The plasma membrane proteome of Medicago truncatula roots as modified by arbuscular mycorrhizal symbiosis.

In arbuscular mycorrhizal (AM) roots, the plasma membrane (PM) of the host plant is involved in all developmental stages of the symbiotic interaction, from initial recognition to intracellular accommodation of intra-radical hyphae and arbuscules. Although the role of the PM as the agent for cellular morphogenesis and nutrient exchange is especially accentuated in endosymbiosis, very little is known regarding the PM protein composition of mycorrhizal roots. To obtain a global overview at the proteome level of the host PM proteins as modified by symbiosis, we performed a comparative protein profiling of PM fractions from Medicago truncatula roots either inoculated or not with the AM fungus Rhizophagus irregularis.

Changes in plastid proteome and structure in arbuscular mycorrhizal roots display a nutrient starvation signature.

During arbuscular mycorrhizal symbiosis, arbuscule-containing root cortex cells display a proliferation of plastids, a feature usually ascribed to an increased plant anabolism despite the lack of studies focusing on purified root plastids. In this study, we investigated mycorrhiza-induced changes in plastidic pathways by performing a label-free comparative subcellular quantitative proteomic analysis targeted on plastid-enriched fractions isolated from Medicago truncatula roots, coupled to a cytological analysis of plastid structure. We identified 490 root plastid protein candidates, among which 79 changed in abundance upon mycorrhization, as inferred from spectral counting.

Direct purification of detergent-insoluble membranes from Medicago truncatula root microsomes: comparison between floatation and sedimentation.

Background: Membrane microdomains are defined as highly dynamic, sterol- and sphingolipid-enriched domains that resist to solubilization by non-ionic detergents. In plants, these so-called Detergent Insoluble Membrane (DIM) fractions have been isolated from plasma membrane by using conventional ultracentrifugation on density gradient (G). In animals, a rapid (R) protocol, based on sedimentation at low speed, which avoids the time-consuming sucrose gradient, has also been developed to recover DIMs from microsomes as starting material.

The membrane proteome of Medicago truncatula roots displays qualitative and quantitative changes in response to arbuscular mycorrhizal symbiosis.

Unlabelled: Arbuscular mycorrhizal (AM) symbiosis that associates roots of most land plants with soil-borne fungi (Glomeromycota), is characterized by reciprocal nutritional benefits. Fungal colonization of plant roots induces massive changes in cortical cells where the fungus differentiates an arbuscule, which drives proliferation of the plasma membrane. Despite the recognized importance of membrane proteins in sustaining AM symbiosis, the root microsomal proteome elicited upon mycorrhiza still remains to be explored.

Protein actors sustaining arbuscular mycorrhizal symbiosis: underground artists break the silence.

The roots of most land plants can enter a relationship with soil-borne fungi belonging to the phylum Glomeromycota. This symbiosis with arbuscular mycorrhizal (AM) fungi belongs to the so-called biotrophic interactions, involving the intracellular accommodation of a microorganism by a living plant cell without causing the death of the host. Although profiling technologies have generated an increasing depository of plant and fungal proteins eligible for sustaining AM accommodation and functioning, a bottleneck exists for their functional analysis as these experiments are difficult to carry out with mycorrhiza.

Gel-based and gel-free quantitative proteomics approaches at a glance.

Two-dimensional gel electrophoresis (2-DE) is widely applied and remains the method of choice in proteomics; however, pervasive 2-DE-related concerns undermine its prospects as a dominant separation technique in proteome research. Consequently, the state-of-the-art shotgun techniques are slowly taking over and utilising the rapid expansion and advancement of mass spectrometry (MS) to provide a new toolbox of gel-free quantitative techniques. When coupled to MS, the shotgun proteomic pipeline can fuel new routes in sensitive and high-throughput profiling of proteins, leading to a high accuracy in quantification.

Influence of arbuscular mycorrhizal colonisation on cadmium induced Medicago truncatula root isoflavonoid accumulation.

Cadmium is a serious environmental pollution threats to the planet. Its accumulation in plants affects many cellular functions, resulting in growth and development inhibition, whose mechanisms are not fully understood. However, some fungi forming arbuscular mycorrhizal symbiosis with the majority of plant species have the capacity to buffer the deleterious effect of this heavy metal.

Optimization of iTRAQ labelling coupled to OFFGEL fractionation as a proteomic workflow to the analysis of microsomal proteins of Medicago truncatula roots.

Background: Shotgun proteomics represents an attractive technical framework for the study of membrane proteins that are generally difficult to resolve using two-dimensional gel electrophoresis. The use of iTRAQ, a set of amine-specific isobaric tags, is currently the labelling method of choice allowing multiplexing of up to eight samples and the relative quantification of multiple peptides for each protein. Recently the hyphenation of different separation techniques with mass spectrometry was used in the analysis of iTRAQ labelled samples.

Arbuscular mycorrhizal symbiosis elicits shoot proteome changes that are modified during cadmium stress alleviation in Medicago truncatula.

Background: Arbuscular mycorrhizal (AM) fungi, which engage a mutualistic symbiosis with the roots of most plant species, have received much attention for their ability to alleviate heavy metal stress in plants, including cadmium (Cd). While the molecular bases of Cd tolerance displayed by mycorrhizal plants have been extensively analysed in roots, very little is known regarding the mechanisms by which legume aboveground organs can escape metal toxicity upon AM symbiosis. As a model system to address this question, we used Glomus irregulare-colonised Medicago truncatula plants, which were previously shown to accumulate and tolerate heavy metal in their shoots when grown in a substrate spiked with 2 mg Cd kg(-1).

Arbuscular mycorrhizal symbiosis elicits proteome responses opposite of P-starvation in SO4 grapevine rootstock upon root colonisation with two Glomus species.

Although plant biotisation with arbuscular mycorrhizal fungi (AMF) is a promising strategy for improving plant health, a better knowledge regarding the molecular mechanisms involved is required. In this context, we sought to analyse the root proteome of grapevine rootstock Selection Oppenheim 4 (SO4) upon colonisation with two AMF. As expected, AMF colonisation stimulates plant biomass.

Transcription of two blue copper-binding protein isogenes is highly correlated with arbuscular mycorrhizal development in Medicago truncatula.

Expression profiling of two paralogous arbuscular mycorrhizal (AM)-specific blue copper-binding gene (MtBcp1a and MtBcp1b) isoforms was performed by real-time quantitative polymerase chain reaction in wild-type Medicago truncatula Jemalong 5 (J5) during the mycorrhizal development with Glomus intraradices for up to 7 weeks. Time-course analysis in J5 showed that expression of both MtBcp1 genes increased continuously and correlated strongly with the colonization intensity and arbuscule content. MtPT4, selected as a reference gene of the functional plant-fungus association, showed a weaker correlation to mycorrhizal development.

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.

Identification of in planta-expressed arbuscular mycorrhizal fungal proteins upon comparison of the root proteomes of Medicago truncatula colonised with two Glomus species.

In the absence of sequenced genomes for arbuscular mycorrhizal (AM) fungi, their obligatory biotrophy makes their intra-radical biology especially recalcitrant to functional analyses. Because tandem mass spectrometry-based proteomics enables fungal gene product identifications in phyla lacking genomic information, we have compared as a way to enlarge the coverage of in planta expressed-mycorrhiza-related proteins, the root proteome responses of Medicago truncatula upon colonisation with two AM fungi, Glomus mosseae and G. intraradices, using two-dimensional electrophoresis.

Differential expression proteomics to investigate responses and resistance to Orobanche crenata in Medicago truncatula.

Background: Parasitic angiosperm Orobanche crenata infection represents a major constraint for the cultivation of legumes worldwide. The level of protection achieved to date is either incomplete or ephemeral. Hence, an efficient control of the parasite requires a better understanding of its interaction and associated resistance mechanisms at molecular levels.

On the mechanisms of cadmium stress alleviation in Medicago truncatula by arbuscular mycorrhizal symbiosis: a root proteomic study.

The arbuscular mycorrhizal (AM) symbiosis belongs to the strategies plants have developed to cope with adverse environmental conditions including contamination by heavy metals such as cadmium (Cd). In the present work, we report on the protective effect conferred by AM symbiosis to the model legume Medicago truncatula grown in presence of Cd, and on the 2-D-based proteomic approach further used to compare the proteomes of M. truncatula roots either colonised or not with the AM fungus Glomus intraradices in Cd-free and Cd-contaminated substrates.

Cadmium effects on populations of root nuclei in two pea genotypes inoculated or not with the arbuscular mycorrhizal fungus Glomus mosseae.

Plants possess a broad range of strategies to cope with cadmium (Cd) stress, including the arbuscular mycorrhizal (AM) symbiosis. In cell responses towards Cd, the contribution of changes in ploidy levels is still unclear. We used flow cytometry to investigate if nuclear ploidy changes are involved in response mechanisms toward Cd and to analyze the effect of the symbiotic status on populations of nuclei.

Mutations in DMI3 and SUNN modify the appressorium-responsive root proteome in arbuscular mycorrhiza.

Modification of the Medicago truncatula root proteome during the early stage of arbuscular mycorrhizal symbiosis was investigated by comparing, using two-dimensional electrophoresis, the protein patterns obtained from non-inoculated roots and roots synchronized for Glomus intraradices appressorium formation. This approach was conducted in wild-type (J5), mycorrhiza-defective (TRV25, dmi3), and autoregulation-defective (TR122, sunn) M. truncatula genotypes.

A mass spectrometric approach to identify arbuscular mycorrhiza-related proteins in root plasma membrane fractions.

One of the most important morphological changes occurring in arbuscular mycorrhizal (AM) roots takes place when the plant plasma membrane (PM) invaginates around the fungal arbuscular structures resulting in the periarbuscular membrane formation. To investigate whether AM symbiosis-specific proteins accumulate at this stage, two complementary MS approaches targeting the root PM from the model legume Medicago truncatula were designed. Membrane extracts were first enriched in PM using a discontinuous sucrose gradient method.

Identification of membrane-associated proteins regulated by the arbuscular mycorrhizal symbiosis.

A sub-cellular proteomic approach was carried out to monitor membrane-associated protein modifications in response to the arbuscular mycorrhizal (AM) symbiosis. Membrane proteins were extracted from Medicago truncatula roots either inoculated or not with the AM fungus Glomus intraradices. Comparative two-dimensional electrophoresis revealed that 36 spots were differentially displayed in response to the fungal colonization including 15 proteins induced, 3 up-regulated and 18 down-regulated.

Impact of sewage sludges on Medicago truncatula symbiotic proteome.

The effects of sewage sludges were investigated on the symbiotic interactions between the model plant Medicago truncatula and the arbuscular mycorrhizal fungus Glomus mosseae or the rhizobial bacteria Sinorhizobium meliloti. By comparison to a control sludge showing positive effects on plant growth and root symbioses, sludges enriched with polycylic aromatic hydrocarbons or heavy metals were deleterious. Symbiosis-related proteins were detected and identified by two-dimensional electrophoresis and matrix-assisted laser desorption ionization mass spectrometry, and image analysis was used to study the effects of sewage sludges on M.

A proteomic approach to studying plant response to crenate broomrape (Orobanche crenata) in pea (Pisum sativum).

Crenate broomrape (Orobanche crenata) is a parasitic plant that threatens legume production in Mediterranean areas. Pea (Pisum sativum) is severely affected, and only moderate levels of genetic resistance have so far been identified. In the present work we selected the most resistant accession available (Ps 624) and compared it with a susceptible (Messire) cultivar.

Sub-cellular proteomic analysis of a Medicago truncatula root microsomal fraction.

Since the last decade, Medicago truncatula has emerged as one of the model plants particularly investigated in the field of plant-microbe interactions. Several genetic and molecular approaches including proteomics have been developed to increase knowledge about this plant species. To complement the proteomic data, which have mainly focused on the total root proteins from M.

Proteomics as a way to identify extra-radicular fungal proteins from Glomus intraradices- RiT-DNA carrot root mycorrhizas.

To identify fungal proteins involved in the arbuscular mycorrhizal symbiosis, root-inducing transferred-DNA transformed roots of carrot (Daucus carota L.) were in vitro inoculated with Glomus intraradices. Proteins extracted from the extra-radical fungus were analysed by two-dimensional gel electrophoresis.

A technical trick for studying proteomics in parallel to transcriptomics in symbiotic root-fungus interactions.

We have developed a protocol in which proteins and mRNA can be analyzed from single root samples. This experimental design was validated in arbuscular mycorrhiza by comparing the proteins profiles obtained with those from a classical protein extraction process. It is a step forward to make simultaneous proteome and transcriptiome profiling possible.