Mitigation of drought stress in chili plants (Capsicum annuum L.) using mango fruit waste biochar, fulvic acid and cobalt.

Drought stress can have negative impacts on crop productivity. It triggers the accumulation of reactive oxygen species, which causes oxidative stress. Limited water and nutrient uptake under drought stress also decreases plant growth. Using cobalt and fulvic acid with biochar in such scenarios can effectively promote plant growth. Cobalt (Co) is a component of various enzymes and co-enzymes. It can increase the concentration of flavonoids, total phenols, antioxidant enzymes (peroxidase, catalase, and polyphenol oxidase) and proline. Fulvic acid (FA), a constituent of soil organic matter, increases the accessibility of nutrients to plants. Biochar (BC) can enhance soil moisture retention, nutrient uptake, and plant productivity during drought stress. That's why the current study explored the influence of Co, FA and BC on chili plants under drought stress. This study involved 8 treatments, i.e., control, 4 g/L fulvic acid (4FA), 20 mg/L cobalt sulfate (20CoSO4), 4FA + 20CoSO4, 0.50%MFWBC (0.50 MFWBC), 4FA + 0.50MFWBC, 20CoSO4 + 0.50MFWBC, 4FA + 20CoSO4 + 0.50MFWBC. Results showed that 4 g/L FA + 20CoSO4 with 0.50MFWBC caused an increase in chili plant height (23.29%), plant dry weight (28.85%), fruit length (20.17%), fruit girth (21.41%) and fruit yield (25.13%) compared to control. The effectiveness of 4 g/L FA + 20CoSO4 with 0.50MFWBC was also confirmed by a significant increase in total chlorophyll contents, as well as nitrogen (N), phosphorus (P), and potassium (K) in leaves over control. In conclusion4g/L, FA + 20CoSO4 with 0.50MFWBC can potentially improve the growth of chili cultivated in drought stress. It is suggested that 4 g/L FA + 20CoSO4 with 0.50MFWBC be used to alleviate drought stress in chili plants.

Transcription factor CaHDZ15 promotes pepper basal thermotolerance by activating HEAT SHOCK FACTORA6a.

High temperature stress (HTS) is a serious threat to plant growth and development and to crop production in the context of global warming, and plant response to HTS is largely regulated at the transcriptional level by the actions of various transcription factors (TFs). However, whether and how homeodomain-leucine zipper (HD-Zip) TFs are involved in thermotolerance are unclear. Herein, we functionally characterized a pepper (Capsicum annuum) HD-Zip I TF CaHDZ15. CaHDZ15 expression was upregulated by HTS and abscisic acid in basal thermotolerance via loss- and gain-of-function assays by virus-induced gene silencing in pepper and overexpression in Nicotiana benthamiana plants. CaHDZ15 acted positively in pepper basal thermotolerance by directly targeting and activating HEAT SHOCK FACTORA6a (HSFA6a), which further activated CaHSFA2. In addition, CaHDZ15 interacted with HEAT SHOCK PROTEIN 70-2 (CaHsp70-2) and glyceraldehyde-3-phosphate dehydrogenase1 (CaGAPC1), both of which positively affected pepper thermotolerance. CaHsp70-2 and CaGAPC1 promoted CaHDZ15 binding to the promoter of CaHSFA6a, thus enhancing its transcription. Furthermore, CaHDZ15 and CaGAPC1 were protected from 26S proteasome-mediated degradation by CaHsp70-2 via physical interaction. These results collectively indicate that CaHDZ15, modulated by the interacting partners CaGAPC1 and CaHsp70-2, promotes basal thermotolerance by directly activating the transcript of CaHSFA6a. Thus, a molecular linkage is established among CaHsp70-2, CaGAPC1, and CaHDZ15 to transcriptionally modulate CaHSFA6a in pepper thermotolerance.

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.

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.

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.

CaHsfA1d Improves Plant Thermotolerance via Regulating the Expression of Stress- and Antioxidant-Related Genes.

Heat shock transcription factor (Hsf) plays an important role in regulating plant thermotolerance. The function and regulatory mechanism of CaHsfA1d in heat stress tolerance of pepper have not been reported yet. In this study, phylogenetic tree and sequence analyses confirmed that CaHsfA1d is a class A Hsf. CaHsfA1d harbored transcriptional function and predicted the aromatic, hydrophobic, and acidic (AHA) motif mediated function of CaHsfA1d as a transcription activator. Subcellular localization assay showed that CaHsfA1d protein is localized in the nucleus. The CaHsfA1d was transcriptionally up-regulated at high temperatures and its expression in the thermotolerant pepper line R9 was more sensitive than that in thermosensitive pepper line B6. The function of CaHsfA1d under heat stress was characterized in CaHsfA1d-silenced pepper plants and CaHsfA1d-overexpression Arabidopsis plants. Silencing of the CaHsfA1d reduced the thermotolerance of the pepper, while CaHsfA1d-overexpression Arabidopsis plants exhibited an increased insensitivity to high temperatures. Moreover, the CaHsfA1d maintained the H2O2 dynamic balance under heat stress and increased the expression of Hsfs, Hsps (heat shock protein), and antioxidant gene AtGSTU5 (glutathione S-transferase class tau 5) in transgenic lines. Our findings clearly indicate that CaHsfA1d improved the plant thermotolerance via regulating the expression of stress- and antioxidant-related genes.

The pepper RING-type E3 ligase, CaATIR1, positively regulates abscisic acid signalling and drought response by modulating the stability of CaATBZ1.

Protein degradation by the ubiquitin/26S proteasome system is a critical process that modulates many eukaryotic cellular processes. E3 ligase usually modulates stress response by adjusting the stability of transcription factors. Previous studies have shown that a RING-type E3 ligase, CaASRF1, positively modulates abscisic acid (ABA) signalling and ABA-mediated drought response by modulating the stability of CaAIBZ1 and CaATBZ1. In this study, we conducted yeast two-hybrid (Y2H) screening with CaATBZ1 to isolate an additional modulator, identified as CaATIR1 (Capsicum annuum ATBZ1 Interacting RING finger protein 1). CaATIR1 has E3 ligase activity and promoted CaATBZ1 degradation using the 26S proteasome system. We investigated the loss-of and gain-of functions of this E3 ligase by using silencing pepper and overexpressing (OX) Arabidopsis plants, respectively. In response to ABA and drought treatments, CaATIR1-silenced pepper plants showed ABA insensitive and drought-sensitive phenotypes, while CaATIR1-OX plants showed the opposite phenotypes. Additionally, CaATBZ1-silencing rescued the ABA insensitive and drought-sensitive phenotypes of CaATIR1-silencing pepper plants. Taken together, these data demonstrate that the stability of CaATBZ1 mediated by CaATIR1 has a crucial role in drought stress signalling in pepper plants.

Pepper CaMLO6 Negatively Regulates Ralstonia solanacearum Resistance and Positively Regulates High Temperature and High Humidity Responses.

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.

Integrative roles of nitric oxide and hydrogen sulfide in melatonin-induced tolerance of pepper (Capsicum annuum L.) plants to iron deficiency and salt stress alone or in combination.

There seems to be no report in the literature on the effect of melatonin (MT) in relieving the detrimental effects of combined application of salt stress (SS) and iron deficiency (ID). Therefore, the effect of MT on the accumulation/synthesis of endogenous nitric oxide (NO) and hydrogen sulphide (H2 S) and how far these molecules are involved in MT-improved tolerance to the combined application of ID and SS in pepper (Capsicum annuum L) were tested. Hence, two individual trials were set up. The treatments in the first experiment comprised: Control, ID (0.1 mM FeSO4 ), SS (100 mM NaCl) and ID + SS. The detrimental effects of combined stresses were more prominent than those by either of the single stress, with respect to growth, oxidative stress and antioxidant defense attributes. Single stress or both in combination improved the endogenous H2 S and NO, and foliar-applied MT (100 µM) led to a further increase in NO and H2 S levels. In the second experiment, 0.1 mM scavenger of NO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) and that of H2 S, hypotuarine (HT) were applied along with MT to get further evidence whether NO and H2 S are involved in MT-induced tolerance to ID and SS. MT combined with cPTIO and HT under a single or combined stress showed that NO effect was reversed by the NO scavenger, cPTIO, alone but the H2 S effect was inhibited by both scavengers. These findings suggested that tolerance to ID and SS induced by MT may be involved in downstream signal crosstalk between NO and H2 S.

A Novel Transcription Factor CaSBP12 Gene Negatively Regulates the Defense Response against Phytophthora capsici in Pepper (Capsicum annuum L.).

SBP-box (Squamosa-promoter binding protein) genes are a type of plant-specific transcription factor and play important roles in plant growth, signal transduction and stress response. However, little is known about the SBP-box genes in pepper (CaSBP), especially in the process of Phytophthora capsici infection. In this study, a novel gene (CaSBP12) was selected from the CaSBP gene family, which was isolated from the pepper genome database in our previous study. The CaSBP12 gene was located in the nucleus of the cell and its silencing in the pepper plant enhanced the defense response against Phytophthora capsici infection. After inoculation with Phytophthora capsici, the root activity of the CaSBP12-silenced plants is compared to control plants, while malondialdehyde (MDA) content is compared viceversa. Additionally, the expression of defense related genes (CaPO1, CaSAR8.2, CaBPR1, and CaDEF1) in the silenced plants were induced to different degrees and the peak of CaSAR8.2 and CaBPR1 were higher than that of CaDEF1. The CaSBP12 over-expressed Nicotiana benthamiana plants were more susceptible to Phytophthora capsici infection with higher EC (electrical conductivity) and MDA contents as compared to the wild-type. The relative expression of defense related genes (NbDEF, NbNPR1, NbPR1a, and NbPR1b) in transgenic and wild-type Nicotiana benthamiana plants were induced, especially the NbPR1a and NbPR1b. In conclusion, these results indicate that CaSBP12 gene negative regulates the defense response against Phytophthora capsici infection which suggests their potentially significant role in plant defense. To our knowledge, this is the first report on CaSBP gene which negative regulate defense response.

Endogenous hydrogen sulfide (H2S) is up-regulated during sweet pepper (Capsicum annuum L.) fruit ripening. In vitro analysis shows that NADP-dependent isocitrate dehydrogenase (ICDH) activity is inhibited by H2S and NO.

Like nitric oxide (NO), hydrogen sulfide (H2S) has been recognized as a new gasotransmitter which plays an important role as a signaling molecule in many physiological processes in higher plants. Although fruit ripening is a complex process associated with the metabolism of reactive oxygen species (ROS) and nitrogen oxygen species (RNS), little is known about the potential involvement of endogenous H2S. Using sweet pepper (Capsicum annuum L.) as a model non-climacteric fruit during the green and red ripening stages, we studied endogenous H2S content and cytosolic l-cysteine desulfhydrase (L-DES) activity which increased by 14% and 28%, respectively, in red pepper fruits. NADPH is a redox compound and key cofactor required for cell growth, proliferation and detoxification. We studied the NADPH-regenerating enzyme, NADP-isocitrate dehydrogenase (NADP-ICDH), whose activity decreased by 34% during ripening. To gain a better understanding of its potential regulation by H2S, we obtained a 50-75% ammonium sulfate-enriched protein fraction containing the NADP-ICDH protein; with the aid of in vitro assays in the presence of H2S, we observed that 2 and 10 mM NaHS used as H2S donors resulted in a decrease of up to 36% and 45%, respectively, in NADP-ICDH activity, which was unaffected by reduced glutathione (GSH). On the other hand, peroxynitrite (ONOO-), S-nitrosocyteine (CysNO) and DETA-NONOate, with the last two acting as NO donors, also inhibited NADP-ICDH activity. In silico analysis of the tertiary structure of sweet pepper NADP-ICDH activity (UniProtKB ID A0A2G2Y555) suggests that residues Cys133 and Tyr450 are the most likely potential targets for S-nitrosation and nitration, respectively. Taken together, the data reveal that the increase in the H2S production capacity of red fruits is due to higher L-DES activity during non-climacteric pepper fruit ripening. In vitro assays appear to show that H2S inhibits NADP-ICDH activity, thus suggesting that this enzyme may be regulated by persulfidation, as well as by S-nitrosation and nitration. NO and H2S may therefore regulate NADPH production and consequently cellular redox status during pepper fruit ripening.

Characterization of the hot pepper (Capsicum frutescens) fruit ripening regulated by ethylene and ABA.

BACKGROUND: Ripening of fleshy fruits has been classically defined as climacteric or non-climacteric. Both types of ripening are controlled by plant hormones, notably by ethylene in climacteric ripening and by abscisic acid (ABA) in non-climacteric ripening. In pepper (Capsicum), fruit ripening has been widely classified as non-climacteric, but the ripening of the hot pepper fruit appears to be climacteric. To date, how to regulate the hot pepper fruit ripening through ethylene and ABA remains unclear. RESULTS: Here, we examined ripening of the hot pepper (Capsicum frutescens) fruit during large green (LG), initial colouring (IC), brown (Br), and full red (FR) stages. We found a peak of ethylene emission at the IC stage, followed by a peak respiratory quotient at the Br stage. By contrast, ABA levels increased slowly before the Br stage, then increased sharply and reached a maximum level at the FR stage. Exogenous ethylene promoted colouration, but exogenous ABA did not. Unexpectedly, fluridone, an inhibitor of ABA biosynthesis, promoted colouration. RNA-sequencing data obtained from the four stages around ripening showed that ACO3 and NCED1/3 gene expression determined ethylene and ABA levels, respectively. Downregulation of ACO3 and NCED1/3 expression by virus-induced gene silencing (VIGS) inhibited and promoted colouration, respectively, as evidenced by changes in carotenoid, ABA, and ethylene levels, as well as carotenoid biosynthesis-related gene expression. Importantly, the retarded colouration in ACO3-VIGS fruits was rescued by exogenous ethylene. CONCLUSIONS: Ethylene positively regulates the hot pepper fruit colouration, while inhibition of ABA biosynthesis promotes colouration, suggesting a role of ABA in de-greening. Our findings provide new insights into processes of fleshy fruit ripening regulated by ABA and ethylene, focusing on ethylene in carotenoid biosynthesis and ABA in chlorophyll degradation.

Crecimiento y eficiencia fotoquímica del fotosistema II en plántulas de 2 variedades de Capsicum annuum L. inoculadas con rizobacterias u hongos micorrícicos arbusculares / Growth and photochemical efficiency of photosystem II in seedlings of two varieties of Capsicum annuum L. inoculated with rhizobacteria and arbuscular mycorrhizal fungi

Una alternativa para el manejo sustentable en el cultivo de Capsicum annuum L. se ha enfocado en el uso de bacterias promotoras del crecimiento vegetal (BPCV) y hongos micorrícicos arbusculares (HMA). Esta investigación seleccionó BPCV y HMA sobre la base de su efecto en plantas de chile Bell Pepper y jalapeño. Se utilizaron 5 cepas bacterianas aisladas de diferentes localidades del estado de México (P61 [Pseudomonas tolaasii], A46 [P. tolaasii], R44 [Bacillus pumilus], BSP1.1 [Paenibacillus sp.] y OLs-Sf5 [Pseudomonas sp.]) y 3 tratamientos con HMA (H1 [consorcio aislado de la rizosfera de chile en el estado de Puebla], H2 [Rhizophagus intraradices]y H3 [consorcio aislado de la rizosfera de limón del estado de Tabasco]). Además, se incluyó un tratamiento fertilizado (solución Steiner 25%) y un testigo absoluto. Plántulas de chile jalapeño «Caloro¼ y pimiento Bell Pepper «California Wonder¼ fueron inoculadas con HMA en el momento de la siembra y con BPCV 15 días después de emerger, y crecidas bajo condiciones de cámara de ambiente controlado. En chile jalapenño, la mejor cepa bacteriana fue P61 y el mejor tratamiento de HMA fue el H1; en Bell Pepper la mejor cepa fue R44 y los mejores HMA fueron el H3 y el H1. Estos microorganismos incrementaron el crecimiento de plántulas de chile jalapenño y Bell Pepper en comparación con el testigo sin fertilizar. Asimismo, P61 y R44 beneficiaron positivamente la capacidad fotosintética del PSII.

Plant growth promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF) are a biological alternative for the sustainable management of Capsicum annuum L. This research work evaluated the effects of both PGPR and AMF on bell pepper and jalapeno pepper plants. Five bacterial strains isolated from several locations in Estado de Mexico were used: [P61 (Pseudomonas tolaasii), A46 (P. tolaasii), R44 (Bacillus pumilus), BSP1.1 (Paenibacillus sp.), and OLs-Sf5 (Pseudomonas sp.)], and three treatments with AMF [H1 (consortium isolated from pepper crops in the State of Puebla), H2 (Rhizophagus intraradices), and H3 (consortium isolated from the rhizosphere of lemon trees, State of Tabasco)]. In addition, a fertilized treatment (Steiner nutrient solution at 25%) and an unfertilized control were included. Seedlings of "Caloro" jalapeno pepper and "California Wonder" bell pepper were inoculated with AMF at seed sowing, and PGPR were inoculated after 15 days of seedling emergence; seedlings were grown under plant growth chamber conditions. P61 bacterium and H1 AMF consortia were the most effective microorganisms for jalapeno pepper whereas R44 bacterium and AMF H3 and H1 were the most effective for bell peppers, when compared to the unfertilized control. Furthermore, P61 and R44 bacteria showed beneficial effects on PSII efficiency.

The Aborted Microspores (AMS)-Like Gene Is Required for Anther and Microspore Development in Pepper (Capsicum annuum L.).

Pepper (Capsicum annuum L.) is an economically important vegetable crop worldwide. Although many genes associated with anther and pollen development have been identified, little is known about the mechanism of pollen abortion in pepper. Here, we identified and isolated two putative aborted microspore (AMS) isoforms from pepper flowers: CaAMS1 and CaAMS2. Sequence analysis showed that CaAMS2 was generated by retention of the fourth intron in CaAMS1 pre-mRNA. CaAMS1 encodes a putative protein with a basic helix-loop-helix (bHLH) domain belonging to the MYC subfamily of bHLH transcription factors, and it is localized to the nucleus. Truncated CaAMS2-1 and CaAMS2-2 are produced by alternative splicing. Quantitative real-time PCR analysis showed that CaAMS (referred to CaAMS1 and CaAMS2-2) was preferentially expressed in stamens and its expression level gradually decreases with flower development. RNA in situ hybridization analysis showed that CaAMS is strongly expressed in the tapetum at the tetrad and uninucleate stages. Downregulation of CaAMS led to partial shortened filaments, shriveled, indehiscent stamens and abortive pollens in pepper flowers. Several genes involved in pollen exine formation were downregulated in defective CaAMS-silenced anthers. Thus, CaAMS seems to play an important role in pepper tapetum and pollen development by regulating a complex genetic network.

Expression and functional evaluation of CaZNF830 during pepper response to Ralstonia solanacearum or high temperature and humidity.

Extensive transcriptional reprogramming after pathogen attack determines immunity to these invaders and plant development. Zinc finger (ZNF) transcription factors regulate important processes in plants such as development, vegetative activities and plant immunity. Despite their immense significance, majority of ZNF transcription factors (TF) involved in pepper immunity and resistance to heat stress have not been focused much. Herein, we identified and functionally characterized CaZNF830 in pepper defense against Ralstonia solanacearum inoculation (RSI) and tolerance to high temperature and high humidity (HTHH). Transient expression analysis of CaZNF830-GFP fusion protein in tobacco leaves revealed its localization to the nucleus. Transcription of CaZNF830 is induced in pepper plants upon RSI or HTHH. Consistent with this, fluorometric GUS enzymatic assay driven by pCaZNF830 presented significantly enhanced activity under RSI and HTHH in comparison with the control plants. The silencing of CaZNF830 by virus induced gene silencing (VIGS) significantly compromised pepper immunity against RSI with enhanced growth of Ralstonia solanacearum in pepper plants. Silencing of CaZNF830 also impaired tolerance to HTHH coupled with decreased expression levels of immunity and thermo-tolerance associated marker genes including CaHIR1, CaNPR1, CaPR1, CaABR1 and CaHSP24. By contrast, the transient over-expression of CaZNF830 in pepper leaves by infiltration of GV3101 cells containing 35S::CaZNF830-HA induced HR mimic cell death, H2O2 accumulation and activated the transcriptions of the tested defense-relative or thermo-tolerance associated marker genes. RT-PCR and immune-blotting assay confirmed the stable expression of HA-tagged CaZNF830 mRNA and protein in pepper. All these results suggest that CaZNF830 acts as a positive regulator of plant immunity against RSI or tolerance to HTHH, it is induced by RSI or HTHH and consequently activate pepper immunity against RSI or tolerance to HTHH by directly or indirectly transcriptional modulation of many defense-linked genes.

A Simple and Useful Method to Apply Exogenous NO Gas to Plant Systems: Bell Pepper Fruits as a Model.

Nitric oxide (NO) is involved many physiological plant processes, including germination, growth and development of roots, flower setting and development, senescence, and fruit ripening. In the latter physiological process, NO has been reported to play an opposite role to ethylene. Thus, treatment of fruits with NO may lead to delay ripening independently of whether they are climacteric or nonclimacteric. In many cases different methods have been reported to apply NO to plant systems involving sodium nitroprusside, NONOates, DETANO, or GSNO to investigate physiological and molecular consequences. In this chapter a method to treat plant materials with NO is provided using bell pepper fruits as a model. This method is cheap, free of side effects, and easy to apply since it only requires common chemicals and tools available in any biology laboratory.

Superoxide serves as a putative signal molecule for plant cell division: overexpression of CaRLK1 promotes the plant cell cycle via accumulation of O2- and decrease in H2 O2.

Reactive oxygen species (ROS) exert both positive and negative effects on plant growth and development and therefore receive a great deal of attention in current research. A hot pepper, Capsicum annuum receptor-like kinase 1 (CaRLK1) was ectopically expressed in Nicotiana tabacum BY-2 cell and Nicotiana benthamiana plants. This ectopic expression of CaRLK1 enhanced cell division and proliferation in both heterologous systems. Apparently, CaRLK1 is involved in controlling the cell cycle, possibly by inducing expressions of cyclin B1, cyclin D3, cyclin-dependent protein kinase 3, condensin complex subunit 2 and anaphase-promoting complex subunit 11 genes. CaRLK1 overexpression also increased transcript accumulation of NADPH oxidase genes, generation of O2- and catalase (CAT) activity/protein levels. In parallel, it decreased cellular H2 O2 levels and cell size. Treatment with Tiron or diphenyleneiodonium (DPI) both decreased the cell division rate and O2- concentrations, but increased cellular H2 O2 levels. Tobacco BY-2 cells overexpressing CaRLK1 were more sensitive to amino-1,2,4-triazole (3-AT), a CAT inhibitor, than control cells, suggesting that the increased H2 O2 levels may not function as a signal for cell division and proliferation. Overexpression of CaRLK1 stimulated progression of the cell cycle from G0 /G1 phase into the S phase. It is concluded that the CaRLK1 protein plays a pivotal role in controlling the level of O2- as signaling molecule which promotes cell division, concomitant with a reduction in H2 O2 by the induction of CAT activity/protein.

CaLecRK-S.5, a pepper L-type lectin receptor kinase gene, confers broad-spectrum resistance by activating priming.

In Arabidopsis, several L-type lectin receptor kinases (LecRKs) have been identified as putative immune receptors. However, to date, there have been few analyses of LecRKs in crop plants. Virus-induced gene silencing of CaLecRK-S.5 verified the role of CaLecRK-S.5 in broad-spectrum resistance. Compared with control plants, CaLecRK-S.5-silenced plants showed reduced hypersensitive response, reactive oxygen species burst, secondary metabolite production, mitogen-activated protein kinase activation, and defense-related gene expression in response to Tobacco mosaic virus pathotype P0 (TMV-P0) infection. Suppression of CaLecRK-S.5 expression significantly enhanced the susceptibility to Pepper mild mottle virus pathotype P1,2,3, Xanthomonas campestris pv. vesicatoria, Phytophthora capsici, as well as TMV-P0 Additionally, ß-aminobutyric acid treatment and a systemic acquired resistance assay revealed that CaLecRK-S.5 is involved in priming of plant immunity. Pre-treatment with ß-aminobutyric acid before viral infection restored the reduced disease resistance phenotypes shown in CaLecRK-S.5-silenced plants. Systemic acquired resistance was also abolished in CaLecRK-S.5-silenced plants. Finally, RNA sequencing analysis indicated that CaLecRK-S.5 positively regulates plant immunity at the transcriptional level. Altogether, these results suggest that CaLecRK-S.5-mediated broad-spectrum resistance is associated with the regulation of priming.

Identification and functional expression of the pepper RING type E3 ligase, CaDTR1, involved in drought stress tolerance via ABA-mediated signalling.

Drought negatively affects plant growth and development, thereby leading to loss of crop productivity. Several plant E3 ubiquitin ligases act as positive or negative regulators of abscisic acid (ABA) and thus play important roles in the drought stress response. Here, we show that the C3HC4-type RING finger E3 ligase, CaDTR1, regulates the drought stress response via ABA-mediated signalling. CaDTR1 contains an amino-terminal RING finger motif and two carboxyl-terminal hydrophobic regions; the RING finger motif functions during attachment of ubiquitins to the target proteins, and the carboxyl-terminal hydrophobic regions function during subcellular localisation. The expression of CaDTR1 was induced by ABA, drought, and NaCl treatments. CaDTR1 localised in the nucleus and displayed in vitro E3 ubiquitin ligase activity. CaDTR1-silenced pepper plants exhibited a drought-sensitive phenotype characterised by high levels of transpirational water loss. On the other hand, CaDTR1-overexpressing (OX) Arabidopsis plants exhibited an ABA-hypersensitive phenotype during the germinative and post-germinative growth stages. Moreover, in contrast to CaDTR1-silenced pepper plants, CaDTR1-OX plants exhibited a drought-tolerant phenotype characterised by low levels of transpirational water loss via increased stomatal closure and high leaf temperatures. Our data indicate that CaDTR1 functions as a positive regulator of the drought stress response via ABA-mediated signalling.

Host preference, population growth and injuries assessment of Polyphagotarsonemus latus (banks) (ACARI: Tarsonemidae) on Capsicum annuum L. Genotypes.

Despite the continued efforts on the search for different genotypes, Capsicum annuum (L.) is quite susceptible to attack by pest arthropods, especially the broad mite Polyphagotarsonemus latus Banks. Thus, the host preference, population growth and the injuries assessment of P. latus was studied on six C. annuum genotypes used in Brazil (Atlantis, California Wonder, Impact, Palloma, Rubia and Tendence). Host preference was accessed in choice tests, pairing the several genotypes, and the population growth was observed through non-choice tests in laboratory. The injuries assessments were evaluated in the greenhouse, comparing the injury level among the six genotypes. The results indicate that California Wonder and Palloma genotypes were more preferred by P. latus, and Impact and Tendence were less preferred. P. latus presented positive population growth rates (ri) on all the genotypes, however, Palloma and California Wonder showed the highest values of population growth rate (ri = 0.344 and ri = 0.340, respectively), while Impact had the lowest value (ri = 0.281). All the evaluated C. annuum genotypes showed low tolerance to P. latus and exhibited several injuries, but there was no statistical difference between them. California Wonder had the highest average number of mites/leaf (57.15), while Impact and Tendence obtained the lowest values (36.67 and 35.12, respectively) at the end of the evaluation period. The total average of injuries notes at the end of the bioassay did not differ between the genotypes. The number of mites/leaf was growing for the injury scale to the note 3.0, but when the injury scale approached the note 4.0, there was observed a decrease in the number of mites/leaf for all the genotypes.