microRNA profiling of root tissues and root forming explant cultures in Medicago truncatula

Plant root architecture is regulated by the initiation and modulation of cell division in regions containing pluripotent stem cells known as meristems. In roots, meristems are formed early in embryogenesis, in the case of the root apical meristem (RAM), and during organogenesis at the site of lateral root or, in legumes, nodule formation. Root meristems can also be generated in vitro from leaf explants cultures supplemented with auxin. microRNAs (miRNAs) have emerged as regulators of many key biological functions in plants including root development. To identify key miRNAs involved in root meristem formation in Medicago truncatula, we used deep sequencing to compare miRNA populations. Comparisons were made between: (1) the root tip (RT), containing the RAM and the elongation zone (EZ) tissue and (2) root forming callus (RFC) and non-root forming callus (NRFC). We identified 83 previously reported miRNAs, 24 new to M. truncatula, in 44 families. For the first time in M. truncatula, members of conserved miRNA families miR165, miR181 and miR397 were found. Bioinformatic analysis identified 38 potential novel miRNAs. Selected miRNAs and targets were validated using Taqman miRNA assays and 5′ RACE. Many miRNAs were differentially expressed between tissues, particularly RFC and NRFC. Target prediction revealed a number of miRNAs to target genes previously shown to be differentially expressed between RT and EZ or RFC and NRFC and important in root development. Additionally, we predict the miRNA/target relationships for miR397 and miR160 to be conserved in M. truncatula. Amongst the predictions, were AUXIN RESPONSE FACTOR 10, targeted by miR160 and a LACCASE-like gene, targeted by miR397, both are miRNA/target pairings conserved in other species.     

Structural changes in Medicago truncatula root nodules caused by short-term aluminum stress

Aluminum in the form of Al³⁺ is one of the most toxic heavy metal pollutants in nature and its effects are primarily root-related. Roots of Medicago truncatula exposed to 50 μM of AlCl₃ for 2 h and 24 h were examined by light and electron microscopy. Changes in the appearance of the host cells, infection threads and bacteroidal tissue occurred during the first 2 h of Al stress. Microscopic observations showed that aluminum: (1) induced thickening of plant cell and infection threads (ITs) walls, (2) stimulated IT enlargement, (3) caused disturbances in bacterial release from the ITs, (4) modified cell vacuolation and induced synthesis of granular material and its deposition in the cytoplasm, (5) and caused structural alterations of organella and bacteroids.     

Peptides isolated from cell walls of Medicago truncatula nodules and uninfected root

The hydroxyproline-rich root nodules of legumes provide a microaerobic niche for symbiotic nitrogen-fixing Rhizobacteria. The contributions of the cell wall and associated structural proteins, particularly the hydroxyproline-rich glycoproteins (HRGPs), are therefore of interest. Our approach involved identification of the protein components by direct chemical analysis of the insoluble wall. Chymotryptic peptide mapping showed a "P3-type" extensin containing the highly arabinosylated Ser-Hyp4-Ser-Hyp-Ser-Hyp4-Tyr3-Lys motif as a major component. Cell wall amino acid analyses and quantitative hydroxyproline arabinoside profiles, predominantly of tri- and tetraarabinosides, confirmed this extensin as the major structural protein in the cell walls of both root nodules and uninfected roots. On the other hand, judging from the Pro, Glu and non-glycosylated Hyp content, the nodule-specific proline-rich glycoproteins, such as the early nodulins (ENOD-PRPs), are present in much lesser amounts. Although we isolated no PRP peptides from nodule cell walls, a single PRP peptide from root cell walls confirmed the presence of a PRP in roots and represented the first direct evidence for a crosslinked PRP in muro. Compared with root cell walls (approximately 7% protein dry weight) nodule cell walls contained significantly more protein (approximately 13% dry weight) with an overall amino acid and peptide composition indicating the presence of structural protein unrelated to the HRGPs.     

Cytochemistry of proteolytic activity and pH status of vacuoles in Medicago truncatula root nodules

The vacuole development in root nodules of Medicago truncatula was analyzed by light and electron microscopy. Histochemistry of protease activity in root nodules was studied using fluorogenic substrates for proteolytic enzymes, 7-amino-4-methylcoumarin, CBZ-L-phenylalanyl-L-arginine amide, hydrochloride (AMC), and rhodamine 110, bis-(CBZ-L-phenylalanyl-L-arginine amide) dihydrochloride (RPA). Furthermore, the topology of acidification of the central vacuoles in infected and noninfected cells in root nodules of Medicago truncatula was analyzed with the fluorescent pH-sensitive acidotropic dye Neutral Red. It was shown that vacuoles were acidic, lytic organelles in noninfected cells and young infected cells of the nodule where they displayed protease activity. Mature vacuoles of infected cells had high pH and did not show any substantial protease activity. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 1, pp. 31–38. The text was submitted by the authors in English.     

Metabolite profiling of mycorrhizal roots of Medicago truncatula

Metabolite profiling of soluble primary and secondary metabolites, as well as cell wall-bound phenolic compounds from roots of barrel medic (Medicago truncatula) was carried out by GC-MS, HPLC and LC-MS. These analyses revealed a number of metabolic characteristics over 56 days of symbiotic interaction with the arbuscular mycorrhizal (AM) fungus Glomus intraradices, when compared to the controls, i.e. nonmycorrhizal roots supplied with low and high amounts of phosphate. During the most active stages of overall root mycorrhization, elevated levels of certain amino acids (Glu, Asp, Asn) were observed accompanied by increases in amounts of some fatty acids (palmitic and oleic acids), indicating a mycorrhiza-specific activation of plastidial metabolism. In addition, some accumulating fungus-specific fatty acids (palmitvaccenic and vaccenic acids) were assigned that may be used as markers of fungal root colonization. Stimulation of the biosynthesis of some constitutive isoflavonoids (daidzein, ononin and malonylononin) occurred, however, only at late stages of root mycorrhization. Increase of the levels of saponins correlated AM-independently with plant growth. Only in AM roots was the accumulation of apocarotenoids (cyclohexenone and mycorradicin derivatives) observed. The structures of the unknown cyclohexenone derivatives were identified by spectroscopic methods as glucosides of blumenol C and 13-hydroxyblumenol C and their corresponding malonyl conjugates. During mycorrhization, the levels of typical cell wall-bound phenolics (e.g. 4-hydroxybenzaldehyde, vanillin, ferulic acid) did not change; however, high amounts of cell wall-bound tyrosol were exclusively detected in AM roots. Principal component analyses of nonpolar primary and secondary metabolites clearly separated AM roots from those of the controls, which was confirmed by an hierarchical cluster analysis. Circular networks of primary nonpolar metabolites showed stronger and more frequent correlations between metabolites in the mycorrhizal roots. The same trend, but to a lesser extent, was observed in nonmycorrhizal roots supplied with high amounts of phosphate. These results indicate a tighter control of primary metabolism in AM roots compared to control plants. Network correlation analyses revealed distinct clusters of amino acids and sugars/aliphatic acids with strong metabolic correlations among one another in all plants analyzed; however, mycorrhizal symbiosis reduced the cluster separation and enlarged the sugar cluster size. The amino acid clusters represent groups of metabolites with strong correlations among one another (cliques) that are differently composed in mycorrhizal and nonmycorrhizal roots. In conclusion, the present work shows for the first time that there are clear differences in development- and symbiosis-dependent primary and secondary metabolism of M. truncatula roots.     

Proteomic analysis of Medicago truncatula root plastids

Despite the recognized importance of non‐photosynthetic plastids in a wide array of plant processes, the root plastid proteome of soil‐grown plants still remains to be explored. In this study, we used a protocol allowing the isolation of Medicago truncatula root plastids with sufficient protein recovery and purity for their subsequent in‐depth analysis by nanoscale capillary LC‐MS/MS. Besides providing the first picture of a root plastid proteome, the results obtained highlighted the identification of 266 protein candidates whose functional distribution mainly resembled that of wheat endosperm amyloplasts and tobacco proplastids together with displaying major differences to those reported for chloroplasts. Most of the identified proteins have a role in nucleic acid‐related processes (16%), carbohydrate (15%) and nitrogen/sulphur (12%) metabolisms together with stress response mechanisms (10%). It is noteworthy that BLAST searches performed against the proteins reported in different plastidomes allowed detecting 30 putative root plastid proteins for which homologues were previously unsuspected as plastid‐located, most of them displaying a common putative role in participating in the plant cell responses against abiotic and/or biotic stresses. Taken together, the data obtained provide new insights into the functioning of root plastids and reinforce the emerging idea for an important role of these organelles in sustaining plant defence reactions.     

Microtubule dynamics in root hairs of Medicago truncatula

The microtubular cytoskeleton plays an important role in the development of tip-growing plant cells, but knowledge about its dynamics is incomplete. In this study, root hairs of the legume Medicago truncatula have been chosen for a detailed analysis of microtubular cytoskeleton dynamics using GFP-MBD and EB1-YFP as markers and 4D imaging. The microtubular cytoskeleton appears mainly to be composed of bundles which form tracks along which new microtubules polymerise. Polymerisation rates of microtubules are highest in the tip of growing root hairs. Treatment of root hairs with Nod factor and latrunculin B result in a twofold decrease in polymerisation rate. Nonetheless, no direct, physical interaction between the actin filament cytoskeleton and microtubules could be observed. A new picture of how the plant cytoskeleton is organised in apically growing root hairs emerges from these observations, revealing similarities with the organisation in other, non-plant, tip-growing cells.     

Nitric oxide (NO): a key player in the senescence of Medicago truncatula root nodules

The ectomycorrhizal fungus Tricholoma populinum is host-specific with Populus species. T. populinum has wind-dispersed progagules and may be capable of long-distance dispersal. In this study, we tested the hypothesis of a panmictic population between Scandinavia and North America. DNA sequences from five nuclear loci were used to assess phylogeographic structure and nucleotide divergence between continents. Tricholoma populinum was composed of Scandinavian and North American lineages with complete absence of shared haplotypes and only one shared nucleotide mutation. Divergence of these lineages was estimated at approx. 1.7-1.0 million yr ago (Ma), which occurred after the estimated divergence of host species Populus tremula and Populus balsamifera/Populus trichocarpa at 5 Ma. Phylogeographic structure was not observed within Scandinavian or North American lineages of T. populinum. Intercontinental divergence appears to have resulted from either allopatric isolation; a recent, rare long-distance dispersal founding event followed by genetic drift; or the response in an obligate mycorrhizal fungus with a narrow host range to contractions and expansion of host distribution during glacial and interglacial episodes within continents. Understanding present genetic variation in populations is important for predicting how obligate symbiotic fungi will adapt to present and future changing climatic conditions.     

GC-MS based metabolite profiling of rice Koji fermentation by various fungi

In this study, Aspergillus kawachii, Aspergillus oryzae, and Rhizopus sp., were utilized for rice Koji fermentation, and the metabolites were analyzed in a time-dependent manner by gas chromatography-mass spectrometry. On Principal Component Analysis, the metabolite patterns were clearly distinguished based on the fungi species. This approach revealed that the quantities of glucose, galactose, and glycerol gradually increased as a function of fermentation time in all trials rice Koji fermentation. The time-dependent changes of these metabolites showed significant increases in glucose in the A. oryzae-treated rice, and in glycerol and galactose in the A. kawachii-treated rice. In addition, glycolysis-related enzyme activities were correlated with the changes in these metabolites. The results indicate that time-dependent metabolite production has the potential to be a valuable tool in selecting inoculant fungi and the optimal fermentation time for rice koji.     

Induction of ammonium assimilation: leguminous roots compared with nodules using a split root system

A split root system for nitrogen uptake, in which one part of the root system was exposed to nitrogen-free nutrient and the other to circulated buffered ammonium, was used to investigate the effects of ammonium per se on the enzyme pathway for its assimilation in nodules and roots of leguminous plants. Plants of Trifolium repens L. cv. Grasslands Huia grown in the system showed similar growth and similar free amino acid content in the NH⁺₄-fed roots and in nodulated plants. Studies of ammonium assimilation using [¹³N]-NH⁺₄, applied to Glycine max [L.] Merr. cv. Amsoy plants, showed the label to be assimilated into amino acids in the NH⁺₄-fed roots and to be transported to the tops before subsequently appearing in the minus-N side of the split root system. Analysis of the xylem sap showed [¹³N]-asparagine to be the principal labelled amino acid component. In these plants, levels of both allantoate and the nodule-specific isoenzyme aspartate aminotransferase-P₂ were at least 10 times higher in the NH⁺₄-fed roots than in the minus-N side of the split root system. These studies strongly suggest that a nodule-type of ammonium assimilation was occurring in the NH⁺₄-fed side of the split root, and that this part of the root was transporting assimilatory products to the tops of the plants in a fashion analogous to that of a nitrogen-fixing nodule. These data implicate the involvement of NH⁺₄ in the induction of its own assimilatory pathway.     

Architecture of infection thread networks in developing root nodules induced by the symbiotic bacterium Sinorhizobium meliloti on Medicago truncatula

During the course of the development of nitrogen-fixing root nodules induced by Sinorhizobium meliloti on the model plant Medicago truncatula, tubules called infection threads are cooperatively constructed to deliver the bacterial symbiont from the root surface to cells in the interior of the root and developing nodule. Three-dimensional reconstructions of infection threads inside M. truncatula nodules showed that the threads formed relatively simple, tree-like networks. Some characteristics of thread networks, such as branch length, branch density, and branch surface-to-volume ratios, were remarkably constant across nodules in different stages of development. The overall direction of growth of the networks changed as nodules developed. In 5-d-old nodules, the overall growth of the network was directed inward toward the root. However, well-defined regions of these young networks displayed an outward growth bias, indicating that they were likely in the process of repolarizing their direction of development in response to the formation of the outward-growing nodule meristem. In 10- and 30-d-old nodules, the branches of the network grew outward toward the meristem and away from the roots on which the nodules developed.