Comparative morpho-physiological and biochemical responses of Capsicum annuum L. plants to multi-walled carbon nanotubes, fullerene C60 and graphene nanoplatelets exposure under water deficit stress.

Water deficit stress is one of the most significant environmental abiotic factors influencing plant growth and metabolism globally. Recently, encouraging outcomes for the use of nanomaterials in agriculture have been shown to reduce the adverse effects of drought stress on plants. The present study aimed to investigate the impact of various carbon nanomaterials (CNMs) on the physiological, morphological, and biochemical characteristics of bell pepper plants subjected to water deficit stress conditions. The study was carried out as a factorial experiment using a completely randomized design (CRD) in three replications with a combination of three factors. The first factor considered was irrigation intensity with three levels [(50%, 75%, and 100% (control) of the field capacity (FC)] moisture. The second factor was the use of carbon nanomaterials [(fullerene C60, multi-walled carbon nanotubes (MWNTs) and graphene nanoplatelets (GNPs)] at various concentrations [(control (0), 100, 200, and 1000 mg/L)]. The study confirmed the foliar uptake of CNMs using the Scanning Electron Microscopy (SEM) technique. The effects of the CNMs were observed in a dose-dependent manner, with both stimulatory and toxicity effects being observed. The results revealed that exposure to MWNTs (1000 mg/L) under well-watered irrigation, and GNPs treatment (1000 mg/L) under severe drought stress (50% FC) significantly (P < 0.01) improved fruit production and fruit dry weight by 76.2 and 73.2% as compared to the control, respectively. Also, a significant decrease (65.9%) in leaf relative water content was obtained in plants subjected to soil moisture of 50% FC over the control. Treatment with GNPs at 1000 mg/L under 50% FC increased electrolyte leakage index (83.6%) compared to control. Foliar applied MWNTs enhanced the leaf gas exchange, photosynthesis rate, and chlorophyll a and b concentrations, though decreased the oxidative shock in leaves which was demonstrated by the diminished electrolyte leakage index and upgrade in relative water content and antioxidant capacity compared to the control. Plants exposed to fullerene C60 at 100 and 1000 mg/L under soil moisture of 100 and 75% FC significantly increased total flavonoids and phenols content by 63.1 and 90.9%, respectively, as compared to the control. A significant increase (184.3%) in antioxidant activity (FRAP) was observed in plants exposed to 200 mg/L MWCNTs under irrigation of 75% FC relative to the control. The outcomes proposed that CNMs could differentially improve the plant and fruit characteristics of bell pepper under dry conditions, however, the levels of changes varied among CNMs concentrations. Therefore, both stimulatory and toxicity effects of employed CNMs were observed in a dose-dependent manner. The study concludes that the use of appropriate (type/dose) CNMs through foliar application is a practical tool for controlling the water shortage stress in bell pepper. These findings will provide the basis for more research on CNMs-plant interactions, and with help to ensure their safe and sustainable use within the agricultural chains.

CaSnRK2.4-mediated phosphorylation of CaNAC035 regulates abscisic acid synthesis in pepper (Capsicum annuum L.) responding to cold stress.

Plant NAC transcription factors play a crucial role in enhancing cold stress tolerance, yet the precise molecular mechanisms underlying cold stress remain elusive. In this study, we identified and characterized CaNAC035, an NAC transcription factor isolated from pepper (Capsicum annuum) leaves. We observed that the expression of the CaNAC035 gene is induced by both cold and abscisic acid (ABA) treatments, and we elucidated its positive regulatory role in cold stress tolerance. Overexpression of CaNAC035 resulted in enhanced cold stress tolerance, while knockdown of CaNAC035 significantly reduced resistance to cold stress. Additionally, we discovered that CaSnRK2.4, a SnRK2 protein, plays an essential role in cold tolerance. In this study, we demonstrated that CaSnRK2.4 physically interacts with and phosphorylates CaNAC035 both in vitro and in vivo. Moreover, the expression of two ABA biosynthesis-related genes, CaAAO3 and CaNCED3, was significantly upregulated in the CaNAC035-overexpressing transgenic pepper lines. Yeast one-hybrid, Dual Luciferase, and electrophoretic mobility shift assays provided evidence that CaNAC035 binds to the promoter regions of both CaAAO3 and CaNCED3 in vivo and in vitro. Notably, treatment of transgenic pepper with 50 µm Fluridone (Flu) enhanced cold tolerance, while the exogenous application of ABA at a concentration of 10 µm noticeably reduced cold tolerance in the virus-induced gene silencing line. Overall, our findings highlight the involvement of CaNAC035 in the cold response of pepper and provide valuable insights into the molecular mechanisms underlying cold tolerance. These results offer promising prospects for molecular breeding strategies aimed at improving cold tolerance in pepper and other crops.

CaRH57, a RNA helicase, contributes pepper tolerance to heat stress.

RNA helicases (RHs) are required for most aspects of RNA metabolism and play an important role in plant stress tolerance. Heat stress (HS) causes the deleterious effects on plant cells, such as membrane disruption and protein misfolding, which results in the inhibition of plant growth and development. In this study, CaRH57 was identified from pepper (Capsicum annuum) and encodes a DEAD-box RH. CaRH57 was induced by HS, and overexpression of CaRH57 in Atrh57-1 rescued the glucose-sensitive phenotype of Atrh57-1, suggesting the functional replacement of CaRH57 to AtRH57. The nucleolus-localized CaRH57 possessed a RH activity in vitro. CaRH57 knockdown impaired pepper heat tolerance, showing severe necrosis and enhanced ROS accumulation in the region of the shoot tip. Additionally, accumulation of aberrant-spliced CaHSFA1d and CaHSFA9d was enhanced, and the corresponding mature mRNA levels were reduced in the TRV2 (Tobacco rattle virus)-CaRH57-infected plants compared with the control plants under HS. Overall, these results suggested that CaRH57 acted as a RH to confer pepper heat tolerance and was required for the proper pre-mRNA splicing of some HS-related genes.

PMT6 Is Required for SWC4 in Positively Modulating Pepper Thermotolerance.

High temperature stress (HTS), with growth and development impairment, is one of the most important abiotic stresses frequently encountered by plants, in particular solanacaes such as pepper, that mainly distribute in tropical and subtropical regions. Plants activate thermotolerance to cope with this stress; however, the underlying mechanism is currently not fully understood. SWC4, a shared component of SWR1- and NuA4 complexes implicated in chromatin remodeling, was previously found to be involved in the regulation of pepper thermotolerance, but the underlying mechanism remains poorly understood. Herein, PMT6, a putative methyltranferase was originally found to interact with SWC4 by co-immunoprecipitation (Co-IP)-combined LC/MS assay. This interaction was further confirmed by bimolecular fluorescent complimentary (BiFC) and Co-IP assay, and PMT6 was further found to confer SWC4 methylation. By virus-induced gene silencing, it was found that PMT6 silencing significantly reduced pepper basal thermotolerance and transcription of CaHSP24 and significantly reduced the enrichment of chromatin-activation-related H3K9ac, H4K5ac, and H3K4me3 in TSS of CaHSP24, which was previously found to be positively regulated by CaSWC4. By contrast, the overexpression of PMT6 significantly enhanced basal thermotolerance of pepper plants. All these data indicate that PMT6 acts as a positive regulator in pepper thermotolerance, likely by methylating SWC4.

A 163-bp insertion in the Capana10g000198 encoding a MYB transcription factor causes male sterility in pepper (Capsicum annuum L.).

Male sterility provides an efficient approach for commercial exploitation of heterosis. Despite more than 20 genic male sterile (GMS) mutants documented in pepper (Capsicum annuum L.), only two causal genes have been successfully identified. Here, a novel spontaneous recessive GMS mutant, designated msc-3, is identified and characterized at both phenotypic and histological levels. Pollen abortion of msc-3 mutant may be due to the delayed tapetum degradation, leading to the non-degeneration of tetrads callosic wall. Then, a modified MutMap method and molecular marker linkage analysis were employed to fine mapping the msc-3 locus, which was delimited to the ~139.91-kb region harboring 10 annotated genes. Gene expression and structure variation analyses indicate the Capana10g000198, encoding a R2R3-MYB transcription factor, is the best candidate gene for the msc-3 locus. Expression profiling analysis shows the Capana10g000198 is an anther-specific gene, and a 163-bp insertion in the Capana10g000198 is highly correlated with the male sterile (MS) phenotype. Additionally, downregulation of Capana10g000198 in male fertile plants through virus-induced gene silencing resulted in male sterility. Finally, possible regulatory relationships of the msc-3 gene with the other two reported pepper GMS genes, msc-1 and msc-2, have been studied, and comparative transcriptome analysis reveals the expression of 16 GMS homologs are significantly downregulated in the MS anthers. Overall, our results reveal that Capana10g000198 is the causal gene underlying the msc-3 locus, providing important theoretical clues and basis for further in-depth study on the regulatory mechanisms of pollen development in pepper.

Mitochondrial protein expression during sweet pepper (Capsicum annuum L.) fruit ripening: iTRAQ-based proteomic analysis and role of cytochrome c oxidase.

The physiological process of fruit ripening is associated with the late developmental stages of plants in which mitochondrial organelles play an important role in the final success of this whole process. Thus, an isobaric tag for relative and absolute quantification (iTRAQ)-based analysis was used to quantify the mitochondrial proteome in pepper fruits in this study. Analysis of both green and red pepper fruits identified a total of 2284 proteins, of which 692 were found to be significantly more abundant in unripe green fruits as compared to red fruits, while 497 showed lower levels as the ripening process proceeded. Of the total number of proteins identified, 2253 (98,6%) were found to share orthologs with Arabidopsis thaliana. Proteomic analysis identified 163 proteins which were categorized as cell components, the major part assigned to cellular, intracellular space and other subcellular locations such as cytosol, plastids and, to a lesser extent, to mitochondria. Of the 224 mitochondrial proteins detected in pepper fruits, 78 and 48 were more abundant in green and red fruits, respectively. The majority of these proteins which displayed differential abundance in both fruit types were involved in the mitochondrial electron transport chain (mETC) and the tricarboxylic acid (TCA) cycle. The abundance levels of the proteins from both pathways were higher in green fruits, except for cytochrome c (CYC2), whose abundance was significantly higher in red fruits. We also investigated cytochrome c oxidase (COX) activity during pepper fruit ripening, as well as in the presence of molecules such as nitric oxide (NO) and hydrogen peroxide (H2O2), which promote thiol-based oxidative post-translational modifications (oxiPTMs). Thus, with the aid of in vitro assays, cytochrome c oxidase (COX) activity was found to be potentially inhibited by the PTMs nitration, S-nitrosation and carbonylation. According to protein abundance data, the final segment of the mETC appears to be a crucial locus with regard to fruit ripening, but also because in this location the biosynthesis of ascorbate, an antioxidant which plays a major role in the metabolism of pepper fruits, occurs.

Genome-wide identification and expression analysis of Raf-like kinase gene family in pepper (Capsicum annuum L.).

As highly conserved signaling pathway modules, mitogen-activated protein kinase (MAPK) cascades play vital roles in a diverse range of stress and hormonal responses in plants. Among the established components of MAPK cascades, Raf-like MAPK kinase kinases (MAPKKKs) are associated with abscisic acid (ABA) signaling and osmotic stress responses. However, despite the availability of a pepper reference genome, few of the Raf-like kinases in pepper plants have been functionally characterized. In this study, we isolated 47 putative Raf-like kinase genes from the pepper genome based on in silico analysis, which were classified into two major categories, namely, groups B and C (further sub-grouped into B1-B4 and C1-C7, respectively) and named sequentially as CaRaf1 to CaRaf47. Subcellular localization prediction analysis revealed that most of the group B CaRaf-like kinases are probably nuclear-localized, whereas a majority of group C members targeted into the cytoplasm. Transcriptional regulation of the 47 CaRaf genes in response to treatment with ABA, drought, NaCl, and mannitol was quantitatively analyzed by reverse-transcription PCR analysis. This revealed a significant induction of subgroup B3, C2, C3, and C5 members, indicating that these genes may be functionally associated with the response to osmotic stress, mediated via both ABA-dependent and -independent pathways. The findings of this study can accordingly serve as a basis for the identification of CaRaf genes associated with the regulation of ABA signaling and osmotic stress response and thus contribute to enhancing our understanding of the biological functions of CaRaf kinases in the responses of plants to different abiotic stresses.

A comprehensive update on Capsicum proteomics: Advances and future prospects.

Capsicum belonging to the family Solanaceae is one of the most widely consumed crops in the world as a vegetable, spice and a raw salad and is distinctly valuable for its spicy pungent flavour. Proteomic investigation of crop plants is an essential step towards deciphering the functional basis of traits in an organism and to deepen our understanding on the regulation of various developmental patterns, biotic, and abiotic stress response and tolerance mechanisms. The differential proteome expression profiling of tissues during different developmental stages and under different conditions may indicate the specific proteome dynamics involved in the developmental programs and under stress conditions. Although substantial progress in proteomics of other Solanaceae plants has been made in the past two decades, a comprehensive review on Capsicum proteomics is still lacking. This review provides updated information on the advancement of Capsicum proteomic study in cytoplasmic male sterility, during fruit development and ripening, and under different biotic and abiotic stresses. Although limited information is available on the post translational protein modifications in Capsicum, a brief outline is given at the end detailing various post translational modifications. This proteomic update on Capsicum will be useful for future studies aimed at Capsicum improvement programs.

Response of anthocyanin biosynthesis to light by strand-specific transcriptome and miRNA analysis in Capsicum annuum.

BACKGROUND: Anthocyanins have distinct biological functions in plant coloring, plant defense against strong light, UV irradiation, and pathogen infection. Aromatic hydroxyl groups and ortho-dihydroxyl groups in anthocyanins are able to inhibit free-radical chain reactions and hydroxyl radicals. Thus, anthocyanins play an antioxidative role by removing various types of ROS. Pepper is one of the solanaceous vegetables with the largest cultivation area in China. The purple-fruited pepper is rich in anthocyanins, which not only increases the ornamental nature of the pepper fruit but also benefits the human body. In this experiment, light-induced regulatory pathways and related specific regulators of anthocyanin biosynthesis were examined through integrative transcriptomic and metabolomic analysis. RESULTS: Results revealed that delphinium 3-O-glucoside significantly accumulated in light exposed surface of pepper fruit after 48 h as compared to shaded surface. Furthermore, through strand-specific sequencing technology, 1341 differentially expressed genes, 172 differentially expressed lncRNAs, 8 differentially expressed circRNAs, and 28 differentially expressed miRNAs were identified significantly different among both surfaces. The flavonoid synthesis pathway was significantly enriched by KEGG analysis including SHT (XM_016684802.1), AT-like (XM_016704776.1), CCoAOMT (XM_016698340.1, XM_016698341.1), CHI (XM_016697794.1, XM_016697793.1), CHS2 (XM_016718139.1), CHS1B (XM_016710598.1), CYP98A2-like (XM_016688489.1), DFR (XM_016705224.1), F3'5'H (XM_016693437.1), F3H (XM_016705025.1), F3'M (XM_016707872.1), LDOX (XM_016712446.1), TCM (XM_016722116.1) and TCM-like (XM_016722117.1). Most of these significantly enriched flavonoid synthesis pathway genes may be also regulated by lncRNA. Some differentially expressed genes encoding transcription factors were also identified including MYB4-like (XM_016725242.1), MYB113-like (XM_016689220.1), MYB308-like (XM_016696983.1, XM_016702244.1), and EGL1 (XM_016711673.1). Three 'lncRNA-miRNA-mRNA' regulatory networks with sly-miR5303, stu-miR5303g, stu-miR7997a, and stu-miR7997c were constructed, including 28 differentially expressed mRNAs and 6 differentially expressed lncRNAs. CONCLUSION: Possible light regulated anthocyanin biosynthesis and transport genes were identified by transcriptome analysis, and confirmed by qRT-PCR. These results provide important data for further understanding of the anthocyanin metabolism in response to light in pepper.

Interactive Effects of Cd and Pb on the Photosynthesis Efficiency and Antioxidant Defense System of Capsicum annuum L.

In this study, the interactive effect of Cd and Pb on the growth of Capsicum annuum L. was studied through pot experiments, and the indicators of photosynthesis efficiency (PE) and antioxidant defense system (ADS) were measured at different plant ages. Single Pb stress on PE and ADS was stronger than single Cd stress at the first month. Both the PE and ADS response showed a significant decrease under the combined stress of Cd and Pb, which was primarily dependent on the Pb concentration. With increasing plant age, the PE and response of non-enzymatic ADS exhibited dramatic decreases under Cd and/or Pb stress, and the activities of enzymatic ADS showed increases to some extent. The factorial analysis showed that Cd and Pb had an interactive effect to reduce PE, while slightly enhanced the activities of enzymatic ADS. Those results are useful to explore the interaction between Cd and Pb in the combined stress and understand their accumulation in the plants.

Mapping of CaPP2C35 involved in the formation of light-green immature pepper (Capsicum annuum L.) fruits via GWAS and BSA.

KEY MESSAGE: Genome-wide association study, bulked segregant analysis, and genetic analysis delimited the LG locus controlling light-green immature pepper fruits into a 35.07 kbp region on chromosome 10. A strong candidate gene, CaPP2C35, was identified in this region. In pepper (Capsicum annuum L.), the common colors of immature fruits are yellowish white, milky yellow, green, purple, and purplish black. Genes related to dark green, white, and purple immature fruits have been cloned; however, only a few studies have investigated light-green immature fruits. Here, we performed a genetic study using light-green (17C827) and green (17C658) immature fruits. The light-green color of immature fruits was controlled by a single locus-dominant genetic trait compared with the green color of immature fruits. We also performed a genome-wide association study and bulked segregant analysis of immature-fruit color and mapped the LG locus to a 35.07 kbp region on chromosome 10. Only one gene, Capana10g001710, was found in this region. A G-A substitution occurred at the 313th base of the Capana10g001710 coding sequence in 17C827, resulting in the conversion of the α-helix of its encoded PP2C35 protein into a ß-fold. The expression of Capana10g001710 (termed CaPP2C35) in 17C827 was significantly higher than in 17C658. Silencing CaPP2C35 in 17C827 resulted in an increase in chlorophyll content in the exocarp and the appearance of green stripes on the surface of the fruit. These results indicate that CaPP2C35 may be involved in the formation of light-green immature fruits by regulating the accumulation of chlorophyll content in the exocarp. Thus, these findings lay the foundation for further studies and genetic improvement of immature-fruit color in pepper.

Bacterial type III effector-induced plant C8 volatiles elicit antibacterial immunity in heterospecific neighbouring plants via airborne signalling.

Upon sensing attack by pathogens and insect herbivores, plants release complex mixtures of volatile compounds. Here, we show that the infection of lima bean (Phaseolus lunatus L.) plants with the non-host bacterial pathogen Pseudomonas syringae pv. tomato led to the production of microbe-induced plant volatiles (MIPVs). Surprisingly, the bacterial type III secretion system, which injects effector proteins directly into the plant cytosol to subvert host functions, was found to prime both intra- and inter-specific defense responses in neighbouring wild tobacco (Nicotiana benthamiana) plants. Screening of each of 16 effectors using the Pseudomonas fluorescens effector-to-host analyser revealed that an effector, HopP1, was responsible for immune activation in receiver tobacco plants. Further study demonstrated that 1-octen-3-ol, 3-octanone and 3-octanol are novel MIPVs emitted by the lima bean plant in a HopP1-dependent manner. Exposure to synthetic 1-octen-3-ol activated immunity in tobacco plants against a virulent pathogen Pseudomonas syringae pv. tabaci. Our results show for the first time that a bacterial type III effector can trigger the emission of C8 plant volatiles that mediate defense priming via plant-plant interactions. These results provide novel insights into the role of airborne chemicals in bacterial pathogen-induced inter-specific plant-plant interactions.

Gene regulatory networks for compatible versus incompatible grafts identify a role for SlWOX4 during junction formation.

Grafting has been adopted for a wide range of crops to enhance productivity and resilience; for example, grafting of Solanaceous crops couples disease-resistant rootstocks with scions that produce high-quality fruit. However, incompatibility severely limits the application of grafting and graft incompatibility remains poorly understood. In grafts, immediate incompatibility results in rapid death, but delayed incompatibility can take months or even years to manifest, creating a significant economic burden for perennial crop production. To gain insight into the genetic mechanisms underlying this phenomenon, we developed a model system using heterografting of tomato (Solanum lycopersicum) and pepper (Capsicum annuum). These grafted plants express signs of anatomical junction failure within the first week of grafting. By generating a detailed timeline for junction formation, we were able to pinpoint the cellular basis for this delayed incompatibility. Furthermore, we inferred gene regulatory networks for compatible self-grafts and incompatible heterografts based on these key anatomical events, which predict core regulators for grafting. Finally, we examined the role of vascular development in graft formation and uncovered SlWOX4 as a potential regulator of graft compatibility. Following this predicted regulator up with functional analysis, we show that Slwox4 homografts fail to form xylem bridges across the junction, demonstrating that indeed, SlWOX4 is essential for vascular reconnection during grafting, and may function as an early indicator of graft failure.

ACC deaminase and indole acetic acid producing endophytic bacterial co-inoculation improves physiological traits of red pepper (Capsicum annum L.) under salt stress.

Salinity induces myriad of physiological and biochemical perturbations in plants and its amelioration can be attained by the use of potential bacterial synthetic communities. The use of microbial consortia in contrast to single bacterial inoculation can additively enhance stress tolerance and productivity of agricultural crops. In this study, co-inoculation of Pseudomonas koreensis S2CB45 and Microbacterium hydrothermale IC37-36 isolated from arbuscular mycorrhizal fungi (AMF) spore and rice seed endosphere, respectively, were used to evaluate the physiological and biochemical effects on red pepper at two salt concentrations (75 mM and 150 mM). Plant growth promoting characteristics particularly 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, indole acetic acid (IAA) and cytokinin production were higher during co-culturing compared to the individual bacterial culture. The higher ACC deaminase activity had resulted in 20% and 22% decrease in stress ethylene emission compared to the non-inoculated plants at 75 mM and 150 mM salt stress, respectively. The decline in ethylene emission had eventually reduced ROS accumulation, and the co-inoculated plants had also harbored enhanced antioxidant enzyme activities and higher sugar accumulation compared to the other treatments suggesting enhanced tolerance to salinity. Collectively, these results put forward a novel consortium of bacterial strains that can be used for sustainable agricultural practices against salinity.

A cysteine-rich receptor-like protein kinase CaCKR5 modulates immune response against Ralstonia solanacearum infection in pepper.

BACKGROUND: Cysteine-rich receptor-like kinases (CRKs) represent a large subfamily of receptor-like kinases and play vital roles in diverse physiological processes in regulating plant growth and development. RESULTS: CaCRK5 transcripts were induced in pepper upon the infection of Ralstonia solanacearum and treatment with salicylic acid. The fusions between CaCRK5 and green fluorescence protein were targeted to the plasma membrane. Suppression of CaCRK5 via virus-induced gene silencing (VIGS) made pepper plants significantly susceptible to R. solanacearum infection, which was accompanied with decreased expression of defense related genes CaPR1, CaSAR8.2, CaDEF1 and CaACO1. Overexpression of CaCRK5 increased resistance against R. solanacearum in Nicotiana benthamiana. Furthermore, electrophoretic mobility shift assay and chromatin immunoprecipitation coupled with quantitative real-time PCR analysis revealed that a homeodomain zipper I protein CaHDZ27 can active the expression of CaCRK5 through directly binding to its promoter. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses suggested that CaCRK5 heterodimerized with the homologous member CaCRK6 on the plasma membrane. CONCLUSIONS: Our data revealed that CaCRK5 played a positive role in regulating immune responses against R. solanacearum infection in pepper.

Shed-Microspore Culture in Ornamental Peppers for Doubled Haploid Plant Production.

The shed-microspore culture technique is an alternative sub-method combining anther and isolated microspore culture to induce microspore embryogenesis. Recently, its effective use in different types of peppers has drawn attention, because it has a higher embryo yield potential compared to anther culture and is more practical than isolated microspore culture. In this chapter, a stepwise protocol for shed-microspore culture of ornamental pepper is described. This protocol includes the steps of donor plant growth conditions, the choice of suitable flower buds based on DAPI staining of microspores, application of a cold pretreatment to flower buds, surface sterilization of the buds, shed-microspore culture of anthers, stress treatments, regeneration of androgenic in vitro plantlets, their acclimatization and ploidy analysis, and in vivo chromosome doubling of the haploid plants.

Fine mapping and candidate gene analysis of the up locus determining fruit orientation in pepper (Capsicum spp.).

KEY MESSAGE: The up locus determining fruit orientation was fine-mapped into a region with a physical length of ~169.51 kb on chromosome P12 in pepper. Capana12g000958, encoding a developmentally regulated G protein 2, was proposed as the strongest candidate via sequence comparison and expression analysis. Fruit orientation is an important horticultural and domesticated trait, which is controlled by a single semi-dominant gene (up) in pepper. However, the gene underlying up locus has not yet been identified. In this study, the previously detected major QTL UP12.1 was firstly verified using a backcross population (n = 225) stem from the cross of BB3 (C. annuum) and its wild relative Chiltepin (C. annuum var. glabriusculum) using BB3 as the recurrent parent. Then, a large BC1F2 population (n = 1827) was used for recombinant screening to delimit the up locus into an interval with ~ 169.51 kb in length. Sequence comparison and expression analysis suggested that Capana12g000958, encoding a developmentally regulated G protein 2, was the most likely candidate gene for the up locus. There is no difference within the coding sequences of Capana12g000958 between BB3 and Chiltepin, while a SNP in the upstream of Capana12g000958 showed a complete correlation with the fruit orientation among a panel of 40 diverse pepper inbred lines. These findings will form a basis for gene isolation and reveal of genetic mechanism underlying the fruit orientation domestication in pepper.

Pepper ubiquitin-specific protease, CaUBP12, positively modulates dehydration resistance by enhancing CaSnRK2.6 stability.

Abscisic acid (ABA) is a plant hormone that activates adaptive mechanisms to environmental stress conditions. Plant adaptive mechanisms are complex and highly modulated processes induced by stress-responsive proteins; however, the precise mechanisms by which these processes function under adverse conditions remain unclear. Here, we isolated CaUBP12 (Capsicum annuum ubiquitin-specific protease 12) from pepper (C. annuum) leaves. We show that CaUBP12 expression is significantly induced after exposure to abiotic stress treatments. We conducted loss-of-function and gain-of-function genetic studies to elucidate the biological functions of CaUBP12 in response to ABA and dehydration stress. CaUBP12-silenced pepper plants and CaUBP12-overexpressing Arabidopsis plants displayed dehydration-sensitive and dehydration-tolerant phenotypes, respectively; these phenotypes were characterized by regulation of transpirational water loss and stomatal aperture. Under dehydration stress conditions, CaUBP12-silenced pepper plants and CaUBP12-overexpressing Arabidopsis plants exhibited lower and higher expression levels of stress-related genes, respectively, than the control plants. We isolated a CaUBP12 interaction protein, CaSnRK2.6, which is a homolog of Arabidopsis OST1; degradation of this protein was partially inhibited by CaUBP12. Similar to CaUBP12-silenced pepper plants and CaUBP12-overexpressing Arabidopsis plants, CaSnRK2.6-silenced pepper plants and CaSnRK2.6-overexpressing Arabidopsis displayed dehydration-sensitive and dehydration-tolerant phenotypes, respectively. Our findings suggest that CaUBP12 positively modulates the dehydration stress response by suppressing CaSnRK2.6 protein degradation.

Pepper NAC-type transcription factor NAC2c balances the trade-off between growth and defense responses.

Plant responses to pathogen attacks and high-temperature stress (HTS) are distinct in nature but generally share several signaling components. How plants produce specific responses through these common signaling intermediates remains elusive. With the help of reverse-genetics approaches, we describe here the mechanism underlying trade-offs in pepper (Capsicum annuum) between growth, immunity, and thermotolerance. The NAC-type transcription factor CaNAC2c was induced by HTS and Ralstonia solanacearum infection (RSI). CaNAC2c-inhibited pepper growth, promoted immunity against RSI by activating jasmonate-mediated immunity and H2O2 accumulation, and promoted HTS responses by activating Heat shock factor A5 (CaHSFA5) transcription and blocking H2O2 accumulation. We show that CaNAC2c physically interacts with CaHSP70 and CaNAC029 in a context-specific manner. Upon HTS, CaNAC2c-CaHSP70 interaction in the nucleus protected CaNAC2c from degradation and resulted in the activation of thermotolerance by increasing CaNAC2c binding and transcriptional activation of its target promoters. CaNAC2c did not induce immunity-related genes under HTS, likely due to the degradation of CaNAC029 by the 26S proteasome. Upon RSI, CaNAC2c interacted with CaNAC029 in the nucleus and activated jasmonate-mediated immunity but was prevented from activating thermotolerance-related genes. In non-stressed plants, CaNAC2c was tethered outside the nucleus by interaction with CaHSP70, and thus was unable to activate either immunity or thermotolerance. Our results indicate that pepper growth, immunity, and thermotolerance are coordinately and tightly regulated by CaNAC2c via its inducible expression and differential interaction with CaHSP70 and CaNAC029.

Uncovering salt tolerance mechanisms in pepper plants: a physiological and transcriptomic approach.

BACKGROUND: Pepper is one of the most cultivated crops worldwide, but is sensitive to salinity. This sensitivity is dependent on varieties and our knowledge about how they can face such stress is limited, mainly according to a molecular point of view. This is the main reason why we decided to develop this transcriptomic analysis. Tolerant and sensitive accessions, respectively called A25 and A6, were grown for 14 days under control conditions and irrigated with 70 mM of NaCl. Biomass, different physiological parameters and differentially expressed genes were analysed to give response to differential salinity mechanisms between both accessions. RESULTS: The genetic changes found between the accessions under both control and stress conditions could explain the physiological behaviour in A25 by the decrease of osmotic potential that could be due mainly to an increase in potassium and proline accumulation, improved growth (e.g. expansins), more efficient starch accumulation (e.g. BAM1), ion homeostasis (e.g. CBL9, HAI3, BASS1), photosynthetic protection (e.g. FIB1A, TIL, JAR1) and antioxidant activity (e.g. PSDS3, SnRK2.10). In addition, misregulation of ABA signalling (e.g. HAB1, ERD4, HAI3) and other stress signalling genes (e.g. JAR1) would appear crucial to explain the different sensitivity to NaCl in both accessions. CONCLUSIONS: After analysing the physiological behaviour and transcriptomic results, we have concluded that A25 accession utilizes different strategies to cope better salt stress, being ABA-signalling a pivotal point of regulation. However, other strategies, such as the decrease in osmotic potential to preserve water status in leaves seem to be important to explain the defence response to salinity in pepper A25 plants.