RESUMO
Floral development is one of the model systems for investigating the mechanisms underlying organogenesis in plants. Floral organ identity is controlled by the well-known ABC model, which has been generalized to many flowering plants. Here, we report a previously uncharacterized MYB-like gene, AGAMOUS-LIKE FLOWER (AGLF), involved in flower development in the model legume Medicago truncatula Loss-of-function of AGLF results in flowers with stamens and carpel transformed into extra whorls of petals and sepals. Compared with the loss-of-function mutant of the class C gene AGAMOUS (MtAG) in M. truncatula, the defects in floral organ identity are similar between aglf and mtag, but the floral indeterminacy is enhanced in the aglf mutant. Knockout of AGLF in the mutants of the class A gene MtAP1 or the class B gene MtPI leads to an addition of a loss-of-C-function phenotype, reflecting a conventional relationship of AGLF with the canonical A and B genes. Furthermore, we demonstrate that AGLF activates MtAG in transcriptional levels in control of floral organ identity. These data shed light on the conserved and diverged molecular mechanisms that control flower development and morphology among plant species.
Assuntos
Flores/genética , Regulação da Expressão Gênica de Plantas , Medicago truncatula/genética , Especificidade de Órgãos/genética , Proteínas de Plantas/genética , Transcrição Gênica , Flores/crescimento & desenvolvimento , Flores/ultraestrutura , Medicago truncatula/ultraestrutura , Mutação/genética , Fenótipo , Proteínas de Plantas/metabolismoRESUMO
BACKGROUND: The intracellular accommodation of arbuscular mycorrhizal (AM) fungi involves a profound molecular reprogramming of the host cell architecture and metabolism, based on the activation of a symbiotic signaling pathway. In analogy with other plant biotrophs, AM fungi are reported to trigger cell cycle reactivation in their host tissues, possibly in support of the enhanced metabolic demand required for the symbiosis. RESULTS: We here compare the efficiency of three Fiji/ImageJ image analysis plugins in localizing and quantifying the increase in nuclear size - a hallmark of recursive events of endoreduplication - in M. truncatula roots colonized by the AM fungus Gigaspora margarita. All three approaches proved to be versatile and upgradeable, allowing the investigation of nuclear changes in a complex tissue; 3D Object Counter provided more detailed information than both TrackMate and Round Surface Detector plugins. On this base we challenged 3D Object Counter with two case studies: verifying the lack of endoreduplication-triggering responses in Medicago truncatula mutants with a known non-symbiotic phenotype; and analysing the correlation in space and time between the induction of cortical cell division and endoreduplication upon AM colonization. Both case studies revealed important biological aspects. Mutant phenotype analyses have demonstrated that the knock-out mutation of different key genes in the symbiotic signaling pathway block AM-associated endoreduplication. Furthermore, our data show that cell divisions occur during initial stages of root colonization and are followed by recursive activation of the endocycle in preparation for arbuscule accommodation. CONCLUSIONS: In conclusion, our results indicate 3D Object Counter as the best performing Fiji/ImageJ image analysis script in plant root thick sections and its application highlighted endoreduplication as a major feature of the AM pre-penetration response in root cortical cells.
Assuntos
Tamanho do Núcleo Celular , Medicago truncatula/ultraestrutura , Micorrizas/ultraestrutura , Processamento de Imagem Assistida por Computador , Raízes de Plantas/ultraestruturaRESUMO
Symbiotic nitrogen fixation in legume root nodules requires a steady supply of molybdenum for synthesis of the iron-molybdenum cofactor of nitrogenase. This nutrient has to be provided by the host plant from the soil, crossing several symplastically disconnected compartments through molybdate transporters, including members of the MOT1 family. Medicago truncatula Molybdate Transporter (MtMOT) 1.2 is a Medicago truncatula MOT1 family member located in the endodermal cells in roots and nodules. Immunolocalization of a tagged MtMOT1.2 indicates that it is associated to the plasma membrane and to intracellular membrane systems, where it would be transporting molybdate towards the cytosol, as indicated in yeast transport assays. Loss-of-function mot1.2-1 mutant showed reduced growth compared with wild-type plants when nitrogen fixation was required but not when nitrogen was provided as nitrate. While no effect on molybdenum-dependent nitrate reductase activity was observed, nitrogenase activity was severely affected, explaining the observed difference of growth depending on nitrogen source. This phenotype was the result of molybdate not reaching the nitrogen-fixing nodules, since genetic complementation with a wild-type MtMOT1.2 gene or molybdate-fortification of the nutrient solution, both restored wild-type levels of growth and nitrogenase activity. These results support a model in which MtMOT1.2 would mediate molybdate delivery by the vasculature into the nodules.
Assuntos
Proteínas de Transporte de Ânions/fisiologia , Medicago truncatula/metabolismo , Molibdênio/metabolismo , Proteínas de Plantas/fisiologia , Nódulos Radiculares de Plantas/metabolismo , Proteínas de Transporte de Ânions/metabolismo , Medicago truncatula/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/ultraestruturaRESUMO
Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondary metabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Here we show that, in the legume Medicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD) quality control system to manage the production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membrane-anchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulation is prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.
Assuntos
Regulação da Expressão Gênica de Plantas , Medicago truncatula/genética , Medicago truncatula/metabolismo , Células Cultivadas , Degradação Associada com o Retículo Endoplasmático , Perfilação da Expressão Gênica , Inativação Gênica , Teste de Complementação Genética , Medicago truncatula/enzimologia , Medicago truncatula/ultraestrutura , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Mutação , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saponinas/biossíntese , Saponinas/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Legumes improve their mineral nutrition through nitrogen-fixing root nodule symbioses with soil rhizobia. Rhizobial infection of legumes is regulated by a number of transcription factors, including ERF Required for Nodulation1 (ERN1). Medicago truncatula plants defective in ERN1 are unable to nodulate, but still exhibit early symbiotic responses including rhizobial infection. ERN1 has a close homolog, ERN2, which shows partially overlapping expression patterns. Here we show that ern2 mutants exhibit a later nodulation phenotype than ern1, being able to form nodules but with signs of premature senescence. Molecular characterization of the ern2-1 mutation reveals a key role for a conserved threonine for both DNA binding and transcriptional activity. In contrast to either single mutant, the double ern1-1 ern2-1 line is completely unable to initiate infection or nodule development. The strong ern1-1 ern2-1 phenotype demonstrates functional redundancy between these two transcriptional regulators and reveals the essential role of ERN1/ERN2 to coordinately induce rhizobial infection and nodule organogenesis. While ERN1/ERN2 act in concert in the root epidermis, only ERN1 can efficiently allow the development of mature nodules in the cortex, probably through an independent pathway. Together, these findings reveal the key roles that ERN1/ERN2 play at the very earliest stages of root nodule development.
Assuntos
Medicago truncatula/metabolismo , Medicago truncatula/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Raízes de Plantas/microbiologia , Rhizobium/fisiologia , Simbiose , Fatores de Transcrição/metabolismo , Alelos , Sequência de Aminoácidos , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Medicago truncatula/genética , Medicago truncatula/ultraestrutura , Mutação/genética , Micorrizas/fisiologia , Fixação de Nitrogênio , Organogênese/genética , Epiderme Vegetal/genética , Epiderme Vegetal/microbiologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/ultraestrutura , Regiões Promotoras Genéticas/genética , Ligação Proteica , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Nódulos Radiculares de Plantas/ultraestrutura , Transdução de Sinais/genética , Simbiose/genética , Fatores de Transcrição/química , Transcrição GênicaRESUMO
During arbuscular mycorrhizal symbiosis, arbuscule-containing root cortex cells display a proliferation of plastids, a feature usually ascribed to an increased plant anabolism despite the lack of studies focusing on purified root plastids. In this study, we investigated mycorrhiza-induced changes in plastidic pathways by performing a label-free comparative subcellular quantitative proteomic analysis targeted on plastid-enriched fractions isolated from Medicago truncatula roots, coupled to a cytological analysis of plastid structure. We identified 490 root plastid protein candidates, among which 79 changed in abundance upon mycorrhization, as inferred from spectral counting. According to cross-species sequence homology searches, the mycorrhiza-responsive proteome was enriched in proteins experimentally localized in thylakoids, whereas it was depleted of proteins ascribed predominantly to amyloplasts. Consistently, the analysis of plastid morphology using transmission electron microscopy indicated that starch depletion associated with the proliferation of membrane-free and tubular membrane-containing plastids was a feature specific to arbusculated cells. The loss of enzymes involved in carbon/nitrogen assimilation and provision of reducing power, coupled to macromolecule degradation events in the plastid-enriched fraction of mycorrhizal roots that paralleled lack of starch accumulation in arbusculated cells, lead us to propose that arbuscule functioning elicits a nutrient starvation and an oxidative stress signature that may prime arbuscule breakdown.
Assuntos
Regulação da Expressão Gênica de Plantas , Medicago truncatula/fisiologia , Micorrizas/fisiologia , Proteoma , Medicago truncatula/microbiologia , Medicago truncatula/ultraestrutura , Micorrizas/ultraestrutura , Proteínas de Plantas/metabolismo , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Raízes de Plantas/ultraestrutura , Plastídeos/metabolismo , Plastídeos/ultraestrutura , Proteômica , SimbioseRESUMO
The symbiotic interaction between legume plants and rhizobia results in the formation of root nodules, in which symbiotic plant cells host and harbor thousands of nitrogen-fixing rhizobia. Here, a Medicago truncatula nodules with activated defense 1 (nad1) mutant was identified using reverse genetics methods. The mutant phenotype was characterized using cell and molecular biology approaches. An RNA-sequencing technique was used to analyze the transcriptomic reprogramming of nad1 mutant nodules. In the nad1 mutant plants, rhizobial infection and propagation in infection threads are normal, whereas rhizobia and their symbiotic plant cells become necrotic immediately after rhizobia are released from infection threads into symbiotic cells of nodules. Defense-associated responses were detected in nad1 nodules. NAD1 is specifically present in root nodule symbiosis plants with the exception of Morus notabilis, and the transcript is highly induced in nodules. NAD1 encodes a small uncharacterized protein with two predicted transmembrane helices and is localized at the endoplasmic reticulum. Our data demonstrate a positive role for NAD1 in the maintenance of rhizobial endosymbiosis during nodulation.
Assuntos
Medicago truncatula/microbiologia , Proteínas de Plantas/metabolismo , Rhizobium/fisiologia , Simbiose/fisiologia , Sequência de Aminoácidos , Reprogramação Celular/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Teste de Complementação Genética , Medicago truncatula/genética , Medicago truncatula/ultraestrutura , Mutação/genética , Fixação de Nitrogênio/genética , Especificidade de Órgãos/genética , Fenóis/metabolismo , Fenótipo , Filogenia , Proteínas de Plantas/genética , Transporte Proteico , Nódulos Radiculares de Plantas/microbiologia , Nódulos Radiculares de Plantas/ultraestrutura , Alinhamento de Sequência , Transcriptoma/genéticaRESUMO
The nitrogen-fixing rhizobia in the symbiotic infected cells of root nodules are kept in membrane compartments derived from the host cell plasma membrane, forming what are known as symbiosomes. These are maintained as individual units, with mature symbiosomes having a specific radial position in the host cell cytoplasm. The mechanisms that adapt the host cell architecture to accommodate intracellular bacteria are not clear. The intracellular organization of any cell depends heavily on the actin cytoskeleton. Dynamic rearrangement of the actin cytoskeleton is crucial for cytoplasm organization and intracellular trafficking of vesicles and organelles. A key component of the actin cytoskeleton rearrangement is the ARP2/3 complex, which nucleates new actin filaments and forms branched actin networks. To clarify the role of the ARP2/3 complex in the development of infected cells and symbiosomes, we analyzed the pattern of actin microfilaments and the functional role of ARP3 in Medicago truncatula root nodules. In infected cells, ARP3 protein and actin were spatially associated with maturing symbiosomes. Partial ARP3 silencing causes defects in symbiosome development; in particular, ARP3 silencing disrupts the final differentiation steps in functional maturation into nitrogen-fixing units.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/ultraestrutura , Proteína 3 Relacionada a Actina/ultraestrutura , Actinas/ultraestrutura , Medicago truncatula/ultraestrutura , Sinorhizobium meliloti/fisiologia , Simbiose , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteína 3 Relacionada a Actina/genética , Proteína 3 Relacionada a Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Citoplasma/metabolismo , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Medicago truncatula/genética , Medicago truncatula/microbiologia , Fixação de Nitrogênio , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/ultraestrutura , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Raízes de Plantas/ultraestrutura , Transporte Proteico , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/microbiologia , Nódulos Radiculares de Plantas/ultraestruturaRESUMO
Nuclear-associated oscillations in calcium act as a secondary messenger in the symbiotic signaling pathway of legumes. These are decoded by a nuclear-localized calcium and calmodulin-dependent protein kinase, the activation of which is sufficient to drive downstream responses. This implies that the calcium oscillations within the nucleus are the predominant signals for legume symbiosis. However, the mechanisms that allow targeted release of calcium in the nuclear region have not been defined. Here we show that symbiosis-induced calcium changes occur in both the nucleoplasm and the perinuclear cytoplasm and seem to originate from the nuclear membranes. Reaction diffusion simulations suggest that spike generation within the nucleoplasm is not possible through transmission of a calcium wave from the cytoplasm alone and that calcium is likely to be released across the inner nuclear membrane to allow nuclear calcium changes. In agreement with this, we found that the cation channel DMI1, which is essential for symbiotic calcium oscillations, is preferentially located on the inner nuclear membrane, implying an essential function for the inner nuclear membrane in symbiotic calcium signaling. Furthermore, a sarco/endoplasmic reticulum calcium ATPase (SERCA) essential for symbiotic calcium oscillations is targeted to the inner nuclear membrane, as well as the outer nuclear membrane and endoplasmic reticulum (ER). We propose that release of calcium across the inner nuclear membrane allows targeted release of the ER calcium store, and efficient reloading of this calcium store necessitates the capture of calcium from the nucleoplasm and nuclear-associated cytoplasm.
Assuntos
Sinalização do Cálcio , Medicago truncatula/citologia , Medicago truncatula/metabolismo , Membrana Nuclear/metabolismo , Simbiose/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Difusão/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inativação Gênica/efeitos dos fármacos , Canais Iônicos/metabolismo , Lipopolissacarídeos/farmacologia , Medicago truncatula/enzimologia , Medicago truncatula/ultraestrutura , Modelos Biológicos , Dados de Sequência Molecular , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/ultraestrutura , Epiderme Vegetal/citologia , Epiderme Vegetal/efeitos dos fármacos , Epiderme Vegetal/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/ultraestrutura , Transporte Proteico/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Simbiose/efeitos dos fármacosRESUMO
In the Medicago genus, saponins are complex mixtures of triterpene pentacyclic glycosides extensively studied for their different and economically relevant biological and pharmaceutical properties. This research is aimed at determining for the first time the tissue and cellular localization of triterpene saponins in vegetative organs of Medicago truncatula, a model plant species for legumes, by histochemistry and transmission electron microscopy. The results showed that saponins are present mainly in the palisade mesophyll layer of leaves, whereas in stems they are mostly located in the primary phloem and the subepidermal cells of cortical parenchyma. In root tissue, saponins occur in the secondary phloem region. Transmission electron microscopy revealed prominent saponin accumulation within the leaf and stem chloroplasts, while in the roots the saponins are found in the vesicular structures. Our results demonstrate the feasibility of using histochemistry and transmission electron microscopy to localize M. truncatula saponins at tissue and cellular levels and provide important information for further studies on biosynthesis and regulation of valuable bioactive saponins on agronomic relevant Medicago spp., such as alfalfa (Medicago sativa L.). RESEARCH HIGHLIGHTS: The Medicago genus represents a valuable rich source of saponins, one of the most interesting groups of secondary plant metabolites, which possess relevant biological and pharmacological properties. Plant tissue and cellular localization of saponins is of great importance to better understand their biological functions, biosynthetic pathway, and regulatory mechanisms. We elucidate the localization of saponins in Medicago truncatula with histochemical and transmission electron microscopy studies.
Assuntos
Medicago truncatula , Microscopia Eletrônica de Transmissão , Folhas de Planta , Raízes de Plantas , Saponinas , Triterpenos , Medicago truncatula/ultraestrutura , Medicago truncatula/metabolismo , Medicago truncatula/química , Saponinas/metabolismo , Triterpenos/metabolismo , Triterpenos/química , Raízes de Plantas/química , Raízes de Plantas/ultraestrutura , Folhas de Planta/química , Folhas de Planta/ultraestrutura , Caules de Planta/química , Caules de Planta/ultraestrutura , Floema/ultraestrutura , Floema/química , Floema/metabolismo , Histocitoquímica , Cloroplastos/ultraestrutura , Cloroplastos/metabolismo , Cloroplastos/químicaRESUMO
⢠The cell and developmental biology of zygotic embryogenesis in the model legume Medicago truncatula has received little attention. We studied M. truncatula embryogenesis from embryo sac until cotyledon maturation, including oil and protein body biogenesis. ⢠We characterized embryo development using light and electron microscopy, measurement of protein and lipid fatty acid accumulation and by profiling the expression of key seed storage genes. ⢠Embryo sac development in M. truncatula is of the Polygonum type. A distinctive multicellular hypophysis and suspensor develops before the globular stage and by the early cotyledon stage, the procambium connects the developing apical meristems. In the storage parenchyma of cotyledons, ovoid oil bodies surround protein bodies and the plasma membrane. Four major lipid fatty acids accumulate as cotyledons develop, paralleling the expression of OLEOSIN and the storage protein genes, VICILIN and LEGUMIN. ⢠Zygotic embryogenesis in M. truncatula features the development of a distinctive multicellular hypophysis and an endopolyploid suspensor with basal transfer cell. A clear procambial connection between the apical meristems is evident and there is a characteristic arrangement of oil bodies in the cotyledons and radicle. Our data help link embryogenesis to the genetic regulation of oil and protein body biogenesis in legume seed.
Assuntos
Medicago truncatula/embriologia , Modelos Biológicos , Óleos de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Cotilédone/citologia , Cotilédone/ultraestrutura , Ácidos Graxos/biossíntese , Fertilização , Flores/citologia , Flores/ultraestrutura , Regulação da Expressão Gênica de Plantas , Medicago truncatula/citologia , Medicago truncatula/genética , Medicago truncatula/ultraestrutura , Microscopia de Fluorescência , Especificidade de Órgãos/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/metabolismo , Sementes/citologia , Sementes/ultraestrutura , Zigoto/citologia , Zigoto/ultraestruturaRESUMO
Medicago truncatula has been developed into a model legume. Its close relative alfalfa (Medicago sativa) is the most widely grown forage legume crop in the United States. By screening a large population of M. truncatula mutants tagged with the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified a mutant line (NF2089) that maintained green leaves and showed green anthers, central carpels, mature pods, and seeds during senescence. Genetic and molecular analyses revealed that the mutation was caused by Tnt1 insertion in a STAY-GREEN (MtSGR) gene. Transcript profiling analysis of the mutant showed that loss of the MtSGR function affected the expression of a large number of genes involved in different biological processes. Further analyses revealed that SGR is implicated in nodule development and senescence. MtSGR expression was detected across all nodule developmental zones and was higher in the senescence zone. The number of young nodules on the mutant roots was higher than in the wild type. Expression levels of several nodule senescence markers were reduced in the sgr mutant. Based on the MtSGR sequence, an alfalfa SGR gene (MsSGR) was cloned, and transgenic alfalfa lines were produced by RNA interference. Silencing of MsSGR led to the production of stay-green transgenic alfalfa. This beneficial trait offers the opportunity to produce premium alfalfa hay with a more greenish appearance. In addition, most of the transgenic alfalfa lines retained more than 50% of chlorophylls during senescence and had increased crude protein content. This study illustrates the effective use of knowledge gained from a model system for the genetic improvement of an important commercial crop.
Assuntos
Agricultura/métodos , Produtos Agrícolas/genética , Genes de Plantas/genética , Medicago sativa/genética , Medicago truncatula/genética , Modelos Biológicos , Clorofila/metabolismo , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Clonagem Molecular , Escuridão , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Medicago sativa/fisiologia , Medicago truncatula/crescimento & desenvolvimento , Medicago truncatula/ultraestrutura , Mutação/genética , Fenótipo , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/ultraestrutura , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Interferência de RNA , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/metabolismoRESUMO
Cellulose is the most abundant biopolymer on earth, and has qualities that make it suitable for biofuel. There are new tools for the visualisation of the cellulose synthase complexes in living cells, but those do not show their product, the cellulose microfibrils (CMFs). In this study we report the characteristics of cell wall textures, i.e. the architectures of the CMFs in the wall, of root hairs of Arabidopsis thaliana, Medicago truncatula and Vicia sativa and compare the different techniques we used to study them. Root hairs of these species have a random primary cell wall deposited at the root hair tip, which covers the outside of the growing and fully grown hair. The secondary wall starts between 10 (Arabidopsis) and 40 (Vicia) µm from the hair tip and the CMFs make a small angle, Z as well as S direction, with the long axis of the root hair. CMFs are 3-4 nm wide in thin sections, indicating that single cellulose synthase complexes make them. Thin sections after extraction of cell wall matrix, leaving only the CMFs, reveal the type of wall texture and the orientation and width of CMFs, but CMF density within a lamella cannot be quantified, and CMF length is always underestimated by this technique. Field emission scanning electron microscopy and surface preparations for transmission electron microscopy reveal the type of wall texture and the orientation of individual CMFs. Only when the orientation of CMFs in subsequent deposited lamellae is different, their density per lamella can be determined. It is impossible to measure CMF length with any of the EM techniques.
Assuntos
Parede Celular/ultraestrutura , Celulose/ultraestrutura , Microfibrilas/ultraestrutura , Células Vegetais/ultraestrutura , Raízes de Plantas/ultraestrutura , Arabidopsis/química , Arabidopsis/ultraestrutura , Parede Celular/química , Celulose/química , Medicago truncatula/química , Medicago truncatula/ultraestrutura , Microfibrilas/química , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Células Vegetais/química , Raízes de Plantas/química , Vicia sativa/química , Vicia sativa/ultraestruturaRESUMO
Phymatotrichopsis omnivora (Duggar) Hennebert causes a destructive root rot in cotton, alfalfa (Medicago sativa), and many other dicot species. No consistently effective control measures or resistant host germplasm for Phymatotrichum root rot (PRR) are known. The relative genetic intractability of cotton and alfalfa precludes their use as model pathosystem hosts for P. omnivora. Therefore, we used the model legume M. truncatula and its available genetic and genomic resources to investigate PRR. Confocal imaging of P. omnivora interactions with M. truncatula roots revealed that the mycelia do not form any specialized structures for penetration and mainly colonize cortical cells and, eventually, form a mycelial mantle covering the root's surfaces. Expression profiling of M. truncatula roots infected by P. omnivora identified several upregulated genes, including the pathogenesis-related class I and class IV chitinases and genes involved in reactive oxygen species generation and phytohormone (jasmonic acid and ethylene) signaling. Genes involved in flavonoid biosynthesis were induced (2.5- to 10-fold over mock-inoculated controls) at 3 days postinoculation (dpi) in response to fungal penetration. However, the expression levels of flavonoid biosynthesis genes returned to the basal levels with the progress of the disease at 5 dpi. These transcriptome results, confirmed by real-time quantitative polymerase chain reaction analyses, showed that P. omnivora apparently evades induced host defenses and may downregulate phytochemical defenses at later stages of infection to favor pathogenesis.
Assuntos
Ascomicetos/fisiologia , Perfilação da Expressão Gênica/métodos , Medicago truncatula/genética , Medicago truncatula/microbiologia , Transdução de Sinais/fisiologia , Ciclopentanos/metabolismo , Etilenos/metabolismo , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Medicago truncatula/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica de Varredura , Análise de Sequência com Séries de Oligonucleotídeos , Oxilipinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genéticaRESUMO
Medicago truncatula was used to characterize resistance to anthracnose and powdery mildew caused by Colletotrichum trifolii and Erysiphe pisi, respectively. Two isolates of E. pisi (Ep-p from pea and Ep-a from alfalfa) and two races of C. trifolii (races 1 and 2) were used in this study. The A17 genotype was resistant and displayed a hypersensitive response after inoculation with either pathogen, while lines F83005.5 and DZA315.16 were susceptible to anthracnose and powdery mildew, respectively. To identify the genetic determinants underlying resistance in A17, two F7 recombinant inbred line (RIL) populations, LR4 (A17 x DZA315.16) and LR5 (A17 x F83005.5), were phenotyped with E. pisi isolates and C. trifolii races, respectively. Genetic analyses showed that i) resistance to anthracnose is governed mainly by a single major locus to both races, named Ct1 and located on the upper part of chromosome 4; and ii) resistance to powdery mildew involves three distinct loci, Epp1 on chromosome 4 and Epa1 and Epa2 on chromosome 5. The use of a consensus genetic map for the two RIL populations revealed that Ct1 and Epp1, although located in the same genome region, were clearly distinct. In silico analysis in this region identified the presence of several clusters of nucleotide binding site leucine-rich repeat genes. Many of these genes have atypical resistance gene analog structures and display differential expression patterns in distinct stress-related cDNA libraries.
Assuntos
Ascomicetos/fisiologia , Colletotrichum/fisiologia , Imunidade Inata/genética , Medicago truncatula/genética , Medicago truncatula/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Cromossomos de Plantas , Etiquetas de Sequências Expressas , Genes de Plantas , Marcadores Genéticos , Genótipo , Imunidade Inata/imunologia , Endogamia , Medicago truncatula/imunologia , Medicago truncatula/ultraestrutura , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Folhas de Planta/ultraestrutura , Locos de Características QuantitativasRESUMO
This review is devoted to the proteomes of the complex developmental stages of dicotyledoneous (dicot) plant materials. The two core technologies, two-dimensional gel electrophoresis (2-DGE) and mass spectrometry (MS), independently or in combination with each other, are propelling dicot plant proteomics to new discoveries and functions, with the establishment of tissue-specific and organelle proteomes, mostly in Arabidopsis thaliana and Medicago truncatula, revealing their complexity and specificity. These experimental proteomes have provided a good start towards the establishment of high-density 2-DGE reference maps and peptide mass fingerprint databases, for not only the model dicot plants, A. thaliana and M. truncatula, but also other important dicot plants, which will serve as a basis for proteomes of many other dicot plants and plant materials.
Assuntos
Desenvolvimento Vegetal , Proteínas de Plantas/genética , Plantas/química , Proteoma , Proteômica/métodos , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Células Cultivadas , Eletroforese em Gel Bidimensional , Espectrometria de Massas , Medicago truncatula/química , Medicago truncatula/genética , Medicago truncatula/crescimento & desenvolvimento , Medicago truncatula/ultraestrutura , Organelas/genética , Organelas/metabolismo , Proteínas de Plantas/química , Plantas/genética , Plantas/ultraestruturaRESUMO
Heavy metal stress affects both, nodulation and nitrogen fixation of legumes. Mercury triggers disturbances in cellular structure and metabolism but its influence on ROS generation is poorly understood. Copper is redox active metal which in opposition to mercury is an essential micronutrient for plants. Excess of copper is cytotoxic, as it participates in ROS generation via Fenton-type reaction. The present work describes changes in hydrogen peroxide (H2O2) accumulation in response to monthly stress caused by mercury (6 mg/L HgCl2) or copper (60 mg/L CuCl2) in root nodules. H2O2 accumulation viewed with a light microscopy was detected by the use of diaminobenzidine (DAB). 2',7'-Dichlorofluorescein diacetate (H2DCF-DA) was used as a probe for the intracellular localization of H2O2 with a confocal laser scanning system. H2O2 detection under transmission electron microscopy was performed by the use of cerium method. Histochemical localization and light and confocal microscopy investigations revealed that under Hg or Cu treatments distinct amount of H2O2 accumulated mainly in the interzone and nitrogen-fixing zone. Under normal conditions H2O2 accumulated predominantly in the interzone. Electron microscopy observations showed H2O2 accumulation under Hg or Cu- treatments around peribacteroid membranes of mature symbiosomes located within nitrogen-fixing zone. It should be underlined that under normal conditions H2O2 was not detected at the peribacteroid membranes. The main result of our observations is increased accumulation of H2O2 in response to mercury and copper treatments at the peribacteroidal membranes, to our knowledge shown for the first time. Therefore, our results revealed that an overproduction of H2O2 in response to copper or mercury-treatment may account for lowering of nitrogen fixation rates in heavy-metal affected root nodules.
Assuntos
Cobre/toxicidade , Peróxido de Hidrogênio/análise , Medicago truncatula/química , Medicago truncatula/efeitos dos fármacos , Mercúrio/toxicidade , Nódulos Radiculares de Plantas/química , Nódulos Radiculares de Plantas/efeitos dos fármacos , Medicago truncatula/citologia , Medicago truncatula/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Nódulos Radiculares de Plantas/citologia , Nódulos Radiculares de Plantas/ultraestruturaRESUMO
Genomics advances in the model legume, Medicago truncatula, have led to an increase in the number of identified genes encoding proteins with unknown biological function. Determining the intracellular location of uncharacterized proteins often aids in the elucidation of biological function. To expedite such localization studies, we have generated a set of intracellular organelle green fluorescence protein (GFP) marker lines in M. truncatula. In addition to fluorescent detection, this set of organelle marker lines can also be used in immunohistochemical and cellular fractionation detection assays. Moreover, this set of marker lines is compatible with both transient and stable expression systems. Thus, this marker set should prove to be a useful resource for the M. truncatula research community.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Substâncias Luminescentes/metabolismo , Medicago truncatula/metabolismo , Organelas/metabolismo , Biomarcadores , Fluorescência , Proteínas de Fluorescência Verde/genética , Medicago truncatula/genética , Medicago truncatula/ultraestrutura , Organelas/ultraestrutura , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Plasmídeos , Transporte ProteicoRESUMO
BACKGROUND: Grain legumes play a worldwide role as a source of plant proteins for feed and food. In the model legume Medicago truncatula, the organisation of protein storage vacuoles (PSV) in maturing seeds remains unknown. FINDINGS: The sub-cellular events accompanying the accumulation of vicilin (globulin7S) were analysed during seed mid-maturation. Immuno-detection of vicilin in light microscopy, allowed a semi-quantitative assessment of the protein body complement. The identified populations of vicilin-containing protein bodies are distinguished by their number and size which allowed to propose a model of their biogenesis. Two distributions were detected, enabling a separation of their processing at early and mid maturation stages. The largest protein bodies, at 16 and 20 days after pollination (DAP), were formed by the fusion of small bodies. They have probably attained their final size and correspond to mature vicilin aggregations. Electron microscopic observations revealed the association of the dense protein bodies with rough endoplasmic reticulum. The presence of a ribosome layer surrounding protein bodies, would support an endoplasmic reticulum-vacuole trafficking pathway. CONCLUSIONS: The stastistic analysis may be useful for screening mutations of candidate genes governing protein content. The definitive evidence for an ER-storage vacuole pathway corresponds to a challenge, for the storage of post-translationally unstable proteins. It was proposed for the accumulation of one class of storage protein, the vicilins. This alternative pathway is a matter of controversy in dicotyledonous seeds.
Assuntos
Medicago truncatula/metabolismo , Proteínas de Armazenamento de Sementes/metabolismo , Sementes/metabolismo , Vacúolos/metabolismo , Cotilédone/metabolismo , Retículo Endoplasmático/metabolismo , Medicago truncatula/embriologia , Medicago truncatula/ultraestrutura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Tamanho das Organelas , Polinização , Transporte Proteico , Ribossomos/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/ultraestrutura , Fatores de Tempo , Vacúolos/ultraestruturaRESUMO
Crystals of calcium oxalate often form in cells adjacent to the vascular bundles in the tissues along the xylem stream. This spatial crystal pattern suggests a role for calcium oxalate formation in regulating calcium transport and partitioning to edible organs such as seeds. To investigate this potential role, microscopic and biochemical comparisons were conducted on the different tissues of Medicago truncatula wild-type and the calcium oxalate defective (cod) 5 which lacks the ability to accumulate prismatic crystals in the cells adjacent to the vascular bundles. Calcium measurements showed that cod5 seeds had more calcium and cod5 pods contained less calcium than the corresponding wild-type tissues. Roots, stems, and leaves from cod5 and wild-type had similar calcium content. Although cod5 was devoid of prismatic crystals, cod5 pods were observed to form druse crystals of calcium oxalate not found in wild-type pods. Taken together these findings suggest a functional role for calcium oxalate formation in regulating calcium transport to the seeds. Regulating calcium uptake at the roots also appeared to be another point of control in determining seed calcium content. Overall, regulating the long distance transport and partitioning of calcium to the seeds appears to be a complex process with multiple points of control.