Eric Boncompagni

Both Plant and Bacterial Nitrate Reductases Contribute to Nitric Oxide Production in Medicago truncatula Nitrogen-Fixing Nodules

Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms. In the model legume Medicago truncatula, NO production has been detected in the nitrogen fixation zone of the nodule, but the systems responsible for its synthesis are yet unknown and its role in symbiosis is far from being elucidated. In this work, using pharmacological and genetic approaches, we explored the enzymatic source of NO production in M. truncatula-Sinorhizobium meliloti nodules under normoxic and hypoxic conditions. When transferred from normoxia to hypoxia, nodule NO production was rapidly increased, indicating that NO production capacity is present in functioning nodules and may be promptly up-regulated in response to decreased oxygen availability. Contrary to roots and leaves, nodule NO production was stimulated by nitrate and nitrite and inhibited by tungstate, a nitrate reductase inhibitor. Nodules obtained with either plant nitrate reductase RNA interference double knockdown (MtNR1/2) or bacterial nitrate reductase-deficient (napA) and nitrite reductase-deficient (nirK) mutants, or both, exhibited reduced nitrate or nitrite reductase activities and NO production levels. Moreover, NO production in nodules was found to be inhibited by electron transfer chain inhibitors, and nodule energy state (ATP-ADP ratio) was significantly reduced when nodules were incubated in the presence of tungstate. Our data indicate that both plant and bacterial nitrate reductase and electron transfer chains are involved in NO synthesis. We propose the existence of a nitrate-NO respiration process in nodules that could play a role in the maintenance of the energy status required for nitrogen fixation under oxygen-limiting conditions.

Identification of new up-regulated genes under drought stress in soybean nodules

Legumes/rhizobium biological N(2) fixation (BNF) is dramatically affected under abiotic stress such as drought, salt, cold and heavy metal stresses. Nodule response to drought stress at the molecular level was analysed using soybean (Glycine max) and Bradyrhizobium japonicum as a model, since this symbiotic partnership is extremely sensitive to this stress. To gain insight into molecular mechanisms involved in drought-induced BNF inhibition, we have constructed a SSH (Suppression Subtractive Hybridisation) cDNA library from nodular tissue of plants irrigated at field capacity or plants water deprived for 5 days. Sequence analysis of the first set of 128 non redundant ESTs using protein databases and the Blastx program indicated that 70% of ESTs could be classified into putative known functions. Using reverse northern hybridization, 56 ESTs were validated as up-regulated genes in response to drought. Interestingly, only a few of them had been previously described as involved in plant response to drought, therefore most of the ESTs could be considered as new markers of drought stress. Here we discuss the potential role of some of these up-regulated genes in response to drought. Our analysis focused on two genes, encoding respectively a ferritin and a metallothionein, which are known to be involved in homeostasis and detoxification of metals and in response to oxidative stress. Their spatiotemporal expression patterns showed a high accumulation of transcripts restricted to infected cells of nodules in response to drought.

Nitric oxide (NO): a key player in the senescence of Medicago truncatula root nodules

Nitric oxide (NO) is a signalling and defence molecule involved in diverse plant developmental processes, as well as in the plant response to pathogens. NO has also been detected at different steps of the symbiosis between legumes and rhizobia. NO is required for an optimal establishment of the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction, but little is known about the role of NO in mature nodules. Here, we investigate the role of NO in the late steps of symbiosis. Genetic and pharmacological approaches were conducted to modulate the NO level inside root nodules, and their effects on nitrogen fixation and root nodule senescence were monitored. An increase in endogenous NO levels led to a decrease in nitrogen fixation and early nodule senescence, characterized by cytological modifications of the nodule structure and the early expression of a specific senescence marker. By contrast, a decrease in NO levels led to a delay in nodule senescence. Together, our results strongly suggest that NO is a signal in developmental as well as stress-induced nodule senescence. In addition, this work demonstrates the pivotal role of the bacterial NO detoxification response in the prevention of early nodule senescence, and hence the maintenance of efficient symbiosis.

Characterization of a Sinorhizobium meliloti ATP-binding cassette histidine transporter also involved in betaine and proline uptake.

The symbiotic soil bacterium Sinorhizobium meliloti uses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli (ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S. meliloti. This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine was used as a nitrogen source, a 30% inhibition of growth was observed in hut mutants (hutX and hutH2). Expression analysis of the hut operon determined using a hutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.

Crucial role of (homo)glutathione in nitrogen fixation in Medicago truncatula nodules

Legumes form a symbiotic interaction with bacteria of the Rhizobiaceae family to produce nitrogen-fixing root nodules under nitrogen-limiting conditions. We examined the importance of glutathione (GSH) and homoglutathione (hGSH) during the nitrogen fixation process. Spatial patterns of the expression of the genes involved in the biosynthesis of both thiols were studied using promoter-GUS fusion analysis. Genetic approaches using the nodule nitrogen-fixing zone-specific nodule cysteine rich (NCR001) promoter were employed to determine the importance of (h)GSH in biological nitrogen fixation (BNF). The (h)GSH synthesis genes showed a tissue-specific expression pattern in the nodule. Down-regulation of the γ-glutamylcysteine synthetase (γECS) gene by RNA interference resulted in significantly lower BNF associated with a significant reduction in the expression of the leghemoglobin and thioredoxin S1 genes. Moreover, this lower (h)GSH content was correlated with a reduction in the nodule size. Conversely, γECS overexpression resulted in an elevated GSH content which was correlated with increased BNF and significantly higher expression of the sucrose synthase-1 and leghemoglobin genes. Taken together, these data show that the plant (h)GSH content of the nodule nitrogen-fixing zone modulates the efficiency of the BNF process, demonstrating their important role in the regulation of this process.

The Sinorhizobium meliloti glycine betaine biosynthetic genes (betICBA) are induced by choline and highly expressed in bacteroids

The symbiotic soil bacterium Sinorhizobium meliloti has the capacity to synthesize the osmoprotectant glycine betaine from choline-O-sulfate and choline. This pathway is encoded by the betICBA locus, which comprises a regulatory gene, betI, and three structural genes, betC (choline sulfatase), betB (betaine aldehyde dehydrogenase), and betA (choline dehydrogenase). Here, we report that betICBA genes constitute a single operon, despite the existence of intergenic regions containing mosaic elements between betI and betC, and betB and betA. The regulation of the bet operon was investigated by using transcriptional lacZ (beta-galactosidase) fusions and has revealed a strong induction by choline at concentrations as low as 25 microM and to a lesser extent by choline-O-sulfate and acetylcholine but not by osmotic stress or oxygen. BetI is a repressor of the bet transcription in the absence of choline, and a nucleotide sequence of dyad symmetry upstream of betI was identified as a putative betI box. Measurements of intracellular pools of choline, well correlated with beta-galactosidase activities, strongly suggested that BetI senses the endogenous choline pool that modulates the intensity of BetI repression. In contrast to Escherichia coli, BetI did not repress choline transport. During symbiosis with Medicago sativa, S. meliloti bet gene expression was observed within the infection threads, in young and in mature nodules. The existence of free choline in nodule cytosol, peribacteroid space, and bacteroids was demonstrated, and the data suggest that bet regulation in planta is mediated by BetI repression, as in free-living cells. Neither Nod nor Fix phenotypes were significantly impaired in a betI::omega mutant, indicating that glycine betaine biosynthesis from choline is not crucial for nodulation and nitrogen fixation.

Peribacteroid space acidification: a marker of mature bacteroid functioning in Medicago truncatula nodules.

Legumes form a symbiotic interaction with Rhizobiaceae bacteria, which differentiate into nitrogen-fixing bacteroids within nodules. Here, we investigated in vivo the pH of the peribacteroid space (PBS) surrounding the bacteroid and pH variation throughout symbiosis. In vivo confocal microscopy investigations, using acidotropic probes, demonstrated the acidic state of the PBS. In planta analysis of nodule senescence induced by distinct biological processes drastically increased PBS pH in the N2 -fixing zone (zone III). Therefore, the PBS acidification observed in mature bacteroids can be considered as a marker of bacteroid N2 fixation. Using a pH-sensitive ratiometric probe, PBS pH was measured in vivo during the whole symbiotic process. We showed a progressive acidification of the PBS from the bacteroid release up to the onset of N2 fixation. Genetic and pharmacological approaches were conducted and led to disruption of the PBS acidification. Altogether, our findings shed light on the role of PBS pH of mature bacteroids in nodule functioning, providing new tools to monitor in vivo bacteroid physiology.

Involvement of papain and legumain proteinase in the senescence process of Medicago truncatula nodules

The symbiotic interaction between legumes and Rhizobiaceae leads to the formation of new root organs called nodules. Within the nodule, Rhizobiaceae differentiate into nitrogen-fixing bacteroids. However, this symbiotic interaction is time-limited as a result of the initiation of a senescence process, leading to a complete degradation of bacteroids and host plant cells. The increase in proteolytic activity is one of the key features of this process. In this study, we analysed the involvement of two different classes of cysteine proteinases, MtCP6 and MtVPE, in the senescence process of Medicago truncatula nodules. Spatiotemporal expression of MtCP6 and MtVPE was investigated using promoter- β-glucuronidase fusions. Corresponding gene inductions were observed during both developmental and stress-induced nodule senescence. Both MtCP6 and MtVPE proteolytic activities were increased during stress-induced senescence. Down-regulation of both proteinases mediated by RNAi in the senescence zone delayed nodule senescence and increased nitrogen fixation, while their early expression promoted nodule senescence. Using green fluorescent protein fusions, in vivo confocal imaging showed that both proteinases accumulated in the vacuole of uninfected cells or the symbiosomes of infected cells. These data enlighten the crucial role of MtCP6 and MtVPE in the onset of nodule senescence.

MtZR1, a PRAF protein, is involved in the development of roots and symbiotic root nodules in Medicago truncatula.

PRAF proteins are present in all plants, but their functions remain unclear. We investigated the role of one member of the PRAF family, MtZR1, on the development of roots and nitrogen-fixing nodules in Medicago truncatula. We found that MtZR1 was expressed in all M. truncatula organs. Spatiotemporal analysis showed that MtZR1 expression in M. truncatula roots was mostly limited to the root meristem and the vascular bundles of mature nodules. MtZR1 expression in root nodules was down-regulated in response to various abiotic stresses known to affect nitrogen fixation efficiency. The down-regulation of MtZR1 expression by RNA interference in transgenic roots decreased root growth and impaired nodule development and function. MtZR1 overexpression resulted in longer roots and significant changes to nodule development. Our data thus indicate that MtZR1 is involved in the development of roots and nodules. To our knowledge, this work provides the first in vivo experimental evidence of a biological role for a typical PRAF protein in plants.

Synthesis and Roles of Glutathione and Homoglutathione in the Nitrogen-Fixing Symbiosis

Glutathione (GSH) is a major antioxidant molecule in plants. It is involved in regulating plant development and responses to abiotic and biotic environment changes. In leguminous plants, a GSH homolog, homoglutathione is also found. Most legumes can develop a symbiotic interaction with soil bacteria of the rhizobium family under nitrogen deficiency. This symbiosis allows the reduction of atmospheric nitrogen by the bacteria in plant organs called root nodules. In this chapter, we summarize studies that describe the synthesis and the roles of GSH and hGSH in the nitrogen-fixing symbiosis.