Purification, cloning and autoproteolytic processing of an aspartic proteinase from Centaurea calcitrapa.

Plant aspartic proteinases (APs) have been isolated from several seed and leaf sources but the only well characterized enzymes from flowers are cardosins and cyprosins from cardoon, Cynara cardunculus L. Here we report a full-length cDNA clone encoding an AP named cenprosin from the flowers of Centaurea calcitrapa L., a thistle related to cardoon. As found for all eukaryotic APs, the deduced primary sequence consists of a signal sequence, a propart and a mature enzyme. In addition, an internal sequence region of 104 residues typical only of plant APs (a plant-specific insert) is present in the primary structure. Northern analysis revealed that the strongest expression is in fresh flowers. The enzyme is also expressed in fairly high amounts in seeds and in leaves, a feature not detected for cardoon APs. The corresponding enzyme was purified in its precursor form from fresh flowers using ammonium-sulfate precipitation followed by ion-exchange and hydrophobic-interaction chromatography. The processing of the precursor into its mature form was studied in vitro. The enzyme underwent autocatalytic processing at pH 3.0 resulting in two chains of 16 and 30 kDa. When dried flowers were used as a starting material for purification, only 16- and 30-kDa chains were obtained, suggesting that autoproteolytic activation of procenprosin in vivo occurs mainly during drying of the flowers. This may indicate a specific degradative role for the enzyme during senescence of the flowers.     

Isolation and characterization of a cDNA from flowers of Cynara cardunculus encoding cyprosin (an aspartic proteinase) and its use to study the organ-specific expression of cyprosin

Poly(A)⁺ RNA isolated from flower buds of Cynara cardunculus has been used to prepare a cDNA library. Screening of the cDNA after expression of cloned DNA with antibodies raised against the large subunit of cyprosin 3 resulted in the isolation of six positive clones. One of these clones (cypro1s; a 1.7 kb Eco RI fragment) codes for cyprosin. The nucleotide sequence contain a 1419 bp open reading frame coding for 473 amino acids (aa) including a putative full-length mature protein (440 aa) and a partial prosequence (33 aa). Cypro1s contains a 162 bp 3 non-coding region followed by a poly(A) tail. The deduced amino acid sequence shows high homology to other plant aspartic proteinases. The homology to mammalian and microbial aspartic proteinases is somewhat lower. Plant aspartic proteinases contain an insert of around 100 aa. We are modelling where this plant-specific insert will appear in the structure of cyprosin. Using cypro1s as a probe in northern blot analysis, the expression of cyprosin in developing flowers and other tissues has been studied. The signal on the northern blot increased for RNA samples from early (flower buds 6 mm in length) to later stages of floral development (flower buds up to 40 mm in length). In late stages of floral development (open flowers 50 mm in length and styles from such flowers) no hybridization signal was visualized showing that the synthesis of mRNA encoding the cyprosin starts in early stages of floral development and switches off at maturation of the flower. Southern blot analysis of genomic DNA showed 4–5 strong hybridizing bands and several minor bands indicating that the cyprosin genes are organized as a multi-gene family in C. cardunculus.     

Molecular cloning and characterization of procirsin,an active aspartic protease precursor from Cirsium vulgare (Asteraceae)

Typical aspartic proteinases from plants of the Astereaceae family like cardosins and cyprosins are well-known milk-clotting enzymes. Their effectiveness in cheesemaking has encouraged several studies on other Astereaceae plant species for identification of new vegetable rennets. Here we report on the cloning, expression and characterization of a novel aspartic proteinase precursor from the flowers of Cirsium vulgare (Savi) Ten. The isolated cDNA encoded a protein product with 509 amino acids, termed cirsin, with the characteristic primary structure organization of plant typical aspartic proteinases. The pro form of cirsin was expressed in Escherichia coli and shown to be active without autocatalytically cleaving its pro domain. This contrasts with the acid-triggered autoactivation by pro-segment removal described for several recombinant plant typical aspartic proteinases. Recombinant procirsin displayed all typical proteolytic features of aspartic proteinases as optimum acidic pH, inhibition by pepstatin, cleavage between hydrophobic amino acids and strict dependence on two catalytic Asp residues for activity. Procirsin also displayed a high specificity towards κ-casein and milk-clotting activity, suggesting it might be an effective vegetable rennet.The findings herein described provide additional evidences for the existence of different structural arrangements among plant typical aspartic proteinases.     

Pistil-Specific and Ethylene-Regulated Expression of 1-Aminocyclopropane-1-Carboxylate Oxidase Genes in Petunia Flowers

The differential expression of the petunia 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene family during flower development and senescence was investigated. ACC oxidase catalyzes the conversion of ACC to ethylene. The increase in ethylene production by petunia corollas during senescence was preceded by increased ACC oxidase mRNA and enzyme activity. Treatment of flowers with ethylene led to an increase in ethylene production, ACC oxidase mRNA, and ACC oxidase activity in corollas. In contrast, leaves did not exhibit increased ethylene production or ACC oxidase expression in response to ethylene. Gene-specific probes revealed that the ACO1 gene was expressed specifically in senescing corollas and in other floral organs following exposure to ethylene. The ACO3 and ACO4 genes were specifically expressed in developing pistil tissue. In situ hybridization experiments revealed that ACC oxidase mRNAs were specifically localized to the secretory cells of the stigma and the connective tissue of the receptacle, including the nectaries. Treatment of flower buds with ethylene led to patterns of ACC oxidase gene expression spatially distinct from the patterns observed during development. The timing and tissue specificity of ACC oxidase expression during pistil development were paralleled by physiological processes associated with reproduction, including nectar secretion, accumulation of stigmatic exudate, and development of the self-incompatible response.     

Purification of cynarases from artichoke (Cynara scolymus L.): enzymatic properties of cynarase A

Aspartic proteinases from flowers of Cynara cardunculus have been extensively studied and long used as coagulants in the manufacture of several traditional Spanish and Portuguese cheeses. These endopeptidases are called cardosins or cynarases, depending on the authors. However, the proteinases of another plant of the genus Cynara, the artichoke (Cynara scolymus), are less known, probably because the flower of this plant is usually consumed as a vegetable. In the study described here, three proteinases (cynarases A, B and C) with milk-clotting properties were purified from the stigma of artichoke. All three proteinases are glycoproteins and composed of a one large and one small subunit. The enzymatic properties of cynarase A, a glycoprotein containing N-linked high mannose type glycans, which express maximum activity at pH 5.0 and 70 degrees C, were studied in detail. Catalytic and inhibition studies indicated that this cynarase is of the aspartic acid type. The results indicate artichoke extract could also be used in the milk industry in the same way as the extract obtained from the flower of C. cardunculus.     

Pepstatin-sensitive proteolytic activity of sorghum seeds

Two novel proteinases were isolated from resting sorghum seeds and purified 100-fold. The activity of the purified enzymes was completely inhibited by pepstatin A and was unaffected by PMSF, leupeptin, EDTA and E-64 (L-trans-epoxysuccinyl leucylamino 4 guanidino butane), which indicates that they belong to the class of aspartic proteinases. SDS-PAGE and native-PAGE revealed a monomeric 29-kDa enzyme and a heterodimeric 61-kDa enzyme with two S-S linked subunits of 49 and 12 kDa. The proteases have maximum activity at 45 °C and pH 3.5, with haemoglobin as substrate. Activity at 60 °C is higher than at 30 °C.     

Photosynthetic carbon fixation in the corollas of Petunia hybrida

Corollas of Petunia hybrida (cv. Hit Parade Rosa) flowers fixed ¹⁴CO₂ under both light and dark conditions. Rates of light fixation were much higher in mature pink corollas than in young, green corollas [57 and 9 nmol (ngchl)¹ min^-1], paralleling the development of chloroplasts in these tissues. Stomatal conductance in corollas was only 12% of that in green leaves, mainly due to the presence of few, and non-functioning stomata in the corolla. The activity and concentration of ribulose bisphosphate carboxylase (EC 4.1.1.39) in corolla extracts were only about 30% (per unit Chi) of those in extracts from green leaves. These results, together with previous results, might indicate a coordinated reduction in activity of systems participating in photosynthesis in corollas. The fixation products following a 6 s pulse with ¹⁴CO₂, were typical of C, plants in both corollas and green leaves, but a higher level of β-carboxylation products was found in the corollas. The activity of phosphoenol-pyruvate carboxylase (EC 4.1.1.31) (per unit protein) was similar in both tissues. Although the total carbon fixed by the corolla constituted only a small part of the metabolites required for flower development, certain photosynthetic metabolites might have a regulatory role in flower development.     

Partial purification of new milk-clotting enzyme produced by Nocardiopsis sp

Numerous attempts have been made to replace calf rennet with other milk clotting proteases because of limited supply and increasingly high prices. The aim of this work was to investigate the characteristic of the milk-clotting enzyme from Nocardiopsis sp. The partial purification extract was obtained by fractional precipitation with ammonium sulphate. Of the fractions obtained by precipitation, 40-60% possessed the milk-clotting activity (156.25 U/mg). The chromatography of 40-100% ammonium sulphate fraction in DEAE-cellulose yielded four fractions (F4, F5, F6, F7) with milk-clotting activity. The F5 yielded the best milk-clotting activity (20 U/ml). Both crude and partially purified extract were active at the range pH 4.5-11.0, however, optimum activity was displayed at pH 11.0 and pH 7.5, respectively. The milk-clotting activity was highest at 55 degrees C for both crude and partially purified extract. The crude and partial purification extract were inactivated at 65 and 75 degrees C after 30 min.     

Screening for Milk-clotting Enzyme from Basidiomycetes

Screening tests for aspartic proteinases with milk-clotting activity were done on basidiomycetes. Crude enzymes from 6 strains had a high ratio of milk-clotting activity to caseinolytic activity. These enzymes showed acidic pH optimum for proteolytic activity and were inhibited considerably by pepstatin, a specific aspartic proteinase inhibitor. Among them, the crude enzyme from Laetiporus sulphureus was more heat-labile than the other enzymes.     

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.     

Marcescent corollas as functional structures: effects on the fecundity of two insect-pollinated plants

Background and aims

Persistence of withered corollas after anthesis (‘corolla marcescence’) is widespread in angiosperms, yet its functional significance does not seem to have been explored for any species. This note reports the results of experiments assessing the fecundity effects of marcescent corollas in two southern Spanish insect-pollinated plants, Lavandula latifolia (Lamiaceae) and Viola cazorlensis (Violaceae).

Methods

The effect of marcescent corollas on seed production was evaluated experimentally on wild-growing plants. Newly open flowers were randomly assigned to either control or treatment groups in experimental plants. After anthesis, withered corollas of treatment flowers were removed and those in control flowers were left in place. Fruits produced by treatment and control flowers were collected shortly before dehiscence and the number of seeds counted.

Key Results

In V. cazorlensis, removal of withered corollas had no effect on percentage of fruit set, but mean seeds per fruit increased from 9·5 to 11·4. In L. latifolia, corolla removal had no effect on the number of seeds per fruit, but reduced the proportion of flowers ripening fruit from 60 % to 40 %. The detrimental effect of corolla removal on L. latifolia fecundity resulted from the drastic increase in fruit infestation by seed-predatory cecidomyiid larvae, which occurred in 4 % and 34 % of control and treatment fruits, respectively.

Conclusions

Because of their potential effects on plant fecundity, marcescent corollas should not be dismissed a priori as biologically irrelevant leftovers from past floral functions. The simplicity of the experimental layout required to test for short-term fecundity effects of corolla marcescence should help to achieve a better understanding of the ecological and evolutionary correlates of this widespread but poorly understood trait.     

Two types of aspartic proteinases from buckwheat seed--gene structure and expression analysis

Two types of aspartic proteinase (AP) genes have been isolated from the cDNA library of developing buckwheat seeds. Analysis of their sequences showed that one of these, FeAP9, resembled the structure and shared high homology with the so-called typical plant APs characterized by the presence of a plant-specific insert (PSI), an element unique among APs. The other cDNA, FeAPL1, encoded an AP-like protein lacking that domain. Different expression profiles were observed for FeAP9 and FeAPL1. FeAPL1 mRNAs were restricted to the seeds only, whereas FeAP9 mRNAs were also present in the other plant tissues - leaves, roots, and flowers. Higher levels of FeAP9 were observed in senescent leaves compared with green leaves. The differential expression pattern of these two unique APs raises the interesting possibility that these proteinases have unique substrate specificity and may have different roles in plant development and other physiological processes.     

Leaf-Mediated Light Responses in Petunia Flowers

In the present work we studied the role of light in the regulation of flavonoid gene expression and anthocyanin synthesis in petunia (Petunia hybrida) corollas. We found that light is required for chalcone synthase gene (chs) expression, anthocyanin synthesis, and growth of detached and attached petunia corollas. Although direct illumination induced chs expression, pigmentation, and elongation of the detached corollas, irradiation of green leaves or sepals played the main role in the attached corollas. The duration, intensity, and spectrum of the light reaction suggest that phytochrome-mediated high-irradiance reactions are involved in the regulation of corolla development. Using the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, we showed that photosynthesis does not significantly contribute to the leaf-mediated light responses. When sepals were removed or covered. [14C]sucrose up-take by the corolla of detached intact flowers was inhibited. The results of this study suggest that light is perceived by leaves and sepals and enhances corolla sink activity, elongation, pigmentation, and chs expression. The role of leaves and sepals in the light regulation of petunia corolla development is discussed.     

Purification and properties of milk-clotting enzyme from Bacillus subtilis K-26

A milk-clotting enzyme from Bacillus subtilis K-26 was purified by gel filtration and ion-exchange chromatography resulting in a 24-fold increase in specific activity with an 80% yield. Polyacrylamide gel electrophoresis and ultracentrifugel analysis revealed that the purified enzyme was homogeneous and had a molecular weight of 27,000 and a K_m of 2.77mg/ml for κ-casein. The enzyme was most stable at pH 7.5 and showed increasing clotting activity with decrease in milk pH up to 5.0. The maximum milk-clotting activity was obtained at 60°C, but the enzyme was inactivated by heating for 30 min at 60°C. The enzyme was irreversibly inhibited by EDTA and unaffected by DFP. Heavy-metal ions (Hg²⁺, Pb²⁺) inactivated the enzyme.     

The three typical aspartic proteinase genes of Arabidopsis thaliana are differentially expressed.

Genomic sequencing has identified three different typical plant aspartic proteinases in the genome of Arabidopsis thaliana, named Pasp-A1, A2 and A3. A1 is identical to a cDNA we had previously isolated and the two others produce proteins 81 and 63% identical to that predicted protein. Sequencing of the aspartic proteinase protein purified from Arabidopsis seeds showed that the peptides are derived from two of these genes, A1 and A2. Using gene specific probes, we have analyzed RNA from different tissues and found these three genes are differentially expressed. A1 mRNA is detected in all tissues analyzed and more abundant in leaves during the light phase of growth. The other two genes are expressed either primarily in flowers (A3) or in seeds (A2). Insitu hybridization demonstrated that all three genes are expressed in many cells of the seeds and developing seed pods. The A1 and A3 genes are expressed in the sepals and petals of flowers as well as the outer layer of the style, but are not expressed in the transmitting tract or on the stigmatal surface. The A2 gene is weakly expressed only in the transmitting tissue of the style. All three genes are also expressed in the guard cells of sepals. These data suggest multiple roles for aspartic proteinases besides those proposed in seeds.     

Distribution of alpha-Galactosidase in Cucurbita pepo

The distribution of α-galactosidase (α-d-galactoside galactohydrolase [EC 3.2.1.22]) in Cucurbita pepo has been determined in an attempt to assess its involvement in hydrolysis of transport sugars of the raffinose oligosaccharide series ([α-1-6-0-galactopyranosyl]_n sucrose). Extracts prepared from leaves and petioles at different stages of development, roots, flowers, dry and germinating seeds, all contained appreciable levels of α-galactosidase activity. Chromatography of these extracts on DEAE-Sephadex resolved the enzyme into three active isozymic forms. These isozymes were present in all regions of the plant analyzed but their relative proportions varied between tissues and changed within leaf and petiole tissues during development and in seeds during their germination. The level of total α-galactosidase activity in the leaf blade measured on a fresh weight or total protein basis remained constant at all developmental stages analyzed. The occurrence of these isozymes in mature exporting leaves indicates an effective intracellular compartmentation between their location and the sites of galactosyl oligosaccharide biosynthesis, accumulation and movement in the tissue. We have used these results to comment on the transport pathway of galactosyl oligosaccharides between the phloem and surrounding tissues in this plant.     

Purification and characterization of milk-clotting enzymes from oyster mushroom (<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> (Fr.) Kumm)

Three enzymes with milk-clotting activity have been isolated from the fruiting bodies of Pleurotus ostreatus (Fr.) Kumm) by (NH₄)₂SO₄ precipitation, gel chromatography on Sephadex G75, and ion exchange chromatography on carboxymethylcellulose (CMC). Isoelectric points of the enzymes, as determined by isoelectrofocusing, equaled 4.2, 6.7, and 8.8. Inhibition analysis showed that the enzymes with isoelectric points of 4.2 and 6.7 belong to the class of metal-dependent proteinases, while the enzyme with the isoelectric point of 8.8 belongs to the serine protease class.     

Processing, activity, and inhibition of recombinant cyprosin, an aspartic proteinase from cardoon (Cynara cardunculus).

The cDNA encoding the precursor of an aspartic proteinase from the flowers of the cardoon, Cynara cardunculus, was expressed in Pichia pastoris, and the recombinant, mature cyprosin that accumulated in the culture medium was purified and characterized. The resultant mixture of microheterogeneous forms was shown to consist of glycosylated heavy chains (34 or 32 kDa) plus associated light chains with molecular weights in the region of 14,000-18,000, resulting from excision of most, but not all, of the 104 residues contributed by the unique region known as the plant specific insert. SDS-polyacrylamide gel electrophoresis under non-reducing conditions indicated that disulfide bonding held the heavy and light chains together in the heterodimeric enzyme forms. In contrast, when a construct was expressed in which the nucleotides encoding the 104 residues of the plant specific insert were deleted, the inactive, unprocessed precursor form (procyprosin) accumulated, indicating that the plant-specific insert has a role in ensuring that the nascent polypeptide is folded properly and rendered capable of being activated to generate mature, active proteinase. Kinetic parameters were derived for the hydrolysis of a synthetic peptide substrate by wild-type, recombinant cyprosin at a variety of pH and temperature values and the subsite requirements of the enzyme were mapped using a systematic series of synthetic inhibitors. The significance is discussed of the susceptibility of cyprosin to inhibitors of human immunodeficiency virus proteinase and particularly of renin, some of which were found to have subnanomolar potencies against the plant enzyme.     

Translocation of 14C-sucrose in Relation to Changes in Carbohydrate Content in Rose Corollas Cut at Different Stages of Development

The dry matter and carbohydrate contents of intact growing ‘Sonia’rose corollas were measured from an immature bud to full expansionof the petals. Reducing sugars and starch, but not sucrose,accumulated throughout most of the corolla development. Thesefindings were compared with the carbohydrate changes in thecorollas of flowers cut at different stages and allowed to agewith their stems either in water or in a sucrose-containingsolution. For a few days after cutting the carbohydrate metabolismof the cut flower roughly paralleled that of the intact floweruntil starch hydrolysed to maintain the soluble carbohydratepool. Feeding with the sucrose solution maintained the solublecarbohydrate levels and retarded the hydrolysis of starch. The cut flowers were fed with ¹⁴C-sucrose and the labelled metabolitesin the leaves and flowers were analysed. Active incorporationof ¹⁴C into ethanol-soluble carbohydrates, starch and ethanol-insolublematerial was found indicating that an active anabolic phaseprecedes the catabolic phase during the senescence of the cutflower. The findings are discussed in relation to the source-sinkhypothesis of flower development, with regard to the senescenceand growth of the corollas of cut and intact flowers respectively.