BIOCHEMICAL DETERMINATION OF Mg 2+ , K + ‐DEPENDENT, DIETHYLSTILBESTROL‐INHIBITED PHOSPHATASE ACTIVITY IN THE OVULES OF SAINTPAULIA IONANTHA BEFORE FERTILIZATION

The occurrence of phosphatase activity that was dependent on Mg2+ and K+ and inhibited by diethylstilbestrol has been detected in unfertilized African violet ovules with the use of biochemical techniques. Since cation-dependent phosphatase activity is believed to be involved in ion transport across cell membranes, these results are interpreted as lending indirect support to cytochemical evidence that suggests the innermost integumentary cells of African violet ovules are involved in active transport of solutes to the embryo sac. THE PRESENCE of what may be transport phosphatase activity in unfertilized African violet ovules has recently been suggested by a study using cytochemical techniques (Mogensen, 1981). However, the existing cytochemical approaches do not unequivocally distinguish between nonspecific and transport phosphatase activity, especially in plant tissues. Even though reaction product may be specifically associated with the plasma membrane, the amount of variability within and between experiments makes it extremely difficult to obtain conclusive results by varying cation concentrations or using specific inhibitors (James Cronshaw, pers. commun.; Mogensen and Rossiter, unpubl. data). Therefore, in the study being reported here, we have used a biochemical approach to determine whether a cation-dependent, diethylstilbestrol-inhibited phosphatase exists in the unfertilized ovules of Saintpaulia ionantha. MATERIALS AND METHODS-For comparative purposes, we selected the same stage of floral development as used in a previous cytochemical study on phosphatase activity in Saintpaulia ionantha ovules (Mogensen, 1981); i.e., flowers with fully opened petals and receptive stigmas, but unpollinated. Our biochemical procedure was modified from that of Ernst (1 972a) and the reaction medium was based on the results of a recent biochemical 1 Received for publication 27 June 1981; revision accepted 21 January 1982. This material is based upon work supported by NSF Grant No. PCM-8009020 and by the Northern Arizona University Organized Research Fund. Assistance with the statistical analyses by Grayden W. Bell and with the biochemical procedures by Gilbert C. Pogany is gratefully acknowledged. study on phosphatase activity in tobacco ovules that tested the effects of variations in kinds and concentrations of buffers and cations and different levels of substrate (Mogensen and Rossiter, 1982). After removal of the pericarp, whole placentae with attached ovules were placed into a given volume of deionized distilled water and gently sonicated for 1 min to detach the ovules. Placental tissue was then removed and the ovules were disrupted by ultrasonication for 45 min. The use of an ice-water bath and brief interruptions of the sonication process prevented overheating of the tissues. Light microscopic examination of the resulting homogenate verified that all cells were disrupted by this technique. Because of variations in flower availability, all experiments did not use the same number of flowers; however, at least three flowers/ml of water were necessary to obtain sufficient absorption readings. The reaction was initiated by adding 0.2 ml of homogenate to 0.75 ml of reaction medium (complete reaction medium consisted of 5 mm of p-nitrophenyl phosphate, di-sodium salt; 2 mM MgCl,; 100 mm KCl and 100 mm Tris-base; final concentrations; at pH 6.0). The reaction was allowed to occur for 30 or 45 min at 20 C on a rotator. After stopping the reaction with 0.2 ml of 37.5% trichloroacetic acid, the entire mixture was centrifuged at 3,600 rpm for 10 min. The absorption of a solution containing 0.75 ml of the supernatant and 0.75 ml of Trisbase (added to restore the high pH at which nitrophenol is yellow) was read at 420 nm. The amount of yellow nitrophenol produced by enzymatic hydrolysis of nitrophenyl phosphate was determined by comparison with known standards of nitrophenol. This method resulted