Rubidium (Potassium) Uptake by Arabidopsis

Abstract

Expriments are reported in which the uptake of 'Rb', used as an analog of K , into cultured cels of Arabidopsis thaiana is investigated. A single transport system is found with KM = 0.34 mliolar and V.. -14 nmoles per miigram of protein per hour. This system is blocked by the metabolic inhibitor carbonyl cyanide n-chlorophenyl hydrazone (CCCP) and by cold. At high concentrations of external K' (above 1 milllar), a significant fraction of total uptake is energy-independent. No evidence is found for more than one energy-dependent uptake system or for concentration-dependent modifications of a carrier as postulated in multiphasic transport models.Rb' uptake was also examined in cultured cells derived from an "osmotic mutant" of Arabidpsis The with NaOH, and 20 g/l glucose. Suspension cultures were grown in Bellco spinner flasks in the dark at 25 C. Large aggregates of cells were removed from suspension by gravity and discarded; the cells remaining in suspension were pelleted by low speed centrifugation and washed three times with HP-7 medium lacking K+ (NH4' substituted for K+). The final suspension used for uptake studies consisted mostly of single cells with some two-and fourcell aggregates. Cells were harvested in log phase at densities between 1 x 105 and 5 x 10' cells/ml. Cell viability was estimated by microscopic inspection and a dye exclusion test using erythrocin B. Viability by these criteria was consistently about 80%Yo.Culture 105 cells/ml. One-ml aliquots of the suspension were withdrawn at appropriate intervals and the cells were collected on Millipore filters and washed three times with 3.0 ml unlabeled HP-7. The amount of isotope taken up was determined by either liquid scintillation or gas flow counting. For experiments in the presence of CCCP2 or at 4 C, there was a 30-min preincubation period before isotope was added. Seedlings were incubated completely immersed with shaking (approximately 100 oscillations/min) at 25 C in medium containing MRbCl (10 mCi/mg) at a concentration of 3 isCi/ml. They were removed from the labeling medium and placed in desorption medium (K+-free growth medium) for 10 min at 4 C. After desorption, seedlings were blotted dry, placed in scintillation vials with scintillation fluid, and the radioactivity measured. Figure 1. Cells were incubated with shaking at 25 C in HP-7 medium containing 10 ,UM KCI and 3 ,uCi/ml 'RbCl. Samples (1.0 ml) were taken at the indicated times and the radioactivity determined. Two observations are apparent: (a) both cold and the metabolic inhibitor, CCCP, at 0.01 mm, strikingly inhibit uptake; this supports the notion that transport is an active process; and (b) there is a lag of 40 to 60 min after which transport is linear. We do not understand the reason for the lag. However, we find that uptake of Rb+ into roots of intact seedlings has a similar lag, which disappears when cells are incubated in the assay medium for 2 h prior to the addition of the radioisotope (Fig. IA)

Potassium Transport in Corn Roots

“…It is dominant in the low concentration range (0-1 mM) and approximates Michaelis-Menten kinetics. Furthermore, it is sensitive to inhibitors such as the sulfhydryl reagents used for these experiments or uncouplers such as CCCP (32). K+ uptake in this concentration range is believed by many workers to be active, accumulating K+ against its electrochemical potential gradient, although this is still subject to debate.…”

“…He argues that many of the frequently cited discontinuous uptake isotherms could be explained by models consisting of one or more Michaelis-Menten terms plus a linear term, particularly if experimental error and biological variability are considered. The existence of this linear term has often been ignored, despite the fact that transport kinetics consisting of Michaelis-Menten and linear terms have been well documented in both plant and animal systems (3,5,8,9,13,20,22,27,28,32 …”

Cellular mechanisms of potassium transport in plants

“…Another aspect of K + transport research that needs to be approached with caution is experimental work that employs rubidium‐86 as a radiotracer for potassium. Justification of the widespread use of 86 Rb + as a potassium analogue stems from several reports suggesting that, at least in some cases, there is little discrimination between the two ions (Kochian and Lucas 1982, Läuchli and Epstein 1970, Polley and Hopkins 1979). However, numerous other studies have shown that, on the contrary, 86 Rb + is a poor tracer for K + in higher plant systems (Behl and Jeschke 1982, Cline and Hungate 1960, de Agazio et al 1983, Jacoby 1975, Jacoby and Nissen 1977, Kannan and Ramani 1973, Maas and Leggett 1968, Marschner and Schimanski 1968, Schachtman et al 1992); substantial discrimination between the two elements has also been observed in Nitellopsis (MacRobbie and Dainty 1958), Chlamydomonas (Malhotra and Glass 1995, Polley and Doctor 1985), Chara (Keifer and Spanswick 1978), Ulva and Chaetomorpha (West and Pitman 1967), as well as in Escherichia coli (Rhoads et al 1977).…”

Eficiência de uso do K em razão do sistema de adubação na rotação aveia-milho

“…Na presente pesquisa, optou-se pelo uso do rubídio ( 85 Rb) para marcar o fertilizante, a fim de avaliar a eficiência em vários sistemas de adubação potássica. O rubídio ( 85 Rb) pode ser usado porque é um elemento análogo ao K, com pequena discriminação entre ambos (KOCHIAN;LUCAS, 1982;LÄUCHLI;EPSTEIN, 1970;POLLEY;HOPKINS, 1979).…”

“…No entanto, as funções metabólicas do K não podem ser substituídas totalmente pelo Rb (BALIGAR; BARBER, 1978; BALIGAR; NIELSEN; BARBER, 1979; ERDEI; TRIVEDI,MARSCHNER, 1995). O Rb comporta-se de maneira semelhante ao K, com pequena discriminação entre ambos(KOCHIAN;LUCAS, 1982; LÄUCHLI;EPSTEIN, 1970;POLLEY;HOPKINS, 1979). O Rb é absorvido pelos mesmos mecanismos do potássio e, aproximadamente, na mesma proporção (MENZEL; HEALD, 1955).…”