NAD

Abstract

Many metabolic processes that occur in living cells involve oxido-reduction (redox) chemistry underpinned by redox compounds such as glutathione, ascorbate and/or pyridine nucleotides. Among these redox carriers, nicotinamide adenine dinucleotide (NAD) is the cornerstone of cellular oxidations along catabolism and is therefore essential for plant growth and development. In addition to its redox role, there is now compelling evidence that NAD is a signal molecule controlling crucial functions like primary and secondary carbon metabolism. Recent studies using integrative -omics approaches combined with molecular pathology have shown that manipulating NAD biosynthesis and recycling lead to an alteration of metabolites pools and developmental processes, and changes in the resistance to various pathogens. NAD levels should now be viewed as a potential target to improve tolerance to biotic stress and crop improvement. In this paper, we review the current knowledge on the key role of NAD (and its metabolism) in plant responses to pathogen infections.

Introduction

Intense efforts are currently devoted to better understand the mechanisms of plant response to pathogenic and non-pathogenic microorganisms. In addition to direct trophic and toxic effects on vegetative and reproductive organs, pathogen infections cause substantial transcriptomic and metabolic reprogramming which may in turn affect growth and development. To recognize and respond to pathogens, plants have evolved innate immunity.

Plant immune system is usually described by two main response networks.¹ First, the plant cell recognizes so-called pathogen-associated molecular patterns (PAMP) which are conserved molecular signatures activating host basal defense responses, referred to as PAMP-triggered immunity (PTI). Second, a specific host response is triggered by the recognition of pathogen-derived effectors that counteract basal defenses. The latter response, called effector-triggered immunity (ETI), is based on earlier and wider molecular effects such as reactive oxygen species (ROS) production and the so-called oxidative burst,^2-4 accumulation of defense phytohormones (e.g. salicylic, jasmonic and abscisic acid, SA, JA and ABA respectively, and ethylene) and local and/or systemic expression of pathogen-related (PR) genes.^5-7 ROS, nitric oxide and SA are key actors of the signaling events which lead to cell death and eventually to the hypersensitive response (HR).^8,9

Up to now, most studies on plant-pathogen interactions have been using forward and reverse genetics (characterization of mutants) or proteomic approaches to infer key regulatory components of pathogen-related signaling pathways.^10,11 There is now a growing interest in small molecules (metabolites) which are believed to play a role in the plant-pathogen interactions. So is the case of defense compounds derived from secondary metabolism.^12,13 Plants synthesize many different secondary metabolites active against a wide variety of pathogens. These metabolites, usually specific to a family or a plant species, are involved indeed as natural pesticides or signals and represent an important part of the plant defense equipment. Immunity-related metabolites are divided into pathogen attack inhibitors (phytoanticipins) and pathogen development inhibitors (phytoalexins).¹⁴ For instance, the indolic compound camalexin is the major phytoalexin in Arabidopsis and other Brassicaceae. Solanaceae rather accumulate scopoletin as an antimicrobial phenolic molecule, while Vitaceae and Fabaceae can contain stilbenoids such as resveratrol.¹⁴

Primary metabolism is also considered to be of importance since it feeds the electron source of the oxidative burst via mitochondrial ROS production,¹⁵ and sustains the production of secondary metabolites. Nicotinamide adenine dinucleotide (NAD) is a primary metabolite synthesized from aspartate in plant (Fig. 1).¹⁶ This pyridine nucleotide is the cornerstone of cellular oxidation since it is the most common electron acceptor in catabolic reactions and serves as a cofactor for a multitude of enzyme involved in primary and secondary metabolisms. In addition to interfacing primary and secondary metabolisms, the content of NAD itself, or a close derivative, appears to participate in cellular responses to pathogen infections.^17-19 In this review, we shall summarize the specific role of NAD in pathogen defense networks, describe the mechanisms and outline current unknowns. In the following, mutants and engineered plant lines referred to are listed in Table 1.

Figure 1. Metabolism of nicotinamide adenine dinucleotide (NAD) in plants and its responses to stress. De novo biosynthesis, recycling, catabolism and utilization of the NAD⁺ molecule. Plain arrows indicate experimentally demonstrated reactions. Dashed arrows refer to condensed biochemical steps or experimentally non-demonstrated reactions. ADP: adenine diphosphate; AMP: adenine monophosphate; AO: aspartate oxidase; ATP: adenine triphosphate; cADPR(P): cyclic ADP-ribose (phospshate); ReT and PeT: respiratory and photosynthetic electron transport chains, respectively; NaAD: nicotinic acid adenine dinucleotide; NADK: NAD kinase; NADP: NAD phosphate; NADP pase: NADP phosphatase; NaMN: nicotinic acid mononucleotide; NaMNAT: NaMN adenylyltransferase; NADS: NAD synthetase; NaPT: nicotinate phosphoribosyltransferase; NDT: NAD⁺ carrier; NIC: nicotinamidase; PARP: poly-ADP-ribose polymerase; PXN: peroxisomal NAD carrier; QPT: quinolinate phosphoribosyltransferase; QS: quinolinate synthase; ROS: reactive oxygen species; TCAP: tricarboxylic acid pathway.

Table 1. Mutants and engineered plants used to illustrate the role of NAD in plant immunity.

Plant line	Species	Genotype	Impact on NAD or its derivative	Pathogen responses	References
CD38 OE	Human enzyme in Arabidopsis	OE	Expressing the human NAD(P)-metabolizing ectoenzyme	altered activation of SAR	[68]
CMSII	Nicotiana	in vitro wild-type protoplast culture	absence of respiratory complex I associated with higher NAD(H)	increased resistance to oxidative stress and a viral pathogen	[56,57]
fin4	Arabidopsis	T-DNA	impaired NAD biosynthesis	affected in several responses to pathogen attacks: PAMP-triggered ROS burst evolved by NADPH oxidases RBOHD and pathogen-related stomatal closure	[71]
gfg1	Arabidopsis	T-DNA	encoded a NUDX increased NADH/NAD+ ratio	increased ROS and NADH improved resistance to bacterial pathogens	[88]
nadC OE	E.coli gene in Arabidopsis	OE	inducible increase in intracellular NAD/P(H) content in leaves	enhanced resistance to avirulent Pst-AvrRpm1 associated with SA accumulation and altered redox dynamics	[18]
NADK2 OE	Arabidopsis gene in Rice	OE	build-up of the NADP(H) pool	enhanced tolerance to oxidative damage	[58]
nudt7	Arabidopsis	T-DNA	increased NADH/NAD ratio	activation of pathways dependent and independent of NPR1 and SA regulation of an EDS1- and PAD4-dependent but SA-independent signaling pathway in plant defense responses	[82,84,86]
parg1, parg2	Arabidopsis	T-DNA	incapable of removing ADPr from poly-ADPr chains	accelerated onset of disease symptoms upon fungal infection	[84,103]
parp1, parp2	Arabidopsis	T-DNA	accumulated poly-ADPr chains	increased resistance to stresses (reducing NAD+ breakdown and consequently energy consumption)	[49,100]
PARP1, PARP2 OE	Arabidopsis	OE	ADPr removed from poly-ADPr chains	reduced incidence of H2O2-induced DNA nicks	[49,99,100,101]
rbohD and F	Arabidopsis	Transposon insertion	impaired NADPH oxidase function in response to stress	extracellular NAD-defense responses (independent of NADPH oxidases encoded by AtrbohD and AtrbohF) the reducing power carried by NADPH could not influence PR genes expression.	[19]
SAG OE	Arabidopsis	OE	redox-related protein	responses to biotic stress via interaction with proteins that participate in mitochondrial ROS signaling.	[67]
srt2	Arabidopsis	T-DNA	impaired function of NAD-dependent histone deacetylation causing altered DNA repair, cell cycle, death and aging	improved resistance to Pst DC3000 associated with the induction of PR1 overexpression of SA biosynthesis-related genes (PAD4, EDS5 and SID2)	[113]
SRT2 OE	Arabidopsis	OE	altered function of NAD-dependent histone deacetylation leading to altered DNA repair, cell cycle, death and aging	bacterial hyper-susceptibility and impaired PR1 induction downregulation of PAD4, EDS5 and SID2	[107]

Basics of NAD Metabolism

Pyridine nucleotides (PNs), including NAD⁺, NADH, NADP⁺ and NADPH, are ubiquitous redox intermediates which are essential to catabolic (and anabolic) reactions, and thus to metabolic fluxes.²⁰ NAD (e.g., total pool of NAD⁺ and NADH) is a common coenzyme carrying electrons through the reversible conversion between oxidized (NAD⁺) and reduced (NADH) forms in all organisms. In plants, NAD is highly oxidized so that NAD⁺ is found at the millimolar concentration while NADH is estimated to be around 1µM only.^21,22 By contrast, phosphorylated NAD (NADP) mainly exists in its reduced form NADPH (around 0.2mM) and is mostly located in chloroplasts where it serves as the terminal electron acceptor in the photosynthetic electron transport chain (Fig. 1).^22,23 Chloroplastic NAD kinase NADK2 catalyzes NAD⁺ phosphorylation to NADP⁺ (Fig. 1).^24-26 Other NADK (NADK1, NADK3) are present in the cytosol and the peroxisome.^27,28

As for other nucleotides metabolism (pyrimidine), pyridines are both synthesized by a de novo biosynthesis and reformed by a recycling pathway (Fig. 1).^16,29,30 Nonetheless, our knowledge of plant pyridine nucleotides metabolism is still fragmentary. It is believed that chloroplastic reactions of NAD synthesis from aspartate^16,18,31 and the cytosolic adenylylation of nicotinic acid mononucleotide (NaMN) by NaMN adenylyltransferase (NaMNAT, Figure 1)^33,34 are the limiting steps of the NAD pathways. Among plant species and organs, the NAD recycling pathway shows some variations in the enzymatic reactions or metabolic intermediates involved,^35-37 suggesting that it can also control the NAD production. However, nicotinamide and nicotinate are constant key intermediates that sustain NAD synthesis along the pathway historically referred to as the “pyridine nucleotide cycle” (Fig. 1).³⁸

NAD⁺ is essential to ensure metabolic energy production in mitochondria (Fig. 1). Indeed, the tricarboxylic acid pathway (TCAP) generates reducing power as NADH which is subsequently used by oxidative phosphorylation to yield ATP.³⁹ The largest NAD pool is believed to be located in mitochondria in terms of concentration (2mM in mitochondria vs. 0.6mM in the cytosol).⁴⁰ Mitochondria have an adenine nucleotide translocator/mitochondrial substrate carrier (NDT2) allowing influx of NAD⁺ and acting as an antiport coupled to ADP or AMP exhaust.^41,42 Plastids also have a NAD carrier, NDT1 (Fig. 1).^36,42 NDT1 and NDT2 genes are notably highly expressed in Arabidopsis tissues under high metabolic activities.⁴² Recently, a peroxisomal NAD⁺ carrier (PXN) has been reported to be important for optimal fatty acid degradation,⁴³ and for providing the peroxisomes with coenzymes A and NAD⁺.⁴⁴ All these organelles-specific carriers are likely to be crucial for an optimal exchange between compartments and thus for the general signaling role of NAD. PNs concentration and localization in plants have been further described elsewhere.^36,45

NAD as a Component of Defense-Related Signaling/Responses

Recent studies indicate that NAD and NADP play a role in cell signaling in plants, animals and fungi.^34,45-48 In particular, PNs are involved in calcium (Ca²⁺) signaling, DNA repair through poly-ADP-ribosylation and proteins deacetylation (Fig. 1).^36,45,49 These reactions consume NAD and NADP, are thus likely influenced by NAD concentration and certainly, require NAD synthesis. Among the signaling events occurring in response to the environment, NAD appears to be a key actor in plant defense responses against pathogens and pests. In the late 60s, phytopathologists already noticed that challenging plants with pathogens could disturb PNs metabolism and NAD levels. In barley, leaves were 2-fold enriched in NAD six days after infection with powdery mildew pathogen Erysiphae gramini var hordei.⁵⁰ More recently, extracellular PNs have been shown to induce PR genes expression and resistance to Pseudomonas syringae pv maculicola (Psm) ES4326 in Arabidopsis.¹⁷

Is NAD an upstream redox signal?

Owing to its very negative redox potential (-0,32V), the couple NAD(P)-NADP(H) is assumed to be the “master” redox soluble compound in the cell.²⁰ Plants exposed to altered environmental conditions (abiotic and biotic stress) usually face secondary oxidative damage⁵¹ and NAD(H)/NADP(H) homeostasis is crucial for cell survival.^34,52 When mitochondrial NADPH pools are depleted, there is an increase in ROS content, DNA damage and lipid peroxidation.⁵³ Plant cells can repair oxidative damage with numerous NADPH-dependent antioxidant systems such as the thiol-disulfide thioredoxin and glutathione reductases.^54,55 In the tobacco CMSII mutant, the absence of respiratory complex I is associated with a clear increase in leaf PNs, and an improved resistance to oxidative stress and a viral pathogen.^56,57 The build-up of the NADP(H) pool by constitutive expression of the Arabidopsis chloroplastic NAD kinase NADK2 gene in rice has been reported to correlate with an enhanced tolerance to oxidative damage.⁵⁸ In Arabidopsis, during both compatible and incompatible interactions with Pst, the expression of the AtNADK1 gene is differentially increased.⁵⁹ However, uncertainty remains on whether this induction is caused by oxidative stress or by pathogen-related signaling directly.

NAD may also be involved in methionine (Met) mediated oxidative signals. In proteins, Met residues can be oxidized by ROS in Met sulfoxide (MetSO). This process can be reversed by Met sulfoxide reductases (MSR) which reduce MetSO back to Met thus participating in defense against oxidative damage.^60-62 MSR can use NADPH either directly or indirectly via thioredoxins. Thus presumably, sufficient NAD(P)H contents are essential to prevent Met oxidation. But quite surprisingly, high intracellular NAD/P(H) content has been shown to cause an accumulation of both Met and MSR transcripts in Arabidopsis (Fig. 2).¹⁸ The large NAD content has stimulated aspartate-derived amino acids production and plausibly MSR activity which has in turn resulted in more Met residues. Met oxidation is further involved in the regulation of calcium binding proteins (CaBP) in animals and plants.^61,63,64 In fact, the oxidation of Met residues in CaMKII (Ca²⁺/calmodulin-dependent protein kinase II) or calcineurin (protein phosphatase) lowers their affinity for Ca²⁺ thus impeding activation of target proteins. Since calcium signaling is involved in defense responses, such a regulation of Ca²⁺ binding proteins might modulate the tolerance to biotic stress.

Figure 2. NAD, a key component of the plant immunity signaling network: a model. Manipulation of NAD biosynthesis (AO, QPT) or utilization (PARP; PARG; NUDIX hydrolases; SIRTUINS) alters redox balance of pyridine nucleotides (PNs) which interferes with plant defense responses. Modified pools of PNs induce calcium binding protein genes (CaBP)¹⁸ and activate Ca²⁺ influx that stimulates NADPH oxidase (RBOH) activity therefore increasing ROS generation. Oxidative burst is believed to favor the signal amplification loop involving salicylic acid (SA).^2,115 PNs also cause induction of SA-biosynthetic gene isochorismate synthase (ICS1) and lead to SA accumulation.¹⁸ SA can activate pathogen-related (PR) genes by pathways that are dependent and independent of the non-repressor of PR1 (NPR1) component.¹⁷ It remains unclear how Ca²⁺ triggers SA-dependent resistance. While it is not totally understood how PNs activate RBOH, the perturbation in NAD/P(H) pools disturbs redox signaling by inducing redox-related genes (glutathione S-transferase: GST; perodixase; PRX; senescence associated gene: SAG). Glutathione (GSH) which is regenerated by the reducing power of NADPH is also potentially altered by modified PNs levels. Another redox-related response upon high NAD content relies on the alteration of the methionine (Met) pools and the induction of Met sulfoxide reductase genes (MSR) that are involved in reducing Met sulfoxide residues (MetSO) back to Met.¹⁸ Dashed arrows refer to possible signaling connections.

Further redox-related transcriptional stimulation was observed under high NAD contents (Fig. 2).¹⁸ This is typically the case of several genes usually expressed upon HR conditions. For instance, α-DOX1 gene encodes a fatty acid dioxygenase that is involved in the protection against oxidative stress and is induced in both incompatible and compatible bacterial infections.⁶⁵ Several genes encoding glutathione S-transferase (GSTs) which underlie peroxidation of GSH and/or glutathionylation are also induced in response to NAD.⁶⁶ The enhancement of the expression of senescence associated genes (SAG) has also been reported upon high and moderate NAD increase.^18,37 Recently, SAG21 has been proposed to be a redox-related protein involved in the response to biotic stress via interaction with proteins that participate in mitochondrial ROS signaling.⁶⁷

Taken as a whole, NAD-mediated redox effects appear to be important to (i) favor the pathogens response when NAD(P) contents are high (e.g., transcriptional stimulation by NAD), and (ii) elicit redox-mediated signaling when the NAD(P)H/NAD(P) ratio is large (e.g., thiols mediated-redox signaling). In fact, an accumulation of reduced forms (NADH and NADPH) has been observed following the infection with the avirulent bacterium Pseudomonas syringae pv tomato (Pst) carrying the AvrRpm1 gene. It is believed that the observed resistance may have resulted from the higher availability of NADPH for ROS production through the NADPH oxidases RBOH (Fig. 2).^2,18 However, mutants impaired in NADPH oxidases showed extracellular NAD-dependent defense responses that were independent of NADPH oxidases encoded by AtrbohD and AtrbohF and furthermore, the reducing power carried by NADPH could not influence PR genes expression.¹⁷ Recently, the application of exogenous NAD(P) has been shown to suppress HR-mediated cell death but it does not affect disease resistance.⁶⁸ Although physiological exogenous NAD(P) concentrations are unable to induce SAR in distal tissues infected with avirulent Pst, the use of a transgenic system depleting external NAD(P) pools reveal that NAD(P) may nevertheless play a critical signaling role in the activation of systemic acquired resistance (SAR).⁶⁸ In addition, exogenous NAD(P) has been suggested to mainly enhance R-gene-independent disease resistance. Quite surprisingly, while there is a link between NADPH-consuming RBOHD and RBOHF in the HR-mediated ROS production,⁹ this recent study has not tested whether reduced forms NAD(P)H might interfere with cell death.⁶⁸ Hitherto, further molecular studies are thus required to remove uncertainty on whether PNs are essential for redox pathogen signaling and oxidative burst and to identify other defense NAD(P)H-dependent ROS production mechanisms than NADPH oxidases.

NAD biosynthesis and its potential roles in defense responses

The use of Arabidopsis mutants and transgenic lines has recently allowed investigating the effect of NAD synthesis alterations on plant defense networks.

First, advantage was taken of Arabidopsis lines overproducing a bacterial quinolinate phosphoribosyltransferase (QPT) to investigate the consequences of an inducible increase in intracellular NAD content in leaves.¹⁸ Large-scale transcriptomics revealed that higher NAD levels directly induced defense-related genes (PR1, GLIP1, RPM1-interacting protein 4, chitinases, several genes involved in glucosinolate metabolism, WRKY transcription factors, etc.) similar to that induced by SA (Fig. 2). Higher NAD levels also lead to an ICS1-dependent accumulation of total and free forms of SA.¹⁸ Consistently, the resistance to Psm following extracellular PNs application has been shown to be accompanied with PR genes expression and a build-up of free SA.¹⁷ However, the specific mechanism by which NAD interacts with SA signaling remains elusive, because the addition of exogenous PNs induce both SA/NPR1-dependent and SA/NPR1-independent defense responses (Fig. 2).¹⁷ The use of plant lines with mutations in SA-metabolism and/or response is now strictly necessary to better understand the role of SA in NAD-related mechanisms. Furthermore, manipulating NAD levels with such an inducible production system can have pleiotropic effects via other phytohormones such as ethylene (ET) and JA, as suggested by the upregulation of ET and JA related genes (like CEJ1, JAZ8, and GLIP1).¹⁸ In fact, one of them, GLIP1, is known to be involved in SA-independent defense responses and to elicit the ET pathway which in turn promotes resistance against several pathogens.⁶⁹

Second, aspartate oxidase (AO), the first enzyme of NAD de novo biosynthesis pathway (see Figure 1) has been shown to be crucial for Arabidopsis defense responses. During incompatible interaction, AO transcripts accumulate.¹⁸ From consideration of Genevestigator predictions,³² transcriptomic data suggest a transcriptional regulation of the gene encoding AO upon bacterial infection. Additionally, the AO gene has been shown to belong to the B4 resistance gene cluster in common bean.⁷⁰ A screening of Arabidopsis flagellin-insensitive (fin) mutants has further reported that FIN4 encodes AO. fin4 mutants have been shown to be viable and fertile, with no morphological phenotype other than a reduction in size despite the strong decrease in AO activity.⁷¹ fin4 mutants are significantly affected in several responses to pathogen attacks including (i) PAMP-triggered ROS burst evolved by NADPH oxidases RBOHD and (ii) pathogen-related stomatal closure. However, the same mutant (SAIL_1145_B10) cultivated under short days conditions exhibits lighter green and modified leaves with a clear developmental phenotype that may contribute to the altered pathogen response (de Bont et al., unpublished).

Involvement of NAD recycling and NAD-derivatives in plant immunity

NAD derivatives are stress mediators in plant defense metabolism against pathogens and pests. NAD is a precursor of pyridine alkaloids (e.g., the well-known nicotine, anabasin, trigonelline and ricinine, see Figure 1) that are presumed to promote plant defense.^72-74 Nicotine accumulates upon foliar wounding in Nicotiana and is an antifeedant compounds for herbivores. In addition, isonicotinamide, which is a structural analog of nicotinamide, has the pyridine structural motif present in many SAR inducers.⁷⁵ In tobacco cells, exogenous isonicotinamide increases the activity of the defense-related phenylalanine ammonia-lyase (PAL) and plant defense secondary metabolites accumulation such as phenolics and sesquiterpenoids.¹⁹ Furthermore, neonicotinoids are well-known insecticides chemically related to nicotine, the most common of which being imidacloprid.⁷⁶

In addition to their toxicity for insects, neonicotinoids have also been associated with an improved resistance to microbial pathogens.^77,78 In fact, Arabidopsis rosettes sprayed with neonicotinoids show an enhanced resistance to powdery mildew and induction of SA-mediated plant defense responses.⁷⁶ The authors suggested that some neonicotinoids stimulate defense responses by mimicking SA tridimentional structure.⁷⁶ Interestingly, under insect-free conditions, SA induction by neonicotinoids is not associated with a lower plant fitness, and neonicotinoids treatments can even promote foliage growth, plant vigor and tolerance to drought.^77,79 However, it has been recently suggested that NAD per se triggers the plant defense signaling and responses to Pst strain carrying AvrRpm1 gene¹⁸ suggesting that plausibly NAD or its derivatives have little direct toxic effect on bacterial pathogens. Accumulated NAD derivatives (nicotinamide and nicotinate) are indeed suspected to have a modest effect or no effect at all on incompatible interactions: for example, neonicotinoids spraying rather promoted NAD recycling and increased NAD(P) pools which in turn improved tolerance against drought or pathogens via molecular mechanisms.^77,78

Nicotinamide is a key metabolite derived from NAD-cleavage pathways.^36,45 As stated before, studies of isonicotinamide (a nicotinamide chemical analog) support a role for nicotinamide-related metabolites in plant defense responses.¹⁹ In addition, NUDIX hydrolases (abbreviated NUDX or NUDT in Arabidopsis)^80,81 can produce nicotinamide. Mutations in genes that encode NUDX have been shown to increase the NADH/NAD⁺ ratio (Fig. 2) and improve plant immunity.^82-85 In Arabidopsis, NUDT7 gene has been shown to regulate an EDS1- and PAD4-dependent but SA-independent signaling pathway in plant defense responses.⁸⁶ NUDT7 is further induced by the flagellin fragment flg22 which is an elicitor of plant basal defenses.⁸⁴ Furthermore, the Arabidopsis nudt7 mutants show a retarded growth and an increased NADH/NAD⁺ ratio (under nutrient-poor conditions), and an induction of SAR-dependent genes (Fig. 2).^82,84 The nudt7 mutation activates simultaneously two pathways, one dependent and one independent of NPR1 and SA.⁸² The characterization of a null mutant impaired in growth factor gene 1, encoding a NUDX, provided evidence for an increase in both ROS and NADH and an improved resistance to virulent bacterial pathogens.⁸⁸ Here again, PNs and redox state appear to be closely linked upon defense responses, that is, it seems that impeding NAD degradation promotes resistance to pathogens. It is plausible therefore that in the natural environment, NAD metabolism operates under a compromise between the maintenance of NAD pools, which participates in plant signaling responses to bacterial or fungal infections, and the consumption of NAD to produce NAD-derivative insecticides.

NAD and calcium signaling

The build-up of intracellular and/or extracellular calcium ions (Ca²⁺) is a well-known component of early signalisation pathways associated with plant defense responses. Coordinated [Ca²⁺] and pH changes are essential during biotic stress since the alkalinization of the cellular medium is necessary to induce the oxidative burst.⁸⁹ It has been clearly demonstrated that [Ca²⁺] increase is upstream of ROS production and R gene-mediated hypersensitive cell death (Fig. 2).⁹⁰ Ca²⁺ signaling is also linked to NAD. In fact, cyclic nucleotides such as the NAD derivative cyclic ADP-ribose (cADPR) enhance the conductance of Ca²⁺ channels leading to a Ca²⁺ influx into the cytoplasm through the plasma membrane, the tonoplast and the membrane of the endoplasmic reticulum.⁴⁵ cADPR has further been reported to induce PAL and PR1 expression in tobacco, which is in agreement with an implication of cyclic nucleotides in plant immunity.⁹¹ In addition to cADPR, the NADP-derivatives nicotinate adenine dinucleotide phosphate (NaADP) and cADPRP (Fig. 1)^92-96 have also been shown to contribute to releasing Ca²⁺ ions into the cytoplasm.^45,97

When NAD(P) pools are large, the induction of PR genes is thought to be mediated by Ca²⁺ signaling (Fig. 2): First, Ca²⁺ trapping with the chelation agent EGTA inhibits PR genes expression upon treatment with exogenous NAD(P).¹⁷ Second, the intracellular build-up of NAD activates several genes encoding various Ca²⁺-binding proteins, thereby suggesting that cADPR and/or NaADP signaling might be a key intermediate between NAD itself and the induction of plant responses.¹⁸ However, recent pharmacological approaches that used tobacco cells treated with a plant defense elicitor (cryptogein) indicate that cADPR has little effect on variations in Ca²⁺ concentrations.⁹⁸ In addition, another report suggested that cADPR and NaADP metabolized through the activity of the human multifunctional ectoenzyme CD38 that catalyzes the synthesis and hydrolysis of cADPR from NAD and NADP, respectively, were not likely to activate immune responses.⁶⁸

Therefore, while NAD signaling involves Ca²⁺ at least partly, no firm conclusion can be drawn on whether the transduction link between NAD and Ca²⁺ is represented by cADPR, NaADP or another molecular species. In support of this, neither plant cADPR cyclase sequence nor endogenous cADPR molecules have been detected so far in Arabidopsis leaves challenged with avirulent Pst-AvrRpm1 (Pétriacq et al., unpublished).

ADP-ribosylation mediated reactions

NAD is a substrate for mono- and poly-ADP-ribosylation (PAR) of proteins, as shown in Figure 1. This important post-translational modification is catalyzed by poly-ADP-ribose polymerases (PARP) which attach long-branched poly-ADP-ribose polymers to nuclear target proteins.^99-102 Poly-ADP ribose residues can be removed by PAR glycohydrolases (PARG), thereby liberating ADP-ribose (ADPr) in the cytoplasm or the nucleoplasm. Thus protein PAR is determined by the balance between PARP and PARG activity.⁴⁹

PAR is believed to be implicated in the regulation of DNA repair, transcription, and programmed cell death, in particular when DNA damage results from oxidative stress.⁹⁹ Our understanding of PARP-mediated processes in biotic stress responses is still at its infancy. However, pioneering works of Adams-Phillips and colleagues showed that PARP inhibitors blocked the effect of the bacterial elicitors flg22 and efu18 (induction of callose deposition), suggesting an apparent role for PAR in plant immunity.⁸⁷ The same study further indicated an induction of the PARG gene At2g31865 upon infection with multiple avirulent Pst. Thus presumably, PARG contributes to ADPr accumulation after the activation of PAR and this may in turn initiate a signaling process. More recently, an increase of PAR was observed when Arabidopsis leaves were infected with avirulent Pst-AvrRpt2⁺.^84,100 Several components of the innate immune responses are also altered by PARP inhibitors (except for the initial ROS generation and early defense genes induction) and parg1 and parg2 mutants show an accelerated onset of disease symptoms upon fungal infection.^84,100

In addition to the crucial role of PARP and PARG to determine the ADPr pool, ADPr degradation might also be involved. ADPr homeostasis involves NUDX hydrolases (see above). Depending on the NUDX considered, the effective enzymatic activity should be NAD-hydrolysing or ADPr-hydrolysing and might influence NADH/NAD⁺ ratio or ADPr contents. Further work is thus needed on the NUDX family in Arabidopsis so as to better understand the substrate specificity of these enzymes and the particular context in which each of them is involved.

Recent studies have further indicated that ADP-ribosylation of molecular actors might be important for neutralizing plant response mechanisms.^104-106 That is, this strongly suggests that ADP-ribosylations (mono- or poly-) play a prominent role in plant-pathogen interactions.

Proteins deacetylation

NAD catabolism further involves histone deacetylation by NAD⁺-dependent histone deacetylases (HDAC), also named “sirtuins” (Fig. 1). HDAC can deacetylate both histone and non-histone substrates as revealed by functional studies in yeast and mammalian cells.^107-109 While it is widely accepted that sirtuins play important roles in chromatin silencing, DNA repair, cell cycle and death, and aging,^110-112 the biological function of NAD-dependent histone deacetylation in plants cellular signaling remains poorly understood.^36,45,48 However, a role in plant defense responses is likely. First, it has been reported that the Arabidopsis sirtuin AtSRT2 negatively regulates plant basal defense against the virulent pathogen Pst DC3000, probably via the inhibition of SA biosynthesis. Second, Pst DC3000 infection leads to a SA-dependent decrease of AtSRT2 expression.^107,113 Third, the knockout mutant of AtSRT2 (srt2) has an improved resistance to Pst DC3000 associated with the induction of PR1 while the overexpression of AtSRT2 causes bacterial hyper-susceptibility and impaired PR1 induction. Fourth, transcriptomics revealed that the srt2 mutation induced the expression of SA biosynthesis-related genes (PAD4, EDS5 and SID2) while the same genes were downregulated when overexpressing AtSRT2.^107,113

Concluding Remarks

Since the discovery of NAD at the beginning of the XX^th century, referred to as cozymase,¹¹⁴ considerable advances have been provided, demonstrating key roles of NAD far beyond metabolism. A growing number of studies clearly indicates interactions between PNs homeostasis and plant defense responses, as explained in this review. However, much uncertainty remains on the intricate mechanism of the maintenance of the intracellular balance between NAD(H) and NADP(H). The dynamics and regulation of NAD(P) biosynthesis, degradation and transport are also poorly documented. In the framework of biotic stress responses, important questions remain unanswered: how does the NAD signal itself interplay with the network of signaling pathways? To what extent does NAD concentration influence plant defense response? What are the mechanisms that potentially control PNs production and recycling upon biotic stress? In this review, we have summarized the current knowledge and arguments in favor of NAD involvement in signaling pathways. Still, unclear is the nature of the prevalent actors in NAD-mediated responses, since both oxidative stress and phytohormone signals appear to be involved. Quite critically, several paths downstream have been highlighted recently (Ca²⁺, PARP, ADPr) and thus many other cell signals may interfere with NAD transduction. Despite the growing evidence for a major role of NAD as a crucial metabolite to initiate plant immune responses, it remains possible that NAD signaling itself is simply a modest component of a redundant hub/pathway elicited by other molecules signals (e.g., SA, avirulent factors, ROS, ADPr, etc.). At present, such a situation appears likely since NAD-related resistance to pathogens has been shown to be essentially SA-mediated¹⁸ although many of the effects reviewed here seem to be NAD-specific. Future studies are warranted to examine more carefully NAD sources and targets along plant-pathogens interactions. It is more than plausible that lab and field-based studies on NAD will provide significant advances to improve crop resistance to pathogens and pests.

Glossary

Abbreviations:

ADP

adenosine diphosphate

ADPr

ADP ribose

AMP

adenosine monophosphate

AO

aspartate oxidase

Arabidopsis

Arabidopsis thaliana

ATP

adenosine triphosphate

AvrRpm1/Rpt²⁺

avirulence gene AvrRpm1/Rpt²⁺

Ca²⁺

calcium ions

CaBP

calcium binding protein

cADPR(P)

cyclic ADP ribose (phosphate)

CaMKII

Ca²⁺/calmodulin-dependent kinase II

CEJ1

cooperatively regulated by ethylene and jasmonate

CMSII

cytoplasmic male sterile II mutant

DNA

deoxyribonucleotide acid

EDS1

5, enhanced disease susceptibility 1, 5

efu18

bacterial elicitor

EGTA

ethylene glycol tetra acetic acid

ET

ethylene

ETI

effector-triggered immunity

FIN4

flagellin-insensitive 4

flg22

bacterial elicitor

GFG1

growth factor G1

GLIP1

lipase G1

GSTs

glutathione S-transferases

HDAC

histone deacetylase

HR

hypersensitive response

ICS1 or SID2

isochorismate synthase 1 or SA-induction deficient 2

JA

jasmonic acid

JAZ8

JA ZIM-domain 8

Met

methionine

MetSO

Met sulfoxide

MSR

MetSO reductase

NaAD(P)

nicotinic acid adenine dinucleotide (phosphate)

NAD(P)⁺

oxidized NAD(phosphate)⁺

NAD(P)H

reduced NAD(phosphate)

NADK

NAD kinase

NaMN

nicotinic acid mononucleotide

NaMNAT

NaMN adenylyltransferase

NDT

NAD carrier

NPR1

non-repressor of pathogen-related1

NUDIX

NUDX or NUDT, nucleoside diphosphate linked to X

OE

overexpression

PAD4

phytoalexin deficient 4

PAL

phenylalanine amonialyase

PAMP

pathogen-associated molecular pattern

PAR

poly-ADP-ribosylation

PARG

poly-ADP-ribose glycohydrolase

PARP

poly-ADP-ribose polymerase

PNs

pyridine nucleotides

PXN

peroxisomal NAD carrier

PR

pathogen-related proteins

Psm

Pseudomonas syringae pv. Maculicola

Pst

Pseudomonas syringae pv. tomato

Pst DC3000

virulent Pst

PTI

PAMP-triggered immunity

pv.

pathovar

QPT

quinolinate phosphoribosyltransferase

R gene

resistance gene

RBOH

respiratory burst oxidase homolog

ROS

reactive oxygen species

SA

salicylic acid

SAG

senescence associated gene

SAR

systemic acquired resistance

SRT2

sirtuin 2

TCAP

tricarboxylic acid pathway

WRKY

transcription factors comprising WRKYGQK domain

α-DOX1

alpha-dioxygenase 1

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.