Mass spectrometry - based imaging techniques for iodine-127 and iodine-129 detection and localization in the brown alga Laminaria digitata.

129I is one of the main radioisotopes of iodine derived from the nuclear fuel cycle that can be found sustainably in the environment due to its long half-life. In coastal marine environment, brown macroalgae, such laminariales (or kelps), are known to naturally feature highest rates of iodine accumulation, and to be an important source of biogenic volatile iodinated compounds released to the atmosphere. These seaweeds are therefore likely to be significantly marked by but also potential vectors of radioactive iodine. In order to better understand the chemical and isotopic speciation of iodine in brown algal tissues, we combined mass spectrometry-based imaging approaches in natural samples of Laminaria digitata young sporophytes, collected at two different locations along the south coast of the English Channel (Roscoff and Goury). Laser desorption ionization (LDI) and desorption electrospray-ionization techniques (DESI), coupled with mass spectrometry, confirmed the predominance of inorganic I- species on the surface of fresh algae, and a peripheral iodine localization when applied on micro-sections. Moreover, radioactive isotope 129I was not detected on plantlet surface or in stipe sections of algal samples collected near Roscoff but was detected in L. digitata samples collected at Goury, near La Hague, where controlled liquid radioactive discharges from the ORANO La Hague reprocessing plant occur. At the subcellular scale, cryo-fixed micro-sections of algal blade samples from both sites were further analyzed by secondary ion mass spectrometry (nano-SIMS), leading to similar results. Even if the signal detected for 129I was much weaker than for 127I in samples from Goury, the chemical imaging revealed some differences in extracellular distribution between radioactive and stable iodine isotopes. Altogether LDI and nano-SIMS are complementary and powerful techniques for the detection and localization of iodine isotopes in algal samples, and for a better understanding of radioactive and stable iodine uptake mechanisms in the marine environment.

Impedance flow cytometry allows the early prediction of embryo yields in wheat (Triticum aestivum L.) microspore cultures.

Haplomethods are key biotechnological tools that make it possible to rapidly produce perfectly homozygous lines, speeding up plant breeding programs. Under specific stress conditions, microspores are reprogrammed toward sporophytic pathways, leading to embryo formation. Various endogenous and exogenous factors affect embryo yield in androgenesis, so the improvement of androgenesis efficiency requires the development of early, reliable and robust reactivity markers. During the last decade, numerous cytological, cellular and biochemical approaches were carried out to finely characterize microspore development and fate during androgenesis. However, the different available markers are often species-dependent, and their development and application are time-consuming and cumbersome. In this study, we show the suitable use of impedance flow cytometry (IFC) to develop new robust, reliable and strong markers of androgenesis reactivity in wheat, leading to: (i) routine monitoring of the viability of heterogeneous cell cultures; (ii) quick and simple evaluation of stress treatment efficiency; and (iii) early prediction of embryo yields from microspore suspensions. IFC can therefore provide the fine characterization of all of the microspore developmental pathways that occur in a cell suspension, for embryogenic microspores as well as pollen-like microspores. IFC technology has become a very useful tool to track and characterize wheat microspores in androgenesis, but can also be adapted to other species and other in vitro cell culture systems.

Algae as nutritional and functional food sources: revisiting our understanding.

Global demand for macroalgal and microalgal foods is growing, and algae are increasingly being consumed for functional benefits beyond the traditional considerations of nutrition and health. There is substantial evidence for the health benefits of algal-derived food products, but there remain considerable challenges in quantifying these benefits, as well as possible adverse effects. First, there is a limited understanding of nutritional composition across algal species, geographical regions, and seasons, all of which can substantially affect their dietary value. The second issue is quantifying which fractions of algal foods are bioavailable to humans, and which factors influence how food constituents are released, ranging from food preparation through genetic differentiation in the gut microbiome. Third is understanding how algal nutritional and functional constituents interact in human metabolism. Superimposed considerations are the effects of harvesting, storage, and food processing techniques that can dramatically influence the potential nutritive value of algal-derived foods. We highlight this rapidly advancing area of algal science with a particular focus on the key research required to assess better the health benefits of an alga or algal product. There are rich opportunities for phycologists in this emerging field, requiring exciting new experimental and collaborative approaches.

Constitutive or Inducible Protective Mechanisms against UV-B Radiation in the Brown Alga Fucus vesiculosus? A Study of Gene Expression and Phlorotannin Content Responses.

A role as UV sunscreens has been suggested for phlorotannins, the phenolic compounds that accumulate in brown algae in response to a number of external stimuli and take part in cell wall structure. After exposure of the intertidal brown alga Fucus vesiculosus to artificial UV-B radiation, we examined its physiological responses by following the transcript level of the pksIII gene encoding a phloroglucinol synthase, likely to be involved in the first step of phlorotannins biosynthesis. We also monitored the expression of three targeted genes, encoding a heat shock protein (hsp70), which is involved in global stress responses, an aryl sulfotransferase (ast), which could be involved in the sulfation of phlorotannins, and a vanadium bromoperoxidase (vbpo), which can potentially participate in the scavenging of Reactive Oxygen Species (ROS) and in the cross-linking and condensation of phlorotannins. We investigated whether transcriptional regulation of these genes is correlated with an induction of phlorotannin accumulation by establishing metabolite profiling of purified fractions of low molecular weight phlorotannins. Our findings demonstrated that a high dose of UV-B radiation induced a significant overexpression of hsp70 after 12 and 24 hours following the exposure to the UV-B treatment, compared to control treatment. The physiological performance of algae quantified by the photosynthetic efficiency (Fv/Fm) was slightly reduced. However UV-B treatment did not induce the accumulation of soluble phlorotannins in F. vesiculosus during the kinetics of four weeks, a result that may be related to the lack of induction of the pksIII gene expression. Taken together these results suggest a constitutive accumulation of phlorotannins occurring during the development of F.vesiculosus, rather than inducible processes. Gene expression studies and phlorotannin profiling provide here complementary approaches to global quantifications currently used in studies of phenolic compounds in brown algae.

In vivo speciation studies and antioxidant properties of bromine in Laminaria digitata reinforce the significance of iodine accumulation for kelps.

The metabolism of bromine in marine brown algae remains poorly understood. This contrasts with the recent finding that the accumulation of iodide in the brown alga Laminaria serves the provision of an inorganic antioxidant - the first case documented from a living system. The aim of this study was to use an interdisciplinary array of techniques to study the chemical speciation, transformation, and function of bromine in Laminaria and to investigate the link between bromine and iodine metabolism, in particular in the antioxidant context. First, bromine and iodine levels in different Laminaria tissues were compared by inductively coupled plasma MS. Using in vivo X-ray absorption spectroscopy, it was found that, similarly to iodine, bromine is predominantly present in this alga in the form of bromide, albeit at lower concentrations, and that it shows similar behaviour upon oxidative stress. However, from a thermodynamic and kinetic standpoint, supported by in vitro and reconstituted in vivo assays, bromide is less suitable than iodide as an antioxidant against most reactive oxygen species except superoxide, possibly explaining why kelps prefer to accumulate iodide. This constitutes the first-ever study exploring the potential antioxidant function of bromide in a living system and other potential physiological roles. Given the tissue-specific differences observed in the content and speciation of bromine, it is concluded that the bromide uptake mechanism is different from the vanadium iodoperoxidase-mediated uptake of iodide in L. digitata and that its function is likely to be complementary to the iodide antioxidant system for detoxifying superoxide.

Differential induction of oxylipin pathway in potato and tobacco cells by bacterial and oomycete elicitors.

KEY MESSAGE: Potato and tobacco cells are differentially suited to study oxylipin pathway and elicitor-induced responses. Synthesis of oxylipins via the lipoxygenase (LOX) pathway provides plant cells with an important class of signaling molecules, related to plant stress responses and innate immunity. The aim of this study was to evaluate the induction of LOX pathway in tobacco and potato cells induced by a concentrated culture filtrate (CCF) from Phytophthora infestans and lipopolysaccharide (LPS) from Pectobacterium atrosepticum. Oxylipin activation was evaluated by the measurement of LOX activity and metabolite quantification. The basal levels of oxylipins and fatty acids showed that potato cells contained higher amounts of linoleic (LA), linolenic (LnA) and stearic acids than tobacco cells. The major oxylipin in potato cells, 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid (9,10,11-THOD), was not detected in tobacco cells. CCF induced a sharp increase of LA and LnA at 8 h in tobacco cells. In contrast they decreased in potato cells. In CCF-treated tobacco cells, colneleic acid increased up to 24 h, colnelenic acid and 9(S)-hydroxyoctadecatrienoic acid (9(S)-HOT) increased up to 16 h. In potato cells, only colneleic acid increased slightly until 16 h. A differential induction of LOX activity was measured in both cells treated by CCF. With LPS treatment, only 9,10,11-THOD accumulation was significantly induced at 16 h in potato cells. Fatty acids were constant in tobacco but decreased in potato cells over the studied time period. These results showed that the two elicitors were differently perceived by the two Solanaceae and that oxylipin pathway is strongly induced in tobacco with the CCF. They also revealed that elicitor-induced responses depended on both cell culture and elicitor.

Proteomic analysis and identification of copper stress-regulated proteins in the marine alga Scytosiphon gracilis (Phaeophyceae).

A proteomic analysis combining peptide de novo sequencing and BLAST analysis was used to identify novel proteins involved in copper tolerance in the marine alga Scytosiphon gracilis (Phaeophyceae). Algal material was cultivated in seawater without copper (control) or supplemented with 100 microg L(-1) for 4 days, and protein extracts were separated by two-dimensional gel electrophoresis (2-DE). From the proteins obtained in the copper treatment, 25 over-expressed, 5 under-expressed and 5 proteins with no changes as compared with the control, were selected for sequencing. Tryptic-peptides obtained from 35 spots were analyzed by capillary liquid chromatography and tandem mass spectroscopy (capLC/MS/MS), and protein identity was determined by BLASTP. We identified 19 over-expressed proteins, including a chloroplast peroxiredoxin, a cytosolic phosphomannomutase, a cytosolic glyceraldehyde-3-phosphate dehydrogenase, 3 ABC transporters, a chaperonine, a subunit of the proteasome and a tRNA synthase, among others. The possible involvement of these over-expressed proteins in buffering oxidative stress and avoiding metal uptake in S. gracilis exposed to copper excess is discussed taking into consideration the information available for other plant models.

Patterns of gene expression induced by oligoguluronates reveal conserved and environment-specific molecular defense responses in the brown alga Laminaria digitata.

* Until now, no information has been available on the transcriptional response to the transduction of defense signals in brown seaweeds that leads to active resistance against pathogens or grazers. * Using a polymerase chain reaction (PCR)-based, subtractive cDNA approach combined with filter arrays we demonstrated that Laminaria digitata exhibits a rapid response to oligoguluronate elicitors. The transcription levels of several genes were validated by quantitative real-time reverse-transcription PCR and further analysed using pharmacological approaches. * Fifty upregulated genes were identified by differential screening in elicited algae over a 24-h time-course. These genes were related to oxidative stress responses, production of antimicrobial secondary compounds or cell wall strengthening. Moreover, pharmacological tests showed that intracellular signal transduction is likely to involve reactive oxygen species. A new oligoguluronate-inducible vanadium-dependent haloperoxidase (vHPO), specific to iodide was also characterized. The transcription of several vHPO genes was shown to be tightly regulated. * Taken together, our data show that early transcriptional defense responses in L. digitata are similar to those in land plants but also include novel defense responses that involve tightly regulated iodine metabolism.

Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry.

Brown algae of the Laminariales (kelps) are the strongest accumulators of iodine among living organisms. They represent a major pump in the global biogeochemical cycle of iodine and, in particular, the major source of iodocarbons in the coastal atmosphere. Nevertheless, the chemical state and biological significance of accumulated iodine have remained unknown to this date. Using x-ray absorption spectroscopy, we show that the accumulated form is iodide, which readily scavenges a variety of reactive oxygen species (ROS). We propose here that its biological role is that of an inorganic antioxidant, the first to be described in a living system. Upon oxidative stress, iodide is effluxed. On the thallus surface and in the apoplast, iodide detoxifies both aqueous oxidants and ozone, the latter resulting in the release of high levels of molecular iodine and the consequent formation of hygroscopic iodine oxides leading to particles, which are precursors to cloud condensation nuclei. In a complementary set of experiments using a heterologous system, iodide was found to effectively scavenge ROS in human blood cells.

Vanadium-dependent iodoperoxidases in Laminaria digitata, a novel biochemical function diverging from brown algal bromoperoxidases.

The brown alga Laminaria digitata features a distinct vanadium-dependent iodoperoxidase (vIPO) activity, which has been purified to electrophoretic homogeneity. Steady-state analyses at pH 6.2 are reported for vIPO (K (m) (I-) = 2.5 mM; k (cat) (I-) = 462 s(-1)) and for the previously characterised vanadium-dependent bromoperoxidase in L. digitata (K (m) (I-) =18.1 mM; k (cat) (I-) = 38 s(-1)). Although the vIPO enzyme specifically oxidises iodide, competition experiments with halides indicate that bromide is a competitive inhibitor with respect to the fixation of iodide. A full-length complementary ANA (cDNA) was cloned and shown to be actively transcribed in L. digitata and to encode the vIPO enzyme. Mass spectrometry analyses of tryptic digests of vIPO indicated the presence of at least two very similar proteins, in agreement with Southern analyses showing that vIPOs are encoded by a multigenic family in L. digitata. Phylogenetic analyses indicated that vIPO shares a close common ancestor with brown algal vanadium-dependent bromoperoxidases. Based on a three-dimensional structure model of the vIPO active site and on comparisons with those of other vanadium-dependent haloperoxidases, we propose a hypothesis to explain the evolution of strict specificity for iodide in L. digitata vIPO.