Regulation of the high-affinity NO3- uptake system by NRT1.1-mediated NO3- demand signaling in Arabidopsis

The NRT2.1 gene of Arabidopsis thaliana encodes a major component of the root high-affinity NO(3)(-) transport system (HATS) that plays a crucial role in NO(3)(-) uptake by the plant. Although NRT2.1 was known to be induced by NO(3)(-) and feedback repressed by reduced nitrogen (N) metabolites, NRT2.1 is surprisingly up-regulated when NO(3)(-) concentration decreases to a low level (<0.5 mm) in media containing a high concentration of NH(4)(+) or Gln (>or=1 mm). The NRT3.1 gene, encoding another key component of the HATS, displays the same response pattern. This revealed that both NRT2.1 and NRT3.1 are coordinately down-regulated by high external NO(3)(-) availability through a mechanism independent from that involving N metabolites. We show here that repression of both genes by high NO(3)(-) is specifically mediated by the NRT1.1 NO(3)(-) transporter. This mechanism warrants that either NRT1.1 or NRT2.1 is active in taking up NO(3)(-) in the presence of a reduced N source. Under low NO(3)(-)/high NH(4)(+) provision, NRT1.1-mediated repression of NRT2.1/NRT3.1 is relieved, which allows reactivation of the HATS. Analysis of atnrt2.1 mutants showed that this constitutes a crucial adaptive response against NH(4)(+) toxicity because NO(3)(-) taken up by the HATS in this situation prevents the detrimental effects of pure NH(4)(+) nutrition. It is thus hypothesized that NRT1.1-mediated regulation of NRT2.1/NRT3.1 is a mechanism aiming to satisfy a specific NO(3)(-) demand of the plant in relation to the various specific roles that NO(3)(-) plays, in addition to being a N source. A new model is proposed for regulation of the HATS, involving both feedback repression by N metabolites and NRT1.1-mediated repression by high NO(3)(-).     

A central role for the nitrate transporter NRT2.1 in the integrated morphological and physiological responses of the root system to nitrogen limitation in Arabidopsis

Up-regulation of the high-affinity transport system (HATS) for NO(3)(-) and stimulation of lateral root (LR) growth are two important adaptive responses of the root system to nitrogen limitation. Up-regulation of the NO(3)(-) HATS by nitrogen starvation is suppressed in the atnrt2.1-1 mutant of Arabidopsis (Arabidopsis thaliana), deleted for both NRT2.1 and NRT2.2 nitrate transporter genes. We then used this mutant to determine whether lack of HATS stimulation affected the response of the root system architecture (RSA) to low NO(3)(-) availability. In Wassilewskija (Ws) wild-type plants, transfer from high to low NO(3)(-) medium resulted in contrasting responses of RSA, depending on the level of nitrogen limitation. Moderate nitrogen limitation (transfer from 10 mm to 1 or 0.5 mm NO(3)(-)) mostly led to an increase in the number of visible laterals, while severe nitrogen stress (transfer from 10 mm to 0.1 or 0.05 mm NO(3)(-)) promoted mean LR length. The RSA response of the atnrt2.1-1 mutant to low NO(3)(-) was markedly different. After transfer from 10 to 0.5 mm NO(3)(-), the stimulated appearance of LRs was abolished in atnrt2.1-1 plants, whereas the increase in mean LR length was much more pronounced than in Ws. These modifications of RSA mimicked those of Ws plants subjected to severe nitrogen stress and could be fully explained by the lowered NO(3)(-) uptake measured in the mutant. This suggests that the uptake rate of NO(3)(-), rather than its external concentration, is the key factor triggering the observed changes in RSA. However, the mutation of NRT2.1 was also found to inhibit initiation of LR primordia in plants subjected to nitrogen limitation independently of the rate of NO(3)(-) uptake by the whole root system and even of the presence of added NO(3)(-) in the external medium. This indicates a direct stimulatory role for NRT2.1 in this particular step of LR development. Thus, it is concluded that NRT2.1 has a key dual function in coordinating root development with external NO(3)(-) availability, both indirectly through its role as a major NO(3)(-) uptake system that determines the nitrogen uptake-dependent RSA responses, and directly through a specific action on LR initiation under nitrogen-limited conditions.     

Soluble sugars—Metabolism,sensing and abiotic stress: A complex network in the life of plants

Plants are autotrophic and photosynthetic organisms that both produce and consume sugars. Soluble sugars are highly sensitive to environmental stresses, which act on the supply of carbohydrates from source organs to sink ones. Sucrose and hexoses both play dual functions in gene regulation as exemplified by the upregulation of growth-related genes and downregulation of stress-related genes. Although coordinately regulated by sugars, these growth- and stress-related genes are upregulated or downregulated through HXK-dependent and/or HXK-independent pathways. Sucrose-non-fermenting-1- (SNF1-) related protein pathway, analogue to the protein kinase (SNF-) yeast-signalling pathway, seems also involved in sugar sensing and transduction in plants. However, even if plants share with yeast some elements involved in sugar sensing, several aspects of sugar perception are likely to be peculiar to higher plants. In this paper, we have reviewed recent evidences how plants sense and respond to environmental factors through sugar-sensing mechanisms. However, we think that forward and reverse genetic analysis in combination with expression profiling must be continued to uncover many signalling components, and a full biochemical characterization of the signalling complexes will be required to determine specificity and cross-talk in abiotic stress signalling pathways.Key words: abiotic stress, gene expression, glucose, metabolism, sucrose, sugar sensing     

Evidence for a nitrate-independent function of the nitrate sensor NRT1.1 in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

NRT1.1 is a putative nitrate sensor and is involved in many nitrate-dependent responses. On the other hand, a nitrate-independent function of NRT1.1 has been implied, but the clear-cut evidence is unknown. We found that NRT1.1 mutants showed enhanced tolerance to concentrated ammonium as sole N source in Arabidopsis thaliana. This unique phenotype was not observed in mutants of NLP7, which has been suggested to play a role in the nitrate-dependent signaling pathway. Our real-time PCR analysis, and evidence from a literature survey revealed that several genes relevant to the aliphatic glucosinolate-biosynthetic pathway were regulated via a nitrate-independent signal from NRT1.1. When taken together, the present study strongly suggests the existence of a nitrate-independent function of NRT1.1.     

Hexokinase as a sugar sensor in higher plants.

The mechanisms by which higher plants recognize and respond to sugars are largely unknown. Here, we present evidence that the first enzyme in the hexose assimilation pathway, hexokinase (HXK), acts as a sensor for plant sugar responses. Transgenic Arabidopsis plants expressing antisense hexokinase (AtHXK) genes are sugar hyposensitive, whereas plants overexpressing AtHXK are sugar hypersensitive. The transgenic plants exhibited a wide spectrum of altered sugar responses in seedling development and in gene activation and repression. Furthermore, overexpressing the yeast sugar sensor YHXK2 caused a dominant negative effect by elevating HXK catalytic activity but reducing sugar sensitivity in transgenic plants. The result suggests that HXK is a dual-function enzyme with a distinct regulatory function not interchangeable between plants and yeast.     

Characterization of a dual-affinity nitrate transporter MtNRT1.3 in the model legume Medicago truncatula

Primary root growth in the absence or presence of exogenous NO(3)(-) was studied by a quantitative genetic approach in a recombinant inbred line (RIL) population of Medicago truncatula. A quantitative trait locus (QTL) on chromosome 5 appeared to be particularly relevant because it was seen in both N-free medium (LOD score 5.7; R(2)=13.7) and medium supplied with NO(3)(-) (LOD score, 9.5; R(2)=21.1) which indicates that it would be independent of the general nutritional status. Due to its localization exactly at the peak of this QTL, the putative NRT1-NO(3)(-) transporter (Medtr5g093170.1), closely related to Arabidopsis AtNRT1.3, a putative low-affinity nitrate transporter, appeared to be a significant candidate involved in the control of primary root growth and NO(3)(-) sensing. Functional characterization in Xenopus oocytes using both electrophysiological and (15)NO(3)(-) uptake approaches showed that Medtr5g093170.1, named MtNRT1.3, encodes a dual-affinity NO(3)(-) transporter similar to the AtNRT1.1 'transceptor' in Arabidopsis. MtNRT1.3 expression is developmentally regulated in roots, with increasing expression after completion of germination in N-free medium. In contrast to members of the NRT1 superfamily characterized so far, MtNRT1.3 is environmentally up-regulated by the absence of NO(3)(-) and down-regulated by the addition of the ion to the roots. Split-root experiments showed that the increased expression stimulated by the absence of NO(3)(-) was not the result of a systemic signalling of plant N status. The results suggest that MtNRT1.3 is involved in the response to N limitation, which increases the ability of the plant to acquire NO(3)(-) under N-limiting conditions.     

The Arabidopsis halophytic relative Thellungiella halophila tolerates nitrogen-limiting conditions by maintaining growth, nitrogen uptake, and assimilation

A comprehensive knowledge of mechanisms regulating nitrogen (N) use efficiency is required to reduce excessive input of N fertilizers while maintaining acceptable crop yields under limited N supply. Studying plant species that are naturally adapted to low N conditions could facilitate the identification of novel regulatory genes conferring better N use efficiency. Here, we show that Thellungiella halophila, a halophytic relative of Arabidopsis (Arabidopsis thaliana), grows better than Arabidopsis under moderate (1 mm nitrate) and severe (0.4 mm nitrate) N-limiting conditions. Thellungiella exhibited a lower carbon to N ratio than Arabidopsis under N limitation, which was due to Thellungiella plants possessing higher N content, total amino acids, total soluble protein, and lower starch content compared with Arabidopsis. Furthermore, Thellungiella had higher amounts of several metabolites, such as soluble sugars and organic acids, under N-sufficient conditions (4 mm nitrate). Nitrate reductase activity and NR2 gene expression in Thellungiella displayed less of a reduction in response to N limitation than in Arabidopsis. Thellungiella shoot GS1 expression was more induced by low N than in Arabidopsis, while in roots, Thellungiella GS2 expression was maintained under N limitation but was decreased in Arabidopsis. Up-regulation of NRT2.1 and NRT3.1 expression was higher and repression of NRT1.1 was lower in Thellungiella roots under N-limiting conditions compared with Arabidopsis. Differential transporter gene expression was correlated with higher nitrate influx in Thellungiella at low (15)NO(3)(-) supply. Taken together, our results suggest that Thellungiella is tolerant to N-limited conditions and could act as a model system to unravel the mechanisms for low N tolerance.