Major alterations of the regulation of root NO(3)(-) uptake are associated with the mutation of Nrt2.1 and Nrt2.2 genes in Arabidopsis

Abstract

The role of AtNrt2.1 and AtNrt2.2 genes, encoding putative NO(3)(-) transporters in Arabidopsis, in the regulation of high-affinity NO(3)(-) uptake has been investigated in the atnrt2 mutant, where these two genes are deleted. Our initial analysis of the atnrt2 mutant (S. Filleur, M.F. Dorbe, M. Cerezo, M. Orsel, F. Granier, A. Gojon, F. Daniel-Vedele [2001] FEBS Lett 489: 220-224) demonstrated that root NO(3)(-) uptake is affected in this mutant due to the alteration of the high-affinity transport system (HATS), but not of the low-affinity transport system. In the present work, we show that the residual HATS activity in atnrt2 plants is not inducible by NO(3)(-), indicating that the mutant is more specifically impaired in the inducible component of the HATS. Thus, high-affinity NO(3)(-) uptake in this genotype is likely to be due to the constitutive HATS. Root (15)NO(3)(-) influx in the atnrt2 mutant is no more derepressed by nitrogen starvation or decrease in the external NO(3)(-) availability. Moreover, the mutant also lacks the usual compensatory up-regulation of NO(3)(-) uptake in NO(3)(-)-fed roots, in response to nitrogen deprivation of another portion of the root system. Finally, exogenous supply of NH(4)(+) in the nutrient solution fails to inhibit (15)NO(3)(-) influx in the mutant, whereas it strongly decreases that in the wild type. This is not explained by a reduced activity of NH(4)(+) uptake systems in the mutant. These results collectively indicate that AtNrt2.1 and/or AtNrt2.2 genes play a key role in the regulation of the high-affinity NO(3)(-) uptake, and in the adaptative responses of the plant to both spatial and temporal changes in nitrogen availability in the environment.

Figure 1

Induction of ¹⁵NO₃⁻ influx (A) and AtNrt2.1 expression (B) by NO₃⁻ in roots of Wamilewskija WT (WS) and atnrt2 mutant (M) plants. The plants were grown on 1 mm NH₄NO₃ until the age of 5 weeks, and were transferred to nitrogen-free solution for 1 week to ensure de-induction of NO₃⁻ transport systems. Thereafter, the plants were supplied with 4 mm NO₃⁻. At the times indicated in the figure, ¹⁵NO₃⁻ influx was measured at 0.2 mm [¹⁵NO₃⁻]_o, and root samples were harvested for northern-blot analysis of AtNrt2.1 mRNA accumulation. Root ¹⁵NO₃⁻ influx data are the means of 12 replicates ± se.

Figure 2

Response of ¹⁵NO₃⁻ influx (A) and AtNrt2.1 expression (B) to nitrogen starvation in roots of WS and atnrt2 plants. The plants were grown on 10 mm NO₃⁻ until the age of 6 weeks, and were transferred to nitrogen-free solution for 24 or 48 h. Root ¹⁵NO₃⁻ influx was measured at 0.2 mm [¹⁵NO₃⁻]_o, and root samples were harvested for northern-blot analysis of AtNrt2.1 mRNA accumulation. Root ¹⁵NO₃⁻ influx data are the means of 12 replicates ± se.

Figure 3

Response of ¹⁵NO₃⁻ influx to localized nitrogen deprivation in split-root WS and atnrt2 plants. The plants were grown on 1 mm NH₄NO₃ until the age of 5 weeks, and were transferred on 1 mm NO₃⁻ 1 week before the experiments. The principle of the experiments (A) was to separate the root system of the plants in two parts. Localized nitrogen deprivation was initiated by the transfer of one side of the split-root system to nitrogen-free solution. Control plants remained supplied with 1 mm NO₃⁻ on both sides of the split-root system. Root ¹⁵NO₃⁻ influx (B) was measured at 0.2 mm [¹⁵NO₃⁻]_o, in either the NO₃⁻-fed side, or both sides of the split root system, of plants under localized nitrogen starvation or control plants, respectively. The data are the means of six replicates ± se.

Figure 4

Kinetics of ¹⁵NO₃⁻ influx in function of [¹⁵NO₃⁻]_o in WS and atnrt2 plants supplied either with 1 (A) or 4 (B) mm NO₃⁻ for 1 week before the measurements. The plants were grown on 1 mm NH₄NO₃ before the treatment at different NO₃⁻ concentrations in the medium. Root ¹⁵NO₃⁻ influx was measured at [¹⁵NO₃⁻]_o ranging from 0.01 to 0.5 mm. The data are the means of 12 replicates ± se. The calculated V_max values are 107 and 37 μmol h⁻¹ g⁻¹ root dry weight for WS plants on 1 and 4 mm NO₃⁻, respectively. The corresponding values for atnrt2 plants are 44 and 33 μmol h⁻¹ g⁻¹ root dry weight on 1 and 4 mm NO₃⁻, respectively.

Figure 5

Repression of ¹⁵NO₃⁻ influx (A) and AtNrt2.1 expression (B) by NH₄⁺ in WS and atnrt2 plants. The plants were grown on 1 mm NH₄NO₃ for 5 weeks, transferred for 1 additional week to 1 mm NO₃⁻, and supplied again with 1 mm NH₄NO₃. At the times indicated in the figure, ¹⁵NO₃⁻ influx was measured at 0.2 mm [¹⁵NO₃⁻]_o, and root samples were harvested for northern-blot analysis of AtNrt2.1 mRNA accumulation in WS plants. Root ¹⁵NO₃⁻ influx data are the means of 12 replicates ± se.

Figure 6

Root ¹⁵NH₄⁺ influx in WS and atnrt2 mutant plants. The plants were grown either on 1 or 5 mm NH₄NO₃. Root ¹⁵NH₄⁺ influx was measured at both 0.2 (A) and 10 (B) mm [¹⁵NH₄⁺]_o to provide estimation of HATS and HATS + LATS activity for NH₄⁺, respectively. The data are the means of 12 replicates ± se.

Figure 7

Reduction of ¹⁵NO₃⁻ influx in the atnrt2 plants, as compared with WS plants, as a function of [¹⁵NO₃⁻]_o. The plants were grown on 1 mm NH₄NO₃ until the age of 5 weeks, and were supplied with 1 mm NO₃⁻ for 1 additional week before the measurements. The experiments were performed as described in Figure 4. The data are the means of those from three independent experiments ± se.