Trends in Plant Science
Volume 22, Issue 2, February 2017, Pages 163-174
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Review
Nitrate Reductase Regulates Plant Nitric Oxide Homeostasis

https://doi.org/10.1016/j.tplants.2016.12.001Get rights and content

Trends

NO synthesis remains a complex picture with many unresolved questions.

NR has been assumed to be the main enzymatic source, but a new and more complex picture for the mechanism of NO synthesis is emerging.

NR, the first enzyme for nitrate assimilation, is a multi-redox protein able to mediate the donation of electrons from NAD(P)H to artificial acceptors and redox proteins. In Chlamydomonas, two of these redox partners are NOFNiR, which efficiently synthesizes NO, and THB1, a truncated hemoglobin, which eliminates NO by its dioxygenase activity.

Homeostasis of the crucial signaling molecule NO in photosynthetic organisms depends on at least two key molybdoenzymes, NR and NOFNiR, as well as on the dioxygenase activity of hemoglobins.

Nitrate reductase (NR) is a key enzyme for nitrogen acquisition by plants, algae, yeasts, and fungi. Nitrate, its main substrate, is required for signaling and is widely distributed in diverse tissues in plants. In addition, NR has been proposed as an important enzymatic source of nitric oxide (NO). Recently, NR has been shown to play a role in NO homeostasis by supplying electrons from NAD(P)H through its diaphorase/dehydrogenase domain both to a truncated hemoglobin THB1, which scavenges NO by its dioxygenase activity, and to the molybdoenzyme NO-forming nitrite reductase (NOFNiR) that is responsible for NO synthesis from nitrite. We review how NR may play a central role in plant biology by controlling the amounts of NO, a key signaling molecule in plant cells.

Section snippets

NO and NR in Plants

The participation of NO in many processes throughout the plant life cycle, including germination, nutrition, growth, development, flowering, programmed cell death, and biotic or abiotic stress, has been increasingly substantiated 1, 2, 3, 4. However, attempts to identify a plant NO synthase catalyzing oxidative synthesis of NO from arginine (as in animals) have led to disappointing results 2, 5, 6. In the plant kingdom, an NO synthase bearing a complete set of protein domains (see Glossary)

Nitrate Is Both a Nutrient and a Signal for Plants

Nitrate and ammonium are the most available inorganic sources for nitrogen (N) acquisition, without considering atmospheric dinitrogen which can only be used by prokaryotic organisms [18]. Most agricultural plants can use both ammonium and nitrate. In aerated soils nitrate is the predominant form of inorganic N, whereas in flooded wetlands or acidic soils ammonium predominates [19]. Nitrate abundance in soils can vary from 0–10 μM, and can be as high as 100 mM in highly fertilized fields [20]. N

Nitrate in Plant Cells

Plants acquire nitrate from soils by the action of membrane transporters which participate in its uptake, allocation, and storage. Nitrate homeostasis in plant cells depends on the influx activity of transporters that belong to the NPF and NRT2 protein families and on the efflux activity mediated by another set of transporters/channels (NAXT1 and SLAH3; reviewed in 23, 26). The first nitrate transporter identified in plants was CHL1/NRT1.1 (also named NPF6.3). High-affinity nitrate transporters

The Nitrate Assimilation Pathway

Inside the cell, nitrate is firstly reduced to nitrite by the cytosolic enzyme NR. This seems to be the rate-limiting step in the assimilation pathway [33]. Then, generated nitrite is transported into the chloroplast by specific systems. In Chlamydomonas this step is carried out by NAR1.1 [28], one of the six members of the formate/nitrite transporter (FNT) family. In plants, NAR1.1 is not conserved and the transporter involved is still unknown. High efficiency of nitrite transport to the

NR Is a Multidomain Protein

The multidomain protein NR catalyzes the reaction:NO3 + NAD(P)H + H+  NO2 + H2O + NAD(P)+

This redox reaction occurs as an electron transport mini-chain across different cofactors (Box 1). In addition to the overall activity of nitrate reduction from NAD(P)H, two partial activities, involving different protein domains, can be measured separately in vitro (Figure 1). These are (i) diaphorase or dehydrogenase activity, assayed by the reduction of acceptors such as ferricyanide (FeCN63−) or cytochrome c

The NO Signal in Plants Is Related to N Metabolism

NO has been demonstrated to be a key signaling molecule in several plant processes including such as whole plant development and different plant stress responses 1, 2, 3, 4, 35.

NO can be produced as a plant response to cope with attacks of different pathogens and promote defense hormones, defense gene expression, and the hypersensitive response mechanism 2, 36. This response is affected by the N nutritional status of the plant, and NO plays an important role in plant immune signaling. Because

Is NR Truly Involved in NO Production?

Although data supporting the relation between NR and NO production in plants have accumulated steadily 11, 54, 55, 56, important issues remain unresolved, as noted earlier, such as the low efficiency of NO production by NR or the generally unclear linkage between NR- and NO-producing activities. In fact, the production of NO by NR in vitro represents a small fraction (1%) of its nitrate reduction activity at saturating NADH and nitrite concentrations, and is strongly and competitively inhibited

A Dual NR–NOFNiR System for NO Synthesis

The molybdoenzyme ARC was first described in humans (mARC, mitochondrial ARC) [70], and only later in E. coli [71] and Chlamydomonas. In this alga, the protein has a similar functional organization to the human mARC [72] and contains a single Moco sulfurase C-terminal (MOSC) domain that is typically found in eukaryotic ARCs and sulfurases that bind Moco. ARC is involved in the reduction of a wide range of N-hydroxylated compounds [70] to generate the corresponding amine compounds, and uses

Regulation of NO levels

Levels of NO in the cytosol depend on the balance between its synthesis [by the dual system NAD(P)H–NR–NOFNiR, among possible others] and the effectiveness of reactions leading to NO removal. Concerning the consumption of synthesized NO, five points need to be considered: (i) NO can react with glutathione (GSH) to produce S-nitrosylated glutathione (GSNO), that is considered to act as a reservoir for NO and which provides the NO signal for nitrosylation of proteins. GSNO reductase (GSNOR)

NO Scavenging by Hemoglobins

Hemoglobins (HB) are ubiquitous proteins present in all kingdoms of life [87]. Plant class 1 (nsHB), and 2 (symbiotic HB) have sequences similar to animal HBs,and class 3 shows conservation with bacterial truncated hemoglobins (THB) [88]. Members of the HB superfamily can dioxygenate NO to give nitrate according to the following reaction:NO + O2 + 1 e  NO3

HB expression is upregulated by nitrate, nitrite, and NO in different plant species 89, 90. In maize roots, HB and NR show a coordinated

A Central Role for NR

Interestingly, half of the mini-ETC of NR, in other words NAD(P)H diaphorase, can supply electrons to (i) the second half of this NR ETC, which bears the Moco cofactor in the active site for nitrate reduction to nitrite, (ii) NOFNiR, a separate molybdoenzyme, which only bears the Moco cofactor, and catalyzes the synthesis of NO from nitrite, and to (iii) THB1, which catalyzes NO oxidation to nitrate. As shown in Figure 4 (Key Figure), one role of NR is the synthesis of nitrite from supplied

Can NO Be Synthesized Independently of NR:NOFNiR?

Under standard photosynthetic conditions, where nitrate is in the medium, NO synthesis takes place predominantly via the dual system NR:NOFNiR. Both enzymes are located in the cytosol where the majority of NO synthesis takes place [16]. However, we do not know in detail about other NO sources under conditions where NR is not synthesized. One such source could involve arginine oxidation, catalyzed possibly by a NOS-like enzyme, but which has not been demonstrated in plants, although this may

Concluding Remarks and Future Outlook

NO and RNS are well-established signaling molecules in plants and are involved in multiple processes required for adaptation to diverse environmental conditions. Research has been carried out for decades to understand the effects of NO and plant hormones in the context of response to different stresses, and for optimizing nutrient utilization and plant growth 1, 2, 4, 35. Notwithstanding, until now the synthesis of NO remained elusive, and NR was considered to be the most likely enzyme

Acknowledgments

This work was funded by MINECO (Ministerio de Economia y Competitividad, Spain, Grant BFU2015-70649-P) with support from the European FEDER program, Junta de Andalucía (P08-CVI-04157 and BIO-502), and the Plan Propio de la Universidad de Córdoba. A.C-A. thanks MINECO for a Formación de Personal Universitario fellowship, and E.S-L. thanks the Alfonso Martín Escudero Fundation for a postdoctoral fellowship.

Glossary

Denitrification
a process mediated by microorganisms, at very low oxygen concentrations, in which oxidized forms of nitrogen are used as the terminal electron acceptors (nitrate, nitrite, nitric oxide, nitrous oxide) to produce dinitrogen gas.
Diaphorase/dehydrogenase
in enzymology these terms refer to enzymes that are able to reduce acceptor molecules with electrons from reduced pyridine nucleotides, NADH or NADPH. These enzymes bear flavins as prosthetic groups.
Dioxygenase
an enzyme able to

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