Abstract
Nitrate taken up by root cells from the soil solution is a major nitrogen source for nutrition of most plant species. It is known for long that root nitrate uptake is feedback regulated by the nutrient status of the whole plant, but the mechanisms responsible for this control are unknown at the molecular level. To unravel these mechanisms, two strategies were developed in Arabidopsis thaliana, using the NRT2.1 nitrate transporter gene as a model. Previous studies have shown that NRT2.1 transcription is repressed by high N supply to the plant. Therefore, (1) we searched for cis-regulatory elements in NRT2.1 promoter (pNRT2.1), and (2) we performed a genetic screen to isolate mutants impaired in the down-regulation of pNRT2.1 activity by high N supply. Although a 150 pb region of pNRT2.1 was confirmed to mediate promoter regulations, neither linkerscan analysis nor in vivo footprinting approaches allowed to clearly identify cis regulatory elements. Three hni mutants (high nitrogen insensitive) displaying a misregulation of pNRT2.1 activity were characterized as Nsignaling mutants, specifically altered in the feedback regulation of root nitrate uptake by the plant N status. Map based cloning allowed to identify the mutated gene in one (hni9) mutant. The HNI9 gene encodes a putative transcription elongation factor that was uncharacterized in A. thaliana. We showed that HNI9 is a nuclear protein which controls the N regulation of more than 400 genes. Concerning specifically NRT2.1, our results support the hypothesis that HNI9 down-regulates pNRT2.1 activity in response to high N supply by increasing the level of repressive histone methylation marks (H3K27me3) on the 150 bp cis-acting region of pNRT2.1. These data point out, for the first time in plants, the possible involvement of histone methylation as a mechanism for modulating gene expression in response to nutrient signaling pathways.