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.     

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.     

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.     

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.     

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.     

The vegetable rennet of Cynara cardunculus L. contains two proteinases with chymosin and pepsin-like specificities

The flowers of cardoon (genus Cynara) are traditionally used in Portugal for cheese making. In this work the vegetable rennet of the species Cynara cardunculus L. was characterized in terms of enzymic composition and proteolytic specificity of its proteinases (cardosin A and cardosin B). Cardosin A was found to cleave insulin B chain at the bonds Leu15-Tyr16, Leu17-Val18 and Phe25-Tyr26. In addition to the bonds mentioned cardosin B cleaves also Glu13-Ala14, Ala14-Leu15 and Phe24-Phe25 indicating that it has a broader specificity. The kinetic parameters for the hydrolysis of the synthetic peptide Leu-Ser-Phe(NO₂)-Nle-Ala-Leu-oMe were also determined and compared to those of chymosin and pepsin. The results obtained indicate that in terms of specificity and kinetic parameters cardosin A is similar to chymosin whereas cardosin B is similar to pepsin. It appears therefore that the enzyme composition of cardoon rennet closely resembles that of calf rennet.     

Insect pollinators improve seed production in globe artichoke (Cynara cardunculus var. scolymus)

The role of many insect species in crop pollination has been widely studied. The use of entomophilous pollination, commonly adopted for other crops, may be of key importance in order to improve seed yields in seed-propagated globe artichoke (Cynara cardunculus var. scolymus) cultivars. In this regard, in a 2-year field experiment, the role of two honeybees (Apis mellifera ligustica and A. mellifera siciliana) and one bumblebee (Bombus terrestris) was evaluated on the seed production of two globe artichoke cultivars in caged-protected environments. Overall, the number and weight of seeds plant⁻¹ and head⁻¹, 1,000 seed weight and fruit setting were higher in caged plants with imposed pollination than open field plants. The pollination efficiency was both insect and cultivar dependent. Both honeybee species performed better on the Mediterranean cultivar ‘Violetto di Sicilia’, while the bumblebee performed better on the Brazilian cultivar ‘NP4’. These results could be very useful to modernise the agronomic management of globe artichoke and reduce the costs of cultivation. In addition, such knowledge can be used to improve the seed production in globe artichoke seed-propagated cultivars and cardoon. This will benefit the bioenergy, nutraceutical, cosmetic and ornamental applications.     

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.     

Biosynthesis and bioactivity of Cynara cardunculus L. guaianolides and hydroxycinnamic acids: a genomic,biochemical and health-promoting perspective

Phytochemistry Reviews - Cynara cardunculus health benefits have aroused much interest, leading to the discovery of valuable bioactive compounds with a crucial role in plant defence. Guaianolides...     

Effectiveness of mycorrhizal fungi on globe artichoke (Cynara cardunculus L. var. scolymus) micropropagation

The effectiveness of two arbuscular mycorrhizal (AM) fungal isolates (Glomus intraradices and Glomus viscosum) in sustaining plant growth and the physiological activities of the micropropagated globe artichoke (Cynara cardunculus L. var. scolymus (L.) Fiori) were investigated during acclimatization and 90 days after plant establishment. All the mycorrhizal microplants survived transplant shock thus confirming the positive role of AM fungi colonization on ex vitro establishment. The growth increased in mycorrhizal plants, especially in plants inoculated with Glomus viscosum. Mycorrhizal plantlets showed higher stomatal conductance, which is probably necessary to supply the carbon needs of fungal symbionts. The SPAD (soil plant analysis development) data could be useful for plant management as a predictor for tissue nitrogen levels. The higher SPAD values in mycorrhizal plants are strictly related to a higher photosynthetic potential, and consequently to their better nitrogen nutrient status due to the symbiotic relationship. Regardless of the mycorrhizal performance in the host–fungus combination, the most efficient fungus for the artichoke microplants was Glomus viscosum.     

Isolation of microsatellite loci in artichoke (Cynara cardunculus L. var. scolymus)

We report the development of nine microsatellite markers in globe artichoke (Cynara cardunculus L. var. scolymus). Four markers were obtained from sequences available in GenBank and five were isolated using a biotin/streptavidin capture technique for (CA)_n and (CT)_n motifs directly from artichoke genomic DNA. Polymorphism was explored in 15 artichoke accessions that represent the genetic variation within cultivated varietal types. Inter‐specific amplification was tested using cultivated cardoon (C. cardunculus L. var. altilis DC.) and wild cardoon (C. cardunculus L. var. sylvestris Lam.). Primers and conditions for polymerase chain reaction (PCR) amplification of detected loci are described.     

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.     

Allelopathy and its coevolutionary implications between native and non‐native neighbors of invasive Cynara cardunculus L.

Invasive plants apply new selection pressures on neighbor plant species by different means including allelopathy. Recent evidence shows allelopathy functions as remarkably influential mediator for invaders to be successful in their invaded range. However, few studies have determined whether native and non‐native species co‐occurring with invaders have evolved tolerance to allelopathy. In this study, we conducted germination and growth experiments to evaluate whether co‐occurring native Juncus pallidus and non‐native Lolium rigidum species may evolve tolerance to the allelochemicals induced by Cyanara cardunculus in Australian agricultural fields. The test species were germinated and grown in pots filled with collected invaded and uninvaded rhizosphere soil of C. cardunculus with and without activated carbon (AC). Additionally, a separate experiment was done to differentiate the direct effects of AC on the test species. The soil properties showed invaded rhizosphere soils had higher total phenolic and lower pH compared with uninvaded soils. We found significant reduction of germination percentage and seedling growth in terms of above‐ and belowground biomass, and maximum plant height and root length of native in the invaded rhizosphere soil of C. cardunculus, but little effect on non‐native grass species. Even soil manipulated with AC showed no significant differences in the measured parameters of non‐native except aboveground biomass. Taken together, the results indicate allelochemicals induced by C. cardunculus exert more suppressive effects on native than non‐native linking the coevolved tolerance of those.     

M‐AFLP‐based protocol for microsatellite loci isolation in Cynara cardunculus L. (Asteraceae)

Nine microsatellite markers for Cynara cardunculus L. were developed using a two‐step ‘primer extension’ procedure, based on microsatellite‐amplified fragment length polymorphism (M‐AFLP) technique. In the first step, highly enriched SSR gel profiles were produced and, from the derived sequences of selected bands, forward primers directed towards the microsatellite motif were designed. In the second step, the opposite microsatellite flanking sequence was isolated using a nested approach on a restricted‐ligated genomic fraction. Polymorphism was explored in 24 plants of wild cardoon (Cynara cardunculus L. var. sylvestris) as well as two accessions of both globe artichoke (Cynara cardunculus L. var. scolymus), and cultivated cardoon (Cynara cardunculus L. var. altilis).     

Crystal structure of cardosin A, a glycosylated and Arg-Gly-Asp-containing aspartic proteinase from the flowers of Cynara cardunculus L.

Aspartic proteinases (AP) have been widely studied within the living world, but so far no plant AP have been structurally characterized. The refined cardosin A crystallographic structure includes two molecules, built up by two glycosylated peptide chains (31 and 15 kDa each). The fold of cardosin A is typical within the AP family. The glycosyl content is described by 19 sugar rings attached to Asn-67 and Asn-257. They are localized on the molecular surface away from the conserved active site and show a new glycan of the plant complex type. A hydrogen bond between Gln-126 and Manbeta4 renders the monosaccharide oxygen O-2 sterically inaccessible to accept a xylosyl residue, therefore explaining the new type of the identified plant glycan. The Arg-Gly-Asp sequence, which has been shown to be involved in recognition of a putative cardosin A receptor, was found in a loop between two beta-strands on the molecular surface opposite the active site cleft. Based on the crystal structure, a possible mechanism whereby cardosin A might be orientated at the cell surface of the style to interact with its putative receptor from pollen is proposed. The biological implications of these findings are also discussed.     

Cardosin A, an abundant aspartic proteinase, accumulates in protein storage vacuoles in the stigmatic papillae of Cynara cardunculus L.

The function of aspartic proteinases (EC 3.4.23) present in flowers of Cynara species is still unknown. Cardosin A, as a highly abundant aspartic proteinase from Cynara cardunculus L., a relative of the artichoke, is synthesised as a zymogen and subsequently undergoes proteolytic processing, yielding the mature and active enzyme. Here we report the study of the expression and localization of cardosin A, as a first approach to address the question of its physiological relevance. A polyclonal antibody specific for cardosin A was raised against a synthetic peptide corresponding to an amino acid sequence of the enzyme. This antibody was used to study the organ-specific, tissue-specific and subcellular localization of cardosin A by immunoblotting, tissue printing and immunogold electron microscopy. The results showed that expression of cardosin A is highly restricted to the pistils, and that the enzyme accumulates mainly in protein storage vacuoles of the stigmatic papillae. Cardosin A is also present, although much less abundantly, in the vacuoles of the cells of the epidermis of the style. In view of these results, the possible physiological roles of cardosin A are discussed, namely an involvement in defense mechanisms or pollen-pistil interaction, as well as in flower senescence. Received: 10 December 1996 / Accepted: 14 March 1997     

Molecular cloning and characterization of cDNA encoding cardosin B,an aspartic proteinase accumulating extracellularly in the transmitting tissue of Cynara cardunculus L.

Cardosins A and B are related aspartic proteinases from the pistils of Cynara cardunculus L., whose milk-clotting activity has been exploited for the manufacture of cheese. Here we report the cloning of cardosin B cDNA and its organ, tissue and cytological localization. The cDNA-derived amino acid sequence has 73% similarity with that of cardosin A and displays several distinguishing features. Cardosin B mRNA was detected in young inflorescences but not in pistils of fully opened inflorescences, indicating that its expression is developmentally regulated. The proteinase, however, accumulates in the pistil until the later stages of floral development. Immunocytochemistry with a monospecific antibody localized cardosin B to the cell wall and extracellular matrix of the floral transmitting tissue. The location of cardosin B in the pistil is therefore clearly different from that of cardosin A, which was found at protein storage vacuoles of the stigmatic papillae and has been suggested to be involved in RGD-mediated proteolytic mechanisms. In view of these results the possible functions of cardosin B in the transmitting tissue are discussed.     

A first linkage map of globe artichoke (Cynara cardunculus var. scolymus L.) based on AFLP, S-SAP, M-AFLP and microsatellite markers

We present the first genetic maps of globe artichoke (Cynara cardunculus var. scolymus L. 2n=2x=34), constructed with a two-way pseudo-testcross strategy. A F₁ mapping population of 94 individuals was generated between a late-maturing, non-spiny type and an early-maturing spiny type. The 30 AFLP, 13 M-AFLP and 9 S-SAP primer combinations chosen identified, respectively, 352, 38 and 41 polymorphic markers. Of 32 microsatellite primer pairs tested, 12 identified heterozygous loci in one or other parent, and 7 were fully informative as they segregated in both parents. The female parent map comprised 204 loci, spread over 18 linkage groups and spanned 1330.5 cM with a mean marker density of 6.5 cM. The equivalent figures for the male parent map were 180 loci, 17 linkage groups, 1239.4 and 6.9 cM. About 3% of the AFLP and AFLP-derived markers displayed segregation distortion with a P value below 0.01, and were not used for map construction. All the SSR loci were included in the linkage analysis, although one locus did show some segregation distortion. The presence of 78 markers in common to both maps allowed the alignment of 16 linkage groups. The maps generated provide a firm basis for the mapping of agriculturally relevant traits, which will then open the way for the application of a marker-assisted selection breeding strategy in this species.