RESUMO
Secondary xylem and phloem originate from a lateral meristem called the vascular cambium that consists of one to several layers of meristematic cells. Recent lineage tracing studies have shown that only one of the cambial cells in each radial cell file functions as the stem cell, capable of producing both secondary xylem and phloem. Here, we first review how phytohormones and signalling peptides regulate vascular cambium formation and activity. We then propose how the stem cell concept, familiar from apical meristems, could be applied to cambium studies. Finally, we discuss how this concept could set the basis for future research.
Assuntos
Câmbio , Células-Tronco , Xilema , Câmbio/citologia , Câmbio/crescimento & desenvolvimento , Câmbio/fisiologia , Células-Tronco/citologia , Xilema/citologia , Floema/citologia , Reguladores de Crescimento de Plantas/metabolismo , Transdução de Sinais , Feixe Vascular de Plantas/crescimento & desenvolvimento , Feixe Vascular de Plantas/citologia , Meristema/citologia , Meristema/crescimento & desenvolvimentoRESUMO
In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Floema/citologia , Floema/crescimento & desenvolvimento , Raízes de Plantas/citologia , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Diferenciação Celular , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Meristema/citologia , Floema/genética , Floema/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , RNA-Seq , Transdução de Sinais , Análise de Célula Única , Fatores de Transcrição/genética , TranscriptomaRESUMO
The process of phloem unloading and post-unloading transport of photoassimilate is critical to crop output. Xanthoceras sorbifolia is a woody oil species with great biomass energy prospects in China; however, underproduction of seeds seriously restricts its development. Here, our cytological studies by ultrastructural observation revealed that the sieve element-companion cell complex in carpellary bundle was symplasmically interconnected with surrounding parenchyma cells at the early and late fruit developmental stages, whereas it was symplasmically isolated at middle stage. Consistently, real-time imaging showed that fluorescent tracer 6(5)carboxyfluorescein was confined to phloem strands at middle stage but released into surrounding parenchymal cells at early and late stages. Enzymatic assay showed that sucrose synthase act as the key enzyme catalyzing the progress of Suc degradation post-unloading pathway whether in pericarp or in seed, while vacuolar acid invertase and neutral invertase play compensation roles in sucrose decomposition. Sugar transporter XsSWEET10 had a high expression profile in fruit, especially at middle stage. XsSWEET10 is a plasma membrane-localized protein and heterologous expression in SUC2-deficient yeast strain SUSY7/ura3 confirmed its ability to uptake sucrose. These findings approved the transition from symplasmic to apoplasmic phloem unloading in Xanthoceras sorbifolia fruit and XsSWEET10 as a key candidate in sugar transport.
Assuntos
Transporte Biológico/fisiologia , Frutas/crescimento & desenvolvimento , Floema/citologia , Floema/metabolismo , Sapindaceae/anatomia & histologia , Sapindaceae/crescimento & desenvolvimento , Sapindaceae/metabolismo , Sacarose/metabolismo , ChinaRESUMO
BACKGROUND: Cassava (Manihot esculenta Crantz) efficiently accumulates starch in its storage roots. However, how photosynthates are transported from the leaves to the phloem (especially how they are unloaded into parenchymal cells of storage roots) remains unclear. RESULTS: Here, we investigated the sucrose unloading pattern and its impact on cassava storage root development using microstructural and physiological analyses, namely, carboxyfluorescein (CF) and C14 isotope tracing. The expression profiling of genes involved in symplastic and apoplastic transport was performed, which included enzyme activity, protein gel blot analysis, and transcriptome sequencing analyses. These finding showed that carbohydrates are transported mainly in the form of sucrose, and more than 54.6% was present in the stem phloem. Sucrose was predominantly unloaded symplastically from the phloem into storage roots; in addition, there was a shift from apoplastic to symplastic unloading accompanied by the onset of root swelling. Statistical data on the microstructures indicated an enrichment of plasmodesmata within sieve, companion, and parenchyma cells in the developing storage roots of a cultivar but not in a wild ancestor. Tracing tests with CF verified the existence of a symplastic channel, and [14C] Suc demonstrated that sucrose could rapidly diffuse into root parenchyma cells from phloem cells. The relatively high expression of genes encoding sucrose synthase and associated proteins appeared in the middle and late stages of storage roots but not in primary fibrous roots, or secondary fibrous roots. The inverse expression pattern of sucrose transporters, cell wall acid invertase, and soluble acid invertase in these corresponding organs supported the presence of a symplastic sucrose unloading pathway. The transcription profile of genes involved in symplastic unloading and their significantly positive correlation with the starch yield at the population level confirmed that symplastic sucrose transport is vitally important in the development of cassava storage roots. CONCLUSIONS: In this study, we revealed that the cassava storage root phloem sucrose unloading pattern was predominantly a symplastic unloading pattern. This pattern is essential for efficient starch accumulation in high-yielding varieties compared with low-yielding wild ancestors.
Assuntos
Manihot/metabolismo , Floema/fisiologia , Fotossíntese/fisiologia , Raízes de Plantas/metabolismo , Amido/metabolismo , Transporte Biológico , Biomassa , Parede Celular/metabolismo , Difusão , Fluoresceínas/metabolismo , Regulação da Expressão Gênica de Plantas , Manihot/genética , Modelos Biológicos , Floema/citologia , Floema/ultraestrutura , Plasmodesmos/metabolismo , Frações Subcelulares/metabolismo , Sacarose/metabolismo , Açúcares/metabolismoRESUMO
Plant microRNAs (miRNAs) guide cytosolic post-transcriptional gene silencing of sequence-complementary transcripts within the producing cells, as well as in distant cells and tissues. Here, we used an artificial miRNA-based system (amiRSUL) in Arabidopsis thaliana to explore the still elusive mechanisms of inter-cellular miRNA movement via forward genetics. This screen identified many mutant alleles of HASTY (HST), the ortholog of mammalian EXPORTIN5 (XPO5) with a recently reported role in miRNA biogenesis in Arabidopsis. In both epidermis-peeling and grafting assays, amiRSUL levels were reduced much more substantially in miRNA-recipient tissues than in silencing-emitting tissues. We ascribe this effect to HST controlling cell-to-cell and phloem-mediated movement of the processed amiRSUL, in addition to regulating its biogenesis. While HST is not required for the movement of free GFP or siRNAs, its cell-autonomous expression in amiRSUL-emitting tissues suffices to restore amiRSUL movement independently of its nucleo-cytosolic shuttling activity. By contrast, HST is dispensable for the movement and activity of amiRSUL within recipient tissues. Finally, HST enables movement of endogenous miRNAs that display mostly unaltered steady-state levels in hst mutant tissues. We discuss a role for HST as a hitherto unrecognized regulator of miRNA movement in relation to its recently assigned nuclear function at the nexus of MIRNA transcription and miRNA processing.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Carioferinas/metabolismo , MicroRNAs/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Transporte Biológico/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Carioferinas/genética , Mutação , Floema/citologia , Floema/genética , Células Vegetais , Raízes de Plantas/citologia , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Interferência de RNA , RNA de Plantas , Xilema/citologia , Xilema/genéticaRESUMO
The shoot apical meristem allows for reiterative formation of new aerial structures throughout the life cycle of a plant. We use single-cell RNA sequencing to define the cellular taxonomy of the Arabidopsis vegetative shoot apex at the transcriptome level. We find that the shoot apex is composed of highly heterogeneous cells, which can be partitioned into 7 broad populations with 23 transcriptionally distinct cell clusters. We delineate cell-cycle continuums and developmental trajectories of epidermal cells, vascular tissue, and leaf mesophyll cells and infer transcription factors and gene expression signatures associated with cell fate decisions. Integrative analysis of shoot and root apical cell populations further reveals common and distinct features of epidermal and vascular tissues. Our results, thus, offer a valuable resource for investigating the basic principles underlying cell division and differentiation in plants at single-cell resolution.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Ciclo Celular , Diferenciação Celular , Gravitropismo/genética , Floema/citologia , Epiderme Vegetal/crescimento & desenvolvimento , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/citologia , Brotos de Planta/genética , Brotos de Planta/metabolismo , Estômatos de Plantas/crescimento & desenvolvimento , RNA-Seq , Análise de Célula Única , Xilema/citologiaRESUMO
Optimal plant growth is hampered by deficiency of the essential macronutrient phosphate in most soils. Plant roots can, however, increase their root hair density to efficiently forage the soil for this immobile nutrient. By generating and exploiting a high-resolution single-cell gene expression atlas of Arabidopsis roots, we show an enrichment of TARGET OF MONOPTEROS 5/LONESOME HIGHWAY (TMO5/LHW) target gene responses in root hair cells. The TMO5/LHW heterodimer triggers biosynthesis of mobile cytokinin in vascular cells and increases root hair density during low-phosphate conditions by modifying both the length and cell fate of epidermal cells. Moreover, root hair responses in phosphate-deprived conditions are TMO5- and cytokinin-dependent. Cytokinin signaling links root hair responses in the epidermis to perception of phosphate depletion in vascular cells.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Meristema/crescimento & desenvolvimento , Floema/crescimento & desenvolvimento , Fosfatos/deficiência , Epiderme Vegetal/crescimento & desenvolvimento , Transativadores/fisiologia , Xilema/crescimento & desenvolvimento , Arabidopsis/citologia , Arabidopsis/genética , Citocininas/biossíntese , Citocininas/genética , Meristema/citologia , Meristema/metabolismo , Floema/citologia , Floema/metabolismo , Epiderme Vegetal/citologia , Epiderme Vegetal/genética , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Xilema/citologia , Xilema/metabolismoRESUMO
The phloem transports photosynthetic assimilates and signalling molecules. It mainly consists of sieve elements (SEs), which act as "highways" for transport, and companion cells (CCs), which serve as "gates" to load/unload cargos. Though SEs and CCs function together, it remains unknown what determines the ratio of SE/CC in the phloem. Here we develop a new culture system for CC differentiation in Arabidopsis named VISUAL-CC, which almost mimics the process of the SE-CC complex formation. Comparative expression analysis in VISUAL-CC reveals that SE and CC differentiation tends to show negative correlation, while total phloem differentiation is unchanged. This varying SE/CC ratio is largely dependent on GSK3 kinase activity. Indeed, gsk3 hextuple mutants possess many more SEs and fewer CCs, whereas gsk3 gain-of-function mutants partially increase the CC number. Taken together, GSK3 activity appears to function as a cell-fate switch in the phloem, thereby balancing the SE/CC ratio.
Assuntos
Arabidopsis/enzimologia , Diferenciação Celular , Quinase 3 da Glicogênio Sintase/metabolismo , Floema/enzimologia , Plantas Geneticamente Modificadas/enzimologia , Arabidopsis/citologia , Arabidopsis/genética , Técnicas de Cultura de Células , Células Cultivadas , Regulação da Expressão Gênica de Plantas , Quinase 3 da Glicogênio Sintase/genética , Mutação , Floema/citologia , Floema/genética , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/genética , Transdução de SinaisRESUMO
Asymmetric cell division (ACD) and positional signals play critical roles in the tissue patterning process. In the Arabidopsis (Arabidopsis thaliana) root meristem, two major phloem cell types arise via ACDs of distinct origins: one for companion cells (CCs) and the other for proto- and metaphloem sieve elements (SEs). The molecular mechanisms underlying each of these processes have been reported; however, how these are coordinated has remained elusive. Here, we report a new phloem development process coordinated via the SHORTROOT (SHR) transcription factor in Arabidopsis. The movement of SHR into the endodermis regulates the ACD for CC formation by activating microRNA165/6, while SHR moving into the phloem regulates the ACD generating the two phloem SEs. In the phloem, SHR sequentially activates NAC-REGULATED SEED MORPHOLOGY 1 (NARS1) and SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 2 (SND2), and these three together form a positive feedforward loop. Under this regulatory scheme, NARS1, generated in the CCs of the root differentiation zone, establishes a top-down signal that drives the ACD for phloem SEs in the meristem. SND2 appears to function downstream to amplify NARS1 via positive feedback. This new regulatory mechanism expands our understanding of the sophisticated vascular tissue patterning processes occurring during postembryonic root development.plantcell;32/5/1519/FX1F1fx1.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Floema/crescimento & desenvolvimento , Raízes de Plantas/crescimento & desenvolvimento , Transdução de Sinais , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Divisão Celular Assimétrica , Diferenciação Celular , Regulação da Expressão Gênica de Plantas , Genoma de Planta , MicroRNAs/genética , MicroRNAs/metabolismo , Floema/citologia , Floema/genética , Raízes de Plantas/citologia , Raízes de Plantas/genética , Fatores de Transcrição/genéticaRESUMO
Stem cells undergo cell division and differentiation to ensure organized tissue development. Because plant cells are immobile, plant stem cells ought to decide their cell fate prior to differentiation, to locate specialized cells in the correct position. In this study, based on a chemical screen, we isolated a novel secondary cell wall indicator BF-170, which binds to lignin and can be used to image in vitro and in situ xylem development. Use of BF-170 to observe the vascular differentiation pattern in the in vitro vascular cell induction system, VISUAL, revealed that adaxial mesophyll cells of cotyledons predominantly generate ectopic xylem cells. Moreover, phloem cells are abundantly produced on the abaxial layer, suggesting the involvement of leaf adaxial-abaxial polarity in determining vascular cell fate. Analysis of abaxial polarity mutants highlighted the role of YAB3, an abaxial cell fate regulator, in suppressing xylem and promoting phloem differentiation on the abaxial domains in VISUAL. Furthermore, YABBY family genes affected in vivo vascular development during the secondary growth. Our results denoted the possibility that such mediators of spatial information contribute to correctly determine the cell fate of vascular stem cells, to conserve the vascular pattern of land plants.
Assuntos
Diferenciação Celular/fisiologia , Imagem Óptica/métodos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Células-Tronco/metabolismo , Compostos de Anilina , Arabidopsis/citologia , Arabidopsis/genética , Parede Celular , Cotilédone/citologia , Cotilédone/genética , Cotilédone/crescimento & desenvolvimento , Cotilédone/metabolismo , Corantes Fluorescentes , Genes de Plantas , Lignina/metabolismo , Floema/citologia , Floema/genética , Floema/crescimento & desenvolvimento , Folhas de Planta/citologia , Raízes de Plantas/citologia , Quinolinas , Xilema/citologia , Xilema/genética , Xilema/crescimento & desenvolvimentoRESUMO
Vascular plants provide most of the biomass, food, and feed on earth, yet the molecular innovations that led to the evolution of their conductive tissues are unknown. Here, we reveal the evolutionary trajectory for the heterodimeric TMO5/LHW transcription factor complex, which is rate-limiting for vascular cell proliferation in Arabidopsis thaliana Both regulators have origins predating vascular tissue emergence, and even terrestrialization. We further show that TMO5 evolved its modern function, including dimerization with LHW, at the origin of land plants. A second innovation in LHW, coinciding with vascular plant emergence, conditioned obligate heterodimerization and generated the critical function in vascular development in Arabidopsis In summary, our results suggest that the division potential of vascular cells may have been an important factor contributing to the evolution of vascular plants.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Transativadores/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proliferação de Células/genética , Floema/citologia , Floema/crescimento & desenvolvimento , Floema/metabolismo , Filogenia , Plantas Geneticamente Modificadas , Multimerização Proteica/genética , Transativadores/metabolismo , Xilema/citologia , Xilema/crescimento & desenvolvimento , Xilema/metabolismoRESUMO
Multiple flowering pathways in Arabidopsis (Arabidopsis thaliana) converge on the transcriptional regulation of FLOWERING LOCUS T (FT), encoding a mobile floral stimulus that moves from leaves to the shoot apex. Despite our progress in understanding FT movement, the mechanisms underlying its transport along the endoplasmic reticulum-plasmalemma pathway in phloem companion cells remain largely unclear. Here, we show that the plasma membrane-resident syntaxin-like glutamine-soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor (Q-SNARE), SYNTAXIN OF PLANTS121 (SYP121), interacts with QUIRKY (QKY), a member of the family of multiple C2 domain and transmembrane region proteins (MCTPs), to mediate FT transport in Arabidopsis. QKY and SYP121 coordinately regulate FT movement to the plasmalemma through the endosomal trafficking pathway and are required for FT export from companion cells to sieve elements, thus affecting FT transport through the phloem to the shoot apical meristem. These findings suggest that MCTP-SNARE complex-mediated endosomal trafficking is essential for the export of florigen from phloem companion cells to sieve elements to induce flowering.plantcell;31/10/2475/FX1F1fx1.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Florígeno/metabolismo , Flores/crescimento & desenvolvimento , Proteínas Q-SNARE/metabolismo , Proteínas de Arabidopsis/genética , Endossomos/metabolismo , Flores/genética , Flores/metabolismo , Flores/efeitos da radiação , Regulação da Expressão Gênica de Plantas , Meristema/citologia , Meristema/metabolismo , Mutação , Floema/citologia , Floema/metabolismo , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Transporte Proteico/genética , Transporte Proteico/fisiologia , Proteínas Q-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Luz SolarRESUMO
The regulation of sugar metabolism and partitioning plays an essential role for a plant's acclimation to its environment, with specific responses in autotrophic and heterotrophic organs. In this work, we analyzed the effects of high salinity on sugar partitioning and vascular anatomy within the floral stem. Stem sucrose and fructose content increased, while starch reduced, in contrast to the response observed in rosette leaves of the same plants. In the stem, the effects were associated with changes in the expression of SWEET and TMT2 genes encoding sugar transporters, SUSY1 encoding a sucrose synthase and several FRK encoding fructokinases. By contrast, the expression of SUC2, SWEET11 and SWEET12, encoding sugar transporters for phloem loading, remained unchanged in the stem. Both the anatomy of vascular tissues and the composition of xylem secondary cell walls were altered, suggesting that high salinity triggered major readjustments of sugar partitioning in this heterotrophic organ. There were changes in the composition of xylem cell walls, associated with the collapse and deformation of xylem vessels. The data are discussed regarding sugar partitioning and homeostasis of sugars in the vascular tissues of the stem.
Assuntos
Floema/metabolismo , Estresse Salino , Açúcares/metabolismo , Xilema/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Flores/crescimento & desenvolvimento , Frutoquinases/genética , Frutoquinases/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Homeostase , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Floema/citologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Xilema/citologiaRESUMO
In the phloem cap region of Arabidopsis plants, sulfur-rich cells (S-cells) accumulate >100 mM glucosinolates (GLS), but are biosynthetically inactive. The source and route of S-cell-bound GLS remain elusive. In this study, using single-cell sampling and scanning electron microscopy with energy-dispersive X-ray analysis we show that two GLS importers, NPF2.10/GTR1 and NPF2.11/GTR2, are critical for GLS accumulation in S-cells, although they are not localized in the S-cells. Comparison of GLS levels in S-cells in multiple combinations of homo- and heterografts of gtr1 gtr2, biosynthetic null mutant and wild-type plants indicate that S-cells accumulate GLS via symplasmic connections either directly from neighboring biosynthetic cells or indirectly to non-neighboring cells expressing GTR1/2. Distinct sources and transport routes exist for different types of GLS, and vary depending on the position of S-cells in the inflorescence stem. Based on these findings, we propose a model illustrating the GLS transport routes either directly from biosynthetic cells or via GTR-mediated import from apoplastic space radially into a symplasmic domain, wherein the S-cells are the ultimate sink. Similarly, we observed accumulation of the cyanogenic glucoside defensive compounds in high-turgor cells in the phloem cap of Lotus japonicus, suggesting that storage of defensive compounds in high-turgor cells may be a general mechanism for chemical protection of the phloem cap.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Glucosinolatos/metabolismo , Inflorescência/citologia , Floema/citologia , Enxofre/metabolismo , Arabidopsis/imunologia , Inflorescência/metabolismo , Modelos Biológicos , Floema/metabolismo , Transporte ProteicoRESUMO
In order to successfully analyze and describe any plant tissue, the first challenge is preparation of good anatomical slides. The challenge is even greater when the target tissue has heterogeneous characteristics, such as the phloem where soft and stiff tissues occur side by side. The goal of this chapter is to present a detailed protocol containing various techniques for optimal preparation of phloem tissue samples for light microscopic analysis. The process typically involves the steps of fixation, softening, embedding, sectioning, staining, and mounting. The protocol can be applied to make samples of phloem and surrounding tissues of stems and roots, from woody to herbaceous plants.
Assuntos
Microscopia , Floema/anatomia & histologia , Floema/citologia , Automação Laboratorial , Técnicas de Preparação Histocitológica , Microscopia/métodos , Floema/químicaRESUMO
X-ray microtomography (µCT) is a three-dimensional imaging technique, which has, over the past decade, established itself as a go-to method for nondestructive visualization of plant tissue with submicrometer resolution. µCT is closely related to medical computed tomography, in that a measurement consists of acquiring a series of radiographs from different directions around the sample. Especially with synchrotron X-ray sources, these radiographs exhibit significant phase contrast. This greatly enhances soft tissue contrast, making it well suited for plant imaging. Tomographic reconstruction techniques are then employed to convert the stack of radiographs into a 3D volumetric image. Compared with the laboratory X-ray tube-based systems, synchrotron tomography beamlines also offer high throughput, with tens of samples scanned over the course of a typical 24-h beam time.Synchrotrons are typically operated as user facilities, with a staff member assisting users in aligning the beamline and all instrumentation-related matters. From the user's point of view, success of a synchrotron µCT experiment is often dependent on secure sample mounting, choice of appropriate beam parameters, and post-processing the data, i.e., extracting scientifically meaningful results from the 3D image. In this chapter, we review the issues to consider in preparation of a µCT experiment from the point of view of a phloem researcher, emphasizing those aspects which are directly under the user's control rather than technical specifics, which vary from one beamline to another.
Assuntos
Imageamento Tridimensional , Floema/citologia , Floema/ultraestrutura , Síncrotrons , Microtomografia por Raio-X , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional/métodos , Síncrotrons/instrumentação , Microtomografia por Raio-X/instrumentação , Microtomografia por Raio-X/métodosRESUMO
Measurements of vein density and foliar minor vein phloem cell numbers, minor vein phloem cell sizes, and transfer cell wall ingrowths provide quantitative proxies for the leaf's capacities to load and export photosynthates. While overall infrastructural capacity for sugar loading and sugar export correlated positively and closely with photosynthetic capacity, the specific targets of the adjustment of minor vein organization varied with phloem-loading mechanism, plant life-cycle characteristics, and environmental growth conditions. Among apoplastic loaders, for which sugar loading into the phloem depends on cell membrane-spanning transport proteins, variation in minor vein density, phloem cell number, and level of cell wall ingrowth (when present) were consistently associated with photosynthetic capacity. Among active symplastic loaders, for which sugar loading into the phloem depends on cytosolic enzymes, variation in vein density and phloem cell size were consistently associated with photosynthetic capacity. All of these anatomical features were also subject to acclimatory adjustment depending on species and environmental conditions, with increased levels of these features supporting higher rates of photosynthesis. We present a procedure for the preparation of leaf tissue for minor vein analysis, using both light and transmission electron microscopy, that facilitates quantification of not only phloem features but also xylem features that provide proxies for foliar water import capacity.
Assuntos
Microscopia , Floema/citologia , Folhas de Planta/citologia , Transporte Biológico , Carboidratos , Parede Celular/metabolismo , Histocitoquímica/métodos , Microscopia/métodos , Microscopia/normas , Floema/metabolismo , Floema/ultraestrutura , Fotossíntese , Folhas de Planta/metabolismo , Folhas de Planta/ultraestruturaRESUMO
There have been exciting new results in phloem research in recent years, at least in part made possible by the rapid advancement of microscopic techniques. Several methods for visualizing phloem cells are available. The suitability of each method depends on the organ and species being studied, and on the scientific question being addressed. This review will briefly explain the specific challenges associated with phloem cell visualization. It will then focus on common methods currently being used for studying phloem anatomy, development, and function. Emphasis will be placed on the most recent improvements in imaging techniques which had, or most certainly will have, an impact on phloem research.