Expression analyses of Nrt2 plant genes have shown a strict correlation with root nitrate influx mediated by the highaffinity transport system (HATS). The precise assignment of NRT2 protein function has not yet been possible due to the absence of heterologous expression studies as well as loss of function mutants in higher plants. Using a reverse genetic approach, we isolated an Arabidopsis thaliana knock-out mutant where the T-DNA insertion led to the complete deletion of the AtNrt2.1 gene together with the deletion of the 3P P region of the AtNrt2.2 gene. This mutant is impaired in the HATS, without being modified in the low-affinity system. Moreover, the deregulated expression of a Nicotiana plumbaginifolia Nrt2 gene restored the mutant nitrate influx to that of the wild-type. These results demonstrate that plant NRT2 proteins do have a role in HATS. ß

Coordination between the activity of ion transport systems in the root and photosynthesis in the shoot is a main feature of the integration of ion uptake in the whole plant. However, the mechanisms that ensure this coordination are largely unknown at the molecular level. Here, we show that the expression of five genes that encode root NO 3 ؊ , NH 4 ؉ , and SO 4 2 ؊ transporters in Arabidopsis is regulated diurnally and stimulated by sugar supply. We also provide evidence that one Pi and one K ؉ transporter also are sugar inducible. Sucrose, glucose, and fructose are able to induce expression of the ion transporter genes but not of the carboxylic acids malate and 2-oxoglutarate. For most genes investigated, induction by light and induction by sucrose are strongly correlated, indicating that they reflect the same regulatory mechanism (i.e., stimulation by photosynthates). The functional importance of this control is highlighted by the phenotype of the atnrt2 mutant of Arabidopsis. In this mutant, the deletion of the sugar-inducible NO 3 ؊ transporter gene AtNrt2.1 is associated with the loss of the regulation of high-affinity root NO 3 ؊ influx by light and sugar. None of the sugar analogs used (3-O -methylglucose, 2-deoxyglucose, and mannose) is able to mimic the inducing effect of sugars. In addition, none of the sugar-sensing mutants investigated ( rsr1-1 , sun6 , and gin1-1 ) is altered in the regulation of AtNrt2.1 expression. These results indicate that the induction of AtNrt2.1 expression by sugars is unrelated to the main signaling mechanisms documented for sugar sensing in plants, such as regulation by sucrose, hexose transport, and hexokinase (HXK) sensing activity. However, the stimulation of AtNrt2.1 transcript accumulation by sucrose and glucose is abolished in an antisense AtHXK1 line, suggesting that HXK catalytic activity and carbon metabolism downstream of the HXK step are crucial for the sugar regulation of AtNrt2.1 expression.

The role of AtNrt2.1 and AtNrt2.2 genes, encoding putative NO 3 Ϫ transporters in Arabidopsis, in the regulation of high-affinity NO 3 Ϫ uptake has been investigated in the atnrt2 mutant, where these two genes are deleted. Our initial analysis of the atnrt2 mutant (S. Filleur, M. Ϫ 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 Ϫ , indicating that the mutant is more specifically impaired in the inducible component of the HATS. Thus, high-affinity NO 3 Ϫ uptake in this genotype is likely to be due to the constitutive HATS. Root 15 NO 3 Ϫ influx in the atnrt2 mutant is no more derepressed by nitrogen starvation or decrease in the external NO 3 Ϫ availability. Moreover, the mutant also lacks the usual compensatory up-regulation of NO 3 Ϫ uptake in NO 3 Ϫ -fed roots, in response to nitrogen deprivation of another portion of the root system. Finally, exogenous supply of NH 4 ϩ in the nutrient solution fails to inhibit 15 NO 3 Ϫ influx in the mutant, whereas it strongly decreases that in the wild type. This is not explained by a reduced activity of NH 4 ϩ 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 Ϫ uptake, and in the adaptative responses of the plant to both spatial and temporal changes in nitrogen availability in the environment. The uptake of NO 3Ϫ by roots cells is a key process for higher plants because it is the first step of the assimilatory pathway providing most of organic nitrogen required for synthesis of biomolecules, including proteins and nucleic acids (Beevers and Hageman, 1980). More than 30 years of physiological investigations have led to the conclusion that at least three uptake systems are responsible for the influx of NO 3 Ϫ into the roots (for review, see Clarkson, 1986;Glass and Siddiqi, 1995;Crawford and Glass, 1998;Daniel-Vedele et al., 1998;Forde, 2000). Two highaffinity transport systems (HATS) are able to take up NO 3 Ϫ at low concentrations in the external medium, and display saturable kinetics as a function of the external NO 3 Ϫ concentration ([NO 3 Ϫ ] o ), with saturation in the range of 0.2 to 0.5 mm [NO 3 Ϫ ] o . One of these systems appears to be present even in plants never supplied with NO 3 Ϫ , and thus is considered as constitutive (cHATS). The other HATS is specifically stimulated by NO 3 Ϫ , and is consequently assumed to be inducible (iHATS). The maximum activity (V max ) recorded for the iHATS is generally much larger than that of the cHATS, suggesting that the former system plays a key role in the root uptake of NO 3 Ϫ from external media where [NO 3 Ϫ ] o does not exceed 1 mm. The iHATS and cHATS appear to be genetically distinct because a mutant defective in the cHATS, but not in the iHATS, has been isolated in Arabidopsis (Wang and Crawford, 1996)....

For an efficient defense response against pathogens, plants must coordinate rapid genetic reprogramming to produce an incompatible interaction. Nitrate Trasnporter2 (NRT2) gene family members are sentinels of nitrate availability. In this study, we present an additional role for NRT2.1 linked to plant resistance against pathogens. This gene antagonizes the priming of plant defenses against the bacterial pathogen Pseudomonas syringae pv tomato DC3000 (Pst). The nrt2 mutant (which is deficient in two genes, NRT2.1 and NRT2.2) displays reduced susceptibility to this bacterium. We demonstrate that modifying environmental conditions that stimulate the derepression of the NRT2.1 gene influences resistance to Pst independently of the total level of endogenous nitrogen. Additionally, hormonal homeostasis seemed to be affected in nrt2, which displays priming of salicylic acid signaling and concomitant irregular functioning of the jasmonic acid and abscisic acid pathways upon infection. Effector-triggered susceptibility and hormonal perturbation by the bacterium seem to be altered in nrt2, probably due to reduced sensitivity to the bacterial phytotoxin coronatine. The main genetic and metabolic targets of coronatine in Arabidopsis (Arabidopsis thaliana) remain largely unstimulated in nrt2 mutants. In addition, a P. syringae strain defective in coronatine synthesis showed the same virulence toward nrt2 as the coronatine-producing strain. Taken together, the reduced susceptibility of nrt2 mutants seems to be a combination of priming of salicylic acid-dependent defenses and reduced sensitivity to the bacterial effector coronatine. These results suggest additional functions for NRT2.1 that may influence plant disease resistance by down-regulating biotic stress defense mechanisms and favoring abiotic stress responses.