Orchestrating plant development, metabolism and plant–microbe interactions – NO problem!


4th Plant Nitric Oxide Meeting, in Edinburgh, UK, July 2012

Nitric oxide (NO) has been shown to orchestrate a plethora of physiological functions in mammals, was the subject of the Noble Prize in 1998 and was named ‘molecule of the year’ in 2002 by the journal Science. Despite the big attention this small molecule has garnered within the animal field, the first report of NO generation within biological systems was actually ascribed to plants (Klepper, 1979). Many years later a potential biological function for this molecule was uncovered when it was implicated in plant pathophysiology, first in potato (Noritake et al., 1996) and subsequently in Arabidopsis (Delledonne et al., 1998; Durner et al., 1998). While the study of NO biology in plants has thus been somewhat of a ‘slow-burn’, the field is now gathering significant momentum. Thus, this gaseous free radical has recently begun to be implicated in a variety of processes in plant growth and development (Beligni & Lamattina, 2000; He et al., 2004; Kwon et al., 2012), as well as in biotic and abiotic stress responses (Zhao et al., 2009; Yun et al., 2011). Further, the application of new tools and technologies to this developing field is facilitating studies addressing the molecular mechanisms underpinning the control of key cellular processes by NO (Forrester et al., 2009; Shi et al., 2012). It was against this backdrop that scientists from five continents gathered in Edinburgh from 26 to 27 July 2012, for the fourth international Plant NO meeting, organized by Gary Loake. This symposium focused on three main aspects of NO function: metabolism and signalling; growth and development; and, plant–microbe interactions. All the participants had the opportunity to present their research, give and receive feedback during the poster sessions and raise any issues during the oral presentations. There was also ample opportunity to exchange scientific information and ideas while climbing the adjacent Arthur's Seat (an extinct volcano), or during the imbibing of single malt whiskies and old ales in the neighbouring medieval taverns of Edinburgh's Old Town.

… NO was found to induce the synthesis of glutathione (GSH), a central component involved in regulating the redox balance in plant cells.’

Nitric oxide signalling and metabolism

In animals NO has been shown to play a key role in many important physiological process such as relaxation of vascular smooth muscle, neurotransmission, inflammation and immune function (Ignarro & Buga, 1987; O'Dell et al., 1991; Eiserich et al., 1998). Similarly, NO signalling in plants modulates a variety of physiological systems, from adaptive responses to germination, root growth and dynamics of stomatal aperture control. A significant emerging theme is the regulation of these processes through post-translational protein modifications, mainly metal nitrosylation, S-nitrosylation and tyrosine nitration (Besson-Bard et al., 2008). Metal nitrosylation is characterized by the formation of a NO-metal-containing protein and is best exemplified by the reaction between NO and haemoglobin (Hb), which controls vascular oxygen distribution. Metal nitrosylation also has a well characterized role during the activation of soluble guanylate cyclase (sGC) in animal cells, a classical route for the transfer of NO bioactivity (Bellamy et al., 2002). The conformational changes triggered by the interaction of NO with the heme ferrous iron of sGC increase the production of the second messenger cyclic guanosine monophosphate (cGMP), which regulates many diverse processes. The application of pharmacological agents has suggested the presence of an NO-dependent sGC in tobacco (Durner et al., 1998). However, a sGC that could be a target for metal nitrosylation and a catalyst for the subsequent synthesis of cGMP has not been uncovered to date. Nevertheless, Diana Bellin and Massimo Delledonne (University of Verona, Verona, Italy) are expressing a heterologous sGC in Arabidopsis and are currently exploring a potential role for this enzyme during plant responses to biotic stress. In addition, they have developed new methodology for the detection of cGMP in plants.

Tyrosine (Tyr) nitration is starting to receive more attention from the NO community. In animals, this process is considered a marker for the nitrosative stress and can also compete with phosphorylation for the Tyr residue, thus modulating kinase-dependent cell signalling (Schopfer et al., 2003). In plants, the reaction between peroxynitrite and Tyr is starting to be uncovered. One example is the work presented by Elodie Vandelle and co-workers (University of Verona, Verona, Italy), where they show a role for Tyr nitration in controlling a MAPK cascade and subsequent cell death in Arabidopsis. In addition, protein Tyr nitration has also been implicated in development and senescence of pea (Pisum sativum L.), as presented by Juan Begara-Morales and co-workers (University of Jaén, Granada, Spain). By using immunological and proteomic approaches, the authors showed an age-dependent increase of Tyr nitration in root cells of pea that intensified during senescence.

S-Nitrosylation, the reaction between a protein cysteine (Cys) thiol and NO, to form an S-nitrosothiol (SNO), is chief among NO mediated post-translational modifications. An increasing number of proteins integral to a wide variety of signalling pathways in mammals have been shown to be modified by NO through this process (Hess et al., 2005). Recently, this redox-based post-translational modification has begun to emerge as an important feature of signal transmission systems in plants (Astier et al., 2012; Yu et al., 2012). Reversibility makes this reaction physiologically relevant and this is achieved through de-S-nitrosylation mediated by glutathione (GSH), leading to the formation of S-nitrosoglutathione (GSNO), an endogenous NO donor and reservoir of NO bioactivity. Levels of total cellular GSNO are controlled by GSNO reductase (GSNOR), which catalyses the oxidation of GSNO to glutathaione disulphide (GSSG) and ammonia. Thus, GSNOR indirectly controls total cellular levels of S-nitrosylation by regulating the turnover of GSNO (Feechan et al., 2005).

A detailed structural and biochemical characterization of GSNOR from tomato (Solanum lycopersicum) was presented by Lucie Kubienová and co-workers (Palacký University, Olomouc, Czech Republic). The crystal structures of the apoenzyme and the enzyme in complex with NAD+ and with NADH and glutathione were solved up to 1.9 Å resolution. The results confirmed that the binding of the co-enzyme is associated with the active site zinc movement and changes in its coordination. In comparison to the human enzyme, plant GSNORs exhibit a different composition of the anion binding pocket, which causes decreased affinity to the carboxyl of ω-hydroxy fatty acids. Further, the kinetic parameters of purified GSNOR, including pH and temperature optima, substrate specificity and inhibition properties were also characterized. This approach will certainly help both the plant and animal communities to better understand the biochemical features of this important enzyme and its role in NO biology.

In animals, NO is synthesized by a small family of nitric oxide synthases (NOSs). Although some biological data has indicated the existence of a NOS-like enzyme in higher plants, to date no similar NOS has been found. However, nitrate reductase (NR), has been implicated in NO production in plants. In a reaction dependent on NADP(H), high nitrite concentrations and small oxygen tensions, NR has been shown to reduce nitrite to NO in a low efficiency ratio (Rockel et al., 2002; Meyer et al., 2005). This reaction is modulated by phosphorylation of NR and may be responsible for the basal level of NO production in plants (Yamasaki & Sakihama, 2000; Lea et al., 2004). Christine Stöhr (University Greifswald, Greifswald, Germany) presented work from her laboratory on a nitrite:nitric oxide reductase (NI-NOR) and plasma membrane-bound nitrate reductase (PM-NR). Interestingly, these enzymes were recently shown to resemble the subunit NarH of the membrane-bound nitrate reductase from denitrifying bacteria. After incubation in the presence of NaNO2 and NaNO3, substrates for these two enzymes, it could be demonstrated that plasma membrane-bound NO production is restricted to particular root cells and seems to play a role in mycorrhizal associations. To determine NO production, a new NO-specific fluorescent probe Fluorescent Nitric Oxide Cheletropic Trap (FNOCT) was employed, with encouraging results.

In addition, Rinukshi Wimalasekera (Leibniz University of Hannover, Hannover, Germany) presented some interesting results implicating COPPER AMINE OXIDASE (CuAO1) and a POLYAMINE OXIDASE2 (PAO2) in ABA-mediated NO accumulation. ABA-induced NO production as investigated by fluorometry, showed that T-DNA insertion mutants of CuAO1 and PAO2 are impaired in NO release in comparison to wild-type plants. Fluorescence microscopic observations indicated that ABA-induced NO production in root tips and shoots of these knockout mutants was lower than in wild-type. Loss of CuAO1 function resulted in less sensitivity to ABA than wild-type as indicated by a higher germination rate and longer primary roots when grown in ABA-supplemented media. In contrast, knockout lines of PAO2 showed increased sensitivity to ABA, marked by inhibition of primary root lengths and lateral root numbers relative to wild-type. These results are consistent with a regulatory role for CuAO1 and PAO2 in ABA-mediated NO biosynthesis and imply a potential contribution of CuAO1 and PAO2 in ABA-mediated root growth regulation.

Also investigating NO production, S. Rasul and co-workers (ERL CNRS, France) provided data supporting a fast and long-lasting NR-mediated, Ca2+-dependent NO production triggered by oligogalacturonides (OGs). Interestingly, the authors also showed that L-NAME, a mammalian NOS inhibitor, affected NO production by interfering with NR activity, thus questioning the specificity of this compound in studying NO synthesis in plants.

Nitric oxide and plant–microbe interactions

The production of NO is a conspicuous feature of the plant defence response, underlying the establishment of plant disease resistance. In this context, S-nitrosylation of cysteine thiols is now considered to be a pivotal regulatory mechanism during plant immune function (Malik et al., 2011; Yu et al., 2012).

Manda Yu and co-workers (University of Edinburgh, Edinburgh, UK) provided evidence for the importance of S-nitrosylation in modulating the activity of an ubiquitin E3 ligase induced upon pathogen infection. S-Nitrosylation of this enzyme at a specific Cys blunted E3 ligase activity in vitro, implying NO might regulate the ubiquitination of certain proteins. Interestingly, this data parallels recent findings in humans, where S-nitrosylation of the related E3 ligase, XIAP, inactivates the activity of this protein leading to cell death development in the brain (Nakamura et al., 2010). The absence of both this plant E3 ligase function together with its most closely related paralogue, impacted the defence response, highlighting a role for these related E3 ligase in plant immunity.

The transcriptional cofactor NPR1 is a central regulator of plant defence signalling mediated by the plant immune activator salicylic acid (SA). NPR1 exists either as a multimer linked by intermolecular disulphide bonds, sequestered in the cytoplasm, or a monomer which is able to move to the nucleus and activate gene expression. Previously, NO has been shown to have an important role in NPR1 function by mediating the S-nitrosylation of this co-activator, promoting multimer formation. Thus, NO helps to maintain the monomer-multimer equilibrium of NPR1. Excessive NO accumulation, however, results in NPR1 existing predominantly in the multimeric form, blunting its nuclear transfer and the associated activation of defence gene expression (Tada et al., 2008). Interestingly, in contrast to this previous data, Kovacs and co-workers (Helmholtz Zentrum, München, Germany) presented findings suggesting that NO triggers the nuclear accumulation of NPR1. Further, NO was found to induce the synthesis of glutathione (GSH), a central component involved in regulating the redox balance in plant cells. Studies on nuclear translocation of the GFP-labelled NPR1 in Arabidopsis mutants with impaired GSH biosynthesis indicated that GSNO-induced accumulation of NPR1 in the nucleus is inhibited in these plants. These data imply possible cross talk between GSNO/NO and GSH, which might be integral to the NPR1-dependent defence signalling pathway.

While NO has been established as a key signalling cue in plant immunity, an important role for this redox molecule during the development of symbiotic interactions has only recently begun to emerge (Meilhoc et al., 2011). Within this landscape, Renaud Brouquisse and co-workers (University of Nice, Nice, France) presented data showing that NO is produced during the early stages of symbiotic interactions between Medicago truncatula and Sinorhizobium meliloti. Further, depletion of this molecule induced nodule formation. Also, it was proposed that the presence of a nitrate-NO respiration process in nodules could maintain the energy status required during nitrogen fixation under low oxygen conditions. Continuing the theme of symbiotic interactions between Medicago truncatula and Sinorhizobium meliloti a transcriptomic analysis by Alexandre Boscari and co-workers (University of Nice, Nice, France) compared the gene expression profile during the symbiotic process of roots in the presence or absence of the NO-scavenger cPTIO. They showed that cPTIO inhibits the up-regulation of genes related to the symbiotic process, including transcription factors implicated in nodule development.

Future studies

Clearly, the source(s) of NO synthesis during plant–microbe interactions and plant growth and development demand serious attention. Is there an enzyme equivalent to an animal NOS in higher plants? Or as seems more likely, does NO accrual occur through the action of one or more of the enzyme systems that have already been identified? Are these sources additive and/or functionally redundant? How might they be regulated? What are the major routes for the transfer of NO bioactivity in plant cells? It will also be important to uncover and catalogue the complete infantry of NO targets. Further, how does NO modulate the function of these proteins? What is the full spectrum of the redox continuum in plant cells? Another important issue is how the information flow through the assumed myriad of NO target proteins is integrated to generate the desired physiological output? Finally, as our depth of appreciation of NO-based redox signalling increases, how can we best exploit this knowledge base to aid the rationale design of crop plants for improved performance?

In keeping with the Scottish theme of this conference, there is a frighteningly steep and winding road in the Scottish Highlands with breathtakingly vertiginous views, not too far from Edinburgh. As your vehicle crawls gradually towards the summit with gears grinding, crying out for relief, there is a small lay-by called ‘stop and be thankful’ where you can pull over for a while, look back, appreciate the journey you have made, admire the landscape stretched out far below and prepare for the next leg of the arduous climb towards the summit. The 4th Plant NO conference was a place where the community could ‘stop and be thank full’, both looking back over the progress we have made during the last 30 yr and preparing ourselves for the long and challenging road ahead.