RESUMEN
Even though Sugars Will Eventually be Exported Transporters (SWEETs) have been found in every sequenced plant genome, a comprehensive understanding of their functionality is lacking. In this study, we focused on the SWEET family of barley (Hordeum vulgare). A radiotracer assay revealed that expressing HvSWEET11b in African clawed frog (Xenopus laevis) oocytes facilitated the bidirectional transfer of not only just sucrose and glucose, but also cytokinin. Barley plants harboring a loss-of-function mutation of HvSWEET11b could not set viable grains, while the distribution of sucrose and cytokinin was altered in developing grains of plants in which the gene was knocked down. Sucrose allocation within transgenic grains was disrupted, which is consistent with the changes to the cytokinin gradient across grains, as visualized by magnetic resonance imaging and Fourier transform infrared spectroscopy microimaging. Decreasing HvSWEET11b expression in developing grains reduced overall grain size, sink strength, the number of endopolyploid endosperm cells, and the contents of starch and protein. The control exerted by HvSWEET11b over sugars and cytokinins likely predetermines their synergy, resulting in adjustments to the grain's biochemistry and transcriptome.
Asunto(s)
Citocininas , Hordeum , Citocininas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Hordeum/genética , Hordeum/metabolismo , Azúcares/metabolismo , Sacarosa/metabolismoRESUMEN
The initial free expansion of the embryo within a seed is at some point inhibited by its contact with the testa, resulting in its formation of folds and borders. Although less obvious, mechanical forces appear to trigger and accelerate seed maturation. However, the mechanistic basis for this effect remains unclear. Manipulation of the mechanical constraints affecting either the in vivo or in vitro growth of oilseed rape embryos was combined with analytical approaches, including magnetic resonance imaging and computer graphic reconstruction, immunolabelling, flow cytometry, transcriptomic, proteomic, lipidomic and metabolomic profiling. Our data implied that, in vivo, the imposition of mechanical restraints impeded the expansion of testa and endosperm, resulting in the embryo's deformation. An acceleration in embryonic development was implied by the cessation of cell proliferation and the stimulation of lipid and protein storage, characteristic of embryo maturation. The underlying molecular signature included elements of cell cycle control, reactive oxygen species metabolism and transcriptional reprogramming, along with allosteric control of glycolytic flux. Constricting the space allowed for the expansion of in vitro grown embryos induced a similar response. The conclusion is that the imposition of mechanical constraints over the growth of the developing oilseed rape embryo provides an important trigger for its maturation.
RESUMEN
Maize (Zea mays) kernels are the largest cereal grains, and their endosperm is severely oxygen deficient during grain fill. The causes, dynamics, and mechanisms of acclimation to hypoxia are minimally understood. Here, we demonstrate that hypoxia develops in the small, growing endosperm, but not the nucellus, and becomes the standard state, regardless of diverse structural and genetic perturbations in modern maize (B73, popcorn, sweet corn), mutants (sweet4c, glossy6, waxy), and non-domesticated wild relatives (teosintes and Tripsacum species). We also uncovered an interconnected void space at the chalazal pericarp, providing superior oxygen supply to the placental tissues and basal endosperm transfer layer. Modeling indicated a very high diffusion resistance inside the endosperm, which, together with internal oxygen consumption, could generate steep oxygen gradients at the endosperm surface. Manipulation of oxygen supply induced reciprocal shifts in gene expression implicated in controlling mitochondrial functions (23.6 kDa Heat-Shock Protein, Voltage-Dependent Anion Channel 2) and multiple signaling pathways (core hypoxia genes, cyclic nucleotide metabolism, ethylene synthesis). Metabolite profiling revealed oxygen-dependent shifts in mitochondrial pathways, ascorbate metabolism, starch synthesis, and auxin degradation. Long-term elevated oxygen supply enhanced the rate of kernel development. Altogether, evidence here supports a mechanistic framework for the establishment of and acclimation to hypoxia in the maize endosperm.
Asunto(s)
Almidón , Zea mays , Embarazo , Femenino , Humanos , Zea mays/metabolismo , Almidón/metabolismo , Placenta/metabolismo , Endospermo/metabolismo , Oxígeno/metabolismo , Hipoxia/metabolismoRESUMEN
Adventitious root (AR) formation is one of the most important developmental processes in vegetative propagation. Although genotypic differences in rose rooting ability are well known, the causal factors are not well understood. The rooting of two contrasting genotypes, 'Herzogin Friederike' and 'Mariatheresia', was compared following a multiscale approach. Using magnetic resonance imaging, we non-invasively monitored the inner structure of stem cuttings during initiation and progression of AR formation for the first time. Spatially resolved Fourier-transform infrared spectroscopy characterized the chemical composition of the tissues involved in AR formation. The results were validated through light microscopy and complemented by immunolabelling. The outcome demonstrated similarity of both genotypes in root primordia formation, which did not result in root protrusion through the shoot cortex in the difficult-to-root genotype 'Mariatheresia'. The biochemical composition of the contrasting genotypes highlighted main differences in cell wall-associated components. Further spectroscopic analysis of 15 contrasting rose genotypes confirmed the biochemical differences between easy- and difficult-to-root groups. Collectively, our data indicate that it is not the lack of root primordia limiting AR formation in these rose genotypes, but the firmness of the outer stem tissue and/or cell wall modifications that pose a mechanical barrier and prevent root extension and protrusion.
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Genotipo , Imagen por Resonancia Magnética , Raíces de Plantas , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/anatomía & histología , Raíces de Plantas/genética , Espectroscopía Infrarroja por Transformada de Fourier , FenotipoRESUMEN
The aquatic Lemnaceae family, commonly called duckweed, comprises some of the smallest and fastest growing angiosperms known on Earth. Their tiny size, rapid growth by clonal propagation, and facile uptake of labeled compounds from the media were attractive features that made them a well-known model for plant biology from 1950 to 1990. Interest in duckweed has steadily regained momentum over the past decade, driven in part by the growing need to identify alternative plants from traditional agricultural crops that can help tackle urgent societal challenges, such as climate change and rapid population expansion. Propelled by rapid advances in genomic technologies, recent studies with duckweed again highlight the potential of these small plants to enable discoveries in diverse fields from ecology to chronobiology. Building on established community resources, duckweed is reemerging as a platform to study plant processes at the systems level and to translate knowledge gained for field deployment to address some of society's pressing needs. This review details the anatomy, development, physiology, and molecular characteristics of the Lemnaceae to introduce them to the broader plant research community. We highlight recent research enabled by Lemnaceae to demonstrate how these plants can be used for quantitative studies of complex processes and for revealing potentially novel strategies in plant defense and genome maintenance.
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Araceae/genética , Genoma de Planta , GenómicaRESUMEN
BACKGROUND: Duckweeds are small, rapidly growing aquatic flowering plants. Due to their ability for biomass production at high rates they represent promising candidates for biofuel feedstocks. Duckweeds are also excellent model organisms because they can be maintained in well-defined liquid media, usually reproduce asexually, and because genomic resources are becoming increasingly available. To demonstrate the utility of duckweed for integrated metabolic studies, we examined the metabolic adaptation of growing Lemna gibba cultures to different nutritional conditions. RESULTS: To establish a framework for quantitative metabolic research in duckweeds we derived a central carbon metabolism network model of Lemna gibba based on its draft genome. Lemna gibba fronds were grown with nitrate or glutamine as nitrogen source. The two conditions were compared by quantification of growth kinetics, metabolite levels, transcript abundance, as well as by 13C-metabolic flux analysis. While growing with glutamine, the fronds grew 1.4 times faster and accumulated more protein and less cell wall components compared to plants grown on nitrate. Characterization of photomixotrophic growth by 13C-metabolic flux analysis showed that, under both metabolic growth conditions, the Calvin-Benson-Bassham cycle and the oxidative pentose-phosphate pathway are highly active, creating a futile cycle with net ATP consumption. Depending on the nitrogen source, substantial reorganization of fluxes around the tricarboxylic acid cycle took place, leading to differential formation of the biosynthetic precursors of the Asp and Gln families of proteinogenic amino acids. Despite the substantial reorganization of fluxes around the tricarboxylic acid cycle, flux changes could largely not be associated with changes in transcripts. CONCLUSIONS: Through integrated analysis of growth rate, biomass composition, metabolite levels, and metabolic flux, we show that Lemna gibba is an excellent system for quantitative metabolic studies in plants. Our study showed that Lemna gibba adjusts to different nitrogen sources by reorganizing central metabolism. The observed disconnect between gene expression regulation and metabolism underscores the importance of metabolic flux analysis as a tool in such studies.
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Araceae , Transcriptoma , Glutamina/genética , Nitratos/metabolismo , Araceae/genética , Nitrógeno/metabolismoRESUMEN
Imaging has long supported our ability to understand the inner life of plants, their development, and response to a dynamic environment. While optical microscopy remains the core tool for imaging, a suite of novel technologies is now beginning to make a significant contribution to visualize plant metabolism. The purpose of this review was to provide the scientific community with an overview of current imaging methods, which rely variously on either nuclear magnetic resonance (NMR), mass spectrometry (MS) or infrared (IR) spectroscopy, and to present some examples of their application in order to illustrate their utility. In addition to providing a description of the basic principles underlying these technologies, the review discusses their various advantages and limitations, reveals the current state of the art, and suggests their potential application to experimental practice. Finally, a view is presented as to how the technologies will likely develop, how these developments may encourage the formulation of novel experimental strategies, and how the enormous potential of these technologies can contribute to progress in plant science.
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Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética/métodos , Espectrometría de MasasRESUMEN
The tradeoff between protein and oil storage in oilseed crops has been tested here in oilseed rape (Brassica napus) by analyzing the effect of suppressing key genes encoding protein storage products (napin and cruciferin). The phenotypic outcomes were assessed using NMR and mass spectrometry imaging, microscopy, transcriptomics, proteomics, metabolomics, lipidomics, immunological assays, and flux balance analysis. Surprisingly, the profile of storage products was only moderately changed in RNA interference transgenics. However, embryonic cells had undergone remarkable architectural rearrangements. The suppression of storage proteins led to the elaboration of membrane stacks enriched with oleosin (sixfold higher protein abundance) and novel endoplasmic reticulum morphology. Protein rebalancing and amino acid metabolism were focal points of the metabolic adjustments to maintain embryonic carbon/nitrogen homeostasis. Flux balance analysis indicated a rather minor additional demand for cofactors (ATP and NADPH). Thus, cellular plasticity in seeds protects against perturbations to its storage capabilities and, hence, contributes materially to homeostasis. This study provides mechanistic insights into the intriguing link between lipid and protein storage, which have implications for biotechnological strategies directed at improving oilseed crops.
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Brassica napus/citología , Brassica napus/metabolismo , Proteínas de Almacenamiento de Semillas/metabolismo , Semillas/citología , Semillas/metabolismo , Albuminas 2S de Plantas/genética , Albuminas 2S de Plantas/metabolismo , Aminoácidos/metabolismo , Antígenos de Plantas/genética , Antígenos de Plantas/metabolismo , Brassica napus/genética , Carbono/metabolismo , Regulación de la Expresión Génica de las Plantas , Espectroscopía de Resonancia Magnética , Lípidos de la Membrana/genética , Lípidos de la Membrana/metabolismo , Nitrógeno/metabolismo , Células Vegetales , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Interferencia de ARN , Proteínas de Almacenamiento de Semillas/genéticaRESUMEN
The development of crop varieties that are resistant to lodging is a top priority for breeding programmes. Herein, we characterize the rye mutant ´Stabilstroh' ('stable straw') possessing an exceptional combination of high lodging resistance, tall posture and high biomass production. Nuclear magnetic resonance imaging displayed the 3-dimensional assembly of vascular bundles in stem. A higher number of vascular bundles and a higher degree of their incline were the features of lodging-resistant versus lodging-prone lines. Histology and electron microscopy revealed that stems are fortified by a higher proportion of sclerenchyma and thickened cell walls, as well as some epidermal invaginations. Biochemical analysis using Fourier-transform infrared spectroscopy and inductively coupled plasma-optical emission spectrometry further identified elevated levels of lignin, xylan, zinc and silicon as features associated with high lodging resistance. Combined effects of above features caused superior culm stability. A simplistic mathematical model showed how mechanical forces distribute within the stem under stress. Main traits of the lodging-resistant parental line were heritable and could be traced back to the genetic structure of the mutant. Evaluation of lodging-resistant wheat 'Babax' ('Baviacora') versus contrasting, lodging-prone, genotype ´Pastor´ agreed with above findings on rye. Our findings on mechanical stability and extraordinary culm properties may be important for breeders for the improvement of lodging resistance of tall posture cereal crops.
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Secale , Triticum , Grano Comestible/metabolismo , Lignina/metabolismo , Fitomejoramiento/métodos , Secale/genética , Secale/metabolismo , Triticum/metabolismoRESUMEN
Plants undergo several developmental transitions during their life cycle. One of these, the differentiation of the young embryo from a meristem-like structure into a highly specialized storage organ, is believed to be controlled by local connections between sugars and hormonal response systems. However, we know little about the regulatory networks underpinning the sugar-hormone interactions in developing seeds. By modulating the trehalose 6-phosphate (T6P) content in growing embryos of garden pea (Pisum sativum), we investigate here the role of this signaling sugar during the seed-filling process. Seeds deficient in T6P are compromised in size and starch production, resembling the wrinkled seeds studied by Gregor Mendel. We show also that T6P exerts these effects by stimulating the biosynthesis of the pivotal plant hormone, auxin. We found that T6P promotes the expression of the auxin biosynthesis gene TRYPTOPHAN AMINOTRANSFERASE RELATED2 (TAR2), and the resulting effect on auxin concentrations is required to mediate the T6P-induced activation of storage processes. Our results suggest that auxin acts downstream of T6P to facilitate seed filling, thereby providing a salient example of how a metabolic signal governs the hormonal control of an integral phase transition in a crop plant.
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Fosfatos de Azúcar , Trehalosa , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos , Fosfatos , Plantas Modificadas Genéticamente , Semillas , SacarosaRESUMEN
Successful biotrophic plant pathogens can divert host nutrition toward infection sites. Here we describe how the protist Plasmodiophora brassicae establishes a long-term feeding relationship with its host by stimulating phloem differentiation and phloem-specific expression of sugar transporters within developing galls. Development of galls in infected Arabidopsis (Arabidopsis thaliana) plants is accompanied by stimulation of host BREVIS RADIX, COTYLEDON VASCULAR PATTERN, and OCTOPUS gene expression leading to an increase in phloem complexity. We characterized how the arrest of this developmental reprogramming influences both the host and the invading pathogen. Furthermore, we found that infection leads to phloem-specific accumulation of SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS11 and 12 facilitating local distribution of sugars toward the pathogen. Utilizing Fourier-transform infrared microspectroscopy to monitor spatial distribution of carbohydrates, we found that infection leads to the formation of a strong physiological sink at the site of infection. High resolution metabolic and structural imaging of sucrose distributions revealed that sweet11 sweet12 double mutants are impaired in sugar transport toward the pathogen, delaying disease progression. This work highlights the importance of precise regulation of sugar partitioning for plant-pathogen interactions and the dependence of P brassicae's performance on its capacity to induce a phloem sink at the feeding site.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Floema/citología , Floema/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Enfermedades de las Plantas , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Maize opaque2 (o2) mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by o2 include pdk1 and pdk2 that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill. pdk1 and pdk2 were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi. pdk2 accounts for the large majority of endosperm PPDK, whereas pdk1 specifies the abundant mesophyll form. The pdk1- mutation is seedling-lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned by o2-. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal that o2- mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression.
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Endospermo/enzimología , Metabolismo Energético/fisiología , Proteínas de Plantas/metabolismo , Piruvato Ortofosfato Diquinasa/metabolismo , Zea mays/enzimología , Endospermo/genética , Mutación , Proteínas de Plantas/genética , Piruvato Ortofosfato Diquinasa/genética , Almidón/biosíntesis , Almidón/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Zea mays/genéticaRESUMEN
Phylogenetically related groups of species contain lineage-specific genes that exhibit no sequence similarity to any genes outside the lineage. We describe here that the Jekyll gene, required for sexual reproduction, exists in two much diverged allelic variants, Jek1 and Jek3. Despite low similarity, the Jek1 and Jek3 proteins share identical signal peptides, conserved cysteine positions and direct repeats. The Jek1/Jek3 sequences are located at the same chromosomal locus and inherited in a monogenic Mendelian fashion. Jek3 has a similar expression as Jek1 and complements the Jek1 function in Jek1-deficient plants. Jek1 and Jek3 allelic variants were almost equally distributed in a collection of 485 wild and domesticated barley accessions. All domesticated barleys harboring the Jek1 allele belong to single haplotype J1-H1 indicating a genetic bottleneck during domestication. Domesticated barleys harboring the Jek3 allele consisted of three haplotypes. Jekyll-like sequences were found only in species of the closely related tribes Bromeae and Triticeae but not in other Poaceae. Non-invasive magnetic resonance imaging revealed intrinsic grain structure in Triticeae and Bromeae, associated with the Jekyll function. The emergence of Jekyll suggests its role in the separation of the Bromeae and Triticeae lineages within the Poaceae and identifies the Jekyll genes as lineage-specific.
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Variación Genética , Proteínas de Plantas/genética , Poaceae/genética , Alelos , Secuencia de Aminoácidos , Evolución Biológica , Geografía , Haplotipos , Hordeum/citología , Hordeum/genética , Imagen por Resonancia Magnética , Familia de Multigenes , Filogenia , Proteínas de Plantas/metabolismo , Poaceae/citología , Reproducción , Semillas/citología , Semillas/genética , Alineación de Secuencia , Especificidad de la Especie , Triticum/citología , Triticum/genéticaRESUMEN
The adaptation strategies of halophytic seaside barley Hordeum marinum to high salinity and osmotic stress were investigated by nuclear magnetic resonance imaging, as well as ionomic, metabolomic, and transcriptomic approaches. When compared with cultivated barley, seaside barley exhibited a better plant growth rate, higher relative plant water content, lower osmotic pressure, and sustained photosynthetic activity under high salinity, but not under osmotic stress. As seaside barley is capable of controlling Na+ and Cl- concentrations in leaves at high salinity, the roots appear to play the central role in salinity adaptation, ensured by the development of thinner and likely lignified roots, as well as fine-tuning of membrane transport for effective management of restriction of ion entry and sequestration, accumulation of osmolytes, and minimization of energy costs. By contrast, more resources and energy are required to overcome the consequences of osmotic stress, particularly the severity of reactive oxygen species production and nutritional disbalance which affect plant growth. Our results have identified specific mechanisms for adaptation to salinity in seaside barley which differ from those activated in response to osmotic stress. Increased knowledge around salt tolerance in halophytic wild relatives will provide a basis for improved breeding of salt-tolerant crops.
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Adaptación Fisiológica , Hordeum/fisiología , Presión Osmótica , Salinidad , Plantas Tolerantes a la Sal/fisiología , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/genética , Aminoácidos/metabolismo , Antioxidantes/metabolismo , Isótopos de Carbono , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Ontología de Genes , Hordeum/efectos de los fármacos , Hordeum/genética , Hordeum/crecimiento & desarrollo , Espectroscopía de Resonancia Magnética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Metabolómica , Minerales/metabolismo , Fotosíntesis/efectos de los fármacos , Fotosíntesis/genética , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Plantas Tolerantes a la Sal/efectos de los fármacos , Plantas Tolerantes a la Sal/genética , Metabolismo Secundario/efectos de los fármacos , Metabolismo Secundario/genética , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Azúcares/metabolismo , Transcriptoma/genéticaRESUMEN
Hypericin is a molecule of high pharmaceutical importance that is synthesized and stored in dark glands (DGs) of St. John's Wort (Hypericum perforatum). Understanding which genes are involved in dark gland development and hypericin biosynthesis is important for the development of new Hypericum extracts that are highly demanded for medical applications. We identified two transcription factors whose expression is strictly synchronized with the differentiation of DGs. We correlated the content of hypericin, pseudohypericin, endocrocin, skyrin glycosides and several flavonoids with gene expression and DG development to obtain a revised model for hypericin biosynthesis. Here, we report for the first time genotypes which are polymorphic for the presence/total absence (G+/G-) of DGs in their placental tissues (PTs). DG development was characterized in PTs using several microscopy techniques. Fourier transform infrared microscopy was established as a novel method to precisely locate polyaromatic compounds, such as hypericin, in plant tissues. In addition, we obtained transcriptome and metabolome profiles of unprecedented resolution in Hypericum. This study addresses for the first time the development of dark glands and identifies genes that constitute strong building blocks for the further elucidation of hypericin synthesis, its manipulation in plants, its engineering in microbial systems and its applications in medical research.
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Hypericum/genética , Hypericum/metabolismo , Perileno/análogos & derivados , Antracenos , Flavonoides , Genes de Plantas , Metaboloma , Perileno/metabolismo , TranscriptomaRESUMEN
Sucrose (Suc) is the major transport sugar in plants and plays a primary role as an energy source and signal in adaptive and stress responses. An ability to quantify Suc over time and space would serve to advance our understanding of these important processes. Current technologies used for Suc mapping are unable to quantitatively visualize its distribution within tissues. Here, we present an infrared-based microspectroscopic method that allows for the quantitative visualization of Suc at a microscopic level of resolution (â¼12 µm). This method can successfully model the sugar concentration in individual vascular bundles and within a complex organ such as the stem, leaf, or seed. The sensitivity of the assay ranges from 20 to 1,000 mm We applied this method to the cereal crop barley (Hordeum vulgare) and the model plant Arabidopsis (Arabidopsis thaliana) to highlight the potential of the procedure for resolving the spatial distribution of metabolites. We also discuss the relevance of the method for studies on carbon allocation and storage in the context of crop improvement.
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Arabidopsis/metabolismo , Hordeum/metabolismo , Imagen Molecular/métodos , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Sacarosa/análisis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Hipocótilo/metabolismo , Procesamiento de Imagen Asistido por Computador , Proteínas de Transporte de Membrana/genética , Mutación , Hojas de la Planta/metabolismo , Tallos de la Planta/metabolismo , Reproducibilidad de los Resultados , Almidón/metabolismo , Sacarosa/metabolismoRESUMEN
The angiosperm embryo and endosperm are limited in space because they grow inside maternal seed tissues. The elimination of cell layers of the maternal seed coat by programmed cell death (PCD) could provide space and nutrition to the filial organs. Using the barley (Hordeum vulgare L.) seed as a model, we elucidated the role of vacuolar processing enzyme 4 (VPE4) in cereals by using an RNAi approach and targeting the enzymatic properties of the recombinant protein. A comparative characterization of transgenic versus wild-type plants included transcriptional and metabolic profiling, flow cytometry, histology and nuclear magnetic imaging of grains. The recombinant VPE4 protein exhibited legumain and caspase-1 properties in vitro. Pericarp disintegration was delayed in the transgenic grains. Although the VPE4 gene and enzymatic activity was decreased in the early developing pericarp, storage capacity and the size of the endosperm and embryo were reduced in the mature VPE4-repressed grains. The persistence of the pericarp in the VPE4-affected grains constrains endosperm and embryo growth and leads to transcriptional reprogramming, perturbations in signalling and adjustments in metabolism. We conclude that VPE4 expression executes PCD in the pericarp, which is required for later endosperm filling, and argue for a role of PCD in maternal control of seed size in cereals.
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Apoptosis , Cisteína Endopeptidasas/metabolismo , Grano Comestible/anatomía & histología , Hordeum/anatomía & histología , Hordeum/citología , Proteínas de Plantas/metabolismo , Semillas/citología , Semillas/metabolismo , Apoptosis/genética , Caspasas/metabolismo , Recuento de Células , Endospermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Hordeum/genética , Hordeum/crecimiento & desarrollo , Espectroscopía de Resonancia Magnética , Tamaño de los Órganos , Especificidad de Órganos , Fenotipo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Ploidias , Proteolisis , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Transcripción Genética , Transcriptoma/genéticaRESUMEN
The regulation of lipid synthesis in oil seeds is still not fully understood. Oilseed rape (Brassica napus) is the third most productive vegetable oil crop on the global market; therefore, increasing our understanding of lipid accumulation in oilseed rape seeds is of great economic, as well as intellectual, importance. Matrix-assisted laser/desorption ionization-mass spectrometry imaging (MALDI-MSI) is a technique that allows the mapping of metabolites directly onto intact biological tissues, giving a spatial context to metabolism. We have used MALDI-MSI to study the spatial distribution of two major lipid species, triacylglycerols and phosphatidylcholines. A dramatic, heterogenous landscape of molecular species was revealed, demonstrating significantly different lipid compositions between the various tissue types within the seed. The embryonic axis was found to be particularly enriched in palmitic acid, while the seed coat/aleurone layer accumulated vaccenic, linoleic, and α-linoleic acids. Furthermore, the lipid composition of the inner and outer cotyledons differed from each other, a remarkable discovery given the supposed identical functionality of these two tissues. Triacylglycerol and phosphatidylcholine molecular species distribution was analyzed through a developmental time series covering early seed lipid accumulation to seed maturity. The spatial patterning of lipid molecular species did not vary significantly during seed development. Data gathered using MALDI-MSI was verified through gas chromatography analysis of dissected seeds. The distinct lipid distribution profiles observed imply differential regulation of lipid metabolism between the different tissue types of the seed. Further understanding of this differential regulation will enhance efforts to improve oilseed rape productivity and quality.
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Brassica napus/metabolismo , Lípidos/biosíntesis , Aceites de Plantas/metabolismo , Semillas/metabolismo , Análisis Espacio-Temporal , Cromatografía de Gases , Cotiledón/metabolismo , Ácido Linoleico/análisis , Lípidos/química , Espectroscopía de Resonancia Magnética , Ácidos Oléicos/análisis , Ácido Palmítico/análisis , Fosfatidilcolinas/biosíntesis , Fosfatidilcolinas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Tiempo , Triglicéridos/biosíntesis , Triglicéridos/químicaRESUMEN
Germination, the process whereby a dry, quiescent seed springs to life, has been a focus of plant biologist for many years, yet the early events following water uptake, during which metabolism of the embryo is restarted, remain enigmatic. Here, the nature of the cues required for this restarting in oilseed rape (Brassica napus) seed has been investigated. A holistic in vivo approach was designed to display the link between the entry and allocation of water, metabolic events and structural changes occurring during germination. For this, we combined functional magnetic resonance imaging with Fourier transform infrared microscopy, fluorescence-based respiration mapping, computer-aided seed modeling and biochemical tools. We uncovered an endospermal lipid gap, which channels water to the radicle tip, from whence it is distributed via embryonic vasculature toward cotyledon tissues. The resumption of respiration is initiated first in the endosperm, only later spreading to the embryo. Sugar metabolism and lipid utilization are linked to the spatiotemporal sequence of tissue rehydration. Together, this imaging study provides insights into the spatial aspects of key events in oilseed rape seeds leading to germination. It demonstrates how seed architecture predetermines the pattern of water intake, which sets the stage for the orchestrated restart of life.