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How many different proteins can be produced from a single spliced transcript? Genome annotation projects overlook the coding potential of reading frames other than that of the reference open reading frames (refORFs). Recently, alternative open reading frames (altORFs) and their translational products, alternative proteins, have been shown to carry out important functions in various organisms. AltORFs overlapping refORFs or other altORFs in a different reading frame may be involved in one fundamental mechanism so far overlooked. A few years ago, it was proposed that altORFs may act as building blocks for chimeric (mosaic) polypeptides, which are produced via multiple ribosomal frameshifting events from a single mature transcript. We adopt terminology from that earlier discussion and call this mechanism mosaic translation. This way of extracting and combining genetic information may significantly increase proteome diversity. Thus, we hypothesize that this mechanism may have contributed to the flexibility and adaptability of organisms to a variety of environmental conditions. Specialized ribosomes acting as sensors probably played a central role in this process. Importantly, mosaic translation may be the main source of protein diversity in genomes that lack alternative splicing. The idea of mosaic translation is a testable hypothesis, although its direct demonstration is challenging. Should mosaic translation occur, we would currently highly underestimate the complexity of translation mechanisms and thus the proteome.

SummaryLegumes establish symbiotic relationships with soil bacteria (rhizobia), housed in nodules on plant roots. The plant supplies carbon substrates and other nutrients to the bacteria in exchange for fixed nitrogen. The exchange occurs across a plant-derived symbiosome membrane (SM), which encloses rhizobia to form a symbiosome. Iron supplied by the plant is crucial for the rhizobial enzyme nitrogenase that catalyses N2 fixation, but the SM iron transporter has not been identified.We use complementation of yeast and plant mutants, real-time PCR, hairy root transformation, microscopy and proteomics to demonstrate the role of soybean GmVTL1 and 2.Both are members of the vacuolar iron transporter family and homologous to Lotus japonicus SEN1 (LjSEN1), previously shown to be essential for N2 fixation. GmVTL1 expression is enhanced in nodule infected cells and both proteins are localised to the SM.GmVTL1 and 2 transport iron in yeast and GmVTL1 restores N2 fixation when expressed in the Ljsen1 mutant.Three GmVTL1 amino acid substitutions that reduce iron transport in yeast also block N2 fixation in Ljsen1 plants.We conclude GmVTL1 is responsible for transport of iron across the SM to bacteroids and plays a crucial role in the N2-fixing symbiosis.

Legumes establish symbiotic relationships with soil bacteria (rhizobia), housed in nodules on roots. The plant supplies carbon substrates and other nutrients to the bacteria in exchange for fixed nitrogen. The exchange occurs across a plant-derived symbiosome membrane (SM), which encloses rhizobia to form a symbiosome. Iron supplied by the plant is crucial for rhizobial enzyme nitrogenase that catalyses nitrogen fixation, but the SM iron transporter has not been identified. We use yeast complementation, real-time PCR and proteomics to study putative soybean (Glycine max) iron transporters GmVTL1a and GmVTL1b and have characterized the role of GmVTL1a using complementation in plant mutants, hairy root transformation and microscopy. GmVTL1a and GmVTL1b are members of the vacuolar iron transporter family and homologous to Lotus japonicus SEN1 (LjSEN1), which is essential for nitrogen fixation. GmVTL1a expression is enhanced in nodule infected cells and both proteins are localized to the SM. GmVTL1a transports iron in yeast and restores nitrogen fixation when expressed in the Ljsen1 mutant. Three GmVTL1a amino acid substitutions that block nitrogen fixation in Ljsen1 plants reduce iron transport in yeast. We conclude GmVTL1a is responsible for transport of iron across the SM to bacteroids and plays a crucial role in the nitrogen-fixing symbiosis.

Iron (Fe) is an essential micronutrient for symbiotic nitrogen fixation in legume nodules, where it is required for the activity of bacterial nitrogenase, plant leghemoglobin, respiratory oxidases, and other Fe proteins in both organisms. Fe solubility and transport within and between plant tissues is facilitated by organic chelators, such as nicotianamine and citrate. We have characterized a nodule-specific citrate transporter of the multidrug and toxic compound extrusion family, MtMATE67 of Medicago truncatula The MtMATE67 gene was induced early during nodule development and expressed primarily in the invasion zone of mature nodules. The MtMATE67 protein was localized to the plasma membrane of nodule cells and also the symbiosome membrane surrounding bacteroids in infected cells. In oocytes, MtMATE67 transported citrate out of cells in an Fe-activated manner. Loss of MtMATE67 gene function resulted in accumulation of Fe in the apoplasm of nodule cells and a substantial decrease in symbiotic nitrogen fixation and plant growth. Taken together, the results point to a primary role of MtMATE67 in citrate efflux from nodule cells in response to an Fe signal. This efflux is necessary to ensure Fe(III) solubility and mobility in the apoplasm and uptake into nodule cells. Likewise, MtMATE67-mediated citrate transport into the symbiosome space would increase the solubility and availability of Fe(III) for rhizobial bacteroids.

Zn deficiency is widespread in traditional areas of chickpea cultivation worldwide. It limits chickpea productivity and causes significant losses to the economies of the world?s largest chickpea exporters. This review may be of interest to researchers who would like to contribute to the improvement of chickpea cultivation on Zn-depleted soils in an environmentally sustainable manner, namely via identification of genotypes with superior symbiotic performance under Zn-limited conditions. The primary aim of the current work is to familiarize the readers with the biology and symbiotic characteristics of chickpea, and also to provide the necessary background on Zn as an essential nutrient for symbiotic nitrogen fixation (SNF). Special attention has been paid to the choice of rhizobial strains compatible with chickpea. Strains that can serve as an inoculum for simultaneous analysis of many genetically diverse chickpea lines have been suggested. The genotypes listed in this work can be good starting material for identification of chickpea lineages useful for unraveling the molecular basis of Zn-use efficiency, SNF efficiency, or both.

Nitrogen-fixing symbiosis is the most successful metabolism-dependent mutualistic symbiosis on earth that influences global nitrogen cycles. Engineering and improvement of this symbiosis is the major avenue towards sustainable agriculture. Root nodule symbiosis (RNS) is restricted to a monophyletic group of angiosperm order. Collectively this group is called nitrogen-fixing symbiosis (NFS) clade. Tremendous multifariousness exists among the infection mechanism, nodule structure, and nitrogen fixation efficiency among nodules. We have gained significant knowledge about the molecular mechanism of RNS in last two decades, using model legumes Medicago truncatula and Lotus japonicus. In this chapter, we present the current status of model legumes used to unveil the molecular mechanisms behind the development of nodules and RNS. We further introduce non-canonical legume species that have been used to expand our understanding of the traits associated with RNS in plants. We have also highlighted the extraordinary variations which came up during the evolution of the RNS. This comparative approach will enable us to identify the variable genetic events that rendered NFS to the RNS clade. In turn, this knowledge will hopefully allow us to carefully engineer it in non-fixing crops.

Optimization of nitrogen fixation by rhizobia in legumes is a key area of research for sustainable agriculture. Symbiotic nitrogen fixation (SNF) occurs in specialized organs called nodules and depends on a steady supply of carbon to both plant and bacterial cells. Here we report the functional characterization of a nodule-specific Suc transporter, MtSWEET11 from Medicago truncatula. MtSWEET11 belongs to a clade of plant SWEET proteins that are capable of transporting Suc and play critical roles in pathogen susceptibility. When expressed in mammalian cells, MtSWEET11 transported sucrose (Suc) but not glucose (Glc). The MtSWEET11 gene was found to be expressed in infected root hair cells, and in the meristem, invasion zone, and vasculature of nodules. Expression of an MtSWEET11-GFP fusion protein in nodules resulted in green fluorescence associated with the plasma membrane of uninfected cells and infection thread and symbiosome membranes of infected cells. Two independent Tnt1-insertion sweet11 mutants were uncompromised in SNF. Therefore, although MtSWEET11 appears to be involved in Suc distribution within nodules, it is not crucial for SNF, probably because other Suc transporters can fulfill its role(s).

One of the most tedious steps in genetic data analyses is the reformatting data generated with one program for use with other applications. This conversion is necessary because comprehensive evaluation of the data may be based on different algorithms included in diverse software, each requiring a distinct input format. A platform-independent and freely available program or a web-based tool dedicated to such reformatting can save time and efforts in data processing. Here, we report widgetcon, a website and a program which has been developed to quickly and easily convert among various molecular data formats commonly used in phylogenetic analysis, population genetics, and other fields. The web-based service is available at https://www.widgetcon.net. The program and the website convert the major data formats in four basic steps in less than a minute. The resource will be a useful tool for the research community and can be updated to include more formats and features in the future.

Copper is an essential nutrient for symbiotic nitrogen fixation. This element is delivered by the host plant to the nodule, where membrane copper (Cu) transporter would introduce it into the cell to synthesize cupro-proteins. COPT family members in the model legume Medicago truncatula were identified and their expression determined. Yeast complementation assays, confocal microscopy and phenotypical characterization of a Tnt1 insertional mutant line were carried out in the nodule-specific M. truncatula COPT family member. Medicago truncatula genome encodes eight COPT transporters. MtCOPT1 (Medtr4g019870) is the only nodule-specific COPT gene. It is located in the plasma membrane of the differentiation, interzone and early fixation zones. Loss of MtCOPT1 function results in a Cu-mitigated reduction of biomass production when the plant obtains its nitrogen exclusively from symbiotic nitrogen fixation. Mutation of MtCOPT1 results in diminished nitrogenase activity in nodules, likely an indirect effect from the loss of a Cu-dependent function, such as cytochrome oxidase activity in copt1-1 bacteroids. These data are consistent with a model in which MtCOPT1 transports Cu from the apoplast into nodule cells to provide Cu for essential metabolic processes associated with symbiotic nitrogen fixation.

Summary• Copper is an essential nutrient for symbiotic nitrogen fixation. This element is delivered by the host plant to the nodule, where membrane copper transporter would introduce it into the cell to synthesize cupro-proteins.• COPT family members in model legumeMedicago truncatulawere identified and their expression determined. Yeast complementation assays, confocal microscopy, and phenotypical characterization of aTnt1insertional mutant line were carried out in the nodule-specificM.truncatulaCOPT family member.•Medicago truncatulagenome encodes eight COPT transporters.MtCOPT1(Medtr4g019870) is the only nodule-specificCOPTgene. It is located in the plasma membrane of the differentiation, interzone and early fixation zones. Loss of MtCOPT1 function results in a copper-mitigated reduction of biomass production when the plant obtains its nitrogen exclusively from symbiotic nitrogen fixation. Mutation ofMtCOPT1results in diminished nitrogenase activity in nodules, likely an indirect effect from the loss of a copper-dependent function, such as cytochrome oxidase activity incopt1-1bacteroids.• These data are consistent with a model in which MtCOPT1 transports copper from the apoplast into nodule cells to provide copper for essential metabolic processes associated with symbiotic nitrogen fixation.

30 páginas, 9 figuras, 1 tabla, 13 páginas de explicaciones suplementarias de las figuras, más otras 10 figuras suplementarias, Iron is critical for symbiotic nitrogen fixation (SNF) as a key component of multiple ferroproteins involved in this biological process. In the model legume Medicago truncatula, iron is delivered by the vasculature to the infection/maturation zone (zone II) of the nodule, where it is released to the apoplast. From there, plasma membrane iron transporters move it into rhizobia-containing cells, where iron is used as the cofactor of multiple plant and rhizobial proteins (e.g. plant leghemoglobin and bacterial nitrogenase). MtNramp1 (Medtr3g088460) is the M. truncatula Natural Resistance-Associated Macrophage Protein family member, with the highest expression levels in roots and nodules. Immunolocalization studies indicate that MtNramp1 is mainly targeted to the plasma membrane. A loss-of-function nramp1 mutant exhibited reduced growth compared with the wild type under symbiotic conditions, but not when fertilized with mineral nitrogen. Nitrogenase activity was low in the mutant, whereas exogenous iron and expression of wild-type MtNramp1 in mutant nodules increased nitrogen fixation to normal levels. These data are consistent with a model in which MtNramp1 is the main transporter responsible for apoplastic iron uptake by rhizobia-infected cells in zone II., This work was supported by the Spanish Ministry of Economy and Competitiveness (grant no. AGL–2012–32974 to M.G.-G. and Formación del Personal Investigador fellowship no. BES–2013– 062674 to R.C.-R.), a Marie Curie International Reintegration grant (grant no. IRG–2010–276771 to M.G.-G.), a European Research Council Starting grant (grant no. ERC–2013–StG–335284 to M.G.-G.), and a Ramón y Cajal fellowship (no. RYC–2010–06363 to M.G.-G.)., ACKNOWLEDGMENTS We thank Dr. Alonso Rodríguez-Navarro (Universidad Politécnica deMadrid) for providing plasmid pYPGE15, Dr. David Eide (University of Wisconsin, Madison) for the strain fet3/fet4 and its parental strain, Dr. Benoit Lefebvre (Laboratoire des Interactions Plantes Microorganismes) for the pBI101 vector, Dr. Florian Frugier (Institut des Sciences du Vegétal) for the pFRN vector, Dr. Rosario Haro (Universidad Politécnica de Madrid) for the organelle-specific markers, Dr. Regla Bustos (Instituto Nacional de Investigaciones Agroalimentarias) for the agroinfiltration protocol, Dr. Larry Peterson for the Casparian strip visualization method, and Dr. Tsuyoshi Nakagawa (Shiimane University) for the pGWB vectors.

A key feature of arbuscular mycorrhizal symbiosis is improved phosphorus nutrition of the host plant via the mycorrhizal pathway, i.e., the fungal uptake of Pi from the soil and its release from arbuscules within root cells. Efficient transport of Pi from the fungus to plant cells is thought to require a proton gradient across the periarbuscular membrane (PAM) that separates fungal arbuscules from the host cell cytoplasm. Previous studies showed that the H+-ATPase gene HA1 is expressed specifically in arbuscule-containing root cells of Medicago truncatula. We isolated a ha1-2 mutant of M. truncatula and found it to be impaired in the development of arbuscules but not in root colonization by Rhizophagus irregularis hyphae. Artificial microRNA silencing of HA1 recapitulated this phenotype, resulting in small and truncated arbuscules. Unlike the wild type, the ha1-2 mutant failed to show a positive growth response to mycorrhizal colonization under Pi-limiting conditions. Uptake experiments confirmed that ha1-2 mutants are unable to take up phosphate via the mycorrhizal pathway. Increased pH in the apoplast of abnormal arbuscule-containing cells of the ha1-2 mutant compared with the wild type suggests that HA1 is crucial for building a proton gradient across the PAM and therefore is indispensible for the transfer of Pi from the fungus to the plant.

Background Genetic crossing is an essential tool in both forward and reverse genetic approaches to understand the biological functions of genes. For Medicago truncatula (barrel medic) various crossing techniques have been used which differ in the methods used to dissect the female parent’s unopened flower bud to remove immature anthers for prevention of self-pollination. Previously described methods including front, side or back incision methods may damage the flower bud, impeding successful fertilization and/or seed development because they may allow pollen to dislodge and floral organs to desiccate after crossing, all of which diminish the success rates of crossing. Results We report the keel petal incision method for genetic crossing in M. truncatula ecotype R108 and demonstrate successful crosses with two other M. truncatula ecotypes, A17 and A20. In the method presented here, an incision is made along the central line of the keel petal from the bottom 1/3rd of the female parent’s flower bud to its distal end. This allows easy removal of anthers from the flower bud and access for cross-pollination. After pollination, the stigma and the deposited pollen from the male donor are covered by the keel petal, wing petals and standard petal, forming a natural pouch. The pouch prevents dislodging of deposited pollen from the stigma and protects the internal floral organs from drying out, without using cling-film or water-containing chambers to maintain a humid environment. The keel petal incision method showed an approximate 80% success rate in the M. truncatula R108 ecotype and also in other ecotypes including Jemalong A17 and A20. Conclusions Our keel petal incision protocol shows marked improvement over existing methods with respect to the ease of crossing and the percentage of successful crosses. Developed for the M. truncatula R108 ecotype, the protocol has been demonstrated with A17 and A20 ecotypes and is expected to work with other ecotypes. Investigators of varying experience have achieved genetic crosses in M. truncatula using this method.