Regulation of High-Affinity Nitrate Uptake in Roots of Arabidopsis Depends Predominantly on Posttranscriptional Control of the NRT2.1/NAR2.1 Transport System    

Abstract

In Arabidopsis (Arabidopsis thaliana), the NRT2.1 gene codes for the main component of the root nitrate (NO3 −) high-affinity transport system (HATS). Due to the strong correlation generally found between high-affinity root NO3 − influx and NRT2.1 mRNA level, it has been postulated that transcriptional regulation of NRT2.1 is a key mechanism for modulation of the HATS activity. However, this hypothesis has never been demonstrated, and is challenged by studies suggesting the occurrence of posttranscriptional regulation at the NRT2.1 protein level. To unambiguously clarify the respective roles of transcriptional and posttranscriptional regulations of NRT2.1, we generated transgenic lines expressing a functional 35S::NRT2.1 transgene in an atnrt2.1 mutant background. Despite a high and constitutive NRT2.1 transcript accumulation in the roots, the HATS activity was still down-regulated in the 35S::NRT2.1 transformants in response to repressive nitrogen or dark treatments that strongly reduce NRT2.1 transcription and NO3 − HATS activity in the wild type. In some treatments, this was associated with a decline of NRT2.1 protein abundance, indicating posttranscriptional regulation of NRT2.1. However, in other instances, NRT2.1 protein level remained constant. Changes in abundance of NAR2.1, a partner protein of NRT2.1, closely followed those of NRT2.1, and thus could not explain the close-to-normal regulation of the HATS in the 35S::NRT2.1 transformants. Even if in certain conditions the transcriptional regulation of NRT2.1 contributes to a limited extent to the control of the HATS, we conclude from this study that posttranscriptional regulation of NRT2.1 and/or NAR2.1 plays a predominant role in the control of the NO3 − HATS in Arabidopsis.

Glucose Elevates NITRATE TRANSPORTER2.1 Protein Levels and Nitrate Transport Activity Independently of Its HEXOKINASE1-Mediated Stimulation of NITRATE TRANSPORTER2.1 Expression

“…Rapid down-regulation of high-affinity nitrate uptake by dark and ammonium nitrate was not accompanied by reduced protein levels, indicating that rapidly acting posttranscriptional mechanisms control the down-regulation of NRT2.1 transport activity while protein levels remain relatively steady. Our observations are consistent with the interpretation of Laugier et al (2012) that NRT2.1 transcriptional responses to nitrate and sugars may be long term and adaptive, while posttranslational regulation of transporter levels and activity, for example by phosphorylation (Ho et al, 2009;Wang et al, 2012a), may modulate nitrate uptake in response to rapid changes in the availability of nitrate and photosynthate.…”

“…Reduced NRT2.1 protein levels were only observed over a period of several days on high nitrate levels. A constitutively expressed 35S::NRT2.1 transgene in the atnrt2.1-2 null mutant has reduced high-affinity nitrate uptake in response to external ammonium nitrate, despite continued high NRT2.1 expression levels in the transgenic lines (Laugier et al, 2012). Rapid down-regulation of high-affinity nitrate uptake by dark and ammonium nitrate was not accompanied by reduced protein levels, indicating that rapidly acting posttranscriptional mechanisms control the down-regulation of NRT2.1 transport activity while protein levels remain relatively steady.…”

“…Microsome purification was according to Laugier et al (2012). Forty roots were harvested, snap frozen in liquid N, and ground in a Genogrinder.…”

“…Repression of NRT2.1 expression by high nitrate is associated with HIGH NITROGEN INSENSITIVE9/ARABIDOPSIS THALIANA INTERACT WITH SPT6 1-mediated increases in promoter histone methylation (Widiez et al, 2011). Constitutive expression of NRT2.1 in the nrt2.1 mutant background showed that repression of high-affinity nitrate uptake by high nitrate was not directly associated with reduced protein levels (Laugier et al, 2012), suggesting that protein modifications may be responsible.…”

Biotechnology of nutrient uptake and assimilation in plants

“…NRT2.1 is the main component of inducible HATS in Arabidopsis roots, as demonstrated in nrt2.1 that lack up to 75% of the high-affinity NO 3 -uptake activity. To be active, NRT2.1 forms a functional unit with NITRATE ACCESSORY PROTEIN2.1 (also called AtNRT3.1) that plays an important role in both constitutive and inducible HATS (Laugier et al, 2012). NRT2.1 is up-regulated by NO 3 -and sugars and down-regulated by N assimilation products (such as Gln) and cytokinin (CK) (Kiba et al, 2011).…”