Pea nodule gradients explain N nutrition and limited symbiotic fixation in hypernodulating mutants

Legumes fix atmospheric nitrogen by symbiosis with soil bacteria in root nodules. Legume yields are limited by the low capacity of N-2 fixation. Hypernodulating mutants have been selected decades ago to try to increase nodule number. However, literature data show that N fixation of hypernodulating mutants was not increased compared to parental lines. Here, we study the functional basis of limited N fixation associated to hypernodulation. We grew two wild type genotypes and nine hypernodulating mutants of pea in hydroponics in three greenhouse experiments. We measured the following traits related to N nutrition during the vegetative period: nodule number, plant N uptake, nodule-specific activity, and plant and nodule concentrations. Genetic and environmental variations induced nodule gradients. These gradients were used to set quantitative relationships between N nutrition traits and nodule number. We compared the relationships obtained for hypernodulating and for wild types. N nutrition traits were analysed together with C nutrition traits, through correlation networks. Our results show that higher nodule number of hypernodulating mutants is correlated with lower levels of nodule activity, from -25 to -60 %, by comparison to the wild type. Higher nodule number of hypernodulating mutants is also correlated with lower total N uptake by symbiotic fixation, from -0 to -60 %, by comparison to the wild type. Findings demonstrate that N nutrition is not a factor limiting growth in hypernodulating mutants, as shown by N nutrition index higher than 1, indicating N nutrition in excess. The correlations suggest that limited N-2 fixation in hypernodulating mutants arises from restricted shoot growth, which limits the plant capacity to accumulate N. Furthermore, symbiotic efficiency decreased with increasing nodule number, down to a minimal value for hypernodulating mutants. Thus, to overcome the trade-off between N benefits from N-2 fixation and carbon nodulation costs, the hypernodulation trait should be associated with high shoot growth capacity in breeding programs