RESUMEN
Cassava (Manihot esculenta) is a deciduous woody perennial shrub that stores large amounts of carbon and water in its storage roots. Previous studies have shown that assimilate unloading into storage roots happens symplasmically once secondary anatomy is established. However, mechanisms controlling phloem loading and overall carbon partitioning to different cassava tissues remain unclear. Here, we used a combination of histological, transcriptional, and biochemical analyses on different cassava tissues and at different timepoints to better understand source-sink carbon allocation. We found that cassava likely utilizes a predominantly passive symplasmic phloem loading strategy, indicated by the lack of expression of genes coding for key players of sucrose transport, the existence of branched plasmodesmata in the companion cell/bundle sheath interface of minor leaf veins, and very high leaf sucrose concentrations. Furthermore, we showed that tissue-specific changes in anatomy and non-structural carbohydrate (NSC) contents are associated with tissue-specific modification in gene expression for sucrose cleavage/synthesis, as well as subcellular compartmentalization of sugars. Overall, our data suggest that carbon allocation during storage root filling is mostly facilitated symplasmically and is likely mostly regulated by local tissue demand and subcellular compartmentalization.
RESUMEN
Symplasmicly connected cells called sieve elements form a network of tubes in the phloem of vascular plants. Sieve elements have essential functions as they provide routes for photoassimilate distribution, the exchange of developmental signals, and the coordination of defense responses. Nonetheless, they are the least understood main type of plant cells. They are extremely sensitive, possess a reduced endomembrane system without Golgi apparatus, and lack nuclei and translation machineries, so that transcriptomics and similar techniques cannot be applied. Moreover, the analysis of phloem exudates as a proxy for sieve element composition is marred by methodological problems. We developed a simple protocol for the isolation of sieve elements from leaves and stems of Nicotiana tabacum at sufficient amounts for large-scale proteome analysis. By quantifying the enrichment of individual proteins in purified sieve element relative to bulk phloem preparations, proteins of increased likelyhood to function specifically in sieve elements were identified. To evaluate the validity of this approach, yellow fluorescent protein constructs of genes encoding three of the candidate proteins were expressed in plants. Tagged proteins occurred exclusively in sieve elements. Two of them, a putative cytochrome b561/ferric reductase and a reticulon-like protein, appeared restricted to segments of the endoplasmic reticulum (ER) that were inaccessible to green fluorescent protein dissolved in the ER lumen, suggesting a previously unknown differentiation of the endomembrane system in sieve elements. Evidently, our list of promising candidate proteins ( SI Appendix, Table S1) provides a valuable exploratory tool for sieve element biology.
Asunto(s)
Retículo Endoplásmico/metabolismo , Nicotiana/metabolismo , Células Vegetales/metabolismo , Hojas de la Planta/metabolismo , Tallos de la Planta/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Proteómica , Retículo Endoplásmico/genética , Hojas de la Planta/citología , Hojas de la Planta/genética , Tallos de la Planta/citología , Tallos de la Planta/genética , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Nicotiana/citología , Nicotiana/genéticaRESUMEN
Carbohydrate partitioning from leaves to sink tissues is essential for plant growth and development. The maize (Zea mays) recessive carbohydrate partitioning defective28 (cpd28) and cpd47 mutants exhibit leaf chlorosis and accumulation of starch and soluble sugars. Transport studies with 14C-sucrose (Suc) found drastically decreased export from mature leaves in cpd28 and cpd47 mutants relative to wild-type siblings. Consistent with decreased Suc export, cpd28 mutants exhibited decreased phloem pressure in mature leaves, and altered phloem cell wall ultrastructure in immature and mature leaves. We identified the causative mutations in the Brittle Stalk2-Like3 (Bk2L3) gene, a member of the COBRA family, which is involved in cell wall development across angiosperms. None of the previously characterized COBRA genes are reported to affect carbohydrate export. Consistent with other characterized COBRA members, the BK2L3 protein localized to the plasma membrane, and the mutants condition a dwarf phenotype in dark-grown shoots and primary roots, as well as the loss of anisotropic cell elongation in the root elongation zone. Likewise, both mutants exhibit a significant cellulose deficiency in mature leaves. Therefore, Bk2L3 functions in tissue growth and cell wall development, and this work elucidates a unique connection between cellulose deposition in the phloem and whole-plant carbohydrate partitioning.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Pared Celular/metabolismo , Proteínas de Plantas/genética , Zea mays/genética , Proteínas de Plantas/metabolismo , Zea mays/metabolismoRESUMEN
In most plant tissues, threads of cytoplasm, or plasmodesmata, connect the protoplasts via pores in the cell walls. This enables symplasmic transport, for instance in phloem loading, transport and unloading. Importantly, the geometry of the wall pore limits the size of the particles that may be transported, and also (co-)defines plasmodesmal resistance to diffusion and convective flow. However, quantitative information on transport through plasmodesmata in non-cylindrical cell wall pores is scarce. We have found conical, funnel-shaped cell wall pores in the phloem-unloading zone in growing root tips of five eudicot and two monocot species, specifically between protophloem sieve elements and phloem pole pericycle cells. 3D reconstructions by electron tomography suggested that funnel plasmodesmata possess a desmotubule but lack tethers to fix it in a central position. Model calculations showed that both diffusive and hydraulic resistance decrease drastically in conical and trumpet-shaped cell wall pores compared with cylindrical channels, even at very small opening angles. Notably, the effect on hydraulic resistance was relatively larger. We conclude that funnel plasmodesmata generally are present in specific cell-cell interfaces in angiosperm roots, where they appear to facilitate symplasmic phloem unloading. Interestingly, cytosolic sleeves of most plasmodesmata reported in the literature do not resemble annuli of constant diameter but possess variously shaped widenings. Our evaluations suggest that widenings too small for unambiguous identification on electron micrographs may drastically reduce the hydraulic and diffusional resistance of these pores. Consequently, theoretical models assuming cylindrical symmetries will underestimate plasmodesmal conductivities.
Asunto(s)
Magnoliopsida , Plasmodesmos , Transporte Biológico , Floema , Raíces de Plantas , Plasmodesmos/metabolismoRESUMEN
Thick glistening cell walls occur in sieve tubes of all major land plant taxa. Historically, these 'nacreous walls' have been considered a diagnostic feature of sieve elements; they represent a conundrum, though, in the context of the widely accepted pressure-flow theory as they severely constrict sieve tubes. We employed the cucurbit Gerrardanthus macrorhizus as a model to study nacreous walls in sieve elements by standard and in situ confocal microscopy and electron microscopy, focusing on changes in functional sieve tubes that occur when prepared for microscopic observation. Over 90% of sieve elements in tissue sections processed for microscopy by standard methods exhibit nacreous walls. Sieve elements in whole, live plants that were actively transporting as shown by phloem-mobile tracers, lacked nacreous walls and exhibited open lumina of circular cross-sections instead, an appropriate structure for Münch-type mass flow of the cell contents. Puncturing of transporting sieve elements with micropipettes triggered the rapid (<1 min) development of nacreous walls that occluded the cell lumen almost completely. We conclude that nacreous walls are preparation artefacts rather than structural features of transporting sieve elements. Nacreous walls in land plants resemble the reversibly swellable walls found in various algae, suggesting that they may function in turgor buffering, the amelioration of osmotic stress, wounding-induced sieve tube occlusion, and possibly local defence responses of the phloem.
Asunto(s)
Cucurbitaceae/crecimiento & desarrollo , Transporte Biológico , Pared Celular/fisiología , Pared Celular/ultraestructura , Cucurbitaceae/fisiología , Cucurbitaceae/ultraestructura , Microscopía Confocal , Microscopía Electrónica , Presión Osmótica , Floema/crecimiento & desarrollo , Floema/fisiología , Floema/ultraestructuraRESUMEN
Soil salinity is an increasingly global problem which hampers plant growth and crop yield. Plant productivity depends on optimal water-use efficiency and photosynthetic capacity balanced by stomatal conductance. Whether and how stomatal behavior contributes to salt sensitivity or tolerance is currently unknown. This work identifies guard cell-specific signaling networks exerted by a salt-sensitive and salt-tolerant plant under ionic and osmotic stress conditions accompanied by increasing NaCl loads. We challenged soil-grown Arabidopsis thaliana and Thellungiella salsuginea plants with short- and long-term salinity stress and monitored genome-wide gene expression and signals of guard cells that determine their function. Arabidopsis plants suffered from both salt regimes and showed reduced stomatal conductance while Thellungiella displayed no obvious stress symptoms. The salt-dependent gene expression changes of guard cells supported the ability of the halophyte to maintain high potassium to sodium ratios and to attenuate the abscisic acid (ABA) signaling pathway which the glycophyte kept activated despite fading ABA concentrations. Our study shows that salinity stress and even the different tolerances are manifested on a single cell level. Halophytic guard cells are less sensitive than glycophytic guard cells, providing opportunities to manipulate stomatal behavior and improve plant productivity.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Transporte Iónico , Estomas de Plantas/metabolismo , Estrés Salino , Plantas Tolerantes a la Sal/metabolismoRESUMEN
Forisomes are protein bodies known exclusively from sieve elements of legumes. Forisomes contribute to the regulation of phloem transport due to their unique Ca2+-controlled, reversible swelling. The assembly of forisomes from sieve element occlusion (SEO) protein monomers in developing sieve elements and the mechanism(s) of Ca2+-dependent forisome contractility are poorly understood because the amino acid sequences of SEO proteins lack conventional protein-protein interaction and Ca2+-binding motifs. We selected amino acids potentially responsible for forisome-specific functions by analyzing SEO protein sequences in comparison to those of the widely distributed SEO-related (SEOR), or SEOR proteins. SEOR proteins resemble SEO proteins closely but lack any Ca2+ responsiveness. We exchanged identified candidate residues by directed mutagenesis of the Medicago truncatula SEO1 gene, expressed the mutated genes in yeast (Saccharomyces cerevisiae) and studied the structural and functional phenotypes of the forisome-like bodies that formed in the transgenic cells. We identified three aspartate residues critical for Ca2+ responsiveness and two more that were required for forisome-like bodies to assemble. The phenotypes observed further suggested that Ca2+-controlled and pH-inducible swelling effects in forisome-like bodies proceeded by different yet interacting mechanisms. Finally, we observed a previously unknown surface striation in native forisomes and in recombinant forisome-like bodies that could serve as an indicator of successful forisome assembly. To conclude, this study defines a promising path to the elucidation of the so-far elusive molecular mechanisms of forisome assembly and Ca2+-dependent contractility.
Asunto(s)
Ácido Aspártico/metabolismo , Calcio/metabolismo , Floema/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Medicago truncatula/genética , Medicago truncatula/metabolismo , Mutagénesis Sitio-Dirigida , Organismos Modificados Genéticamente , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de SecuenciaRESUMEN
Cassava (Manihot esculenta) is one of the most important staple food crops worldwide. Its starchy tuberous roots supply over 800 million people with carbohydrates. Yet, surprisingly little is known about the processes involved in filling of those vital storage organs. A better understanding of cassava carbohydrate allocation and starch storage is key to improving storage root yield. Here, we studied cassava morphology and phloem sap flow from source to sink using transgenic pAtSUC2::GFP plants, the phloem tracers esculin and 5(6)-carboxyfluorescein diacetate, as well as several staining techniques. We show that cassava performs apoplasmic phloem loading in source leaves and symplasmic unloading into phloem parenchyma cells of tuberous roots. We demonstrate that vascular rays play an important role in radial transport from the phloem to xylem parenchyma cells in tuberous roots. Furthermore, enzymatic and proteomic measurements of storage root tissues confirmed high abundance and activity of enzymes involved in the sucrose synthase-mediated pathway and indicated that starch is stored most efficiently in the outer xylem layers of tuberous roots. Our findings form the basis for biotechnological approaches aimed at improved phloem loading and enhanced carbohydrate allocation and storage in order to increase tuberous root yield of cassava.
Asunto(s)
Manihot/metabolismo , Floema/metabolismo , Raíces de Plantas/metabolismo , Transporte Biológico , Esculina/metabolismo , Regulación de la Expresión Génica de las Plantas , Manihot/fisiología , Floema/fisiología , Proteínas de Plantas/metabolismo , Raíces de Plantas/fisiología , Xilema/metabolismo , Xilema/fisiologíaRESUMEN
Recent studies have revealed that some responses of fern stomata to environmental signals differ from those of their relatives in seed plants. However, it is unknown whether the biophysical properties of guard cells differ fundamentally between species of both clades. Intracellular micro-electrodes and the fluorescent Ca2+ reporter FURA2 were used to study voltage-dependent cation channels and Ca2+ signals in guard cells of the ferns Polypodium vulgare and Asplenium scolopendrium. Voltage clamp experiments with fern guard cells revealed similar properties of voltage-dependent K+ channels as found in seed plants. However, fluorescent dyes moved within the fern stomata, from one guard cell to the other, which does not occur in most seed plants. Despite the presence of plasmodesmata, which interconnect fern guard cells, Ca2+ signals could be elicited in each of the cells individually. Based on the common properties of voltage-dependent channels in ferns and seed plants, it is likely that these key transport proteins are conserved in vascular plants. However, the symplastic connections between fern guard cells in mature stomata indicate that the biophysical mechanisms that control stomatal movements differ between ferns and seed plants.
Asunto(s)
Calcio/metabolismo , Helechos/citología , Células Vegetales/metabolismo , Plasmodesmos/metabolismo , Transporte Biológico , Citosol/metabolismo , Helechos/metabolismo , Estomas de Plantas/citología , Estomas de Plantas/metabolismo , Polypodium/citología , Polypodium/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismoRESUMEN
In the 1920s, the German forestry scientist Ernst Münch postulated that photo-assimilate transport is a mass flow driven by osmotically induced pressure gradients between source organs (high turgor) and sink organs (lower turgor). Two crucial components of Münch's hypothesis, the translocation by mass flow from sources to sinks and the osmotic mechanism of pressure flow, were established notions at the time, but had been developed by two institutionally separated groups of scholars. A conceptual separation of whole-plant biology from cellular physiology had followed the institutional separation of forestry science from botany in German-speaking central Europe during the so-called Humboldtian reforms, and was reinforced by the delayed institutionalization of plant physiology as an academic discipline. Münch did not invent a novel concept, but accomplished an integration of the organism-focused and the cell-focused research traditions, reducing the polarization that had evolved when research universities emerged in central Europe. Post-Münch debates about the validity of his hypothesis focused increasingly on the suitability of available research methodologies, especially the electron microscope and the proper interpretation of the results it produced. The present work reconstructs the influence of the dynamic scientific and non-scientific context on the history of the Münch hypothesis.
Asunto(s)
Modelos Teóricos , Transporte Biológico/fisiología , Fenómenos Fisiológicos de las PlantasRESUMEN
Using Arabidopsis (Arabidopsis thaliana) seedlings, we identified a range of small fluorescent probes that entered the translocation stream and were unloaded at the root tip. These probes had absorbance/emission maxima ranging from 367/454 to 546/576 nm and represent a versatile toolbox for studying phloem transport. Of the probes that we tested, naturally occurring fluorescent coumarin glucosides (esculin and fraxin) were phloem loaded and transported in oocytes by the sucrose transporter, AtSUC2. Arabidopsis plants in which AtSUC2 was replaced with barley (Hordeum vulgare) sucrose transporter (HvSUT1), which does not transport esculin in oocytes, failed to load esculin into the phloem. In wild-type plants, the fluorescence of esculin decayed to background levels about 2 h after phloem unloading, making it a suitable tracer for pulse-labeling studies of phloem transport. We identified additional probes, such as carboxytetraethylrhodamine, a red fluorescent probe that, unlike esculin, was stable for several hours after phloem unloading and could be used to study phloem transport in Arabidopsis lines expressing green fluorescent protein.
Asunto(s)
Arabidopsis/metabolismo , Colorantes Fluorescentes/metabolismo , Glucósidos/metabolismo , Hordeum/genética , Floema/metabolismo , Animales , Arabidopsis/genética , Transporte Biológico , Cumarinas/química , Cumarinas/metabolismo , Esculina/metabolismo , Expresión Génica , Genes Reporteros , Glucósidos/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Oocitos , Floema/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Plantones/genética , Plantones/metabolismo , XenopusRESUMEN
Kelps, brown algae (Phaeophyceae) of the order Laminariales, possess sieve tubes for the symplasmic long-distance transport of photoassimilates that are evolutionarily unrelated but structurally similar to the tubes in the phloem of vascular plants. We visualized sieve tube structure and wound responses in fully functional, intact Bull Kelp (Nereocystis luetkeana [K. Mertens] Postels & Ruprecht 1840). In injured tubes, apparent slime plugs formed but were unlikely to cause sieve tube occlusion as they assembled at the downstream side of sieve plates. Cell walls expanded massively in the radial direction, reducing the volume of the wounded sieve elements by up to 90%. Ultrastructural examination showed that a layer of the immediate cell wall characterized by circumferential cellulose fibrils was responsible for swelling and suggested that alginates, abundant gelatinous polymers of the cell wall matrix, were involved. Wall swelling was rapid, reversible and depended on intracellular pressure, as demonstrated by pressure-injection of silicon oil. Our results revive the concept of turgor generation and buffering by swelling cell walls, which had fallen into oblivion over the last century. Because sieve tube transport is pressure-driven and controlled physically by tube diameter, a regulatory role of wall swelling in photoassimilate distribution is implied in kelps.
Asunto(s)
Pared Celular/fisiología , Kelp/fisiología , Agua/fisiología , Pared Celular/ultraestructura , Kelp/ultraestructuraRESUMEN
BACKGROUND AND AIMS: In vascular plants, important questions regarding phloem function remain unanswered due to problems with invasive experimental procedures in this highly sensitive tissue. Certain brown algae (kelps; Laminariales) also possess sieve tubes for photoassimilate transport, but these are embedded in large volumes of a gelatinous extracellular matrix which isolates them from neighbouring cells. Therefore, we hypothesized that kelp sieve tubes might tolerate invasive experimentation better than their analogues in higher plants, and sought to establish Nereocystis luetkeana as an experimental system. METHODS: The predominant localization of cellulose and the gelatinous extracellular matrix in N. luetkeana was verified using specific fluorescent markers and confocal laser scanning microscopy. Sieve tubes in intact specimens were loaded with fluorescent dyes, either passively (carboxyfluorescein diacetate; CFDA) or by microinjection (rhodamine B), and the movement of the dyes was monitored by fluorescence microscopy. KEY RESULTS: Application of CFDA demonstrated source to sink bulk flow in N. luetkeana sieve tubes, and revealed the complexity of sieve tube structure, with branches, junctions and lateral connections. Microinjection into sieve elements proved comparatively easy. Pulsed rhodamine B injection enabled the determination of flow velocity in individual sieve elements, and the direct visualization of pressure-induced reversals of flow direction across sieve plates. CONCLUSIONS: The reversal of flow direction across sieve plates by pressurizing the downstream sieve element conclusively demonstrates that a critical requirement of the Münch theory is satisfied in kelp; no such evidence exists for tracheophytes. Because of the high tolerance of its sieve elements to experimental manipulation, N. luetkeana is a promising alternative to vascular plants for studying the fluid mechanics of sieve tube networks.
Asunto(s)
Matriz Extracelular/metabolismo , Kelp/metabolismo , Floema/metabolismo , Presión , Reología , Transporte BiológicoRESUMEN
Intracellular pressure has a multitude of functions in cells surrounded by a cell wall or similar matrix in all kingdoms of life. The functions include cell growth, nastic movements, and penetration of tissue by parasites. The precise measurement of intracellular pressure in the majority of cells, however, remains difficult or impossible due to their small size and/or sensitivity to manipulation. Here, we report on a method that allows precise measurements in basically any cell type over all ranges of pressure. It is based on the compression of nanoliter and picoliter volumes of oil entrapped in the tip of microcapillaries, which we call pico gauges. The production of pico gauges can be accomplished with standard laboratory equipment, and measurements are comparably easy to conduct. Example pressure measurements are performed on cells that are difficult or impossible to measure with other methods.
Asunto(s)
Presión Hidrostática , Técnicas Analíticas Microfluídicas/instrumentación , Citoplasma/metabolismo , Diseño de Equipo , Técnicas Analíticas Microfluídicas/métodosRESUMEN
The phloem provides a network of sieve tubes for long-distance translocation of photosynthates. For over a century, structural proteins in sieve tubes have presented a conundrum since they presumably increase the hydraulic resistance of the tubes while no potential function other than sieve tube or wound sealing in the case of injury has been suggested. Here we summarize and critically evaluate current speculations regarding the roles of these proteins. Our understanding suffers from the suggestive power of images; what looks like a sieve tube plug on micrographs may not actually impede translocation very much. Recent reports of an involvement of SEOR (sieve element occlusion-related) proteins, a class of P-proteins, in the sealing of injured sieve tubes are inconclusive; various lines of evidence suggest that, in neither intact nor injured plants, are SEORs determinative of translocation stoppage. Similarly, the popular notion that P-proteins serve in the defence against phloem sap-feeding insects is unsupported by empirical facts; it is conceivable that in functional sieve tubes, aphids actually could benefit from inducing a plug. The idea that rising cytosolic Ca(2+) generally triggers sieve tube blockage by P-proteins appears widely accepted, despite lacking experimental support. Even in forisomes, P-protein assemblages restricted to one single plant family and the only Ca(2+)-responsive P-proteins known, the available evidence does not unequivocally suggest that plug formation is the cause rather than a consequence of translocation stoppage. We conclude that the physiological roles of structural P-proteins remain elusive, and that in vivo studies of their dynamics in continuous sieve tube networks combined with flow velocity measurements will be required to (hopefully) resolve this scientific roadblock.
Asunto(s)
Áfidos/fisiología , Floema/fisiología , Fenómenos Fisiológicos de las Plantas , Proteínas de Plantas/genética , Plantas/genética , Animales , Conducta Alimentaria , Proteínas de Plantas/metabolismoRESUMEN
Since the first ultrastructural investigations of sieve tubes in the early 1960s, their structure has been a matter of debate. Because sieve tube structure defines frictional interactions in the tube system, the presence of P protein obstructions shown in many transmission electron micrographs led to a discussion about the mode of phloem transport. At present, it is generally agreed that P protein agglomerations are preparation artifacts due to injury, the lumen of sieve tubes is free of obstructions, and phloem flow is driven by an osmotically generated pressure differential according to Münch's classical hypothesis. Here, we show that the phloem contains a distinctive network of protein filaments. Stable transgenic lines expressing Arabidopsis thaliana Sieve-Element-Occlusion-Related1 (SEOR1)-yellow fluorescent protein fusions show that At SEOR1 meshworks at the margins and clots in the lumen are a general feature of living sieve tubes. Live imaging of phloem flow and flow velocity measurements in individual tubes indicate that At SEOR1 agglomerations do not markedly affect or alter flow. A transmission electron microscopy preparation protocol has been generated showing sieve tube ultrastructure of unprecedented quality. A reconstruction of sieve tube ultrastructure served as basis for tube resistance calculations. The impact of agglomerations on phloem flow is discussed.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Floema/ultraestructura , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Clonación Molecular , Colorantes Fluorescentes/metabolismo , Substitución por Congelación , Genes de Plantas , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica de Transmisión , Mutagénesis Insercional , Floema/crecimiento & desarrollo , Floema/metabolismo , Células Vegetales/metabolismo , Fenómenos Fisiológicos de las Plantas , Proteínas de Plantas/genética , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Populus/crecimiento & desarrollo , Populus/metabolismo , Presión , Transporte de Proteínas , Nicotiana/crecimiento & desarrollo , Nicotiana/metabolismo , Transformación GenéticaRESUMEN
The secluded lifestyle of endoparasitic plant nematodes hampers progress toward a comprehensive understanding of plant-nematode interactions. A novel technique that enables nondestructive, long-term observations of a wide range of live nematodes in planta is presented here. As proof of principle, Pratylenchus penetrans, Heterodera schachtii, and Meloidogyne chitwoodi were labeled fluorescently with PKH26 and used to infect Arabidopsis thaliana grown in microscopy rhizosphere chambers. Nematode behavior, development, and morphology were observed for the full duration of each parasite's life cycle by confocal microscopy for up to 27 days after inoculation. PKH26 accumulated in intestinal lipid droplets and had no negative effect on nematode infectivity. This technique enabled visualization of Meloidogyne gall formation, nematode oogenesis, and nematode morphological features, such as the metacorpus, vulva, spicules, and cuticle. Additionally, microscopy rhizosphere chambers were used to characterize plant organelle dynamics during M. chitwoodi infection. Peroxisome abundance strongly increased in early giant cells but showed a marked decrease at later stages of feeding site development, which suggests a modulation of plant peroxisomes by root-knot nematodes during the infection process. Taken together, this technique facilitates studies aimed at deciphering plant-nematode interactions at the cellular and subcellular level and enables unprecedented insights into nematode behavior in planta.
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Arabidopsis/parasitología , Interacciones Huésped-Parásitos , Compuestos Orgánicos , Enfermedades de las Plantas/parasitología , Tylenchoidea/fisiología , Animales , Arabidopsis/citología , Colorantes Fluorescentes , Peroxisomas/parasitología , Peroxisomas/ultraestructura , Raíces de Plantas/citología , Raíces de Plantas/parasitología , Plantas Modificadas Genéticamente , Plantones/parasitología , Tylenchoidea/crecimiento & desarrollo , Tylenchoidea/ultraestructuraRESUMEN
Long-distance assimilate distribution in higher plants takes place in the enucleate sieve-tube system of the phloem. It is generally accepted that flow of assimilates is driven by an osmotically generated pressure differential, as proposed by Ernst Münch more than 80 years ago. In the period between 1960 and 1980, the pressure flow hypothesis was challenged when electron microscopic images suggested that sieve tubes contain obstructions that would prevent passive flow. This led to the proposal of alternative translocation mechanisms. However, most investigators came to the conclusion that obstructions in the sieve-tube path were due to preparation artifacts. New developments in bioimaging have vastly enhanced our ability to study the phloem. Unexpectedly, in vivo studies challenge the pressure-flow hypothesis once again. In this review we summarize current investigations of phloem structure and function and discuss their impact on our understanding of long-distance transport in the phloem.
Asunto(s)
Transporte Biológico , Floema/fisiología , Fenómenos Fisiológicos de las Plantas , Imagen por Resonancia Magnética/métodos , Microscopía Confocal/métodos , Microscopía Electrónica de Rastreo/métodos , Microscopía Electrónica de Transmisión/métodosRESUMEN
The storage of light energy in chemical form through photosynthesis is the key process underlying life as we know it. To utilize photosynthates efficiently as structural materials or as fuel to drive endergonic processes, they have to be transported from where they are produced to where they are needed. In this primer, we provide an overview of basic biophysical concepts underlying our current understanding of the mechanisms of photosynthate long-distance transport, and briefly discuss current developments in the field.
Asunto(s)
Fotosíntesis , Plantas/metabolismo , Transporte Biológico , Modelos Biológicos , Plantas/ultraestructuraRESUMEN
Sieve elements are one of the least understood cell types in plants. Translocation velocities and volume flow to supply sinks with photoassimilates greatly depend on the geometry of the microfluidic sieve tube system and especially on the anatomy of sieve plates and sieve plate pores. Several models for phloem translocation have been developed, but appropriate data on the geometry of pores, plates, sieve elements, and flow parameters are lacking. We developed a method to clear cells from cytoplasmic constituents to image cell walls by scanning electron microscopy. This method allows high-resolution measurements of sieve element and sieve plate geometries. Sieve tube-specific conductivity and its reduction by callose deposition after injury was calculated for green bean (Phaseolus vulgaris), bamboo (Phyllostachys nuda), squash (Cucurbita maxima), castor bean (Ricinus communis), and tomato (Solanum lycopersicum). Phloem sap velocity measurements by magnetic resonance imaging velocimetry indicate that higher conductivity is not accompanied by a higher velocity. Studies on the temporal development of callose show that small sieve plate pores might be occluded by callose within minutes, but plants containing sieve tubes with large pores need additional mechanisms.