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1.
Physiol Plant ; 175(6): e14079, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38148229

RESUMO

All land-plant cell walls possess hemicelluloses, cellulose and anionic pectin. The walls of their cousins, the charophytic algae, exhibit some similarities to land plants' but also major differences. Charophyte 'pectins' are extractable by conventional land-plant methods, although they differ significantly in composition. Here, we explore 'pectins' of an early-diverging charophyte, Chlorokybus atmophyticus, characterising the anionic polysaccharides that may be comparable to 'pectins' in other streptophytes. Chlorokybus 'pectin' was anionic and upon acid hydrolysis gave GlcA, GalA and sulphate, plus neutral sugars (Ara≈Glc>Gal>Xyl); Rha was undetectable. Most Gal was the l-enantiomer. A relatively acid-resistant disaccharide was characterised as ß-d-GlcA-(1→4)-l-Gal. Two Chlorokybus 'pectin' fractions, separable by anion-exchange chromatography, had similar sugar compositions but different sulphate-ester contents. No sugars were released from Chlorokybus 'pectin' by several endo-hydrolases [(1,5)-α-l-arabinanase, (1,4)-ß-d-galactanase, (1,4)-ß-d-xylanase, endo-polygalacturonase] and exo-hydrolases [α- and ß-d-galactosidases, α-(1,6)-d-xylosidase]. 'Driselase', which hydrolyses most land-plant cell wall polysaccharides to mono- and disaccharides, released no sugars except traces of starch-derived Glc. Thus, the Ara, Gal, Xyl and GalA of Chlorokybus 'pectin' were not non-reducing termini with configurations familiar from land-plant polysaccharides (α-l-Araf, α- and ß-d-Galp, α- and ß-d-Xylp and α-d-GalpA), nor mid-chain residues of α-(1→5)-l-arabinan, ß-(1→4)-d-galactan, ß-(1→4)-d-xylan or α-(1→4)-d-galacturonan. In conclusion, Chlorokybus possesses anionic 'pectic' polysaccharides, possibly fulfilling pectic roles but differing fundamentally from land-plant pectin. Thus, the evolution of land-plant pectin since the last common ancestor of Chlorokybus and land plants is a long and meandering path involving loss of sulphate, most l-Gal and most d-GlcA; re-configuration of Ara, Xyl and GalA; and gain of Rha.


Assuntos
Embriófitas , Polissacarídeos , Pectinas , Plantas , Poligalacturonase , Sulfatos
2.
NMR Biomed ; 34(3): e4456, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33398876

RESUMO

Apoptosis maintains an equilibrium between cell proliferation and cell death. Many diseases, including cancer, develop because of defects in apoptosis. A known metabolic marker of apoptosis is a notable increase in 1 H NMR-observable resonances associated with lipids stored in lipid droplets. However, standard one-dimensional NMR experiments allow the quantification of lipid concentration only, without providing information about physical characteristics such as the size of lipid droplets, viscosity of the cytosol, or cytoskeletal rigidity. This additional information can improve monitoring of apoptosis-based cancer treatments in intact cells and provide us with mechanistic insight into why these changes occur. In this paper, we use high-resolution magic angle spinning (HRMAS) 1 H NMR spectroscopy to monitor lipid concentrations and apparent diffusion coefficients of mobile lipid in intact cells treated with the apoptotic agents cisplatin or etoposide. We also use solution-state NMR spectroscopy to study changes in lipid profiles of organic solvent cell extracts. Both NMR techniques show an increase in the concentration of lipids but the relative changes are 10 times larger by HRMAS 1 H NMR spectroscopy. Moreover, the apparent diffusion rates of lipids in apoptotic cells measured by HRMAS 1 H NMR spectroscopy decrease significantly as compared with control cells. Slower diffusion rates of mobile lipids in apoptotic cells correlate well with the formation of larger lipid droplets as observed by microscopy. We also compared the mean lipid droplet displacement values calculated from the two methods. Both methods showed shorter displacements of lipid droplets in apoptotic cells. Our results demonstrate that the NMR-based diffusion experiments on intact cells discriminate between control and apoptotic cells. Apparent diffusion measurements in conjunction with 1 H NMR spectroscopy-derived lipid signals provide a novel means of following apoptosis in intact cells. This method could have potential application in enhancing drug discovery by monitoring drug treatments in vitro, particularly for agents that cause portioning of lipids such as apoptosis.


Assuntos
Apoptose , Espectroscopia de Prótons por Ressonância Magnética , Animais , Linhagem Celular , Cisplatino/farmacologia , Citoplasma/metabolismo , Difusão , Etoposídeo/farmacologia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Metabolômica , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Viscosidade
3.
Curr Biol ; 28(18): 2921-2933.e5, 2018 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-30220504

RESUMO

We report here the 98.5 Mbp haploid genome (12,924 protein coding genes) of Ulva mutabilis, a ubiquitous and iconic representative of the Ulvophyceae or green seaweeds. Ulva's rapid and abundant growth makes it a key contributor to coastal biogeochemical cycles; its role in marine sulfur cycles is particularly important because it produces high levels of dimethylsulfoniopropionate (DMSP), the main precursor of volatile dimethyl sulfide (DMS). Rapid growth makes Ulva attractive biomass feedstock but also increasingly a driver of nuisance "green tides." Ulvophytes are key to understanding the evolution of multicellularity in the green lineage, and Ulva morphogenesis is dependent on bacterial signals, making it an important species with which to study cross-kingdom communication. Our sequenced genome informs these aspects of ulvophyte cell biology, physiology, and ecology. Gene family expansions associated with multicellularity are distinct from those of freshwater algae. Candidate genes, including some that arose following horizontal gene transfer from chromalveolates, are present for the transport and metabolism of DMSP. The Ulva genome offers, therefore, new opportunities to understand coastal and marine ecosystems and the fundamental evolution of the green lineage.


Assuntos
Evolução Biológica , Genoma , Características de História de Vida , Ulva/genética , Mapeamento Cromossômico , Família Multigênica , Ulva/crescimento & desenvolvimento
4.
Sci Rep ; 6: 30728, 2016 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-27470705

RESUMO

We describe an integrated process that can be applied to biomass of the green seaweed, Ulva fasciata, to allow the sequential recovery of four economically important fractions; mineral rich liquid extract (MRLE), lipid, ulvan, and cellulose. The main benefits of our process are: a) its simplicity and b) the consistent yields obtained from the residual biomass after each successive extraction step. For example, dry Ulva biomass yields ~26% of its starting mass as MRLE, ~3% as lipid, ~25% as ulvan, and ~11% as cellulose, with the enzymatic hydrolysis and fermentation of the final cellulose fraction under optimized conditions producing ethanol at a competitive 0.45 g/g reducing sugar. These yields are comparable to those obtained by direct processing of the individual components from primary biomass. We propose that this integration of ethanol production and chemical feedstock recovery from macroalgal biomass could substantially enhance the sustainability of marine biomass use.


Assuntos
Biocombustíveis/microbiologia , Clorófitas/química , Clorófitas/metabolismo , Extratos Vegetais/análise , Biomassa , Celulose/análise , Fermentação , Hidrólise , Lipídeos/análise , Minerais/análise , Extratos Vegetais/química , Polissacarídeos/análise , Alga Marinha/química , Alga Marinha/metabolismo
5.
PLoS One ; 10(7): e0130789, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26214806

RESUMO

Similar environmental driving forces can produce similarity among geographically distant ecosystems. Coastal oceanic upwelling, for example, has been associated with elevated biomass and abundance patterns of certain functional groups, e.g., corticated macroalgae. In the upwelling system of Northern Chile, we examined measures of intertidal macrobenthic composition, structure and trophic ecology across eighteen shores varying in their proximity to two coastal upwelling centres, in a hierarchical sampling design (spatial scales of >1 and >10 km). The influence of coastal upwelling on intertidal communities was confirmed by the stable isotope values (δ13C and δ15N) of consumers, including a dominant suspension feeder, grazers, and their putative resources of POM, epilithic biofilm, and macroalgae. We highlight the utility of muscle δ15N from the suspension feeding mussel, Perumytilus purpuratus, as a proxy for upwelling, supported by satellite data and previous studies. Where possible, we used corrections for broader-scale trends, spatial autocorrelation, ontogenetic dietary shifts and spatial baseline isotopic variation prior to analysis. Our results showed macroalgal assemblage composition, and benthic consumer assemblage structure, varied significantly with the intertidal influence of coastal upwelling, especially contrasting bays and coastal headlands. Coastal topography also separated differences in consumer resource use. This suggested that coastal upwelling, itself driven by coastline topography, influences intertidal communities by advecting nearshore phytoplankton populations offshore and cooling coastal water temperatures. We recommend the isotopic values of benthic organisms, specifically long-lived suspension feeders, as in situ alternatives to offshore measurements of upwelling influence.


Assuntos
Ecologia , Ecossistema , Chile
6.
Front Plant Sci ; 6: 72, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25745427

RESUMO

Green macroalgae, mostly represented by the Ulvophyceae, the main multicellular branch of the Chlorophyceae, constitute important primary producers of marine and brackish coastal ecosystems. Ulva or sea lettuce species are some of the most abundant representatives, being ubiquitous in coastal benthic communities around the world. Nonetheless the genus also remains largely understudied. This review highlights Ulva as an exciting novel model organism for studies of algal growth, development and morphogenesis as well as mutualistic interactions. The key reasons that Ulva is potentially such a good model system are: (i) patterns of Ulva development can drive ecologically important events, such as the increasing number of green tides observed worldwide as a result of eutrophication of coastal waters, (ii) Ulva growth is symbiotic, with proper development requiring close association with bacterial epiphytes, (iii) Ulva is extremely developmentally plastic, which can shed light on the transition from simple to complex multicellularity and (iv) Ulva will provide additional information about the evolution of the green lineage.

7.
Curr Biol ; 24(17): 1945-57, 2014 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-25176635

RESUMO

BACKGROUND: A common feature of most genetic sex-determination systems studied so far is that sex is determined by nonrecombining genomic regions, which can be of various sizes depending on the species. These regions have evolved independently and repeatedly across diverse groups. A number of such sex-determining regions (SDRs) have been studied in animals, plants, and fungi, but very little is known about the evolution of sexes in other eukaryotic lineages. RESULTS: We report here the sequencing and genomic analysis of the SDR of Ectocarpus, a brown alga that has been evolving independently from plants, animals, and fungi for over one giga-annum. In Ectocarpus, sex is expressed during the haploid phase of the life cycle, and both the female (U) and the male (V) sex chromosomes contain nonrecombining regions. The U and V of this species have been diverging for more than 70 mega-annum, yet gene degeneration has been modest, and the SDR is relatively small, with no evidence for evolutionary strata. These features may be explained by the occurrence of strong purifying selection during the haploid phase of the life cycle and the low level of sexual dimorphism. V is dominant over U, suggesting that femaleness may be the default state, adopted when the male haplotype is absent. CONCLUSIONS: The Ectocarpus UV system has clearly had a distinct evolutionary trajectory not only to the well-studied XY and ZW systems but also to the UV systems described so far. Nonetheless, some striking similarities exist, indicating remarkable universality of the underlying processes shaping sex chromosome evolution across distant lineages.


Assuntos
Haploidia , Phaeophyceae/crescimento & desenvolvimento , Phaeophyceae/genética , Processos de Determinação Sexual
8.
Proc Natl Acad Sci U S A ; 110(13): 5247-52, 2013 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-23503846

RESUMO

Red seaweeds are key components of coastal ecosystems and are economically important as food and as a source of gelling agents, but their genes and genomes have received little attention. Here we report the sequencing of the 105-Mbp genome of the florideophyte Chondrus crispus (Irish moss) and the annotation of the 9,606 genes. The genome features an unusual structure characterized by gene-dense regions surrounded by repeat-rich regions dominated by transposable elements. Despite its fairly large size, this genome shows features typical of compact genomes, e.g., on average only 0.3 introns per gene, short introns, low median distance between genes, small gene families, and no indication of large-scale genome duplication. The genome also gives insights into the metabolism of marine red algae and adaptations to the marine environment, including genes related to halogen metabolism, oxylipins, and multicellularity (microRNA processing and transcription factors). Particularly interesting are features related to carbohydrate metabolism, which include a minimalistic gene set for starch biosynthesis, the presence of cellulose synthases acquired before the primary endosymbiosis showing the polyphyly of cellulose synthesis in Archaeplastida, and cellulases absent in terrestrial plants as well as the occurrence of a mannosylglycerate synthase potentially originating from a marine bacterium. To explain the observations on genome structure and gene content, we propose an evolutionary scenario involving an ancestral red alga that was driven by early ecological forces to lose genes, introns, and intergenetic DNA; this loss was followed by an expansion of genome size as a consequence of activity of transposable elements.


Assuntos
Chondrus/genética , Evolução Molecular , Genes de Plantas , Sequência de Bases , MicroRNAs/genética , Dados de Sequência Molecular , Proteínas de Plantas/genética , RNA de Plantas/genética
10.
Biol Rev Camb Philos Soc ; 86(2): 493-510, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-20969720

RESUMO

Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful analytical techniques available to biology. This review is an introduction to the potential of this method and is aimed at readers who have little or no experience in acquiring or analyzing NMR spectra. We focus on spectroscopic applications of the magnetic resonance effect, rather than imaging ones, and explain how various aspects of the NMR phenomenon make it a versatile tool with which to address a number of biological problems. Using detailed examples, we discuss the use of (1) H NMR spectroscopy in mixture analysis and metabolomics, the use of (13) C NMR spectroscopy in tracking isotopomers and determining the flux through metabolic pathways ('fluxomics') and the use of (31) P NMR spectroscopy in monitoring ATP generation and intracellular pH homeotasis in vivo. Further examples demonstrate how NMR spectroscopy can be used to probe the physical environment of a cell by measuring diffusion and the tumbling rates of individual metabolites and how it can determine macromolecular structures by measuring the bonds and distances which separate individual atoms. We finish by outlining some of the key challenges which remain in NMR spectroscopy and we highlight how recent advances-such as increased magnet field strengths, cryogenic cooling, microprobes and hyperpolarisation-are opening new avenues for today's biological NMR spectroscopists.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteômica/métodos , Animais , Isótopos de Carbono/análise , Concentração de Íons de Hidrogênio , Isótopos de Fósforo/análise
11.
Plant Signal Behav ; 5(11): 1473-5, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21057192

RESUMO

The recent announcement of the first genome sequence of a brown macroalga, the filamentous Ectocarpus, has been accompanied by a number of companion papers in New Phytologist. In a paper which contributes to this special issue, we classified the core cell cycle components of Ectocarpus, comparing them to the previously studied cell cycle components of diatoms. We then carried out fluorescence microscopy experiments to show that the Ectocarpus cell cycle could be deregulated during early development to give endopolyploid adults. We discuss here how our findings complement recent studies on endopolyploidy in plant and algal systems.


Assuntos
Ciclo Celular/fisiologia , Phaeophyceae/citologia , Phaeophyceae/fisiologia , Ciclo Celular/genética , Microscopia de Fluorescência , Phaeophyceae/genética , Poliploidia
12.
New Phytol ; 188(1): 111-21, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20618911

RESUMO

• The filamentous brown alga Ectocarpus has a complex life cycle, involving alternation between independent and morphologically distinct sporophyte and gametophyte generations. In addition to this basic haploid-diploid life cycle, gametes can germinate parthenogenetically to produce parthenosporophytes. This article addresses the question of how parthenosporophytes, which are derived from a haploid progenitor cell, are able to produce meiospores in unilocular sporangia, a process that normally involves a reductive meiotic division. • We used flow cytometry, multiphoton imaging, culture studies and a bioinformatics survey of the recently sequenced Ectocarpus genome to describe its life cycle under laboratory conditions and the nuclear DNA changes which accompany key developmental transitions. • Endoreduplication occurs during the first cell cycle in about one-third of parthenosporophytes. The production of meiospores by these diploid parthenosporophytes involves a meiotic division similar to that observed in zygote-derived sporophytes. By contrast, meiospore production in parthenosporophytes that fail to endoreduplicate occurs via a nonreductive apomeiotic event. • Our results highlight Ectocarpus's reproductive and developmental plasticity and are consistent with previous work showing that its life cycle transitions are controlled by genetic mechanisms and are independent of ploidy.


Assuntos
Duplicação Gênica , Meiose/genética , Modelos Biológicos , Partenogênese/genética , Phaeophyceae/citologia , Phaeophyceae/genética , Núcleo Celular/metabolismo , DNA/metabolismo , Evolução Molecular , Geografia , Células Germinativas Vegetais/citologia , Células Germinativas Vegetais/metabolismo , Ploidias , Distribuição de Poisson , Reprodução/genética
13.
Nature ; 465(7298): 617-21, 2010 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-20520714

RESUMO

Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related. These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity (Fig. 1). We report the 214 million base pair (Mbp) genome sequence of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for brown algae, closely related to the kelps (Fig. 1). Genome features such as the presence of an extended set of light-harvesting and pigment biosynthesis genes and new metabolic processes such as halide metabolism help explain the ability of this organism to cope with the highly variable tidal environment. The evolution of multicellularity in this lineage is correlated with the presence of a rich array of signal transduction genes. Of particular interest is the presence of a family of receptor kinases, as the independent evolution of related molecules has been linked with the emergence of multicellularity in both the animal and green plant lineages. The Ectocarpus genome sequence represents an important step towards developing this organism as a model species, providing the possibility to combine genomic and genetic approaches to explore these and other aspects of brown algal biology further.


Assuntos
Proteínas de Algas/genética , Evolução Biológica , Genoma/genética , Phaeophyceae/citologia , Phaeophyceae/genética , Animais , Eucariotos , Evolução Molecular , Dados de Sequência Molecular , Phaeophyceae/metabolismo , Filogenia , Pigmentos Biológicos/biossíntese , Transdução de Sinais/genética
14.
New Phytol ; 179(2): 378-385, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19086288

RESUMO

Calcium (Ca2+)-permeable plasma membrane ion channels are critical to root hair elongation and signalling. Arabidopsis thaliana root hair plasma membrane contains a hyperpolarization-activated Ca2+ channel (HACC) conductance. Here, the co-residence of HACC with a depolarization-activated Ca2+ channel (DACC) conductance has been investigated. Whole-cell patch-clamping of apical plasma membrane has been used to study Ca2+ conductances and reveal the negative slope conductance typical of DACCs. Specific voltage protocols, Ba(2+)-permeation and inhibition by the cation channel blocker Gd3+ have been used to identify the DACC conductance. The Gd3+ sensitive DACC conductance was identified in only a minority of cells. DACC activity was quickly masked by the development of the HACC conductance. However, in the period between the disappearance of the negative slope conductance and the predominance of HACC, DACC activity could still be detected. A DACC conductance coexists with HACC in the root hair apical plasma membrane and could provide Ca2+ influx over a wide voltage range, consistent with a role in signalling.


Assuntos
Arabidopsis/metabolismo , Canais de Cálcio/metabolismo , Membrana Celular/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Cálcio/metabolismo , Canais de Cálcio/genética , Eletrofisiologia , Gadolínio , Regulação da Expressão Gênica de Plantas/fisiologia , Potenciais da Membrana
15.
Development ; 135(12): 2173-81, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18480164

RESUMO

Zygotes of the fucoid brown algae provide excellent models for addressing fundamental questions about zygotic symmetry breaking. Although the acquisition of polarity is tightly coordinated with the timing and orientation of the first asymmetric division--with zygotes having to pass through a G1/S-phase checkpoint before the polarization axis can be fixed--the mechanisms behind the interdependence of polarization and cell cycle progression remain unclear. In this study, we combine in vivo Ca2+ imaging, single cell monitoring of S-phase progression and multivariate analysis of high-throughput intracellular Ca2+ buffer loading to demonstrate that Ca2+ signals coordinate polarization and cell cycle progression in the Fucus serratus zygote. Consistent with earlier studies on this organism, and in contrast to animal models, we observe no fast Ca2+ wave following fertilization. Rather, we show distinct slow localized Ca2+ elevations associated with both fertilization and S-phase progression, and we show that both S-phase and zygotic polarization are dependent on pre-S-phase Ca2+ increases. Surprisingly, this Ca2+ requirement cannot be explained by co-dependence on a single G1/S-phase checkpoint, as S phase and zygotic polarization are differentially sensitive to pre-S-phase Ca2+ elevations and can be uncoupled. Furthermore, subsequent cell cycle progression through M phase is independent of localized actin polymerization and zygotic polarization. This absence of a morphogenesis checkpoint, together with the observed Ca2+-dependences of S phase and polarization, show that the regulation of zygotic division in the brown algae differs from that in other eukaryotic model systems, such as yeast and Drosophila.


Assuntos
Sinalização do Cálcio , Ciclo Celular , Polaridade Celular , Phaeophyceae/citologia , Zigoto/citologia , Zigoto/fisiologia , Animais , Modelos Biológicos , Phaeophyceae/genética , Phaeophyceae/fisiologia
16.
Planta ; 227(5): 1037-46, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18087716

RESUMO

We report the existence of a tip-high reactive oxygen species (ROS) gradient in growing Fucus serratus zygotes, using both 5-(and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein and nitroblue tetrazolium staining to report ROS generation. Suppression of the ROS gradient inhibits polarized zygotic growth; conversely, exogenous ROS generation can redirect zygotic polarization following inhibition of endogenous ROS. Confocal imaging of fluo-4 dextran distributions suggests that the ROS gradient is interdependent on the tip-high [Ca(2+)](cyt) gradient which is known to be associated with polarized growth. Our data support a model in which localized production of ROS at the rhizoid tip stimulates formation of a localized tip-high [Ca(2+)](cyt) gradient. Such modulation of intracellular [Ca(2+)](cyt) signals by ROS is a common motif in many plant and algal systems and this study extends this mechanism to embryogenesis.


Assuntos
Cálcio/metabolismo , Fucus/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fucus/citologia , Fucus/crescimento & desenvolvimento , Microscopia Confocal , Reprodução , Fosfolipases Tipo C/metabolismo
17.
Plant Signal Behav ; 3(8): 570-2, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19704472

RESUMO

In a recent paper in Planta, we combined novel confocal reflectance imaging of intracellular reactive oxygen species (ROS) with inhibition-of-growth experiments to show that ROS help to direct polarized growth in brown algal zygotes. Using confocal fluorescence imaging of intracellular Ca(2+) distributions, we were also able to show an interaction between ROS and Ca(2+) signaling. The modulation of intracellular Ca(2+) signals by reactive oxygen species (ROS) is a common motif in many plant and algal systems, but our Planta paper is its first demonstration during early development. We explain here how our findings complement a number of recent studies on polarized growth in plant and algal systems.

19.
Plant J ; 46(2): 327-35, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16623894

RESUMO

In eukaryotes, changes in cytosolic Ca2+ concentrations ([Ca2+]cyt) are associated with a number of environmental and developmental stimuli. However, measuring [Ca2+]cyt changes in single plant or algal cells is often problematic. Although a wide range of Ca2+-sensitive fluorescent dyes is available, they are often difficult to introduce into plant cells. Micro-injection is the most robust method for dye loading, but is time-consuming, technically demanding, and unsuitable in many cell types. To overcome these problems, we have adapted biolistic techniques to load Ca2+-sensitive dyes into guard cells of the flowering plant, Commelina communis, cells of the green alga Chlamydomonas reinhardtii, and zygotes of the brown alga, Fucus serratus. Using this approach, we have been able to monitor [Ca2+]cyt changes in response to various stimuli, including a novel [Ca2+]cyt response in C. reinhardtii. The method allows the use of free acid and dextran-conjugated dyes. Biolistic loading of differentiated plant cells is easier, quicker, and more widely applicable than micro-injection, and should broaden the study of plant signal transduction.


Assuntos
Biolística/métodos , Cálcio/metabolismo , Commelina/genética , Eucariotos/genética , Corantes Fluorescentes/farmacocinética , Animais , Transporte Biológico , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Commelina/crescimento & desenvolvimento , Commelina/metabolismo , Citosol/metabolismo , Eucariotos/crescimento & desenvolvimento , Eucariotos/metabolismo
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