Molecular Characterization of Carbonic Anhydrase Genes in Lotus japonicus and Their Potential Roles in Symbiotic Nitrogen Fixation

Carbonic anhydrase (CA) plays a vital role in photosynthetic tissues of higher plants, whereas its non-photosynthetic role in the symbiotic root nodule was rarely characterized. In this study, 13 CA genes were identified in the model legume Lotus japonicus by comparison with Arabidopsis CA genes. Using qPCR and promoter-reporter fusion methods, three previously identified nodule-enhanced CA genes (LjαCA2, LjαCA6, and LjβCA1) have been further characterized, which exhibit different spatiotemporal expression patterns during nodule development. LjαCA2 was expressed in the central infection zone of the mature nodule, including both infected and uninfected cells. LjαCA6 was restricted to the vascular bundle of the root and nodule. As for LjβCA1, it was expressed in most cell types of nodule primordia but only in peripheral cortical cells and uninfected cells of the mature nodule. Using CRISPR/Cas9 technology, the knockout of LjβCA1 or both LjαCA2 and its homolog, LjαCA1, did not result in abnormal symbiotic phenotype compared with the wild-type plants, suggesting that LjβCA1 or LjαCA1/2 are not essential for the nitrogen fixation under normal symbiotic conditions. Nevertheless, the nodule-enhanced expression patterns and the diverse distributions in different types of cells imply their potential functions during root nodule symbiosis, such as CO2 fixation, N assimilation, and pH regulation, which await further investigations.

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

Root nodule-infected cells have defects in K+ balance, as compared with non-infected cells, probably due to variation in the location of K+ channel proteins 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.

Soybean Yellow Stripe-like7 is a symbiosome membrane peptide transporter essential for nitrogen fixation

ABSTRACTLegumes form a symbiosis with rhizobia that convert atmospheric nitrogen (N2) to ammonia which they provide to the plant in return for a carbon and nutrient supply. Nodules, developed as part of the symbiosis, harbor rhizobia which are enclosed in the plant-derived symbiosome membrane (SM), to form a symbiosome. In the mature nodule all exchanges between the symbionts occur across the SM. Here we characterize GmYSL7, a member of Yellow stripe-like family which is localized to the SM in soybean nodules. It is expressed specifically in nodule infected cells with expression peaking soon after nitrogenase becomes active. Although most members of the family transport metal complexed with phytosiderophores, GmYSL7 does not. It transports oligopeptides of between four and 12 amino acids. Silencing of GmYSL7 reduces nitrogenase activity and blocks development when symbiosomes contain a single bacteroid. RNAseq of nodules in which GmYSL7 is silenced suggests that the plant initiates a defense response against the rhizobia. There is some evidence that metal transport in the nodules is dysregulated, with upregulation of genes encoding ferritin and vacuolar iron transporter family and downregulation of a gene encoding nicotianamine synthase. However, it is not clear whether the changes are a result of the reduction of nitrogen fixation and the requirement to store excess iron or an indication of a role of GmYSL7 in regulation of metal transport in the nodules. Further work to identify the physiological substrate for GmYSL7 will allow clarification of this role.One sentence summaryGmYSL7 is a symbiosome membrane peptide transporter that is essential for symbiotic nitrogen fixation that when silenced blocks symbiosome development.

Refined analysis of early symbiotic steps of the Rhizobium-Medicago interaction in relationship with microtubular cytoskeleton rearrangements

In situ immunolocalization of tubulin revealed that important rearrangements occur during all the early symbiotic steps in the Medicago/R. meliloti symbiotic interaction. Microtubular cytoskeleton (MtC) reorganizations were observed in inner tissues, first in the pericycle and then in the inner cortex where the nodule primordium forms. Subsequently, major MtC changes occurred in outer tissues, associated with root hair activation and curling, the formation of preinfection threads (PITs) and the initiation and the growth of an infection network. From the observed sequence of MtC changes, we propose a model which aims to better define, at the histological level, the timing of the early symbiotic stages. This model suggests the existence of two opposite gradients of cell differentiation controlling respectively the formation of division centers in the inner cortex and plant preparation for infection. It implies that (i) MtC rearrangements occur in pericycle and inner cortex earlier than in the root hair, (ii) the infection process proceeds prior to the formation of the nodule meristem, (iii) the initial primordium prefigures the future zone II of the mature nodule and (iv) the nodule meristem derives from the nodule primordium. Finally, our data also strongly suggest that in alfalfa PIT differentiation, a stage essential for successful infection, requires complementary signaling additional to Nod factors.

Early development of Rhizobium-induced root nodules of Parasponia rigida. II. Nodule morphogenesis and symbiotic development

The Rhizobium-induced root nodules of Parasponia rigida (Ulmaceae) outwardly resemble those formed on actinorhizal plants, being coralloid in shape and consisting of multiple, branched lobes. The details of nodule morphogenesis also resemble more closely those which occur in an actinorhizal association than a typical Rhizobium–legume association and include prenodule formation, initiation of modified lateral roots which are termed nodule lobe primordia, and rhizobial colonization of tissues derived from the nodule lobe primordia to form the primary nodule lobes. Mature nodule lobe structure is actinorhizallike. Each lobe has an apical meristem and a central vascular cylinder which is surrounded by an uninfected inner cortex and then a zone of infected tissue. Peripheral to the infected tissue is an uninfected outer cortex. Infection threads and intercellular rhizobia progress continuously toward the apical meristem but do not infect the meristem itself. The establishment of the symbiosis in the host cells involves continuous thread formation after the initial infection until the host cells are nearly filled with rhizobia enclosed in threads. The rhizobia remain in threads throughout the symbiotic relationship and are not released from the threads as occurs in bacteroid formation in legumes.

Preliminary evidence for the involvement of suberization in infection of Casuarina

Histochemistry of infected cells in mature nodule lobes of Casuarina showed that walls of infected host cells had a ligninlike component (ultraviolet-stimulated autofluorescence and staining with auramine O, phloroglucinol staining, and resistance to degradation by hydrolytic enzymes). Cytoplasm of infected cells had a pronounced affinity for lipid stains (Sudan black B, Rose Bengal fluorescence), though walls of infected cells were less clearly stained. When nodules were digested several days in cold 50% chromic acid, the walls of infected cells and suberized host tissue (epidermis, endodermis) were not degraded. Endophyte cell wall components were also found to be resistant to chromic acid digestion. The digested tissue retained the capacity to adsorb lipid dyes. These observations suggested that walls of infected host cells had become impregnated with a suberinlike compound. The hydrophobic quality of this wall was evident when its ultrastructure was examined after en bloc staining with the polar stain KMnO4. This stain did not penetrate the walls of mature infected cells, perhaps because of the presence of aliphatic compounds similar to those found in suberin. As is known for suberizing tissue, peroxidase activity (via diaminobenzidine oxidation) was high in nodule cortical tissue cell walls. The peroxidase stain was also localized on endophyte hyphae. This report is the first instance associating a suberizationlike host reaction with infection of an actinorhizal plant.

Histological studies of ectomycorrhizae and root nodules from Cercocarpus montanus and Cercocarpus paucidentatus

The ectomycorrhizae of Cercocarpus montanus Raf. and Cercocarpus paucidentatus Britt. displayed morphologies ranging from single swollen short lateral roots on long roots to terminal pyramidal clusters. Most short roots appeared to be mycorrhizal, although C. paucidentatus was only infected under growth chamber conditions. Histological sections revealed a conspicuous fungal mantle, averaging 30 μ in thickness, and a Hartig's net.The root nodules appeared as swellings on lateral roots, and later formed compact coralloid orange-colored masses several centimeters in diameter. Histological analyses indicated that both species of Cercocarpus harbored a similar endophyte. Three developmental stages were noted in cortical tissue, including (a) hyphal masses in apical nodule cells; (b) hyphae terminating in 3 × 4 μ club-shaped vesicular swellings; and (c) older structureless hyphal masses in cells of mature nodule branches. The older hyphal masses did not appear to be absorbed by the host plant. The endophyte possessed branching filaments 0.5 μ in diameter and was considered to be an actinomycete.