Phylogenetic relationships within Aglaopheniidae (Cnidaria,Hydrozoa) reveal unexpected generic diversity

Morphology can be misleading in the representation of phylogenetic relationships, especially in simple organisms like cnidarians and particularly in hydrozoans. These suspension feeders are widely distributed in many marine ecosystems, and the family Aglaopheniidae Marktanner‐Turneretscher, 1890 is among the most diverse and visible, especially on tropical coral reefs. The taxonomy of this family is based on morphological characters with emphasis on reproductive structures for the identification of genera. This study is the most comprehensive molecular phylogeny of the Aglaopheniidae to date, including six genera and 38 species, of which 13 were investigated for the first time and sampled on tropical coral reefs throughout the Indo‐Pacific region. For newly sampled individuals, we sequenced the 16S rRNA, the nuclear locus comprising the complete ITS1‐5.8S rRNA gene‐ITS2 and the first intron of the calmodulin nuclear gene. Phylogenetic analyses of the data revealed and confirmed a general polyphyly, or doubtful monophyly, of all sampled genera in tropical regions based on both the mitochondrial and nuclear markers. Our results revealed that several morphological characters used today are unsuited to resolve phylogenetic relationships between species and genera, as well as the high phyletic diversity within this family. Future revision of the classification of this family will require extensive geographic sampling and the use of an integrative approach.     

Stimulus-induced downregulation of root water transport involves reactive oxygen species-activated cell signalling and plasma membrane intrinsic protein internalization

The water uptake capacity of plant roots (i.e. their hydraulic conductivity, Lp(r)) is determined in large part by aquaporins of the plasma membrane intrinsic protein (PIP) subfamily. In the present work, we investigated two stimuli, salicylic acid (SA) and salt, because of their ability to induce an accumulation of reactive oxygen species (ROS) and an inhibition of Lp(r) concomitantly in the roots of Arabidopsis plants. The inhibition of Lp(r) by SA was partially counteracted by preventing the accumulation of hydrogen peroxide (H(2)O(2)) with exogenous catalase. In addition, exogenous H(2)O(2) was able to reduce Lp(r) by up to 90% in <15 min. Based on the lack of effects of H(2)O(2) on the activity of individual aquaporins in Xenopus oocytes, and on a pharmacological dissection of the action of H(2)O(2) on Lp(r), we propose that ROS do not gate Arabidopsis root aquaporins through a direct oxidative mechanism, but rather act through cell signalling mechanisms. Expression in transgenic roots of PIP-GFP fusions and immunogold labelling indicated that external H(2)O(2) enhanced, in <15 min, the accumulation of PIPs in intracellular structures tentatively identified as vesicles and small vacuoles. Exposure of roots to SA or salt also induced an intracellular accumulation of the PIP-GFP fusion proteins, and these effects were fully counteracted by co-treatment with exogenous catalase. In conclusion, the present work identifies SA as a novel regulator of aquaporins, and delineates an ROS-dependent signalling pathway in the roots of Arabidopsis. Several abiotic and biotic stress-related stimuli potentially share this path, which involves an H(2)O(2)-induced internalization of PIPs, to downregulate root water transport.     

Small localized breeding populations in a widely distributed coastal shark species

Identifying the geographical scale at which natural populations structure themselves is essential for conservation. One way to gauge this structure is by estimating local effective population size (N_e) and the associated measure of effective number of breeders (N_b), as the smaller and more isolated natural populations are, the smaller N_e and N_b they will present. However, as N_e and N_b are greatly influenced by demographic events and by both species’ behavior and biology, assessing the effectiveness of sample design is necessary to ensure the reliability of said estimates. Here, we first test the sample size effect on yearly N_b and generational N_e estimates from a lemon shark Negaprion brevirostris nursery in Bimini (The Bahamas) and subsequently compare these parameters to estimates of the minimal number of breeders based on pedigree reconstruction. We found that yearly estimates of N_b are positively correlated to annual variations in number of breeders estimated via pedigree reconstructions. Moreover, we measured that 30 individuals from a single cohort were sufficient to obtain reliable yearly estimates of N_b in Bimini’s lemon sharks. We then estimated generational N_e in 10 lemon shark nurseries across the Western Atlantic. Almost every nursery sampled represents an independent population on a generational time scale, with N_e rarely higher than 100 individuals. Our study reveals strong local population structure in lemon sharks, and thus their exposure to localized depletion or extirpation, suggesting that studies of coastal shark nursery areas could routinely estimate N_e and N_b to obtain management-relevant information on adult populations.

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Expression and inhibition of aquaporins in germinating Arabidopsis seeds

Extensive and kinetically well-defined water exchanges occur during germination of seeds. A putative role for aquaporins in this process was investigated in Arabidopsis. Macro-arrays carrying aquaporin gene-specific tags and antibodies raised against aquaporin subclasses revealed two distinct aquaporin expression programs between dry seeds and young seedlings. High expression levels of a restricted number of tonoplast intrinsic protein (TIP) isoforms (TIP3;1 and/or TIP3;2, and TIP5;1) together with a low expression of all 13 plasma membrane aquaporin (PIP) isoforms was observed in dry and germinating materials. In contrast, prevalent expression of aquaporins of the TIP1, TIP2 and PIP subgroups was induced during seedling establishment. Mercury (5 microM HgCl(2)), a general blocker of aquaporins in various organisms, reduced the speed of seed germination and induced a true delay in maternal seed coat (testa) rupture and radicle emergence, by 8-9 and 25-30 h, respectively. Most importantly, mercury did not alter seed lot homogeneity nor the seed germination developmental sequence, and its effects were largely reversed by addition of 2 mM dithiothreitol, suggesting that these effects were primarily due to oxidation of cell components, possibly aquaporins, without irreversible alteration of cell integrity. Measurements of water uptake in control and mercury-treated seeds suggested that aquaporin functions are not involved in early seed imbibition (phase I) but would rather be associated with a delayed initiation of phase III, i.e. water uptake accompanying expansion and growth of the embryo. A possible role for aquaporins in germinating seeds and more generally in plant tissue growth is discussed.     

Phylogeography and population genetics of the cryptic bonnethead shark Sphyrna aff. tiburo in Brazil and the Caribbean inferred from mtDNA markers

Resolving the identity, phylogeny and distribution of cryptic species within species complexes is an essential precursor to management. The bonnethead shark, Sphyrna tiburo, is a small coastal shark distributed in the Western Atlantic from North Carolina (U.S.A.) to southern Brazil. Genetic analyses based on mitochondrial markers revealed that bonnethead sharks comprise a species complex with at least two lineages in the Northwestern Atlantic and the Caribbean (S. tiburo and Sphyrna aff. tiburo, respectively). The phylogeographic and phylogenetic analysis of two mitochondrial markers [control region (mtCR) and cytochrome oxidase I (COI)] showed that bonnethead sharks from southeastern Brazil correspond to S. aff. tiburo, extending the distribution of this cryptic species >5000 km. Bonnethead shark populations are only managed in the U.S.A. and in the 2000s were considered to be regionally extinct or collapsed in southeast Brazil. The results indicate that there is significant genetic differentiation between S. aff. tiburo from Brazil and other populations from the Caribbean (Φ_ST = 0.9053, P < 0.000), which means that collapsed populations in the former are unlikely to be replenished from Caribbean immigration. The species identity of bonnethead sharks in the Southwest Atlantic and their relationship to North Atlantic and Caribbean populations still remains unresolved. Taxonomic revision and further sampling are required to reevaluate the status of the bonnethead shark complex through its distribution range.     

The response of Arabidopsis root water transport to a challenging environment implicates reactive oxygen species- and phosphorylation-dependent internalization of aquaporins

Aquaporins, which facilitate the diffusion of water across biological membranes, are key molecules for the regulation of water transport at the cell and organ levels. We recently reported that hydrogen peroxide (H₂O₂) acts as an intermediate in the regulation of Arabidopsis root water transport and aquaporins in response to NaCl and salicylic acid (SA).¹ Its action involves signaling pathways and an internalization of aquaporins from the cell surface. The present addendum connects these findings to another recent work which describes multiple phosphorylations in the C-terminus of aquaporins expressed in the Arabidopsis root plasma membrane.² A novel role for phosphorylation in the process of salt-induced relocalization of AtPIP2;1, one of the most abundant root aquaporins, was unraveled. Altogether, the data delineate reactive oxygen species (ROS)-dependent signaling mechanisms which, in response to a variety of abiotic and biotic stresses, can trigger phosphorylation-dependent PIP aquaporin intracellular trafficking and root water transport downregulation.Key words: reactive oxygen species, aquaporin, phosphorylation, cell signaling, stress, protein relocalization, root water transportPlants can regulate their water uptake capacity i.e. their root hydraulic conductivity (Lp_r) on a short term (minutes to hour) basis through regulation of plasma membrane (PM) aquaporins of the Plasma membrane Intrinsic Protein (PIP) subfamily.³ It has been known for a long time that salt stress (NaCl), as many other abiotic stresses such as cold, anoxia or nutrient deprivation, induces an inhibition of Lp_r in many plant species.³ In the recent study by Boursiac et al. (2008),¹ we identified SA as a new inhibitory increased the accumulation of ROS in roots, it was hypothesized that H₂O₂ or other ROS may have a central role in the regulation of root water transport in response to various biotic or abiotic stimuli. When Arabidopsis roots were treated with mM concentrations of exogenous H₂O₂, Lp_r was inhibited within minutes by up to 90%. These findings are consistent with previous reports showing that ROS can downregulate water transport in cucumber and maize roots or in the algae Chara corallina.^4–7 H₂O₂ and possibly other derived ROS may modulate the Lp_r through signaling mechanisms or by a direct oxidative gating of aquaporins. The latter hypothesis, which has been favored in previous studies by Steudle and colleagues,^6,7 was investigated by Boursiac et al., by functionally expressing aquaporins in Xenopus oocytes and by testing their sensitivity to external H₂O₂. The results show that Arabidopsis aquaporins are insensitive to direct oxidation by H₂O₂ or hydroxyl radicals. Thus, these and complementary pharmacological analyses on excised roots rather support a role for H₂O₂ as a second messenger that connects environmental stimulus perception to water transport regulation in plant roots. The additional finding that H₂O₂ can be transported by aquaporins^8,9 opens the possibility of intricate loop mechanisms whereby these proteins may interfere with their own regulation. For example, active PIP aquaporins could facilitate the diffusion within the cell of NADPH-oxidase derived apoplastic H₂O₂, which in turn would activate signaling pathways acting on PIP activity and/or subcellular localization.In a previous study, we monitored the subcellular localization of AtPIP1;2 and AtPIP2;1, two of the most abundant PIPs in roots, by expression in transgenic Arabidopsis of fusions with the green fluorescent protein (GFP).¹⁰ We observed that a 100 mM NaCl treatment induced in 2–4 hours an increased intracellular labeling which was interpreted as an intracellular relocalization of the two aquaporins.¹⁰ In our more recent study, both a 150 mM NaCl and a 0.5 mM SA treatments induced an intracellular labeling by GFP-PIP1;2 and PIP2;1-GFP fusions, with a “fuzzy” pattern or at the level of spherical bodies. Preventing the NaCl- or SA-dependent accumulation of ROS with exogenous catalase was able to almost completely counteract the effects of the two stimuli on the localization pattern of the PIP2;1-GFP fusion. In addition, the inhibition of Lp_r by SA was also counteracted at 33% by the catalase treatment. Altogether, the data stress the importance of an ROS-induced relocalization of aquaporins in the regulation of root water transport. Yet, we still miss quantitative data and complementary pharmacological evidence to determine the exact contribution of aquaporin relocalization with respect to other aquaporin regulatory mechanisms.Another recent work by our group has, however, provided deeper insights into the mechanisms of stress-induced relocalization of aquaporins in plants.² Our group identified by mass spectrometry multiple adjacent phosphorylation sites (up to 4 in the case of AtPIP2;4) in the C-terminus of aquaporins expressed at the root plasma membrane.² Phosphorylation of AtPIP2;1, which shows a simpler profile with only two sites at Ser280 and Ser283, was studied in closer detail by site-directed mutagenesis and expression in transgenic Arabidopsis of GFP-PIP2;1 fusions. A Ser283Ala mutation, which mimics a constitutively dephosphorylated Ser283, induced a marked intracellular accumulation of GFP-PIP2;1 in resting conditions. Because no phenotype was observed after a Ser280Ala mutation, the data suggest a specific role for Ser283 phosphorylation in the proper targeting of the protein. When plants were treated by 100 mM NaCl for 2 to 4 hours, the wild type (WT) and Ser280Ala mutant forms of GFP-PIP2;1 showed similar intracellular staining, in both “fuzzy” structures or spherical bodies. On the contrary, the Ser283Ala mutant did not label any spherical body. Interestingly, a Ser283Asp mutation that mimics a constitutively phosphorylated Ser283 resulted in a salt-induced labeling of spherical bodies similar to the one observed with WT GFP-PIP2;1 whereas no “fuzzy” staining was observed. Therefore, the phosphorylation status of Ser283 seems to determine the redistribution of AtPIP2;1 towards fuzzy structures (non-phosphorylated Ser283) or spherical bodies (phosphorylated Ser283). Although the nature of these intracellular structures remains to be identified, we now consider the possibility that the spherical bodies correspond to the late endosome/prevacuolar compartment that orientates aquaporins towards a degradation pathway whereas the fuzzy structures may act as a storage compartment for subsequent relocalization of PIP aquaporins to the PM, and rapid recovery of the PM water permeability. Although we favor the idea that the intracellular labeling shown by GFP-PIP2;1 in response to salt originates from aquaporins relocalized from the PM, newly synthesized proteins may also contribute to this pattern.Prak et al., also developed an absolute quantification method to show that the phosphorylation profile of AtPIP2;1 at the root plasma membrane was altered upon 100 mM NaCl and 2 mM H₂O₂ treatments. Whereas NaCl decreased the abundance of phosphorylated Ser283, H₂O₂ enhanced the overall phosphorylation of the AtPIP2;1 C-terminus. These observations add another level of complexity to the mechanisms of stimulus-induced and phosphorylation- dependent relocalisation of plant aquaporins uncovered in our group. Although one of the primary effects of NaCl is undoubtedly an accumulation of ROS, the difference in phosphorylation patterns observed in response to H₂O₂ and NaCl treatments may come from quantitative and kinetic differences in ROS patterns between the two treatments or from additional regulations activated by salt.We note that phosphorylation of PIP aquaporins had already been investigated in detail.^11–13 In particular, studies with spinach SoPIP2;1 has pointed to two phosphorylation sites, Ser115 in the first cytoplasmic loop (loop B) and Ser274 at the C-terminus, as important for modulating the water transport activity of this aquaporin after expression in Xenopus oocytes. A role for these two sites in aquaporin gating was also deduced from the atomic structure of SoPIP2;1.¹⁴ Whereas Ser280 in AtPIP2;1 corresponds to Ser274 in SoPIP2;1, the functional role of sites equivalent to Ser283 in AtPIP2;1 had not been considered previously in any other PIP. To our knowledge, the study by Prak et al., provides the first evidence in plants for a role of phosphorylation on the relocalization of aquaporins and highlights the importance of multiple phosphorylations sites in the C-terminus of aquaporins, as has been recently shown in human Aquaporin-2.^15,16Overall, the advance provided by our two recent studies delineates a working model (Fig. 1), whereby multiple abiotic and biotic stresses, which all induce an accumulation of ROS, activate common signaling pathways to downregulate root water transport. We have provided evidence that some of these pathways are calcium- and/ or protein kinase-dependent. One regulatory mechanism triggered by these pathways is the relocalization of aquaporins into intracellular “fuzzy” structures or bigger spherical bodies. For AtPIP2;1, the sorting between these structures is determined in part by the phosphorylation status of Ser283, which ultimately may control the cellular fate of the protein for degradation or remobilization to the PM. A coming challenge will be to determine how this and other cellular mechanisms quantitatively contribute to the integrated regulation of water transport at the cell and tissue (whole root) levels. Another avenue for future research will be to identify the molecular components involved in upstream ROS-dependent cell signaling and aquaporin phosphorylation. These studies will tell us how the regulation of root water uptake in parallel to the regulation of transpiration allows the plant to preserve its water status when it is continuously challenged by multiple stresses.Open in a separate windowFigure 1Tentative model of regulation of root hydraulic conductivity (Lp_r) through reactive oxygen species (ROS) signaling. Multiple biotic and abiotic stimuli such as NaCl or salicylic acid can induce an intra- and/or extracellular accumulation of ROS by acting on their production, degradation or transport. The stimulus-induced ROS in turn activate signaling pathways involving protein kinases and cytosolic calcium. These events result in changes in the phosphorylation and subcellular localization patterns of plasma membrane (PM) aquaporins (PIPs). In particular, endocytosis can direct PIPs towards various intracellular compartments for subsequent recycling at the PM or degradation. Phosphorylation can interfere with this routing process, but also determines the intrinsic water transport activity (gating) of PM localized PIPs. The possibility exists that signaling components directly act on PIP gating, recycling or degradation through phosphorylation- and endocytosis-independent pathways (not shown). In addition, transport of H₂O₂ by PIP aquaporins may provide retroactive effects of aquaporins on upstream signaling events. Aquaporin activity at the PM determines root cell water permeability, which contributes to most of Lp_r in Arabidopsis. The overall scheme shows how stress-induced ROS signaling results in an inhibition of PIP aquaporin activity and, as a consequence, in an overall downregulation of Lp_r.     

Zinc and diabetes mellitus: is there a need of zinc supplementation in diabetes mellitus patients?

Diabetes mellitus is a group of metabolic disorders, the incidence of which varies widely throughout the world. The treatment of diabetes mellitus includes insulin, oral antidiabetic agents, and dietary regimens. Although the emphasis is on macronutrients intakes, there is strong evidence that there is an abnormal metabolism of several micronutrients in diabetic individuals. Zinc is one of the essential micronutrients of which status and metabolism is altered in this condition. This work is a short review about the close relation among zinc, glucose metabolism, and insulin physiology, as well as about the few experimental data about zinc absorption and zinc supplementation in diabetes mellitus patients.