From the *Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia; and †Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), Victoria, Australia.
Received March 6, 2013.
Accepted for publication March 10, 2013.
Reprints: Chris Gehring, Division of Biological and Environmental Science and Engineering, Building 2, Room 4222, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia. E-mail: christoph.gehring@KAUST.edu.sa.
Supported, in part, by a grant from the National Center for Research Resources (R13 RR023236).
First indications that plants might also have a natriuretic peptide (NP) signaling system came from immunoassays on tissue extracts from Florida beauty (Dracena godseffiana).1 In this study, antibodies against the N-terminus (the atrial natriuretic peptide [ANP] prohormone, 1–98), midportion (ANP, 31–67), and the C-terminus (ANP, 99–126) of ANP prohormone detected molecules in leaves and stems. A follow-up study presented further immunologic evidence for NP-like proteins in several other plant classes including the Euglenophyceae, and data from high performance gel permeation chromatography also predicted that pro-NPs and NPs from plants may share similar molecular masses with vertebrate ANPs.2 It was also reported that the rate of transpiration, solute flow, and solute uptake in carnation and chrysanthemum was rapidly and significantly increased after exogenous application of synthetic human ANP.2 Subsequently, it was demonstrated that synthetic rat ANP can induce stomatal opening in Tradescantia species in a concentration dependent manner.3,4 It was noted that, unlike animal systems, Na+ was not required in the medium for this response, indicating that effects other than on Na+ ion movements are essential for this effect. Additional evidence for specific plant NP (PNP) receptor-ligand interactions came from competitive in vitro binding assays where isolated leaf membranes were incubated with radiolabeled rat (3-[125I]iodotyrosol28) ANP, and results showed that increasing concentrations of unlabelled rat ANP effectively displaced unlabeled ligand.3 Much like in the vertebrate NP response, the second messenger cyclic guanosine 3′,5′-monophosphate (cGMP) also appeared to have an essential role in plant signal transduction.5,6
ISOLATION AND PHYSIOLOGIC CHARACTERIZATION OF A PNP IMMUNOANALOG
In the late 90s, we extracted and then purified by immunoaffinity chromatography biologically active PNP immunoanalogs from a number of different species including ivy and potato7,8 and showed that exogenous application caused stomatal opening and activation of the membrane H+-ATPase.9 Moreover, the immunoanalog was shown to rapidly and specifically induce transient elevation of cGMP levels in the conductive stele tissue of maize roots10 and stomatal guard cell protoplasts6 and to enhance osmoticum-dependent volume changes in isolated leaf mesophyll protoplasts.11 The immunoreactants also modulated ion fluxes across plant membranes leading to a rapid (<60 seconds) net influx of H+ and a delayed (>20 minutes) net influx of K+ and Na+ in maize stele tissue.12 These physiologic effects are entirely consistent with in situ hybridization results that localize molecules recognized by anti-ANP in the conductive tissue of plants.13
ARABIDOPSIS THALIANA PNP—STRUCTURE AND EVOLUTION
The limited amino acid sequence information from the N-terminal fragments of potato immunoanalogs obtained by Edman sequencing proved sufficient to identify A. thaliana orthologs.8,14Arabidopsis thaliana PNP (AtPNP-A) is a small protein of 126 amino acids (molecular weight: 14.016 kilo Dalton; isoelectric point: 9.22) that is encoded by a gene with a single intron of 100 bp (accession no.: AT2G18660). The protein contains a 24-amino-acid signal peptide (MW: 2249; Fig. 1). AtPNP-A and its related sequence AtPNP-B have orthologs in many species, both monocots and dicots as well as moss. AtPNP-A is distantly related to the cell wall loosening expansins15 (Fig. 2). Expansins in turn are distantly related to glucanases and cellulases, both hydrolytic enzymes that use the carbohydrates of cell walls as substrates. The C-termini of the glucanases and cellulases have been suggested to bind the carbohydrate scaffold of the cell wall.16 Because PNPs do not contain the C-terminus, unlike the expansisn and the ancestral glucanases and cellulases, it is reasonable to argue that PNPs have lost this domain. This is in keeping with the fact that the domain is delineated by an intron-exon border. Loss of the wall-binding domain is likely to result in the increased mobility of PNPs, a precondition for the observed systemic actions of PNPs.17–19 The hypothesis is also entirely compatible with the physiologic observations in protoplasts where the cell walls have been removed for experimental studies.
Perhaps astonishingly, the bacterial citrus biotrophic pathogen Xanthomonas axonopodis pv. citri str. 306, but not Xanthomonas campestris, also contains a gene encoding a PNP–like protein,20 and we found that the X. axonopodis PNP–like protein shares significant sequence similarity and an identical domain organization with PNP.20 We also noted a significant excess of conserved residues within the domain identified as being necessary and sufficient to induce biological activity.21 At the time of this discovery, no significant similarity between the X. axonopodis PNP–like protein (XacPNP) and other bacterial proteins from GenBank (2004) was found, and given that the XacPNP and AtPNP-A also share their domain organization, this incongruent phylogeny suggested that the bacteria may have acquired the plant molecule in an ancient lateral gene transfer event.20 It was speculated that the pathogen uses the plant-like protein during host infection to signal to its host or to modulate host homeostasis to its own advantage.20 If so, this would be a case of molecular mimicry.
PLANT NP AND THE HOST–PATHOGEN INTERACTIONS
A systems level transcriptomics analysis22 of AtPNP-A has indeed implicated PNP in Arabidopsis stress responses and in particular responses to biotic stresses.23 With this in mind, we tested the role of XacPNP, particularly during host infection. First, we showed that recombinant XacPNP, much like At PNP-A, does elicit physiologic responses (eg, the modulation of photosynthesis) in plants.24 Second, we also demonstrated that XacPNP is not induced when the bacteria are grown in a rich medium but highly induced in a medium that mimics the ionic and osmotic conditions in planta. Third, we showed that a XacPNP deletion mutant causes lesions on the host that were more necrotic than those observed with the wild type and that bacterial cell death occurs earlier in the mutant.24 This clearly indicates that release of XacPNP mimics host PNP in situ and results in a suppressed host response and consequently better survival of the biotrophic pathogen in the lesions, much to the detriment of the host.25,26 Given the role of PNP in the regulation of plant homeostasis, it was also proposed that X. axonopodis pv. citri host interactions can serve as a model system for the study of the role of host homeostasis in plant defense against biotrophic pathogens.
There has been significant progress in our understanding of the molecular mechanisms that govern plant-pathogen interactions. One aspect is that fungal plant pathogens secrete effector molecules to cause disease in their hosts, whereas the host plants use immune receptors to try to intercept these effectors. For example, in tomato, the immune receptor Ve1 governs resistance to race 1 strains of the soil-borne vascular wilt fungi Verticillium dahliae and Verticillium alboatrum, whereas the corresponding Verticillium effector has until recently remained elusive. High-throughput population genome sequencing has identified Ave1 (for Avirulence on Ve1 tomato), and functional analyses have established Ave1 as ligand of the Ve1 receptor.27 Remarkably, Ave1 is a homolog of PNP, and recent searches of the increasing genomic data have shown that PNPs are also present in the plant pathogenic fungi Colletotrichum higginsianum, Cercospora beticola, and Fusarium oxysporum f. sp. Lycopersici.27 It was also reported that transient expression of the Ave1 homologs from F. oxysporum and C. beticola can activate Ve1-mediated resistance,27 and Ve1 mediates resistance to F. oxysporum in tomato, thus showing that the receptor has a key role in resistance against multiple fungal pathogens.
PLANT NP EFFECTS IN VERTEBRATES
To date, we are still awaiting extensive and detailed investigations of the physiologic effects of PNP and bacterial or fungal PNP-like molecules in animal systems. It will be particularly interesting to learn if PNPs are indeed exerting natriuretic effects in vertebrates and if some natriuretic effects induced by plants are due to the presence and abundance of their endogenous PNPs. Interestingly, recombinant AtPNP-A has been shown to induce apoptosis in a dose-dependent manner in several cell lines. Rat cardiac myoblasts (H9c2 cells) are more susceptible to the apoptosis-promoting effects of PNP and ANP than HEK-293T cells where PNP had a protective effect at lower concentrations.28 Similarly, rat thoracic myoblasts (A-10) were less responsive to both PNP and ANP than the H9c2 cells. Because PNP is eliciting similar effects to ANP in this instance, it has been speculated that PNP may prove to be a useful lead molecule for developing novel therapeutic peptides.28
An increasing amount of evidence suggests that PNPs are a key systemic regulator of plant homeostasis and defense against abiotic and biotic stress in plants. We are beginning to understand the molecular mechanism(s) that govern PNP interactions with plant PNP receptors and downstream signaling events. Progress is also being made at the systems level, where AtPNP-A –dependent transcriptome and (phosphor-)proteome data suggest that these peptidic signaling molecules elicit complex cellular responses including the regulation cellular homeostasis and photosynthesis. The study of PNP-dependent responses during host-pathogen interactions also affords insights into how plants use metabolic changes as a means of the host to defend itself against attacks from biotrophic pathogens, for example, through starving them of nutrients or by osmotic shock. Finally, it can be conceivable that the study of PNP responses in vertebrates will afford new insights into the complex role of this peptide signaling system.
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