• Background and Aims White lupin is highly adapted to growth in a low-P environment. The objective of the present study was to evaluate whether white lupin grown under P-stress has adaptations in nodulation and N₂ fixation that facilitate continued functioning. • Methods Nodulated plants were grown in silica sand supplied with N-free nutrient solution containing 0 to 0·5 mm P. At 21 and 37 d after inoculation (DAI) growth, nodulation, P and N concentration, N₂ fixation (¹⁵N₂ uptake and H₂ evolution), root/nodule net CO₂ evolution and CO₂ fixation (¹⁴CO₂ uptake) were measured. Furthermore, at 21 DAI in-vitro activities and transcript abundance of key enzymes of the C and N metabolism in nodules were determined. Moreover, nodulation in cluster root zones was evaluated. • Key Results Treatment without P led to a lower P concentration in shoots, roots, and nodules. In both treatments, with or without P, the P concentration in nodules was greater than that in the other organs. At 21 DAI nitrogen fixation rates did not differ between treatments and the plants displayed no symptoms of P or N deficiency on their shoots. Although nodule number at 21 DAI increased in response to P-deficiency, total nodule mass remained constant. Increased nodule number in P-deficient plants was associated with cluster root formation. A higher root/nodule CO₂ fixation in the treatment without P led to a lower net CO₂ release per unit fixed N, although the total CO₂ released per unit fixed N was higher in the treatment without P. The higher CO₂ fixation was correlated with increased transcript abundance and enzyme activities of phosphoenolpyruvate carboxylase and malate dehydrogenase in nodules. Between 21 and 37 DAI, shoots of plants grown without P developed symptoms of N- and P-deficiency. By 37 DAI the P concentration had decreased in all organs of the plants treated with no P. At 37 DAI, nitrogen fixation in the treatment without P had almost ceased. • Conclusions Enhanced nodulation in cluster root zones and increased potential for organic acid production in root nodules appear to contribute to white lupin's resilience to P-deficiency.

In most legume nodules, the di-nitrogen (N₂)-fixing rhizobia are present as organelle-like structures inside their root host cells. Many processes operate and interact within the symbiotic relationship between plants and nodules, including nitrogen (N)/carbon (C) metabolisms, oxygen flow through nodules, oxidative stress, and phosphorous (P) levels. These processes, which influence the regulation of N₂ fixation and are finely tuned on a whole-plant basis, are extensively reviewed in this paper. The carbonic anhydrase (CA)-phosphoenolpyruvate carboxylase (PEPC)-malate dehydrogenase (MDH) is a key pathway inside nodules involved in this regulation, and malate seems to play a crucial role in many aspects of symbiotic N₂ fixation control. How legumes specifically sense N-status and how this stimulates all of the regulatory factors are key issues for understanding N₂ fixation regulation on a whole-plant basis. This must be thoroughly studied in the future since there is no unifying theory that explains all of the aspects involved in regulating N₂ fixation rates to date. Finally, high-throughput functional genomics and molecular tools (i.e., miRNAs) are currently very valuable for the identification of many regulatory elements that are good candidates for accurately dissecting the particular N₂ fixation control mechanisms associated with physiological responses to abiotic stresses. In combination with existing information, utilizing these abundant genetic molecular tools will enable us to identify the specific mechanisms underlying the regulation of N₂ fixation.

The mechanism through which nitrate reduces the activity of legume nodules is controversial. The objective of the study was to follow Medicago truncatula nodule activity after nitrate provision continuously and to identify molecular mechanisms, which down-regulate the activity of the nodules. Nodule H₂ evolution started to decline after about 4 h of nitrate application. At that point in time, a strong shift in nodule gene expression (RNA sequencing) had occurred (1,120 differentially expressed genes). The most pronounced effect was the down-regulation of 127 genes for nodule-specific cysteine-rich peptides. Various other nodulins were also strongly down-regulated, in particular all the genes for leghemoglobins. In addition, shifts in the expression of genes involved in cellular iron allocation and mitochondrial ATP synthesis were observed. Furthermore, the expression of numerous genes for the formation of proteins and glycoproteins with no obvious function in nodules (e.g. germins, patatin, and thaumatin) was strongly increased. This occurred in conjunction with an up-regulation of genes for proteinase inhibitors, in particular those containing the Kunitz domain. The additionally formed proteins might possibly be involved in reducing nodule oxygen permeability. Between 4 and 28 h of nitrate exposure, a further reduction in nodule activity occurred, and the number of differentially expressed genes almost tripled. In particular, there was a differential expression of genes connected with emerging senescence. It is concluded that nitrate exerts rapid and manifold effects on nitrogenase activity. A certain degree of nitrate tolerance might be achieved when the down-regulatory effect on late nodulins can be alleviated.

Legume nodules are plant tissues with an exceptionally high concentration of phosphorus (P), which, when there is scarcity of P, is preferentially maintained there rather than being allocated to other plant organs. The hypothesis of this study was that nodules are affected before the P concentration in the organ declines during whole-plant P depletion. Nitrogen (N₂) fixation and P concentration in various organs were monitored during a whole-plant P-depletion process in Medicago truncatula. Nodule gene expression was profiled through RNA-seq at day 5 of P depletion. Until that point in time P concentration in leaves reached a lower threshold but was maintained in nodules. N₂-fixation activity per plant diverged from that of fully nourished plants beginning at day 5 of the P-depletion process, primarily because fewer nodules were being formed, while the activity of the existing nodules was maintained for as long as two weeks into P depletion. RNA-seq revealed nodule acclimation on a molecular level with a total of 1140 differentially expressed genes. Numerous genes for P remobilization from organic structures were increasingly expressed. Various genes involved in nodule malate formation were upregulated, while genes involved in fermentation were downregulated. The fact that nodule formation was strongly repressed with the onset of P deficiency is reflected in the differential expression of various genes involved in nodulation. It is concluded that plants follow a strategy to maintain N₂ fixation and viable leaf tissue as long as possible during whole-plant P depletion to maintain their ability to react to emerging new P sources (e.g. through active P acquisition by roots).

Nodulated alfalfa (Medicago sativa L. cv. Saranac) plants were grown in hydroponics at P-sufficient and P-deficient supply levels. After 5 weeks of growth, dry matter accumulation, nodulation, total N and P accumulation, as well as ¹⁵N₂ uptake, were measured. Moreover, the response of nodule O₂-uptake to raising external pO₂ was determined in an open-flow measurement system and nodule permeability was calculated. Plants in the P-deficient supply treatment had a lower P concentration in all organs. In both treatments the highest P concentration was found in nodules. In the P-deficient supply treatment plants formed less dry matter, had a lower shoot/root ratio, less nodulation, decreased total N accumulation, and lower ¹⁵N₂ uptake per dry matter nodule. Nodules in the P-deficient treatment were, on average, smaller and had a higher O₂ uptake per N₂ reduced, coinciding with increased nodule permeability and conductance. Thus increased oxygen uptake appears to be a mechanism to adjust nodule metabolism to P deficiency in indeterminate N₂-fixing nodules such as in alfalfa, as has previously been shown for determinate nodule forms.