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Nitric oxide (NO) is involved in diverse physiological processes in plants, including growth, development, response to pathogens, and interactions with beneficial microorganisms. In this work, a dedicated microarray representing the widest database available of NO-related transcripts in plants has been produced with 999 genes identified by a cDNA amplified fragment length polymorphism analysis as modulated in Medicago truncatula roots treated with two NO donors. The microarray then was used to monitor the expression of NO-responsive genes in M. truncatula during the incompatible interaction with the foliar pathogen Colletotrichum trifolii race 1 and during the symbiotic interaction with Sinorhizobium meliloti 1021. A wide modulation of NO-related genes has been detected during the hypersensitive reaction or during nodule formation and is discussed with special emphasis on the physiological relevance of these genes in the context of the two biotic interactions. This work clearly shows that NO-responsive genes behave differently depending on the plant organ and on the type of interaction, strengthening the need to consider regulatory networks, including different signaling molecules.

Nitric oxide (NO) has recently gained interest as a major signaling molecule during plant development and response to environmental cues. Its role is particularly crucial for plant-pathogen interactions, during which it participates in the control of plant defense response and resistance. Indication for the presence of NO during symbiotic interactions has also been reported. Here, we defined when and where NO is produced during Medicago truncatula-Sinorhizobium meliloti symbiosis. Using the NO-specific fluorescent probe 4,5-diaminofluorescein diacetate, NO production was detected by confocal microscopy in functional nodules. NO production was localized in the bacteroid-containing cells of the nodule fixation zone. The infection of Medicago roots with bacterial strains impaired in nitrogenase or nitrite reductase activities lead to the formation of nodules with an unaffected NO level, indicating that neither nitrogen fixation nor denitrification pathways are required for NO production. On the other hand, the NO synthase inhibitor N-methyl-L-arginine impaired NO detection, suggesting that a NO synthase may participate to NO production in nodules. These data indicate that a NO production occurs in functional nodules. The location of such a production in fully metabolically active cells raises the hypothesis of a new function for NO during this interaction unrelated to defense and cell-death activation.

Legumes form a symbiotic interaction with bacteria of the Rhizobiaceae family toproduce nitrogen-fixing root nodules under nitrogen-limiting conditions. This process involves the recognition of the bacterial Nod factors by the plant which mediates the entry of the bacteria into the root and nodule organogenesis. We have examined the importance of the low molecular weight thiols, glutathione (GSH) and homoglutathione (hGSH), during the nodulation process in the model legume Medicago truncatula. Using both buthionine sulfoximine, a specific inhibitor of GSH and hGSH synthesis, and transgenic roots expressing GSH synthetase and hGSH synthetase in an antisense orientation, we showed that deficiency in GSH and hGSH synthesis inhibited the formation of the root nodules. This inhibition was not correlated to a modification in the number of infection events or to a change in the expression of the Rhizobium sp.-induced peroxidase rip1, indicating that the low level of GSH or hGSH did not alter the first steps of the infection process. In contrast, a strong diminution in the number of nascent nodules and in the expression of the early nodulin genes, Mtenod12 and Mtenod40, were observed in GSHand hGSH-depleted plants. In conclusion, GSH and hGSH appear to be essential for proper development of the root nodules during the symbiotic interaction.

Sinorhizobium meliloti possesses three distinct catalases to cope with oxidative stress: two monofunctional catalases (KatA and KatC) and one bifunctional catalase-peroxydase (KatB). The katB gene is constitutively expressed during growth in batch culture and is not induced under oxidative stress conditions. In contrast, the expression of katA and katC genes is mainly regulated at the transcription level in these conditions. A differential expression of kat genes was observed during the development of the nodule. A high expression of katA gene was detected in bacteroids, suggesting that the nitrogen-fixation process induces a strong oxidative stress. In contrast, bacteria express katB and katC genes and not the H2O2-inducible katA gene in infection threads despite the detection of H2O2 around the bacteria. A katB katC double mutant nodulated poorly and displayed abnormal infection. After nonefficient release into plant cells, bacteria failed to differentiate into bacteroids and rapidly underwent senescence. Our results indicate that these two catalases are essential for the establishment of the symbiosis. They also suggest that the bacteria are in a nonexponential growth phase in infection threads and corroborate previous studies on the growth rate of bacteria inside the plant.

Reactive oxygen species are produced as an early event in plant defense response against avirulent pathogens. We show here that alfalfa responds to infection with Sinorhizobium meliloti by production of superoxide and hydrogen peroxide. This similarity in the early response to infection by pathogenic and symbiotic bacteria addresses the question of which mechanism rhizobia use to counteract the plant defense response.