Acid Phosphatase Development during Ripening of Avocado

Abstract

The activity and subcellular distribution of acid phosphatase were assayed during ethylene-induced ripening of whole fruit or thick slices of avocado (Persea americana Mill. var. Fuerte and Hass). The activity increased up to 30-fold during ripening in both the supernatant fraction and the Triton X-100 extract of the precipitate of a 30,000g centrifugation of tissue homogenates from whole fruit or slices ripening in moist air. Enzyme activity in the residual precipitate after Triton extraction remained constant. The development of acid phosphatase in thick slices ripened in moist air was similar to that in intact fruit, except that enzyme development and ripening were accelerated about 24 hours in the slices. The increase in enzvme activity that occurs in slices ripening in moist air was inhibited when tissue sections were infiltrated with solutions, by aspiration for 2 minutes or by soaking for 2 hours, anytime 22 hours or more after addition of ethylene. This inhibition was independent of the presence or absence of cycloheximide or sucrose (0.3-0.5M).However, the large decline in enzyme activity in the presence of cycloheximide, as compared with the controls, indicated that synthesis of acid phosphatase was occurring at all stages of ripening.Ripening of fleshy fruits is an irreversible process of senescence (1,6,8,(16)(17)(18) that is dependent on synthesis of both RNA and protein (2,8,15). Although there is no consistent correlation among different kinds of fruit between changes in the levels of total RNA and protein and the ripening process, there is ample evidence for an increase in activity of manv enzymes during ripening (12,14,19 (22 C), in a large desiccator, at high relative humidity, with an opening to allow for gas exchange. Alternatively, 12-mm thick, cross-sectional slices from the midregion of avocados were similarily treated with ethylene. After cutting, the slices were rinsed briefly in running tap water, blotted, and then stored in a humidity chamber until assayed.Enzyme Extraction. For assay of acid phosphatase, duplicate samples (500 mg fresh weight) of inner mesocarp tissue were taken daily from the midregion of different intact fruits. Most experiments, however, were conducted by aging thick (12 mm) slices and similarly taking daily tissue samples from the slices, after first removing about 2 mm from the outer surfaces. Tissue samples were homogenized in 10 ml of pH 5, 0.1 M acetate buffer containing 7% carbowax 4000, and were centrifuged at 2 C for 15 mmn at 15,000g. Carbowax was added to prevent inactivation of enzyme by tannins (20). The supernatant fraction was used directly as a source of soluble enzyme. The precipitate was washed once in 10 ml of grinding medium by centrifugation as above, and the supernatant fraction was discarded. The precipitate was resuspended with gentle stirring in 10 ml of grinding medium containing 1lcTriton X-100, and was centrifuged similarly; the supernatant fraction is referred to as the Triton extract. The residual precipitate was also retained ...

Hydrolytic Enzyme Activities and Protein Pattern of Avocado Fruit Ripened in Air and in Low Oxygen, with and without Ethylene

“…5), with the possible exception of polypeptides j(51.5 kD) and v(13.5 kD) whose levels may have been enhanced by ethylene. DISCUSSION The data demonstrate that ripening of avocado fruit is associated with substantial increases in the activities of polygalacturonase, acid phosphatase and cellulose, consistent with previously published reports (3,21,23). Furthermore, it is shown that the development of these enzyme activities is suppressed to different degrees in fruits stored in low 02 atmospheres.…”

“…Five mL of the reaction mixture containing 50 ,gL cell-free extract, 80 ,ul of 0.3 mg-ml' p-nitrophenyl phosphate solution, 0.5 itmol Naacetate (pH 5), were incubated for 10 min in a shaking water bath at 40°C (23). One mL aliquot of the reaction mixture was removed at intervals of 10 min and added to test tubes containing 3 mL of 0.02 N NaOH.…”

“…Softening in avocado fruits is accompanied by an extensive breakdown of the cell wall (22), which is associated with an increase in the activities of hydrolytic enzymes such as cellulase, polygalacturonase, and acid phosphatase (3,21,23). Information concerning the synthesis and regulation of polygalacturonase and acid phosphatase during avocado ripening is very limited.…”

Cloning and characterization of avocado fruit mRNAs and their expression during ripening and low-temperature storage

“…As with other climacteric fruit, avocado ripening is related to an increase in ethylene synthesis due to an increase in the activity of two enzymes, 1-aminocyclopropane-l-carboxylic acid synthase (ACC-synthase) and EFE [9]. Changes in the activities of several enzymes including cellulase, PG, pectinmethylesterase and acid phosphatase have been described during avocado ripening [5,31,45] and changes in the expression levels of a number of mRNAs have been demonstrated [15], including cellulase [ 16], cytochrome P-450 oxidase [7], EFE [39] and others of unknown function [151.…”

The Increase in Activities of Acid Phosphatase and Ribonuclease during Ripening of Banana Fruit

“…He also demonstrated that the increase in membrane permeability, as determined by free space, preceded the onset of the climacteric rise in respiration. On the other hand, Brady et al (2) De Leo and Sacher (5) and Sacher (15) showed for banana and for avocado that the development of APase activity was reduced in the presence of cycloheximide, which suggested that the increase in APase activity may have resulted from de novo synthesis of enzyme protein.…”