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Molecular and physiological studies of nitrate and sulfate uptake in roots of barley Vidmar, Joseph John


Molecular and physiological approaches were employed to characterize nitrate and sulfate transporters from roots of Hordeum vulgare cv. Klondike. A cDNA, hvstl (accession no. U52867), by heterologous complementation in E. coli. This cDNA encodes a high-affinity sulfate transporter, that is 2442 bp in length and encodes a protein of 660 amino acids. Under steadystate conditions of sulfate supply, ranging from 2.5 to 250 μM, sulfate influx (measured at 100 uM external sulfate concentration) and hvst1 transcript levels were inversely correlated with sulfate concentrations in the culture solution. A time-course study, designed to investigate effects of S-withdrawal on the abundance of hvst1 transcript, showed a 5-fold increase of the latter within the first two hours after removing external sulfate, followed by a further slight increase during the period up to 48 h. These changes were accompanied by a parallel increase in sulfate influx and a decrease of root glutathione concentrations. When plants that had been deprived of sulfate for 24 h were exposed to 1 mM L-cysteine, or glutathione, for a period of 3 h, glutathione was the more effective down regulator of hvstl transcript levels, reducing the latter to a level that was below that of unstarved controls. Both hvstl transcript abundance and sulfate influx increased as a function of N-supply to N-starved plants. Two new cDNAs, bch3 and bch4 (homologous with an Aspergillus nidulans gene encoding a nitrate transporter, crnA) were isolated from barley roots by RACE PCR. Bch3 and bch4 are 1822 and 1705 bp, encode putative polypeptides of 507 amino acids, with a predicted m.w. of 54.6 kDa. Predicted BCH3 and BCH4 proteins are members of a nitrate/nitrite transporter subfamily of the major facilitator superfamily. Northern blot analysis, revealed that supplying NO3" to N-deprived plants increased both the abundance of bch transcripts and NO3" influx. All four bch genes (bch1, bch2, bch3, bch4) are co-ordinately up-regulated in response to NO3⁻ treatment. Plants provided with 50 pM NO3⁻ showed the highest bch transcript abundance and , JN03⁻ influx. The effects of exogenous provision of various amino acids on bch transcript levels was investigated, when plants were co-supplied with nitrate. Asparagine, aspartate, glutamate and glutamine decreased transcript levels by >60% and ¹³NO₃ influx by 50-80%. Analysis of amino acid concentrations of roots showed that the decrease of bch transcript was correlated with increased glutamine levels.

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