Pepper catalase: a broad analysis of its modulation during fruit ripening and by nitric oxide.

Catalase is a major antioxidant enzyme located in plant peroxisomes that catalyzes the decomposition of H2O2. Based on our previous transcriptomic (RNA-Seq) and proteomic (iTRAQ) data at different stages of pepper (Capsicum annuum L.) fruit ripening and after exposure to nitric oxide (NO) enriched atmosphere, a broad analysis has allowed us to characterize the functioning of this enzyme. Three genes were identified, and their expression was differentially modulated during ripening and by NO gas treatment. A dissimilar behavior was observed in the protein expression of the encoded protein catalases (CaCat1-CaCat3). Total catalase activity was down-regulated by 50% in ripe (red) fruits concerning immature green fruits. This was corroborated by non-denaturing polyacrylamide gel electrophoresis, where only a single catalase isozyme was identified. In vitro analyses of the recombinant CaCat3 protein exposed to peroxynitrite (ONOO-) confirmed, by immunoblot assay, that catalase underwent a nitration process. Mass spectrometric analysis identified that Tyr348 and Tyr360 were nitrated by ONOO-, occurring near the active center of catalase. The data indicate the complex regulation at gene and protein levels of catalase during the ripening of pepper fruits, with activity significantly down-regulated in ripe fruits. Nitration seems to play a key role in this down-regulation, favoring an increase in H2O2 content during ripening. This pattern can be reversed by the exogenous NO application. While plant catalases are generally reported to be tetrameric, the analysis of the protein structure supports that pepper catalase has a favored quaternary homodimer nature. Taken together, data show that pepper catalase is down-regulated during fruit ripening, becoming a target of tyrosine nitration, which provokes its inhibition.

Pepper SUMO protease CaDeSI2 positively modulates the drought responses via deSUMOylation of clade A PP2C CaAITP1.

Posttranslational modification of multiple ABA signaling components is an essential process for the adaptation and survival of plants under stress conditions. In our previous study, we established that the pepper group A PP2C protein CaAITP1, one of the core components of ABA signaling, undergoes ubiquitination mediated by the RING-type E3 ligase CaAIRE1. In this study, we discovered an additional form of regulation mediated via the SUMOylation of CaAITP1. Pepper plants subjected to drought stress were characterized by reductions in both the stability and SUMOylation of CaAITP1 protein. Moreover, we identified a SUMO protease, Capsicum annuum DeSUMOylating Isopeptidase 2 (CaDeSI2), as a new interacting partner of CaAITP1. In vitro and in vivo analyses revealed that CaAITP1 is deSUMOylated by CaDeSI2. Silencing of CaDeSI2 in pepper plants led to drought-hypersensitive and ABA-hyposensitive phenotypes, whereas overexpression of CaDeSI2 in transgenic Arabidopsis plants resulted in the opposite phenotypes. Importantly, we found that the CaAITP1 protein was stabilized in response to the silencing of CaDeSI2, and CaDeSI2 and CaAITP1 co-silenced pepper plants were characterized by drought-tolerant phenotypes similar to those observed in CaAITP1-silenced pepper. Collectively, our findings indicate that CaDeSI2 reduces the stability of CaAITP1 via deSUMOylation, thereby positively regulating drought tolerance.

Characterization of GPX Gene Family in Pepper (Capsicum annuum L.) under Abiotic Stress and ABA Treatment.

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.

The CBL-interacting protein kinase CaCIPK13 positively regulates defence mechanisms against cold stress in pepper.

Cold stress is one of the main factors limiting growth and development in pepper. Calcineurin B-like proteins (CBLs) are specific calcium sensors with non-canonical EF-hands to capture calcium signals, and interact with CBL-interacting protein kinases (CIPKs) in the regulation of various stresses. In this study, we isolated a cold-induced CIPK gene from pepper named CaCIPK13, which encodes a protein of 487 amino acids. In silico analyses indicated that CaCIPK13 is a typical CIPK family member with a conserved NAF motif, which consists of the amino acids asparagine, alanine, and phenylalanine. The CaCIPK13 protein was located in the nucleus and plasma membrane. Knock down of CaCIPK13 resulted in enhanced sensitivity to cold stress in pepper, with increased malondialdehyde content, H2O2 accumulation, and electrolyte leakage, while the catalase, peroxidase, superoxide dismutase activities and anthocyanin content were decreased. The transcript level of cold and anthocyanin-related genes was substantially decreased in CaCIPK13-silenced pepper leaves relative to the empty vector control. On the contrary, overexpression of CaCIPK13 in tomato improved cold tolerance via increasing anthocyanin content and activities of reactive oxygen species scavenging enzymes. Furthermore, the interaction of CaCIPK13 with CaCBL1/6/7/8 was Ca2+-dependent. These results indicate that CaCIPK13 plays a positive role in cold tolerance mechanism via CBL-CIPK signalling.

Crystal Structures of the Plant Phospholipase A1 Proteins Reveal a Unique Dimerization Domain.

Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the ester bonds they cleave off: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). Among the four phospholipase groups, PLA1 has been less studied than the other phospholipases. Here, we report the first molecular structures of plant PLA1s: AtDSEL and CaPLA1 derived from Arabidopsis thaliana and Capsicum annuum, respectively. AtDSEL and CaPLA1 are novel PLA1s in that they form homodimers since PLAs are generally in the form of a monomer. The dimerization domain at the C-terminal of the AtDSEL and CaPLA1 makes hydrophobic interactions between each monomer, respectively. The C-terminal domain is also present in PLA1s of other plants, but not in PLAs of mammals and fungi. An activity assay of AtDSEL toward various lipid substrates demonstrates that AtDSEL is specialized for the cleavage of sn-1 acyl chains. This report reveals a new domain that exists only in plant PLA1s and suggests that the domain is essential for homodimerization.

A pepper RING-type E3 ligase, CaASRF1, plays a positive role in drought tolerance via modulation of CaAIBZ1 stability.

Plants have evolved complex defense mechanisms to adapt and survive under adverse growth conditions. Abscisic acid (ABA) is a phytohormone that plays a pivotal role in the stress response, especially regulation of the stomatal aperture in response to drought. Here, we identified the pepper CaASRF1 (Capsicum annuum ABA Sensitive RING Finger E3 ligase 1) gene, which modulates drought stress tolerance via ABA-mediated signaling. CaASRF1 contains a C3H2C3-type RING finger domain, which functions as an E3 ligase by attaching ubiquitins to the target proteins. CaASRF1 expression was enhanced after exposure to ABA, drought and NaCl. Loss-of-function in pepper plants and gain-of-function in Arabidopsis plants revealed that CaASRF1 positively modulates ABA signaling and the drought stress response. Moreover, CaASRF1 interacted with and was associated with degradation of the bZIP transcription factor CaAIBZ1 (Capsicum annuum ASRF1-Interacting bZIP transcription factor 1). Contrary to CaASRF1 phenotypes, CaAIBZ1-silenced pepper and CaAIBZ1-overexpressing Arabidopsis exhibited drought-tolerant and drought-sensitive phenotypes, respectively. Taken together, our data indicate that CaASRF1 positively modulates ABA signaling and the drought stress response via modulation of CaAIBZ1 stability.

Inhibition of NADP-malic enzyme activity by H2 S and NO in sweet pepper (Capsicum annuum L.) fruits.

NADPH is an essential cofactor in many physiological processes. Fruit ripening is caused by multiple biochemical pathways in which, reactive oxygen and nitrogen species (ROS/RNS) metabolism is involved. Previous studies have demonstrated the differential modulation of nitric oxide (NO) and hydrogen sulfide (H2 S) content during sweet pepper (Capsicum annuum L.) fruit ripening, both of which regulate NADP-isocitrate dehydrogenase activity. To gain a deeper understanding of the potential functions of other NADPH-generating components, we analyzed glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), which are involved in the oxidative phase of the pentose phosphate pathway (OxPPP) and NADP-malic enzyme (NADP-ME). During fruit ripening, G6PDH activity diminished by 38%, while 6PGDH and NADP-ME activity increased 1.5- and 2.6-fold, respectively. To better understand the potential regulation of these NADP-dehydrogenases by H2 S, we obtained a 50-75% ammonium-sulfate-enriched protein fraction containing these proteins. With the aid of in vitro assays, in the presence of H2 S, we observed that, while NADP-ME activity was inhibited by up to 29-32% using 2 and 5 mM Na2 S as H2 S donor, G6PDH and 6PGDH activities were unaffected. On the other hand, NO donors, S-nitrosocyteine (CysNO) and DETA NONOate also inhibited NADP-ME activity by 35%. These findings suggest that both NADP-ME and 6PGDH play an important role in maintaining the supply of NADPH during pepper fruit ripening and that H2 S and NO partially modulate the NADPH-generating system.

The role of nitrate reductase in brassinosteroid-induced endogenous nitric oxide generation to improve cadmium stress tolerance of pepper plants by upregulating the ascorbate-glutathione cycle.

A study was performed to assess if nitrate reductase (NR) participated in brassinosteroid (BR)-induced cadmium (Cd) stress tolerance primarily by accelerating the ascorbate-glutathione (AsA-GSH) cycle. Prior to initiating Cd stress (CdS), the pepper plants were sprayed with 0.5 µM 24-epibrassinolide (EBR) every other day for 10 days. Thereafter the seedlings were subjected to control or CdS (0.1 mM CdCl2) for four weeks. Cadmium stress decreased the plant growth related attributes, water relations as well as the activities of monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR), but enhanced proline content, leaf Cd2+ content, oxidative stress-related traits, activities of ascorbate peroxidase (APX) and glutathione reductase (GR), and the activities of antioxidant defence system-related enzymes as well as NR activity and endogenous nitric oxide content. EBR reduced leaf Cd2+ content and oxidative stress-related parameters, enhanced plant growth, regulated water relations, and led to further increases in proline content, AsA-GSH cycle-related enzymes' activities, antioxidant defence system-related enzymes as well as NR activity and endogenous nitric oxide content. The EBR and the inhibitor of NR (tungstate) reversed the positive effects of EBR by reducing NO content, showing that NR could be a potential contributor of EBR-induced generation of NO which plays an effective role in tolerance to CdS in pepper plants by accelerating the AsA-GSH cycle and antioxidant enzymes.

Chitinase Gene Positively Regulates Hypersensitive and Defense Responses of Pepper to Colletotrichum acutatum Infection.

Anthracnose caused by Colletotrichum acutatum is one of the most devastating fungal diseases of pepper (Capsicum annuum L.). The utilization of chitin-binding proteins or chitinase genes is the best option to control this disease. A chitin-binding domain (CBD) has been shown to be crucial for the innate immunity of plants and activates the hypersensitive response (HR). The CaChiIII7 chitinase gene has been identified and isolated from pepper plants. CaChiIII7 has repeated CBDs that encode a chitinase enzyme that is transcriptionally stimulated by C. acutatum infection. The knockdown of CaChiIII7 in pepper plants confers increased hypersensitivity to C. acutatum, resulting in its proliferation in infected leaves and an attenuation of the defense response genes CaPR1, CaPR5, and SAR8.2 in the CaChiIII7-silenced pepper plants. Additionally, H2O2 accumulation, conductivity, proline biosynthesis, and root activity were distinctly reduced in CaChiIII7-silenced plants. Subcellular localization analyses indicated that the CaChiIII7 protein is located in the plasma membrane and cytoplasm of plant cells. The transient expression of CaChiIII7 increases the basal resistance to C. acutatum by significantly expressing several defense response genes and the HR in pepper leaves, accompanied by an induction of H2O2 biosynthesis. These findings demonstrate that CaChiIII7 plays a prominent role in plant defense in response to pathogen infection.

Variability and Phylogeny of the Pepper Phytoene Synthase Paralogs PSY1 and PSY2 in Species of Various Capsicum Complexes.

Genes homologous to PSY1 and PSY2 that encode phytoene synthase isoforms in Capsicum species C. baccatum, C. chinense, C. frutescens, C. tovarii, C. eximium, and C. chacoense were identified. High conservatism of functionally significant sites of phytoene synthases of the analyzed accessions was revealed. It was found that only PSY1-based clustering of pepper species corresponds to the traditional Capsicum phylogeny; C. eximium was a part of the Purple corolla complex, and C. chacoense was equidistant from Annuum and Baccatum clades. The absence of significant differences between PSY1 and PSY2 of yellow-fruited C. chinense and red-fruited pepper accessions was shown. The yellow color of C. chinense fruit may be the result of both decreased PSY1 expression and increased PSY2 transcription. Thus, it was demonstrated that the acquired fruit pigmentation retains strict phylogenetic limitations, which, however, can be overcome using artificial selection for the activity of phytoene synthase PSY1.

Roles of pepper bZIP protein CaDILZ1 and its interacting partner RING-type E3 ligase CaDSR1 in modulation of drought tolerance.

Abscisic acid (ABA) is a plant hormone that plays a key role in the environmental stress response, especially the induction of ABA-responsive and stress-responsive genes and modulation of the stomatal aperture in response to drought stress. Here, we identified CaDILZ1 (Capsicum annuum Drought-Induced Leucine Zipper 1) belonging to subgroup D of the bZIP protein family; gene functions of this family in response to ABA and drought signaling still remain unknown. CaDILZ1 expression was significantly induced in pepper leaves after exposure to ABA, drought, and NaCl. The CaDILZ1 protein localized in the nucleus of plant cells. In response to drought stress, CaDILZ1-silenced pepper and CaDILZ1-overexpressing Arabidopsis plants exhibited drought-sensitive and drought-tolerant phenotypes, respectively, via altered ABA content, stomatal closure, and expression of ABA-responsive and drought-responsive marker genes. We isolated the RING finger protein CaDSR1 (Capsicum annuum Drought Sensitive RING finger protein 1), which interacted with CaDILZ1 in the nucleus. The CaDSR1 protein exhibited E3 ligase activity and promoted CaDILZ1 degradation via the 26S proteasome pathway. Under drought stress conditions, CaDSR1-silenced pepper and CaDSR1-overexpressing Arabidopsis plants exhibited contrasting phenotypes to those of CaDILZ1-silenced pepper and CaDILZ1-overexpressing Arabidopsis plants. Taken together, our data suggest that CaDSR1 and CaDILZ1 function in ABA-mediated drought stress signaling in pepper plants.

ChiIV3 Acts as a Novel Target of WRKY40 to Mediate Pepper Immunity Against Ralstonia solanacearum Infection.

ChiIV3, a chitinase of pepper (Capsicum annuum), stimulates cell death in pepper plants. However, there are only scarce reports on its role in resistance against bacterial wilt disease such as that caused by Ralstonia solanacearum and their transcriptional regulation. In this study, the silencing of ChiIV3 in pepper plants significantly reduced the resistance to R. solanacearum. The transcript of ChiIV3 was induced by R. solanacearum inoculation (RSI) as well as exogenous application of methyl jasmonate and abscisic acid. The bioinformatics analysis revealed that the ChiIV3 promoter consists of multiple stress-related cis elements, including six W-boxes and one MYB1AT. With the 5' deletion assay in the ChiIV3 promoter, the W4-box located from -640 to -635 bp was identified as the cis element that is required for the response to RSI. In addition, the W4-box element was shown to be essential for the binding of the ChiIV3 promoter by the WRKY40 transcription factor, which is known to positively regulate the defense response to R. solanacearum. Site-directed mutagenesis in the W4-box sequence impaired the binding of WRKY40 to the ChiIV3 promoter. Subsequently, the transcription of ChiIV3 decreased in WRKY40-silenced pepper plants. These results demonstrated that the expression of the defense gene ChiIV3 is controlled through multiple modes of regulation, and WRKY40 directly binds to the W4-box element of the ChiIV3 promoter region for its transcriptional regulation.

CaLRR-RLK1, a novel RD receptor-like kinase from Capsicum annuum and transcriptionally activated by CaHDZ27, act as positive regulator in Ralstonia solanacearum resistance.

BACKGROUND: Bacterial wilt caused by Ralstonia solanacearum is one of the most important diseases in pepper worldwide, however, the molecular mechanism underlying pepper resistance to bacterial wilt remains poorly understood. RESULTS: Herein, a novel RD leucine-rich repeat receptor-like kinase, CaLRR-RLK1, was functionally characterized in immunity against R. solanacearum. CaLRR-RLK1 was targeted exclusively to plasma membrane and was up-regulated by R. solanacearum inoculation (RSI) as well as by the exogenous application of salicylic acid (SA), methyl jasmonate (MeJA) or ethephon (ETH). The silencing of CaLRR-RLK1 led to enhanced susceptibility of pepper plants to RSI, accompanied by down-regulation of immunity-related genes including CaACO1, CaHIR1, CaPR4 and CaPO2. In contrast, transient overexpression of CaLRR-RLK1 triggered hypersensitive response (HR)-like cell death and H2O2 accumulation in pepper leaves, manifested by darker trypan blue and DAB staining respectively. In addition, the ectopic overexpression of CaLRR-RLK1 in tobacco plants enhanced resistance R. solanacearum, accompanied with the immunity associated marker genes including NtPR2, NtPR2, NtHSR203 and NtHSR515. Furthermore, it was found that CaHDZ27, a positive regulator in pepper response to RSI in our previous study, transcriptionally activated CaLRR-RLK1 by direct targeting its promoter probably in a CAATTATTG dependent manner. CONCLUSION: The study revealed that CaLRR-RLK1 confers pepper resistance to R. solanacearum as the direct targeting of CaHDZ27.

Mutation in the putative ketoacyl-ACP reductase CaKR1 induces loss of pungency in Capsicum.

KEY MESSAGE: A putative ketoacyl-ACP reductase (CaKR1) that was not previously known to be associated with pungency of Capsicum was identified from map-based cloning and functional characterization. The pungency of chili pepper fruits is due to the presence of capsaicinoids, which are synthesized through the convergence of the phenylpropanoid and branched-chain fatty acid pathways. The extensive, global use of pungent and non-pungent peppers underlines the importance of understanding the genetic mechanism underlying capsaicinoid biosynthesis for breeding pepper cultivars. Although Capsicum is one of the earliest domesticated plant genera, the only reported genetic causes of its loss of pungency are mutations in acyltransferase (Pun1) and putative aminotransferase (pAMT). In this study, a single recessive gene responsible for the non-pungency of pepper No.3341 (C. chinense) was identified on chromosome 10 using an F2 population derived from a cross between Habanero and No.3341. Five candidate genes were identified in the target region, within a distance of 220 kb. A candidate gene, a putative ketoacyl-ACP reductase (CaKR1), of No.3341 had an insertion of a 4.5-kb transposable element (TE) sequence in the first intron, resulting in the production of a truncated transcript missing the region coding the catalytic domain. Virus-induced gene silencing of CaKR1 in pungent peppers resulted in the decreased accumulation of capsaicinoids, a phenotype consistent with No.3341. Moreover, GC-MS analysis of 8-methyl-6-nonenoic acid, which is predicted to be synthesized during the elongation cycle of branched-chain fatty acid biosynthesis, revealed that its deficiency in No.3341. Genetic, genomic, transcriptional, silencing, and biochemical precursor analyses performed in combination provide a solid ground for the conclusion that CaKR1 is involved in capsaicinoid biosynthesis and that its disruption results in a loss of pungency.

A Fungus-Inducible Pepper Carboxylesterase Exhibits Antifungal Activity by Decomposing the Outer Layer of Fungal Cell Walls.

Colletotrichum species are major fungal pathogens that cause devastating anthracnose diseases in many economically important crops. In this study, we observed the hydrolyzing activity of a fungus-inducible pepper carboxylesterase (PepEST) on cell walls of C. gloeosporioides, causing growth retardation of the fungus by blocking appressorium formation. To determine the cellular basis for the growth inhibition, we observed the localization of PepEST on the fungus and found the attachment of the protein on surfaces of conidia and germination tubes. Moreover, we examined the decomposition of cell-wall materials from the fungal surface after reaction with PepEST, which led to the identification of 1,2-dithiane-4,5-diol (DTD) by gas chromatography mass spectrometry analysis. Exogenous DTD treatment did not elicit expression of defense-related genes in the host plant but did trigger the necrosis of C. gloeosporioides. Furthermore, the DTD compound displayed protective effects on pepper fruits and plants against C. gloeosporioides and C. coccodes, respectively. In addition, DTD was also effective in preventing other diseases, such as rice blast, tomato late blight, and wheat leaf rust. Therefore, our results provide evidence that PepEST is involved in hydrolysis of the outmost layer of the fungal cell walls and that DTD has antifungal activity, suggesting an alternative strategy to control agronomically important phytopathogens.

Genome-wide identification, classification and expression of lipoxygenase gene family in pepper.

KEY MESSAGE: Lipoxygenases mediate important biological processes. Through comparative genomics, domain-scan analysis, sequence analysis, phylogenetic analysis, homology modelling and transcriptional analysis the lipoxygenase gene family of pepper (Capsicum annuum) has been identified. Lipoxygenases (LOXs) are non-heme, iron-containing dioxygenases playing a pivotal role in diverse biological processes in plants, including defence and development. Here, we exploited the recent sequencing of the pepper genome to investigate the LOX gene family in pepper. Two LOX classes are recognized, the 9- and 13-LOXs that oxygenate lipids at the 9th and 13th carbon atom, respectively. Using two main in-silico approaches, we identified a total of eight LOXs in pepper. Phylogenetic analysis classified four LOXs (CaLOX1, CaLOX3, CaLOX4 and CaLOX5) as 9-LOXs and four (CaLOX2, CaLOX6, CaLOX7 and CaLOX8) as 13-LOXs. Furthermore, sequence similarity/identity and subcellular localization analysis strengthen the classification predicted by phylogenetic analysis. Pivotal amino acids together with all domains and motifs are highly conserved in all pepper LOXs. Expression of 13-LOXs appeared to be more dynamic compared to 9-LOXs both in response to exogenous JA application and to thrips feeding. Bioinformatic and expression analyses predict the putative functions of two 13-LOXs, CaLOX6 and CaLOX7, in the biosynthesis of Green Leaf Volatiles, involved in indirect defence. The data are discussed in the context of LOX families in solanaceous plants and plants of other families.

The Pepper RING-Type E3 Ligase CaAIRF1 Regulates ABA and Drought Signaling via CaADIP1 Protein Phosphatase Degradation.

Ubiquitin-mediated protein modification occurs at multiple steps of abscisic acid (ABA) signaling. Here, we sought proteins responsible for degradation of the pepper (Capsicum annuum) type 2C protein phosphatase CaADIP1 via the 26S proteasome system. We showed that the RING-type E3 ligase CaAIRF1 (Capsicum annuum ADIP1 Interacting RING Finger Protein 1) interacts with and ubiquitinates CaADIP1. CaADIP1 degradation was slower in crude proteins from CaAIRF1-silenced peppers than in those from control plants. CaAIRF1-silenced pepper plants displayed reduced ABA sensitivity and decreased drought tolerance characterized by delayed stomatal closure and suppressed induction of ABA- and drought-responsive marker genes. In contrast, CaAIRF1-overexpressing Arabidopsis (Arabidopsis thaliana) plants exhibited ABA-hypersensitive and drought-tolerant phenotypes. Moreover, in these plants, CaADIP1-induced ABA hyposensitivity was strongly suppressed by CaAIRF1 overexpression. Our findings highlight a potential new route for fine-tune regulation of ABA signaling in pepper via CaAIRF1 and CaADIP1.

Genome-Wide Identification, Expression, and Functional Analysis of the Alkaline/Neutral Invertase Gene Family in Pepper.

Alkaline/neutral invertase (NINV) proteins irreversibly cleave sucrose into fructose and glucose, and play important roles in carbohydrate metabolism and plant development. To investigate the role of NINVs in the development of pepper fruits, seven NINV genes (CaNINV1-7) were identified. Phylogenetic analysis revealed that the CaNINV family could be divided into α and ß groups. CaNINV1-6 had typical conserved regions and similar protein structures to the NINVs of other plants, while CaNINV7 lacked amino acid sequences at the C-terminus and N-terminus ends. An expression analysis of the CaNINV genes in different tissues demonstrated that CaNINV5 is the dominant NINV in all the examined tissues (root, stem, leaf, bud, flower, and developmental pepper fruits stage). Notably, the expression of CaNINV5 was found to gradually increase at the pre-breaker stages, followed by a decrease at the breaker stages, while it maintained a low level at the post-breaker stages. Furthermore, the invertase activity of CaNINV5 was identified by functional complementation of the invertase-deficient yeast strain SEY2102, and the optimum pH of CaNINV5 was found to be ~7.5. The gene expression and enzymatic activity of CaNINV5 suggest that it might be the main NINV enzyme for hydrolysis of sucrose during pepper fruit development.

Physiological effects of the brown seaweed Ascophyllum nodosum) and humic substances on plant growth, enzyme activities of certain pepper plants grown under salt stress.

This study was aimed to examine the effects of seaweed extract (SW) and humic acid on the fruit yield, dry weight (DW%), protein, proline, lipid peroxidation (LPO) and antioxidative enzyme activity of pepper plants (Capsicum annuum L.) grown under saline conditions (100 mM). The obtained results indicated that salinity stress affected deleteriously plant growth and all other parameters. Besides, the treatment of seaweed (SW) and humic acid (HA) improved vegetative growth in the plant at all concentration levels applied under salinity conditions. Leaf fresh and dry weight was increased by all SW and HA applications in salinity stressed plant compared to those of control. Furthermore, there was a significant improvement in antioxidant enzyme activity, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) activities in the plant under salt stress and treated with SW and HA compounds. It suggests that seaweed and humic acid can enhance salt stress tolerance and leads to conservation of pepper plant against oxidative stress.

Engineering a bacterial platform for total biosynthesis of caffeic acid derived phenethyl esters and amides.

Caffeic acid has been widely recognized as a versatile pharmacophore for synthesis of new chemical entities, among which caffeic acid derived phenethyl esters and amides are the most extensively-investigated bioactive compounds with potential therapeutical applications. However, the natural biosynthetic routes for caffeic acid derived phenethyl esters or amides remain enigmatic, limiting their bio-based production. Herein, product-directed design of biosynthetic schemes allowed the development of thermodynamically favorable pathways for these compounds via acyltransferase (ATF) mediated trans-esterification. Production based screening identified a microbial O-ATF from Saccharomyces cerevisiae and a plant N-ATF from Capsicum annuum capable of forming caffeic acid derived esters and amides, respectively. Subsequent combinatorial incorporation of caffeic acid with various aromatic alcohol or amine biosynthetic pathways permitted the de novo bacterial production of a panel of caffeic acid derived phenethyl esters or amides in Escherichia coli for the first time. Particularly, host strain engineering via systematic knocking out endogenous caffeoyl-CoA degrading thioesterase and pathway optimization via titrating co-substrates enabled production enhancement of five caffeic acid derived phenethyl esters and amides, with titers ranging from 9.2 to 369.1mg/L. This platform expanded the capabilities of bacterial production of high-value natural aromatic esters and amides from renewable carbon source via tailoring non-natural biosynthetic pathways.