Glutathione Deficiency in Sinorhizobium meliloti Does Not Impair Bacteroid Differentiation But Induces Early Senescence in the Interaction With Medicago truncatula

Abstract

Under nitrogen-limiting conditions, legumes are able to interact symbiotically with bacteria of the Rhizobiaceae family. This interaction gives rise to a new organ, named a root nodule. Root nodules are characterized by an increased glutathione (GSH) and homoglutathione (hGSH) content compared to roots. These low molecular thiols are very important in the biological nitrogen fixation. In order to characterize the modification of nodule activity induced by the microsymbiont glutathione deficiency, physiological, biochemical, and gene expression modifications were analyzed in nodules after the inoculation of Medicago truncatula with the SmgshB mutant of Sinorhizobium meliloti which is deficient in GSH production. The decline in nitrogen fixation efficiency was correlated to the reduction in plant shoot biomass. Flow cytometry analysis showed that SmgshB bacteroids present a higher DNA content than free living bacteria. Live/dead microscopic analysis showed an early bacteroid degradation in SmgshB nodules compared to control nodules which is correlated to a lower bacteroid content at 20 dpi. Finally, the expression of two marker genes involved in nitrogen fixation metabolism, Leghemoglobin and Nodule Cysteine Rich Peptide 001, decreased significantly in mutant nodules at 20 dpi. In contrast, the expression of two marker genes involved in the nodule senescence, Cysteine Protease 6 and Purple Acid Protease, increased significantly in mutant nodules at 10 dpi strengthening the idea that an early senescence process occurs in SmgshB nodules. In conclusion, our results showed that bacterial GSH deficiency does not impair bacterial differentiation but induces an early nodule senescence.

How Rhizobia Adapt to the Nodule Environment

“…Further examples of stress responses include glutathione S-transferases, which were consistently upregulated in bacteroids. Mutants unable to produce glutathione, an important antioxidant molecule, showed reduced symbiotic efficiency (121,122). Similarly, enzymes involved in detoxification of reactive oxygen species, such as superoxide dismutases and catalases, were commonly upregulated in agreement with the presence of reactive oxygen species due to autooxidation of leghemoglobin and ferredoxin (123).…”

Redox Regulation in Diazotrophic Bacteria in Interaction with Plants

“…Furthermore, GSH has been shown to be crucial in nodule functioning. In most cases, gshB mutants produced abnormal nodules with a strong nitrogen-fixation deficiency, correlated with the early senescence of nodules and bacteroids [74][75][76][77][78]. In line with these findings, the recycling of GSH by glutathione reductase was also shown to be involved in nodulation and in nitrogen fixation ability [79].…”

“…vulgaris interaction, GSH deficiency was also associated with increased production of superoxide anion [77]. Regarding the interaction between S. meliloti and M. truncatula, a combination of physiological, and molecular markers, microscopic and flow cytometry analyses, showed that a gshB mutant undergoes complete differentiation before being engaged in an early senescence process [75]. Totally, these results underline the importance of the bacterial GSH/GSSG redox couple for adaptation to the host-plant environment and survival inside the host cells (Table 1).…”

Sinorhizobium melilotifunctions required for resistance to antimicrobial NCR peptides and bacteroid differentiation

“…Many additional S. meliloti mutants are described with symbiotic phenotypes that are suggestive for a similar contribution to NCR resistance. (26,(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53). In addition, other genes that could be important for symbiosis, were discovered in a Tn-seq screen with the peptide NCR247 (25).…”