Transcript profiling in the chl1-5 mutant of Arabidopsis reveals a role of the nitrate transporter NRT1.1 in the regulation of another nitrate transporter, NRT2.1.

Arabidopsis thaliana mutants deficient for the NRT1.1 NO(3)(-) transporter display complex phenotypes, including lowered NO(3)(-) uptake, altered development of nascent organs, and reduced stomatal opening. To obtain further insight at the molecular level on the multiple physiological functions of NRT1.1, we performed large-scale transcript profiling by serial analysis of gene expression in the roots of the chl1-5 deletion mutant of NRT1.1 and of the Columbia wild type. Several hundred genes were differentially expressed between the two genotypes, when plants were grown on NH(4)NO(3) as N source. Among these genes, the N satiety-repressed NRT2.1 gene, encoding a major component of the root high-affinity NO(3)(-) transport system (HATS), was found to be strongly derepressed in the chl1-5 mutant (as well as in other NRT1.1 mutants). This was associated with a marked stimulation of the NO(3)(-) HATS activity in the mutant, suggesting adaptive response to a possible N limitation resulting from NRT1.1 mutation. However, derepression of NRT2.1 in NH(4)NO(3)-fed chl1-5 plants could not be attributed to lowered production of N metabolites. Rather, the results show that normal regulation of NRT2.1 expression is strongly altered in the chl1-5 mutant, where this gene is no more repressible by high N provision to the plant. This indicates that NRT1.1 plays an unexpected but important role in the regulation of both NRT2.1 expression and NO(3)(-) HATS activity. Overexpression of NRT2.1 was also found in wild-type plants supplied with 1 mM NH(4)(+) plus 0.1 mM NO(3)(-), a situation where NRT1.1 is likely to mediate very low NO(3)(-) transport. Thus, we suggest that it is the lack of NRT1.1 activity, rather than the absence of this transporter, that derepresses NRT2.1 expression in the presence of NH(4)(+). Two hypotheses are discussed to explain these results: (1) NRT2.1 is upregulated by a NO(3)(-) demand signaling, indirectly triggered by lack of NRT1.1-mediated uptake, which overrides feedback repression by N metabolites, and (2) NRT1.1 plays a more direct signaling role, and its transport activity generates an unknown signal required for NRT2.1 repression by N metabolites. Both mechanisms would warrant that either NRT1.1 or NRT2.1 ensure significant NO(3)(-) uptake in the presence of NH(4)(+) in the external medium, which is crucial to prevent the detrimental effects of pure NH(4)(+) nutrition.

The Arabidopsis root transcriptome by serial analysis of gene expression. Gene identification using the genome sequence.

Large-scale identification of genes expressed in roots of the model plant Arabidopsis was performed by serial analysis of gene expression (SAGE), on a total of 144,083 sequenced tags, representing at least 15,964 different mRNAs. For tag to gene assignment, we developed a computational approach based on 26,620 genes annotated from the complete sequence of the genome. The procedure selected warrants the identification of the genes corresponding to the majority of the tags found experimentally, with a high level of reliability, and provides a reference database for SAGE studies in Arabidopsis. This new resource allowed us to characterize the expression of more than 3,000 genes, for which there is no expressed sequence tag (EST) or cDNA in the databases. Moreover, 85% of the tags were specific for one gene. To illustrate this advantage of SAGE for functional genomics, we show that our data allow an unambiguous analysis of most of the individual genes belonging to 12 different ion transporter multigene families. These results indicate that, compared with EST-based tag to gene assignment, the use of the annotated genome sequence greatly improves gene identification in SAGE studies. However, more than 6,000 different tags remained with no gene match, suggesting that a significant proportion of transcripts present in the roots originate from yet unknown or wrongly annotated genes. The root transcriptome characterized in this study markedly differs from those obtained in other organs, and provides a unique resource for investigating the functional specificities of the root system. As an example of the use of SAGE for transcript profiling in Arabidopsis, we report here the identification of 270 genes differentially expressed between roots of plants grown either with NO3- or NH4NO3 as N source.