Trehalose and the nitrogen fixing nodule symbiosis of legumes : studies on rhizobia deficient in the trehalose-6-phosphate synthase gene "ots"A

The non-reducing disaccharide trehalose (a-D-glucopyranosyl-1,1-a-D-glucopyranoside) is widespread in nature, but is normally not present in higher plants. With respect to plant-microbe interactions, it is interesting that trehalose is regularly found in plant roots interacting with antagonistic fungi, mycorrhizal fungi, and in nitrogen-fixing root nodules, probably as a microbial substance. The impact of trehalose on plant metabolism and its role in nitrogen fixing symbiosis is unclear. This work focuses on the nodule symbiosis. It represents a genetic approach to study the role of trehalose synthesis by the microsymbiont. One pathway for trehalose synthesis is the OtsA/B pathway. Trehalose is synthesized from UDP-glucose and glucose-6-phosphate in a two-step process by the action of trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB). Homologues of the genes coding for these two enzymes in Escherichia coli, otsA and otsB, have been localized on the symbiotic plasmid of Rhizobium sp. NGR234 (pNGR234a). To study the significance of rhizobial trehalose synthesis in free living and symbiotic rhizobia, an Ω-cassette was inserted into the otsA homologue. Phenotypically, the deletion of the rhizobial otsA-homologue strongly reduced trehalose synthesis under microaerobic growth conditions. Thus, there are strong indications that the rhizobial trehalose synthesis induced under hypoxic conditions is directed by the symbiotic plasmid encoded otsA-homologue in conjunction with otsB. The functionality of otsA has therefore indirectly been demonstrated in Rhizobium sp. NGR234, which is the first time in aproteobacteria in general. In addition, the induction of otsA and its homologues by low oxygen conditions has not been previously reported. The natural environment inside nodules is characterized by low oxygen. In contrast, trehalose synthesis under salt stress was not influenced by the mutation of otsA. This indicates that Rhizobium sp. NGR234 exhibits a second trehalose pathway. Activities of maltooligosyltrehalose synthase and maltooligosyl trehalohydrolase (MOS – pathway) had been demonstrated in Rhizobium sp. NGR234 in previous work. To study the role of rhizobial otsA in symbiosis, various host plants were infected with Rhizobium sp. NGRWotsA. In a number of hosts, average nodule size was reduced, nodule number was increased (up to 30 %) and nitrogen fixation was reduced compared to control plants infected with the wildtype strain NGR234. Analysis of the carbohydrate content of these nodules revealed significant increases in the levels of sucrose, hexoses and starch. Thus the deletion of the potential rhizobial otsA-homologue has a severe impact on rhizobium-legume symbiosis, and a signal function of trehalose in carbohydrate partitioning and root nodule development is proposed.