article addendum
article addendum
Plant Signaling & Behavior 5:12, 1679-1681; December 2010; © 2010 Landes Bioscience
Endogenous NO levels regulate nodule functioning Potential role of cGMP in nodule functioning? Marshall Keyster, Ashwil Klein and Ndiko Ludidi* Institute for Plant Biotechnology; Stellenbosch University; Matieland, South Africa
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Key words: cyclic guanosine monophosphate, nitric oxide synthase, nitric oxide, nitrogen fixation, nodulation efficiency, nodule functioning, reactive oxygen species Abbreviations: ANOVA, analysis of variance; cGMP, cyclic guanosine monophosphate; sGC, soluble guanylate cyclise; L-NNA, Nω -nitro-L-arginine; NO, nitric oxide; NOS, nitric oxide synthase; ROS, reactive oxygen species Submitted: 10/28/10 Accepted: 10/28/10 Previously published online: www.landesbioscience.com/journals/psb/ article/14041 DOI: 10.4161/psb.5.12.14041 *Correspondence to: Ndiko Ludidi; Email:
[email protected] Addendum to: Leach J, Keyster M, du Plessis M, Ludidi N. Nitric oxide synthase activity is required for development of functional nodules in soybean. J Plant Physiol 2010; 167:1584–91; PMID: 20709426; DOI:10.1016/j.jplph.2010.06.019.
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itric oxide is a small gaseous signaling molecule which functions in the regulation of plant development and responses to biotic and abiotic stresses. Recently, we have shown that nitric oxide is required for development of functional nodules. Here, we show that inhibition of nitric oxide synthase enzymatic activity (using Nω -nitro-L-arginine) reduces nitric oxide content in soybean root nodules and this is coupled by reduction of endogenous cyclic guanosine monophosphate content in the nodules. We postulate that the regulation of soybean nodule development by nitric oxide is transduced via cyclic guanosine monophosphate through activation of nitric oxide-responsive soluble guanylate cyclase. Furthermore, we hypothesize that this signaling cascade is mediated via modulation of the activities of antioxidant metabolic pathways. Introduction
Treatment of soybean nodules with the nitric oxide synthase (NOS) inhibitor Nω -nitro-L-arginine (L-NNA) inhibits nodulation intensity and nodule development if applied before inoculation of soybean seedlings with Bradyrhizobium japonicum and such inhibition is sustained by repeated treatment with L-NNA.1 It is widely accepted that cyclic guanosine monophosphate (cGMP) exists in plants2-5 and there are several reports providing evidence for the existence of genes encoding proteins with guanylyl cyclase activity.6-9 However, very scant reports on cGMP content in legume nodules exist10,11 and the role of cGMP in regulation of
nodulation/nodule functioning is not yet known. It is also known that exogenously applied nitric oxide (NO) can raise cGMP content in plant cells.12 We present data demonstrating that treatment of nodulated plants with L-NNA reduces both the endogenous NO and cGMP content in the nodules. Furthermore, we show that endogenous NO emanating from NOS enzymatic activity regulates cGMP content in root nodules. Nitric Oxide Synthase Inhibitor Reduces NO Content in Root Nodules: Alteration in cGMP Content and Nitrogen Fixation The existence of NOS enzymatic activity has been reported for various legume species13,14 but the enzyme responsible for this activity has not yet been identified. We inoculated soybean seeds with Bradyrhizobium japonicum and germinated them as previously described in reference 1. When the seedlings were at the V2 stage of vegetative growth, we treated them every three days with nutrient solution1 only or nutrient solution supplemented with the following: 1 mM L-NNA or 200 μM DETA/NO or a combination of 1 mM L-NNA and 200 μM DETA/NO. The treatments were continued until the plants reached the V5 stage of vegetative growth (21 days after the initial treatment) and repeated once again at 3 hours before any assays were performed. At this stage, the plants were used to measure root nodule NO-based pixel intensities estimated as the level of fluorescence of DAF-2T on a Zeiss LSM 510 Meta Confocal
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Figure 1. (A) NO and (B) cGMP content in soybean nodules in response to 1 mM L-NNA, 200 μM DETA/NO and 1 mM L-NNA + 200 μM DETA/NO. Inhibition in both NO and cGMP content was observed upon treatment with 1 mM L-NNA. Increases in both NO and cGMP content were observed in response to 200 μM DETA/NO. Error bar values represent the mean (±SE; n = 3) and the data are representative of three independent experiments.
Laser Scanning Microscope15 using the AlphaEase FC imaging software (Alpha Innotech Corporation, USA) and as NO concentration measured via a hemoglobin-based assay.16 Furthermore, these plants were used to measure root nodule cGMP content using the cGMP Enzyme Immunoassay Kit according to the manufacturer’s instruction (SigmaAldrich, St. Louis, MO). One-way analysis of variance (ANOVA) test was used to evaluate all data, with means compared by the Tukey-Kramer test at 5% level of significance, using GraphPad Prism 5.03 software. Application of 1 mM L-NNA reduced NO content in soybean nodules, whereas application of 200 μM DETA/NO increased nitric oxide content in comparison to the controls supplied only with nutrient solution (Figs. 1A and 2). Evidence for a role of NOS in regulating cGMP content in root nodules was provided by the observation that inhibition of NOS enzymatic activity upon application of 1 mM L-NNA reduced cGMP content in soybean nodules by ±42%, whereas application of 200 μM DETA/NO increased cGMP content by ±52% in comparison to the controls supplied only with nutrient solution (Fig. 1B). The role of cGMP in nodulation/nodule functioning has not been studied widely except for a few contradicting reports in which cGMP appears to support nodule functioning on the one hand10,11 and suppress nitrogen fixation on the other hand.17,18 How could NOS Activity Regulate Nodule Functioning?
Figure 2. NO-based pixel intensities in soybean nodules in response to 1 mM L-NNA, 200 μM DETA/NO and 1 mM L-NNA + 200 μM DETA/NO. Inhibition of NO-based pixel intensity was observed in response to 1 mM L-NNA. DETA/NO (200 μM) caused an increase in NO-based pixel intensity in soybean root nodules. Error bars represent the mean (±SE; n = 3) from data that are representative of three independent experiments. 1680
Plant Signaling & Behavior
Amongst several possibilities, it is likely that nodule development/functioning is maintained via regulation of the levels of nodule reactive oxygen species (ROS) such as superoxide (O2-) and hydrogen peroxide (H2O2). It is known that NO can regulate ROS levels by modulating the activities of antioxidant enzymes, both in response to biotic and abiotic stresses in plants.19-21 The fact that changes in ROS levels occur upon infection of legume roots with rhizobia and the ROS fluxes are constantly regulated during nodule development and functioning22,23 means
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that the plant antioxidant system must be crucial in maintaining nodule functioning. If NOS enzymatic activity truly plays a role in modulating antioxidant enzyme activity during nodule development and in nodule functioning, then it follows that inhibition of NOS enzymatic activity would lead to dysfunction in antioxidant enzymatic activity. The result of such dysfunction would be accumulation of ROS to levels that would trigger nodule cell death (thus loss of nodule cell viability), possibly via cysteine endopeptidase enzymatic activity since these enzymes regulate programmed cell death.24,25 The fact that we previously presented evidence for a role of NOS activity in regulating cysteine endopeptidase enzymatic activity1 supports such hypothesis but needs to be substantiated with evidence showing a role of NOS enzymatic activity in regulating some of the plant antioxidant enzymatic activities and ROS accumulation. It remains to be studied if such redox systems and pathways for cell viability maintenance are regulated by cGMP and how soluble guanylate cyclase, which is regulated by NO, affects nodule development and functioning. Acknowledgements
This work was supported by Stellenbosch University and the National Research Foundation (South Africa).
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