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Effects of nutrients on N-fixation of Lotus spp. and Rhizobium strains:

Supervisor: Stefanie Wienkoop

PhD student: Sebastian Schneider

Group: Plant-Systems Interactions, Division of Molecular Systems Biology, Department of Ecogenomics and Systems Biology

Defended on June 19th 2019


Legume-Rhizobia symbioses play a major role in food production for an ever growing human population. In this symbiosis, dinitrogen is reduced (“fixed“) to ammonia by the rhizobial nitrogenase enzyme complex and is secreted to the plant host cells, whereas dicarboxylic acids derived from photosynthetically produced sucrose are transported towards the bacterial symbionts and serve as respiratory substrates for the them. This enables plants to grow in nitrogen (N)-deficient soils as N is the most important nutrient for plant growth.
Early research found that sulfate availability of soils play a crucial role for symbiotic N-fixation (SNF). Clover, grown under sulfate deficiency, showed drastically reduced SNF activity. Furthermore, it was found that SNF was very sensitive to sulfate deficiency because the addition of sulfate to pea plants growing on sulfate-deficient soil doubled the amount of fixed N at all growth stages of the plant. Whether this was caused by the general lack of sulfate in the host legume or specifically affecting N-fixation remained unclear. Interestingly, the symbiosome membrane contains high levels of a sulfate transporter. Sulfate is an essential nutrient for all living organisms, but its importance for SNF and nodule metabolism has long been underestimated. In this project, the following questions could be answered and have been published (Schneider et al. 2019):

Do the micro-symbionts take up the sulfate coming from the plant?
What do the micro-symbionts use the sulfate for?
Is sulfate directly influencing SNF efficiency?

The results of this study gave new insights into the important role of sulfate within plant-microbe interaction. For the first time chemical imaging (NanoSIMS) in root nodules was used and we could demonstrate that the microbial symbionts take up 20-fold more sulfate than the plant nodule cells themselfs. Furthermore, we could show that production of the bacterial key enzyme complex necessary for SNF, the  nitrogenase, relies on high levels of imported sulfate from the plant, making sulfur as essential as carbon for the regulation and functioning of SNF. Our findings thus establish the importance of sulfate and its active transport for the plant-microbe interaction that is most relevant for agriculture and soil fertility.

The project was additionally leading to the analysis of another functionally still uncharacterized symbiosome membrane protein, a Hypersensitive Induced Response (HIR) protein, we termed Lotus japonius HIR1. HIR proteins belong to Proliferation, Ion and Death family (PID) of proteins, some of which are involved in defense responses and cell-death. Identification of HIR1 on the symbiosome membrane poses therefore a highly relevant question for its role during symbiosis and N-fixation. No insertion mutants (LORE1) in the coding region of HIR1 were present and therefore, HIR1 silencing and overexpression constructs were created. The effect was analyzed in mutated roots (hairy roots) of Lotus japonicus during symbiosis with M.loti. Microscopic analysis revealed candidate phenotypes in early stages of the symbiosis during infection thread formation and progression, and no morphological differences during later stages of nodule formation. The roots with overexpression of HIR1 showed opposite phenotypes to those transformed with down-regulation constructs, when the number of nodules and the proteome profile was analysed in a comparative study. Interestingly, a number of proteins involved in N-fixation, nitrogen and carbon metabolism were affected by HIR1 downregulation indicating a serious effect on symbiosis. Furthermore proteins involved in signaling (jasmonic acid, calcium and flavonoid biosynthesis) were differentially expressed in the roots with HIR1 downregulation, which is especially interesting since these processes were already shown to be important for the establishment of the symbiosis, but little is known about their role at later stages, during nodule maintenance.


Please find a list of publications here.

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