Transcriptome profiling of Capsicum annuum using Illumina- and PacBio SMRT-based RNA-Seq for in-depth understanding of genes involved in trichome formation.

Trichomes, specialized epidermal cells located in aerial parts of plants, play indispensable roles in resisting abiotic and biotic stresses. However, the regulatory genes essential for multicellular trichrome development in Capsicum annuum L. (pepper) remain unclear. In this study, the transcript profiles of peppers GZZY-23 (hairy) and PI246331 (hairless) were investigated to gain insights into the genes responsible for the formation of multicellular trichomes. A total of 40,079 genes, including 4743 novel genes and 13,568 differentially expressed genes (DEGs), were obtained. Functional enrichment analysis revealed that the most noticeable pathways were transcription factor activity, sequence-specific DNA binding, and plant hormone signal transduction, which might be critical for multicellular trichome formation in hairy plants. We screened 11 DEGs related to trichome development; 151 DEGs involved in plant hormone signal transduction; 312 DEGs belonging to the MYB, bHLH, HD-Zip, and zinc finger transcription factor families; and 1629 DEGs predicted as plant resistance genes (PRGs). Most of these DEGs were highly expressed in GZZY-23 or trichomes. Several homologs of trichome regulators, such as SlCycB2, SlCycB3, and H, were considerably upregulated in GZZY-23, especially in the trichomes. The transcriptomic data generated in this study provide a basis for future characterization of trichome formation in pepper.

Is Autophagy Involved in Pepper Fruit Ripening?

Autophagy is a universal self-degradation process involved in the removal and recycling of cellular constituents and organelles; however, little is known about its possible role in fruit ripening, in which the oxidation of lipids and proteins and changes in the metabolism of different cellular organelles occur. In this work, we analyzed several markers of autophagy in two critical maturation stages of pepper (Capsicum annuum L.) fruits where variations due to ripening become clearly visible. Using two commercial varieties that ripen to yellow and red fruits respectively, we studied changes in the gene expression and protein content of several autophagy (ATG) components, ATG4 activity, as well as the autophagy receptor NBR1 and the proteases LON1 and LON2. Additionally, the presence of intravacuolar vesicles was analyzed by electron microscopy. Altogether, our data reveal that autophagy plays a role in the metabolic changes which occur during ripening in the two studied varieties, suggesting that this process may be critical to acquiring final optimal quality of pepper fruits.

The Proteome of Fruit Peroxisomes: Sweet Pepper (Capsicum annuum L.) as a Model.

Despite of their economical and nutritional interest, the biology of fruits is still little studied in comparison with reports of other plant organs such as leaves and roots. Accordingly, research at subcellular and molecular levels is necessary not only to understand the physiology of fruits, but also to improve crop qualities. Efforts addressed to gain knowledge of the peroxisome proteome and how it interacts with the overall metabolism of fruits will provide tools to be used in breeding strategies of agricultural species with added value. In this work, special attention will be paid to peroxisomal proteins involved in the metabolism of reactive oxygen species (ROS) due to the relevant role of these compounds at fruit ripening. The proteome of peroxisomes purified from sweet pepper (Capsicum annuum L.) fruit is reported, where an iron-superoxide dismutase (Fe-SOD) was localized in these organelles, besides other antioxidant enzymes such as catalase and a Mn-SOD, as well as enzymes involved in the metabolism of carbohydrates, malate, lipids and fatty acids, amino acids, the glyoxylate cycle and in the potential organelles' movements.

Holistic protocol for callus culture optimization using statistical modelling.

Plants endue a key role against illnesses caused by oxidative stress. These attributes are frequently associated with polyphenolic compounds. However, presence and concentration of secondary metabolites are affected by abiotic factors. The in vitro culture techniques can solve these drawbacks. Peppers can be a suitable alternative to obtain polyphenols. Aiming to optimise the callus culture stage from Capsicum baccatum to produce polyphenols, this work evaluated systemically the effects of the explant's origin (root, hypocotyl and cotyledon), growth hormone type (2,4-dichlorophenoxyacetic acid (2,4-D), benzylaminopurine (BAP) and a combination of 2,4-D/BAP at five-to-one ratio) and concentration (0.023-10.000 mg L-1) on callus culture efficiency parameters using a multilevel factorial design. The root explant in combination with BAP at 1.138 mg L-1 ensured the optimal values of the assessed responses; ​callus mass (225.03 mg), antioxidant activity (35.95%), total phenols (11.48 mg of GAE/g DE) and flavonoids (15.92 mg of RU/g DE) production.

Difference in capsaicinoid biosynthesis gene expression in the pericarp reveals elevation of capsaicinoid contents in chili peppers (Capsicum chinense).

KEY MESSAGE: This research reveals that the up-regulated expression of multiple capsaicinoid biosynthetic genes in pericarp tissue leads to the elevation of total capsaicinoid content in chili pepper fruit. Capsaicinoids are health-functional compounds that are produced uniquely in chili pepper fruits. A high capsaicinoid level is one of the major parameters determining the commercial quality and health-promoting properties of chili peppers. To investigate the mechanisms responsible for its high contents, we compared an extremely pungent cultivar 'Trinidad Moruga Scorpion Yellow' (MY) with other cultivars of different pungency levels (Fushimi-amanaga, Takanotsume, Red Habanero). Capsaicinoid concentrations were markedly higher in MY fruit (23.9 mg/g DW) than in other pungent cultivars including 'Red Habanero' (HB) fruit (14.3 mg/g DW). Comparative analysis of MY and HB reveals that both cultivars accumulated similar capsaicinoid concentrations in the placental septum, with that in the HB pericarp (1.8 mg/g DW) being markedly lower than that in the placental septum (69.1 mg/g DW). The capsaicinoid concentration in HB fruit is dependent on the placental septum, as reported in other accessions. Therefore, even though placental septum tissue contains high capsaicinoid concentrations, those in the pericarp and seeds attenuated its total content. In contrast, the MY pericarp exhibited a markedly higher concentration (23.2 mg/g DW). A qRT-PCR analysis revealed that multiple capsaicinoid biosynthetic pathway genes (Pun1, pAMT, KAS, and BCAT) were strongly up-regulated in placental septum of pungent cultivars. The genes were expressed exclusively in the MY pericarp, but were barely detected in the pericarps of other pungent cultivars. Collectively, the present study indicates that the up-regulated expression of these genes not only in placental septum but also in pericarp plays an important role in driving capsaicinoid accumulation in the whole fruit.

Impedance Flow Cytometry: A Novel Technique in Pollen Analysis.

INTRODUCTION: An efficient and reliable method to estimate plant cell viability, especially of pollen, is important for plant breeding research and plant production processes. Pollen quality is determined by classical methods, like staining techniques or in vitro pollen germination, each having disadvantages with respect to reliability, analysis speed, and species dependency. Analysing single cells based on their dielectric properties by impedance flow cytometry (IFC) has developed into a common method for cellular characterisation in microbiology and medicine during the last decade. The aim of this study is to demonstrate the potential of IFC in plant cell analysis with the focus on pollen. METHOD: Developing and mature pollen grains were analysed during their passage through a microfluidic chip to which radio frequencies of 0.5 to 12 MHz were applied. The acquired data provided information about the developmental stage, viability, and germination capacity. The biological relevance of the acquired IFC data was confirmed by classical staining methods, inactivation controls, as well as pollen germination assays. RESULTS: Different stages of developing pollen, dead, viable and germinating pollen populations could be detected and quantified by IFC. Pollen viability analysis by classical FDA staining showed a high correlation with IFC data. In parallel, pollen with active germination potential could be discriminated from the dead and the viable but non-germinating population. CONCLUSION: The presented data demonstrate that IFC is an efficient, label-free, reliable and non-destructive technique to analyse pollen quality in a species-independent manner.

Biotechnological enhancement of capsaicin biosynthesis in cell suspension cultures of Naga King Chili (Capsicum chinense Jacq.).

Cell suspension cultures were initiated from hypocotyl derived callus to induce capsaicin biosynthesis in suspension cultures of Naga King Chili (Capsicum chinense Jacq.). Efficient capsaicin production with high growth index (GI) was obtained by exposing cells to salicylic acid (SA) and calcium channel modulators in suspension cultures. The time course of capsaicin formation is related to the cell growth profile in a batch culture. Cells cultivated in the standard medium (SM) initially showed low level of capsaicin yield during active growth. When the cells approached stationary phase, cell growth and cell viability decreased whereas capsaicin production increased continuously. In the fed-batch cultures, the highest capsaicin yield (567.4 ± 8.1 µgg(1) fresh weight) (f.wt) was obtained by feeding the cells with 1 mM SA. However, SA feeding during cultivation repressed the cell growth. Enhanced cell growth (3.1 ± 0.1 GI/culture) and capsaicin yield (534 ± 7.8 µgg(-1)f.wt) were obtained when the cells were fed with calcium ionophore A23187 (0.5 mM) on day 25 as compared to the control. Addition of the calcium channel blocker verapamil hydrochloride (100 mM) inhibited cell growth and capsaicin production in Naga King Chili suspension cell cultures.

Novel genetic male sterility developed in (Capsicum annuum x C. chinense) x C. pubescens and induced by HNO2 showing Mendelian inheritance and aborted at telophase of microspore mother cell stage.

A novel genetic male sterile germplasm was developed by successively crossing of (C. annuum x C. chinense) x C. pubescens and by chemical mutagenesis in pepper. The sterile anthers showed morphological abnormalities, but pistils developed normally with fine pollination capability. We investigated fertility segregation through sib-crossing of the same strains and test crossing by male sterile plants with 6 advanced inbred lines. The results showed that male fertility in the pepper was dominant in the F1 generation and segregated at a rate of 3:1 in the F2 generation, suggesting that monogenic male sterility was recessive and conformed to Mendelian inheritance. Cyto-anatomy analysis revealed that microspore abortion of sterile anthers occurred during telophase in the microspore mother cell stage when tapetal cells showed excessive vacuolation, resulting in occupation of the loculi. The microspore mother cells self-destructed and autolyzed with the tapetum so that meiosis in pollen mother cells could not proceed past the tetrad stage.

Characterization of Cell-Death-Inducing Members of the Pectate Lyase Gene Family in Phytophthora capsici and Their Contributions to Infection of Pepper.

Pectate lyases (PL) play a critical role in pectin degradation. PL have been extensively studied in major bacterial and fungal pathogens of a wide range of plant species. However, the contribution of PL to infection by oomycete pathogens remains largely unknown. Here, we cloned 22 full-length pectate lyase (PcPL) genes from a highly aggressive strain of Phytophthora capsici SD33. Of these, PVX agroinfiltration revealed that 12 PcPL genes were found to be highly induced during infection of pepper by SD33 but the induction level was twofold less in a mildly aggressive strain, YN07. The four genes with the highest transcript levels as measured by by quantitative reverse-transcription polymerase chain reaction (PcPL1, PcPL15, PcPL16, and PcPL20) also produced a severe cell death response following transient expression in pepper leaves but the other eight PcPL genes did not. Overexpression of these four genes increased the virulence of SD33 on pepper slightly, and increased it more substantially during infection of tobacco. Overexpression of the genes in YN07 restored its aggressiveness to near that of SD33. Gene silencing experiments with the 12 PcPL genes produced diverse patterns of silencing of PcPL genes, from which it could be inferred from regression analysis that PcPL1, PcPL16, and PcPL20 could account for nearly all of the contributions of the PcPL genes to virulence.

Pepper pathogenesis-related protein 4c is a plasma membrane-localized cysteine protease inhibitor that is required for plant cell death and defense signaling.

Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis-related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT-triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease-inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H2 O2 and significant induction of some defense-response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT-triggered resistance responses, and enabled Xcv proliferation in infected leaves. H2 O2 accumulation, cell-death induction, and defense-response gene expression were distinctly reduced in CaPR4c-silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling.

GLYCINE-RICH RNA-BINDING PROTEIN1 interacts with RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 and suppresses cell death and defense responses in pepper (Capsicum annuum).

Plants use a variety of innate immune regulators to trigger cell death and defense responses against pathogen attack. We identified pepper (Capsicum annuum) GLYCINE-RICH RNA-BINDING PROTEIN1 (CaGRP1) as a RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 (CaPIK1)-interacting partner, based on bimolecular fluorescence complementation and coimmunoprecipitation analyses as well as gene silencing and transient expression analysis. CaGRP1 contains an N-terminal RNA recognition motif and a glycine-rich region at the C-terminus. The CaGRP1 protein had DNA- and RNA-binding activity in vitro. CaGRP1 interacted with CaPIK1 in planta. CaGRP1 and CaGRP1-CaPIK1 complexes were localized to the nucleus in plant cells. CaPIK1 phosphorylated CaGRP1 in vitro and in planta. Transient coexpression of CaGRP1 with CaPIK1 suppressed the CaPIK1-triggered cell death response, accompanied by a reduced CaPIK1-triggered reactive oxygen species (ROS) burst. The RNA recognition motif region of CaGRP1 was responsible for the nuclear localization of CaGRP1 as well as the suppression of the CaPIK1-triggered cell death response. CaGRP1 silencing in pepper conferred enhanced resistance to Xanthomonas campestris pv vesicatoria (Xcv) infection; however, CaPIK1-silenced plants were more susceptible to Xcv. CaGRP1 interacts with CaPIK1 and negatively regulates CaPIK1-triggered cell death and defense responses by suppressing ROS accumulation.

Pepper heat shock protein 70a interacts with the type III effector AvrBsT and triggers plant cell death and immunity.

Heat shock proteins (HSPs) function as molecular chaperones and are essential for the maintenance and/or restoration of protein homeostasis. The genus Xanthomonas type III effector protein AvrBsT induces hypersensitive cell death in pepper (Capsicum annuum). Here, we report the identification of the pepper CaHSP70a as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirm the specific interaction between CaHSP70a and AvrBsT in planta. The CaHSP70a peptide-binding domain is essential for its interaction with AvrBsT. Heat stress (37°C) and Xanthomonas campestris pv vesicatoria (Xcv) infection distinctly induce CaHSP70a in pepper leaves. Cytoplasmic CaHSP70a proteins significantly accumulate in pepper leaves to induce the hypersensitive cell death response by Xcv (avrBsT) infection. Transient CaHSP70a overexpression induces hypersensitive cell death under heat stress, which is accompanied by strong induction of defense- and cell death-related genes. The CaHSP70a peptide-binding domain and ATPase-binding domain are required to trigger cell death under heat stress. Transient coexpression of CaHSP70a and avrBsT leads to cytoplasmic localization of the CaHSP70a-AvrBsT complex and significantly enhances avrBsT-triggered cell death in Nicotiana benthamiana. CaHSP70a silencing in pepper enhances Xcv growth but disrupts the reactive oxygen species burst and cell death response during Xcv infection. Expression of some defense marker genes is significantly reduced in CaHSP70a-silenced leaves, with lower levels of the defense hormones salicylic acid and jasmonic acid. Together, these results suggest that CaHSP70a interacts with the type III effector AvrBsT and is required for cell death and immunity in plants.

Efficacy of bioconversion of paper mill bamboo sludge and lime waste by composting and vermiconversion technologies.

Paper mill bamboo sludge (PMBS) and Paper mill lime waste (PMLW) are extensively produced as solid wastes in paper mills. Untreated PMBS and PMLW contain substantial amount of heavy metals (Zn, Pb, Ni, Cd, Cr) in soluble forms. Efficiency of vermiconversion and aerobic composting with these wastes is reported here. Adopted bioconversion systems enhanced the availability of some essential nutrients (N, P, K and Zn) in various combinations of cow dung (CD) with PMBS and PMLW. Colonization of nitrogen fixing bacteria and phosphate solubilizing bacteria considerably intensified under the vermiconversion system. Moreover, significant metal detoxification occurred due to vermiconversion. Various combinations of bioconverted PMBS and PMLW were applied to tissue cultured bamboo (Bambusa tulda) and chilli (Capsicum annum). Accelerated nutrient uptake coupled with improved soil quality resulted in significant production of chilli. Furthermore, vermiconverted PMBS+CD (1:1) and PMLW+CD (1:3) confirmed as potential enriching substrate for tissue cultured bamboo.

Pepper suppressor of the G2 allele of skp1 interacts with the receptor-like cytoplasmic kinase1 and type III effector AvrBsT and promotes the hypersensitive cell death response in a phosphorylation-dependent manner.

Xanthomonas campestris pv vesicatoria type III effector protein, AvrBsT, triggers hypersensitive cell death in pepper (Capsicum annuum). Here, we have identified the pepper SGT1 (for suppressor of the G2 allele of skp1) as a host interactor of AvrBsT and also the pepper PIK1 (for receptor-like cytoplasmic kinase1). PIK1 specifically phosphorylates SGT1 and AvrBsT in vitro. AvrBsT specifically binds to the CHORD-containing protein and SGT1 domain of SGT1, resulting in the inhibition of PIK1-mediated SGT1 phosphorylation and subsequent nuclear transport of the SGT1-PIK1 complex. Liquid chromatography-tandem mass spectrometry of the proteolytic peptides of SGT1 identified the residues serine-98 and serine-279 of SGT1 as the major PIK1-mediated phosphorylation sites. Site-directed mutagenesis of SGT1 revealed that the identified SGT1 phosphorylation sites are responsible for the activation of AvrBsT-triggered cell death in planta. SGT1 forms a heterotrimeric complex with both AvrBsT and PIK1 exclusively in the cytoplasm. Agrobacterium tumefaciens-mediated coexpression of SGT1 and PIK1 with avrBsT promotes avrBsT-triggered cell death in Nicotiana benthamiana, dependent on PIK1. Virus-induced silencing of SGT1 and/or PIK1 compromises avrBsT-triggered cell death, hydrogen peroxide production, defense gene induction, and salicylic acid accumulation, leading to the enhanced bacterial pathogen growth in pepper. Together, these results suggest that SGT1 interacts with PIK1 and the bacterial effector protein AvrBsT and promotes the hypersensitive cell death associated with PIK1-mediated phosphorylation in plants.

Manipulation of culture strategies to enhance capsaicin biosynthesis in suspension and immobilized cell cultures of Capsicum chinense Jacq. cv. Naga King Chili.

Manipulation of culture strategies was adopted to study the influence of nutrient stress, pH stress and precursor feeding on the biosynthesis of capsaicin in suspension and immobilized cell cultures of C. chinense. Cells cultured in the absence of one of the four nutrients (ammonium and potassium nitrate for nitrate and potassium stress, potassium dihydrogen orthophosphate for phosphorus stress, and sucrose for sugar stress) influenced the accumulation of capsaicin. Among the stress factors studied, nitrate stress showed maximal capsaicin production on day 20 (505.9 ± 2.8 µg g(-1) f.wt) in immobilized cell, whereas in suspension cultures the maximum accumulation (345.5 ± 2.9 µg g(-1) f.wt) was obtained on day 10. Different pH affected capsaicin accumulation; enhanced accumulation of capsaicin (261.6 ± 3.4 µg g(-1) f.wt) was observed in suspension cultures at pH 6 on day 15, whereas in case of immobilized cultures the highest capsaicin content (433.3 ± 3.3 µg g(-1) f.wt) was obtained at pH 5 on day 10. Addition of capsaicin precursors and intermediates significantly enhanced the biosynthesis of capsaicin, incorporation of vanillin at 100 µM in both suspension and immobilized cell cultures resulted in maximum capsaicin content with 499.1 ± 5.5 µg g(-1) f.wt on day 20 and 1,315.3 ± 10 µg g(-1) f.wt on day 10, respectively. Among the different culture strategies adopted to enhance capsaicin biosynthesis in cell cultures of C. chinense, cells fed with vanillin resulted in the maximum capsaicin accumulation. The rate of capsaicin production was significantly higher in immobilized cells as compared to freely suspended cells.

The pepper patatin-like phospholipase CaPLP1 functions in plant cell death and defense signaling.

Phospholipases hydrolyze phospholipids into fatty acids and other lipophilic substances. Phospholipid signaling is crucial for diverse cellular processes in plants. However, the precise role of phospholipases in plant cell death and defense signaling is not fully understood. Here, we identified a pepper (Capsicum annuum) patatin-like phospholipase (CaPLP1) gene that is transcriptionally induced in pepper leaves by avirulent Xanthomonas campestris pv. vesicatoria (Xcv) infection. CaPLP1 containing an N-terminal signal peptide localized to the cytoplasm and plasma membrane, leading to the secretion into the apoplastic regions. Silencing of CaPLP1 in pepper conferred enhanced susceptibility to Xcv infection. Defense responses to Xcv, including the generation of reactive oxygen species (ROS), hypersensitive cell death and the expression of the salicylic acid (SA)-dependent marker gene CaPR1, were compromised in the CaPLP1-silenced pepper plants. Transient expression of CaPLP1 in pepper leaves induced the accumulation of fluorescent phenolics, expression of the defense marker genes CaPR1 and CaSAR82A, and generation of ROS, ultimately leading to the hypersensitive cell death response. Overexpression (OX) of CaPLP1 in Arabidopsis also conferred enhanced resistance to Pseudomonas syringae pv. tomato (Pst) and Hyaloperonospora arabidopsidis infection. CaPLP1-OX leaves showed reduced Pst growth, enhanced ROS burst and electrolyte leakage, induction of the defense response genes AtPR1, AtRbohD and AtGST, as well as constitutive activation of both the SA-dependent gene AtPR1 and the JA-dependent gene AtPDF1.2. Together, these results suggest that CaPLP1 is involved in plant defense and cell death signaling in response to microbial pathogens.

Pathogenesis-related protein 4b interacts with leucine-rich repeat protein 1 to suppress PR4b-triggered cell death and defense response in pepper.

To control defense and cell-death signaling, plants contain an abundance of pathogen recognition receptors such as leucine-rich repeat (LRR) proteins. Here we show that pepper (Capsicum annuum) LRR1 interacts with the pepper pathogenesis-related (PR) protein 4b, PR4b, in yeast and in planta. PR4b is synthesized in the endoplasmic reticulum, interacts with LRR1 in the plasma membrane, and is secreted to the apoplast via the plasma membrane. Binding of PR4b to LRR1 requires the chitin-binding domain of PR4b. Purified PR4b protein inhibits spore germination and mycelial growth of plant fungal pathogens. Transient expression of PR4b triggers hypersensitive cell death. This cell death is compromised by co-expression of LRR1 as a negative regulator in Nicotiana benthamiana leaves. LRR1/PR4b silencing in pepper and PR4b over-expression in Arabidopsis thaliana demonstrated that LRR1 and PR4b are necessary for defense responses to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis (Hpa) infection. The mutant of the PR4b Arabidopsis ortholog, pr4, showed enhanced susceptibility to Hpa infection. Together, our results suggest that PR4b functions as a positive modulator of plant cell death and defense responses. However, the activity of PR4b is suppressed by interaction with LRR1.

Transcriptome sequencing and de novo analysis of a cytoplasmic male sterile line and its near-isogenic restorer line in chili pepper (Capsicum annuum L.).

BACKGROUND: The use of cytoplasmic male sterility (CMS) in F1 hybrid seed production of chili pepper is increasingly popular. However, the molecular mechanisms of cytoplasmic male sterility and fertility restoration remain poorly understood due to limited transcriptomic and genomic data. Therefore, we analyzed the difference between a CMS line 121A and its near-isogenic restorer line 121C in transcriptome level using next generation sequencing technology (NGS), aiming to find out critical genes and pathways associated with the male sterility. RESULTS: We generated approximately 53 million sequencing reads and assembled de novo, yielding 85,144 high quality unigenes with an average length of 643 bp. Among these unigenes, 27,191 were identified as putative homologs of annotated sequences in the public protein databases, 4,326 and 7,061 unigenes were found to be highly abundant in lines 121A and 121C, respectively. Many of the differentially expressed unigenes represent a set of potential candidate genes associated with the formation or abortion of pollen. CONCLUSIONS: Our study profiled anther transcriptomes of a chili pepper CMS line and its restorer line. The results shed the lights on the occurrence and recovery of the disturbances in nuclear-mitochondrial interaction and provide clues for further investigations.

A pepper (Capsicum annuum†L.) metacaspase 9 (Camc9) plays a role in pathogen-induced cell death in plants.

Metacaspases, which belong to the cysteine-type C14 protease family, are most structurally similar to mammalian caspases than any other caspase-like protease in plants. Atmc9 (Arabidopsis thaliana metacaspase 9) has a unique domain structure, and distinct biochemical characteristics, such as Ca²âº binding, pH, redox status, S-nitrosylation and specific protease inhibitors. However, the biological roles of Atmc9 in plant-pathogen interactions remain largely unknown. In this study, a metacaspase gene present as a single copy in the pepper genome, and sharing 54% amino acid sequence identity with Atmc9, was isolated and named Capsicum annuum metacaspase 9 (Camc9). Camc9 encodes a 318-amino-acid polypeptide with an estimated molecular weight of 34.6 kDa, and shares approximately 40% amino acid sequence identity with known type II metacaspases in plants. Quantitative reverse transcription-polymerase chain reaction analyses revealed that the expression of Camc9 was induced by infections of Xanthomonas campestris pv. vesicatoria race 1 and race 3 and treatment with methyl jasmonate. Suppression of Camc9 expression using virus-induced gene silencing enhanced disease resistance and suppressed cell death symptom development following infection with virulent bacterial pathogens. By contrast, overexpression of Camc9 by transient or stable transformation enhanced disease susceptibility and pathogen-induced cell death by regulation of reactive oxygen species production and defence-related gene expression. These results suggest that Camc9 is a possible member of the metacaspase gene family and plays a role as a positive regulator of pathogen-induced cell death in the plant kingdom.

CaHAM is autoregulated and regulates CaSTM expression and is required for shoot apical meristem organization in pepper.

The angiosperm shoot apical meristem (SAM) is characterized by tightly organized cell layers and zones. The SAM's organization allows it to maintain its indeterminate nature while producing determinate lateral organs. Alterations in SAM gene expression partly account for the immense diversity in plant architecture. The GRAS protein family gene HAIRY MERISTEM (HAM) is an important regulator of SAM organization in Petunia and Arabidopsis. Here we describe CaHAM loss-of-function pepper mutants characterized by an arrested SAM following the formation of several leaves on the primary stem, complete inhibition of axillary meristem development, an expanded tunica domain and trichome formation on the SAM epidermis. CaHAM is expressed in the periphery of the SAM and in the vasculature of young leaves throughout plant development, reaching its highest level in the reproductive growth stage. Analysis of the effect of CaHAM loss-of-function on its own expression showed that CaHAM is negatively autoregulated. Furthermore, CaHAM negatively regulates the expression level and pattern of pepper SHOOT MERISTEMLESS (CaSTM), which is required to maintain the SAM in an undifferentiated state. We conclude that CaHAM is regulated to achieve adjusted functional levels and has a conserved role in controlling SAM maintenance, organization and axillary meristem formation.