Two new aspartic proteinases have been isolated from stigmas of the cardoon Cynara cardunculus L. by a two-step purification procedure including extraction at low pH, gel filtration on Superdex 200, and ion-exchange chromatography on Mono Q. To follow the conventional nomenclature for aspartic proteinases, we have named these proteinases cardosin A and cardosin B. On SDS/PAGE, cardosin A migrated as two bands with apparent molecular masses of 31 000 Da and 15000 Da where as the chains of cardosin B migrated as bands of 34000 Da and 14000 Da. The partial amino acid sequences of the two cardosins revealed that they are similar but not identical, and that they differ horn the previously reported cardoon proteinases named cynarases, which were assumed to be derived from a common precursor. Although the cardosins show some degree of similarity to each other, we could detect no immunological cross-reactivity between them. Both cardosins were active at low pH and were inhibited by pepstatin, with Ki values of 3 nM for cardosin A and 1 nM for cardosin B, indicating that they belong to the class of aspartic proteinases. Significant differences between the two enzymes were also found for the Kcat/Km values for the hydrolysis of two chromophoric synthetic peptides. The active-site ionization constants, pKe1 and pKe2, for cardosin A are 2.5±0.2 and 5.3±20.2, whereas for cardosin R they are 3.73±10.09 and 6.7±50.1. The results herein described on the structural and kinetic properties of the cardosins indicate that they are the products of distinct genes which have probably arisen by gene duplication. A scheme for the proteolytic processing of the two enzymes is also proposed.

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Purification, characterization and partial amino acid sequencing of two new aspartic proteinases from fresh flowers of Cynara cardunculus L.

Previous work suggested that the aspartic proteinase from Hordeum vulgare (HvAP) would be a vacuolar protein in plant cells Based on N-terminal sequencing we show that the in vitro-translated protein was translocated into the lumen of microsomal membranes, causing a concomitant removal of 25 amino acid residues from the protein Vacuoles were purified from barley leaf protoplasts and were shown to contain all of the aspartic proteinase activity found in the protoplasts This vacuolar localization of HvAP was confirmed with immunocytochemical electron microscopy using antibodies to HvAP in both barley leaf and root cells In an attempt to discern a function for this protease, we investigated the ability of HvAP to process the C-terminal proregion of barley lectin (BL) in vitro Prolectin (proBL), expressed in bacteria, was processed rapidly when HvAP was added Using several means, we were able to determine that 13 amino acid residues at the C terminus of proBL were cleaved off, whereas the N terminus stayed intact during this incubation Immunohistochemical electron microscopy showed that HvAP and BL are co-localized in the root cells of developing embryos and germinating seedlings Thus, we propose that the vacuolar HvAP participates in processing the C terminus of BL

The Aspartic Proteinase of Barley Is a Vacuolar Enzyme That Processes Probarley Lectin in Vitro

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.

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

The development of techniques for somatic embryogenesis in conifers has led to rapid advances in the ability to culture conifer tissue in vitro. Somatic embryogenesis now provides a means to clonally propagate commercially valuable conifers of several species and offers an in vitro experimental system for studying embryogenesis. As a result of these developments, the conifer zygotic and somatic embryo system has recently attracted the attention not only of the cell biologist but also of biochemists and molecular biologists. In this review I have attempted to consolidate the recent information on ultrastructure, biochemical and molecular aspects of conifer zygotic and somatic embryogenesis. In the first section, salient features of zygotic embryogenesis are highlighted followed by biochemical and ultrastructural characterization of development with reference to storage reserve deposition. In the second section, detailed characterization of seed storage proteins, changes in gene expression with emphasis on storage protein genes and Lea genes during zygotic and somatic embryogenesis are described. Effect of culture treatments such as abscisic acid, osmoticum and desiccation on storage protein gene expression in somatic embryos is discussed. Based on these studies, comparisons are presented between angiosperm and gymnosperm embryogeny regarding the unique morphological and developmental patterns of conifer embryogenesis. Finally, a brief discussion of recent progress in biochemical and molecular characterization of conifer seed germination is presented.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S0960258500002439

Conifer zygotic embryogenesis, somatic embryogenesis, and seed germination: Biochemical and molecular advances

Aspartic proteases (APs) one of the main proteinase classes, have different physiological functions in animals, fungi and viruses. In plants, knowledge of the biological roles of APs is less well developed. An AP has been purified from potato tuber and leaves (Guevara et al., 1999, 2001). In this paper, the changes in the level of AP in response to infection by Phytophthora infestans (P. infestans) and wounding were studied in intercellular washing fluids (IWFs) from tuber disks of two potato cultivars differing in their susceptibility to P. infestans. A differential induction was observed between both cultivars: in the resistant cultivar, induction was higher and faster in infected tissues than in wounded ones. In the susceptible cultivar, a lower and later accumulation was observed than in the resistant cultivar. In addition, AP had a direct inhibitory effect on the germination of cysts of P. infestans and conidia of Fusarium solani. The pattern of accumulation and in vitro activity of AP suggest that this enzyme may have a role in the defense response of potato.

An Aspartic Protease With Antimicrobial Activity is Induced after Infection and Wounding in Intercellular Fluids of Potato Tubers

A high-molecular-weight aspartyl proteinase complex, sensitive to pepstatin A was purified to near electrophoretic homogeneity from dry seeds ofjack pine (Pinus banksiana, Lamb.). Two partial activities, endo-and exo-proteinolytic, were associated with the complex, judged by the analysis of haemoglobin hydrolysis products. The high activity of this enzyme in dry jack pine seed suggests a possible function in early germination.

Jacqueline Bourgeois

Papers

Purification and characterization of an aspartyl proteinase from dry jack pine seeds