Nodulating white lupins take advantage of the reciprocal interplay between N and P nutritional responses

Abstract

The low bioavailability of nutrients, especially nitrogen (N) and phosphorus (P), is one of the most limiting factors for crop production. In this study, under N- and P-free nutrient solution (-N-P), nodulating white lupin plants developed some nodules and analogous cluster root structures characterized by different morphological, physiological, and molecular responses than those observed upon single nutrient deficiency (strong acidification of external media, a better nutritional status than -N+P and +N-P plants). The multi-elemental analysis highlighted that the concentrations of nutrients in white lupin plants were mainly affected by P availability. Gene-expression analyses provided evidence of interconnections between N and P nutritional pathways that are active to promote N and P balance in plants. The root exudome was mainly characterized by N availability in nutrient solution, and, in particular, the absence of N and P in the nutrient solution triggered a high release of phenolic compounds, nucleosides monophosphate and saponines by roots. These morphological, physiological, and molecular responses result from a close interplay between N and P nutritional pathways. They contribute to the good development of nodulating white lupin plants when grown on N- and P-free media. This study provides evidence that limited N and P availability in the nutrient solution can promote white lupin-Bradyrhizobium symbiosis, which is favourable for the sustainability of legume production.

FIGURE 1

Representative pictures of shoots of white lupin plants after 28 days after inoculation (DAI) under the different nutrient conditions (+N+P, −N+P, +N−P, −N−P) and relative SPAD values (Holm–Sidak analysis of variance [anova], for each condition 20 plants were analysed n = 20, p‐value <0.05). Different letters indicate statistically significant differences

FIGURE 2

Acidification activity of white lupin roots using a pH gel indicator (bromocresol purple) at 28 days after inoculation (DAI) under the different nutrient conditions (+N+P, −N+P, +N−P, −N−P)

FIGURE 3

(A) Fresh and dry weights of nodulating white lupin plants at 28 days after inoculation (DAI) under different nutritional conditions (+N+P, −N+P, +N−P, −N−P). (B) pH values of root exudates of white lupin plants at 28 DAI. (C,D) Nitrogen (N) and phosphorus (P) concentration in white lupin plants at 28 DAI. (E) Relative quantification of Bradyrhizobium in white lupin roots by real‐time RT‐PCR at 14, 21, and 28 DAI using primers specific for 16S rRNA gene of Bradyrhizobium, using LaHEL expression for normalization (E). Letters refer to statistical significance (Holm–Sidak analysis of variance [anova], n = 3, p‐value <0.05)

FIGURE 4

Ionomic analyses in white lupin plants at 28 days after inoculation (DAI) under the different nutrient conditions (+N+P, −N+P, +N−P, −N−P). (A) Principal component analysis (PCA) scatterplot representing the 12 samples of nodule (R2: 0.977; Q2: 0.871). (B) PCA scatterplot representing the 12 samples of roots (R2: 0.0097; Q2: 0.773). (C) PCA scatterplot representing the 12 samples of shoots (R2: 0.977; Q2: 0.861). Green circle: +N+P samples; rose box: +N−P; grey diamond: −N+P; purple hexagon: −N−P

FIGURE 5

(A) Schematic representation of C, N, and P metabolism in nodulated root cell (modified from Liu et al., 2018). (B) Expression of genes involved in N and P acquisition in whole white lupin roots at 14, 21, and 28 days after inoculation (DAI) by real‐time RT‐PCR. Data indicate the fold change in comparison to one replicate of +N+P roots used as reference (value = 1); red, upregulated genes; blue, downregulated ones. Averages and standard deviations of real‐time RT‐PCR data are reported in Table S5

FIGURE 6

Orthogonal partial least square discriminant analysis, score plot (OPLS‐DA, on the left) and loading plot (pq(corr)1 vs. pq(corr)2, on the right) from untargeted metabolomic analyses of white lupin root exudates at 28 days after inoculation (DAI). Analyses obtained by negative (A) and positive (B) ionization. In the loading plots, black dots refer to metabolites; red dots refer to four treatments

FIGURE 7

Schematic representation of changes occurring at white lupin root system (lupin plant was drawn by Müller et al., 2015)