Genome-scale metabolic reconstruction of the symbiosis between a leguminous plant and a nitrogen-fixing bacterium

The mutualistic association between leguminous plants and endosymbiotic rhizobial bacteria is a paradigmatic example of a symbiosis driven by metabolic exchanges. Here, we report the reconstruction and modelling of a genome-scale metabolic network of Medicago truncatula (plant) nodulated by Sinorhizobium meliloti (bacterium). The reconstructed nodule tissue contains five spatially distinct developmental zones and encompasses the metabolism of both the plant and the bacterium. Flux balance analysis (FBA) suggests that the metabolic costs associated with symbiotic nitrogen fixation are primarily related to supporting nitrogenase activity, and increasing N2-fixation efficiency is associated with diminishing returns in terms of plant growth. Our analyses support that differentiating bacteroids have access to sugars as major carbon sources, ammonium is the main nitrogen export product of N2-fixing bacteria, and N2 fixation depends on proton transfer from the plant cytoplasm to the bacteria through acidification of the peribacteroid space. We expect that our model, called ‘Virtual Nodule Environment’ (ViNE), will contribute to a better understanding of the functioning of legume nodules, and may guide experimental studies and engineering of symbiotic nitrogen fixation. The association between leguminous plants and rhizobial bacteria is a paradigmatic example of a symbiosis driven by metabolic exchanges. Here, diCenzo et al. report the reconstruction and modelling of a genome-scale metabolic network of the plant Medicago truncatula nodulated by the bacterium Sinorhizobium meliloti.


Summary
This study is a computational exploration of symbiotic nitrogen fixation which is realized in the metabolic interactions between the legume plant Medicago truncatula and its rhizobial symbiotic bacterial partner Sinorhizobium meliloti. The bacteria invade and colonize Medicago roots and differentiate into forms that are specialized to fix atmospheric nitrogen. Fixed nitrogen is traded with the host plant against reduced carbon, which establishes a metabolic interaction of mutual benefit. Both M truncatula and S. meliloti have fully sequenced genomes. Three interacting plant tissues and four different stages of internalized bacteria residing within nodules are represented as cellular metabolic models. The integrated plant model allows to study aspects of resource allocation between the plant and the rhizobia / symbionts. First, the authors improved genome-scale metabolic reconstructions for S. meliloti and M truncatula, which were then used to make leaf, root, and nodule sub-models into an integrated model called Virtual Nodule Environment (ViNE). Simulated variations in N2 fixation efficiency and/or rate of nodulation showed there were diminishing returns in plant growth rate increases due to improvements in fixation efficiency, and that the plant benefitted from lower nodulation rates (lower biomass proportion) because they did not need to invest in nodule maintenance costs. Differences in energy production and other metabolic activities associated with nodule development were identified by comparing reaction essentialities in nodule zones specific for uninfected cells, differentiation, and nitrogen fixation. Further examination of the nitrogen fixation zone led to the finding that limitations in mitochondrial terminal oxidase activity could be responsible for C4-dicarboxlyates being traded to the bacterial partner from the plant instead of sucrose. 1. Altogether the integrated model is a very complex multicellular set-up. The authors put much emphasis on detailing the nodule into 5 different cell types / zones. However, the motivation to do this could be clearer. Which literature background could be cited to justify differentiating these zones?
2. The simulations shown in Figure 5 are explained in the supplement (Text S5), but the very complex iterative optimization procedures are hard to conceptualize. Could there be a supplemental figure to help understanding this? In the main text, some details on description could be made clearer: There seems to be some confusion with regards to how biomass ratios are referred to. There is "plant to nodule ratio" and "rate of nodulation". These definitions are the same? If yes, they could be clearer standardized throughout the manuscript. (In line 383: "We next evaluated the relationship between the rate of N2-fixation (without modifying the plant to nodule ratio) …". Supplemental Text S5 then talks about "rate of nodulation", which is "gram of nodule per gram of plant". ) Figure 5A: Here biomass formation (growth rate) is shown in response to an increasing rate of N2 fixation forced onto the model. Is N2 the only possible nitrogen source? After the point where the biomass production (growth rate) doesn't increase further, where does the reduced nitrogen end up? Are there effluxes allowed, like NH4 or organic forms of nitrogen? If yes, does this make physiological sense that N2 fixation products can leak out? Also, the text says that in figure 5A the ratio of nodule biomass to plant biomass is kept constant. The value used could be shortly mentioned in the text. Is it 98:2 as mentioned as "default" in the supplement? 98:2 would mean 2% nodule biomass. Is 2 % where the growth rate reaches its maximum in figure 5B? Figure 5C: N2 fixation efficiency. Figure 5C defines N2 fixation efficiency as "rate of N2-fixation per gram nodule". In Figure 5B this ratio is held constant? What's the value? The legend in Figure 5C spell out N2 fixation efficiency differently: "… with a constant rate of N2-fixation per gram of nodule)." For better understanding, again, one definition could be used throughout.
3. The objective function(s) and model constraints (e.g., light uptake, carbon metabolite able to be traded) should be declared in the main text 4. Lines 261-263 -What fixed rates of nodulation and nitrogen fixation were used here? How do they compare to literature findings? 5. Lines 261-263-The nodulation rate should be defined here 6. Table 2, third row: if the nitrogen source is "N2-fixation" (i.e. N2, not ammonium) and the nodulation State is "Non-nodulated" (i.e. no symbiotic bacteria present), how can the plant grow? 7. Lines 450 -460 discuss metabolic consequences of limitations of free oxygen in the N2 fixation zone. Helpful context for the reader might be here to mention the oxygen sensitivity of nitrogenase, which dictates the physiological low oxygen conditions in the N2 fixation zone.

FURTHER COMMENTS
7. Line 30 -"protons transfer protons" 8. Line 121 -"will to" 9. Table 1 -why are the model properties of Zone III not listed?
10. Table S1 -What is number/units of "trace" metabolites/elements for the model?
11. Figure S3 -The legend refers to Figure 5D, but there is no Figure 5D; Was Figure 5C