Dissecting cardosin B trafficking pathways in heterologous systems

In cardoon pistils, while cardosin A is detected in the vacuoles of stigmatic papillae, cardosin B accumulates in the extracellular matrix of the transmitting tissue. Given cardosins’ high homology and yet different cellular localisation, cardosins represent a potentially useful model to understand and study the structural and functional plasticity of plant secretory pathways. The vacuolar targeting of cardosin A was replicated in heterologous species so the targeting of cardosin B was examined in these systems. Inducible expression in transgenic Arabidopsis and transient expression in tobacco epidermal cells were used in parallel to study cardosin B intracellular trafficking and localisation. Cardosin B was successfully expressed in both systems where it accumulated mainly in the vacuole but it was also detected in the cell wall. The glycosylation pattern of cardosin B in these systems was in accordance with that observed in cardoon high-mannose-type glycans, suggesting that either the glycans are inaccessible to the Golgi processing enzymes due to cardosin B conformation or the protein leaves the Golgi in an early step before Golgi-modifying enzymes are able to modify the glycans. Concerning cardosin B trafficking pathway, it is transported through the Golgi in a RAB-D2a-dependent route, and is delivered to the vacuole via the prevacuolar compartment in a RAB-F2b-dependent pathway. Since cardosin B is secreted in cardoon pistils, its localisation in the vacuoles in cardoon ovary and in heterologous systems, suggests that the differential targeting of cardosins A and B in cardoon pistils results principally from differences in the cells in which these two proteins are expressed.     

Ultrastructural and physiological differences between buds and mature flowers of Petunia hybrida prior to and following pollination

The structural events accompanying the maturation of the pistil of Petunia hybrida have been studied in detail, together with the changes in the protein spectrum of the transmitting tissue that occur over this period. These events have been considered in terms of the acquisition of the self-incompatibility response, which occurs while the pistil is enclosed in the bud. Apart from several minor differences, the young pistils differ only from the mature in that their transmitting tissue cells fail to respond to pollination by undergoing characteristic ultrastructural changes. Data from electrofocusing indicates that several proteins, mobilised in the mature transmitting tissue some three hours after pollination, are absent from bud pistils. It is proposed that the pollination-stimulated release of certain polypeptides, believed to be involved in the self-incompatibility response, does not take place in young pistils. These observations are considered with reference to currently-accepted models of the operation of the self-incompatibility mechanism in Petunia.     

Pistil induction by hormones from callus of <Emphasis Type="Italic">Oryza sativa</Emphasis> in vitro

This study describes the successful formation of floral organ pistil from the callus of pistil explants of Oryza sativa L. For induction of floral organs, different explants—including young embryo, lemma, palea and pistil—were used for callus induction with different combinations of N⁶-benzyladenine and 2,4-dichlorophenoxyacetic acid (2,4-D). High frequencies of callus formation from pistil and young embryo explants were achieved. Floral organs were induced after calli from pistils were transferred to medium containing both zeatin and 2,4-D. The morphological characteristics of the pistil-like organs are very similar to those formed in planta though with minor differences. Further histological study revealed that the in vitro pistil contains an ovule within its ovary. Furthermore, a pistil-specific gene, OsMADS3 used as a molecular marker for pistil identity, was expressed in the pistil-like organs as it was in pistils in the flower of the plant.     

Action of the Tunicate locus on maize floral development

The co-dominant Tunicate (Tu) mutation in maize causes nonreproductive structures in both the male and female inflorescences to be enlarged. This mutation also affects sex determination, permitting the development of pistils in the normally staminate tassel. In order to characterize the role of the normal tu gene product, we have analysed genetic interaction between Tu and other mutations that perturb specific stages of floral development. Synergistic interactions observed suggested that the tu product functions in at least three stages of floral development–determination of spikelet primordia, differentiation of non-reproductive organs and pistil abortion in the tassel. © 1994 Wiley-Liss, Inc.     

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.     

Cardosin A endocytosis mediated by integrin leads to lysosome leakage and apoptosis of epithelial cells

Cardosin A is an aspartic protease present in large amount in the pistils of cardoon flowers. This protease is known to contain an -Arg-Gly-Asp- (RGD) motif located on the molecular surface. In this study, we found that isolated recombinant cardosin A attached to human epithelial cells A549, mediated by the binding of its RGD motif to cell surface integrins. The cell bound cardosin A was internalized to endosomes and lysosomes and triggered the permeability of lysosomal membrane leading to apoptosis of the epithelial cells. These events are identical to those observed for three RGD-containing aspartic proteases, Saps 4-6, secreted by Candida albicans. Such a process, which has been called the Trojan Horse mechanism, is believed to benefit the invasion of C. albican into the epithelium of the host. The location of the RGD motifs of cardosin A and Saps 4-6 are on the opposite ends of the homologous three-dimensional structures, suggesting that the Trojan Horse mechanism is insensitive to the RGD position. Current finding also suggests that cardosin A may have a defensive function against the ingestion of cardoon flowers by human, insects, and other herbivores.     

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     

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.     

AN ULTRASTRUCTURAL STUDY OF TRANSMITTING TISSUE DEVELOPMENT IN THE PISTIL OF LILIUM LEUCANTHUM

A study of developing transmitting tissue of Lilium Leucanthum pistils was undertaken in order to correlate structure with function. Lining the stylar canal are stigmatoid cells which contain a secretory zone consisting of a labyrinth of wall ingrowths characteristic of transfer cells. The functional feature of the labyrinth is a high surface-to-volume ratio that facilitates an intensive transmembrane flux of solutes. Stigmatoid cells in various stages of development and maturation have been investigated with the aid of electron and light optics in conjunction with cytochemical techniques. During development of the secretory zone, vesicles, formed by hypersecretory dictyosomes, fuse with the plasma membrane and contribute their contents to the growing wall. The pattern of secretory zone development is basipetal and is associated with initiation of chemotropism. In a mature pistil large crystals, having a basipetal pattern of development, and sensitive to protease, can be observed in the cytoplasm of stigmatoid cells. At anathesis, degradation of the crystal can be observed in the cells of the stigma surface and progresses basipetally as the pistil ages. The role of the crystal is uncertain. Immature pistils cultured in the presence of labeled proline take up the label which at maturity of the pistil is transferred to the canal of the pistil. The label is found in the crystals and the secretory zone of the stigmatoid cells. Pollen tubes growing in the canal of a labeled pistil take up the label.     

Cardosins in postembryonic development of cardoon: towards an elucidation of the biological function of plant aspartic proteinases

Summary. Following on from previous work, the temporal and spatial accumulation of the aspartic proteinases (EC 3.4.23) cardosin A and cardosin B during postembryonic seed development of cardoon (Cynara cardunculus) was studied. mRNA and protein analyses of both cardosins suggested that the proteins accumulate during seed maturation, and that cardosin A is later synthesised de novo at the time of radicle emergence. Immunocytochemistry revealed that the precursor form of cardosin A accumulates in protein bodies and cell walls. This localisation in seeds is different from that previously described for cardoon flowers, suggesting a tissue-dependent targeting of the protein. It is known that procardosins are active and may have a role in proteolysis and processing of storage proteins. However, the presence of procardosin A in seeds could be related to the proposed role of the plant-specific insert in membrane lipid conversion during water uptake and solute leakage in actively growing tissues. This is in accordance with the recently proposed bifunctional role of aspartic proteinase precursor molecules that possess a membrane-destabilising domain in addition to a protease domain. Mature cardosin B, but not its mRNA, was detected in the first hours after seed imbibition and disappeared at the time of radicle emergence. This extracellular aspartic protease has already been implicated in cell wall loosening and remodelling, and its role in seed germination could be related to loosening tissue constraints for radicle protusion. The described pattern of cardosin A and B expression suggests a finely tuned developmental regulation and prompts an analysis of their possible roles in the physiology of postembryonic development. Correspondence: C. S. Pereira, Institute for Molecular and Cell Biology, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.     

Isolation of Tissue-Specific cDNAs from Tomato Pistils

We have used a differential plaque hybridization screening procedure to isolate cDNA clones for genes that show elevated or exclusive expression in tomato pistils. Clones that showed maximal expression in immature pistils (premeiotic to early meiosis) and mature pistils (at anthesis) were isolated. Of nine clones that were characterized, four were found also to express at some stage of anther development. In situ hybridization experiments showed that expression of the genes we have identified is very tightly regulated both spatially and temporally within the pistil. One gene was identified that is expressed in the pistil only in the transmitting tissue of the style. A second gene was found to express exclusively in two to three cell layers of the ovules for a period of less than eight days.     

Cloning and characterization of cDNA encoding cardosin A, an RGD-containing plant aspartic proteinase.

Cardosin A is an abundant aspartic proteinase from pistils of Cynara cardunculus L. whose milk-clotting activity has been exploited for the manufacture of cheese. Here we report the cloning and characterization of cardosin A cDNA. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant-specific insertion (PSI), and revealed the presence of an Arg-Gly-Asp (RGD) motif, which is known to function in cell surface receptor binding by extracellular proteins. Cardosin A mRNA was detected predominantly in young flower buds but not in mature or senescent pistils, suggesting that its expression is likely to be developmentally regulated. Procardosin A, the single chain precursor, was found associated with microsomal membranes of flower buds, whereas the active two-chain enzyme generated upon removal of PSI is soluble. This result implies a role for PSI in promoting the association of plant aspartic proteinase precursors to cell membranes. To get further insights about cardosin A, the functional relevance of the RGD motif was also investigated. A 100-kDa protein that interacts specifically with the RGD sequence was isolated from octyl glucoside pollen extracts by affinity chromatography on cardosin A-Sepharose. This result suggests that the 100-kDa protein is a cardosin A receptor and indicates that the interaction between these two proteins is apparently mediated through RGD recognition. It is possible therefore that cardosin A may have a role in adhesion-mediated proteolytic mechanisms involved in pollen recognition and growth.     

青秆竹花的解剖观察与分析

为明确自然状态下青秆竹(Bambusa tuldoides)不同发育阶段花器官的形态以及雌雄配子体的发育状态,总结其败育类型,该文通过采用解剖和切片等方法对青秆竹花器官的各部分外观形态以及雌雄配子体的发育过程进行观察,并分析其结实率低下的原因。结果表明:(1)青秆竹小穗为无限花序,下部的小花先发育,但基部具有潜伏芽,因此又具有有限花序的特征;小穗柄不发达,簇生花枝节部。(2)每朵小花拥有内、外稃各1枚,花药6枚,浆片3枚,雌蕊1枚;浆片透明,边缘具有发达的纤毛;子房具棱,子房上部具绒毛,子房1室,侧膜胎座,倒生胚珠,三分枝羽状柱头。(3)青秆竹花药具有4个药室,花药壁由表皮、药室内壁、中层、绒毡层4层结构组成;绒毡层为腺质型,花药发育后期极度退化;小孢子母细胞分裂类型为连续型,产生两边对称型小孢子,花粉粒细胞成熟后为3核。(4)雄蕊和雌蕊出现多种败育类型,可能是导致结实率低的主要原因。综上结果表明,青秆竹花器官的形态结构发育正常,而雌雄配子体发育过程中出现异常,造成了其结实率低。     

Characterization of the pollen growth transition in self-incompatible <Emphasis Type="Italic">Petunia inflata</Emphasis>

In Petunia inflata, as in other species that shed bicellular pollen, early pollen tube growth in the pistil is slow, then increases 2- to 5-fold depending on the genotype of the female parent. We refer to the time point at which pollen tubes enter the accelerated phase of growth as the pollen growth transition (PGT). Here, we present evidence that pre-PGT and post-PGT growth are quantitatively and qualitatively different, and that the PGT is triggered when pollen tubes reach the transition zone (TZ) below the stigma. The capacity of various pistil zones to precipitate the PGT was tested through 'stump' pollinations: varying lengths of the pistil apex were excised, the cut surface of the remaining pistil (the stump) coated with stigmatic exudates then dusted with compatible pollen. Pollen applied to TZ tissues entered the PGT earlier than pollen growing in intact control pistils; the PGT was delayed in stylar stumps, largely because of delayed germination and reduced pre-PGT growth. In immature pistils, the PGT was delayed by several hours relative to its onset in mature pistils. The PGT fails to occur in pollen cultured in vitro. Collectively, the data suggest that pollen tubes become competent to enter the PGT when they reach a critical size, but the physicochemical environment of the transmitting tissue is necessary for triggering the cellular changes that result in accelerated growth. An analysis of the distribution of pollen tube tips before and after the PGT suggests that pollen competition is most intense during the pre-PGT phase.     

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.     

Different regulatory processes control pollen hydration and germination in Arabidopsis

To elucidate the functional differences in how Arabidopsis stigmas regulate pollen hydration and germination, we analyzed receptivity of stigmas, epidermal surfaces (leaves, stems of inflorescence bolts, and floral organs), and an abiotic surface (cover glass) for pollen hydration and germination. Using 65% relative humidity (RH), we found that mature pollen grains were able to hydrate and germinate on stigmas at flower developmental stages 9–13, but not on the distal end of pistils at stage 8, epidermal surfaces, or glass. Furthermore, under 100% RH, pollen grains could hydrate on all tested surfaces, but pollen germination was observed only on the young floral organs (stages 9–12) and the stigmas at stages 9–13. The distal ends of pistils at stage 8, the epidermal surfaces, and the cover glass did not support pollen germination even under 100% RH. Our results indicate that pistil factors regulating pollen hydration and germination are synthesized at stage 9 when stigmatic papillar cells begin to develop. Although pistil factors involved in pollen hydration may only be present on the stigma, the factors involved in pollen germination may localize on both the stigma and surfaces of unopened floral organs.     

Some Aspects of Growth Characteristics of Tobacco Pistils in vitro

In order to learn more about how young pistil primordia assimilateor produce the regulatory information influencing further growthand development and how the information is collated and processedwithin the primordium, young pistil primordia from two speciesof Nicotiana were excised and placed on the surface of a developmentallyneutral medium. The pistils of one type of tobacco seemed tobe almost fully autonomous for its growth and development becauseit typically grew to a size and stage of development comparableto in vivo grown pistils. Pistils of the other type did notfare so well. Other potential influences were tested; the degreeof injury to, or exposure of, the placentas, the effects ofculturing while attached to other floral members in every combination,and the effects of kinetin were investigated. Each of theseprovided clues relating to how the plant regulated pistil development.