Same but different - pseudo-pectin in the charophytic alga Chlorokybus atmophyticus.

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.

Monitoring apoptosis in intact cells by high-resolution magic angle spinning 1 H NMR spectroscopy.

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.

Insights into the Evolution of Multicellularity from the Sea Lettuce Genome.

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.

Coastal Upwelling Drives Intertidal Assemblage Structure and Trophic Ecology.

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.

The green seaweed Ulva: a model system to study morphogenesis.

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.

Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida.

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.

An introduction to biological nuclear magnetic resonance spectroscopy.

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.

Cell cycles and endocycles in the model brown seaweed, Ectocarpus siliculosus.

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.

Role of endoreduplication and apomeiosis during parthenogenetic reproduction in the model brown alga Ectocarpus.

• 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.

The Ectocarpus genome and the independent evolution of multicellularity in brown algae.

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.

Two voltage-dependent calcium channels co-exist in the apical plasma membrane of Arabidopsis thaliana root hairs.

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.

Ca2+ signals coordinate zygotic polarization and cell cycle progression in the brown alga Fucus serratus.

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.

A tip-high, Ca(2+) -interdependent, reactive oxygen species gradient is associated with polarized growth in Fucus serratus zygotes.

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.

Biolistic delivery of Ca2+ dyes into plant and algal cells.

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.

The evolution of Ca2+ signalling in photosynthetic eukaryotes.

It is likely that cytosolic Ca2+ elevations have played a part in eukaryotic signal transduction for about the last 2 Gyr, being mediated by a group of molecules which are collectively known as the [Ca2+]cyt signalling toolkit. Different eukaryotes often display strikingly similar [Ca2+]cyt signalling elevations, which may reflect conservation of toolkit components (homology) or similar constraints acting on different toolkits (homoplasy). Certain toolkit components, which are presumably ancestral, are shared by plants and animals, but some components are unique to photosynthetic organisms. We propose that the structure of modern plant [Ca2+]cyt signalling toolkits may be explained by their modular adaptation from earlier pathways.

Reactive oxygen species produced by NADPH oxidase regulate plant cell growth.

Cell expansion is a central process in plant morphogenesis, and the elongation of roots and root hairs is essential for uptake of minerals and water from the soil. Ca2+ influx from the extracellular store is required for (and sets the rates of) cell elongation in roots. Arabidopsis thaliana rhd2 mutants are defective in Ca2+ uptake and consequently cell expansion is compromised--rhd2 mutants have short root hairs and stunted roots. To determine the regulation of Ca2+ acquisition in growing root cells we show here that RHD2 is an NADPH oxidase, a protein that transfers electrons from NADPH to an electron acceptor leading to the formation of reactive oxygen species (ROS). We show that ROS accumulate in growing wild-type (WT) root hairs but their levels are markedly decreased in rhd2 mutants. Blocking the activity of the NADPH oxidase with diphenylene iodonium (DPI) inhibits ROS formation and phenocopies Rhd2-. Treatment of rhd2 roots with ROS partly suppresses the mutant phenotype and stimulates the activity of plasma membrane hyperpolarization-activated Ca2+ channels, the predominant root Ca2+ acquisition system. This indicates that NADPH oxidases control development by making ROS that regulate plant cell expansion through the activation of Ca2+ channels.

Abnormalities in brain biochemistry associated with lack of dystrophin: studies of the mdx mouse.

Biochemical abnormalities have been reported in dystrophin-deficient muscle of boys with Duchenne (severe Xp21) muscular dystrophy or in the murine (mdx) model of the disease. These abnormalities include altered energy metabolism and responses to osmotic shock. In contrast, the situation in brain is less well understood and it is probable that dystrophin is playing a different role (or roles) in this organ. In this study we conclude that the elevation in choline-containing compounds reported in mdx brain is confined to cerebellum and hippocampus in older (> 6 months) mice. We report alterations in glucose metabolism in mdx brain under normal, awake conditions, and a reduced response of brain metabolism to the gamma-aminobutyric acid(A) receptor agonist muscimol. Using brain cortical slices we found no difference in the response of dystrophic tissue to hypoosmotic shock, but increased, substrate-dependent oxygen consumption rates at low oxygen partial pressures.