Trends in Plant Science
ReviewNitrate Reductase Regulates Plant Nitric Oxide Homeostasis
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
References (100)
- et al.
New frontiers in nitric oxide biology in plant
Plant Sci.
(2011) Plant nitric oxide synthase: a never-ending story?
Trends Plant Sci.
(2006)Nitric oxide in marine photosynthetic organisms
Nitric Oxide
(2015)The emerging roles of nitric oxide (NO) in plant mitochondria
Plant Sci.
(2011)The increasing importance of distinguishing among plant nitrogen sources
Curr. Opin. Plant Biol.
(2015)Integration of local and systemic signaling pathways for plant N responses
Curr. Opin. Plant Biol.
(2012)Uptake, allocation and signaling of nitrate
Trends Plant Sci.
(2012)The Arabidopsis nitrate transporter AtNRT2.1 is targeted to the root plasma membrane
Plant Physiol. Biochem.
(2007)Characterization of Chlamydomonas 102 and 104 mutants reveals intermolecular complementation in the molybdenum cofactor protein CNX1E
Protist
(2013)- et al.
Xanthine oxidoreductase-catalyzed reduction of nitrite to nitric oxide: insights regarding where, when and how
Nitric Oxide
(2013)
Characterization of the magnitude and mechanism of aldehyde oxidase-mediated nitric oxide production from nitrite
J. Biol. Chem.
Nitrite reductase and nitric-oxide synthase activity of the mitochondrial molybdopterin enzymes mARC1 and mARC2
J. Biol. Chem.
On the origins of nitric oxide
Trends Plant Sci.
An alternative pathway for nitric oxide production in plants: new features of an old enzyme
Trends Plant Sci.
Reduction of nitrite to nitric oxide by enteric bacteria
Biochem. Biophys. Res. Commun.
Nitrate reductase (NR)-dependent NO production mediates ABA- and H2O2-induced antioxidant enzymes
Plant Physiol. Biochem.
Identification of the missing component in the mitochondrial benzamidoxime prodrug-converting system as a novel molybdenum enzyme
J. Biol. Chem.
Detection of peroxynitrite accumulation in Arabidopsis thaliana during the hypersensitive defense response
Nitric Oxide
Evaluation of nitroalkenes as nitric oxide donors
Bioorg. Med. Chem. Lett.
Nitroalkylation – a redox sensitive signaling pathway
Biochim. Biophys. Acta
Hypothesis: nitro-fatty acids play a role in plant metabolism
Plant Sci.
Plant hemoglobins: important players at the crossroads between oxygen and nitric oxide
FEBS Lett.
Nitric oxide and nitric oxide synthase activity in plants
Phytochemistry
Functional domains of assimilatory nitrate reductases and nitrite reductases
Trends Biochem. Sci.
Structural studies on corn nitrate reductase: refined structure of the cytochrome b reductase fragment at 2.5 Å, its ADP complex and an active-site mutant and modeling of the cytochrome b domain
J. Mol. Biol.
Haemoglobin modulates salicylate and jasmonate/ethylene-mediated resistance mechanisms against pathogens
J. Exp. Bot.
When bad guys become good ones: the key role of reactive oxygen species and nitric oxide in the plant responses to abiotic stress
Front. Plant Sci.
Understanding nitrate assimilation and its regulation in microalgae
Front. Plant Sci.
Identification of a plant nitric oxide synthase gene involved in hormonal signaling
Science
Occurrence, structure, and evolution of nitric oxide synthase-like proteins in the plant kingdom
Sci. Signal.
Production and scavenging of nitric oxide by barley root mitochondria
Plant Cell Physiol.
Nitric oxide in plants: an assessment of the current state of knowledge
AoB Plants
Nitric oxide production in plants: facts and fictions
Plant Signal. Behav.
Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro
J. Exp. Bot.
The Chlamydomonas Sourcebook
Establishing Chlamydomonas reinhardtii as an industrial biotechnology host
Plant J.
A dual system formed by the ARC and NR molybdoenzymes mediates nitrite-dependent NO production in Chlamydomonas
Plant Cell Environ.
THB1, a truncated hemoglobin, modulates nitric oxide levels and nitrate reductase activity
Plant J.
Plant nitrogen assimilation and use efficiency
Annu. Rev. Plant Biol.
An updated model for nitrate uptake modelling in plants. I. Functional component: cross-combination of flow-force interpretation of nitrate uptake isotherms, and environmental and in planta regulation of nitrate influx
Ann. Botany
Improving nitrogen use efficiency in crops for sustainable agriculture
Sustainability
Nitrate transport and signalling in Arabidopsis
J. Exp. Bot.
Nitrate and glutamate as environmental cues for behavioural responses in plant roots
Plant Cell Environ.
Nitrate, a signal relieving seed dormancy in Arabidopsis
Plant Cell Environ.
The role of Ynt1 in nitrate and nitrite transport in the yeast Hansenula polymorpha
Yeast
Inorganic nitrogen assimilation in Chlamydomonas
J. Exp. Bot.
Members of the NPF3 transporter subfamily encode pathogen-inducible nitrate/nitrite transporters in grapevine and Arabidopsis
Plant Cell Physiol.
The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles
Nature
Cytosolic nitrate ion homeostasis: could it have a role in sensing nitrogen status?
Ann. Bot.
Structure and function of eukaryotic NAD(P)H:nitrate reductase
Cell Mol. Life Sci.
Cited by (310)
Electroenzymatic tandem catalysis for the conversion of nitrate into ammonia
2024, Chemical CommunicationsInterspecific variations in growth, physiology and Cd accumulation between Populus deltoides and P. × canadensis in response to Cd pollution under two soil types
2024, Ecotoxicology and Environmental SafetyNitro-oxidative response to internalized multi-walled carbon nanotubes in Brassica napus and Solanum lycopersicum
2023, Ecotoxicology and Environmental SafetyThe mitochondrial amidoxime reducing component—from prodrug-activation mechanism to drug-metabolizing enzyme and onward to drug target
2023, Journal of Biological ChemistryNitric oxide: An emerging warrior of plant physiology under abiotic stress
2023, Nitric Oxide - Biology and Chemistry
- 3
These authors contributed equally