Two Modes of Rubidium Uptake in Sunflower Plants1 - NCBI

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ABSTRACT. The Rb+-uptake kinetics in K+-starved sunflower (Helianthus annuus) plants can be explained by the addition of two Michaelis-. Menten equations.
Received for publication March 29, 1988 and in revised form January 31, 1989

Plant Physiol. (1989) 90, 939-942

0032-0889/89/90/0939/04/$01 .00/0

Two Modes of Rubidium Uptake in Sunflower Plants1 Manuel Beniloch*, Ines Moreno, and Alonso Rodriguez-Navarro Departamento de Agronomia, Escuela Tecnica Superior de Ingenieros Agr6nomos, E-14071 C6rdoba, Spain (M.B., I.M.), and Departamento de Microbiologia, Escuela Tecnica Superior de Ingenieros Agronomos, E-28040 Madrid, Spain (A.R.-N.) (16 and unpublished data of these authors). This system is a 180 kD protein with 12 potential membrane-spanning domains (8). Considering the similarities between plants and fungi in some of the basic transport processes (24), and the similarities between N. crassa and plants in the kinetics of Rb+ uptake, some experiments were designed to disclose more extensive similarities between K+ and Rb+ uptake in plants and fungi.

ABSTRACT The Rb+-uptake kinetics in K+-starved sunflower (Helianthus annuus) plants can be explained by the addition of two MichaelisMenten equations. In contrast, Rb+ uptake can be described by a single Michaelis-Menten equation in normal-K plants. Differences in the Kms and in the Arrhenius plots of Rb+ uptake in the two types of plants suggest two uptake modes.

MATERIALS AND METHODS Plant Material Helianthus annuus seeds (cv Sun-Gro 380 Eruosemillas, S.A. C6rdoba, Spain) were surface-sterilized in 0.5% NaOCl (1 min) and germinated in perlite irrigated with 5 mM CaCl2. Five-day-old seedlings were transferred to a K+ free nutrient solution complemented with the amount of KC1 required in each experiment and were grown for an additional 9-d period, unless otherwise stated. The K+-free nutrient solution was a slight modification of one previously described (5): 2.5 mm Ca(NO3)2, 1.0 mM MgSO4, 0.25 mM Ca(H2PO4)2, 12.5 ,uM H3BO3, 1.0 AM MnSO4, 1.0 AM ZnSO4, 0.25 MM CuSO4, 0.2 Mm (NH4)6Mo7024, and 10 AM Fe-ethylenediamine-di-o-hydrosyphenylacetic acid. Ca(OH)2 was used to adjust the pH to 5.5. Plants were grown under constant aeration of the nutrient solution in a chamber at 22°C day/l 8°C night, with a 13-h photoperiod and a photosynthetic photon flux density of 350 Mmol m-2s-' at the height of the plant canopy (fluorescent tubes, Sylvania cool white VHO). Two types of plants were used: normal plants and K+starved plants. Normal plants were grown in nutrient solution with different K+ concentrations, ranging from 20 AM to 5 mm. The growth techniques used depended on the K+ level. Plants grown at 5 mm K+ or 0.5 mM K+ were distributed individually or in groups of 20 in 700 ml or 25 L vessels, respectively. The K+ concentration of 0.5 mM K+ nutrient solution was controlled daily, but this was unnecessary in the 5 mM K+-grown plants. On the 8th d, the plants, grown at either 5 mM K+ or 0.5 mM K+, were transferred to a fresh solution. Plants grown at both K+ concentrations were identical in weight, K+ content and Rb+ uptake kinetics. In some experiments, plants were grown at 0.5 mm K+, because in uptake experiments at low Rb+, K+ had to be exactly controlled at 0.25 mm, and the transfer of plants from 5 mm K+ nutrient solutions carried too much K+ to the test solution. In other experiments, at 30 mm Rb+, slight deviations from 0.25 mm K+ did not affect the results, and plants were grown at 5 mM K+.

K+ uptake in plants does not exhibit simple MichaelisMenten kinetics. Instead, complex K+ uptake kinetics are usually observed, which have been explained by many different hypotheses. Some authors have proposed the existence of two transport systems (7), or a transport system plus a diffusion component (11), operating in parallel in the plasmalemma or in series in the plasmalemma and tonoplast (12). For some others, the kinetics of a system which intrinsically obeys Michaelis-Menten kinetics is modified by external factors, such as the presence of microorganisms in the roots (2) or the unstirred layers outside the membrane (4). Finally, for others the cause of the complex kinetics is intrinsic to the transport protein(s), which either experience conformational changes at different substrate concentrations (14) or changes in transport activity due to a sequential binding of K+ (22). The negative effect of the K+ content on K+ uptake (9, 10), another observable characteristic of these kinetics, has been explained by an allosteric regulation of the system operating at the lower range of K+ concentrations (9, 15) or by longdistance regulation (6). In the last few years, in addition to research conducted on plants and on plant cells, work on K+ and Rb+ uptake in fungi has produced some interesting data. In Saccharomyces cerevisiae (18, 19) and in Neurospora crassa (20), Rb+ uptake occurs in two modes, that can be distinguished by differences in temperature dependence. One is characteristic of normal K+ cells and the other of low K+ cells. In low K+ cells of N. crassa, K+ uptake at low K+ (0-200 Mm) is mediated by a K+H+ symport (21), and Rb+ uptake in an extended range (0 100 mM) presents a biphasic kinetics similar to some of those observed in plants (17). The biphasic kinetics can be explained by random binding (23) or by the displacement of the H+ by Rb+ in the Rb+-H+ symport (20). However, in S. cerevisiae, based on results obtained with a transport mutant, only one system has been proposed to explain both modes of transport -

'Supported by a grant from Junta de Andalucia. 939

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To grow normal-K+ plants at 20 and 40 AM, seedlings were transferred to a vessel with 25 L of nutrient solution aerated at three points. The 20 plants were supported by an opaque Perspex tray, and a Perspex frame in the vessel avoided tangling of the roots without obstructing the circulation of the solution. The K+ level was maintained by the continuous addition, during the light period, of nutrient solution containing 5 mM KCl. The pump making the addition was adjusted manually according to previous experiences. Maximum deviation from the selected concentration at any time in any part of the vessel was less than 10%. Rb+ uptake experiments with K+-starved plants were performed with plants which had received small amounts of K+ because plants grown in K+-free medium exhibited K+ deficiency symptoms. Individual seedlings were transferred to 700 mL of 0.1 mm K+ nutrient solution, which was changed for a fresh one on the 7th d. The seedlings exhausted the K+ of the first solution in 5 d but, after the transfer, K+ was exhausted in only 6 h. These K+-starved plants were smaller than those grown with K+ and their K+ content was 20 ,umol g-' and 50 Mmol g-1 in roots and shoots, respectively.

Uptake Experiments In normal K+ plants, individual plants were removed from the growth medium and transferred to 700 mL of a fresh nutrient solution with the required concentrations of Rb+ and K+. At intervals, the plants were removed from the solution, rinsed 5 min in 150 mL of 5 mM CaSO4 at 5° to 10°C, and the roots and shoots weighed, and independently frozen. To extract the cations, roots or shoots were thawed, immersed in 10% acetic acid (approximately 10 mL/g) for 12 h, and then filtered and washed with boiling water. The liquids were combined and analyzed by atomic absorption spectrophotometry (nitric digestion of the acetic-acid-treated plant material demonstrated a total extraction). Preliminary experiments with this method showed that the plots of the time courses of Rb+ uptake during 30 min were linear, but crossed the Rb+ axis at a small but significant positive value. Therefore, the rates of Rb+ uptake determined between 2 and 20 min were taken as the initial rates of uptake. In K+-starved plants, Rb+ was added to the K+-exhausted medium (less than 0.2 AM K+) where the plants were growing, and then Rb+ uptake was determined as described above. However, in the tests at low Rb+ (