Sodium Accumulation in Infected Cells and Ion Transporters Mistargeting in Nodules of Medicago truncatula: Two Ugly Items That Hinder Coping with Salt Stress Effects

Abstract

The maintenance of intracellular nitrogen-fixing bacteria causes changes in proteins' location and in gene expression that may be detrimental to the host cell fitness. We hypothesized that the nodule's high vulnerability toward salt stress might be due to alterations in mechanisms involved in the exclusion of Na⁺ from the host cytoplasm. Confocal and electron microscopy immunolocalization analyses of Na⁺/K⁺ exchangers in the root nodule showed the plasma membrane (MtNHX7) and endosome/tonoplast (MtNHX6) signal in non-infected cells; however, in mature infected cells the proteins were depleted from their target membranes and expelled to vacuoles. This mistargeting suggests partial loss of the exchanger's functionality in these cells. In the mature part of the nodule 7 of the 20 genes encoding ion transporters, channels, and Na⁺/K⁺ exchangers were either not expressed or substantially downregulated. In nodules from plants subjected to salt treatments, low temperature-scanning electron microscopy and X-ray microanalysis revealed the accumulation of 5-6 times more Na⁺ per infected cell versus non-infected one. Hence, the infected cells' inability to withstand the salt may be the integral result of preexisting defects in the localization of proteins involved in Na⁺ exclusion and the reduced expression of key genes of ion homeostasis, resulting in premature senescence and termination of symbiosis.

Keywords: Medicago truncatula; NHX Na+/K+ exchangers; ion transport; potassium; root nodule; salt stress; sodium; symbiosis.

Figure 1

Confocal microscopy immunolocalization of the MtNHX7 protein in the transgenic nodules carrying the ProMtNHX7:MtMTNHX7:GFP construct (A–E), and the scheme of protein distribution in infected and non-infected cells as per immunosignal (in green color) to explain the localization pattern and its changes (F). Color code: red fluorescence—propidium iodide counterstained bacteria and nuclei, green fluorescence—protein labeling. MtNHX7 distribution was visualized by GFP signal in the sections carrying the ProMtNHX7:MtNHX7:GFP construct. Green fluorescence in the nodule meristem and distal infection zone (close to the meristem) was detected in the plasma membrane (arrowhead). (B) High magnification of fraction of (A). Note the immunosignal is distributed in double rows decorating the plasma membranes of neighboring cells (arrows), separated by the cell wall (arrowhead). (C) Distal infection zone and fully infected cells situated below the infection zone, MtNHX7 is detected in the plasma membrane in meristematic cells and in cells containing freshly released bacteria. (D) Infected cells with MtNHX7 signal present in a dot-like pattern in the cytoplasm. (E) Localization of MtNHX7 in the vascular bundles; note the strong membrane signal. (F) Scheme of protein distribution in infected and non-infected cells. The arrow is pointing to the green immunosignal. Green rectangular box is representing the plasma membrane (PM) of non-infected cell, that retained the immunosignal, black rectangle is representing the PM of infected cell, with no green signal present, the green labelled protein is located in the cytoplasm in do-like pattern. IC: infected cell, NiC: non-infected cell, M: meristem, It: infection thread, Vac: vacuole, VB: vascular bundle, T: tonoplast. Arrowhead: signal over the plasma membrane (PM). Bars: (A,B,E): 50 μm; (C): 25 μm; (D): 10 μm.

Figure 2

MtNHX6 (green fluorescence) was visualized by using a specific custom-made antibody in wt nodules. Color code: red fluorescence—propidium iodide counterstained bacteria and nuclei, green fluorescence—protein, labeling. For the immunolocalization of MtNHX6, an antibody developed in rabbit and a secondary antibody developed in goat were used. (A) In the meristem and distal infection zone, the signal is present over the cytoplasm and tonoplast of young vacuoles in non-infected and freshly infected cells. (B) Freshly infected young cell, note the strong signal on the tonoplast, and the small dots in the cytoplasm (arrowhead). (C) High magnification of a fraction of (B). The signal is in small dots, early endosomes. (D) (The mature infected cells are filled with symbiosomes with the signal represented as big dots. (E) The vascular bundle with the labeling over the tonoplast; note the infected cells with dot-like pattern of labeling. (F) The scheme of NHX6 protein distribution as per immunosignal (in green color) to explain the pattern and its changes in infected and non-infected cells. Bact: bacteroid, IC: infected cell, NiC: non-infected cell, M: meristem, It: infection thread, VB: vascular bundle, N: nucleus, T: tonoplast. Arrowhead: signal over the plasma membrane (PM). Bars: (A,E): 25 μm; (B): 12.5 μm; (C): 1.1 μm; (D): 10 μm.

Figure 3

Electron microscopy immunolocalization of MtNHX7 (A) and MtNHX6 (B). Note the immunogold signal in the small vacuoles (~1000 nm). Bar: 200 nm. Vac: vacuole, B: bacteroid, arrow: immunogold signal.

Figure 4

Scanning microscopy image of root nodule cells (A) and distribution of Na⁺ (B) and K⁺ (C) in cells of the nitrogen-fixing zone from control and salt-treated nodules of M. truncatula. B, bacteroid; IC, infected cell; NIC, non-infected cell; V, vacuole. (*) p-value ≤ 0.01, (#) p-value ≤ 0.05.

Figure 5

qPCR gene expression analysis of M. truncatula genes in nodules of control plants and nodules from the plants under salt stress. (A) the expression of Na⁺/K⁺ (NHX) exchangers; (B) the expression of high-affinity K⁺ transporters 6 and 7, exchanger CHX18 and K⁺ channels AKT1 and SKOR/GORK; (C) expression of the nifH gene to estimate the nitrogen -fixing activity. Reference genes: MtMtc27, MtGAPDH, SMc00128. Asterisks (*) indicate significant differences between control and salt treatment.

Figure 6

Evolutionary analysis of NHX genes in M. truncatula, other legume species, and Arabidopsis thaliana. Other plant species where NHX6-7-8 genes have been characterized also have been included. The M. truncatula NHX6-7-8 homologous genes are contained in boxes. The corresponding A. thaliana homologs are underlined. Bootstrap values are shown next to the branches (1000 pseudoreplicates). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. For each gene, the gene name is indicated. Plant orders (for non-legume species) and legume clades are also indicated.