Evolution of Capsaicinoids in Peter Pepper (Capsicum annuum var. annuum) During Fruit Ripening

The evolution of individual and total contents of capsaicinoids present in Peter peppers (Capsicum annuum var. annuum) at different ripening stages has been studied. Plants were grown in a glasshouse and the new peppers were marked in a temporal space of ten days. The extraction of capsaicinoids was performed by ultrasound‐assisted extraction with MeOH. The capsaicinoids nordihydrocapsaicin (n‐DHC), capsaicin, dihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin were analyzed by ultraperformance liquid chromatography (UHPLC)‐fluorescence and identified by UHPLC‐Q‐ToF‐MS. The results indicate that the total capsaicinoids increase in a linear manner from the first point of harvest at ten days (0.283 mg/g FW) up to 90 days, at which point they reach a concentration of 1.301 mg/g FW. The evolution as a percentage of the individual capsaicinoids showed the initial predominance of capsaicin, dihydrocapsaicin, and n‐DHC. Dihydrocapsaicin was the major capsaicinoid up to day 50 of maturation. After 50 days, capsaicin became the major capsaicinoid as the concentration of dihydrocapsaicin fell slightly. The time of harvest of Peter pepper based on the total capsaicinoids content should be performed as late as possible. In any case, harvesting should be performed before overripening of the fruit is observed.     

Shikimate dehydrogenase from pepper (Capsicum annuum) seedlings. Purification and properties

Shikimate dehydrogenase (SKDH, EC 1.1.1.25) was extracted from seedlings of pepper ( Capsicum annuum L.) and purified 347-fold. The purification procedure included precipitation with ammonium sulphate and chromatography in columns of Reactive Red-agarose, Q-Sepharose and Sephadex G-100. Pepper SKDH isozymes are separable only using PAGE. The purified enzyme has a relative molecular mass of 67 000 as estimated by gel filtration. The optimum pH of enzyme activity is 10.5 and the optimum temperature is 50°C, but the enzyme is quickly inactivated at temperatures higher than 40°C. The purified enzyme exhibited typical Michaelis-Menten kinetics and K_m values are 0.087 m M for shikimic acid and 0.017 m M for NADP. The mechanism of reaction is sequential considering NADP as a cosubstrate. Ions such as Ca²⁺, Mg²⁺ and Mn²⁺ activate the enzyme, but Zn²⁺ and Cu²⁺ are strong inhibitors. Some phenolic compounds such as guaiacol, protocatechuic acid and 2,4-D are competitive inhibitors of pepper SKDH, showing K_i values of 0.38 m M , 0.27 m M and 0.16 m M , respectively.     

Isolation and structure elucidation of flavonoid and phenolic acid glycosides from pericarp of hot pepper fruit Capsicum annuum L

Hot pepper fruits (Capsicum annuum L.) var. Bronowicka Ostra have been studied with regard to content of flavonoids and other phenolics. Nine compounds were isolated from pericarp of pepper fruits by preparative HPLC. Their structures were identified by chromatographic (analytical HPLC) and spectroscopic (UV, NMR) techniques. Two of the identified compounds, trans-p-ferulylalcohol-4-O-(6-(2-methyl-3-hydroxypropionyl) glucopyranoside and luteolin-7-O-(2-apiofuranosyl-4-glucopyranosyl-6-malonyl)-glucopyranoside were found for the first time in the plant kingdom. Additionally compounds: trans-p-feruloyl-beta-D-glucopyranoside, trans-p-sinapoyl-beta- D-glucopyranoside, quercetin 3-O-alpha-L-rhamnopyranoside-7-O-beta-D-glucopyranoside, luteolin 6-C-beta-D-glucopyranoside-8-C-alpha-L-arabinopyranoside, apigenin 6-C-beta-D-glucopyranoside-8-C-alpha-L-arabinopyranoside and luteolin 7-O-[2-(beta-D-apiofuranosyl)-beta-D-glucopyranoside] were found for the first time in pepper fruit Capsicum annuum L.     

Preparation and properties of L-asparaginase from green chillies (Capsicum annum L.)

Green chillies(Capsicum annum L.) and tamarind (Tamarindus indica) contain appreciable amount of L-asparaginase. The enzyme was purified 400-fold from green chillies, by successive precipitations with ammonium sulphate and sodium sulphate, Sephadex-gel filtration and affinity chromatography and the purified enzyme was homogenous on gel electrophoresis. The enzyme exists in two forms, only one having antitumour activity. The purified enzyme has a molecular weight of 120,000 ±500. The N-terminal and the C-terminal amino acids are alanine and phenylalanine, respectively. The enzyme has a sharp optimum pH of 8.5 and a temperature optimum of 37‡C. It is stable upto 40‡C. The energy of activation is 3 kilo calories. The K_m value for the enzyme is 3.3. mm. The enzyme has little action on D-asparagine, which is a strong inhibitor. The enzyme has inseparable glutaminase ctivity and is thus an asparaginase—glutaminase. In addition, it possesses urease activity.     

Genetic structure and differentiation of wild and domesticated populations of Capsicum annuum (Solanaceae) from Mexico

 Genetic variation and structure of ten wild, three domesticated and one wild-cultivated populations of pepper (Capsicum annuum) from northwestern Mexico were studied in order to find out if the domestication process has reduced the genetic variation of the modern cultivars of this species. The analysis was based on 12 polymorphic loci from nine isozymes. Wild populations were sampled in different habitats along a latitudinal gradient of ca. 500 km. All populations had high genetic variation (i.e. wild: A = 2.72, P = 90.8%, He = 0.445; wild-cultivated: A = 2.50, P = 92.3%, He = 0.461; domesticated: A = 2.60, P = 84.6%, He = 0.408), indicating little genetic erosion in modern cultivars of pepper. Genetic diversity estimated by Nei's method showed that most genetic variation is found within, rather than among populations. However, genetic differentiation is greater among cultivated (G _ST=0.167) than among wild (G _ST=0.056) populations. Wild populations had an average genetic identity (I) of 0.952, indicating little differentiation and high gene flow (Nm=4.21) among these populations. Average genetic identity between wild and domesticated populations was of I=0.818, revealing that the domestication process has modified the genetic composition of commercial varieties of pepper. Changes in genetic composition among commercial varieties seem to have occurred in different directions, as indicated by the average value of I = 0.817 among these populations. The high level of diversity found in wild populations of C. annuum suggests that the wild relatives of cultivated peppers are a valuable genetic resource which must be conserved. Received May 5, 1999 Accepted October 30, 2000     

Ripening of pepper (Capsicum annuum) fruit is characterized by an enhancement of protein tyrosine nitration

Background and Aims Pepper (Capsicum annuum, Solanaceae) fruits are consumed worldwide and are of great economic importance. In most species ripening is characterized by important visual and metabolic changes, the latter including emission of volatile organic compounds associated with respiration, destruction of chlorophylls, synthesis of new pigments (red/yellow carotenoids plus xanthophylls and anthocyanins), formation of pectins and protein synthesis. The involvement of nitric oxide (NO) in fruit ripening has been established, but more work is needed to detail the metabolic networks involving NO and other reactive nitrogen species (RNS) in the process. It has been reported that RNS can mediate post-translational modifications of proteins, which can modulate physiological processes through mechanisms of cellular signalling. This study therefore examined the potential role of NO in nitration of tyrosine during the ripening of California sweet pepper.Methods The NO content of green and red pepper fruit was determined spectrofluorometrically. Fruits at the breaking point between green and red coloration were incubated in the presence of NO for 1 h and then left to ripen for 3 d. Profiles of nitrated proteins were determined using an antibody against nitro-tyrosine (NO₂-Tyr), and profiles of nitrosothiols were determined by confocal laser scanning microscopy. Nitrated proteins were identified by 2-D electrophoresis and MALDI-TOF/TOF analysis.Key Results Treatment with NO delayed the ripening of fruit. An enhancement of nitrosothiols and nitroproteins was observed in fruit during ripening, and this was reversed by the addition of exogenous NO gas. Six nitrated proteins were identified and were characterized as being involved in redox, protein, carbohydrate and oxidative metabolism, and in glutamate biosynthesis. Catalase was the most abundant nitrated protein found in both green and red fruit.Conclusions The RNS profile reported here indicates that ripening of pepper fruit is characterized by an enhancement of S-nitrosothiols and protein tyrosine nitration. The nitrated proteins identified have important functions in photosynthesis, generation of NADPH, proteolysis, amino acid biosynthesis and oxidative metabolism. The decrease of catalase in red fruit implies a lower capacity to scavenge H₂O₂, which would promote lipid peroxidation, as has already been reported in ripe pepper fruit.     

Response of Resistant and Susceptible Bell Pepper (Capsicum annuum) to a Southern California Meloidogyne incognita Population from a Commercial Bell Pepper Field

To determine the presence and level of root-knot nematode (Meloidogyne spp.) infestation in Southern California bell pepper (Capsicum annuum) fields, soil and root samples were collected in April and May 2012 and analyzed for the presence of root-knot nematodes. The earlier samples were virtually free of root-knot nematodes, but the later samples all contained, sometimes very high numbers, of root-knot nematodes. Nematodes were all identified as M. incognita. A nematode population from one of these fields was multiplied in a greenhouse and used as inoculum for two repeated pot experiments with three susceptible and two resistant bell pepper varieties. Fruit yields of the resistant peppers were not affected by the nematodes, whereas yields of two of the three susceptible pepper cultivars decreased as a result of nematode inoculation. Nematode-induced root galling and nematode multiplication was low but different between the two resistant cultivars. Root galling and nematode reproduction was much higher on the three susceptible cultivars. One of these susceptible cultivars exhibited tolerance, as yields were not affected by the nematodes, but nematode multiplication was high. It is concluded that M. incognita is common in Southern California bell pepper production, and that resistant cultivars may provide a useful tool in a nonchemical management strategy.     

Tapetum Dimorphism and Its Histochemical Study in Sweet Pepper (Capsicum annuum L. Var. grossum)

In sweet pepper, the portion of tapetum toward the interior of the anther comprising large cells is derived from cells of connective of anther whereas the remaining tapetum on the outside of the anther comprising comparatively small cells is derived from the parietal layer. Those ceils of the former prosessing large vacuoles and large nuclei are stained weaker than the cells of the latter by methyl green-pyronin and mercuric-bromophenol blue staining. Large spherical grains which contain acid phosphatase appear in the vacuoles in both kinds of tapetum at sporogenesis stage. During meiosis of pollen mother cells, DNA, RNA and protein sysntheses increase in tapetum. The tapetum derived from connective accumulates more DNA than that derived from parietal layer. The activity of acid phosphatase becomes higher in tapetum when it degenerates. The degeneration of two kinds of tapetum is similar. There are no starch grains in tapetum through its whole course of development.     

Limonoids from the stems of Toona ciliata var. henryi (Meliaceae)

Ten limonoids, toonacilianins A-J, and two norlimonoids, toonacilianins K and L, together with seven known compounds were isolated from the stems of Toona ciliata var. henryi (Meliaceae). Their structures were elucidated by spectroscopic analysis. Two compounds showed strong cytotoxic activities.     

Identification and development of polymorphic EST-SSR markers by sequence alignment in pepper, Capsicum annuum (Solanaceae)

? Premise of the study: The redundancies in expressed sequence tags (ESTs) in the National Center for Biotechnology Information sequence database were used to identify and develop polymorphic simple sequence repeat (SSR) markers for pepper (Capsicum annuum). ? Methods and Results: Sixty-eight polymorphic SSR loci were identified in the contigs (containing redundant ESTs) generated by assembling 118060 pepper ESTs from the public sequence database. Thirty-three SSR markers exhibited polymorphism among 31 pepper varieties, with alleles per SSR marker ranging from two to six. The mean observed and expected heterozygosity were 0.28 and 0.39, respectively. There were 18 SSR markers with a motif repeat number of less than five, accounting for 55% of the total. ? Conclusions: We demonstrated the value of mining the redundant sequences in public sequence databases for the development of polymorphic SSR markers, which can be used for marker-assisted breeding in pepper.     

Isolation of coniferyl esters from Capsicum baccatum L., and their enzymatic preparation and agonist activity for TRPV1

Coniferyl esters--capsiconiate and dihydrocapsiconiate--were isolated from the fruits of the pepper, Capsicum baccatum L. var. praetermissum. Their structures were determined by spectroscopic methods to be coniferyl (E)-8-methyl-6-nonenoate (capsiconiate) and coniferyl 8-methylnonanoate (dihydrocapsiconiate). This finding was further confirmed by the lipase-catalyzed condensation of coniferyl alcohol with its corresponding fatty acid derivative. The agonist activity of the esters for transient receptor potential vanilloid 1 (TRPV1) was evaluated by conducting an analysis of the intracellular calcium concentrations in TRPV1-expressing HEK293 cells. The EC50 values of capsiconiate and dihydrocapsiconiate were 3.2 and 4.2 microM, respectively.     

Functional study of Capsicum annuum fatty acid desaturase 1 cDNA clone induced by Tobacco mosaic virus via microarray and virus-induced gene silencing

A series of microarray analyses employing the expressed sequence tags (ESTs) of hot pepper was conducted in an effort to elucidate the molecular mechanisms inherent to hypersensitive response (HR) by viral or bacterial pathogens. There were 2535 ESTs exhibiting differential expression (over 2-fold changes) among about 5000 ESTs during viral or bacterial response. Further, via virus-induced gene silencing (VIGS) and TMV-infection studies, we were able to isolate several ESTs, which may be relevant to defense response against TMV. Of these ESTs, Capsicum annuum fatty acid desaturase 1 (CaFAD1) showed the distinct phenotype against TMV infection and thus was subjected to further study. CaFAD1-silenced plants showed weaker resistance against TMV-P0 infection compared to TRV2 control plants. Also the suppression of FAD1 expression caused blocking of cell death induced by Bcl2-associated X (Bax) protein in tobacco plants. Therefore, this report presents that both microarray and VIGS approaches are feasible in hot pepper plants and the TMV-induced CaFAD1 plays a role in HR response.     

Cornutins C-L,neo-clerodane-type diterpenoids from Cornutia grandifolia var. intermedia

Ten novel neo-clerodane diterpenoids, named cornutins C-L, have been isolated from the leaves of Cornutia grandifolia var. intermedia. Their structures have been elucidated by detailed spectroscopic analysis. In addition, the in vitro antiplasmodial activity of four isolated compounds (cornutin C-F) has been evaluated, revealing only a marginal activity.     

Terpenoids from Juniperus polycarpus var. seravschanica

Two eudesmanes, two abietanes, two podocarpanes and other nine known compounds were isolated from the dried fruits of Juniperus polycarpus var. seravschanica. Their structures were established on the basis of analysis of spectroscopic evidence. Some of the isolated terpenoids showed antimalarial activity.