Biochemistry and Molecular Biology of Carotenoid Biosynthesis in Chili Peppers (Capsicum spp.)

Capsicum species produce fruits that synthesize and accumulate carotenoid pigments, which are responsible for the fruits’ yellow, orange and red colors. Chili peppers have been used as an experimental model for studying the biochemical and molecular aspects of carotenoid biosynthesis. Most reports refer to the characterization of carotenoids and content determination in chili pepper fruits from different species, cultivars, varieties or genotypes. The types and levels of carotenoids differ between different chili pepper fruits, and they are also influenced by environmental conditions. Yellow-orange colors of chili pepper fruits are mainly due to the accumulation of α- and β-carotene, zeaxanthin, lutein and β-cryptoxanthin. Carotenoids such as capsanthin, capsorubin and capsanthin-5,6-epoxide confer the red colors. Chromoplasts are the sites of carotenoid pigment synthesis and storage. According to the most accepted theory, the synthesis of carotenoids in chili peppers is controlled by three loci: c1, c2 and y. Several enzymes participating in carotenoid biosynthesis in chili pepper fruits have been isolated and characterized, and the corresponding gene sequences have been reported. However, there is currently limited information on the molecular mechanisms that regulate this biosynthetic pathway. Approaches to gain more knowledge of the regulation of carotenoid biosynthesis are discussed.     

Identification and Expression Analysis of Candidate Genes Associated with Defense Responses to Phytophthora capsici in Pepper Line “PI 201234”

Phytophthora capsici (Leonian), classified as an oomycete, seriously threatens the production of pepper (Capsicum annuum). Current understanding of the defense responses in pepper to P. capsici is limited. In this study, RNA-sequencing analysis was utilized to identify differentially expressed genes in the resistant line “PI 201234”, with 1220 differentially expressed genes detected. Of those genes, 480 were up-regulated and 740 were down-regulated, with 211 candidate genes found to be involved in defense responses based on the gene annotations. Furthermore, the expression patterns of 12 candidate genes were further validated via quantitative real-time PCR (qPCR). These genes were found to be significantly up-regulated at different time points post-inoculation (6 hpi, 24 hpi, and 5 dpi) in the resistant line “PI 201234” and susceptible line “Qiemen”. Seven genes were found to be involved in cell wall modification, phytoalexin biosynthesis, symptom development, and phytohormone signaling pathways, thus possibly playing important roles in combating exogenous pathogens. The genes identified herein will provide a basis for further gene cloning and functional verification studies and will aid in an understanding of the regulatory mechanism of pepper resistance to P. capsici.     

Characteristic of the Pepper CaRGA2 Gene in Defense Responses against Phytophthora capsici Leonian

The most significant threat to pepper production worldwide is the Phytophthora blight, which is caused by the oomycete pathogen, Phytophthora capsici Leonian. In an effort to help control this disease, we isolated and characterized a P. capsici resistance gene, CaRGA2, from a high resistant pepper (C. annuum CM334) and analyzed its function by the method of real-time PCR and virus-induced gene silencing (VIGS). The CaRGA2 has a full-length cDNA of 3,018 bp with 2,874 bp open reading frame (ORF) and encodes a 957-aa protein. The protein has a predicted molecular weight of 108.6 kDa, and the isoelectric point is 8.106. Quantitative real-time PCR indicated that CaRGA2 expression was rapidly induced by P. capsici. The gene expression pattern was different between the resistant and susceptible cultivars. CaRGA2 was quickly expressed in the resistant cultivar, CM334, and reached to a peak at 24 h after inoculation with P. capsici, five-fold higher than that of susceptible cultivar. Our results suggest that CaRGA2 has a distinct pattern of expression and plays a critical role in P. capsici stress tolerance. When the CaRGA2 gene was silenced via VIGS, the resistance level was clearly suppressed, an observation that was supported by semi-quantitative RT-PCR and detached leave inoculation. VIGS analysis revealed their importance in the surveillance to P. capsici in pepper. Our results support the idea that the CaRGA2 gene may show their response in resistance against P. capsici. These analyses will aid in an effort towards breeding for broad and durable resistance in economically important pepper cultivars.     

Thinopyrum ponticum Chromatin-Integrated Wheat Genome Shows Salt-Tolerance at Germination Stage

A wild wheatgrass, Thinopyrum ponticum (2n = 10x = 70), which exhibits substantially higher levels of salt tolerance than cultivated wheat, was employed to transfer its salt tolerance to common wheat by means of wide hybridization. A highly salt-tolerant wheat line S148 (2n = 42) was obtained from the BC₃F₂ progenies between Triticum aestivum (2n = 42) and Th. ponticum. In the cross of S148 × salt-sensitive wheat variety Chinese Spring, the BC₄F₂ seeds at germination stage segregated into a ratio of 3 salt tolerant to 1 salt sensitive, indicating that the salt tolerance was conferred by a dominant gene block. Genomic in situ hybridization analysis revealed that S148 had a single pair of Th. ponticum–T. aestivum translocated chromosomes bearing the salt-tolerance. This is an initial step of molecular breeding for salt-tolerant wheat.     

The Effect of the Aerial Part of Lindera akoensis on Lipopolysaccharides (LPS)-Induced Nitric Oxide Production in RAW264.7 Cells

Four new secondary metabolites, 3α-((E)-Dodec-1-enyl)-4β-hydroxy-5β-methyldihydrofuran-2-one (1), linderinol (6), 4′-O-methylkaempferol 3-O-α-l-(4″-E-p-coumaroyl)rhamnoside (11) and kaempferol 3-O-α-l-(4″-Z-p-coumaroyl) rhamnoside (12) with eleven known compounds—3-epilistenolide D₁ (2), 3-epilistenolide D₂ (3), (3Z,4α,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (4), (3E,4β,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (5), matairesinol (7), syringaresinol (8), (+)-pinoresinol (9), salicifoliol (10), 4″-p-coumaroylafzelin (13), catechin (14) and epicatechin (15)—were first isolated from the aerial part of Lindera akoensis. Their structures were determined by detailed analysis of 1D- and 2D-NMR spectroscopic data. All of the compounds isolated from Lindera akoensis showed that in vitro anti-inflammatory activity decreases the LPS-stimulated production of nitric oxide (NO) in RAW 264.7 cell, with IC₅₀ values of 4.1–413.8 μM.     

Two New Quinochalcone C-Glycosides from the Florets of Carthamus tinctorius

Two new quinochalcone C-glycosides, named hydroxysafflor yellow B (1) and hydroxysafflor yellow C (2), along with two known quinochalcone C-glycosides, safflomin C (3) and saffloquinoside C (4), and one known flavanone, (2R)-4'',5-dihydroxyl-6,7-di-O-β-d-glucopyranosyl flavanone (5), were isolated from the florets of Carthamus tinctorius. Their structures were determined by extensive spectroscopic (UV, IR, HR-ESI-MS, 1D and 2D NMR) analyses. In addition, these quinochalcone C-glycosides together with hydroxysafflor yellow A and anhydrosafflor yellow B were evaluated for their anti-oxidative effects against H₂O₂-induced cytotoxicity in cultured H9c2 cells. Among them, compound 2 exhibited significant anti-oxidative effects.     

New Benzo[c]phenanthridine and Benzenoid Derivatives,and Other Constituents from Zanthoxylum ailanthoides: Effects on Neutrophil Pro-Inflammatory Responses

A new benzo[c]phenanthridine, oxynorchelerythrine (1), and two new benzenoid derivatives, methyl 4-(2-hydroxy-4-methoxy-3-methyl-4-oxobutoxy)benzoate (2) and (E)-methyl 4-(4-((Z)-3-methoxy-3-oxoprop-1-enyl)phenoxy)-2-methylbut-2-enoate (3), have been isolated from the twigs of Zanthoxylum ailanthoides, together with 11 known compounds (4–14). The structures of these new compounds were determined through spectroscopic and MS analyses. Among the isolated compounds, decarine (4), (−)-syringaresinol (6), (+)-episesamin (8), glaberide I (9), (−)-dihydrocubebin (10), and xanthyletin (11) exhibited potent inhibition (IC₅₀ values ≤ 4.79 μg/mL) of superoxide anion generation by human nutrophils in response to N-formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB). Compounds 4, 8, and 11 also inhibited fMLP/CB-induced elastase release with IC₅₀ values ≤ 5.48 μg/mL.     

Identification of Novel Pepper Genes Involved in Bax- or INF1-Mediated Cell Death Responses by High-Throughput Virus-Induced Gene Silencing

Hot pepper is one of the economically important crops in Asia. A large number of gene sequences, including expressed sequence tag (EST) and genomic sequences are publicly available. However, it is still a daunting task to determine gene function due to difficulties in genetic modification of a pepper plants. Here, we show the application of the virus-induced gene silencing (VIGS) repression for the study of 459 pepper ESTs selected as non-host pathogen-induced cell death responsive genes from pepper microarray experiments in Nicotiana benthamiana. Developmental abnormalities in N. benthamiana plants are observed in the 32 (7%) pepper ESTs-silenced plants. Aberrant morphological phenotypes largely comprised of three groups: stunted, abnormal leaf, and dead. In addition, by employing the combination of VIGS and Agrobacterium-mediated transient assays, we identified novel pepper ESTs that involved in Bax or INF1-mediated cell death responses. Silencing of seven pepper ESTs homologs suppressed Bax or INF1-induced cell death, five of which suppressed both cell death responses in N. benthamiana. The genes represented by these five ESTs encode putative proteins with functions in endoplasmic reticulum (ER) stress and lipid signaling. The genes represented by the other two pepper ESTs showing only Bax-mediated cell death inhibition encode a CCCH-type zinc finger protein containing an ankyrin-repeat domain and a probable calcium-binding protein, CML30-like. Taken together, we effectively isolated novel pepper clones that are involved in hypersensitive response (HR)-like cell death using VIGS, and identified silenced clones that have different responses to Bax and INF1 exposure, indicating separate signaling pathways for Bax- and INF1-mediated cell death.     

Polyoxygenated Cembrane Diterpenoids from the Soft Coral Sarcophyton ehrenbergi

Five new polyoxygenated cembranoids, named (+)-1,15-epoxy-2-methoxy-12-methoxycarbonyl-11E-sarcophytoxide (1), (+)-2-epi-12-methoxycarbonyl-11E-sarcophine (2), 3,4-epoxyehrenberoxide A (3), ehrenbergol D (4) and ehrenbergol E (5), were obtained from the soft coral Sarcophyton ehrenbergi. The structures of 1–5 were established on the basis of comprehensive NMR and HR-ESI-MS analyses and by comparison with reported data in the literature. Compounds 4 and 5 showed moderate cytotoxicity against P-388 (mouse lymphocytic leukemia) cancer cell line with EC₅₀ values of 2.0 and 3.0 μM, respectively. Compound 2 exhibited slight antiviral activity against HCMV (human cytomegalovirus) with IC₅₀ values of 25.0 μg/mL.     

In Vitro Antiprotozoal Activity of Abietane Diterpenoids Isolated from Plectranthus barbatus Andr.

Chromatographic separation of the n-hexane extract of the aerial part of Plectranthus barbatus led to the isolation of five abietane-type diterpenes: dehydroabietane (1); 5,6-didehydro-7-hydroxy-taxodone (2); taxodione (3); 20-deoxocarnosol (4) and 6α,11,12,-trihydroxy-7β,20-epoxy-8,11,13-abietatriene (5). The structures were determined using spectroscopic methods including one- and two-dimensional NMR methods. Compounds (1)–(3) and (5) are isolated here for the first time from the genus Plectranthus. The isolated abietane-type diterpenes tested in vitro for their antiprotozoal activity against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi and free trypomastigotes of T. brucei. Cytotoxicity was determined against fibroblast cell line MRC-5. Compound (2) 5,6-didehydro-7-hydroxy-taxodone showed remarkable activity with acceptable selectivity against P. falciparum (IC₅₀ 9.2 μM, SI 10.4) and T. brucei (IC₅₀ 1.9 μM, SI 50.5). Compounds (3)–(5) exhibited non-specific antiprotozoal activity due to high cytotoxicity. Compound (1) dehydroabietane showed no antiprotozoal potential.     

Core RNAi Machinery and Sid1, a Component for Systemic RNAi,in the Hemipteran Insect,Aphis glycines

RNA interference (RNAi) offers a novel tool to manage hemipteran pests. For the success of RNAi based pest control in the field, a robust and systemic RNAi response is a prerequisite. We identified and characterized major genes of the RNAi machinery, Dicer2 (Dcr2), Argonaute2 (Ago2), and R2d2 in Aphis glycines, a serious pest of soybean. The A. glycines genome encodes for at least one copy of Dcr2, R2d2 and Ago2. Comparative and molecular evolution analyses (dN/dS) showed that domain regions of encoded proteins are highly conserved, whereas linker (non-domain) regions are diversified. Sequence homology and phylogenetic analyses suggested that the RNAi machinery of A. glycines is more similar to that of Tribolium casteneum as compared to that of Drosophila melanogaster. We also characterized Sid1, a major gene implicated in the systemic response for RNAi-mediated gene knockdown. Through qPCR, Dcr2, R2d2, Ago2, and Sid1 were found to be expressed at similar levels in various tissues, but higher expression of Dcr2, R2d2, and Ago2 was seen in first and second instars. Characterization of RNAi pathway and Sid1 in A. glycines will provide the foundation of future work for controlling one of the most important insect pests of soybean in North America.     

Characterization of Two Homogalacturonan Pectins with Immunomodulatory Activity from Green Tea

Two natural homogalacturonan (HG) pectins (M_W ca. 20 kDa) were isolated from green tea based on their immunomodulatory activity. The crude tea polysaccharides (TPS1 and TPS2) were obtained from green tea leaves by hot water extraction and followed by 40% and 70% ethanol precipitation, respectively. Two homogenous water soluble polysaccharides (TPS1-2a and TPS1-2b) were obtained from TPS1 after purification with gel permeation, which gave a higher phagocytic effect than TPS2. A combination of composition, methylation and configuration analyses, as well as NMR (nuclear magnetic resonance) spectroscopy revealed that TPS1-2a and TPS1-2b were homogalacturonan (HG) pectins consisting of a backbone of 1,4-linked α-d-galacturonic acid (GalA) residues with 28.4% and 26.1% of carboxyl groups as methyl ester, respectively. The immunological assay results demonstrated that TPS1-2, which consisted mainly of HG pectins, showed phagocytosis-enhancing activity in HL-60 cells.     

Differential Proteomic Analysis of Anthers between Cytoplasmic Male Sterile and Maintainer Lines in Capsicum annuum L

Cytoplasmic male sterility (CMS), widely used in the production of hybrid seeds, is a maternally inherited trait resulting in a failure to produce functional pollen. In order to identify some specific proteins associated with CMS in pepper, two-dimensional gel electrophoresis (2-DE) was applied to proteomic analysis of anthers/buds between a CMS line (designated NA3) and its maintainer (designated NB3) in Capsicum annuum L. Thirty-three spots showed more than 1.5-fold in either CMS or its maintainer. Based on mass spectrometry, 27 spots representing 23 distinct proteins in these 33 spots were identified. Proteins down-regulated in CMS anthers/buds includes ATP synthase D chain, formate dehydrogenase, alpha-mannosidas, RuBisCO large subunit-binding protein subunit beta, chloroplast manganese stabilizing protein-II, glutathione S-transferase, adenosine kinase isoform 1T-like protein, putative DNA repair protein RAD23-4, putative caffeoyl-CoA 3-O-methyltransferase, glutamine synthetase (GS), annexin Cap32, glutelin, allene oxide cyclase, etc. In CMS anthers/buds, polyphenol oxidase, ATP synthase subunit beta, and actin are up-regulated. It was predicted that male sterility in NA3 might be related to energy metabolism turbulence, excessive ethylene synthesis, and suffocation of starch synthesis. The present study lays a foundation for future investigations of gene functions associated with pollen development and cytoplasmic male sterility, and explores the molecular mechanism of CMS in pepper.