Potassium content diminishes in infected cells of Medicago truncatula nodules due to the mislocation of channels MtAKT1 and MtSKOR/GORK

Abstract

Rhizobia establish a symbiotic relationship with legumes that results in the formation of root nodules, where bacteria encapsulated by a membrane of plant origin (symbiosomes), convert atmospheric nitrogen into ammonia. Nodules are more sensitive to ionic stresses than the host plant itself. We hypothesize that such a high vulnerability might be due to defects in ion balance in the infected tissue. Low temperature SEM (LTSEM) and X-ray microanalysis of Medicago truncatula nodules revealed a potassium (K⁺) decrease in symbiosomes and vacuoles during the life span of infected cells. To clarify K⁺ homeostasis in the nodule, we performed phylogenetic and gene expression analyses, and confocal and electron microscopy localization of two key plant Shaker K⁺ channels, AKT1 and SKOR/GORK. Phylogenetic analyses showed that the genome of some legume species, including the Medicago genus, contained one SKOR/GORK and one AKT1 gene copy, while other species contained more than one copy of each gene. Localization studies revealed mistargeting and partial depletion of both channels from the plasma membrane of M. truncatula mature nodule-infected cells that might compromise ion transport. We propose that root nodule-infected cells have defects in K⁺ balance due to mislocation of some plant ion channels, as compared with non-infected cells. The putative consequences are discussed.

Keywords: Medicago truncatula; AKT1; GORK; SKOR; X-ray microanalysis; potassium channels; symbiosis; symbiosome.

Fig. 1.

(A, B) LTSEM images of M. truncatula nodules and potassium distribution. (A) Image of a freeze-fractured nodule and root of M. truncatula. (B) High magnification of infected and non-infected cells. (C) Relative distribution of K⁺ in vacuoles and symbiosomes in the different nodule zones for nodules prepared by LTSEM. The elemental analysis was carried out focusing the excitation X-ray beam into the vacuoles or the symbiosomes exposed in fractured cells. The number and the energy of the X-rays re-emitted from the specimens were measured by an energy-dispersive spectrometer. Data represent means ±SD of atomic (%) (n=10–13). * indicate significant differences between infected cell vacuoles in z2, and in z3 and z4; # indicate differences between symbiosomes in z2, and in z3 and z4. C, cortex zone, CW, cell wall; IC, infected cell; N, nodule; NIC, non-infected cell; R, root; S, symbiosome; V, vacuole; M (zone1), meristem; z2, young infected zone; z3, mature infected zone; z4, senescent zone. Scale bars: (A) 500 µm; (B) 50 µm.

Fig. 2.

Evolutionary analyses of AKT1 and SKOR/GORK homologues of M. truncatula and other legumes. (A) The evolutionary history of AKT1 was inferred by using the maximum likelihood method and Tamura–Nei model. A discrete Gamma distribution was used to model evolutionary rate differences among sites. The tree with the highest log likelihood is shown. The M. truncatula AKT1 reference gene is contained in a box. The A. thaliana AKT1 reference is underlined. Arabidopsis thaliana AKT5, AKT6, and two M. truncatula homologue sequences were added as the outgroup (OG). (B) Evolutionary history of SKOR/GORK homologues in M. truncatula and other legumes was inferred by using the maximum likelihood method based on the general time reversible model. A discrete Gamma distribution was used to model evolutionary rate differences among sites. The tree with the highest log likelihood is shown. The M. truncatula reference gene is contained in a box. Arabidopsis thaliana GORK and SKOR references are underlined. Clusters containing legume and Brassicales homologues are identified to the right. The A. thaliana AKT1, AKT2/3, KAT1, and M. truncatula AKT2/3 gene sequences were added as the OG. In both trees, bootstrap values are shown next to the branches (1000 pseudoreplicates). The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. For each gene, the GenBank accession number and/or gene name (in parentheses) are indicated.

Fig. 3.

MtAKT1 and MtSKOR/GORK expression in nodules. (A, B) Relative transcription of MtAKT1 (A) and MtSKOR/GORK (B) in the different nodule developmental zones as analysed by qPCR. Data represent means ±SD (n=3). (C–F) Histochemical staining of GUS activity in the transgenic roots and nodules carrying the construct ProAKT1:GUS (C) Root and root primordium. Expression detected in vascular bundles and meristem. (D) Nodule promordium and young root nodule. Expression in the meristematic region and in the zone of cell growth. (E) Developed nodule showing expression in all nodule zones. (F) Senescing nodule. Expression was detected in all nodule developmental zones and vascular bundles. R, root primordium; M, meristem; N, nodule primordium; VB, vascular bundles; z2, infection zone; z3, nitrogen-fixing zone; z4, senescence zone. Scale bars: (C–F) 100 µm.

Fig. 4.

Localization of the potassium transporters MtAKT1 and MtSKOR/GORK in the cells of M. truncatula root nodules. (A–C) Confocal imaging of the GFP-fused protein MtAKT1 in roots and nodules from transgenic roots carrying the construct ProMtAKT1:MtAKT1:GFP. (A) MtAKT1 (green fluorescence) in the root meristem was detected in the plasma membrane. Note the strong signal in the plasma membrane between the freshly divided cells (*). (B) Distal infection zone (close to the meristem), MtAKT1 was detected in the plasma membrane. Note the infection threads. Rhizobia were counterstained with propidium iodide (red). (C) MtAKT1 in the mature infected zone, the signal was visible as green dots (arrow) in the cytoplasm of infected cells, but not in the plasma membrane; in contrast to infected cells, the non-infected cells showed the signal over the plasma membrane. (D–F) Immunolocalization of MtSKOR/GORK by anti-SKOR antibody in root nodules. (D) Apical part of the root nodule and infection zone. The signal for MtSKOR/GORK (green) was present in the plasma membrane region in non-infected cells. Rhizobia were counterstained with propidium iodide (red). (E) Mature part of the nodule. Note the MtSKOR/GORK signal in the plasma membrane region of non-infected cells (arrowhead) versus the nearly absent signal in the plasma membrane of infected cells. Strong green labelling in the symbiosome membranes. (F) Magnification of infected cells. MtSKOR/GORK labelling in the symbiosome membrane (arrowhead). (G) Double localization of MtAKT1 (red fluorescence) and the endosome marker Rab7 (green fluorescence) in mature infected cells. Rhizobia were not counterstained. Note the same pattern of MtAKT1 (red dots) distribution in the cytoplasm as in (C) where MtAKT1 was displayed as green dots. The green signal for the endosomal marker Rab7 was present as very small dots encompassing individual symbiosomes. Co-localization of green and red signals provided a yellow signal in the endosomes (arrow). IC, infected cell; IT, infection thread; M, meristem; NI, non-infected cell. Scale bars: (A) 10 µm; (B) 12.5 µm; (C) 25 µm; (D) 25 µm; (E–G) 10 µm.

Fig. 5.

Electron microscopy analysis of localization of MtAKT1 and MtSKOR/GORK by immunogold labelling. (A) Immunolabelling of MtAKT1 performed in transgenic nodules carrying the construct ProMtAKT1:MtAKT1:GFP. The immunosignal is revealed by anti-GFP antibody. Note the immunogold particles (arrow) over the endoplasmic reticulum (ER), the plasma membrane region surrounding the cell wall, and in the endosomes (arrowhead). (B) Immunolabelling of MtSKOR/GORK. The signal was revealed by custom-made anti-SKOR antibody. Note the gold particles over the ER and the symbiosome membranes (arrow). CW, cell wall; ER, endoplasmic reticulum; S, symbiosome; VB, vascular bundles. Scale bars: (A, B) 1µm.