Effect of acetonitrile on Cynara cardunculus L. cardosin A stability

The kinetics of the structural changes affecting cardosin A, a plant aspartic proteinase (AP) from Cynara cardunculus L., in the presence of a mixture of acetonitrile (AN) in water (W) was studied. Incubation of cardosin A with 10% (v/v) AN resulted in a gradual increase in protein helicity, accompanied by changes in the tertiary structure, seen by changes in the intrinsic fluorescence of tryptophan. Differential scanning calorimetry (DSC) revealed that the temperature of denaturation of cardosin A decreased upon the addition of AN. With longer incubation times, the small chain of cardosin A denatured completely, consequent exposure of the single tryptophan residue accounting well for the observed spectral shift intrinsic fluorescence of the protein. Enzymatic activity assays demonstrated that the kinetically determined unfolding of the small chain of cardosin A does not result in loss of the activity of this enzyme.     

红豆杉细胞悬浮培养结构化数学模型的探讨

用１０Ｌ机械搅拌式生物反应器悬浮培养红豆杉细胞,得到细胞生长、基质消耗和紫杉醇合成动力学曲线。经过代谢动力学分析建立了结构化数学模型。并将模型值与实验值进行比较,结果表明模型预测值与实验值较吻合。     

Multiplicity of aspartic proteinases from Cynara cardunculus L.

Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC–MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO₂)AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Allocation of proteolytic activity in the seedling of Cynara cardunculus L. in the initial growth stages

Cynara cardunculus L. seeds were germinated in vitro under environmentally controlled conditions. Seeds showed a 60% germination rate, and three growth stages were established based on the seedling mean relative growth rate (RGR). Root, stem and cotyledons were compared in these stages with respect to the emergence of total proteases and cardosin activity and its allocation in the seedling. In growth stage I (1st-5th post-germinative days), seedlings grew very slowly. Total proteases and cardosins were already active at the onset of seedlings in the stem. Total soluble protein remained constant in cardoon seedlings during stage I, and the content of all free amino acids (aa) but proline (Pro) was equally allocated on the 1st post-germinative day. In growth stage II (5th-10th post-germinative days), seedlings grew intensively and exhibited fully developed cotyledons. A pronounced increase in the content of all free aa up to the middle of growth stage II in both stems and roots was observed. In addition, the allocation of the total proteolytic activity and cardosins followed a gradient from the root to the seedling shoot. However, the whole seedling soluble protein remained constant up to the 7th day in and tended to peak on the 10th post-germinative day, being allocated mainly to the seedling stem. In growth stage III (10th-15th post-germinative days), cardoon seedlings exhibited the lowest mean RGR and the highest R/S growth ratio. An intensive degradation of total soluble protein present in the whole seedling except for cotyledons (ca. 5-fold) was observed. Nevertheless, in growth stage III, both the gradients exhibited by total proteases and cardosins activities between the root and the seedling shoot were enhanced, as were contents of all aa except Pro, exhibiting the highest levels in cotyledons on the 15th post-germinative day.     

Development and characterization of microsatellite markers in Cynara cardunculus L.

Cynara cardunculus L. is a species native to the Mediterranean basin that comprises 2 crops, globe artichoke (var. scolymus L.) and cultivated cardoon (var. altilis DC), as well as wild cardoon (var. sylvestris (Lamk) Fiori). Globe artichoke represents an important component of the South European agricultural economy but is also cultivated in North Africa, the Near East, South America, the United States, and China. Breeding activities and molecular marker studies have been, to date, extremely limited. Better knowledge of the genome of the species might be gained by developing a range of molecular markers. Here, we report on the development of 14 microsatellites (simple sequence repeats (SSRs)) through a novel approach that we have defined as the microsatellite amplified library (MAL). The approach represents a combination of amplified fragment length polymorphism and a primer extension based enriched library, is rapid, and requires no hybridization enrichment steps. The technique provided a approximately 40-fold increase in the efficiency of SSR identification compared with conventional library procedures. The developed SSRs were applied for genotyping 36 accessions of C. cardunculus, including a core of 27 varietal types of globe artichoke, 3 accessions of cultivated cardoon, and 6 Sicilian accessions of wild cardoon. Principal coordinates analysis made it possible to differentiate both cultivated and wild forms from each other.     

Cynara cardunculus L. alkaline pulps: alternatives fibres for paper and paperboard production

The pulping of Cynara cardunculus L. (cardoon) was performed under conditions for kraft, kraft-AQ and soda-AQ processes. The best results in terms of delignification degree, expressed as kappa number, pulp viscosity and screened yield, were obtained for the kraft-AQ process with 0.20% of anthraquinone (AQ). The papermaking potential of the selected pulp was studied attending to biometric fibre characterisation, refining aptitude, optical and strength properties. All properties were compared against a Eucalyptus globulus pulp at different refining degrees. The cardoon pulp was also evaluated concerning its potential to board manufacture, alone and in mixtures with pine pulp, giving rise to promising results for liner manufacture.     

A structured model for Thiobacillus ferrooxidans growth on ferrous iron

A structured model for Thiobacillus ferrooxidans growth dependence on ferrous and ferric iron, arsenic, oxygen, carbon dioxide, pH, and temperature is presented. A new kinetic mechanism for ferrous oxidation by T. ferrooxidans is introduced. Data from several earlier experimental studies of T. ferroaxidans growth are used for model development. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 310-319, 1997.     

Phenolic composition of Cynara cardunculus L. organs, and their biological activities

Polyphenols are bioactive molecules exhibiting a lot of scientific attention due to their multiple biological activities. This study compared phenolic contents and antioxidant activity in Cynara cardunculus L. organs and focus on leaf phenolic compounds identification by RP-HPLC and their antibacterial activity. The analyzed organs exhibited different total polyphenol contents (7-14.8 mg GAE g(-1) DW). Leaf and seed phenolic contents were similar and two times higher than those in flowers. The same tendency was observed for the amount of flavonoids and tannins. However, seed extracts displayed the highest DPPH. scavenging ability with the lowest IC50 value (23 microg ml(-1)), followed by leaves and flowers (over 50 microg ml(-1)). In contrast, leaves showed the highest capacity to quench superoxide (IC50: 1 microg ml(-1)) as compared to seeds (6 microg ml(-1)). In addition, cardoon leaves were efficient to inhibit growth of pathogenic bacteria mainly against Staphylococcus aureus and Escherichia coli. The identification of phenolic compounds from leaves revealed that syringic and trans-cinnamic acids were the major molecules.     

Plant cell suspension cultures as model systems for investigating growth regulating compounds

Several plant growth regulators were investigated for their activity in cell suspension cultures of Glycine max, Gossypium hirsutum and Zea mays. The effect on the growth of the cell cultures was traced by means of cell counting and determining packed cell volume and turbidity of the suspensions. The growth retardant 5-(4-chlorophenyl)-3,4,5,9,10-pentaaza-tetracyclo-5,4,10^2,6 ,0^8,11-dodeca-3,9-diene (NDA) and, to a slightly lesser extent, ancymidol proved to be the compounds with the greatest inhibitory action on cell division growth of all three cell cultures. In the case of cotton this effect was accompanied by increased synthesis and secretion of cell-wall material. Staining methods showed that, especially in the case of NDA, a high percentage of cells could be considered as viable, and showed thus that NDA inhibits the cell division process while the cells remain metabolically active. The effects of 1,1-Dimethyl-piperidiniumchloride (DPC), a genuine growth retardant of cell propagation, and, with less efficiency, N-trimethyl-(-chloroethyl)-ammoniumchloride (CCC) in cotton, the triazole LAB 117 682 in soybean and maize, and, to a lesser extent, (2-isopropyl-5-methyl-4-trimethyl-ammoniumchloride)-phenyl-l-piperidiniumcarboxylate (AM0-1618) in soybean can be regarded as species-specific. Otherwise, CCC and particularly daminozide exhibited no action at the concentrations used. A comparison of the data from hydroculture studies with soybean and maize seedlings showed considerable agreement with the effectiveness of the substances in the corresponding cell cultures. Thus, cell cultures can be used to identify and screen substances with growth-influencing activity, and may also offer new ways to elucidate the mode of action of plant growth regulators.     

Analysis of molecular genetic diversity of cardoon (Cynara cardunculus L.) in Tunisia

The objective of this study is to investigate the genetic diversity, the relationships among six Tunisian wild cardoon populations (Cynara cardunculus var. sylvestris) and a Tunisian's population of cultivated cardoon (Cynara cardunculus var. altilis DC) in seven different geographical locations (Tiurif, Bahra, Zriba, Bouficha, Enfidha, Beja and Wad mliz) from semi-arid and wet regions of Tunisia. Twenty-three selected microsatellite markers are used for a sample of 98 cardoon genotypes. The total of 243 alleles is detected in the studied populations and the number of alleles per locus ranged from six to 23. The dendrogram based on Nei's (1972) UPGMA method divides the seven studied populations to five clusters. These preliminary results show that microsatellites are effective tools for plant species characterization and the analysed populations have a high genetic variability and will be suitable as genetic stocks for conservation and sustainable utilization programs of Cynara cardunculus L. in Tunisia.     

Characterization and expression analysis of the aspartic protease gene family of Cynara cardunculus L

Cardosin A and cardosin B are two aspartic proteases mainly found in the pistils of cardoon Cynara cardunculus L., whose flowers are traditionally used in several Mediterranean countries in the manufacture of ewe's cheese. We have been characterizing cardosins at the biochemical, structural and molecular levels. In this study, we show that the cardoon aspartic proteases are encoded by a multigene family. The genes for cardosin A and cardosin B, as well as those for two new cardoon aspartic proteases, designated cardosin C and cardosin D, were characterized, and their expression in C. cardunculus L. was analyzed by RT-PCR. Together with cardosins, a partial clone of the cyprosin B gene was isolated, revealing that cardosin and cyprosin genes coexist in the genome of the same plant. As a first approach to understanding what dictates the flower-specific pattern of cardosin genes, the respective gene 5' regulatory sequences were fused with the reporter beta-glucuronidase and introduced into Arabidopsis thaliana. A subsequent deletion analysis of the promoter region of the cardosin A gene allowed the identification of a region of approximately 500 bp essential for gene expression in transgenic flowers. Additionally, the relevance of the leader intron of the cardosin A and B genes for gene expression was evaluated. Our data showed that the leader intron is essential for cardosin B gene expression in A. thaliana. In silico analysis revealed the presence of potential regulatory motifs that lay within the aforementioned regions and therefore might be important in the regulation of cardosin expression.     

Key phenological events in globe artichoke (Cynara cardunculus var.scolymus) development

A priority for the field vegetable grower is to be able to schedule a regular supply of product throughout the growing season. This requires a predictive framework, based on the identification of key developmental events of the crop, and an understanding of how genotypic and environmental factors interact to determine plant development. Four globe artichoke (Cynara cardunculus var. scolymus) cultivars, representing the existing phenological range, were grown in a field experiment, and a range of environmental conditions was imposed by varying both the timing of the first irrigation (which determines the initiation of regrowth) and by repeating the experiment across two locations and 2 years. The timing of the appearance of the main stem capitulum was sensitive to both the growing environment and the cultivar. These differences persisted till flowering and were correlated with final leaf number. As the plant developed, the phyllochron decreased, resulting in three values of phyllochron, each of which was responsive to genotype, and hardly to environment. The timing of the first change in phyllochron was associated with the final leaf number and the appearance of the capitulum. For all the cultivars, the rate of development fell and the final leaf number increased as the length of the photoperiod increased. The later flowering cultivars shared a similar vernalisation requirement, but ‘Spinoso sardo’ did not require a cold period to flower. Leaf length reached a peak before the beginning of stem elongation, and maximum leaf length was correlated with final leaf number. The sensitiveness of the phyllochron to the genotype, and of the number of leaves and the timing of the appearance of the capitulum to both genotype and environment makes them suitable as variables in developmental models. The importance of the final number of leaves is not only because of its phenological significance, but also because of its effect on the ability of the canopy to intercept radiation.     

Peptide synthesis catalyzed by Cynara cardunculus L. Acid protease in aqueous-organic biphasic systems

The ability of Cynara cardunculus L. protease to perform peptide synthesis was investigated using an aqueous organic biphasic system and the amino acid derivatives CBZ.Phe and Met.OMe. The reaction products were separated by RP-HPLC and identified by amino acid analysis and by EI-MS. Significant amounts of the dipeptide CBZ.Phe.Met.OMe was produced within 24h by Cynara cardunculus L. protease as compared with the amount produced by pepsin under the same conditions.     

A model that links growth and secondary metabolite production in plant cell suspension cultures

Plant cell suspensions of grape cells (Vitis vinifera L. cv. Gamay Fréaux) were grown in shake flasks operated both in the batch and semicontinuous mode. A mathematical model was developed to describe grape cell growth, sucrose uptake, and secondary metabolite (anthocyanin) production. Parameters were estimated from batch studies data. The model was able to predict results for semicontinuous experiments by only modifying the value of four of these parameters. The modified parameters (maximum specific rate of biomass production, maximum specific rate of substrate consumption for maintenance, maximum specific rate of anthocyanin production, and degradation constant of anthocyanins) were related to the kinetics rather than to the yield of the process. The model introduces the concept of primary and secondary metabolism substrate concentration-dependent competition for precursors. Further, the model was able to predict the evolution of the cell system when substrate is scarce, as the value of the different kinetic constants determines the portion of substrate that is used for biomass production, secondary metabolite production, and cell maintenance. (c) 1995 John Wiley & Sons, Inc.     

Callus and suspension cultures for biomass production ofCynara cardunculus (Compositae)

Summary Friable calli, obtained from hypocotyl and leaf segments ofCynara cardunculus, were used for the production of cell suspension cultures in liquid Gamborg B₅ medium supplemented as for callus obtention. The low inoculum cell suspension cultures (6.9×10⁴ cells.ml^–1) presented a td=136.8 hours while those obtained with a high inoculum (15.6×10⁴ cells.ml^–1) reached a td=33.6 hours.