Borate Exchanges of Lemna minor L. as Studied with the Help of the Enriched Stable Isotopes and of a (n,α) Nuclear Reaction

Abstract

Despite the lack of a convenient radioisotope of boron, it is possible to measure unidirectional fluxes of borate between cellular systems and their external medium. It was accomplished by using the two purified stable isotopes ('OB and "B), with '0B specifically detected by a (n,a) nuclear reaction. The method was applied to compartmental analysis of borate with intact plants of Lemna minor L. Four compartments were suggested. Three of them apparently correspond to the three classical ones: free space (including easily dissociable borate monoesters), cytoplasm, and vacuole. The fourth one was interpreted as corresponding to very stable borate diesters in the cell wails. The method allows the determination of the borate capacities of the various compartments and of the borate unidirec-tional fluxes between the different compartments, at borate flux equilibrium. Other physicochemical data (mono and diester mass action constants, turn over numbers) were evaluated. The results are consistent with what is known of pure substances. Boron was recognized long ago as an essential element for the growth of higher plants (1, 14). Studies of the cellular transport of borate in plant systems have been limited (4, 21); the reason is probably the lack of a convenient radioisotope, which makes it very difficult to determine unidirectional fluxes. We have under-taken a study of borate exchanges with the help of the stable isotopes ('0B and "B), one of which ('°B) can be specifically detected by the nuclear reaction 1 OB(nba)7Li I) Boron effluxes were thus measured with plants preequilibrated with '0B-boric acid and exchanging it against "B-boric acid. In previous experiments, we have used photographic emulsions to detect the nuclear reaction (7, 20, 21); we now prefer the use of homogeneous detectors (13, 22) which allow a much better sensitivity of detection (23). MATERIALS AND METHODS Plants. The experiments were performed on Lemna minor L., a plant particularly well adapted to this type of work. The plants were grown in aseptic conditions (10) at constant temperature (20 C), under continuous light (4,000 lux), in flasks each containing 250 ml solution. The composition of the nutrient solution is given in Table I. It was modified from (11) for the macroelements. The boron concentration was supraoptimal, although it was in the ' This research was supported by Centre National de la Recherche Scientifique (L.A. 203, RCP 285) and by Delegation Generale a la Recherche Scientifique et Technique (Contract 77 7 1272). range of concentrations which allow a normal growth (20). The nutrient and absorption solutions were prepared free of natural boron; water was quartz-twice-distilled; mineral salts were boron-purified by two successive recrystallizations (9); and Kavalier, quartz or plastic vessels were used, in order to avoid any possible boron contamination as might occur with ordinary or Pyrex glass. Boron was provided as °B-boric acid or "B-boric acid. The relative isotopic purities are 90 and 98.5%. These isotopes were obtained from Detection of '0B-Isotope in a Liquid Sample by (n,a) Reaction. The methodology of the detection has already been discussed in detail (6, 22). The calibration curves are reliable over a scale of concentrations ranging from 1o-6 to 1O-2 M H3BO3 (Fig. 1). These curves are given here on a log/log scale, in order to include all of the data on the same figure; but, operationally, it is better to draw the calibration curves in arithmetic coordinates over a more limited scale of concentrations. Experimental Procedure. The Lemna plants were grown and pretreated in the nutrient medium supplied with 0.16 mm 10B-boric acid. After they were blotted rapidly on a fritted glass, Lemna samples were deposited (time 0) on well stirred efflux media (5 ml each) for increasing intervals, at 20 C, under continuous light. Two different efflux media were used simultaneously: quartz-distilled H20, and a 0.16 mm "B-boric acid solution. After each efflux period, the '0B content of the efflux medium was determined by the (n,a) method. At any time (t), the remaining '0B in the plants, Q*(t), is obtained by subtracting the exchanged quantitr from the initial content of the plants. The °B content, both ofthe pretreated plants at time 0 and of the plants after the longest efflux period (1,288 min), was determined on plants ashed as follows. The samples (10-20 plants) were taken up, and very rapidly rinsed with quartz-twice-distilled H20 on fritted glass, then dried at 110 C for 24 hr, weighed (about 10 mg), and incinerated at 400 C (until gray ashes were obtained). The ashes were treated by 5 ml HCI 0.1 N. After addition of gelatin up to 2%, 10-pl aliquots of the solutions were deposited and dried on the detectors, irradiated, and the '0B content of each sample of Lemna was evaluated from the corresponding number of tracks on the detectors. The '0B determinations in ashed samples fluctuate much more than those performed on solutions. Accuracy can be regained by making several droplets per sample and averaging the results. However, the counting becomes very time-consuming when the method has to be applied to more than a few samples. This has prevented us from obtaining reliable influx measurements, to complete the efflux ones as given in this paper. RESULTS As measured from the ashed samples, the '°B content of the plants was 93.4 umol g-' dry weiFht at time 0; at the end of the efflux period, it was 49.6 ,umol g-when the efflux was done into 283