Methylammonium Transport in Phaseolus vulgaris Leaf Slices

Abstract

Methylammonium (as a nonmetabolized analog of ammonium) transport was studied in leaf slices of Phaseolus vulgaris L. var. 'Hawkesbury Wonder.' The relationship of influx to external pH (6.0-10.5) shows that the influx at low external pH is a larger fraction of that at high external pH than would be expected from the pK. of methylammonium and the assumption that only CH3NH2 is entering the cells. The relationship between methylammonium influx and external methylammonium concentration shows some evidence of saturation; this is a function of the transport system rather than of the (limited) methylammonium metabolism in the cells. The "equilibrium" concentration ratio for methylammonium between leaf slices and bathing medium is far higher than can be explained by the transport of CH3NH2 alone and the pH of the compartments involved. when all of the nitrate assimilation in nitrate-grown plants occurs in the leaves (2). The immediate application of the work described here relates to the control of gaseous NH3 fluxes between Phaseolus vulgaris leaves and the aerial environment (2). A major determinant of the NH3 concentration in the intracellular gas spaces in equilibrium with the leaf apoplast solution is the ammonium concentration in the cell wall solution; other important factors are the temperature of the leaf and the apoplastic pH.The work reported here deals with the mechanism(s) of ammonium transport at the plasmalemma of leaf of P. vulgaris; the other important parameter (apoplastic pH) will be dealt with elsewhere (J. A. Raven, manuscript in preparation). Work on a wide range of other cell types has shown that, in addition to the unfacilitated movement of NH3 through the lipid portion of the cell membranes, the plasmalemma commonly possesses an NH4' uniporter system (4, 16, 18, 20, 22, 23, 25-27; cf. ref. 13). The NH3 diffusion system on its own leads to an ammonium distribution related to the pH on each side of the membrane. The NH4' uniport leads to an equilibrium distribution related to the electrical potential difference across the membrane; however, the involvement of a specific uniporter permits control of net NH4+ fluxes (and, hence, of ammonium levels in phases of restricted volume separated by the membrane) via induction/repression and feedback inhibition. The occurrence of the NH4' uniporter system would permit a more precise control of ammonium (and NH3) concentration in the leaf apoplast than would the occurrence solely of NH3 diffusion. An elegant demonstration of NH4+ transport come from parallel voltage-clamp and net ammonium (methylammonium) flux measurements (25,26)

Cytosolic Concentrations and Transmembrane Fluxes of NH4 +/NH3. An Evaluation of Recent Proposals

“…Howitt and Udvardi (2000), however, propose that LATS transport occurs via a reversible NH 3 transporter. In support of this proposal, the authors cite a study of 14 C-methylamine (CH 3 NH 2 /CH 3 NH 3 ϩ ) uptake by Phaseolus vulgaris leaves (Raven and Farquhar, 1981), but in fact this study showed that the relationship of influx to external pH and the equilibrium concentration of methylamine were "far higher than could be explained by the transport of CH 3 NH 2 alone." Raven and Farquhar concluded that methylamine uptake, at least at pH values below 7, was predominantly as CH 3 NH 3 ϩ , and driven by the membrane electrical potential difference (⌬).…”

Transcriptomic Analysis of the Role of Carboxylic Acids in Metabolite Signaling in Arabidopsis Leaves

“…To investigate the transcript response to alterations in organic acid levels, an established leaf slice system was applied (Raven and Farquhar, 1981;Muller et al, 2001;Horling et al, 2003) that allows quick and efficient feeding of organic acids and other effector solutions to leaves. The microarray experiments from feeding low levels of citrate and malate (1 mM each) revealed that both organic acids convey an overall very different transcriptome response and that only a small set of transcripts such as AOX1a is regulated by both organic acids (Fig.…”

“…The leaf slice system has been used for gene expression analysis in several previous studies (Raven and Farquhar, 1981;Horling et al, 2003). It allows a fast and homogenous application of effector solutions.…”

Apoplastic pH and Ammonium Concentration in Leaves of Brassica napus L

“…Transport of NH4+ across plasmalemma of mesophyll cells thus seems to be rapid relative to that across plasmalemma of root cells. Raven and Farquhar (1981) and Karasawa et al (1994) suggested that NH4+ was transported across the leaf plasmalemma by an uniport, actively controlling the NH,+ and NH3(aq) concentration in the apoplast via induction/ repression and feedback inhibition. The NH,' concentration in leaves of B. napus ranged from 0.4 to 1.3 mM in the present experiments.…”

“…3) Hours from start of experiment Farquhar (1981) (Wang et al, 199313). Transport of NH4+ across plasmalemma of mesophyll cells thus seems to be rapid relative to that across plasmalemma of root cells.…”