Identification of an Isoflavonoid Transporter Required for the Nodule Establishment of the Rhizobium- Fabaceae Symbiotic Interaction

Abstract

Nitrogen (N) as well as Phosphorus (P) are key nutrients determining crop productivity. Legumes have developed strategies to overcome nutrient limitation by, for example, forming a symbiotic relationship with N-fixing rhizobia and the release of P-mobilizing exudates and are thus able to grow without supply of N or P fertilizers. The legume-rhizobial symbiosis starts with root release of isoflavonoids that act as signaling molecules perceived by compatible bacteria. Subsequently, bacteria release nod factors, which induce signaling cascades allowing the formation of functional N-fixing nodules. We report here the identification and functional characterization of a plasma membrane-localized MATE-type transporter (LaMATE2) involved in the release of genistein from white lupin roots. The LaMATE2 expression in the root is upregulated under N deficiency as well as low phosphate availability, two nutritional deficiencies that induce the release of this isoflavonoid. LaMATE2 silencing reduced genistein efflux and even more the formation of symbiotic nodules, supporting the crucial role of LaMATE2 in isoflavonoid release and nodulation. Furthermore, silencing of LaMATE2 limited the P-solubilization activity of lupin root exudates. Transport assays in yeast vesicles demonstrated that LaMATE2 acts as a proton-driven isoflavonoid transporter.

Keywords: Bradyrhizobium; Lupinus albus; MATE transporter; genistein; nitrogen; phosphorus; plant-microbe interaction.

Figure 1

LaMATE2 expression level and genistein release in roots of P-deficient white lupin. LaMATE2 expression analyses in white lupin grown under control (+N + P, black bars) condition or P deficiency (+N-P, white bars) (A). Gene expression was evaluated in control apex (+N + P), P-deficient apex and cluster roots (separated depending on developing stages: Juvenile, Immature, Mature, Senescent cluster-root stages; example of cluster root shown in Supplementary Figure S1). Expression levels are shown relative to the LaMATE2 expression level of the root apex under control conditions (+N + P). Release of genistein from different root tissues of 4-week-old P-deficient plants, release from the control apex was below detectable value <LOD (B). Data are mean + SD (Asterisks refer to statistically significant differences among the mean value of the sample vs control or -P apex for genistein release, ANOVA Holm–Sidak, N=6, ^*p<0.05, ^**p<0.01; with 3 technical replicates for each real-time PCR).

Figure 2

LaMATE2 expression analyses and genistein release in roots of N-deficient white lupin. LaMATE2 expression analyses in white lupin grown under control (+N + P, black bars) condition or after 2 weeks of N-deficiency (-N + P, dark grey bars) (A). Gene expression was evaluated in control and N-deficient (other) root, root apex and nodules. Expression level is shown relative to LaMATE2 expression in control (+N+P) root. Root release of genistein from white lupin plants grown under 1- or 2-week-old N-sufficient and -deficient condition (B). Genistein release in 7 and 14 days of N deficiency (-N+P) or sufficiency (+N + P). Data are mean + SD (Asterisks refers to statistically significant differences among the mean value of the sample vs control, ANOVA Holm–Sidak, N=6–8, ^** p<0.01; with 3 technical replicates for each real-time PCR).

Figure 3

Phylogenetic tree of MATE transporters. A phylogenetic analysis was performed using the LaMATE2 amino acid sequences of Lupinus albus (LaMATE1, LaMATE2, Lalb_Chr17g0335991, Lalb_Chr06g0165721, Lalb_Chr02g0145611, LaALMT1); AtFRD3 AtEDS5, AtTT12, AtDTX1,10,11,12,13,14, AtALF5 and AtFFT of Arabidopsis thaliana; Glyma.10G267700 of Glycine max; HvAACT1 of Hordeum vulgare; MtMATE1, MtMATE2, Medtr1g108840, Medtr1g108990, Medtr1g109060.2, Medtr1g109060.1 of Medicago truncatula; NtJAT1, NtMATE1 and NtMATE2 of Nicotiana tabacum; SlMTP77 of Solanum lycopersicum; SbMATE1 of Sorghum bicolor; VvAM1 and VvAM3 of Vitis vinifera; VcMATE1 and VcMATE4 of Vaccinium corymbosum. The tree was constructed by aligning the protein sequences by Clustal-W and the evolutionary history was inferred using the Neighbour-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site.

Figure 4

Subcellular localization of LaMATE2. Co-localization of LaMATE2 fused with green fluorescent protein (GFP) and mCherry-labeled plasma membrane marker AtPIP2A in an Arabidopsis mesophyll protoplast. White scale bars=20 μM. Fluorescence intensity over distance plot of LaMATE2-GFP (green) and AtPIP2a-mCherry (red).

Figure 5

Effect of LaMATE2 silencing in N and P deficient roots. Alteration of LaMATE2 expression, flavonoid content and release, nodule number or P mobilization due to LaMATE2 silencing in N (A-F) or P (G-I) deficiency. LaMATE2 relative expression (a, g; 6 biological replicates), genistein release (B,H), number of nodules per plant (pictures shown in Supplementary Figure S4) (C), cell content: genistein (D), genistein 7-O-glucoside (E), genistein malonylglucoside (F), P mobilization from a poorly soluble P source (I). The analyses were performed on roots of N- or P-deficient lupin plants independently transformed with either pRedRoot::LaMATE2 RNAi (LaMATE2 RNAi) or empty-vector pRedRoot (Empty vector). All data are expressed relative to Empty-vector-transformed roots, with the exceptions of the number of nodule per plants and P mobilization (μmol P h⁻¹). Data are mean + SD (Asterisks refers to statistically significant differences among the mean value of RNAi and Empty vector, ANOVA Holm–Sidak, N=6–21, ^*p<0.05; for ^**p<0.01; with 3 technical replicates for each real-time PCR).

Figure 6

Effect of exogenous genistein on LaMATE2 expression and nodule numbers. LaMATE2 relative expression (A) and number of nodules per plant (B) in roots of N-deficient lupin plants independently transformed with either pRedRoot::LaMATE2 RNAi (LaMATE2 RNAi) or empty-vector pRedRoot (Empty vector) or RNAi roots treated with 1μM genistein (LaMATE2 RNAi + genistein). Expression data are shown relative to LaMATE2 expression level in empty vector-transformed roots. Data are mean + SD (Asterisks refers to statistically significant differences among the mean value of RNAi vs EV or RNAi+G vs RNAi values, ANOVA Holm–Sidak, N=6, ^*p<0.05; ^** for p<0.01).

Figure 7

Transport of phenylpropanoid compounds mediated by LaMATE2. LaMATE2-mediated transport rate of phenylpropanoids in yeast microsomal membrane vesicles. (A) Time-dependent accumulation of ³H-genistein in yeast microsomal membrane vesicles. Membrane vesicles were isolated from yeast transformed with the empty vector (pNEV empty vector) or transformed with pNEV-LaMATE2_ORF. The yeast vesicles were incubated up to 120 s in presence of 5 μM ³H-genistein: To determine the effect of the pH gradient, 25 mM NH₄Cl was added in an assay solution (pNEV-LaMATE2_ORF + NH₄Cl), as uncoupler of the proton gradient. (B) Concentration-dependent transport of ³H-genistein by LaMATE2 in yeast vesicles. Vesicles were incubated for 30s in the assay solution containing ³H-genistein at different concentrations (from 5 to 100 μM). The kinetic parameters of ³H-genistein uptake were calculated by subtracting uptake rates recorded in the empty-vector vesicles using the Hanes–Woolf plot. (C) Substrate specificity of LaMATE2 transporter was evaluated in presence of genistein, hydroxygenistein, genistin (glycosylated genistein), biochanin A (methylgenistein), daidzein or kaempferol (5 μM, 30 s). All data are expressed relative to pNEV-LaMATE2_ORF-transformed yeast. Data are mean ± SD of three independent experiments (Asterisks refers to statistically significant differences among the mean value in comparison to empty vector sample within each time point; capital letters refer to statistically significant differences among the mean value, ANOVA Holm–Sidak, N=6, ^**p<0.01, <LOD: below detectable value).