Medicago Truncatula Roots Research Articles

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

BackgroundShotgun 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. OFFGEL electrophoresis has proved its effectiveness in isoelectric point-based peptide and protein separation in solution. Here we describe the first application of iTRAQ-OFFGEL-LC-MS/MS on microsomal proteins from plant material. The investigation of the iTRAQ labelling effect on peptide electrofocusing in OFFGEL fractionator was carried out on Medicago truncatula membrane protein digests.ResultsIn-filter protein digestion, with easy recovery of a peptide fraction compatible with iTRAQ labelling, was successfully used in this study. The focusing quality in OFFGEL electrophoresis was maintained for iTRAQ labelled peptides with a higher than expected number of identified peptides in basic OFFGEL-fractions. We furthermore observed, by comparing the isoelectric point (pI) fractionation of unlabelled versus labelled samples, a non-negligible pI shifts mainly to higher values.ConclusionsThe present work describes a feasible and novel protocol for in-solution protein digestion in which the filter unit permits protein retention and buffer removal. The data demonstrates an impact of iTRAQ labelling on peptide electrofocusing behaviour in OFFGEL fractionation compared to their native counterpart by the induction of a substantial, generally basic pI shift. Explanations for the occasionally observed acidic shifts are likewise presented.

Gene expression profiling of Macrophomina phaseolina infected Medicago truncatula roots reveals a role for auxin in plant tolerance against the charcoal rot pathogen

The soil-borne necrotrophic fungal pathogen Macrophomina phaseolina causes charcoal rot disease in a wide range of plant species. There are no effective control methods for this disease and no genetic resistance for M. phaseolina has been identified in most field crops. Currently, little is known about the molecular mechanisms involved in host susceptibility and resistance to M. phaseolina. Using Medicago truncatula as a model, we analyzed the global gene expression profile of M. truncatula roots infected by M. phaseolina. MAPMAN analyses identified genes involved in jasmonic acid and ethylene pathways, which are important for plant resistance against necrotrophs. In addition, genes involved in auxin homeostasis, polar auxin transport and auxin signalling were also regulated by the infection process. The differential expression patterns of these auxin-related genes suggested that the host susceptibility maybe partly due to the suppression of auxin response in the host by M. phaseolina. In additional, M. truncatula plants treated with exogenous auxin gained partial resistance against M. phaseolina, further suggesting the potential role for auxin in plant defense against this necrotrophic pathogen.

A switch in Ca2+ spiking signature is concomitant with endosymbiotic microbe entry into cortical root cells of Medicago truncatula

Ca(2+) spiking is a central component of a common signaling pathway that is activated in the host epidermis during initial recognition of endosymbiotic microbes. However, it is not known to what extent Ca(2+) signaling also plays a role during subsequent root colonization involving apoplastic transcellular infection. Live-tissue imaging using calcium cameleon reporters expressed in Medicago truncatula roots has revealed that distinct Ca(2+) oscillatory profiles correlate with specific stages of transcellular cortical infection by both rhizobia and arbuscular mycorrhizal fungi. Outer cortical cells exhibit low-frequency Ca(2+) spiking during the extensive intracellular remodeling that precedes infection. This appears to be a prerequisite for the formation of either pre-infection threads or the pre-penetration apparatus, both of which are fully reversible processes. A transition from low- to high-frequency spiking is concomitant with the initial stages of apoplastic cell entry by both microbes. This high-frequency spiking is of limited duration in the case of rhizobial infection and is completely switched off by the time transcellular infection by both microsymbionts is completed. The Ca(2+) spiking profiles associated with both rhizobial and arbuscular mycorrhizal cell entry are remarkably similar in terms of periodicity, suggesting that microbe specificity is unlikely to be encoded by the Ca(2+) signature during this particular stage of host infection in the outer cortex. Together, these findings lead to the proposal that tightly regulated Ca(2+) -mediated signal transduction is a key player in reprogramming root cell development at the critical stage of commitment to endosymbiotic infection.

Ethylene-responsive miRNAs in roots of Medicago truncatula identified by high-throughput sequencing at whole genome level

Ethylene is one of the classical plant hormones with a diverse function in plant growth and development. Root elongation is sensitive to ethylene such that treatments with ethylene and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) inhibit root growth. MicroRNA as one type of endogenous, non-coding small RNAs, plays an important role in regulation of plant growth, development and hormonal signaling by affecting expression of target genes. However, there has been no detailed study to evaluate the role of microRNAs in mediation of ethylene-dependent physiological processes in plants. Medicago truncatula is a model plant widely used for investigation of molecular biology in legume species. In this study, we constructed two small RNA libraries from roots of M. truncatula treated with and without ACC. High-throughput sequencing was employed to sequence the small RNA libraries, and more than 30M raw reads were obtained. We annotated 301 known miRNAs and identified 3 new miRNAs in the two libraries. Treatment of M. truncatula with 10μM ACC led to changes in expression of 8 miRNAs. The targets of the ethylene-responsive miRNAs were predicted by bioinformatic approach. The potential role of the ethylene-responsive miRNAs in the ethylene-induced inhibition of root elongation is discussed. These results are useful for functional characterization of miRNAs in mediation of ethylene-dependent physiological processes in general and root elongation in particular.

Pseudonodule Formation by Wild-Type and Symbiotic Mutant Medicago truncatula in Response to Auxin Transport Inhibitors

Rhizobium and allied bacteria form symbiotic nitrogen-fixing nodules on legume roots. Plant hormones play key roles in nodule formation. We treated Medicago truncatula roots with auxin transport inhibitors (ATI) N-(1-naphthyl)phthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA) to induce the formation of pseudonodules. M. truncatula mutants defective for rhizobial Nod factor signal transduction still formed pseudonodules in response to ATI. However, a M. truncatula ethylene-insensitive supernodulator, sickle 1-1, did not form pseudonodules in response to TIBA, suggesting that the ethylene response pathway is involved in ATI-induced pseudonodule formation. We compared the transcriptional responses of M. truncatula roots treated with ATI to roots inoculated with Sinorhizobium meliloti. Some genes showed consistently parallel expression in ATI-induced and Rhizobium-induced nodules. For other genes, the transcriptional response of M. truncatula roots 1 and 7 days after ATI treatment was in the opposite direction to roots treated with S. meliloti; then, by 21 days, the transcriptional patterns for the two conditions became similar. We silenced 17 genes that were upregulated in both ATI and S. meliloti treatments to determine their effect on nodule formation. Some gene-silenced roots showed a decrease in nodulation efficiency, suggesting a role in nodule formation but not in later nodule functions.

Laser microdissection unravels cell-type-specific transcription in arbuscular mycorrhizal roots, including CAAT-box transcription factor gene expression correlating with fungal contact and spread.

Arbuscular mycorrhizae (AM) are the most widespread symbioses on Earth, promoting nutrient supply of most terrestrial plant species. To unravel gene expression in defined stages of Medicago truncatula root colonization by AM fungi, we here combined genome-wide transcriptome profiling based on whole mycorrhizal roots with real-time reverse transcription-PCR experiments that relied on characteristic cell types obtained via laser microdissection. Our genome-wide approach delivered a core set of 512 genes significantly activated by the two mycorrhizal fungi Glomus intraradices and Glomus mossae. Focusing on 62 of these genes being related to membrane transport, signaling, and transcriptional regulation, we distinguished whether they are activated in arbuscule-containing or the neighboring cortical cells harboring fungal hyphae. In addition, cortical cells from nonmycorrhizal roots served as a reference for gene expression under noncolonized conditions. Our analysis identified 25 novel arbuscule-specific genes and 37 genes expressed both in the arbuscule-containing and the adjacent cortical cells colonized by fungal hyphae. Among the AM-induced genes specifying transcriptional regulators were two members encoding CAAT-box binding transcription factors (CBFs), designated MtCbf1 and MtCbf2. Promoter analyses demonstrated that both genes were already activated by the first physical contact between the symbionts. Subsequently, and corresponding to our cell-type expression patterns, they were progressively up-regulated in those cortical areas colonized by fungal hyphae, including the arbuscule-containing cells. The encoded CBFs thus represent excellent candidates for regulators that mediate a sequential reprogramming of root tissues during the establishment of an AM symbiosis.

Stars and Symbiosis: MicroRNA- and MicroRNA*-Mediated Transcript Cleavage Involved in Arbuscular Mycorrhizal Symbiosis

The majority of plants are able to form the arbuscular mycorrhizal (AM) symbiosis in association with AM fungi. During symbiosis development, plant cells undergo a complex reprogramming resulting in profound morphological and physiological changes. MicroRNAs (miRNAs) are important components of the regulatory network of plant cells. To unravel the impact of miRNAs and miRNA-mediated mRNA cleavage on root cell reprogramming during AM symbiosis, we carried out high-throughput (Illumina) sequencing of small RNAs and degradome tags of Medicago truncatula roots. This led to the annotation of 243 novel miRNAs. An increased accumulation of several novel and conserved miRNAs in mycorrhizal roots suggest a role of these miRNAs during AM symbiosis. The degradome analysis led to the identification of 185 root transcripts as mature miRNA and also miRNA*-mediated mRNA cleavage targets. Several of the identified miRNA targets are known to be involved in root symbioses. In summary, the increased accumulation of specific miRNAs and the miRNA-mediated cleavage of symbiosis-relevant genes indicate that miRNAs are an important part of the regulatory network leading to symbiosis development.

Hydrogen peroxide scavenging mechanisms are components of Medicago truncatula partial resistance to Aphanomyces euteiches

The biochemical processes underlying the expression of resistance in the roots of Medicago truncatula against Aphanomyces euteiches infection was investigated, with emphasis on oxidative stress. The levels of H2O2, superoxide dismutase, peroxidase, ascorbate peroxidase, catalase, soluble phenolics and lignin were measured in the roots of two lines, A17 partially resistant and F83005.5 susceptible to A. euteiches at three infection stages; penetration of the epidermis (1 dpi), colonization of the cortex (3 dpi) and invasion of the root stele (6 dpi). A rapid and large decrease of the H2O2 levels in A17 roots occurred. However, in F83005.5 roots, the decrease in H2O2 levels was delayed until 3 dpi. In A17 roots, the activities of ascorbate peroxidase, peroxidase and catalase were induced as early as 1 dpi, whereas a general decrease in the activity of the four antioxidant enzymes was observed in F83005.5 roots. The levels of soluble phenolics and lignin were increased in A17 roots at 3 and 6 dpi, respectively. The H2O2 levels were negatively correlated to ascorbate peroxidase, catalase and lignin production at 1, 3 and 6 dpi, respectively in A17 roots. Physiological concentrations of H2O2 found in M. truncatula infected roots had no detrimental effect on the in vitro growth of this oomycete. Our data suggest that H2O2 does not have a direct antimicrobial effect on M. truncatula resistance to A. euteiches, but is involved in cell wall strengthening around the root stele, preventing pathogen invasion of the vascular tissues.

THE EFFECT OF MYCORRHIZA GROWTH AND EXPRESSION OF SOME GENES IN BARLEY

Barley is one of the most important crops for food consumption. Soil fertilization using minerals and chemical compounds are not safe for human health beside beingas expensive. Therefore, It is important to find suitable alternatives. The arbuscular mycorrhiza (AM) is characterized by the formation of arbuscules (sometimes vesicles). The fungus occupies various proportions of the volume of host cells but is separated from the cell cytoplasm by the elaboration of the host plasma membrane forming a fungal membrane and an apoplectic compartment between the fungus and host cytoplasm. Mycorrhiza changes whatever source of phosphorus into phosphoric acid which acidifies the rhizosphere and thus increases the availability of elements for plants uptake. The increase of the availability of elements specially, calcium which plays the main role in cell signaling which could lead to switch on or accelerate the function of several genes such as protein kinases and other genes related with photosynthesis and eventially, affect the plant growth. Inoculation with mycorrhiza increased all plant growth variables such as number of leaves, stem fresh and dry weights and root dry weight Shockley et al. (2004). Gahoonia and Nielsen (2004) reported that the inoculation with mycorrhiza enhanced the release of organic acids and higher acid phosphatase activity in the rhizosphere which may be useful for increasing phosphorus acquisition from inorganic and organic phosphorus pools to the plant. Al-Karaki et al. (2004) found that biomass and grain yields were higher in mycorrhizal than non-mycorrhizal plants. Wu et al. (2005) reported biofertilizer as an alternative to chemical one to increase soil fertility and crop production. Glassop et al. (2005) reported that a very large number of plant species are capable of forming symbiotic associations with arbuscular mycorrhizal (AM) fungi. Moreover, they reported that a large number of phosphate (P) transporters have been identified in plants; tissue expression patterns and kinetic information supports the roles of some of these in the direct root uptake pathways. Chen et al. (2007) indicated that the beneficial impacts of mycorrhizal colonization on plant growth could be largely explained by improved P nutrition. On the other hand, Baltruschat et al. (2008) found that the root endophytic basidiomycete Piriformospora indica increase the tolerance against abiotic stress as in many plants. Gryndler et al. (2009) concluded that mycelia of AM fungi are influenced by organic matter decomposition via release of the compounds during the decomposition process and also by secondary metabolites produced by microorganisms involved in organic matter decomposition. Araim et al. (2009) reported that AM colonization significantly increased the mass of shoots and roots and the concentrations of proteins and most of the phenolics in the roots. Hence, the selected trait of mycorrhiza could play an important role in optimizing the growth of E. purpurea by inducing the production of secondary phytomedicinal metabolites. The impact of mycorrhizaon gene expression has been addressed by some recent studies, Hause et al. (2002) reported that colonization of barley (Hordeum vulgare Cv. Salome) roots by an arbuscular mycorrhizal fungus, led to elevated levels of endogenous jasmonic acid (JA) and its amino acid conjugate JA-isoleucine, whereas the level of the JA precursor remained constant. The rise in jasmonate was accompanied by the expression of genes coding for an enzyme of JA biosynthesis. Delp et al. (2003) performed relative quantitative RT-PCR and western blotting to investigate the expression of three genes with potentially regulatory functions from the arbuscular mycorrhizal fungus Glomus intraradices in symbiosis with barley. Weidmann et al. (2004) identified 29 plant genes which were upregulated in response to Medicago truncatula root inoculation with mycorrhiza. Twenty-two protein spots, 14 upregulated and 8 downregulated were found changed in mycorrhiza inoculated plants as compared with the non inoculated ones by Liang et al. (2007). Abo Doma et al. (2008) indicated that the treatment with mycorrhiza significantly increased plant growth in two cultivars of wheat using different yield related traits. Moreover, transcriptome analysis using real timereverse transcriptase indicated that mycorrhiza treatment resulted in an increase in the gene expression of proline 5 carboxilate synthetase (P5CS) on the transcription level. Roldan et al. (2008) indicated that differential gene expression for genes conferring superoxide dismutase and peroxidases were recorded between mycorrhizal and non-mycorrhizal shoots of juniperus oxycedrus seedling. This investigation aimed to: 1- study the effect of barley root inoculation with mycorrhiza on some plant traits and 2-determine the changes in gene expression of the genes conferring calcium dependent protein kinases (CDPKs), phosphoenol pyrovate carboxylases (PEPCs) and proline-5 carboxylate synthetase (P-5CS) in barley plants in response to their inoculation with mycorrhiza at the molecular level as quantified using RT-PCR.

Metabolite Profiling of Triterpene Saponins in Medicago truncatula Hairy Roots by Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Triterpenes are one of the largest classes of plant natural products, with an enormous variety in structure and bioactivities. Here, triterpene saponins from hairy roots of the model legume Medicago truncatula were profiled with reversed-phase liquid chromatography coupled to negative-ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS). Owing to the accuracy of the FT-ICR MS, reliable molecular formulas of the detected compounds could be predicted, which, together with the generated MS(n) spectra, allowed the tentative identification of 79 different saponins, of which 61 had not been detected previously in M. truncatula. Upon collision-induced dissociation of saponins that contain a uronic acid residue in the sugar chain, fragment ions resulting from cross-ring cleavages of the uronic acid residues were observed. The identified saponins are glycosides of 10 different sapogenins, of which three were not detected before in M. truncatula. Zanhic acid glycosides, which are prevalent in the aerial parts of M. truncatula, were absent in the hairy root extracts. This metabolite compendium will facilitate future functional genomic studies of triterpene saponin biosynthesis in M. truncatula.

LC/MS profiling of flavonoid glycoconjugates isolated from hairy roots, suspension root cell cultures and seedling roots of Medicago truncatula

Hairy roots and suspension cell cultures are commonly used in deciphering different problems related to the biochemistry and physiology of plant secondary metabolites. Here, we address about the issue of possible differences in the profiles of flavonoid compounds and their glycoconjugates derived from various plant materials grown in a standard culture media. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and suspension root cell cultures of a model legume plant, Medicago truncatula. The analyses were conducted with plant isolates as well as the media. The LC/MS profiles of target natural products obtained from M. truncatula seedling roots, hairy roots, and suspension root cell cultures differed substantially. The most abundant compounds in seedlings roots were mono- and diglucuronides of isoflavones and/or flavones. This type of glycosylation was not observed in hairy roots or suspension root cell cultures. The only recognized glycoconjugates in the latter samples were glucose derivatives of isoflavones. Application of a high-resolution mass spectrometer helped evaluate the elemental composition of protonated molecules, such as [M + H]+. Comparison of collision-induced dissociation MS/MS spectra registered with a quadrupole time-of-flight analyzer for tissue extracts and standards allowed us to estimate the aglycone structure on the basis of the pseudo-MS3 experiment. Structures of these natural products were described according to the registered mass spectra and literature data. The analyses conducted represent an overview of flavonoids and their conjugates in different types of plant material representing the model legume, M. truncatula.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-011-0287-2) contains supplementary material, which is available to authorized users.

The LOB-like transcription factor MtLBD1 controls Medicago truncatula root architecture under salt stress

Lateral root (LR) formation and emergence are influenced by the environment and determines the architecture of the root system in the soil. Whereas auxins appear as the main hormone controlling LR initiation, patterning and emergence, abscisic acid (ABA) is the key hormone mediating the effect of the environment on root architecture. Hormone signaling act through transcription factors (TFs) and the Medicago truncatula LOB-like TF LBD1 was shown to be auxin-inducible but repressed by the HD-Zip I TF MtHB1 in response to salt stress and ABA during LR formation. Here, we demonstrate that the constitutive expression of Mt LBD1 in Medicago roots alters their global architecture when the plant is subjected to salt stress. Hence, LBD1 may control the final form of the root system in the soil environment.

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.

Phymatotrichum (cotton) root rot caused by Phymatotrichopsis omnivora: retrospects and prospects.

Phymatotrichopsis omnivora (Duggar) Hennebert [synonym Phymatotrichum omnivorum (Shear) Duggar] is an asexual fungus with no known sexual stage. Mitosporic botryoblastospores occasionally form on epigeous spore mats in nature, but perform no known function and do not contribute to the disease cycle. The fungus has been affiliated erroneously with the polypore basidiomycete Sistotrema brinkmannii (Bres.) J. Erikss. Recent phylogenetic studies have placed this fungus in the ascomycete order Pezizales. HOST RANGE AND DISEASE SYMPTOMS: The fungus infects most dicotyledonous field crops, causing significant losses to cotton, alfalfa, grape, fruit and nut trees and ornamental shrubs in the south-western USA, northern Mexico and possibly parts of central Asia. However, this fungus does not cause disease in monocotyledonous plants. Symptoms include an expanding tissue collapse (rot) of infected taproots. In above-ground tissues, the root rot results in vascular discoloration of the stem and rapid wilting of the leaves without abscission, and eventually the death of the plant. Characteristic mycelial strands of the pathogen are typically present on the root's surface, aiding diagnosis. Confocal imaging of P. omnivora interactions with Medicago truncatula roots revealed that infecting hyphae do not form any specialized structures for penetration and mainly colonize cortical cells and eventually form a mycelial mantle covering the root's surfaces. Cell wall-degrading enzymes have been implicated in penetration and symptom development. Global gene expression profiling of infected M. truncatula revealed roles for jasmonic acid, ethylene and the flavonoid pathway during disease development. Phymatotrichopsis omnivora apparently evades induced host defences and may suppress the host's phytochemical defences at later stages of infection to favour pathogenesis. No consistently effective control measures are known. The long-lived sclerotia and facultative saprotrophism of P. omnivora make crop rotation ineffective. Chemical fumigation methods are not cost-effective for most crops. Interestingly, no genetic resistance has been reported in any of the susceptible crop species.

Differential gel electrophoresis (DIGE) to quantitatively monitor early symbiosis- and pathogenesis-induced changes of the Medicago truncatula root proteome

Symbiosis- and pathogenesis-related early protein induction patterns in the model legume Medicago truncatula were analysed with two-dimensional differential gel electrophoresis. Two symbiotic soil microorganisms (Glomus intraradices, Sinorhizobium meliloti) were used in single infections and in combination with a secondary pathogenic infection by the oomycete Aphanomyces euteiches. Proteomic analyses performed 6 and 24h after inoculations led to identification of 87 differentially induced proteins which likely represent the M. truncatula root 'interactome'. A set of proteins involved in a primary antioxidant defense reaction was detected during all associations investigated. Symbiosis-related protein induction includes a typical factor of early symbiosis-specific signalling (CaM-2), two Ran-binding proteins of nucleocytoplasmic signalling, and a set of energy-related enzymes together with proteins involved in symbiosis-initiated C- and N-fixation. Pathogen-associated protein induction consists of mainly PR proteins, Kunitz-type proteinase inhibitors, a lectin, and proteins related to primary carbohydrate metabolism and phytoalexin synthesis. Absence of PR proteins and decreased pathogen-induced protein patterns during mixed symbiotic and pathogenic infections indicate bioprotective effects due to symbiotic co-infection. Several 14-3-3 proteins were found as predominant proteins during mixed infections. With respect to hormone-regulation, A. euteiches infection led to induction of ABA-related pathways, while auxin-related pathways are induced during symbiosis.

Gene expression profiling of Medicago truncatula roots in response to the parasitic plant Orobanche crenata

Summary Orobanche crenata is a root parasitic weed that is a major constraint for grain and forage legume cultivation in Mediterranean and West Asia. Only moderate to low levels of incomplete resistance of complex inheritance has been identified so far in legume crops, which has hampered genetic and genomic analysis. In the present study, we provide a gene expression profile of roots of the model legume Medicago truncatula in response to infection by O. crenata. M. truncatula accessions SA27774 (complete resistance acting at early penetration stages) and SA4087 (incomplete late acting resistance mediated by necrosis of parasite tubercle) were inoculated with O. crenata seeds in a semi‐sterile dish system. Roots were harvested at 15 (first contacts of the parasitism structures with the host roots), 21 (initial stage of parasite tubercle formation on SA4087) and 35 (prior necrosis of well‐developed parasite tubercle of on SA4087) days post‐inoculation. Array hybridisations revealed several hundred genes up‐regulated in response to O. crenata infection. Gene expression patterns suggest that resistance mechanisms activated in both genotypes are temporally and spatially different and resemble those associated with plant resistance to microbial pathogens. Regulated genes identified here represent a comprehensive resource that can be used as a support to breeding strategies for resistance.

Global and cell‐type gene expression profiles in tomato plants colonized by an arbuscular mycorrhizal fungus

*Arbuscular mycorrhizal symbiosis develops in roots; extensive cellular reorganizations and specific metabolic changes occur, which are mirrored by local and systemic changes in the transcript profiles. *A TOM2 microarray (c. 12 000 probes) has been used to obtain an overview of the transcriptional changes that are triggered in Solanum lycopersicum roots and shoots, as a result of colonization by the arbuscular mycorrhizal fungus Glomus mosseae. The cell-type expression profile of a subset of genes was monitored, using laser microdissection, to identify possible plant determinants of arbuscule development,. *Microarrays revealed 362 up-regulated and 293 down-regulated genes in roots. Significant gene modulation was also observed in shoots: 85 up- and 337 down-regulated genes. The most responsive genes in both organs were ascribed to primary and secondary metabolism, defence and response to stimuli, cell organization and protein modification, and transcriptional regulation. Six genes, preferentially expressed in arbusculated cells, were identified. *A comparative analysis only showed a limited overlap with transcript profiles identified in mycorrhizal roots of Medicago truncatula, probably as a consequence of the largely nonoverlapping probe sets on the microarray tools used. The results suggest that auxin and abscisic acid metabolism are involved in arbuscule formation and/or functioning.

A Nuclear-Targeted Cameleon Demonstrates Intranuclear Ca2+ Spiking in Medicago truncatula Root Hairs in Response to Rhizobial Nodulation Factors

Lipochitooligosaccharide nodulation factors (NFs) secreted by endosymbiotic nitrogen-fixing rhizobia trigger Ca(2+) spiking in the cytoplasmic perinuclear region of host legume root hairs. To determine whether NFs also elicit Ca(2+) responses within the plant cell nucleus we have made use of a nucleoplasmin-tagged cameleon (NupYC2.1). Confocal microscopy using this nuclear-specific calcium reporter has revealed sustained and regular Ca(2+) spiking within the nuclear compartment of Medicago truncatula root hairs treated with Sinorhizobium meliloti NFs. Since the activation of Ca(2+) oscillations is blocked in M. truncatula nfp, dmi1, and dmi2 mutants, and unaltered in a dmi3 background, it is likely that intranuclear spiking lies on the established NF-dependent signal transduction pathway, leading to cytoplasmic calcium spiking. A semiautomated mathematical procedure has been developed to identify and analyze nuclear Ca(2+) spiking profiles, and has revealed high cell-to-cell variability in terms of both periodicity and spike duration. Time-lapse imaging of the cameleon Förster resonance energy transfer-based ratio has allowed us to visualize the nuclear spiking variability in situ and to demonstrate the absence of spiking synchrony between adjacent growing root hairs. Finally, spatio-temporal analysis of the asymmetric nuclear spike suggests that the initial rapid increase in Ca(2+) concentration occurs principally in the vicinity of the nuclear envelope. The discovery that rhizobial NF perception leads to the activation of cell-autonomous Ca(2+) oscillations on both sides of the nuclear envelope raises major questions about the respective roles of the cytoplasmic and nuclear compartments in transducing this key endosymbiotic signal.

Time course induction of several key enzymes in Medicago truncatula roots in response to Fe deficiency

Medicago truncatula constitutes a good model for Strategy I plants, since when this plant is challenged with Fe shortage the most important root physiological responses induced by Fe deficiency are developed, including the yellowing of root tips. A better understanding of the mechanisms involved in root adaptation to Fe deficiency in M. truncatula may strengthen our ability to enhance Fe efficiency responses in other plant species, especially in different agronomically relevant legumes. Riboflavin concentration, phosphoenolpyruvate carboxylase (EC 4.1.1.31) and Fe reductase activities, and acidification capacity have been determined in M. truncatula roots at different time points after imposing Fe deficiency. Root riboflavin concentrations increased with Fe deficiency and concomitantly MtDMRL was upregulated at the transcriptional level, supporting a role for flavins in the Fe deficiency response. Root Fe reductase and phosphoenolpyruvate carboxylase activities as well as acidification capacity were higher in roots of Fe-deficient than in control plants, and the corresponding genes, MtFRO1, MtPEPC1 and MtHA1 were also upregulated by Fe deficiency. Expression of these genes and their corresponding physiological activities followed different patterns over time, suggesting the existence of both transcriptional and post-transcriptional regulation.

Composite Medicago truncatula plants harbouring Agrobacterium rhizogenes-transformed roots reveal normal mycorrhization by Glomus intraradices.

Composite plants consisting of a wild-type shoot and a transgenic root are frequently used for functional genomics in legume research. Although transformation of roots using Agrobacterium rhizogenes leads to morphologically normal roots, the question arises as to whether such roots interact with arbuscular mycorrhizal (AM) fungi in the same way as wild-type roots. To address this question, roots transformed with a vector containing the fluorescence marker DsRed were used to analyse AM in terms of mycorrhization rate, morphology of fungal and plant subcellular structures, as well as transcript and secondary metabolite accumulations. Mycorrhization rate, appearance, and developmental stages of arbuscules were identical in both types of roots. Using Mt16kOLI1Plus microarrays, transcript profiling of mycorrhizal roots showed that 222 and 73 genes exhibited at least a 2-fold induction and less than half of the expression, respectively, most of them described as AM regulated in the same direction in wild-type roots. To verify this, typical AM marker genes were analysed by quantitative reverse transcription-PCR and revealed equal transcript accumulation in transgenic and wild-type roots. Regarding secondary metabolites, several isoflavonoids and apocarotenoids, all known to accumulate in mycorrhizal wild-type roots, have been found to be up-regulated in mycorrhizal in comparison with non-mycorrhizal transgenic roots. This set of data revealed a substantial similarity in mycorrhization of transgenic and wild-type roots of Medicago truncatula, validating the use of composite plants for studying AM-related effects.