RESUMEN
The maize ligule is an epidermis-derived structure that arises from the preligule band (PLB) at a boundary between the blade and sheath. A hinge-like auricle also develops immediately distal to the ligule and contributes to blade angle. Here, we characterize the stages of PLB and early ligule development in terms of topography, cell area, division orientation, cell wall rigidity and auxin response dynamics. Differential thickening of epidermal cells and localized periclinal divisions contributed to the formation of a ridge within the PLB, which ultimately produces the ligule fringe. Patterns in cell wall rigidity were consistent with the subdivision of the PLB into two regions along a distinct line positioned at the nascent ridge. The proximal region produces the ligule, while the distal region contributes to one epidermal face of the auricles. Although the auxin transporter PIN1 accumulated in the PLB, observed differential auxin transcriptional response did not underlie the partitioning of the PLB. Our data demonstrate that two zones with contrasting cellular properties, the preligule and preauricle, are specified within the ligular region before ligule outgrowth.
Asunto(s)
Ácidos Indolacéticos , Zea mays , Zea mays/genéticaRESUMEN
Introduction: During proliferative plant cell division, the new cell wall, called the cell plate, is first built in the middle of the cell and then expands outward to complete cytokinesis. This dynamic process requires coordinated movement and arrangement of the cytoskeleton and organelles. Methods: Here we use live-cell markers to track the dynamic reorganization of microtubules, nuclei, endoplasmic reticulum, and endomembrane compartments during division and the formation of the cell plate in maize leaf epidermal cells. Results: The microtubule plus-end localized protein END BINDING1 (EB1) highlighted increasing microtubule dynamicity during mitosis to support rapid changes in microtubule structures. The localization of the cell-plate specific syntaxin KNOLLE, several RAB-GTPases, as well as two plasma membrane localized proteins was assessed after treatment with the cytokinesis-specific callose-deposition inhibitor Endosidin7 (ES7) and the microtubule-disrupting herbicide chlorpropham (CIPC). While ES7 caused cell plate defects in Arabidopsis thaliana, it did not alter callose accumulation, or disrupt cell plate formation in maize. In contrast, CIPC treatment of maize epidermal cells occasionally produced irregular cell plates that split or fragmented, but did not otherwise disrupt the accumulation of cell-plate localized proteins. Discussion: Together, these markers provide a robust suite of tools to examine subcellular trafficking and organellar organization during mitosis and cell plate formation in maize.
RESUMEN
Grass leaves develop from a ring of primordial initial cells within the periphery of the shoot apical meristem, a pool of organogenic stem cells that generates all of the organs of the plant shoot. At maturity, the grass leaf is a flattened, strap-like organ comprising a proximal supportive sheath surrounding the stem and a distal photosynthetic blade. The sheath and blade are partitioned by a hinge-like auricle and the ligule, a fringe of epidermally derived tissue that grows from the adaxial (top) leaf surface. Together, the ligule and auricle comprise morphological novelties that are specific to grass leaves. Understanding how the planar outgrowth of grass leaves and their adjoining ligules is genetically controlled can yield insight into their evolutionary origins. Here we use single-cell RNA-sequencing analyses to identify a 'rim' cell type present at the margins of maize leaf primordia. Cells in the leaf rim have a distinctive identity and share transcriptional signatures with proliferating ligule cells, suggesting that a shared developmental genetic programme patterns both leaves and ligules. Moreover, we show that rim function is regulated by genetically redundant Wuschel-like homeobox3 (WOX3) transcription factors. Higher-order mutations in maize Wox3 genes greatly reduce leaf width and disrupt ligule outgrowth and patterning. Together, these findings illustrate the generalizable use of a rim domain during planar growth of maize leaves and ligules, and suggest a parsimonious model for the homology of the grass ligule as a distal extension of the leaf sheath margin.
Asunto(s)
Hojas de la Planta , Poaceae , Poaceae/genética , Hojas de la Planta/metabolismo , Zea mays , Mutación , Meristema , Regulación de la Expresión Génica de las PlantasRESUMEN
Although plants are able to withstand a range of environmental conditions, spikes in ambient temperature can impact plant fertility causing reductions in seed yield and notable economic losses1,2. Therefore, understanding the precise molecular mechanisms that underpin plant fertility under environmental constraints is critical to safeguarding future food production3. Here, we identified two Argonaute-like proteins whose activities are required to sustain male fertility in maize plants under high temperatures. We found that MALE-ASSOCIATED ARGONAUTE-1 and -2 associate with temperature-induced phased secondary small RNAs in pre-meiotic anthers and are essential to controlling the activity of retrotransposons in male meiocyte initials. Biochemical and structural analyses revealed how male-associated Argonaute activity and its interaction with retrotransposon RNA targets is modulated through the dynamic phosphorylation of a set of highly conserved, surface-located serine residues. Our results demonstrate that an Argonaute-dependent, RNA-guided surveillance mechanism is critical in plants to sustain male fertility under environmentally constrained conditions, by controlling the mutagenic activity of transposons in male germ cells.
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Elementos Transponibles de ADN/genética , Zea mays/genética , Producción de Cultivos , Elementos Transponibles de ADN/fisiología , Fertilidad , Respuesta al Choque Térmico , Plantas Modificadas Genéticamente , Polen/crecimiento & desarrollo , Polen/fisiología , Proteómica , Zea mays/crecimiento & desarrollo , Zea mays/fisiologíaRESUMEN
Crop productivity depends on activity of meristems that produce optimized plant architectures, including that of the maize ear. A comprehensive understanding of development requires insight into the full diversity of cell types and developmental domains and the gene networks required to specify them. Until now, these were identified primarily by morphology and insights from classical genetics, which are limited by genetic redundancy and pleiotropy. Here, we investigated the transcriptional profiles of 12,525 single cells from developing maize ears. The resulting developmental atlas provides a single-cell RNA sequencing (scRNA-seq) map of an inflorescence. We validated our results by mRNA in situ hybridization and by fluorescence-activated cell sorting (FACS) RNA-seq, and we show how these data may facilitate genetic studies by predicting genetic redundancy, integrating transcriptional networks, and identifying candidate genes associated with crop yield traits.
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Estudios de Asociación Genética , Sitios de Carácter Cuantitativo/genética , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Zea mays/crecimiento & desarrollo , Zea mays/genética , Secuencia de Bases , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Protoplastos/metabolismo , Reproducibilidad de los Resultados , Transcriptoma/genéticaRESUMEN
The formation of developmental boundaries is a common feature of multicellular plants and animals, and impacts the initiation, structure and function of all organs. Maize leaves comprise a proximal sheath that encloses the stem, and a distal photosynthetic blade that projects away from the plant axis. An epidermally derived ligule and a joint-like auricle develop at the blade/sheath boundary of maize leaves. Mutations disturbing the ligule/auricle region disrupt leaf patterning and impact plant architecture, yet it is unclear how this developmental boundary is established. Targeted microdissection followed by transcriptomic analyses of young leaf primordia were utilized to construct a co-expression network associated with development of the blade/sheath boundary. Evidence is presented for proximodistal gradients of gene expression that establish a prepatterned transcriptomic boundary in young leaf primordia, before the morphological initiation of the blade/sheath boundary in older leaves. This work presents a conceptual model for spatiotemporal patterning of proximodistal leaf domains, and provides a rich resource of candidate gene interactions for future investigations of the mechanisms of blade/sheath boundary formation in maize.
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Transcriptoma , Zea mays , Biología Computacional , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transcriptoma/genética , Zea mays/genética , Zea mays/metabolismoRESUMEN
Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.
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Regulación de la Expresión Génica de las Plantas , Captura por Microdisección con Láser/métodos , Hojas de la Planta/genética , Proteínas de Plantas/genética , ARN/metabolismo , Análisis de Secuencia de ARN/métodos , Zea mays/genética , Secuencia de Bases , Perfilación de la Expresión Génica/métodos , Hibridación in Situ , Hojas de la Planta/metabolismo , Proteínas de Plantas/biosíntesis , Zea mays/metabolismoRESUMEN
A sensitive and dynamically responsive auxin signaling reporter based on the DII domain of the INDOLE-3-ACETIC ACID28 (IAA28, DII) protein from Arabidopsis (Arabidopsis thaliana) was modified for use in maize (Zea mays). The DII domain was fused to a yellow fluorescent protein and a nuclear localization sequence to simplify quantitative nuclear fluorescence signal. DII degradation dynamics provide an estimate of input signal into the auxin signaling pathway that is influenced by both auxin accumulation and F-box coreceptor concentration. In maize, the DII-based marker responded rapidly and in a dose-dependent manner to exogenous auxin via proteasome-mediated degradation. Low levels of DII-specific fluorescence corresponding to high endogenous auxin signaling occurred near vasculature tissue and the outer layer and glume primordia of spikelet pair meristems and floral meristems, respectively. In addition, high DII levels were observed in cells during telophase and early G1, suggesting that low auxin signaling at these stages may be important for cell cycle progression.
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Proteínas de Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Telofase/fisiología , Factores de Transcripción/metabolismo , Zea mays/citología , Proteínas de Arabidopsis/genética , Fase G1/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ácidos Indolacéticos/farmacología , Meristema/genética , Meristema/metabolismo , Plantas Modificadas Genéticamente , Dominios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Imagen de Lapso de Tiempo , Factores de Transcripción/genética , Zea mays/efectos de los fármacos , Zea mays/genética , Zea mays/metabolismoRESUMEN
Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbri1 complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbri1-RNAi transgenic lines have compromised BR signaling. zmbri1-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbri1-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbri1-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbri1-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling.
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Brasinoesteroides/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas de Plantas/genética , Interferencia de ARN , Transducción de Señal , Esteroides Heterocíclicos/metabolismo , Zea mays/anatomía & histología , Zea mays/genética , Secuencia de Aminoácidos , Brasinoesteroides/farmacología , División Celular/efectos de los fármacos , Datos de Secuencia Molecular , Mutación/genética , Fenotipo , Hojas de la Planta/citología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de los fármacos , Esteroides Heterocíclicos/farmacología , Zea mays/efectos de los fármacosRESUMEN
Pre-mitotic establishment of polarity is a key event in the preparation of mother cells for asymmetric cell divisions that produce daughters of distinct fates, and ensures correct cellular patterning of tissues and eventual organ function. Previous work has shown that two receptor-like kinases, PANGLOSS2 (PAN2) and PAN1, and the small GTPase RHO GTPASE OF PLANTS (ROP) promote mother cell polarity and subsequent division asymmetry in developing maize stomata. PAN proteins become polarized prior to asymmetric cell division, however, the mechanism of this polarization is unknown. Here we show that the SCAR/WAVE regulatory complex, which activates the actin-nucleating ARP2/3 complex, is the first known marker of polarity in this asymmetric division model and is required for PAN polarization. These findings implicate actin, and specifically branched actin networks, in PAN polarization and asymmetric cell division.
RESUMEN
Development of multicellular organisms proceeds via the correct interpretation of positional information to establish boundaries that separate developmental fields with distinct identities. The maize (Zea mays) leaf is an ideal system to study plant morphogenesis as it is subdivided into a proximal sheath and a distal blade, each with distinct developmental patterning. Specialized ligule and auricle structures form at the blade-sheath boundary. The auricles act as a hinge, allowing the leaf blade to project at an angle from the stem, while the ligule comprises an epidermally derived fringe. Recessive liguleless1 mutants lack ligules and auricles and have upright leaves. We used laser microdissection and RNA sequencing to identify genes that are differentially expressed in discrete cell/tissue-specific domains along the proximal-distal axis of wild-type leaf primordia undergoing ligule initiation and compared transcript accumulation in wild-type and liguleless1-R mutant leaf primordia. We identified transcripts that are specifically upregulated at the blade-sheath boundary. A surprising number of these "ligule genes" have also been shown to function during leaf initiation or lateral branching and intersect multiple hormonal signaling pathways. We propose that genetic modules utilized in leaf and/or branch initiation are redeployed to regulate ligule outgrowth from leaf primordia.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Zea mays/genética , Biología Computacional , Perfilación de la Expresión Génica , ARN Mensajero/metabolismo , Transducción de Señal , Zea mays/anatomía & histología , Zea mays/crecimiento & desarrolloRESUMEN
Mechanisms governing the polarization of plant cell division are poorly understood. Previously, we identified pangloss1 (PAN1) as a leucine-rich repeat-receptor-like kinase (LRR-RLK) that promotes the polarization of subsidiary mother cell (SMC) divisions toward the adjacent guard mother cell (GMC) during stomatal development in maize (Zea mays). Here, we identify pangloss2 (PAN2) as a second LRR-RLK promoting SMC polarization. Quantitative proteomic analysis identified a PAN2 candidate by its depletion from membranes of pan2 single and pan1;pan2 double mutants. Genetic mapping and sequencing of mutant alleles confirmed the identity of this protein as PAN2. Like PAN1, PAN2 has a catalytically inactive kinase domain and accumulates in SMCs at sites of GMC contact before nuclear polarization. The timing of polarized PAN1 and PAN2 localization is very similar, but PAN2 acts upstream because it is required for polarized accumulation of PAN1 but is independent of PAN1 for its own localization. We find no evidence that PAN2 recruits PAN1 to the GMC contact site via a direct or indirect physical interaction, but PAN2 interacts with itself. Together, these results place PAN2 at the top of a cascade of events promoting the polarization of SMC divisions, potentially functioning to perceive or amplify GMC-derived polarizing cues.
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División Celular , Polaridad Celular , Fosfotransferasas/metabolismo , Proteómica , Zea mays/metabolismo , Mapeo Cromosómico , Leucina , Proteínas Repetidas Ricas en Leucina , Proteínas de la Membrana , Mutación , Fenotipo , Fosfotransferasas/genética , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estomas de Plantas/citología , Estomas de Plantas/genética , Estomas de Plantas/crecimiento & desarrollo , Estomas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Estructura Terciaria de Proteína , Proteínas/genética , Proteínas/metabolismo , Técnicas del Sistema de Dos Híbridos , Zea mays/citología , Zea mays/genética , Zea mays/crecimiento & desarrolloRESUMEN
Maize (Zea mays) transformation routinely produces stable transgenic lines essential for functional genomics; however, transient expression of target proteins in maize cells is not yet routine. Such techniques are critical for rapid testing of transgene constructs and for experimental studies. Here, we report bombardment methods that depend on leaf developmental stage and result in successful expression with broad applications. Fluorescent marker genes were constructed and bombarded into five developmental regions in a growing maize leaf. Expression efficiency was highest in the basal-most 3 cm above the ligule of an approximately 50-cm growing adult leaf. Straightforward dissection procedures provide access to the receptive leaf regions, increasing efficiency from less than one transformant per cm(2) to over 21 transformants per cm(2). Successful expression was routine for proteins from full genomic sequences driven by native regulatory regions and from complementary DNA sequences driven by the constitutive maize polyubiquitin promoter and a heterologous terminator. Four tested fusion proteins, maize PROTEIN DISULFIDE ISOMERASE-Yellow Fluorescent Protein, GLOSSY8a-monomeric Red Fluorescent Protein and maize XYLOSYLTRANSFERASE, and maize Rho-of-Plants7-monomeric Teal Fluorescent Protein, localized as predicted in the endoplasmic reticulum, Golgi, and plasma membrane, respectively. Localization patterns were similar between transient and stable modes of expression, and cotransformation was equally successful. Coexpression was also demonstrated by transiently transforming cells in a stable line expressing a second marker protein, thus increasing the utility of a single stable transformant. Given the ease of dissection procedures, this method replaces heterologous expression assays with a more direct, native, and informative system, and the techniques will be useful for localization, colocalization, and functional studies.
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Genómica/métodos , Células Vegetales/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/genética , Transformación Genética , Zea mays/citología , Zea mays/genética , Expresión Génica , Genes de Plantas/genética , Proteínas Luminiscentes/metabolismo , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Multifocal multiphoton microscopy (MMM) in the biological and medical sciences has become an important tool for obtaining high resolution images at video rates. While current implementations of MMM achieve very high frame rates, they are limited in their applicability to essentially those biological samples that exhibit little or no scattering. In this paper, we report on a method for MMM in which imaging detection is not necessary (single element point detection is implemented), and is therefore fully compatible for use in imaging through scattering media. Further, we demonstrate that this method leads to a new type of MMM wherein it is possible to simultaneously obtain multiple images and view differences in excitation parameters in a single shot.
RESUMEN
The Cellulose Synthase-Like D (CslD) genes have important, although still poorly defined, roles in cell wall formation. Here, we show an unexpected involvement of CslD1 from maize (Zea mays) in cell division. Both division and expansion were altered in the narrow-organ and warty phenotypes of the csld1 mutants. Leaf width was reduced by 35%, due mainly to a 47% drop in the number of cell files across the blade. Width of other organs was also proportionally reduced. In leaf epidermis, the deficiency in lateral divisions was only partially compensated by a modest, uniform increase in cell width. Localized clusters of misdivided epidermal cells also led to the formation of warty lesions, with cell clusters bulging from the epidermal layer, and some cells expanding to volumes 75-fold greater than normal. The decreased cell divisions and localized epidermal expansions were not associated with detectable changes in the cell wall composition of csld1 leaf blades or epidermal peels, yet a greater abundance of thin, dense walls was indicated by high-resolution x-ray tomography of stems. Cell-level defects leading to wart formation were traced to sites of active cell division and expansion at the bases of leaf blades, where cytokinesis and cross-wall formation were disrupted. Flow cytometry confirmed a greater frequency of polyploid cells in basal zones of leaf blades, consistent with the disruption of cytokinesis and/or the cell cycle in csld1 mutants. Collectively, these data indicate a previously unrecognized role for CSLD activity in plant cell division, especially during early phases of cross-wall formation.
Asunto(s)
División Celular , Glucosiltransferasas/metabolismo , Hojas de la Planta/crecimiento & desarrollo , Zea mays/crecimiento & desarrollo , Evolución Biológica , Glucosiltransferasas/genética , Mutación , Filogenia , ARN Mensajero/genética , Zea mays/citología , Zea mays/enzimologíaRESUMEN
Plant Rho family GTPases (ROPs) have been investigated primarily for their functions in polarized cell growth. We previously showed that the maize (Zea mays) Leu-rich repeat receptor-like protein PANGLOSS1 (PAN1) promotes the polarization of asymmetric subsidiary mother cell (SMC) divisions during stomatal development. Here, we show that maize Type I ROPs 2 and 9 function together with PAN1 in this process. Partial loss of ROP2/9 function causes a weak SMC division polarity phenotype and strongly enhances this phenotype in pan1 mutants. Like PAN1, ROPs accumulate in an asymmetric manner in SMCs. Overexpression of yellow fluorescent protein-ROP2 is associated with its delocalization in SMCs and with aberrantly oriented SMC divisions. Polarized localization of ROPs depends on PAN1, but PAN1 localization is insensitive to depletion and depolarization of ROP. Membrane-associated Type I ROPs display increased nonionic detergent solubility in pan1 mutants, suggesting a role for PAN1 in membrane partitioning of ROPs. Finally, endogenous PAN1 and ROP proteins are physically associated with each other in maize tissue extracts, as demonstrated by reciprocal coimmunoprecipitation experiments. This study demonstrates that ROPs play a key role in polarization of plant cell division and cell growth and reveals a role for a receptor-like protein in spatial localization of ROPs.
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División Celular/fisiología , Polaridad Celular , Proteínas de Plantas/metabolismo , Zea mays/citología , Zea mays/enzimología , Zea mays/fisiología , Proteínas de Unión al GTP rho/metabolismo , Aminoquinolinas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Fenotipo , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Estomas de Plantas/citología , Estomas de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Pirimidinas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rho/genéticaRESUMEN
A challenge for nonlinear imaging in living tissue is to maximize the total fluorescent yield from each fluorophore. We investigated the emission rates of three fluorophores-rhodamine B, a red fluorescent protein, and CdSe quantum dots-while manipulating the phase of the laser excitation pulse at the focus. In all cases a transform-limited pulse maximized the total yield to insure the highest signal-to-noise ratio. Further, we find evidence of fluorescence antibleaching in quantum dot samples.
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Proteínas Luminiscentes/química , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Puntos Cuánticos , Rodaminas/química , Biología/instrumentación , Compuestos de Cadmio/química , Modelos Teóricos , Fotoblanqueo , Células Vegetales , Compuestos de Selenio/química , Proteína Fluorescente RojaRESUMEN
First discovered in jellyfish, fluorescent proteins (FPs) have been successfully optimized for use as effective biomarkers within living plant cells. When exposed to light, FPs fused to a protein or regulatory element will fluoresce, and non-invasively mark expression and protein localization, which allows for the in vivo monitoring of diverse cellular processes. In this review, we discuss how FP technology has evolved from small-scale analysis of individual genes to more high-throughput techniques for global expression and functional profiling in plants.
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Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Luminiscentes/metabolismo , Plantas/metabolismo , Elementos de Facilitación Genéticos/genética , Fluorescencia , Plantas/genética , Transporte de ProteínasRESUMEN
We present the application of remote focusing to multiphoton laser scanning microscopy and utilize this technology to demonstrate simultaneous, programmable multi-layer imaging. Remote focusing is used to independently control the axial location of multiple focal planes that can be simultaneously imaged with single element detection. This facilitates volumetric multiphoton imaging in scattering specimens and can be practically scaled to a large number of focal planes. Further, it is demonstrated that the remote focusing control can be synchronized with the lateral scan directions, enabling imaging in orthogonal scan planes.
RESUMEN
We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences.