarding its B3 superfamily in pepper. Through the utilization of genome-wide sequence analysis, we identified a total of 106 B3 genes from pepper (Capsicum annuum), they are categorized into four subfamilies: RAV, ARF, LAV, and REM. Chromosome distribution, genetic structure, motif, and cis-acting element of the pepper B3 protein were analyzed. Conserved gene structure and motifs outside the B3 domain provided strong evidence for phylogenetic relationships, allowing potential functions to be deduced by comparison with homologous genes from Arabidopsis. According to the high-throughput transcriptome sequencing analysis, expression patterns differ during different phases of fruit development in the majority of the 106 B3 pepper genes. By using qRT-PCR analysis, similar expression patterns in fruits from various time periods were discovered. In addition, further analysis of the CaRAV4 gene showed that its expression level decreased with fruit ripening and located in the nucleus. B3 transcription factors have been genome-wide characterized in a variety of crops, but the present study is the first genome-wide analysis of the B3 superfamily in pepper. More importantly, although B3 transcription factors play key regulatory roles in fruit development, it is uncertain whether B3 transcription factors are involved in the regulation of the fruit development and ripening process in pepper and their specific regulatory mechanisms because the molecular mechanisms of the process have not been fully explained. The results of the study provide a foundation and new insights into the potential regulatory functions and molecular mechanisms of B3 genes in the development and ripening process of pepper fruits, and provide a solid theoretical foundation for the enhancement of the quality of peppers and their selection and breeding of high-yield varieties.

Comparative transcriptome analysis and Arabidopsis thaliana overexpression reveal key genes associated with cadmium transport and distribution in root of two Capsicum annuum cultivars.

The molecular mechanisms underlying high and low cadmium (Cd) accumulation in hot pepper cultivars remain unclear. In this study, comparative transcriptome analysis of root between high-Cd (J) and low-Cd (Z) cultivars was conducted under hydroponic cultivation with 0 and 0.4 mg/L Cd, respectively. The results showed that J enhanced the root uptake of Cd by elevating the expression of Nramp5 and counteracting Cd toxicity by increasing the expression of genes, such as NIR1, GLN1, and IAA9. Z reduced Cd accumulation by enhancing the cell wall lignin synthesis genes PAL, COMT, 4CL, LAC, and POD and the Cd transporters ABC, MTP1, and DTX1. Elevated expression of genes related to sulfur metabolism was observed in Z, potentially contributing to its ability to detoxify Cd. To investigate the function of CaCOMT1, an Arabidopsis thaliana overexpression line (OE-CaCOMT1) was constructed. The results revealed that OE-CaCOMT1 drastically increased the lignin content by 38-42% and reduced the translocation of Cd to the aboveground parts by 32%. This study provides comprehensive insights into the mechanisms underlying Cd accumulation in hot pepper cultivars using transcriptome analysis. Moreover, this study elucidates the critical function of CaCOMT1, providing a theoretical foundation for the production of low-Cd vegetables for food safety.

Effect of dietary intervention with Capsicum annuum extract on growth performance, physiological status, innate immune response, and related gene expression in Nile tilapia.

The red pepper (Capsicum annuum) has gained great attention recently because of its biological and pharmacological characteristics. The present approach aimed to evaluate the effects of C. annuum alcoholic extract (CAE) supplementation on Nile tilapia (Oreochromis niloticus) growth performance, physiological status, some metabolic, immune, and regulatory genes expression, and resistance against Streptococcus agalactiae infection. Fish (22.26 ± 0.19 g) were assigned to four treatments (five replicates, each with 10 fish replicate-1) and fed tested diets for 60 days. The experimental diets were supplemented with CAE at 0, 0.4, 0.8, and 1.6 g kg-1, expressed as CAE0, CAE0.4, CAE0.8, and CAE1.6, respectively. The findings exhibited that CAE dietary supplementation improved growth performance, feed utilization, elevated growth hormone level, and digestive enzyme activities (amylase and protease), and lowered leptin hormone in a level-dependent manner. Boosting the mRNA expression of the transporter proteins (solute carrier family 15 member 2 and solute carrier family 26 member 6) and insulin-like growth factor-1 genes with a decrease in the myostatin gene expression was noticed in the CAE-fed groups. The innate immune (serum bactericidal activity %, complement 3, and phagocytic activity %) and antioxidant (glutathione peroxidase and total antioxidant capacity) parameters were significantly (p < 0.05) improved, and the serum malondialdehyde level was significantly decreased by CAE dietary inclusion. A marked upregulation in the mRNA expression of interleukins (il-1ß, il-6, il-8, and il-10), transforming growth factor-ß, glutathione peroxidase, and glutathione synthetase genes were observed in CAE-fed groups. Dietary CAE decreased the cumulative mortalities after the challenge with S. agalactiae by 20, 13.33, and 10% in CAE0.4, CAE0.8, and CAE1.6, respectively, compared to the control (40%). Overall, dietary supplementation with CAE could improve growth performance and physiological status, and modulate the expression of several regulatory genes in Nile tilapia. The recommended level of CAE is 1.6 g kg-1 to augment growth and health status.

The fate of sulfonamides in microenvironments of rape and hot pepper rhizosphere soil system.

Sulfonamides (SAs) in agricultural soils can be degraded in rhizosphere, but can also be taken up by vegetables, which thereby poses human health and ecological risks. A glasshouse experiment was conducted using multi-interlayer rhizoboxes to investigate the fate of three SAs in rape and hot pepper rhizosphere soil systems to examine the relationship between the accumulation and their physicochemical processes. SAs mainly entered pepper shoots in which the accumulation ranged from 0.40 to 30.64 mg kg-1, while SAs were found at high levels in rape roots ranged from 3.01 to 16.62 mg kg-1. The BCFpepper shoot exhibited a strong positive linear relationship with log Dow, while such relationship was not observed between other bioconcentration factors (BCFs) and log Dow. Other than lipophilicity, the dissociation of SAs may also influence the uptake and translocation process. Larger TF and positive correlation with log Dow indicate preferential translocation of pepper SAs. There was a significant (p < 0.05) dissipation gradient of SAs observed away from the vegetable roots. In addition, pepper could uptake more SAs under solo exposure, while rape accumulated more SAs under combined exposure. When SAs applied in mixture, competition between SAs might occur to influence the translocation and dissipation patterns of SAs.

The phloem and xylem structure of plants and the neutral and ionic partitioning of sulfonamides (SAs) influence the uptake and translocation of SAs.A significant (p < 0.05) dissipation gradient of SAs was observed away from the vegetable roots.Combined exposure could promote the correlation between log BCF and log D_ow.

Effect of a blend of cinnamaldehyde, eugenol, and Capsicum oleoresin on methane emission and lactation performance of Holstein-Friesian dairy cows.

This study aimed to investigate the effect of administering a standardized blend of cinnamaldehyde, eugenol, and Capsicum oleoresin (CEC) to lactating dairy cattle for 84 d (i.e., 12 wk) on enteric CH4 emission, feed intake, milk yield and composition, and body weight. The experiment involved 56 Holstein-Friesian dairy cows (145 ± 31.1 d in milk at the start of the trial; mean ± standard deviation) in a randomized complete block design. Cows were blocked in pairs according to parity, lactation stage, and current milk yield, and randomly allocated to 1 of the 2 dietary treatments: a diet including 54.5 mg of CEC/kg of DM or a control diet without CEC. Diets were provided as partial mixed rations in feed bins, which automatically recorded individual feed intake. Additional concentrate was fed in the GreenFeed system that was used to measure emissions of CO2, CH4, and H2. Feeding CEC decreased CH4 yield (g/kg DMI) by on average 3.4% over the complete 12-wk period and by on average 3.9% from 6 wk after the start of supplementation onward. Feeding CEC simultaneously increased feed intake and body weight, and tended to increase milk protein content, whereas no negative responses were observed. These results must be further investigated and confirmed in longer-term in vivo experiments.

Ralstonia solanacearum effector RipAK suppresses homodimerization of the host transcription factor ERF098 to enhance susceptibility and the sensitivity of pepper plants to dehydration.

Plants have evolved a sophisticated immune system to defend against invasion by pathogens. In response, pathogens deploy copious effectors to evade the immune responses. However, the molecular mechanisms used by pathogen effectors to suppress plant immunity remain unclear. Herein, we report that an effector secreted by Ralstonia solanacearum, RipAK, modulates the transcriptional activity of the ethylene-responsive factor ERF098 to suppress immunity and dehydration tolerance, which causes bacterial wilt in pepper (Capsicum annuum L.) plants. Silencing ERF098 enhances the resistance of pepper plants to R. solanacearum infection not only by inhibiting the host colonization of R. solanacearum but also by increasing the immunity and tolerance of pepper plants to dehydration and including the closure of stomata to reduce the loss of water in an abscisic acid signal-dependent manner. In contrast, the ectopic expression of ERF098 in Nicotiana benthamiana enhances wilt disease. We also show that RipAK targets and inhibits the ERF098 homodimerization to repress the expression of salicylic acid-dependent PR1 and dehydration tolerance-related OSR1 and OSM1 by cis-elements in their promoters. Taken together, our study reveals a regulatory mechanism used by the R. solanacearum effector RipAK to increase virulence by specifically inhibiting the homodimerization of ERF098 and reprogramming the transcription of PR1, OSR1, and OSM1 to boost susceptibility and dehydration sensitivity. Thus, our study sheds light on a previously unidentified strategy by which a pathogen simultaneously suppresses plant immunity and tolerance to dehydration by secreting an effector to interfere with the activity of a transcription factor and manipulate plant transcriptional programs.

Melatonin improves drought stress tolerance of pepper (Capsicum annuum) plants via upregulating nitrogen metabolism.

While ameliorating effects of melatonin (MT) on abiotic stress tolerance in plants are widely reported, the mechanism that underlies this process remains elusive. This work investigated mechanisms by which MT improved drought tolerance in pepper (Capsicum annuum ) plants. A foliar spray of 0.1mM MT treatment was applied to plants grown at 80% and 40% of full field capacity for 3days. Drought stress caused a significant decrease in plant dry weight, relative water content, leaf water potential, PSII efficiency (F v /F m ratio), chlorophyll, soluble protein, leaf and root nitrogen content. Drought increased hydrogen peroxide, malondialdehyde (MDA), nitrate, ammonium, free amino acids, soluble sugars, proline and glycine betaine. Drought also increased peroxidase (POD), glutathione S-transferase (GST) and catalase (CAT) activities, electrolyte leakage (EL) and methylglyoxal (MG). MT pre-treatment reduced oxidative stress and improved nitrogen metabolism by activating various enzymes such as nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthetase (GOGAT) and glutamine dehydrogenase (GDH) activities. It also activated enzymes related to the glyoxalase system (Gly I and Gly II) and decreased NO3 - , NH4 + and free amino acid content. Our study suggests a cost-effective and sustainable solution to improve crop productivity in water-limited conditions, by enhancing plant growth, photosynthesis and nitrogen content.

An evolutionary view of vanillylamine synthase pAMT, a key enzyme of capsaicinoid biosynthesis pathway in chili pepper.

Pungent capsaicinoid is synthesized only in chili pepper (Capsicum spp.). The production of vanillylamine from vanillin is a unique reaction in the capsaicinoid biosynthesis pathway. Although putative aminotransferase (pAMT) has been isolated as the vanillylamine synthase gene, it is unclear how Capsicum acquired pAMT. Here, we present a phylogenetic overview of pAMT and its homologs. The Capsicum genome contained 5 homologs, including pAMT, CaGABA-T1, CaGABA-T3, and two pseudogenes. Phylogenetic analysis indicated that pAMT is a member of the Solanaceae cytoplasmic GABA-Ts. Comparative genome analysis found that multiple copies of GABA-T exist in a specific Solanaceae genomic region, and the cytoplasmic GABA-Ts other than pAMT are located in the region. The cytoplasmic GABA-T was phylogenetically close to pseudo-GABA-T harboring a plastid transit peptide (pseudo-GABA-T3). This suggested that Solanaceae cytoplasmic GABA-Ts occurred via duplication of a chloroplastic GABA-T ancestor and subsequent loss of the plastid transit signal. The cytoplasmic GABA-T may have been translocated from the specific Solanaceae genomic region during Capsicum divergence, resulting in the current pAMT locus. A recombinant protein assay demonstrated that pAMT had higher vanillylamine synthase activity than those of other plant GABA-Ts. pAMT was expressed exclusively in the placental septum of mature green fruit, whereas tomato orthologs SlGABA-T2/4 exhibit a ubiquitous expression pattern in plants. These findings suggested that both the increased catalytic efficiency and transcriptional changes in pAMT may have contributed to establish vanillylamine synthesis in the capsaicinoid biosynthesis pathway. This study provides insights into the establishment of pungency in the evolution of chili peppers.

Biochemical Aspects of Putative Aminotransferase Responsible for Converting Vanillin to Vanillylamine in the Capsaicinoid Biosynthesis Pathway in Capsicum Plants.

The biosynthesis pathway of capsaicinoids includes the conversion of vanillin to vanillylamine, where putative aminotransferase (pAMT) is thought to be the enzyme responsible in Capsicum plants. The objectives of this study were to prove that pAMT is the enzyme responsible for this conversion in plants and to clarify its catalytic properties using biochemical methods. Both an extract of habanero placenta and recombinant pAMT (rpAMT) constructed by using an Escherichia coli expression system were able to convert vanillin to vanillylamine in the presence of γ-aminobutyric acid as an amino donor and pyridoxal phosphate as a coenzyme. Conversion from vanillin to vanillylamine by the placenta extract was significantly attenuated by adding an anti-pAMT antibody to the reaction system. The amino donor specificity and affinity for vanillin of rpAMT were similar to those of the placenta extract. We thus confirmed that pAMT is the enzyme responsible for the conversion of vanillin to vanillylamine in capsaicinoid synthesis in Capsicum fruits. Therefore, we propose that pAMT should henceforth be named vanillin aminotransferase (VAMT).

Genome-Wide Identification and Expression Analysis of the SWEET Gene Family in Capsicum annuum L.

Sugars will eventually be exported transporters (SWEETs) are a novel class of sugar transport proteins that play a crucial role in plant growth, development, and response to stress. However, there is a lack of systematic research on SWEETs in Capsicum annuum L. In this study, 33 CaSWEET genes were identified through bioinformatics analysis. The Ka/Ks analysis indicated that SWEET genes are highly conserved not only among peppers but also among Solanaceae species and have experienced strong purifying selection during evolution. The Cis-elements analysis showed that the light-responsive element, abscisic-acid-responsive element, jasmonic-acid-responsive element, and anaerobic-induction-responsive element are widely distributed in the promoter regions of CaSWEETs. The expression pattern analysis revealed that CaSWEETs exhibit tissue specificity and are widely involved in pepper growth, development, and stress responses. The post-transcription regulation analysis revealed that 20 pepper miRNAs target and regulate 16 CaSWEETs through cleavage and translation inhibition mechanisms. The pathogen inoculation assay showed that CaSWEET16 and CaSWEET22 function as susceptibility genes, as the overexpression of these genes promotes the colonization of pathogens, whereas CaSWEET31 functions as a resistance gene. In conclusion, through systematic identification and characteristic analysis, a comprehensive understanding of CaSWEET was obtained, which lays the foundation for further studies on the biological functions of SWEET genes.

Identification and expression analysis of LEA gene family members in pepper (Capsicum annuum L.).

Pepper (Capsicum annuum L.) is an economically important crop containing capsaicinoids in the seed and placenta, which has various culinary, medical, and industrial applications. Late embryogenesis abundant (LEA) proteins are a large group of hydrophilic proteins participating in the plant stress response and seed development. However, to date there have been no genome-wide analyses of the LEA gene family in pepper. In the present study, 82 LEA genes were identified in the C. annuum genome and classified into nine subfamilies. Most CaLEA genes contain few introns (≤ 2) and are unevenly distributed across 10 chromosomes. Eight pairs of tandem duplication genes and two pairs of segmental duplication genes were identified in the LEA gene family; these duplicated genes were highly conserved and may have performed similar functions during evolution. Expression profile analysis indicated that CaLEA genes exhibited different tissue expression patterns, especially during embryonic development and stress response, particularly in cold stress. Three out of five CaLEA genes showed induced expression upon cold treatment. In summary, we have comprehensively reviewed the LEA gene family in pepper, offering a new perspective on the evolution of this family.