Evolution of the B3 DNA Binding Superfamily: New Insights into REM Family Gene Diversification

Background

The B3 DNA binding domain includes five families: auxin response factor (ARF), abscisic acid-insensitive3 (ABI3), high level expression of sugar inducible (HSI), related to ABI3/VP1 (RAV) and reproductive meristem (REM). The release of the complete genomes of the angiosperm eudicots Arabidopsis thaliana and Populus trichocarpa, the monocot Orysa sativa, the bryophyte Physcomitrella patens,the green algae Chlamydomonas reinhardtii and Volvox carteri and the red algae Cyanidioschyzon melorae provided an exceptional opportunity to study the evolution of this superfamily.

Methodology

In order to better understand the origin and the diversification of B3 domains in plants, we combined comparative phylogenetic analysis with exon/intron structure and duplication events. In addition, we investigated the conservation and divergence of the B3 domain during the origin and evolution of each family.

Conclusions

Our data indicate that showed that the B3 containing genes have undergone extensive duplication events, and that the REM family B3 domain has a highly diverged DNA binding. Our results also indicate that the founding member of the B3 gene family is likely to be similar to the ABI3/HSI genes found in C. reinhardtii and V. carteri. Among the B3 families, ABI3, HSI, RAV and ARF are most structurally conserved, whereas the REM family has experienced a rapid divergence. These results are discussed in light of their functional and evolutionary roles in plant development.     

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.     

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。     

Photoinactivation of photosystem II in leaves of Capsicum annuum

Leaf discs of Capsicum annuum L. were illuminated in air enriched with 1% CO₂ in the absence or presence of lincomycin, an inhibitor of chloroplast-encoded protein synthesis. The loss of functional photosystem (PS) II complexes with increase in cumulative light dose (photon exposure), assessed by the O₂ yield per single-turnover flash, was greater in leaves of plants grown in low light than those in high light; it was also exacerbated in the presence of lincomycin. A single exponential decay can describe the relationship between the loss of functional PSII and increase in cumulative photon exposure. From this relationship we obtained both the maximum quantum yield of photoinactivation of PSII at limiting photon exposures and the coefficient k, interpreted as the probability of photoinactivation of PSII per unit photon exposure. Parallel measurements of chlorophyll fluorescence after light treatment showed that 1/F_o−1/F_m was linearly correlated with the functionality of PSII, where F_o and F_m are the chlorophyll fluorescence yields corresponding to open and closed PSII reaction centers, respectively. Using 1/F_o−1/F_m as a convenient indicator of PSII functionality, it was found that PSII is present in excess; only after the loss of about 40% functional PSII complexes did PSII begin to limit photosynthetic capacity in capsicum leaves.     

Daily changes in nitrate uptake and metabolism in Capsicum annuum

The diurnal pattern of nitrate uptake by Capsicum annuum L. cv. California Wonder in a constant environment is described by a Fourier harmonic, with the maximum uptake in the middle of the photoperiod and the minimum in the middle of the dark period. Comparison of the uptake pattern with that of nitrate reductase (EC 1.6.6.1.) activity suggests against a direct control of one process by the other. This was confirmed by the observation that the pattern of nitrate reductase activity was not altered by restricting nitrate uptake to one hour per day. Translocation of ¹⁵N from the roots is much greater in the lightperiod than in the dark period. Reduction of ¹⁵N in the leaves occurs in the lightperiod but very little is reduced in the dark period. Amino acid levels showed marked daily fluctuations but in the roots neither amino acids, sucrose, fructose, glucose nor malate showed fluctuations. The amino acid composition of roots and leaves differed: glutamine+glutamate were relatively more important in leaves than in roots whereas alanine was a more important constituent of roots than of leaves.Abbreviation NR nitrate reductase     

Inheritance of a novel flaccid mutant in Capsicum annuum

A mutant that causes a novel flaccidity phenotype in bell pepper (Capsicum annuum L.) was generated by treating seeds with ethyl methanesulfonate (EMS). Inheritance studies indicated that the mutant was controlled by a single recessive gene. It is proposed that the gene designation representing this mutation be flc (flaccid). The mutation may be useful for investigations of the genetic basis for turgor maintenance and drought stress physiology.     

Thidiazuron induced somatic embryogenesis and plant regeneration in Capsicum annuum

An efficient protocol of direct somatic embryogenesis (without involving intermediate callus) has been developed from stem segments and shoot tips of Capsicum annuum L. Explants were cultured on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ). Among the various concentration of TDZ tested, 0.5 μM was proved to be best for induction of somatic embryos. Induction, maturation and germination were achieved on the same medium. The shoots developed from somatic embryos were transferred for rooting to MS medium supplemented with indole-3-butyric acid (IBA). All the regenerated plants with 85 % survival rate were normal with respect to morphology and growth characteristics.     

Three New Constituents with Anti-Inflammatory and Antimicrobial Activities in Vitro from the Roots of Capsicum annuum L.

Three new compounds, including two new sesquiterpenes ( 1–2 ), named Annuumine E−F, and one new natural product, 3-hydroxy-2,6-dimethylbenzenemethanol ( 3 ), together with seventeen known compounds ( 4–20 ) were isolated from the ethanol extract of the roots of Capsicum annuum L. Among them, five compounds ( 4 , 5 , 9 , 10 and 20 ) were isolated from this plant for the first time. The structures of new compounds ( 1–3 ) were determined via detailed analysis of the IR, HR-ESI-MS and 1D and 2D NMR spectra. The anti-inflammatory activities of the isolated compounds were evaluated by their ability to reduce NO release by LPS-induced RAW 264.7 cells. Notably, compound 11 exhibited moderate anti-inflammatory activity (IC₅₀=21.11 μM). Moreover, the antibacterial activities of the isolated compounds were also evaluated.     

辣椒抗病基因工程研究进展(综述)

辣椒易发生由细菌、真菌和病毒等引致的病害,基因工程技术为辣椒抗病育种开辟了新途径。本文就晚近以离体再生植株和转基因技术等为基础的辣椒抗病基因工程研究进展进行综述。     

Verticillium Wilt of Pepper (Capsicum annuum) in Morocco

Pepper crops in Morocco formerly thought to be free from attack by Verticillium were found to be susceptible to this pathogen. Verticillium wilt of pepper was observed in plots where the crop was cultivated in rotation with tomatoes. Strains of Verticillium isolated from pepper possessed different morphological and pathogenic characteristics.     

Population Densities of Meloidogyne incognita and Yield of Capsicum annuum

Two microplot experiments in 1981 and 1983 provided information on the effect of different population densities of Meloidogyne incognita race 1 and yield of sweet pepper. Microplots were square concrete pipes (30 × 30 cm and 50 cm long) filled with 40 liters of soil infested with 0, 0.062, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256, and 512 eggs and juveniles/cm³ soil. Tolerance limits of 2.2 and 0.165 eggs and juveniles/cm³ soil and minimum yields of 58% and 20% of the controls were obtained in 1981 and 1983, respectively. Maximum reproduction rates of the nematode were 274 and 1,498 at the lowest initial population density. The population of the nematode declined rapidly after harvest, and only 13% and 6.5% of eggs and juveniles were detected in the soil after 1 and 6 months, respectively.     

Genetic diversity of Chile (Capsicum annuum var. annuum L.) landraces from Northern New mexico, Colorado, and Mexico

Four hundred years of chile(Capsicum annuum var.annuum) cultivation, together with concerns about losing genetic resources in their native agrohabitats, provide the pretext for collecting and preserving landraces of this species in New Mexico. The molecular analysis of these accessions provided a powerful means by which their genetic structures were characterized. Random amplified polymorphic DNA (RAPD) molecular markers were used to compare the relative genetic diversity of native chile landraces to the genetic diversity found in commercially available cultivars in the United States as well as landraces from Mexico.     

ChrA Is a Carotenoid-Binding Protein in Chromoplasts of Capsicum annuum

Chromoplasts of Capsicum annuum var Albino contain a carotenoid-protein complex, which migrates as a brilliant orange band in gels under conditions of nondenaturing electrophoresis. In a second, denaturing separation, the complex resolves into a principal protein (ChrA) of 58 kilodaltons and several minor proteins of 20 to 55 kilodaltons, which may be adventitiously associated. Analysis of Western blots of both one- and two-dimensional gels showed that the principal protein component of the carotenoid complex is ChrA, a protein previously shown to be located specifically in chromoplast membranes. The identification of ChrA as a carotenoid-binding protein appears to be the first instance of a nonthylakoid, carotenoid-binding protein in higher plants.     

Purification and characterization of farnesyl pyrophosphate synthase from Capsicum annuum

Farnesyl pyrophosphate synthase (FPP) displaying dimethylallyl transferase activity (EC 2.5.1.1) and geranyl transferase activity (EC 2.5.1.10) was purified from Capsicum fruits. This prenyltransferase has a molecular mass of 89,000 +/- 5000 Da resulting from the association of two apparently identical subunits having a molecular mass of 43,000 +/- 2000 Da. Antibodies raised against Capsicum FPP synthase selectively blocked the transferase activity. Analysis of the immunological relationships between FPP synthase and geranylgeranyl pyrophosphate synthase (EC 2.5.1.1, EC 2.5.1.10 and EC 2.5.1.30) revealed that these two enzymes though performing the same mechanism of catalysis and accepting identical substrates have different antigenic determinants. Thus, in connection to previous work, this immunological study suggests that Capsicum FPP is strictly located in the extraplastidial compartment.     

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.     

Molecular AFLP Analysis of the Genotypes of Pepper Capsicum annuum Cultivars

The results of AFLP study of 14 Capsicum annuum cultivars are presented. In spite of the known low genomic variation of large-fruited sweet pepper, AFLP analysis proved to be suitable for detecting polymorphism and genotyping pepper cultivars. Nine primer pairs were selected to allow identification of the cultivars under study. Among-cultivar polymorphism detectable with these primers was estimated at 16.5%. A characteristic AFLP pattern was obtained for each cultivar. Several cultivar-specific fragments were revealed for seven cultivars. On the basis of the AFLP data, genetic distances between cultivars were determined and a tree was constructed by means of hierarchic cluster analysis (UPGMA) with the Jacquard coefficient. It was assumed that this information is useful in breeding programs involving the cultivars examined.