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Presents a method for microscopically examining cytoplasmic streaming in root cells, and physiologically examining the delivery of ions to the xylem exudate. The expected result does not occur and the authors believe that students learn to re-examine the assumptions, thereby increasing their skills in scientific inquiry. (Authors/SA)

Most higher plants develop severe toxicity symptoms when grown on ammonium ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]) as the sole nitrogen source. Recently, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] toxicity has been implicated as a cause of forest decline and even species extinction. Although mechanisms underlying [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] toxicity have been extensively sought, the primary events conferring it at the cellular level are not understood. Using a high-precision positron tracing technique, we here present a cell-physiological characterization of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] acquisition in two major cereals, barley (Hordeum vulgare), known to be susceptible to toxicity, and rice (Oryza sativa), known for its exceptional tolerance to even high levels of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. We show that, at high external [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] concentration ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]), barley root cells experience a breakdown in the regulation of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] influx, leading to the accumulation of excessive amounts of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] in the cytosol. Measurements of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] efflux, combined with a thermodynamic analysis of the transmembrane electrochemical potential for [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], reveal that, at elevated [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], barley cells engage a high-capacity [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]-efflux system that supports outward [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] fluxes against a sizable gradient. Ammonium efflux is shown to constitute as much as 80% of primary influx, resulting in a never-before-documented futile cycling of nitrogen across the plasma membrane of root cells. This futile cycling carries a high energetic cost (we record a 40% increase in root respiration) that is independent of N metabolism and is accompanied by a decline in growth. In rice, by contrast, a cellular defense strategy has evolved that is characterized by an energetically neutral, near-Nernstian, equilibration of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] at high [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. Thus our study has characterized the primary events in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] nutrition at the cellular level that may constitute the fundamental cause of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] toxicity in plants.

The high-affinity nitrate transporter of green plants is composed of two polypeptides, NRT2.1 and NAR2.1, while in fungi it appears that nitrate influx is mediated by NRT2 alone. Another difference between plants and fungi is that the central (cytoplasmic) loop of the 12 membrane spanning regions of NRT is quite large in fungi, consisting of 91 amino acid residues, compared with the relatively short (21 amino acid residues) plant NRT2.1. Here we examine potential amino acid residues involved in the plant NRT2.1:NAR2.1 association by mutation of conserved amino acids in Arabidopsis thaliana AtNRT2.1. Only the replacement of leucine 85 by glutamine disrupted the association between AtNRT2.1 and AtNAR2.1, as examined using the yeast two-hybrid system. Further, to investigate the nitrate-transporting function of AtNRT2.1 in a context free of other members of the NRT2 family, we expressed AtNRT2.1 in Aspergillus nidulans. In the fungal context the plant NRT alone was capable of restoring nitrate transport to a nitrate transport defective mutant, but only when the AtNRT2.1 central loop was replaced by its fungal counterpart. [ABSTRACT FROM AUTHOR]

In plants that have been deprived of nitrate for a significant length of time, a constitutive high- affinity nitrate transport system ( cHATS) is responsible for initial nitrate uptake. This absorbed nitrate leads to the induction of the major nitrate transporters and enzymes involved in nitrate assimilation. By use of 13 NO3- influx measurements and Blue Native polyacrylamide gel electrophoresis we examined the role of AtNRT2.5 in cHATS in wild type ( WT) and various T-DNA mutants of A rabidopsis thaliana. We demonstrate that AtNRT 2.5 is predominantly expressed in roots of nitrate-deprived WT plants as a 150 kDa molecular complex with AtNAR2.1. This complex represents the major contributor to cHATS influx, which is reduced by 63% compared with WT in roots of A tnrt2.5 mutants. The remaining cHATS nitrate influx in these mutants is due to a residual contribution by the inducible high-affinity transporter encoded by AtNRT2.1/ AtNAR2.1. Estimates of the kinetic properties of the NRT2.5 transporter reveal that its low Km for nitrate makes this transporter ideally suited to detect and respond to trace quantities of nitrate in the root environment. [ABSTRACT FROM AUTHOR]