Cross-Regulation between N Metabolism and Nitric Oxide (NO) Signaling during Plant Immunity

Abstract

Plants are sessile organisms that have evolved a complex immune system which helps them cope with pathogen attacks. However, the capacity of a plant to mobilize different defense responses is strongly affected by its physiological status. Nitrogen (N) is a major nutrient that can play an important role in plant immunity by increasing or decreasing plant resistance to pathogens. Although no general rule can be drawn about the effect of N availability and quality on the fate of plant/pathogen interactions, plants' capacity to acquire, assimilate, allocate N, and maintain amino acid homeostasis appears to partly mediate the effects of N on plant defense. Nitric oxide (NO), one of the products of N metabolism, plays an important role in plant immunity signaling. NO is generated in part through Nitrate Reductase (NR), a key enzyme involved in nitrate assimilation, and its production depends on levels of nitrate/nitrite, NR substrate/product, as well as on L-arginine and polyamine levels. Cross-regulation between NO signaling and N supply/metabolism has been evidenced. NO production can be affected by N supply, and conversely NO appears to regulate nitrate transport and assimilation. Based on this knowledge, we hypothesized that N availability partly controls plant resistance to pathogens by controlling NO homeostasis. Using the Medicago truncatula/Aphanomyces euteiches pathosystem, we showed that NO homeostasis is important for resistance to this oomycete and that N availability impacts NO homeostasis by affecting S-nitrosothiol (SNO) levels and S-nitrosoglutathione reductase activity in roots. These results could therefore explain the increased resistance we noted in N-deprived as compared to N-replete M. truncatula seedlings. They open onto new perspectives for the studies of N/plant defense interactions.

Keywords: Aphanomyces euteiches; Medicago truncatula; nitric oxide homeostasis; nitrogen metabolism; plant immunity.

FIGURE 1

Transformed root validation. (A) Transcript levels of MtNIA1 and MtNIA2 in RNAi::NIA1/2-transformed roots were compared to control transformed roots (control NR). SNO quantification using the Saville–Griess assay and NO quantification using the fluorophore DAF (10 μM). Control NR and RNAi::NIA1/2-transformed roots extracts were pre-incubated or not with 500 μM cPTIO as an NO scavenger. (B) Transcript levels of MtGSNOR in 35S::GSNOR-transformed roots were compared to control transformed roots (control LacZ). SNO quantification using the Saville–Griess assay and NO quantification using the fluorophore DAF (10 μM). Control LacZ and 35S::GSNOR-transformed roots extracts were pre-incubated or not with 500 μM cPTIO as an NO scavenger. Error bars indicate standard errors (n = 4 for transcripts and NO levels; n = 8 for SNO levels), and ^∗ indicates significant differences (p < 0.05).

FIGURE 2

Quantification of Aphanomyces euteiches in extracts from inoculated transformed roots. RNAi::NIA1/2-transformed roots (A) and 35S::GSNOR-transformed roots (B) were extracted for ELISA tests. Roots were cultivated in vitro for 7 days on Fahraeus medium and then inoculated with A. euteiches. The background signal in non-inoculated roots was subtracted from the signal detected in inoculated roots. Error bars indicate standard errors (n = 4), and ^∗ indicates significant differences (p < 0.05). Data from one representative experiment out of four independent experiments.

FIGURE 3

Nitrate reductase (NR) activity and NO₃^- contents in transformed roots. (A) NR activity in control transformed roots (Control LacZ roots transformed with pK7GWG2D-GFP) and in transformed roots overexpressing GSNOR (35S::GSNOR). Transformed roots were cultivated in vitro on Shb10 medium. (B) NO₃^- concentrations in control transformed roots (Control LacZ) and GSNOR-overexpressing roots. Transformed roots were cultivated in vitro for 7 days on Fahraeus medium, and inoculated with A. euteiches. Error bars indicate standard errors (n = 4), and letters or ^∗ indicate significant differences (p < 0.05). Data from one representative experiment out of three independent experiments for both NR activity and NO₃^- contents (n = 12).

FIGURE 4

H₂O₂, NO, ONOO^-, and SNO quantification in Medicago truncatula roots 7 dpi. M. truncatula was cultivated on complete medium or NØ medium, and roots were harvested 7 days after inoculation with A. euteiches and used to detect H₂O₂, NO, and ONOO^- concentrations using fluorescent probes, and SNO concentrations using the Saville–Griess assay. (A) H₂O₂ quantification using 10 μM Amplex Red^® fluorophore and 0.2 U/mL of peroxidase. Catalase (1 U/μL), used as an H₂O₂ scavenger, abolished Amplex Red^® fluorescence. (B) NO quantification using the fluorophore DAF (10 μM). Root extracts were pre-incubated or not with 500 μM cPTIO as an NO scavenger. (C) ONOO^- quantification using the fluorophore APF (5 μM). Root extracts were pre-incubated or not with 1 mM of the ONOO^- scavenger epicatechin. (D) SNO quantification by the Saville–Griess assay. Error bars indicate standard errors (n = 4 for A–C; n = 14 for D), and letters indicate significant differences (p < 0.05). Data from one representative experiment out of three independent experiments for H₂O₂, NO, and ONOO^-concentrations, and data corresponding to two independent experiments pooled together for SNO concentrations. RFU, relative fluorescence units.

FIGURE 5

GSNO reductase activity in M. truncatula roots 7 dpi. M. truncatula seedlings were inoculated or not with A. euteiches, and cultivated on complete medium or NØ medium for 7 days. Root extracts were used to measure GSNOR activity. Error bars indicate standard errors (n = 4), and letters indicate significant differences (p < 0.05). Data from one representative experiment out of four independent experiments.

FIGURE 6

Working model. Results from the present work indicate that RNAi::MtNIA1/2 and 35S::GSNOR transformed roots are, respectively, more susceptible and more resistant to A. euteiches (1). NR activity and NO₃^- content were impacted by GSNOR overexpression, indicating a possible effect of GSNOR on basal NO₃^- transport/assimilation (2). NO₃^- availability in the medium causes quantitative modulation of ROS/RNS/NO content and affects their balance (3). Infection by A. euteiches decreases root NO₃^- content (4) and induces higher ROS levels (5). According to the literature superoxyde (O₂^⋅-), by reacting with NO to form peroxynitrite, can influence the concentration of NO available for signaling (6). ^∗: GSNO was shown to regulate NO₃^- uptake through transcriptional regulation of NRT2.1 (Frungillo et al., 2014).