Accumulation of Capsaicin in Seed, Pericarp and Placenta of Capsicum annuum L Fruit

The accumulation of capsaicin in different parts of fruit viz, placenta, pericarp and seeds of Capsicum annuum L cv Punjab Lal was compared with the activities of first four enzymes of capsaicin biosynthetic pathway at various physiological stages. Capsaicin accumulation (mg g^?1 DW) was about ten fold higher in placenta (63.96), than in pericarp (7.12) and seeds (5.06) in ripe fruits. Capsaicin accumulation was 5.79 mg g^?1 DW at 28 DAF in whole fruit. The specific activity of PAL was also ten times higher in placenta, whereas the specific activities of Ca4H, Ca3H and CaOMT were about two times higher in placenta than in other parts of fruit. The trend CaOMT > PAL > Ca4H > Ca3H was observed with peak activity at 28 DAF for Ca3H and CaOMT and at 35 DAF for PAL and Ca4H in placenta. These four enzymes showed low activity during the period up to 21 DAF, and peak activities for these enzymes were obtatined at the time of maximum growth of fruit in length and thereafter.     

Toxicity of Some Terpenoids Against Fungi Infesting Fruits and Seeds of Capsicum annuum L. During Storage

On mycoflora analysis of dried fruits and seeds of chilli stored for six months, Aspergillus flavus and A. niger were the more frequent out of eighteen fungi isolated. Some terpenoids viz, Citral, Citronellal, Citronellol, Eugenol, Farnesol and Nerol showed absolute toxicity against A. flavus and A. niger at 0.5 per cent concentration. The minimum inhibitory concentration (MIC) and nature (fungistatic/fungicidal) of all the terpenoids against both these fungi were determined, Citral, Eugenol and Nerol exhibited a broad range of toxicity and were found to be more efficacious than some synthetic fungicides.     

In Vivo and In Vitro Formation of Dihydrocapsaicin in Sweet Pepper Fruits,Capsicum annuum L. var. grossum

Dihydrocapsaicin, one of pungent principles in Capsicum fruits, was formed and accumulated in sweet pepper fruits after 6 days’ post-harvest ripening under continuous light in a medium containing vanillylamine and isocapric acid. No capsaicinoids were formed in sweet pepper fruits ripened in the dark even in the presence of both vanillylamine and isocapric acid. The capsaicinoid newly formed during the ripening was almost exclusively dihydrocapsaicin, as much as 92.8% of the total capsaicinoids. Dihydrocapsaicin was also formed by cell-free extracts prepared from the sweet pepper fruits in a reaction mixture containing vanillylamine and isocapric acid. Dihydrocapsaicin formed was quantified by TLC, GLC, GC-MS and MF.     

Properties of chlorophyllase from Capsicum annuum L. fruits

The in vitro properties of semi-purified chlorophyllase (chlorophyll-chlorophyllido hydrolase, EC 3.1.1.14) from Capsicum annuum fruits have been studied. The enzyme showed an optimum of activity at pH 8.5 and 50 degrees C. Substrate specificity was studied for chlorophyll (Chl) a, Chl b, pheophytin (Phe) a and Phe b, with Km values of 10.70, 4.04, 2.67 and 6.37 microM respectively. Substrate inhibition was found for Phe b at concentrations higher than 5 microM. Chlorophyllase action on Chl a' and Chl b' was also studied but no hydrolysis was observed, suggesting that the mechanism of action depends on the configuration at C-13(2) in the chlorophyll molecule, with the enzyme acting only on compounds with R132 stereochemistry. The effect of various metals (Mg2+, Hg2+, Cu2+, Zn2+ Co2+, Fe2+ and Fe3+) was also investigated, and a general inhibitory effect was found, this being more marked for Hg2+ and Fe2+. Functional groups such as -SH and -S-S- seemed to participate in the formation of the enzyme-substrate complex. Chelating ion and the carbonyl group at C3 appeared to be important in substrate recognition by the enzyme. The method for measuring Chlase activity, including HPLC separation of substrate and product, has been optimized.     

Peroxidases and the metabolism of capsaicin in Capsicum annuum L.

Capsaicinoids are acid amides of C₉ - C₁₁ branched-chain fatty acids and vanillylamine. These compounds are responsible for the pungency of the Capsicum species and of cultivars regarded as hot peppers. Moreover, it has been suggested that these compounds play an ecological role in seed dispersal. Because they are used in the pharmacological, food and pesticide industries, much attention has been paid on knowing how their accumulation is controlled, both in the fruit and in cell cultures. Such control involves the processes of biosynthesis, conjugation and catabolism. Recent progress has been made on the biosynthetic pathway, and several of the genes coding for biosynthetic enzymes have been cloned and expression studies performed. With regard to catabolism, cumulative evidence supports that capsaicinoids are oxidized in the pepper by peroxidases. Peroxidases are efficient in catalyzing in vitro oxidation of both capsaicin and dihydrocapsaicin. These enzymes are mainly located in placental and the outermost epidermal cell layers of pepper fruits, as occurs with capsaicinoids, and some peroxidases are present in the organelle of capsaicinoid accumulation, that is, the vacuole. Hence, peroxidases are in the right place for this function. The products of capsaicin oxidation by peroxidases have been characterized in vitro, and some of them have been found to appear in vivo in the Capsicum fruit. Details on the kinetics and catalytic cycle for capsaicin oxidation by peroxidases are also discussed.     

Linkage disequilibrium and population-structure analysis among Capsicum annuum L. cultivars for use in association mapping

Knowledge of population structure and linkage disequilibrium among the worldwide collections of peppers currently classified as hot, mild, sweet and ornamental types is indispensable for applying association mapping and genomic selection to improve pepper. The current study aimed to resolve the genetic diversity and relatedness of Capsicum annuum germplasm by use of simple sequence repeat (SSR) loci across all chromosomes in samples collected in 2011 and 2012. The physical distance covered by the entire set of SSRs used was 2,265.9 Mb from the 3.48-Gb hot-pepper genome size. The model-based program STRUCTURE was used to infer five clusters, which was further confirmed by classical molecular-genetic diversity analysis. Mean heterozygosity of various loci was estimated to be 0.15. Linkage disequilibrium (LD) was used to identify 17 LD blocks across various chromosomes with sizes from 0.154 Kb to 126.28 Mb. CAMS-142 of chromosome 1 was significantly associated with both capsaicin (CA) and dihydrocapsaicin (DCA) levels. Further, CAMS-142 was located in an LD block of 98.18 Mb. CAMS-142 amplified bands of 244, 268, 283 and 326 bp. Alleles 268 and 283 bp had positive effects on both CA and DCA levels, with an average R ² of 12.15 % (CA) and 12.3 % (DCA). Eight markers from seven different chromosomes were significantly associated with fruit weight, contributing an average effect of 15 %. CAMS-199, HpmsE082 and CAMS-190 are the three major quantitative trait loci located on chromosomes 8, 9, and 10, respectively, and were associated with fruit weight in samples from both years of the study. This research demonstrates the effectiveness of using genome-wide SSR-based markers to assess features of LD and genetic diversity within C. annuum.     

观赏辣椒一步成苗诱导条件的筛选

采用正交试验设计方法探讨6-BA、NAA和2,4-D对观赏辣椒一步成苗影响的结果表明:6-BA与NAA的浓度比对丛生芽诱导的影响最大。筛选出的观赏辣椒一步成苗的最适培养基为1/2MS 1.0mg·L-16-BA 0.5mg·L-1NAA 0.2mg·L-12,4-D,并获得移栽后的成活植株。     

Phytoalexin Induction as a Factor in the Protection of Capsicum annuum L. Fruits Against Infection by Botrytis cinerea Pers.

Immature Capsicum annuum fruits treated at wound-sites with glucans from the hyphal walls of Glomerella cingulata and incubated for 24 h or longer accumulated levels of capsicannol phytoalexins sufficient to inhibit rot development by Botrytis cinerea. Elicitor application to intact fruits induced the accumulation of relatively low levels of capsicannol compounds but further phytoalexin accumulation in fruits after wound-inoculation with B. cinerea was much more rapid than in untreated fruits and rot development was suppressed. Capsicannol phytoalexins were produced in the live cells of the epidermis and little or no necrosis was associated with quantities produced in intact tissues. Elicitor treatment of intact fruits to protect against rot development by B. cinerea was achieved without the fruits sustaining unsightly damage.     

辣椒花粉形态特征及分类初探

利用扫描电镜对辣椒(Capsicum annuum L.)6个变种植株的花粉形态进行了系统观察和比较分析.研究结果表明:6个变种的花粉呈矩圆形、椭圆形或圆形;极面观均为三裂片圆形;具三萌发沟,沟长达两极,沟的末端在极面上不连接形成合沟;花粉外壁纹饰在扫描电子显微镜下以刺-颗粒状复合纹饰为主.变种间在花粉形状和外壁纹饰方面有一定差异.根据其特征可以对辣椒进行分类,并反映变种间的亲缘关系.     

The Precursors Affecting the Composition of Capsaicin and Its Analogues in the Fruits of Capsicum annuum var. annuum cv. Karayatsubusa

The effects of tropomyosin and troponin on the heat-induced gelation of myosin were investigated by SDS-polyacrylamide gel electrophoresis, scanning electron microscopy and gel rigidity assay, in comparisons with natural and desensitized actomyosin. SDS-polyacrylamide gel electrophoretograms revealed that tropomyosin was almost completely removed from each desensitized actomyosin samples while it was retained in natural actomyosin samples. In spite of this, no significant differences were found in rigidity between natural and desensitized actomyosin gels. No differences could be observed in the microstructure of either actomyosin gel. It may, therefore, be concluded that tropomyosin does not affect the gel texture of the actomyosin system.     

生物技术在辣椒育种研究上的应用

综述了国内外组织及器官培养、分子标记和基因工程三大生物技术在辣椒育种的研究与利用，并进行了展望。     

Improved plant regeneration in Capsicum annuum L. from nodal segments

Multiple shoots were induced by culturing nodal explants excised from 1-month-old aseptic seedlings of red pepper (Capsicum annuum L. cv. Pusa Jwala) on Murashige and Skoog (MS) medium supplemented with (0.1–10 μM) thidiazuron (TDZ). The rate of multiple shoot induction per explant was maximum (14.4 ± 0.06) on MS medium supplemented with 1.0 μM TDZ. Regenerated shoots were elongated well on growth regulator free MS medium. Adventitious roots were induced two weeks after transfer of elongated shoots to MS medium supplemented with auxins (IAA, IBA or NAA) in different concentrations. Optimum root formation frequency was obtained in medium containing 1.0 μM IBA. Ex-vitro rooting was also achieved by pulse treatment with 300 μM IBA for 10 min. Rooted shoots were transplanted in plastic pots containing garden soil (with 90 % survival rate), where they grew well and attained maturity. Regenerated plants were phenotypically and cytologically normal.     

Effect of {alpha}-Naphthalene Acetic Acid on Variations in Free Amino Acid Contents in Developing Fruits of Capsicum annuum L.

Capsicum annuum seeds were pretreated with three concentrationsof -naphthaleneacetic acid (NAA). The fruits of the controland treated plants showed differences in the quantities of freeamino acids during three developmental stages. From a relativelyhigh amount of total free amino acids in the early phase offruit development, there was lowering in the mid-phase and furtherlowering in the terminal phase. There were eight prominent ninhydrin-reactingspots which were identified as tryptophan, glycine, glutamicacid, valine-methionine, -aminobutyric acid, leucine(s), alanine,and aspartic acid and eleven trace spots attributable to tyrosine,serine, lysine, asparagine, cystine, thrconine histidine, phenylalanine,arginine, glutamine, and ornithine. Hormone treatment tendedto decrease the total amino acids in the early- and mid-phasesof fruit development compared with the control fruits. Asparticacid was present in greater quantities in the treatments thanin the controls. In the terminal phase, the treated fruit didnot show any significant change in the total free amino acidcontents. The significance of the NAA-included variation infree amino acids is discussed. Capsicum annuum, amino acids, -naphthaleneacetic acid     

Differential expression patterns of genes containing microsatellites in Capsicum annuum L.

Microsatellites are important for research in both basic and applied sciences. Understanding how genes containing microsatellites are expressed and regulated in different tissues and developmental stages is a fundamental and challenging question. However, there has been limited study of differential expression of genes containing microsatellites in different plant tissues and developmental stages. We studied microsatellite-containing gene expression profiles in 16 different tissues and at different developmental stages in pepper (Capsicum annuum L.). Results indicated that tissue-specific genes contained more dinucleotide microsatellites, and housekeeping genes contained more trinucleotide microsatellites. Differential expression patterns of microsatellite-containing genes indicated that, in the longer term, development of allele-specific markers for genes controlling agronomic traits will be useful for advancing the science of plant breeding. This is the first report on differential expression of microsatellite-containing genes within tissues and developmental stages in pepper.     

Assessment of mutagenicity induced by MMS and DES in Capsicum annuum L.

Seeds of Capsicum annuum L. var. G4 were subjected to different concentrations of methyl methane sulphonate (MMS) and diethyl sulphate (DES). The effects of different mutagenic treatments on meiosis, chiasma frequency, and pollen fertility have been studied in M₁ generation. Various types of meiotic aberrations such as univalent, multivalent, stickiness, bridge, laggards, cytomixis etc. were observed in all the treatments. However, the MMS treatments proved to be more effective in inducing meiotic aberrations as compared to DES. Moreover, the frequency of meiotic aberrations was at its maximum at metaphase followed by anaphase and telophase stages. As the concentrations increase, reduction in chiasma frequency and pollen fertility was observed in all the treatments and, MMS again was found to be more effective than DES treatments.     

Glycerolipid-fatty-acid desaturase deficiencies in chloroplasts from fruits of Capsicum annuum L.

Chloroplasts from fruits and leaves of Capsicum annuum cv. Bell Tower were purified on sucrose gradients, and the lipids were separated by column and thin-layer chromatography. The glycerolipids mono- and digalactosyldiacylglycerol (MGDG, DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol (PG) were quantified, and the fatty-acid composition at the 1 and 2 positions of the glycerol moiety (sn-1 and sn-2) was determined after hydrolysis with position-specific lipases. In fruit chloroplasts, ³-trans hexadecenoate (trans-3-161) was absent and replaced by palmitate (160) at sn-2 of PG, and ^7,10,13-hexadecatrienoate (163) at sn-2 of MGDG was greatly reduced and largely replaced by linoleate (182). The ratio of 182 to linolenate (183) was consistently greater in glycerolipids from fruit compared with leaf chloroplasts. The lower percentage of C-16 fatty acids at sn-2 indicated that prokaryotic molecular species were reduced by 15% in DGDG, 40% in SQDG, and 90% in MGDG, in fruit compared with leaf chloroplasts. The MGDGDGDG ratios in fruit and leaf chloroplasts were 1.21 and 2.21, respectively. Taken together, the data indicate that chloroplasts in Capsicum fruit are deficient in three desaturases: those that convert 1) 160 to ³-trans-161 at sn-2 of PG, 2) 160 to ⁷–cis-161 at sn-2 of MGDG, and 3) 182 to 183 at both sn-1 and sn-2 of various chloroplast glycerolipids.Abbreviations Chl chlorophyll - DGDG digalactosyldiacylglycerol - FS free sterol - GL galactolipid - MGDG monogalactosyldiacylglycerol - PE phosphatidyl ethanolamine - PG phosphatidylglycerol - PL phospholipid - SQDG sulfoquinovosyldiacylglycerol We are grateful to Dr. Roger Calza for providing us with the tobacco gt11 cDNA expression library and to Dr. Eric Huttner for his advice throughout the screening procedure. We also wish to thank M. Gosse for his assistance in growing and maintaining our plants. T.W.B. was supported by a BAP research grant from the Commission of the European Communities.     

Cytogenetic markers for the characterization of Capsicum annuum L. cultivars

Karyotypic studies with conventional staining have been unsuccessful due to the uniformity of Capsicum chromosomes. In this study, we found diagnostic chromosome characters that permit to characterize cultivars; this is the first cytological characterization of both rDNAs (18S and 5S) in a species of Capsicum using a genus-specific probe and the most exhaustive in C. annuum to date. The heterochromatic banding patterns enabled us to identify cultivars, and fluorescent in situ hybridization (FISH) showed one 5S rDNA locus largely conserved within the cultivars, whereas high variation in the number of 18S rDNA loci was observed. One of the most obvious differences is the presence of an additional active nucleolar organizer region in pair #12 and the dispersal of inactive 18S rDNA signals. These results indicate that fluorochrome banding together with silver impregnation and FISH procedures are very useful for the identification of chromosomes and the interpretation of chromosomal variation between cultivars. The functional role of this variation is still uncertain, but our results show that copy number variation of repetitive DNA during the course of evolution might provide an excellent experimental system for studying genome rearrangements accompanying functional divergence in domesticated C. annuum.     

Characterization of gamma-tocopherol methyltransferases from Capsicum annuum L and Arabidopsis thaliana.

Tocopherols are essential micronutrients in human and animal nutrition due to their function as lipophilic antioxidants. They are exclusively synthesized by photosynthetic organisms including higher plants. Despite the attributed beneficial health effects and many industrial applications, research on the tocopherol biosynthetic pathway and its regulation in plants is still limited. In the work presented here we performed a detailed biochemical characterization of a gamma-tocopherol methyltransferase (gamma-TMT) from Arabidopsis thaliana and of a gamma-TMT purified from Capsicum annuum fruits, a tissue with high accumulation of tocopherols. The biochemical characteristics of both enzyme preparations were remarkably similar including substrate specificities. Both enzymes converted delta- and gamma- into beta- and alpha-tocopherol, respectively, but beta-tocopherol was not accepted as a substrate, pointing to a specific methylation at the C(5)-position of the tocopherol aromatic head group. A kinetic analysis performed with the Arabidopsis enzyme was consistent with an iso-ordered bi-bi type reaction mechanism. Our results emphasize the role of gamma-TMT in regulating the spectrum of accumulated tocopherols in plants.