A cytochemical and biochemical study of acid hydrolases in developing plant cells, with particular reference to non-specific esterases
James, David John
mesheuropmc: food and beverages
Investigations have been carried out into the occurrence and functions of acid hydrolases during plant cell division, differentiation and senescence using histochemical and biochemical procedures. Statistical analyses of histochemical data have shown that particles staining for naphthol AS BI phosphatase in dividing unfixed root cells of Vicia f aba do not act as a 'trigger 1 in the way suggested for animal cells, since there were significantly greater numbers of particles in mitotic cells as opposed to interphase cells in all 3 root tissues, apical initials, procortex and central cylinder. Increasing the length of incubation time markedly increased the number of particles in all 3 tissues.\ud \ud The procedure for determining ft -glycerophosphatase activity in glutaraldehyde-fixed root tips was found to be unsatisfactory since cells in sections of tissues incubated without substrate contained lead-staining particles and in both test and control sections diffuse cytoplasmic and nuclear staining were apparent. \ud \ud Using the histochemical reagents, naphthol AS D acetate and the diazonium salt, fast red violet LB salt, a biochemical study of non-specific esterases extracted from broad bean root tips, has shown that these enzymes have an estimated Km of 0.07 mM, a pH optimum of 5.5 and that they lose substantial activity when dialysed against distilled water as opposed to 5mM magnesium chloride and 10mM phosphate buffer. However, adding magnesium and/or chloride ions to the assay system reduced enzyme activity.\ud \ud A combined cytochemical and biochemical analysis of non-specific esterases in differentiating root tissues of the broad bean showed that maximal enzyme activity, expressed on a per segment, per unit protein or per unit area of average-sized section basis, occurred within the zone of cell expansion, elongation and differentiation of procambial tissues.\ud \ud Cytochemical studies showed that in glutaraldehyde-fixed roots activity was only slightly inhibited when compared with unfixed material, whereas biochemical studies revealed that only about ⅛ of the activity of enzyme extracted from unfixed roots was 8 present in homogenates of fixed roots.\ud \ud In a study of non-specific esterases in soft fruit ripening and senescence, cytochemical studies demonstrated that enzyme activity was associated with subcellular structures in the pulp cells of the ripening strawberry. The fleshy mesocarp tissues of raspberry and blackberry, however contained no enzyme end-product at any of the stages of ripening examined.\ud \ud In older strawberry receptacles, also examined biochemically, enzyme end-product was associated with large spherical lipid-dropletlike bodies absent in younger receptacles. Assays of non-specific esterases in immature, maturing and mature pulp tissues showed that these enzymes increased several fold during ripening when expressed on a per g fresh weight or per berry basis. Although esterase activity changed little during the transition from 'maturing 1 to 'mature 1 when expressed on either basis, the specific activity of the enzymes fell markedly. The Km values of enzyme extracts from the 3 stages of berry development decreased as ripening proceeded denoting an increased affinity of these enzymes for their substrates possibly due to the removal of an inhibitor or a change in iso-enzyme pattern. Esterases from the three ripening stages exhibited broad pH optima (pH 5-5 - 6.5) although limitations of the assay procedure prevented the detection of possible alkaline optima.\ud \ud Ultrastructural studies showed that changes in the subcellular organelles of ripening strawberry pulp cells were similar to that reported for other senescihg.plant material, vesiculation of many organelles and loss of ribosomal material being the most characteristic. Attempts to localise esterases in fruit cells at the ultrastructural level were unsuccessful since electron dense end-product of diazotised lead phthalocyanin bound to a variety of structures even in the absence of the enzymes’ substrate. In contrast the use of the diazotate of LPED in Vicia faba root cells was more promising in that in test tissue end-product was found in small vacuoles and rough endoplasmic reticulum, whilst control cells incubated in the absence of substrate lacked enzyme end-product.\ud \ud The water-soluble epoxy resin, Durcupan, was assessed for its effect on plant cell organelle morphology by comparing similar tissue conventionally dehydrated in alcohol. No gross changes in ultrastructure were observed.
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