Genetic Variation in Host-Specific Competitiveness of the Symbiont Rhizobium leguminosarum Symbiovar viciae

Legumes of the Fabeae tribe form nitrogen-fixing root nodules resulting from symbiotic interaction with the soil bacteria Rhizobium leguminosarum symbiovar viciae (Rlv). These bacteria are all potential symbionts of the Fabeae hosts but display variable partner choice when co-inoculated in mixture. Because partner choice and symbiotic nitrogen fixation mostly behave as genetically independent traits, the efficiency of symbiosis is often suboptimal when Fabeae legumes are exposed to natural Rlv populations present in soil. A core collection of 32 Rlv bacteria was constituted based on the genomic comparison of a collection of 121 genome sequences, representative of known worldwide diversity of Rlv. A variable part of the nodD gene sequence was used as a DNA barcode to discriminate and quantify each of the 32 bacteria in mixture. This core collection was co-inoculated on a panel of nine genetically diverse Pisum sativum, Vicia faba, and Lens culinaris genotypes. We estimated the relative Early Partner Choice (EPC) of the bacteria with the Fabeae hosts by DNA metabarcoding on the nodulated root systems. Comparative genomic analyses within the bacterial core collection identified molecular markers associated with host-dependent symbiotic partner choice. The results revealed emergent properties of rhizobial populations. They pave the way to identify genes related to important symbiotic traits operating at this level.

In vivo imaging and quantification of carbon tracer dynamics in nodulated root systems of pea plants

Legumes associate with root colonizing rhizobia that provide fixed nitrogen to its plant host in exchange for recently fixed carbon. There is a lack in understanding how individual plants modulate carbon allocation to a nodulated root system as a dynamic response to abiotic stimuli. One reason is that most approaches are based on destructive sampling, making quantification of localized carbon allocation dynamics in the root system difficult. We established an experimental workflow for routinely using non-invasive Positron Emission Tomography (PET) to follow the allocation of leaf-supplied 11C tracer towards individual nodules in a three-dimensional (3D) root system of pea (Pisum sativum). Nitrate was used for triggering the shutdown of biological nitrogen fixation (BNF) expected to rapidly affect carbon allocation dynamics in the root-nodule system. This nitrate treatment lead to a reduction of 11C tracer allocation to nodules by 40% - 47% in 5 treated plants while the variation in control plants was less than 11%. The established experimental pipeline enabled for the first time that several plants could consistently be labelled and measured using 11C tracer in a PET approach to quantify C-allocation to individual nodules following a BNF shutdown. This demonstrates the strength of using 11C tracers in a PET approach for non-invasive quantification of dynamic carbon allocation in several growing plants over several days. A major advantage of the approach is the possibility to investigate carbon dynamics in small regions of interest in a 3D system such as nodules in comparison to whole plant development.

Visualizing Glutamine Accumulation in Root Systems Involved in the Legume–Rhizobia Symbiosis by Placement on Agar Embedded with Companion Biosensor Cells

Microbial symbiotic nitrogen fixation (SNF) occurs inside root nodules, where fixed-N (NH4+) from rhizobia is first assimilated into the amino acid glutamine (Gln). Visualization of Gln dynamics in nodulated root systems of different plant species would require re-engineering transgenic Gln reporters specific for each rhizobia/host genotype. Here we demonstrate the use of companion biosensor cells called GlnLux (Escherichia coli auxotrophic for Gln and constitutively expressing lux) to image Gln accumulation in nodulated root systems across a diversity of legume/rhizobia species. Companion GlnLux cells are embedded into agar (GlnLux agar) upon which legume root systems are placed following freeze-thawing to cause Gln leakage. Photons released from nearby activated biosensor cells are captured using a photon capture camera. Using split root systems, we demonstrate that in diverse amide-exporting legumes (alfalfa, lentil, and green pea) and a ureide-exporting legume (soybean) that GlnLux agar imaging is sufficiently sensitive to detect Gln release from individual nodules and can differentiate root systems with active nif+ from inactive nif− nodules. The assay permits visualization of both source and sink dynamics of nodule Gln, specifically, Gln import into nodules from roots (for nodule growth and/or amino acid cycling), Gln assimilated from fixed nitrogen that accumulates inside nodules, and Gln export from nodules into roots from this assimilatory-N. GlnLux agar-based imaging is thus a new research tool to localize the accumulation and transfer of a critical amino acid required for rhizobia symbionts within legume phytobiomes. We discuss the ability of this technology to open new frontiers in basic research and its limitations.

Nitrogenase Activity and Associated Carbon Budgets in Seedlings of Acacia mangium Measured With a Flow-Through System of the Acetylene Reduction Assay

Following introduction of acetylene into the flow-through gas system, nitrogenase activity of young seedlings of Acacia mangiurn Willd. increased during the initial gas mixing period but declined thereafter. Nitrogenase-linked respiration also declined rapidly for 3-4 min and slowly thereafter following introduction of acetylene. Seedlings of 12-42 weeks showed a decline in nitrogenase-linked respiration of 15-40%. Nitrogenase activity and nodulated root respiration declined further when the oxygen concentration in the gas flowing past the nodules was reduced. These concomitant declines in nodulated root respiration and nitrogenase activity were used to explore the relation between nodulated root respiration and nitrogenase activity, and to provide an estimate of the carbon cost of nitrogenase activity, and the growth and maintenance respiration of nodulated roots. The carbon cost of nitrogenase activity was 2-8 μmol CO2 μmol-1 C2H2 reduced for young seedlings (12-20 weeks) and was lower (1.3) for older seedlings (30-42 weeks). Nitrogenase activity was 1.2 μmol C2H4 g-1 nod. dw min-1 for plants at 12 weeks, but was 0.3 μmol C2H4 g-1 nod. dw min-1 for plants at 42 weeks after transplanting. The proportion of nitrogenase-linked respiration in nodulated root respiration also declined rapidly with plant age, being 70% at 12 weeks and only 6% at 42 weeks after transplanting.

Physiological acclimations to chilling temperature in symbiotically grown alfalfa

An apparent shoot rest period was induced in the 2nd month of growth of alfalfa (Medicago sativa L.) seedlings by a drop in growth temperature from 25:20 °C to 10:7 °C. After prolonged chilling the shoots were replaced by new shoots. Temperature profiles of nodulated root respiration and nitrogenase activity (acetylene reduction and H2 evolution) were measured simultaneously in experiments with a flow-through gassing system during 3 months of cold treatment. Net photosynthesis of whole plants was measured in a closed system. More than half the total initial nitrogenase activity and relative efficiency (RE) were lost during the rest period and recovered during regrowth. Acetylene reduction by chilled plants was insensitive to temperature in the 5 – 15 °C range, unlike the temperature dependence of respiration and H2 evolution in air. In all temperature profiles of RE the RE was highest at 5 – 10 °C. The RE was minimum 10 – 15 °C during the rest period. The optimum temperature for whole plant net photosynthesis also declined to 10 – 15 °C during chilling and it later flattened out in the cold-acclimated regrowth. Possible mechanisms are discussed.

Nitrogenase Activity by Subterranean Clover and Other Temperate Pasture Legumes

Nitrogenase (C2H2-reduction) activity and nodulated root respiration of intact plants of subterranean clover (Trifolium subterraneum L.) cv. Seaton Park nodulated by Rhizobium trifolii WU95 were measured in a flow-through system. Simultaneous declines in nitrogenase activity and respiration were exhibited 2 min after 10% C2H2 had been introduced into the gas stream. Declines in nitrogenase activity and nodulated root respiration provided an estimate of the efficiency of nitrogenase activity (mol CO2 evolved/mol C2H4 produced). The pre-decline rate of nitrogenase activity at time zero was thus calculated as the product of the respiration associated with nitrogenase activity and the reciprocal of the efficiency of nitrogenase activity. Pre-decline rates of nitrogenase activity were similar to peak rates for several pasture legumes. However, post-decline rates of activity were as much as 70% lower than the pre-decline rate. The age of subterranean clover plants had an important influence on the magnitude of the C2H2-induced decline; young plants exhibited the largest C2H2-induced inhibition of nitrogenase activity. Neither sainfoin (Onobrychis viciifolia Scop.) cv. Othello nodulated by Rhizobium sp. CC1108 nor yellow serradella (Ornithopus compressus L.) cv. Pitman nodulated by R. lupini WU425 exhibited C2H2-induced declines in nitrogenase activity. Nitrogenase-linked respiration of subterranean clover at the 14-leaf stage accounted for 50% of total nodulated root respiration. The oxygen diffusion resistance of the nodules increased in the presence of C2H2 but the effect was reversible once C2H2 was removed from the gas atmosphere. The pre-decline rate of acetylene reduction activity of subterranean clover reached a maximum at 10% C2H2. The C2H2-induced decline in nitrogenase activity was lower at subsaturating pC2H2 and was not detected at 0.4% C2H2.