The Nitrate Transporter MtNPF6.8 Is a Master Sensor of Nitrate Signal in the Primary Root Tip of Medicago truncatula

Abstract

Nitrate is not only an essential nutrient for plants, but also a signal involved in plant development. We have previously shown in the model legume Medicago truncatula, that the nitrate signal, which restricts primary root growth, is mediated by MtNPF6.8, a nitrate transporter. Nitrate signal also induces changes in reactive oxygen species accumulation in the root tip due to changes in cell wall peroxidase (PODs) activity. Thus, it was interesting to determine the importance of the role of MtNPF6.8 in the regulation of the root growth by nitrate and identify the POD isoforms responsible for the changes in POD activity. For this purpose, we compared in M. truncatula a npf6.8 mutant and nitrate insensitive line deficient in MtNPF6.8 and the corresponding wild and sensitive genotype for their transcriptomic and proteomic responses to nitrate. Interestingly, only 13 transcripts and no protein were differently accumulated in the primary root tip of the npf6.8-3 mutant line in response to nitrate. The sensitivity of the primary root tip to nitrate appeared therefore to be strongly linked to the integrity of MtNPF6.8 which acts as a master mediator of the nitrate signal involved in the control of the root system architecture. In parallel, 7,259 and 493 genes responded, respectively, at the level of transcripts or proteins in the wild type, 196 genes being identified by both their transcript and protein. By focusing on these 196 genes, a concordance of expression was observed for most of them with 143 genes being up-regulated and 51 being down-regulated at the two gene expression levels. Their ontology analysis uncovered a high enrichment in POD genes, allowing the identification of POD candidates involved in the changes in POD activity previously observed in response to nitrate.

Integration of reactive oxygen species and nutrient signalling to shape root system architecture

“…The authors found a maize orthologue of AtUPBEAT1 transcription factor to be responsible for regulating ROS levels by repressing the expression of a POD gene (Zm00001d024119) in a similar fashion to what was described in Arabidopsis, suggesting the existence of an evolutionarily conserved signalling module common to both monocots and eudicots(Trevisan et al, 2019;Tsukagoshi et al, 2010). Accordingly, in M. truncatula, the orthologue of AtUPBEAT1, MtUPBEAT1, was recently found to be downregulated in response to 5 mM NO 3 − treatment(Zang et al, 2022).In M. truncatula, the NO 3 − transporter MtNPF1.7 controls ROS homoeostasis in the primary root tip and promote cell elongation through an increased RBOH activity(Zhang et al, 2014). A link between ROS accumulation and NO 3 − availability was proposed although conclusive evidence was lacking.…”

“…In the following sections, we focus specifically on their roles in regulating the root system architecture in response to nutrient stress. Interestingly, no change in ROS accumulation or POD activity was observed in the primary root tip of the mutant npf6.8 deficient in the NO 3 − transporter MtNPF6.8 that has lost the sensitivity to NO 3 − (Zang et al, 2022). It is noteworthy that such a mechanism does not occur in lateral roots, in which growth rate is enhanced in the presence of NO 3 − .…”

Pisum sativum Response to Nitrate as Affected by Rhizobium leguminosarum-Derived Signals

“…For this aim, sequences of genes determined in our previous studies as being nitrate-inducible in M. truncatula roots at early stages of development [ 9 , 20 , 21 , 22 ] were used to browse the P. sativum genome database ( (accessed on 8 June 2022)) and search for sequences of their orthologs in this species. Chosen genes were: MtNR1 (nitrate reductase), MtGS1 (cytosolic glutamine synthetase), MtGS2 (plastidic glutamine synthetase), MtASNS (asparagine synthetase), MtNPF6 .…”

“…In the present work we studied the regulation of PsNPF6.4 , an ortholog of M. truncatula nitrate transporter MtNPF6.7 , identified in one of our previous works as a nitrate-inducible gene [ 22 ]. In here, PsNPF6.4 expression was not detected in young (2-day-old) seedlings in all conditions and in 12-day-old seedling in the absence of nitrate, but it was slightly detected in 5-day-old seedlings in the absence of nitrate ( Figure 3 ).…”

“…Once NO 3 − has been taken up, it is reduced by nitrate reductase, an enzyme encoded by NR1 that is known to be nitrate-inducible, as we have shown recently in M. truncatula [ 22 ]. In the present work, we found that in pea, also induction of PsNR1 by nitrate held true, and it was observed at all three stages of development ( Figure 4 ).…”