Proteome analysis of bell pepper (Capsicum annuum L.) chromoplasts

We report a comprehensive proteome analysis of chromoplasts from bell pepper (Capsicum annuum L.). The combination of a novel strategy for database-independent detection of proteins from tandem mass spectrometry (MS/MS) data with standard database searches allowed us to identify 151 proteins with a high level of confidence. These include several well-known plastid proteins but also novel proteins that were not previously reported from other plastid proteome studies. The majority of the identified proteins are active in plastid carbohydrate and amino acid metabolism. Among the most abundant individual proteins are capsanthin/capsorubin synthase and fibrillin, which are involved in the synthesis and storage of carotenoids that accumulate to high levels in chromoplasts. The relative abundances of the identified chromoplast proteins differ remarkably compared with their abundances in other plastid types, suggesting a chromoplast-specific metabolic network. Our results provide an overview of the major metabolic pathways active in chromoplasts and extend existing knowledge about prevalent metabolic activities of different plastid types.     

Cytomorphology of induced octoploid Chili pepper (Capsicum annuum L.)

Summary Octoploidy was induced in Chili pepper (Capsicum annuum cultivar cerasiformis) through the application of colchicine and the cytomorphological features of two octoploid plants were described. In general, the octoploids did not exhibit gigas characters when compared to the tetraploids; on the contrary they were less vigorous, suggesting that the optimum and desirable ploidy level for Capsicum is probably tetraploid. Chromosome associations such as octovalents and hexavalents, in addition to IVs, IIIs, IIs and Is, were recorded at diakinesis and metaphase I. Meiosis was highly irregular and the pollen and seed fertility was very low. Cytological features of octoploid Chili peppers are compared with octoploids of Physalis and Petunia.     

EMS-induced lincomycin resistance in red pepper (Capsicum annuum L.)

Summary Ethyl methane sulphonate (EMS) is a potential mutagen to induce lincomycin resistance in Capsicum annuum. Mutagenized cotyledons were cultured on shoot regenerating medium containing lincomycin (100 mgl⁻¹). Approximately 14% of regenerated shoots were chlorophyll deficient and about 4% of regenerated shoots were green from mutaganized cotyledons. The regenerated green plants were resistant to lincomycin but sensitive to chloramphenicol, kanamycin, spectinomycin, and streptomycin. Reciprocal crosses were made between resistant and sensitive plants. Inheritance of lincomycin resistance was transmitted as a non-Mendelian trait. Lincomycin resistance is a first selectable and maternally inherited organelle encoded genetic marker described in chili pepper. Such mutants should be useful in designing biochemical selection schemes to recover somatic hybrids and cybrids.     

Plant regeneration from sweet pepper (Capsicum annuum L.) hypocotyl explants

Morphogenetic potential of hypocotyl and cotyledon explants of the three Polish Capsicum annuum L. cultivars (Kujawianka, Passat and Zorza) was studied to develop a reliable plant regeneration protocol. Out of 8 and 15 combinations of growth regulators used in the first and second series respectively, the best medium contained BAP (5 mg·l⁻¹) and IAA (1 mg·l⁻¹). Media containing thidiazuron (TDZ) and IAA proved to be worse than those with BAP and IAA. Additionally, it was indicated that hypocotyl explants placed upside-down developed more adventious buds. ‘Passat’ was the most responsive variety (approximately 40 % of both types of explants produced buds). In the second part of experiment the aim was to stimulate shoot induction in the conditions most adapted to Agrobacterium transformation. ‘Bryza’ replaced cv ‘Kujawianka’ and proved to be better than ‘Passat’, previously distinguished as a highly responsive cultivar. The experiments confirmed a significant effect of the hypocotyl explant length and induction period on shoot regeneration. Finally, the optimum concentration of carbenicillin and kanamycin was determined.     

Cytokinins and leaf development in sweet pepper (Capsicum annuum L.)

Stimulation of leaf expansion by an exogenous cytokinin was studied in isolated leaf discs of sweet pepper with emphasis on the assimilate utilization of the tissue. Leaf discs were floated on solutions containing sucrose and plant growth regulators. Benzyladenine (BA) promoted the area expansion rate of the leaf discs. Sucrose at 100 mM resulted in increased area expansion rate compared with 10 mM sucrose. However, the increased sucrose concentration had no influence on the effect of BA. Over a period of 24 h, treatment with BA did not result in any change of sucrose uptake nor of the partitioning of assimilated carbon in the leaf discs. Neither did BA treatment affect the activity of acid invertase (EC 3.2.1.26) or pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) in the leaf discs. We conclude that the observed promotion of leaf area expansion by exogenous BA is not mediated through the uptake of sucrose or the carbohydrate metabolism of the leaf tissue.Abbreviations BA N⁶-benzyladenine - GA₃ gibberellic acid - PPi-PFK pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) This study was supported by grants from the Danish Research Counsil (SJVF 13-4148 and 13-4547 to P.U. SJVF 13-4146 and 13-4494 to T.H.N.) and from The Research Center for Plant Biotechnology to P.U.     

Androgenesis induction in microspore culture of sweet pepper (Capsicum annuum L.)

Isolated microspore culture experiments were carried out in sweet pepper (Capsicum annuum L.) F₁ hybrid genotypes. In the first experiment, four culture media (W14, B5, MS and NLN) were compared to test their effectiveness in inducing the formation of microspore-derived structures in two genotypes. The experiments revealed the superiority of B5 medium. In the second experiment, the effects of different ratios of 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.1, 0.2 and 0.5 mg l⁻¹) and kinetin (0, 0.2 and 0.5 mg l⁻¹) were also investigated in B5 medium with two genotypes. The effect of growth regulators were investigated on the production of microspore-derived calli and embryo-like structures (ELSs), the ratio of the two and plant regeneration (number of regenerated plantlets) in microspore culture. The histological experiments revealed the differences between the microspore-derived ELSs and calli. The most promising results were obtained on the investigated parameters in the presence of 0.1 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ kinetin producing the highest number of plantlets in both genotypes tested. In the response of 11 genotypes, the androgenesis induction was successful in each sweet pepper genotypes tested using the best basic medium and growth regulators combination. In case of 11 genotypes, the number of ELSs ranged from 20 to 100/Petri dish (an average of 48.1 ELS/Petri dish), while the number of green plantlets varied from 0 to 8 plantlets/Petri dish (an average of 1.5 plantlets/Petri dish) depending on the genotype. The spontaneous rediploidization rate obtained was 25% in isolated microspore.     

Suppression of ethylene levels promotes morphogenesis in pepper (Capsicum annuum L.)

Ethylene and polyamines (PAs) are two phytohormones that play important roles during in vitro morphogenesis of several plant species. The interaction between ethylene and PAs has been of interest because both have S-adenosylmethionine as a precursor. To study the influence of ethylene and PAs on in vitro morphogenesis of an ornamental pepper, we added an ethylene scavenger, PAs, a PA inhibitor, and compounds that affect ethylene biosynthesis and activity to the regeneration medium. Regeneration frequencies increased in response to treatment with ethylene inhibitors (aminoethoxyvinylglycine and silver thiosulfate) and an ethylene scavenger (mercury perchlorate). Treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid reduced the regeneration frequency, increased callus formation, and increased ethylene levels; similar results were obtained in response to treatment with the PA inhibitor methylglyoxal-bis(guanylhydrazone). By contrast, treatment with PAs (particularly spermidine and spermine) decreased ethylene levels, increased the regeneration frequency, and increased shoot bud formation. These results suggest a coordinated regulation of ethylene and polyamines because the suppression of ethylene levels using ethylene inhibitors, polyamines, or mercury perchlorate increased the in vitro regeneration frequency and morphogenic responses of Capsicum annuum L.     

Increased capsaicin content in PFP-resistant cells of chili pepper (Capsicum annuum L.)

Cell suspensions of chili pepper (Capsicum annuum L.) were subjected to a selection process on semisolid medium containing the amino acid analog p-fluorophenylalanine (PFP). Four cell lines with different degrees of resistance were selected and suspension cultures were established from each of them. Resistance was retained even after 75 days of culture in the absence of PFP. PFP-resistant cell lines accumu lated higher levels of capsaicin than sensitive lines even after prolonged culture in PFP-free medium. Capsaicin production in non-selected cells was only 26.8% of that found in one cell line resistant to 500 M PFP. The capsaicin content in the non-selected cell suspension and in one of the resis tant cell lines was 6.7% and 24.9% respectively, that of fruits.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - PFP p-fluorophenylalanine - d. wt. dry weight - f. wt. fresh weight     

Alleviation of low-temperature photoinhibition in gamma-irradiated red pepper (Capsicum annuum L.) plants

We studied the radiation-induced stress resistance in red pepper leaves under conditions of low-temperature photoinhibition or artificially induced photo-oxidative stress. Plants irradiated with 4, 8, or 16-Gy gamma rays were more resistant to both stress factors than were the controls. However, exposure to a low temperature for 12 h with illumination or photo-oxidative treatment for 1 h differentially affected the irradiated leaves, although they had similar stress intensities as defined by their maximal photochemical efficiencies (Fv/Fm). Decreases in Fv/Fm induced by the two stress factors were instead alleviated, dose-dependently, by as much as 22 to 41% (low temperature) or 14 to 29% (photo-oxidation) in the irradiated groups. In contrast, non-photochemical quenching (NPQ) and the de-epoxidation state of xanthophyll cycle pigments could not be correlated with this enhanced stress resistance in the irradiated groups. These results suggest that the adaptive response of plants exposed to gamma radiation is more effective in protecting against low-temperature photoinhibition than against photo-oxidative stress. We also discuss here the involvement of antioxidative defense systems for increased resistance against low-temperature photoinhibition in irradiated red pepper.     

Establishment of a highly efficient transformation system for pepper (Capsicum annuum L.)

Application of modern genetic manipulation has been limited in pepper ( Capsicum annuum L.) due to the lack of an efficient transformation system. Following the development of an efficient protocol for in vitro regeneration of pepper cotyledons, we investigated the key factors affecting transformation and established a highly efficient genetic transformation system using the pepper cotyledon as starting material. In this system, cotyledon explants are preconditioned for 2 days on kanamycin (km)-free DM1 medium [Murashige and Skoog (MS) salts/Gamborg B5 vitamins basal medium supplemented with 20 g/l sucrose, 5,000 mg/l DJ nutrients and a hormone combination of 1.0 mg/l indoleacetic acid (IAA) and 5.0 mg/l 6-benzyladenine (BA) solidified with 0.7% agar, pH 5.8], followed by co-cultivation with Agrobacterium tumefaciens on DM1 for 2 days and delay selection on DM1 with 500 mg/l carbenicillin (carb) for 2 days. The explants are then placed on DM1 containing 10 mg/l AgNO(3), 50 mg/l km-sulfate and 500 mg/l carb. After 4-5 weeks, the explants with buds are transferred to EM1 medium (MS salts/Gamborg B5 vitamins basal medium supplemented with 20 g/l sucrose, 5,000 mg/l DJ nutrients, 10 mg/l AgNO(3) and a hormone combination of 1.0 mg/l IAA, 3.0 mg/l BA and 2.0 mg/l gibberellic acid, solidified with 0.7% agar, pH 5.8) with 50 mg/l kanamycin and 500 mg/l carbenicillin for the elongation of buds. After 3-6 weeks, 1- to 2-cm-long elongated shoots are excised and planted on RM1 medium (MS basal medium supplemented with a hormone combination of 0.2 mg/l NAA and 0.1 mg/l IAA, solidified with 0.8% agar, pH 5.8) with 25 mg/l km and 200 mg/l carb for rooting. We tested four genotypes of pepper, and all presented a high differentiation efficiency (81.3% on average), elongation rate (61.5%) and rooting efficiency (89.5%). Polymerase chain reaction analysis results showed that 40.8% of the regenerated plantlets were transgenic plants.     

Isolation and characterization ofo-diphenol-O-methyltransferase cDNA clone in hot pepper (Capsicum annuum L.)

A cDNA clone,CaOMTl encoding ano-diphenol-O-methyltransferase (OMT), which is involved in capsaicin biosynthesis, was isolated by screening of a cDNA library prepared from the mRNA of pepper (Capsicum annuum L.) pericarp. Nucleotide sequence analysis ofCaOMTl revealed that it had an open reading frame of 1080 bp which encodes a polypeptide with a predicted molecular weight of 39,430 D, corresponding well with the size of the known OMT’s of tobacco, poplar, aspen, alfalfa, and cabbage. It also had five conserved boxes which appear in all known OMT’s. The nucleotide sequence ofCaOMTl had 89–74% identity with the OMT cDNA’s of tobacco, aspen, alfalfa, and poplar, but a relatively lower identity of 59% with the OMT cDNA of maize. Amino acid sequence analysis also revealed that CaOMT1 has high identity with the known OMT’s which have a substrate ofo-diphenolic compounds, especially 5-hydroxyferulic acid and caffeic acid. It supportsCaOMTl which encodes an OMT. Southern blot analysis suggested thatCaOMTl might exist in the form of multiple copies in the pepper genome.CaOMTl is expressed preferentially in pepper fruit and its expression levels increased during pepper fruit development, but decreased during fruit ripening, suggesting that theCaOMTl gene is fruit development-related.CaOMTl is the first reported cDNA clone for enzymes related to the phenlypropanoid pathway in pepper.     

An improved and reliable chili pepper (Capsicum annuum L.) plant regeneration method

In this work we report a new method forin vitro chili pepper (Capsicum annuum L.) plant regeneration based on shoot formation from wounded hypocotyls. Chili pepper seeds were surface sterilized and germinated on agar (0.8%) at 25 ± 2°C in the dark. Five factors that may influence shoot regeneration were studied: age of seedlings, hypocotyl wounding site, time elapsed between wounding the hypocotyls and decapitation of seedlings, culture media and cultivars. In order to study the influence of the first three factors on shoot regeneration, the apical, middle or basal hypocotyl regions of seedlings of cv. Mulato Bajio at different stages of development (9, 15, 16, 21 and 28 d old) were wounded with a syringe needle, and the seedlings were cultured on MS semisolid medium without growth regulators at 25 ± 2°C under a 16/8 h light/dark photoperiod (daylight fluorescent lamps; 35 mol m-² s-^-1) until decapitation. The seedlings were decapitated (3 mm below the cotyledons) at different times after wounding (0, 2, 4, 10, 12 and 14 d), and each explant was evaluated for bud and shoot formation ( 5 mm in length) at the wounded site after 30 d of incubation. In general, seedlings at the stage of curved hypocotyl (9 d old) wounded in the apical region of hypocotyl were the best explants for shoot regeneration when inoculated on culture medium without growth regulators. Decapitation after wounding also influenced the shoot regeneration efficiency, with 10–14 d being the best period. Up to 90% shoot regeneration in cv. Mulato Bajio was obtained under these conditions. Statistically significant differences were observed for shoot formation among 21 cultivars tested. Regeneration of whole plants was achieved by rooting the shoots with indole-3-butyric acid pulses of 60 mg L^–1 for 3 h and then subculturing on MS medium without growth regulators.     

Microspore-derived embryogenesis in pepper (Capsicum annuum L.): subcellular rearrangements through development

Background information. In vitro-cultured microspores, after an appropriate stress treatment, can switch towards an embryogenic pathway. This process, known as microspore embryogenesis, is an important tool in plant breeding. Basic studies on this process in economically interesting crops, especially in recalcitrant plants, are very limited and the sequence of events is poorly understood. In situ studies are very convenient for an appropriate dissection of microspore embryogenesis, a process in which a mixture of different cell populations (induced and non-induced) develop asynchronically.Results. In the present study, the occurrence of defined subcellular rearrangements has been investigated during early microspore embryogenesis in pepper, an horticultural crop of agronomic interest, in relation to proliferation and differentiation events. Haploid plants of Capsicum annuum L. (var. Yolo Wonder B) have been regenerated from in vitro anther cultures by a heat treatment at 35 degrees C for 8 days. Morphogenesis of microspore-derived embryos has been analysed, at both light and electron microscopy levels, using low-temperature-processed, well-preserved specimens. The comparison with the normal gametophytic development revealed changes in cell organization after embryogenesis induction, and permitted the characterization of the time sequence of a set of structural events, not previously defined in pepper, related to the activation of proliferative activity and differentiation. These changes mainly affected the plastids, the vacuolar compartment, the cell wall and the nucleus. Further differentiation processes mimicked that of the zygotic development.Conclusions. The reported changes can be considered as markers of the microspore embryogenesis. They have increased the understanding of the mechanisms controlling the switch and progression of the microspore embryogenesis, which could help to improve its efficiency and to direct strategies, especially in agronomically interesting crops.     

Induction of direct somatic embryogenesis and plant regeneration in pepper (Capsicum annuum L.)

Summary Pepper (cv. New Mexico — 6 and Rajur Hirapur) plants were regenerated from immature zygotic embryos via direct somatic embryogenesis. Somatic embryos were formed directly, without any intervening callus, on the zygotic embryo apex, embryo axis and cotyledons on Murashige and Skoog's (MS) medium containing 2,4-D (418 M), thidiazuron (10 M) and a high concentration of sucrose (6–10%). The best response was observed on MS medium containing 2,4-D (9 M), coconut water (10%) and high sucrose (8%). The entire process of induction and maturation of the embryos was completed on the same medium. Histological examination indicated that secondary embryogenesis also occurred directly from the primary somatic embryos. Differentiation of embryos was nonsynchronous, and some embryos were swollen and distorted with fasciation. More than 70% of the mature normal somatic embryos germinated readily on MS medium containing GA₃ or TDZ, alone and in combination, and following transfer to pots developed into normal plants.Abbreviations CM Coconut milk - 2,4-D 2,4-dichlorophonoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) medium - NAA napthaleneacetic acid - TDZ thidiazuron     

辣椒31个优良自交系的亲本类群分析

以包含我国重要尖椒品种的亲本材料在内的31份优良自交系为材料, 利用SRAP标记和基因型值分析技术开展了辣椒自交系间遗传差异的分析与类群划分研究。结果表明: 在30个引物组合中, 27个引物组合可以 在自交系间扩增出多态性条带, 共扩增出310个多态性条带, 平均每个引物组合产生11.5个多态性条带, 显示出SRAP技术具有较强的分析效率; 基于SRAP标记和Yule相似系数对这些自交系进行的聚类分析中, 可以基本区分辣椒的2个变种(C. annuum var. grossum和C. annuum var. longum), 而且可以反映出自交系间的亲缘及系谱关系; 在相似系数为0.67处, 可将这31个自交系分为4个类群; 基于基因型值和标准Euclidean距离对这些自交系进行的聚类分析可成功地将辣椒的两个变种完全区分; 在遗传距离约4.5处, 可将这31个自交系分为4个类群; 自交系间基于SRAP标记与基因型值的遗传距离存在一定的相关性。     

Bacterial endophytes contribute to abiotic stress adaptation in pepper plants (Capsicum annuum L.)

Endophytes are nonpathogenic plant-associated bacteria that can play an important role in plant vitality and may confer resistance to abiotic or biotic stress. The effects of 5 endophytic bacterial strains isolated from pepper plants showing 1-aminocyclopropane-1-carboxylate deaminase activity were studied in sweet pepper under in vitro conditions. Four of the strains tested showed production of indole acetic acid. Plant growth, osmotic potential, free proline content, and gene expression were monitored in leaves and roots under control and mild osmotic stress conditions. All indole acetate producers promoted growth in Capsicum annuum L. 'Ziegenhorn Bello', from which they were isolated. Osmotic stress caused an increase in the content of free proline in the leaves of both inoculated and noninoculated plants. Inoculated control plants also revealed higher proline levels in comparison with noninoculated control plants. Differential gene expression patterns of CaACCO, CaLTPI, CaSAR82A, and putative P5CR and P5CS genes during moderate stress were observed, depending on the bacterium applied. Inoculation with 2 bacterial strains, EZB4 and EZB8 (Arthrobacter sp. and Bacillus sp., respectively), resulted in a significantly reduced upregulation or even downregulation of the stress-inducible genes CaACCO and CaLTPI, as compared with the gene expression in noninoculated plants. This indicates that both strains reduced abiotic stress in pepper under the conditions tested.     

Development of SCAR markers for early identification of cytoplasmic male sterility genotype in chili pepper (Capsicum annuum L.)

We previously used Southern blot analysis to detect restriction-length polymorphisms between male fertile and cytoplasmic male sterile (CMS) cytoplasms at the coxII and atp6 loci of the mtDNA of Capsicum annuum L. Two copies of atp6 were found in each male fertile and CMS pepper lines. Interestingly, one of the copies of atp6 in CMS pepper was a 3'-truncated pseudogene. The open reading frame of the coxII gene was the same in the fertile (N-) and CMS (S-) lines. However, the nucleotide sequence in the S-cytoplasm diverged from that in the N-cytoplasm 41 bp downstream of the stop codon. To develop CMS-specific sequence-characterized amplified region (SCAR) markers, inverse PCR was performed to characterize the nucleotide sequences of the 5' and 3' flanking regions of mitochondrial atp6 and coxII from the cytoplasms of male fertile (N-) and CMS (S-) pepper plants. Based on these data, two CMS-specific SCAR markers, 607 and 708 bp long, were developed to distinguish N-cytoplasm from S-cytoplasm by PCR. The CMS-specific PCR bands were verified for 20 cultivars containing either N- or S-cytoplasm. PCR amplification of CMS-specific mitochondrial nucleotide sequences will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of F1 seed lots. The strategy used in this report for identifying CMS-specific markers could be adopted for many other crops where CMS is used for F1 seed production.