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* Background and Aims Pepper (Capsicum annuum) contains high levels of antioxidants, such as vitamins A and C and flavonoids. However, information on the role of these beneficial compounds in the physiology of pepper fruit remains scarce. Recent studies have shown that antioxidants in ripe pepper fruit play a key role in responses to temperature changes, and the redox state at the time of harvest affects the nutritional value for human consumption. In this paper, the role of antioxidant metabolism of pepper fruit during ripening and in the response to low temperature is addressed, paying particular attention to ascorbate, NADPH and the superoxide dismutase enzymatic system. The participation of chloroplasts, mitochondria and peroxisomes in the ripening process is also investigated. * Scope and Results Important changes occur at a subcellular level during ripening of pepper fruit. Chloroplasts turn into chromoplasts, with drastic conversion of their metabolism, and the role of the ascorbate-glutathione cycle is essential. In mitochondria from red fruits, higher ascorbate peroxidase (APX) and Mn-SOD activities are involved in avoiding the accumulation of reactive oxygen species in these organelles during ripening. Peroxisomes, whose antioxidant capacity at fruit ripening is substantially affected, display an atypical metabolic pattern during this physiological stage. In spite of these differences observed in the antioxidative metabolism of mitochondria and peroxisomes, proteomic analysis of these organelles, carried out by 2-D electrophoresis and MALDI-TOF/TOF and provided here for the first time, reveals no changes between the antioxidant metabolism from immature (green) and ripe (red) fruits. * Conclusions Taken together, the results show that investigation of molecular and enzymatic antioxidants from cell compartments, especially chloroplasts, mitochondria and peroxisomes, is a useful tool to study the physiology of pepper fruit, particularly in the context of expanding their shelf-life after harvest and in maintaining their nutritional value. [ABSTRACT FROM AUTHOR]

There is growing evidence to suggest that epigenetic tags, especially DNA methylation, are critical regulators of fruit ripening. To examine whether this is the case in sweet pepper (Capsicum annuum) we conducted experiments at the transcriptional, epigenetic, and physiological levels. McrBC PCR, bisulfite sequencing, and real-time PCR demonstrated that DNA hypomethylation occurred in the upstream region of the transcription start site of some genes related to pepper ripening at the turning stage, which may be attributed to up-regulation of CaDML2-like and down-regulation of CaMET1-like1 , CaMET1-like2 , CaCMT2-like , and CaCMT4-like. Silencing of CaMET1-like1 by virus-induced gene silencing led to DNA hypomethylation, increased content of soluble solids, and accumulation of carotenoids in the fruit, which was accompanied by changes in expression of genes involved in capsanthin/capsorubin biosynthesis, cell wall degradation, and phytohormone metabolism and signaling. Endogenous ABA increased during fruit ripening, whereas endogenous IAA showed an opposite trend. No ethylene signal was detected during ripening. DNA hypomethylation repressed the expression of auxin and gibberellin biosynthesis genes as well as cytokinin degradation genes, but induced the expression of ABA biosynthesis genes. In mature-green pericarp, exogenous ABA induced expression of CaDML2-like but repressed that of CaCMT4-like. IAA treatment promoted the transcription of CaMET1-like1 and CaCMT3-like. Ethephon significantly up-regulated the expression of CaDML2-like. Treatment with GA3 and 6-BA showed indistinct effects on DNA methylation at the transcriptional level. On the basis of the results, a model is proposed that suggests a high likelihood of a role for DNA methylation in the regulation of ripening in the non-climacteric pepper fruit. [ABSTRACT FROM AUTHOR]

Interspecies grafting is an economically relevant technique that allows beneficial shoot and root combinations from separate species to be combined. One hypothesis for the basis of graft compatibility revolves around taxonomic relatedness. To test how phylogenetic distance affects interspecific graft compatibility within the economically important Solanaceae subfamily, Solanoideae, we characterized the anatomical and biophysical integrity of graft junctions between four species: tomato (Solanum lycopersicum), eggplant (Solanum melongena), pepper (Capsicum annuum), and groundcherry (Physalis pubescens). We analyzed the survival, growth, integrity, and cellular composition of the graft junctions. Utilizing various techniques, we were able to quantitatively assess compatibility among the interspecific grafts. Even though most of our graft combinations could survive, we show that only intrageneric combinations between tomato and eggplant are compatible. Unlike incompatible grafts, the formation of substantial vascular reconnections between tomato and eggplant in the intrageneric heterografts likely contributed to biophysically stable grafts. Furthermore, we identified 10 graft combinations that show delayed incompatibility, providing a useful system to pursue deeper work into graft compatibility. This work provides new evidence that graft compatibility may be limited to intrageneric combinations within the Solanoideae subfamily. Further research amongst additional Solanaceous species can be used to test the extent to which our hypothesis applies to this family. [ABSTRACT FROM AUTHOR]

Background Stomatal pores in many species are separated from the atmosphere by different anatomical obstacles produced by leaf epidermal cells, especially by sunken stomatal crypts, stomatal antechambers and/or hairs (trichomes). The evolutionary driving forces leading to sunken or 'hidden' stomata whose antechambers are filled with hairs or waxy plugs are not fully understood. The available hypothetical explanations are based mainly on mathematical modelling of water and CO2 diffusion through superficial vs. sunken stomata, and studies of comparative autecology. A better understanding of this phenomenon may result from examining the interactions between the leaf cuticle and stomata and from functional comparisons of sunken vs. superficially positioned stomata, especially when transpiration is low, for example at night or during severe drought. Scope I review recent ideas as to why stomata are hidden and test experimentally whether hidden stomata may behave differently from those not covered by epidermal structures and so are coupled more closely to the atmosphere. I also quantify the contribution of stomatal vs. cuticular transpiration at night using four species with sunken stomata and three species with superficial stomata. Conclusions Partitioning of leaf conductance in darkness (g tw) into stomatal and cuticular contributions revealed that stomatal conductance dominated g tw across all seven investigated species with antechambers with different degrees of prominence. Hidden stomata contributed, on average, less to g tw (approx. 70 %) than superficial stomata (approx. 80 %) and reduced their contribution dramatically with increasing g tw. In contrast, species with superficial stomata kept their proportion in g tw invariant across a broad range of g tw. Mechanisms behind the specific behaviour of hidden stomata and the multipurpose origin of sunken stomata are discussed. [ABSTRACT FROM AUTHOR]

The pepper receptor-like cytoplasmic protein kinase, CaPIK1, which mediates signalling of plant cell death and defence responses was previously identified. Here, the identification of a class IV chitinase, CaChitIV, from pepper plants (Capsicum annuum), which interacts with CaPIK1 and promotes CaPIK1-triggered cell death and defence responses, is reported. CaChitIV contains a signal peptide, chitin-binding domain, and glycol hydrolase domain. CaChitIV expression was up-regulated by Xanthomonas campestris pv. vesicatoria (Xcv) infection. Notably, avirulent Xcv infection rapidly induced CaChitIV expression in pepper leaves. Bimolecular fluorescence complementation and co-immunoprecipitation revealed that CaPIK1 interacts with CaChitIV in planta, and that the CaPIK1-CaChitIV complex is localized mainly in the cytoplasm and plasma membrane. CaChitIV is also localized in the endoplasmic reticulum. Transient co-expression of CaChitIV with CaPIK1 enhanced CaPIK1-triggered cell death response and reactive oxygen species (ROS) and nitric oxide (NO) bursts. Co-silencing of both CaChitIV and CaPIK1 in pepper plants conferred enhanced susceptibility to Xcv infection, which was accompanied by a reduced induction of cell death response, ROS and NO bursts, and defence response genes. Ectopic expression of CaPIK1 in Arabidopsis enhanced basal resistance to Hyaloperonospora arabidopsidis infection. Together, the results suggest that CaChitIV positively regulates CaPIK1-triggered cell death and defence responses through its interaction with CaPIK1. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

APETALA2 (AP2) subfamily transcription factors participate in plant growth and development, but their roles in plant immunity remain unclear. Here, we discovered that the AP2 transcription factor CaAIL1 functions in immunity against Ralstonia solanacearum infection (RSI) in pepper (Capsicum annuum). CaAIL1 expression was upregulated by RSI, and loss- and gain-of-function assays using virus-induced gene silencing and transient overexpression, respectively, revealed that CaAIL1 plays a positive role in immunity to RSI in pepper. Chromatin immunoprecipitation sequencing (ChIP-seq) uncovered a subset of transcription-factor-encoding genes, including CaRAP2-7, CaGATA17, CaGtf3a and CaTCF25 , that were directly targeted by CaAIL1 via their cis -elements, such as GT or AGGCA motifs. ChIP-qPCR and electrophoretic mobility shift assays confirmed these findings. These genes, encoding transcription factors with negative roles in immunity, were repressed by CaAIL1 during pepper response to RSI, whereas genes encoding positive immune regulators such as CaEAS were derepressed by CaAIL1. Importantly, we showed that the atypical EAR motif (LXXLXXLXX) in CaAIL1 is indispensable for its function in immunity. These findings indicate that CaAIL1 enhances the immunity of pepper against RSI by repressing a subset of negative immune regulators during the RSI response through its binding to several cis -elements in their promoters. [ABSTRACT FROM AUTHOR]

Optical films that alter light transmittance may reduce energy consumption in high-tech greenhouses, but their impact on crop physiology remains unclear. We compared the stomatal responses of Capsicum plants grown hydroponically under control glass (70% diffuse light) or the smart glass (SG) film ULR-80, which blocked >50% of short-wave radiation and ~9% of photosynthetically active radiation (PAR). SG had no significant effects on steady-state (g s) or maximal (g max) stomatal conductance. In contrast, SG reduced stomatal pore size and sensitivity to exogenous abscisic acid (ABA), thereby increasing rates of leaf water loss, guard cell K+ and Cl– efflux, and Ca2+ influx. SG induced faster stomatal closing and opening rates on transition between low (100 µmol m–2 s–1) and high PAR (1500 µmol m–2 s–1), which compromised water use efficiency relative to control plants. The fraction of blue light (0% or 10%) did not affect g s in either treatment. Increased expression of stomatal closure and photoreceptor genes in epidermal peels of SG plants is consistent with fast stomatal responses to light changes. In conclusion, stomatal responses of Capsicum to SG were more affected by changes in light intensity than spectral quality, and re-engineering of the SG should maximize PAR transmission, and hence CO2 assimilation. [ABSTRACT FROM AUTHOR]

Resistance (R) gene-mediated resistance is a robust and efficient antiviral immune system in the plants. Thus, when R -mediated resistance was suppressed at elevated temperatures, resistance towards viruses was expected to be completely collapsed. Nonetheless, the multiplication of Tobacco mosaic virus pathotype P0 (TMV-P0) was inhibited, and TMV-P0 particles were only occasionally present in the systemic leaves of pepper plants (Capsicum annuum). RNAi-mediated RNA silencing is a well-known antiviral immune mechanism. At elevated temperatures, RNAi-mediated antiviral resistance was induced and virus-derived siRNAs (vsiRNAs) were dramatically increased. Through sRNA-sequencing (sRNA-Seq) analysis, we revealed that vsiRNAs derived from TMV-P0 were greatly increased. Intriguingly, virus-infected plants could select the temperature-specific vsiRNAs for antiviral resistance from the amplified vsiRNAs at elevated temperatures. Pre-application of these temperature-specific vsiRNAs endowed antiviral resistance of the plants. Therefore, plants sustain antiviral resistance by activating RNAi-mediated resistance, based on temperature-specific vsiRNAs at elevated temperatures. [ABSTRACT FROM AUTHOR]

CabZIP63 acts positively in the resistance of pepper (Capsicum annuum) to bacterial wilt caused by Ralstonia solanacearum or tolerance to high-temperature/high-humidity stress, but it is unclear how CabZIP63 achieves its functional specificity against R. solanacearum. Here, CaASR1, an abscisic acid-, stress-, and ripening-inducible protein of C. annuum , was functionally characterized in modulating the functional specificity of CabZIP63 during the defense response of pepper to R. solanacearum. In pepper plants inoculated with R. solanacearum , CaASR1 was up-regulated before 24 h post-inoculation but down-regulated thereafter, and was down-regulated by high-temperature/high-humidity stress. Data from gene silencing and transient overexpression experiments indicated that CaASR1 acts as a positive regulator in the immunity of pepper against R. solanacearum and a negative regulator of thermotolerance. Pull-down combined with mass spectrometry revealed that CaASR1 interacted with CabZIP63 upon R. solanacearum infection; the interaction was confirmed by microscale thermophoresis and bimolecular fluorescence complementation assays. CaASR1 silencing upon R. solanacearum inoculation repressed CabZIP63-mediated transcription from the promoters of the salicylic acid (SA)-dependent CaPR1 and CaNPR1 , but derepressed transcription of CaHSP24 and the jasmonic acid (JA)-dependent CaDEF1. Our findings suggest that CaASR1 acts as a positive regulator of the defense response of pepper to R. solanacearum by interacting with CabZIP63, enabling it to promote SA-dependent but repress JA-dependent immunity and thermotolerance during the early stages of infection. [ABSTRACT FROM AUTHOR]

Plant mildew-resistance locus O (MLO) proteins influence susceptibility to powdery mildew. However, their roles in plant responses to other pathogens and heat stress remain unclear. Here, we showed that CaMLO6, a pepper (Capsicum annuum) member of MLO clade V, is a protein targeted to plasma membrane and probably endoplasmic reticulum. The transcript expression level of CaMLO6 was upregulated in the roots and leaves of pepper plants challenged with high temperature and high humidity (HTHH) and was upregulated in leaves but downregulated in roots of plants infected with the bacterial pathogen Ralstonia solanacearum. CaMLO6 was also directly upregulated by CaWRKY40 upon HTHH but downregulated by CaWRKY40 upon R. solanacearum infection. Virus-induced gene silencing of CaMLO6 significantly decreased pepper HTHH tolerance and R. solanacearum susceptibility. Moreover, CaMLO6 overexpression enhanced the susceptibility of Nicotiana benthamiana and pepper plants to R. solanacearum and their tolerance to HTHH, effects that were associated with the expression of immunity- and thermotolerance-associated marker genes, respectively. These results suggest that CaMLO6 acts as a positive regulator in response to HTHH but a negative regulator in response to R. solanacearum. Moreover, CaMLO6 is transcriptionally affected by R. solanacearum and HTHH; these transcriptional responses are at least partially regulated by CaWRKY40. [ABSTRACT FROM AUTHOR]

The exploitation of diverse natural variation has been a key progenitor of crop breeding over the last decade. However, commercial practice is now turning to the use of accessions with less extreme phenotypes as genetic donors. In the present study, the carotenoid formation in a red-fruited discovery panel of Capsicum annuum (chilli pepper) has been characterized. The data indicated that colour intensity correlated with the amount of capsanthin and its esters, along with transcript levels of the 1-deoxy- d -xylulose 5-phosphate synthase (DXS) and phytoene synthase-1 (PSY -1) genes. Quantification of carotenoids through development and ripening suggested the presence of separate biosynthesis and accumulation phases. Subplastid fractionation demonstrated the differential sequestration of pigments in high- and low-intensity lines and revealed the PSY protein to be most active in the membrane fractions when abundance was highest in the fibril fractions. Carotenoid accumulation was associated with the esterification of xanthophylls, expression of a putative carotenoid acyl transferase, and increased fibril content within the plastid. Interrogation of TEM images and carotenoid analysis of subplastid fractions suggest that the plastoglobuli are likely to be the progenitor of the characteristic fibrils found in pepper fruit. Collectively, these data provide an insight into the underpinning molecular, biochemical, and cellular mechanisms associated with the synthesis and sequestration of carotenoids in chromoplast-containing fruits, in addition to providing potential tools and resources for the breeding of high red colour intensity pepper varieties. [ABSTRACT FROM AUTHOR]

WRKY transcription factors have been implicated in both plant immunity and plant responses to cadmium (Cd); however, the mechanism underlying the crosstalk between these processes is unclear. Here, we characterized the roles of CaWRKY41, a group III WRKY transcription factor, in immunity against the pathogenic bacterium Ralstonia solanacearum and Cd stress responses in pepper (Capsicum annuum). CaWRKY41 was transcriptionally up-regulated in response to Cd exposure, R. solanacearum inoculation, and H2O2 treatment. Virus-induced silencing of CaWRKY41 increased Cd tolerance and R. solanacearum susceptibility, while heterologous overexpression of CaWRKY41 in Arabidopsis impaired Cd tolerance, and enhanced Cd and zinc (Zn) uptake and H2O2 accumulation. Genes encoding reactive oxygen species-scavenging enzymes were down-regulated in CaWRKY41 -overexpressing Arabidopsis plants, whereas genes encoding Zn transporters and enzymes involved in H2O2 production were up-regulated. Consistent with these findings, the ocp3 (overexpressor of cationic peroxidase 3) mutant, which has elevated H2O2 levels, displayed enhanced sensitivity to Cd stress. These results suggest that a positive feedback loop between H2O2 accumulation and CaWRKY41 up-regulation coordinates the responses of pepper to R. solanacearum inoculation and Cd exposure. This mechanism might reduce Cd tolerance by increasing Cd uptake via Zn transporters, while enhancing resistance to R. solanacearum. [ABSTRACT FROM AUTHOR]

The responses of pepper (Capsicum annuum) plants to inoculation with the pathogenic bacterium Ralstonia solanacearum and to high-temperature-high-humidity (HTHH) conditions were previously found to be coordinated by the transcription factors CaWRKY6 and CaWRKY40 ; however, the underlying molecular mechanism was unclear. Herein, we identified and functionally characterized CaHsfB2a, a nuclear-localized heat shock factor involved in pepper immunity to R. solanacearum inoculation (RSI) and tolerance to HTHH. CaHsfB2a is transcriptionally induced in pepper plants by RSI or HTHH and by exogenous application of salicylic acid (SA), methyl jasmonate (MeJA), ethylene (ETH), or abscisic acid (ABA). Virus-induced gene silencing (VIGS) of CaHsfB2a significantly impaired pepper immunity to RSI, hampered HTHH tolerance, and curtailed expression of immunity- and thermotolerance-associated marker genes such as CaHIR1, CaNPR1, CaABR1, and CaHSP24. Likewise, transient overexpression of CaHsfB2a in pepper leaves induced hypersensitive response (HR)-like cell death and H2O2 accumulation and upregulated the above-mentioned marker genes as well as CaWRKY6 and CaWRKY40. Chromatin immunoprecipitation (ChIP) and microscale thermophoresis (MST) analysis revealed that CaHsfB2a bound the promoters of both CaWRKY6 and CaWRKY40. In a parallel experiment, we determined by ChIP-PCR and MST that CaHsfB2a was regulated directly by CaWRKY40 but indirectly by CaWRKY6. Cumulatively, our results suggest that CaHsfB2a positively regulates plant immunity against RSI and tolerance to HTHH, via transcriptional cascades and positive feedback loops involving CaWRKY6 and CaWRKY40. [ABSTRACT FROM AUTHOR]

Several E3 ubiquitin ligases have been associated with the response to abiotic and biotic stresses in higher plants. Here, we report that the hot pepper (Capsicum annuum) ABA-Insensitive RING protein 1 gene (CaAIR1) is essential for a hypersensitive response to drought stress. CaAIR1 contains a C3HC4-type RING finger motif, which plays a role for attachment of ubiquitins to the target protein, and a putative transmembrane domain. The expression levels of CaAIR1 are up-regulated in pepper leaves by ABA treatments, drought and NaCl, suggesting its role in the response to abiotic stress. Our analysis showed that CaAIR1 displays self-ubiquitination and is localized in the nucleus. We generated CaAIR1-silenced peppers via virus-induced gene silencing (VIGS) and CaAIR1- overexpressing (OX) transgenic Arabidopsis plants to evaluate their responses to ABA and drought. VIGS of CaAIR1 in pepper plants conferred an enhanced tolerance to drought stress, which was accompanied by low levels of transpirational water loss in the drought-treated leaves. CaAIR1-OX plants displayed an impaired sensitivity to ABA during seed germination, seedling and adult stages. Moreover, these plants showed enhanced sensitivity to drought stress because of reduced stomatal closure and decreased expression of stress-responsive genes. Thus, our data indicate that CaAIR1 is a negative regulator of the ABA-mediated drought stress tolerance mechanism. [ABSTRACT FROM AUTHOR]

High temperature (HT), high humidity (HH), and pathogen infection often co-occur and negatively affect plant growth. However, these stress factors and plant responses are generally studied in isolation. The mechanisms of synergistic responses to combined stresses are poorly understood. We isolated the subgroup IIb WRKY family member CaWRKY6 from Capsicum annuum and performed quantitative real-time PCR analysis. CaWRKY6 expression was upregulated by individual or simultaneous treatment with HT, HH, combined HT and HH (HTHH), and Ralstonia solanacearum inoculation, and responded to exogenous application of jasmonic acid (JA), ethephon, and abscisic acid (ABA). Virus-induced gene silencing of CaWRKY6 enhanced pepper plant susceptibility to R. solanacearum and HTHH, and downregulated the hypersensitive response (HR), JA-, ethylene (ET)-, and ABA-induced marker gene expression, and thermotolerance-associated expression of CaHSP24, ER-small CaSHP, and Chl-small CaHSP. CaWRKY6 overexpression in pepper attenuated the HTHH-induced suppression of resistance to R. solanacearum infection. CaWRKY6 bound to and activated the CaWRKY40 promoter in planta, which is a pepper WRKY that regulates heatstress tolerance and R. solanacearum resistance. CaWRKY40 silencing significantly blocked HR-induced cell death and reduced transcriptional expression of CaWRKY40. These data suggest that CaWRKY6 is a positive regulator of R. solanacearum resistance and heat-stress tolerance, which occurs in part by activating CaWRKY40. [ABSTRACT FROM AUTHOR]