Vacuoles were prepared from endosperm tissue of 4-day-old castor bean seedlings (Ricinus communis var. Hale) and purified on a stepped sucrose gradient. It was shown by assays of marker enzymes that there was only trace contamination of the final preparation by other organelles (mitochondria, glyoxysomes, nuclei, spherosomes, and plastids) and by cytoplasmic components. Hydrolytic enzymes (acid protease, carboxypeptidase, phosphodiesterase, RNAase, phytase and β-glucosidase) were present in the isolated vacuoles in amounts indicating a primarily vacuolar localization in vivo. The vacuoles also contained storage protein and high concentrations of sucrose. The over-all results indicate that the vacuoles from castor bean endosperm are the site of hydrolysis of the constituents of the protein bodies and are a temporary storage compartment for the sucrose produced from fat and protein reserves.

Hydrolases in Vacuoles from Castor Bean Endosperm

Publisher Summary The varietal specificity in gene–for–gene interactions as a surface–surface interaction biochemically is similar to many other biological recognition phenomena This chapter discusses the recognition, resistance, and the role of plant lectins in host–parasite interactions Plant lectins posses certain similar properties to proteinaceous inhibitors and appear to be associated with plant cell walls The antigenic determinants may be recognized by plant carbohydrate binding proteins known as lectins which are able to discriminate between various blood groups Lectins are generally extracted from plant tissues by homogenizing the tissue in physiological saline They are used to investigate egg surface in sea urchins and mammals They have the ability to interact with different types of animal cells to produce various effects Genetic analysis of many instances of varietal specificity in host plant–fungal parasite interactions reveals that the resistance and virulence characteristics are under oligogenic control and the genes are interdependent for resistance susceptibility and virulence–avirulence Therefore, a given avirulent fungal race will differ from its virulent counterpart in that a surface–localized carbohydrate–containing macromolecule will have been modified Perhaps by the addition of a specific sugar in a certain configuration, it is now recognized as a parasite signal by the products of host resistance genes, or sensors

Recognition, Resistance and the Role of Plant Lectins in Host-Parasite Interactions

Cell death is as difficult to define as clinical death. The research scientist, like the medical practitioner, often finds himself probing the remains. Cell death itself is a dynamic process, and the end point or the point of commitment may be both fleeting and controversial. Much of the scientific evidence is still largely histological or cytological so that as D. H. Lawrence (1918) surmized, ‘We have only a hardened bit of the past the bygone life under our observation’. In such a situation we have to select profiles of previously dying cells from amongst a host of artificially killed or fixed cells.

Techniques for demonstrating cell death

In order to elucidate the events that lead to cellular autolysis, and thus better understand the mechanism of cellular incompatibility betweenSedum telephoides andSolanum pennellii stems, we have followed the appearance and fate of the hydrolytic enzyme acid phosphatase in both the compatibleSedum autograft and the incompatibleSedum/Solanum heterograft. Acid phosphatase was localized by a modified Gomori-type reaction. Following an initial association with the endoplasmic reticulum and dictyosomes by 6–10 hours after grafting, acid phosphatase activity in the compatibleSedum autograft was associated primarily with the plasmalemma, tonoplast, and vacuole. This strict compartmentation in membranes or organelles and absence of enzyme from the cytosol was maintained throughout the development of the compatible autograft inSedum. Although acid phosphatase activity in the incompatible heterograft betweenSedum andSolanum was initially similar to the compatible autograft inSedum, a marked difference in enzyme localization occurred in the two graft partners over time.Solanum cells accumulated increased amounts of acid phosphatase, but the enzyme remained sequestered in the plasmalemma, tonoplast, and vacuole. In comparableSedum cells, however, there was a dramatic increase in acid phosphatase activity in the cytosol, often without any prior compartmentation within the vacuole. This high activity of acid phosphatase in theSedum cytosol was correlated with cellular autolysis, death, and eventual cell collapse to form the characteristic necrotic layer that insulates the stock from the scion. These results suggest that the lethal cellular senescence associated withSedum cells of the incompatible heterograft is correlated with a cytoplasmic release of acid phosphatase. A similar release of the enzyme does not occur in theSolanum stock or in the compatibleSedum autograft. Thus, while acid phosphatase synthesis and/or activation is induced in both the compatible and incompatible grafts, incompatibility betweenSedum andSolanum involves a failure ofSedum cells to isolate hydrolytic enzymes from the cytosol, which subsequently leads to cellular necrosis.

Studies of vegetative compatibility-incompatibility in higher plants: III. The involvement of acid phosphatase in the lethal cellular senescence associated with an incompatible heterograft

The lysosomal concept plays an important role in explaining numerous pathological processes in animals (27). For some reason plant pathologists have been reluctant to borrow or even consider concepts used in animal pa­ thology. However, evidence is mounting that lysosomes may be just as impor­ tant in plant pathology as in animal pathology. A rationale for the existence of lysosomes in both animal and plant cells can be developed without having to examine cells directly. Anabolism and catabolism take place simultaneously in living cells. These two processes are antagonistic if allowed to occur at the same site. The compartmentalization of hydrolytic enzymes into membrane-bound organelles would provide a mechanism whereby enzymes and substrates of biosynthesis in the cell would not be destroyed during anabolism. Such organelles would also provide loci for the hydrolysis of macromolecules. Since the original discovery of lysosomes in animals by DeDuve & Novi­ koff the lysosomal concept has been continually expanded (26). Originally DeDuve emphasized the autOlytic function of lysosomes, calling them "sui­ cide bags." Subsequent research has shown that lysosomes are important or­ ganelles in normal metabolism and that a number of pathological processes can be explained by their malfunction (27). A lysosome can be defined as a single-membrane-bounded body that con­ tains more than one acid hydrolytic enzyme (34). Because there is now in­ formation on the origin and disposition of lysosomes, we can think in terms of a lysosomal system. LysosQmes are involved essentially with catabolic pro-

A Lysosomal Concept for Plant Pathology

SUMMARY: Histochemical evidence showed that infection of tuber tissues and tissue-culture cells of Solanum tuberosum by Phytophthora erythroseptica, Phytophthora infestans and Fusarium caeruleum caused swelling and disruption of host cytoplasmic particles containing acid phosphatase, esterases and proteases. Heavy diffuse cytoplasmic staining for acid phosphatase was a consistent feature of infection by all three fungi, but staining reactions for esterases and proteases showed much less diffuse staining and a lesser degree of particle swelling. Biochemical assays showed that acid phosphatase was liberated from the particulate fraction to the supernatant fluid fraction of infected callus cells; acid ribonuclease behaved similarly. The biochemical evidence suggested that esterase activity of infected cells remained associated with a sedimentable fraction from cell homogenates. Assay methods for proteolytic enzymes did not confirm the histochemical evidence of a particulate localization of these enzymes in healthy or infected callus tissues. An excess recovery of ribonuclease from tissues infected with P. infestans and F. caeruleum was found; the significance of this is discussed. Attempts to separate a particulate fraction rich in hydrolases from homogenates of healthy or infected potato tubers were unsuccessful.

/pdf/release-of-hydrolytic-enzymes-from-cytoplasmic-particles-of-203po1137x.pdf

Release of Hydrolytic Enzymes from Cytoplasmic Particles of Solanum Tuber Tissues During Infection by Tuber-rotting Fungi

SUMMARY: Histochemical methods showed that tuber tissue and tissue culture cells of Solanum tuberosum contained phase-dense particles which absorbed neutral red and fluorescent dye, and particles which contained acid phosphatase and a non-specific esterase. It is suggested that these structures may be comparable to the lysosomes of animal tissues and the lysosome-like structures of plant cells which possess similar and additional properties. During infection of Solanum tissues by Phytophthora erythroseptica there was swelling and disruption of these host cell particles, accompanied by the release of acid phosphatase and esterase. Biochemical assay for acid phosphatase confirmed that infection of host cells resulted in the liberation of acid phosphatase from a particulate to the supernatant fluid fraction of cell homogenates.

Clare Coombes

Papers

Release of Hydrolytic Enzymes from Cytoplasmic Particles of Solanum Tuber Tissues During Infection by Tuber-rotting Fungi

The disruption of lysosome-like particles of Solanum tuberosum cells during infection by Phytophthora erythroseptica Pethybr