The gene space of European mistletoe (Viscum album).

European mistletoe (Viscum album) is a hemiparasitic flowering plant that is known for its very special life cycle and extraordinary biochemical properties. Particularly, V. album has an unusual mode of cellular respiration that takes place in the absence of mitochondrial complex I. However, insights into the molecular biology of V. album so far are very limited. Since the genome of V. album is extremely large (estimated 600 times larger than the genome of the model plant Arabidopsis thaliana) it has not been sequenced up to now. We here report sequencing of the V. album gene space (defined as the space including and surrounding genic regions, encompassing coding as well as 5' and 3' non-coding regions). mRNA fractions were isolated from different V. album organs harvested in summer or winter and were analyzed via single-molecule real-time sequencing. We determined sequences of 39 092 distinct open reading frames encoding 32 064 V. album proteins (designated V. album protein space). Our data give new insights into the metabolism and molecular biology of V. album, including the biosynthesis of lectins and viscotoxins. The benefits of the V. album gene space information are demonstrated by re-evaluating mass spectrometry-based data of the V. album mitochondrial proteome, which previously had been evaluated using the A. thaliana genome sequence. Our re-examination allowed the additional identification of nearly 200 mitochondrial proteins, including four proteins related to complex I, which all have a secondary function not related to respiratory electron transport. The V. album gene space sequences are available at the NCBI.

Pre-announcement of symbiotic guests: transcriptional reprogramming by mycorrhizal lipochitooligosaccharides shows a strict co-dependency on the GRAS transcription factors NSP1 and RAM1.

BACKGROUND: More than 80 % of all terrestrial plant species establish an arbuscular mycorrhiza (AM) symbiosis with Glomeromycota fungi. This plant-microbe interaction primarily improves phosphate uptake, but also supports nitrogen, mineral, and water aquisition. During the pre-contact stage, the AM symbiosis is controled by an exchange of diffusible factors from either partner. Amongst others, fungal signals were identified as a mix of sulfated and non-sulfated lipochitooligosaccharides (LCOs), being structurally related to rhizobial nodulation (Nod)-factor LCOs that in legumes induce the formation of nitrogen-fixing root nodules. LCO signals are transduced via a common symbiotic signaling pathway (CSSP) that activates a group of GRAS transcription factors (TFs). Using complex gene expression fingerprints as molecular phenotypes, this study primarily intended to shed light on the importance of the GRAS TFs NSP1 and RAM1 for LCO-activated gene expression during pre-symbiotic signaling. RESULTS: We investigated the genome-wide transcriptional responses in 5 days old primary roots of the Medicago truncatula wild type and four symbiotic mutants to a 6 h challenge with LCO signals supplied at 10(-7/-8) M. We were able to show that during the pre-symbiotic stage, sulfated Myc-, non-sulfated Myc-, and Nod-LCO-activated gene expression almost exclusively depends on the LysM receptor kinase NFP and is largely controled by the CSSP, although responses independent of this pathway exist. Our results show that downstream of the CSSP, gene expression activation by Myc-LCOs supplied at 10(-7/-8) M strictly required both the GRAS transcription factors RAM1 and NSP1, whereas those genes either co- or specifically activated by Nod-LCOs displayed a preferential NSP1-dependency. RAM1, a central regulator of root colonization by AM fungi, controled genes activated by non-sulfated Myc-LCOs during the pre-symbiotic stage that are also up-regulated in areas with early physical contact, e.g. hyphopodia and infecting hyphae; linking responses to externally applied LCOs with early root colonization. CONCLUSIONS: Since both RAM1 and NSP1 were essential for the pre-symbiotic transcriptional reprogramming by Myc-LCOs, we propose that downstream of the CSSP, these GRAS transcription factors act synergistically in the transduction of those diffusible signals that pre-announce the presence of symbiotic fungi.

Spatial gene expression analysis in tomato hypocotyls suggests cysteine as key precursor of vascular sulfur accumulation implicated in Verticillium dahliae defense.

Verticillium dahliae is a prominent generator of plant vascular wilting disease and sulfur (S)-enhanced defense (SED) mechanisms contribute to its in-planta elimination. The accumulation of S-containing defense compounds (SDCs) including elemental S (S(0) ) has been described based on the comparison of two near-isogenic tomato (Solanum lycopersicum) lines differing in fungal susceptibility. To better understand the effect of S nutrition on V. dahliae resistance both lines were supplied with low, optimal or supraoptimal sulfate-S. An absolute quantification demonstrated a most effective fungal elimination due to luxury plant S nutrition. High-pressure liquid chromatography (HPLC) showed a strong regulation of Cys levels and an S-responsive GSH pool rise in the bulk hypocotyl. High-frequency S peak accumulations were detected in vascular bundles of resistant tomato plants after fungal colonization by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). Global transcriptomic analysis suggested that early steps of the primary S metabolism did not promote the SDCs synthesis in the whole hypocotyl as gene expression was downregulated after infection. Enhanced S fertilization mostly alleviated the repressive fungal effect but did not reverse it. Upregulation of glutathione (GSH)-associated genes in bulk hypocotyls but not in vascular bundles indicated a global antioxidative role of GSH. To finally assign the contribution of S metabolism-associated genes to high S(0) accumulations exclusively found in the resistant tomato line, a spatial gene expression approach was applied. Laser microdissection of infected vascular bundles revealed a switch toward transcription of genes connected with cysteine (Cys) synthesis. The upregulation of LeOASTLp1 suggests a role for Cys as key precursor for local S accumulations (possibly S(0) ) in the vascular bundles of the V. dahliae-resistant tomato line.

Through the doors of perception to function in arbuscular mycorrhizal symbioses.

The formation of an arbuscular mycorrhizal (AM) symbiosis is initiated by the bidirectional exchange of diffusible molecules. While strigolactone hormones, secreted from plant roots,stimulate hyphal branching and fungal metabolism, fungal short-chain chitin oligomers as well assulfated and nonsulfated lipochitooligosaccharides (s/nsMyc-LCOs) elicit pre-symbiosis responses in the host. Fungal LCO signals are structurally related to rhizobial Nod-factor LCOs. Genome-wide expression studies demonstrated that defined sets of genes were induced by Nod-, sMyc- and nsMyc-LCOs, indicating LCO-specific perception in the pre-symbiosis phase. During hyphopodium formation and the subsequent root colonization, cross-talk between plant roots and AM fungi also involves phytohormones. Notably, gibberellins control arbuscule formation via DELLA proteins, which themselves serve as positive regulators of arbuscule formation. The establishment of arbuscules is accompanied by a substantial transcriptional and post-transcriptional reprogramming of host roots, ultimately defining the unique protein composition of arbuscule-containing cells. Based on cellular expression profiles, key check points of AM development as well as candidate genes encoding transcriptional regulators and regulatory microRNAs were identified. Detailed functional analyses of promoters specified short motifs sufficient for cell-autonomous gene regulation in cells harboring arbuscules, and suggested simultaneous, multi-level regulation of the mycorrhizal phosphate uptake pathway by integrating AM symbiosis and phosphate starvation response signaling.

A roadmap of cell-type specific gene expression during sequential stages of the arbuscular mycorrhiza symbiosis.

BACKGROUND: About 80% of today's land plants are able to establish an arbuscular mycorrhizal (AM) symbiosis with Glomeromycota fungi to improve their access to nutrients and water in the soil. On the molecular level, the development of AM symbioses is only partly understood, due to the asynchronous development of the microsymbionts in the host roots. Although many genes specifically activated during fungal colonization have been identified, genome-wide information on the exact place and time point of their activation remains limited. RESULTS: In this study, we relied on a combination of laser-microdissection and the use of Medicago GeneChips to perform a genome-wide analysis of transcription patterns in defined cell-types of Medicago truncatula roots mycorrhized with Glomus intraradices. To cover major stages of AM development, we harvested cells at 5-6 and at 21 days post inoculation (dpi). Early developmental stages of the AM symbiosis were analysed by monitoring gene expression in appressorial and non-appressorial areas from roots harbouring infection units at 5-6 dpi. Here, the use of laser-microdissection for the first time enabled the targeted harvest of those sites, where fungal hyphae first penetrate the root. Circumventing contamination with developing arbuscules, we were able to specifically detect gene expression related to early infection events. To cover the late stages of AM formation, we studied arbusculated cells, cortical cells colonized by intraradical hyphae, and epidermal cells from mature mycorrhizal roots at 21 dpi. Taken together, the cell-specific expression patterns of 18014 genes were revealed, including 1392 genes whose transcription was influenced by mycorrhizal colonization at different stages, namely the pre-contact phase, the infection of roots via fungal appressoria, the subsequent colonization of the cortex by fungal hyphae, and finally the formation of arbuscules. Our cellular expression patterns identified distinct groups of AM-activated genes governing the sequential reprogramming of host roots towards an accommodation of microsymbionts, including 42 AM-activated transcription factor genes. CONCLUSIONS: Our genome-wide analysis provides novel information on the cell-specific activity of AM-activated genes during both early and late stages of AM development, together revealing the road map of fine-tuned adjustments of transcript accumulation within root tissues during AM fungal colonization.

Transcriptional responses toward diffusible signals from symbiotic microbes reveal MtNFP- and MtDMI3-dependent reprogramming of host gene expression by arbuscular mycorrhizal fungal lipochitooligosaccharides.

The formation of root nodules and arbuscular mycorrhizal (AM) roots is controlled by a common signaling pathway including the calcium/calmodulin-dependent kinase Doesn't Make Infection3 (DMI3). While nodule initiation by lipochitooligosaccharide (LCO) Nod factors is well characterized, diffusible AM fungal signals were only recently identified as sulfated and nonsulfated LCOs. Irrespective of different outcomes, the perception of symbiotic LCOs in Medicago truncatula is mediated by the LysM receptor kinase M. truncatula Nod factor perception (MtNFP). To shed light on transcriptional responses toward symbiotic LCOs and their dependence on MtNFP and Ca(2+) signaling, we performed genome-wide expression studies of wild-type, Nod-factor-perception mutant1, and dmi3 mutant roots challenged with Myc- and Nod-LCOs. We show that Myc-LCOs lead to transient, quick responses in the wild type, whereas Nod-LCOs require prolonged incubation for maximal expression activation. While Nod-LCOs are most efficient for an induction of persistent transcriptional changes, sulfated Myc-LCOs are less active, and nonsulfated Myc-LCOs display the lowest capacity to activate and sustain expression. Although all symbiotic LCOs up-regulated a common set of genes, discrete subsets were induced by individual LCOs, suggesting common and specific functions for these in presymbiotic signaling. Surprisingly, even sulfated fungal Myc-LCOs and Sinorhizobium meliloti Nod-LCOs, having very similar structures, each elicited discrete subsets of genes, while a mixture of both Myc-LCOs activated responses deviating from those induced by single treatments. Focusing on the precontact phase, we identified signaling-related and transcription factor genes specifically up-regulated by Myc-LCOs. Comparative gene expression studies in symbiotic mutants demonstrated that transcriptional reprogramming by AM fungal LCOs strictly depends on MtNFP and largely requires MtDMI3.

The Viscum album Gene Space database.

The hemiparasitic flowering plant Viscum album (European mistletoe) is known for its very special life cycle, extraordinary biochemical properties, and extremely large genome. The size of its genome is estimated to be 30 times larger than the human genome and 600 times larger than the genome of the model plant Arabidopsis thaliana. To achieve insights into the Gene Space of the genome, which is defined as the space including and surrounding protein-coding regions, a transcriptome project based on PacBio sequencing has recently been conducted. A database resulting from this project contains sequences of 39,092 different open reading frames encoding 32,064 distinct proteins. Based on 'Benchmarking Universal Single-Copy Orthologs' (BUSCO) analysis, the completeness of the database was estimated to be in the range of 78%. To further develop this database, we performed a transcriptome project of V. album organs harvested in summer and winter based on Illumina sequencing. Data from both sequencing strategies were combined. The new V. album Gene Space database II (VaGs II) contains 90,039 sequences and has a completeness of 93% as revealed by BUSCO analysis. Sequences from other organisms, particularly fungi, which are known to colonize mistletoe leaves, have been removed. To evaluate the quality of the new database, proteome data of a mitochondrial fraction of V. album were re-analyzed. Compared to the original evaluation published five years ago, nearly 1000 additional proteins could be identified in the mitochondrial fraction, providing new insights into the Oxidative Phosphorylation System of V. album. The VaGs II database is available at https://viscumalbum.pflanzenproteomik.de/. Furthermore, all V. album sequences have been uploaded at the European Nucleotide Archive (ENA).

Involvement of bacterial TonB-dependent signaling in the generation of an oligogalacturonide damage-associated molecular pattern from plant cell walls exposed to Xanthomonas campestris pv. campestris pectate lyases.

BACKGROUND: Efficient perception of attacking pathogens is essential for plants. Plant defense is evoked by molecules termed elicitors. Endogenous elicitors or damage-associated molecular patterns (DAMPs) originate from plant materials upon injury or pathogen activity. While there are comparably well-characterized examples for DAMPs, often oligogalacturonides (OGAs), generated by the activity of fungal pathogens, endogenous elicitors evoked by bacterial pathogens have been rarely described. In particular, the signal perception and transduction processes involved in DAMP generation are poorly characterized. RESULTS: A mutant strain of the phytopathogenic bacterium Xanthomonas campestris pv. campestris deficient in exbD2, which encodes a component of its unusual elaborate TonB system, had impaired pectate lyase activity and caused no visible symptoms for defense on the non-host plant pepper (Capsicum annuum). A co-incubation of X. campestris pv. campestris with isolated cell wall material from C. annuum led to the release of compounds which induced an oxidative burst in cell suspension cultures of the non-host plant. Lipopolysaccharides and proteins were ruled out as elicitors by polymyxin B and heat treatment, respectively. After hydrolysis with trifluoroacetic acid and subsequent HPAE chromatography, the elicitor preparation contained galacturonic acid, the monosaccharide constituent of pectate. OGAs were isolated from this crude elicitor preparation by HPAEC and tested for their biological activity. While small OGAs were unable to induce an oxidative burst, the elicitor activity in cell suspension cultures of the non-host plants tobacco and pepper increased with the degree of polymerization (DP). Maximal elicitor activity was observed for DPs exceeding 8. In contrast to the X. campestris pv. campestris wild type B100, the exbD2 mutant was unable to generate elicitor activity from plant cell wall material or from pectin. CONCLUSIONS: To our knowledge, this is the second report on a DAMP generated by bacterial features. The generation of the OGA elicitor is embedded in a complex exchange of signals within the framework of the plant-microbe interaction of C. annuum and X. campestris pv. campestris. The bacterial TonB-system is essential for the substrate-induced generation of extracellular pectate lyase activity. This is the first demonstration that a TonB-system is involved in bacterial trans-envelope signaling in the context of a pathogenic interaction with a plant.

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.

The 2-oxoglutarate/malate translocator mediates amino acid and storage protein biosynthesis in pea embryos.

Heterotrophic plastids of seeds perform many biosynthetic reactions. Understanding their function in crop plants is crucial for seed production. Physiological functions depend on the uptake of precursors by a range of different metabolite translocators. The 2-oxoglutarate/malate translocator gene (PsOMT), which is highly expressed during pea (Pisum sativum) embryo maturation, has an important role during seed storage. PsOMT functions have been studied by antisense repression in maturing pea embryos, and were found to reduce mRNA levels and transport rates of 2-oxoglutarate and malate by 50-70%. Combined metabolite and transcript profiling revealed that OMT repression affects the conversion of carbohydrates from sucrose into amino acids and proteins, decreases seed weight and delays maturation. OMT-repressed pea embryos have increased levels of organic acids, ammonia, and higher ratios of Asn : Asp and Gln : Glu. Decreased levels of most other amino acids indicate the reduced usage of organic acids and ammonia for amino acid biosynthesis in plastids, possibly caused by substrate limitation of the plastidial glutamine synthetase/glutamine-2-oxoglutarate aminotransferase cycle. Expression of storage proteins is delayed, and mature seeds have reduced protein content. Downregulated gene expression of starch biosynthesis and plastidial glucose-6-phosphate transport in asOMT embryos reveals that decreased 2-oxoglutarate/malate transport capacity affects other pathways of central carbon metabolism. Gene expression analysis related to plastid physiology revealed that OMT repression delays differentiation of storage plastids, thereby maintaining gene expression associated with green chloroplasts. We conclude that OMT is important for protein-storing crop seeds, and is necessary for amino acid biosynthesis in pea seeds. In addition, carbon supply as mediated by OMT controls plastid differentiation during seed maturation.

Abscisic acid deficiency of developing pea embryos achieved by immunomodulation attenuates developmental phase transition and storage metabolism.

The transition of pea embryos from pre-storage to maturation is partially controlled by abscisic acid (ABA). Immunomodulation in pea embryos specifically reduces free ABA levels during transition stages. Such seeds are, therefore, suitable models for studying ABA deficiency by global transcript and metabolite analysis. Compared with the wild type, anti-ABA seeds are smaller, contain fewer globulins and show lower dry matter accumulation and delayed differentiation. Free sugars are decreased, indicating lower uptake and/or elevated mobilisation. Lower levels of trans-zeatins suggest that ABA reduction influences rates of cytokinin synthesis and/or its level of accumulation. Abscisic acid deficiency leads to a general downregulation of gene expression related to transcription and translation. At the transcriptional level, anti-ABA embryos reveal a wide-range repression of carbohydrate oxidation, downregulated sucrose mobilisation, glycolysis and the tricarboxylic acid cycle/Krebs cycle (TCA cycle). Genes related to starch, amino acid and storage protein biosynthesis are downregulated, indicating a general decrease in metabolic fluxes. We conclude that during embryo differentiation ABA triggers broad upregulation of gene activity and genetic reprogramming, involving regulated protein degradation via the ubiquitin/proteasome system. Abscisic acid deficiency affects gene expression associated with transport processes and stimulation of membrane energisation. Our study identified mediators and downstream signalling elements of ABA during embryo differentiation, such as the transcription factor FUSCA3, SnRK1 kinase and Ca(2+) signalling processes. This suggests that ABA interacts with SnRK1 complexes, thus connecting SnRK1, sugar and stress signalling with ABA. Certain protein kinases/phosphatases known to negatively respond to ABA are upregulated in the modulated line, whilst those which respond positively are downregulated, pointing to a highly coordinated response of the gene network to ABA levels.

Identification of genes differentially expressed in a resistant reaction to Mycosphaerella pinodes in pea using microarray technology.

BACKGROUND: Ascochyta blight, caused by Mycosphaerella pinodes is one of the most important pea pathogens. However, little is known about the genes and mechanisms of resistance acting against M. pinodes in pea. Resistance identified so far to this pathogen is incomplete, polygenic and scarce in pea, being most common in Pisum relatives. The identification of the genes underlying resistance would increase our knowledge about M. pinodes-pea interaction and would facilitate the introgression of resistance into pea varieties. In the present study differentially expressed genes in the resistant P. sativum ssp. syriacum accession P665 comparing to the susceptible pea cv. Messire after inoculation with M. pinodes have been identified using a M. truncatula microarray. RESULTS: Of the 16,470 sequences analysed, 346 were differentially regulated. Differentially regulated genes belonged to almost all functional categories and included genes involved in defense such as genes involved in cell wall reinforcement, phenylpropanoid and phytoalexins metabolism, pathogenesis- related (PR) proteins and detoxification processes. Genes associated with jasmonic acid (JA) and ethylene signal transduction pathways were induced suggesting that the response to M. pinodes in pea is regulated via JA and ET pathways. Expression levels of ten differentially regulated genes were validated in inoculated and control plants using qRT-PCR showing that the P665 accession shows constitutively an increased expression of the defense related genes as peroxidases, disease resistance response protein 39 (DRR230-b), glutathione S-transferase (GST) and 6a-hydroxymaackiain methyltransferase. CONCLUSIONS: Through this study a global view of genes expressed during resistance to M. pinodes has been obtained, giving relevant information about the mechanisms and pathways conferring resistance to this important disease. In addition, the M. truncatula microarray represents an efficient tool to identify candidate genes controlling resistance to M. pinodes in pea.

Knockdown of the symbiotic sucrose synthase MtSucS1 affects arbuscule maturation and maintenance in mycorrhizal roots of Medicago truncatula.

The relevance of the symbiosis-induced Medicago truncatula sucrose synthase gene MtSucS1 for an efficient arbuscular mycorrhiza (AM) was studied using two independent antisense lines that displayed up to 10-fold reduced SucS1 levels in roots. Mycorrhizal MtSucS1-reduced lines exhibited an overall stunted aboveground growth under inorganic phosphorus limitation. Apart from a reduced plant height, shoot weight, and leaf development, a delayed flowering, resulting in a lower seed yield, was observed. In addition, the root-to-shoot and root weight ratios increased significantly. Gene expression studies demonstrated a major reversion of AM-associated transcription, exhibiting a significant repression of well-known plant AM marker and mycosymbiont genes, together indicating a diminished AM fungus colonization of MtSucS1-antisense lines. Concomitantly, gas chromatography-mass spectrometry-based metabolite profiling revealed that mycorrhizal MtSucS1-reduced lines were affected in important nodes of the carbon, nitrogen, and phosphorus metabolism, accentuating a physiological significance of MtSucS1 for AM. In fact, antisensing MtSucS1 provoked an impaired fungal colonization within the less abundant infected regions, evident from strongly reduced frequencies of internal hyphae, vesicles, and arbuscules. Moreover, arbuscules were early senescing, accompanied with a reduced development of mature arbuscules. This defective mycorrhiza status correlated with reduced phosphorus and nitrogen levels and was proportional to the extent of MtSucS1 knockdown. Together, our results point to an important role for MtSucS1 in the establishment and maintenance of arbuscules in the AM symbiosis.

Membrane steroid-binding protein 1 induced by a diffusible fungal signal is critical for mycorrhization in Medicago truncatula.

Arbuscular mycorrhiza (AM) is a mutualistic biotrophic association that requires a complex exchange of signals between plant and fungus to allow accommodation of the mycosymbiont in the root cortex. Signal exchange happens even before physical contact, activating the plant symbiotic program. We investigated very early transcriptional responses in Medicago truncatula to inoculation with Glomus intraradices and identified four genes induced by diffusible AM fungal signals before contact. Three of them were previously shown to be mycorrhiza induced at later stages of the symbiosis, while MtMSBP1, encoding a membrane-bound steroid-binding protein, is a novel mycorrhizal marker. Expression analyses in plants defective in the symbiotic receptor kinase DMI2 allowed discrimination of two different signaling cascades involved in the perception of the diffusible signals. Thus, while some of the genes are activated in a DMI2-dependent manner, the induction of one of them encoding a proteinase inhibitor is DMI2-independent. Downregulation of MtMSBP1 by RNAi led to an aberrant mycorrhizal phenotype with thick and septated appressoria, decrease number of arbuscules and distorted arbuscule morphology. This provides genetic evidence that MtMSBP1 is critical for mycorrhiza development. We hypothesize that MtMSBP1 plays a role in sterol homeostasis in the root.

EMMA 2--a MAGE-compliant system for the collaborative analysis and integration of microarray data.

BACKGROUND: Understanding transcriptional regulation by genome-wide microarray studies can contribute to unravel complex relationships between genes. Attempts to standardize the annotation of microarray data include the Minimum Information About a Microarray Experiment (MIAME) recommendations, the MAGE-ML format for data interchange, and the use of controlled vocabularies or ontologies. The existing software systems for microarray data analysis implement the mentioned standards only partially and are often hard to use and extend. Integration of genomic annotation data and other sources of external knowledge using open standards is therefore a key requirement for future integrated analysis systems. RESULTS: The EMMA 2 software has been designed to resolve shortcomings with respect to full MAGE-ML and ontology support and makes use of modern data integration techniques. We present a software system that features comprehensive data analysis functions for spotted arrays, and for the most common synthesized oligo arrays such as Agilent, Affymetrix and NimbleGen. The system is based on the full MAGE object model. Analysis functionality is based on R and Bioconductor packages and can make use of a compute cluster for distributed services. CONCLUSION: Our model-driven approach for automatically implementing a full MAGE object model provides high flexibility and compatibility. Data integration via SOAP-based web-services is advantageous in a distributed client-server environment as the collaborative analysis of microarray data is gaining more and more relevance in international research consortia. The adequacy of the EMMA 2 software design and implementation has been proven by its application in many distributed functional genomics projects. Its scalability makes the current architecture suited for extensions towards future transcriptomics methods based on high-throughput sequencing approaches which have much higher computational requirements than microarrays.

Exploring the nuclear proteome of Medicago truncatula at the switch towards seed filling.

Despite its importance in determining seed composition, and hence quality, regulation of the development of legume seeds is incompletely understood. Because of the cardinal role played by the nucleus in gene expression and regulation, we have characterized the nuclear proteome of Medicago truncatula at the 12 days after pollination (dap) stage that marks the switch towards seed filling. Nano-liquid chromatography-tandem mass spectrometry analysis of nuclear protein bands excised from one-dimensional SDS-PAGE identified 179 polypeptides (143 different proteins), providing an insight into the complexity and distinctive feature of the seed nuclear proteome and highlighting new plant nuclear proteins with possible roles in the biogenesis of ribosomal subunits (PESCADILLO-like) or nucleocytoplasmic trafficking (dynamin-like GTPase). The results revealed that nuclei of 12-dap seeds store a pool of ribosomal proteins in preparation for intense protein synthesis activity, occurring subsequently during seed filling. Diverse proteins of the molecular machinery leading to the synthesis of ribosomal subunits were identified along with proteins involved in transcriptional regulation, RNA processing or transport. Some had already been shown to play a role during the early stages of seed formation whereas for others the findings are novel (e.g. the DIP2 and ES43 transcriptional regulators or the RNA silencing-related ARGONAUTE proteins). This study also revealed the presence of chromatin-modifying enzymes and RNA interference proteins that have roles in RNA-directed DNA methylation and may be involved in modifying genome architecture and accessibility during seed filling and maturation.

Increasing amino acid supply in pea embryos reveals specific interactions of N and C metabolism, and highlights the importance of mitochondrial metabolism.

SUMMARY: The application of nitrogen to legumes regulates seed metabolism and composition. We recently showed that the seed-specific overexpression of amino acid permease VfAAP1 increases amino acid supply, and the levels of N and protein in the seeds. Two consecutive field trials using Pisum sativum AAP1 lines confirmed increases in the levels of N and globulin in seed; however, compensatory changes of sucrose/starch and individual seed weight were also observed. We present a comprehensive analysis of AAP1 seeds using combinatorial transcript and metabolite profiling to monitor the effects of nitrogen supply on seed metabolism. AAP1 seeds have increased amino acids and stimulated gene expression associated with storage protein synthesis, maturation, deposition and vesicle trafficking. Transcript/metabolite changes reveal the channelling of surplus N into the transient storage pools asparagine and arginine, indicating that asparagine synthase is transcriptionally activated by high N levels and/or C limitation. Increased C-acceptor demand for amino acid synthesis, resulting from elevated levels of N in seeds, initiates sucrose mobilization and sucrose-dependent pathways via sucrose synthase, glycolysis and the TCA cycle. The AAP1 seeds display a limitation in C, which leads to the catabolism of arginine, glutamic acid and methionine to putrescine, beta-alanine and succinate. Mitochondria are involved in the coordination of C/N metabolism, with branched-chain amino acid catabolism and a gamma-amino-butyric acid shunt. AAP1 seeds contain higher levels of ABA, which is possibly involved in storage-associated gene expression and the N-dependent stimulation of sucrose mobilization, indicating that a signalling network of C, N and ABA is operating during seed maturation. These results demonstrate that legume seeds have a high capacity to regulate N:C ratios, and highlight the importance of mitochondria in the control of N-C balance and amino acid homeostasis.

Knock-down of the MEP pathway isogene 1-deoxy-D-xylulose 5-phosphate synthase 2 inhibits formation of arbuscular mycorrhiza-induced apocarotenoids, and abolishes normal expression of mycorrhiza-specific plant marker genes.

The first step of the plastidial methylerythritol phosphate (MEP) pathway is catalyzed by two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase (DXS1 and DXS2). In Medicago truncatula, MtDXS1 and MtDXS2 genes exhibit completely different expression patterns. Most prominently, colonization by arbuscular mycorrhizal (AM) fungi induces the accumulation of certain apocarotenoids (cyclohexenone and mycorradicin derivatives) correlated with the expression of MtDXS2 but not of MtDXS1. To prove a distinct function of DXS2, a selective RNAi approach on MtDXS2 expression was performed in transgenic hairy roots of M. truncatula. Repression of MtDXS2 consistently led to reduced transcript levels in mycorrhizal roots, and to a concomitant reduction of AM-induced apocarotenoid accumulation. The transcript levels of MtDXS1 remained unaltered in RNAi plants, and no phenotypical changes in non-AM plants were observed. Late stages of the AM symbiosis were adversely affected, but only upon strong repression with residual MtDXS2-1 transcript levels remaining below approximately 10%. This condition resulted in a strong decrease in the transcript levels of MtPT4, an AM-specific plant phosphate transporter gene, and in a multitude of other AM-induced plant marker genes, as shown by transcriptome analysis. This was accompanied by an increased proportion of degenerating and dead arbuscules at the expense of mature ones. The data reveal a requirement for DXS2-dependent MEP pathway-based isoprenoid products to sustain mycorrhizal functionality at later stages of the symbiosis. They further validate the concept of a distinct role for DXS2 in secondary metabolism, and offer a novel tool to selectively manipulate the levels of secondary isoprenoids by targeting their precursor supply.

The signal peptide of the Medicago truncatula modular nodulin MtNOD25 operates as an address label for the specific targeting of proteins to nitrogen-fixing symbiosomes.

The nodule-specific MtNOD25 gene of the model legume Medicago truncatula encodes a modular nodulin composed of different repetitive modules flanked by distinct N- and C-termini. Although similarities are low with respect to all repetitive modules, both the N-terminal signal peptide (SP) and the C-terminus are highly conserved in modular nodulins from different legumes. On the cellular level, MtNOD25 is only transcribed in the infected cells of root nodules, and this activation is mediated by a 299-bp minimal promoter containing an organ-specific element. By expressing mGFP6 translational fusions in transgenic nodules, we show that MtNOD25 proteins are exclusively translocated to the symbiosomes of infected cells. This specific targeting only requires an N-terminal MtNOD25 SP that is highly conserved across a family of legume-specific symbiosome proteins. Our finding sheds light on one possible mechanism for the delivery of host proteins to the symbiosomes of infected root nodule cells and, in addition, defines a short molecular address label of only 24 amino acids whose N-terminal presence is sufficient to translocate proteins across the peribacteroid membrane.