Mathematical kinetic modelling followed by in vitro and in vivo assays reveal the bifunctional rice GTPCHII/DHBPS enzymes and demonstrate the key roles of OsRibA proteins in the vitamin B2 pathway.

BACKGROUND: Riboflavin is the precursor of several cofactors essential for normal physical and cognitive development, but only plants and some microorganisms can produce it. Humans thus rely on their dietary intake, which at a global level is mainly constituted by cereals (> 50%). Understanding the riboflavin biosynthesis players is key for advancing our knowledge on this essential pathway and can hold promise for biofortification strategies in major crop species. In some bacteria and in Arabidopsis, it is known that RibA1 is a bifunctional protein with distinct GTP cyclohydrolase II (GTPCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) domains. Arabidopsis harbors three RibA isoforms, but only one retained its bifunctionality. In rice, however, the identification and characterization of RibA has not yet been described. RESULTS: Through mathematical kinetic modeling, we identified RibA as the rate-limiting step of riboflavin pathway and by bioinformatic analysis we confirmed that rice RibA proteins carry both domains, DHBPS and GTPCHII. Phylogenetic analysis revealed that OsRibA isoforms 1 and 2 are similar to Arabidopsis bifunctional RibA1. Heterologous expression of OsRibA1 completely restored the growth of the rib3∆ yeast mutant, lacking DHBPS expression, while causing a 60% growth improvement of the rib1∆ mutant, lacking GTPCHII activity. Regarding OsRibA2, its heterologous expression fully complemented GTPCHII activity, and improved rib3∆ growth by 30%. In vitro activity assays confirmed that both OsRibA1 and OsRibA2 proteins carry GTPCHII/DHBPS activities, but that OsRibA1 has higher DHBPS activity. The overexpression of OsRibA1 in rice callus resulted in a 28% increase in riboflavin content. CONCLUSIONS: Our study elucidates the critical role of RibA in rice riboflavin biosynthesis pathway, establishing it as the rate-limiting step in the pathway. By identifying and characterizing OsRibA1 and OsRibA2, showcasing their GTPCHII and DHBPS activities, we have advanced the understanding of riboflavin biosynthesis in this staple crop. We further demonstrated that OsRibA1 overexpression in rice callus increases its riboflavin content, providing supporting information for bioengineering efforts.

The bHLH transcription factor OsPRI1 activates the Setaria viridis PEPC1 promoter in rice.

Photosynthetic efficiency is reduced by the dual role of Rubisco, which acts either as a carboxylase or as an oxygenase, the latter leading to photorespiration. C4 photosynthesis evolved as a carbon-concentrating mechanism to reduce photorespiration. To engineer C4 into a C3 plant, it is essential to understand how C4 genes, such as phosphoenolpyruvate carboxylase (PEPC1), are regulated to be expressed at high levels and in a cell-specific manner. Yeast one-hybrid screening was used to show that OsPRI1, a rice bHLH transcription factor involved in iron homeostasis, binds to the Setaria viridis PEPC1 promoter. This promoter drives mesophyll-specific gene expression in rice. The role of OsPRI1 in planta was characterized using a rice line harbouring SvPEPC1pro ::GUS. We show that OsPRI1 activates the S. viridis PEPC1 promoter by binding to an N-box in the proximal promoter, and that GUS activity is highly reduced in SvPEPC1pro ::GUS lines when OsPRI1 is mutated. Cross-species comparisons showed that the SvPRI1 homolog binds to the SvPEPC1 promoter but the maize ZmPRI1 does not bind to the ZmPEPC1 promoter. Our results suggest that elements of the iron homeostasis pathway were co-opted to regulate PEPC1 gene expression during the evolution of some but not all C4 species.

SUMO E3 ligase SIZ1 connects sumoylation and reactive oxygen species homeostasis processes in Arabidopsis.

The ubiquitin-like modifying peptide SMALL UBIQUITIN-LIKE MODIFIER (SUMO) has become a known modulator of the plant response to multiple environmental stimuli. A common feature of many of these external stresses is the production of reactive oxygen species (ROS). Taking into account that SUMO conjugates rapidly accumulate in response to an external oxidative stimulus, it is likely that ROS and sumoylation converge at the molecular and regulatory levels. In this study, we explored the SUMO-ROS relationship, using as a model the Arabidopsis (Arabidopsis thaliana) null mutant of the major SUMO-conjugation enhancer, the E3 ligase SAP AND MIZ 1 (SIZ1). We showed that SIZ1 is involved in SUMO conjugate increase when primed with both exogenous and endogenous ROS. In siz1, seedlings were sensitive to oxidative stress imposition, and mutants accumulated different ROS throughout development. We demonstrated that the deregulation in hydrogen peroxide and superoxide homeostasis, but not of singlet O2 (1O2), was partially due to SA accumulation in siz1. Furthermore, transcriptomic analysis highlighted a transcriptional signature that implicated siz1 with 1O2 homeostasis. Subsequently, we observed that siz1 displayed chloroplast morphological defects and altered energy dissipation activity and established a link between the chlorophyll precursor protochlorophyllide and deregulation of PROTOCHLOROPHYLLIDE OXIDOREDUCTASE A (PORA), which is known to drive overproduction of 1O2. Ultimately, network analysis uncovered known and additional associations between transcriptional control of PORA and SIZ1-dependent sumoylation. Our study connects sumoylation, and specifically SIZ1, to the control of chloroplast functions and places sumoylation as a molecular mechanism involved in ROS homeostatic and signaling events.

A novel panel of yeast assays for the assessment of thiamin and its biosynthetic intermediates in plant tissues.

Thiamin (or thiamine), known as vitamin B1, represents an indispensable component of human diets, being pivotal in energy metabolism. Thiamin research depends on adequate vitamin quantification in plant tissues. A recently developed quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is able to assess the level of thiamin, its phosphorylated entities and its biosynthetic intermediates in the model plant Arabidopsis thaliana, as well as in rice. However, their implementation requires expensive equipment and substantial technical expertise. Microbiological assays can be useful in deter-mining metabolite levels in plant material and provide an affordable alternative to MS-based analysis. Here, we evaluate, by comparison to the LC-MS/MS reference method, the potential of a carefully chosen panel of yeast assays to estimate levels of total vitamin B1, as well as its biosynthetic intermediates pyrimidine and thiazole in Arabidopsis samples. The examined panel of Saccharomyces cerevisiae mutants was, when implemented in microbiological assays, capable of correctly assigning a series of wild-type and thiamin biofortified Arabidopsis plant samples. The assays provide a readily applicable method allowing rapid screening of vitamin B1 (and its biosynthetic intermediates) content in plant material, which is particularly useful in metabolic engineering approaches and in germplasm screening across or within species.

ZmbHLH80 and ZmbHLH90 transcription factors act antagonistically and contribute to regulate PEPC1 cell-specific gene expression in maize.

Compartmentation of photosynthetic reactions between mesophyll and bundle sheath cells is a key feature of C4 photosynthesis and depends on the cell-specific accumulation of major C4 enzymes, such as phosphoenolpyruvate carboxylase 1. The ZmPEPC1 upstream region, which drives light-inducible and mesophyll-specific gene expression in maize, has been shown to keep the same properties when introduced into rice (C3 plant), indicating that rice has the transcription factors (TFs) needed to confer C4 -like gene expression. Using a yeast one-hybrid approach, we identified OsbHLH112, a rice basic Helix-Loop-Helix (bHLH) TF that interacts with the maize ZmPEPC1 upstream region. Moreover, we found that maize OsbHLH112 homologues, ZmbHLH80, and ZmbHLH90, also interact with the ZmPEPC1 upstream region, suggesting that these C4 regulators were co-opted from C3 plants. A transactivation assay in maize mesophyll protoplasts revealed that ZmbHLH80 represses, whereas ZmbHLH90 activates, ZmPEPC1 expression. In addition, ZmbHLH80 was shown to impair the ZmPEPC1 promoter activation caused by ZmbHLH90. We showed that ZmbHLH80 and ZmbHLH90 bind to the same cis-element within the ZmPEPC1 upstream region either as homodimers or heterodimers. The formation of homo- and heterodimers with higher oligomeric forms promoted by ZmbHLH80 may explain its negative effect on gene transcription. Gene expression analysis revealed that ZmbHLH80 is preferentially expressed in bundle sheath cells, whereas ZmbHLH90 does not show a clear cell-specific expression pattern. Altogether, our results led us to propose a model in which ZmbHLH80 contributes to mesophyll-specific ZmPEPC1 gene expression by impairing ZmbHLH90-mediated ZmPEPC1 activation in the bundle sheath cells.

Arabidopsis thaliana SPF1 and SPF2 are nuclear-located ULP2-like SUMO proteases that act downstream of SIZ1 in plant development.

Post-translational modifiers such as the small ubiquitin-like modifier (SUMO) peptide act as fast and reversible protein regulators. Functional characterization of the sumoylation machinery has determined the key regulatory role that SUMO plays in plant development. Unlike components of the SUMO conjugation pathway, SUMO proteases (ULPs) are encoded by a relatively large gene family and are potential sources of specificity within the pathway. This study reports a thorough comparative genomics and phylogenetic characterization of plant ULPs, revealing the presence of one ULP1-like and three ULP2-like SUMO protease subgroups within plant genomes. As representatives of an under-studied subgroup, Arabidopsis SPF1 and SPF2 were subjected to functional characterization. Loss-of-function mutants implicated both proteins with vegetative growth, flowering time, and seed size and yield. Mutants constitutively accumulated SUMO conjugates, and yeast complementation assays associated these proteins with the function of ScUlp2 but not ScUlp1. Fluorescence imaging placed both proteins in the plant cell nucleoplasm. Transcriptomics analysis indicated strong regulatory involvement in secondary metabolism, cell wall remodelling, and nitrate assimilation. Furthermore, developmental defects of the spf1-1 spf2-2 (spf1/2) double-mutant opposed those of the major E3 ligase siz1 mutant and, most significantly, developmental and transcriptomic characterization of the siz1 spf1/2 triple-mutant placed SIZ1 as epistatic to SPF1 and SPF2.

Rice phytochrome-interacting factor protein OsPIF14 represses OsDREB1B gene expression through an extended N-box and interacts preferentially with the active form of phytochrome B.

DREB1/CBF genes, known as major regulators of plant stress responses, are rapidly and transiently induced by low temperatures. Using a yeast one-hybrid screening, we identified a putative Phytochrome-Interacting bHLH Factor (OsPIF14), as binding to the OsDREB1B promoter. bHLH proteins are able to bind to hexameric E-box (CANNTG) or N-box (CACG(A/C)G) motifs, depending on transcriptional activity. We have shown that OsPIF14 binds to the OsDREB1B promoter through two N-boxes and that the flanking regions of the hexameric core are essential for protein-DNA interaction and stability. We also showed that OsPIF14 down-regulates OsDREB1B gene expression in rice protoplasts, corroborating the OsPIF14 repressor activity observed in the transactivation assays using Arabidopsis protoplasts. In addition, we showed that OsPIF14 is indeed a phytochrome interacting factor, which preferentially binds to the active form (Pfr) of rice phytochrome B. This raises the possibility that OsPIF14 activity might be modulated by light. However, we did not observe any regulation of the OsDREB1B gene expression by light under control conditions. Moreover, OsPIF14 gene expression was shown to be modulated by different treatments, such as drought, salt, cold and ABA. Interestingly, OsPIF14 showed also a specific cold-induced alternative splicing. All together, these results suggest the possibility that OsPIF14 is involved in cross-talk between light and stress signaling through interaction with the OsDREB1B promoter. Although in the absence of stress, OsDREB1B gene expression was not regulated by light, given previous reports, it remains possible that OsPIF14 has a role in light modulation of stress responses.

The Rice E3-Ubiquitin Ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 Modulates the Expression of ROOT MEANDER CURLING, a Gene Involved in Root Mechanosensing, through the Interaction with Two ETHYLENE-RESPONSE FACTOR Transcription Factors.

Plant roots can sense and respond to a wide diversity of mechanical stimuli, including touch and gravity. However, little is known about the signal transduction pathways involved in mechanical stimuli responses in rice (Oryza sativa). This work shows that rice root responses to mechanical stimuli involve the E3-ubiquitin ligase rice HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 (OsHOS1), which mediates protein degradation through the proteasome complex. The morphological analysis of the roots in transgenic RNA interference::OsHOS1 and wild-type plants, exposed to a mechanical barrier, revealed that the OsHOS1 silencing plants keep a straight root in contrast to wild-type plants that exhibit root curling. Moreover, it was observed that the absence of root curling in response to touch can be reverted by jasmonic acid. The straight root phenotype of the RNA interference::OsHOS1 plants was correlated with a higher expression rice ROOT MEANDER CURLING (OsRMC), which encodes a receptor-like kinase characterized as a negative regulator of rice root curling mediated by jasmonic acid. Using the yeast two-hybrid system and bimolecular fluorescence complementation assays, we showed that OsHOS1 interacts with two ETHYLENE-RESPONSE FACTOR transcription factors, rice ETHYLENE-RESPONSIVE ELEMENT BINDING PROTEIN1 (OsEREBP1) and rice OsEREBP2, known to regulate OsRMC gene expression. In addition, we showed that OsHOS1 affects the stability of both transcription factors in a proteasome-dependent way, suggesting that this E3-ubiquitin ligase targets OsEREBP1 and OsEREBP2 for degradation. Our results highlight the function of the proteasome in rice response to mechanical stimuli and in the integration of these signals, through hormonal regulation, into plant growth and developmental programs.

Transcriptomics and physiological analyses reveal co-ordinated alteration of metabolic pathways in Jatropha curcas drought tolerance.

Jatropha curcas, a multipurpose plant attracting a great deal of attention due to its high oil content and quality for biofuel, is recognized as a drought-tolerant species. However, this drought tolerance is still poorly characterized. This study aims to contribute to uncover the molecular background of this tolerance, using a combined approach of transcriptional profiling and morphophysiological characterization during a period of water-withholding (49 d) followed by rewatering (7 d). Morphophysiological measurements showed that J. curcas plants present different adaptation strategies to withstand moderate and severe drought. Therefore, RNA sequencing was performed for samples collected under moderate and severe stress followed by rewatering, for both roots and leaves. Jatropha curcas transcriptomic analysis revealed shoot- and root-specific adaptations across all investigated conditions, except under severe stress, when the dramatic transcriptomic reorganization at the root and shoot level surpassed organ specificity. These changes in gene expression were clearly shown by the down-regulation of genes involved in growth and water uptake, and up-regulation of genes related to osmotic adjustments and cellular homeostasis. However, organ-specific gene variations were also detected, such as strong up-regulation of abscisic acid synthesis in roots under moderate stress and of chlorophyll metabolism in leaves under severe stress. Functional validation further corroborated the differential expression of genes coding for enzymes involved in chlorophyll metabolism, which correlates with the metabolite content of this pathway.

SIZ1-Dependent Post-Translational Modification by SUMO Modulates Sugar Signaling and Metabolism in Arabidopsis thaliana.

Post-translational modification mechanisms function as switches that mediate the balance between optimum growth and the response to environmental stimuli, by regulating the activity of key proteins. SUMO (small ubiquitin-like modifier) attachment, or sumoylation, is a post-translational modification that is essential for the plant stress response, also modulating hormonal circuits to co-ordinate developmental processes. The Arabidopsis SUMO E3 ligase SAP and Miz 1 (SIZ1) is the major SUMO conjugation enhancer in response to stress, and is implicated in several aspects of plant development. Here we report that known SUMO targets are over-represented in multiple carbohydrate-related proteins, suggesting a functional link between sumoylation and sugar metabolism and signaling in plants. We subsequently observed that SUMO-conjugated proteins accumulate in response to high doses of sugar in a SIZ1-dependent manner, and that the null siz1 mutant displays increased expression of sucrose and starch catabolic genes and shows reduced starch levels. We demonstrated that SIZ1 controls germination time and post-germination growth via osmotic and sugar-dependent signaling, respectively. Glucose was specifically linked to SUMO-sugar interplay, with high levels inducing root growth inhibition and aberrant root hair morphology in siz1. The use of sugar analogs and sugar marker gene expression analysis allowed us to implicate SIZ1 in a signaling pathway dependent on glucose metabolism, probably involving modulation of SNF1-related kinase 1 (SnRK1) activity.

High resolution magic angle spinning NMR as a tool for unveiling the molecular enantiorecognition of omeprazole by amylose-based chiral phase.

Polysaccharide-based chiral stationary phases (CSP) demonstrate great versatility and higher chiral selectivity for a variety of chiral compounds in multimodal elution modes (normal, reverse and polar organic). The main role of CSP phenyl carbamate based derivatives as chiral selectors is the formation of diastereoisomeric complexes by means of π-π interaction, dipole-dipole, hydrogen bonding and/or inclusion complex mechanisms. Nevertheless, the mechanism behind their enantioselectivity requires clarification. High resolution magic angle spinning nuclear magnetic resonance spectroscopy ((1)H HR/MAS NMR) has provided key information on the recognition process at the binding sites of the CSP surface. Herein we report the results obtained using omeprazole as a probe for these investigations.

A Proof-of-Concept for a Hypolipidemic Brown Trout Model.

The impacts of hypolipidemic pharmaceuticals on fish lipid metabolism remain unexplored. However, data points to similar effects and mechanisms of action between fish and humans. Therefore, fish may be a strong model for screening hypolipidemic drug candidates and water pollution by lipid-modulating agents. This study aimed to test a new hypolipidemic model assay with juvenile brown trout using atorvastatin (ATV)-a hypolipidemic chemical. We selected 17α-ethinylestradiol (EE2), known to cause hyperlipidemia in fish, to ensure model functionality. Fish received intramuscular injections of 4 µL/g for two weeks under the following experimental conditions: control-C (0.7% NaCl), solvent control-SC (0.7% NaCl, 0.9% ethanol, 0.1% dimethyl sulfoxide), ATV (0.3 µg/g), EE2 (2 µg/g), and a mixture of both compounds-MIX (0.3 µg/g ATV and 2 µg/g EE2). Endpoints included blood lipid biochemistry, hepatic lipid droplet quantification, and liver mRNA expression of lipid-related target genes (related to lipogenesis, lipid transport, and ß-oxidation pathways). ATV lowered blood total cholesterol, high-density lipoproteins (HDL), and low-density lipoproteins (LDL) levels, whilst triglycerides and very-low-density lipoproteins (VLDL) were highest under EE2. Hepatic lipid droplet deposition significantly increased in the ATV, EE2, and MIX groups. ATV and MIX caused a significant downregulation of the peroxisome proliferator-activated receptor γ (pparγ) and acetyl Co-A oxidase 3 (acox3). EE2 upregulated acyl-CoA long-chain synthetase 1 (acsl1) and downregulated both fatty acid binding protein 1 (fabp1) and acetyl Co-A oxidase 1-3I (acox1-3I). ATV caused hypolipidemic effects in juvenile brown trout and could even counteract EE2-stimulated hyperlipidemia, reinforcing the potential of fish hypo- and hyperlipidemic models.

OsTH1 is a key player in thiamin biosynthesis in rice.

Thiamin is a vital nutrient that acts as a cofactor for several enzymes primarily localized in the mitochondria. These thiamin-dependent enzymes are involved in energy metabolism, nucleic acid biosynthesis, and antioxidant machinery. The enzyme HMP-P kinase/thiamin monophosphate synthase (TH1) holds a key position in thiamin biosynthesis, being responsible for the phosphorylation of HMP-P into HMP-PP and for the condensation of HMP-PP and HET-P to form TMP. Through mathematical kinetic model, we have identified TH1 as a critical player for thiamin biofortification in rice. We further focused on the functional characterization of OsTH1. Sequence and gene expression analysis, along with phylogenetic studies, provided insights into OsTH1 bifunctional features and evolution. The indispensable role of OsTH1 in thiamin biosynthesis was validated by heterologous expression of OsTH1 and successful complementation of yeast knock-out mutants impaired in thiamin production. We also proved that the sole OsTH1 overexpression in rice callus significantly improves B1 concentration, resulting in 50% increase in thiamin accumulation. Our study underscores the critical role of OsTH1 in thiamin biosynthesis, shedding light on its bifunctional nature and evolutionary significance. The significant enhancement of thiamin accumulation in rice callus upon OsTH1 overexpression constitutes evidence of its potential application in biofortification strategies.

Isolation and characterization of rice (Oryza sativa L.) E3-ubiquitin ligase OsHOS1 gene in the modulation of cold stress response.

Plants can cope with adverse environmental conditions through the activation of stress response signalling pathways, in which the proteasome seems to play an important role. However, the mechanisms underlying the proteasome-mediated stress response in rice are still not fully understood. To address this issue, we have identified a rice E3-ubiquitin ligase, OsHOS1, and characterized its role in the modulation of the cold stress response. Using a RNA interference (RNAi) transgenic approach we found that, under cold conditions, the RNAi::OsHOS1 plants showed a higher expression level of OsDREB1A. This was correlated with an increased amount of OsICE1, a master transcription factor of the cold stress signalling. However, the up-regulation of OsDREB1A was transient and the transgenic plants did not show increased cold tolerance. Nevertheless, we could confirm the interaction of OsHOS1 with OsICE1 by Yeast-Two hybrid and bi-molecular fluorescence complementation in Arabidopsis protoplasts. Moreover, we could also determine through an in vitro degradation assay that the higher amount of OsICE1 in the transgenic plants was correlated with a lower amount of OsHOS1. Hence, we could confirm the involvement of the proteasome in this response mechanism. Taken together our results confirm the importance of OsHOS1, and thus of the proteasome, in the modulation of the cold stress signalling in rice.

OsRMC, a negative regulator of salt stress response in rice, is regulated by two AP2/ERF transcription factors.

High salinity causes remarkable losses in rice productivity worldwide mainly because it inhibits growth and reduces grain yield. To cope with environmental changes, plants evolved several adaptive mechanisms, which involve the regulation of many stress-responsive genes. Among these, we have chosen OsRMC to study its transcriptional regulation in rice seedlings subjected to high salinity. Its transcription was highly induced by salt treatment and showed a stress-dose-dependent pattern. OsRMC encodes a receptor-like kinase described as a negative regulator of salt stress responses in rice. To investigate how OsRMC is regulated in response to high salinity, a salt-induced rice cDNA expression library was constructed and subsequently screened using the yeast one-hybrid system and the OsRMC promoter as bait. Thereby, two transcription factors (TFs), OsEREBP1 and OsEREBP2, belonging to the AP2/ERF family were identified. Both TFs were shown to bind to the same GCC-like DNA motif in OsRMC promoter and to negatively regulate its gene expression. The identified TFs were characterized regarding their gene expression under different abiotic stress conditions. This study revealed that OsEREBP1 transcript level is not significantly affected by salt, ABA or severe cold (5 °C) and is only slightly regulated by drought and moderate cold. On the other hand, the OsEREBP2 transcript level increased after cold, ABA, drought and high salinity treatments, indicating that OsEREBP2 may play a central role mediating the response to different abiotic stresses. Gene expression analysis in rice varieties with contrasting salt tolerance further suggests that OsEREBP2 is involved in salt stress response in rice.

In vitro analysis of albendazole sulfoxide enantiomers shows that (+)-(R)-albendazole sulfoxide is the active enantiomer against Taenia solium.

Albendazole is an anthelmintic drug widely used in the treatment of neurocysticercosis (NCC), an infection of the brain with Taenia solium cysts. However, drug levels of its active metabolite, albendazole sulfoxide (ABZSO), are erratic, likely resulting in decreased efficacy and suboptimal cure rates in NCC. Racemic albendazole sulfoxide is composed of ABZSO (+)-(R)- and (-)-(S) enantiomers that have been shown to differ in pharmacokinetics and activity against other helminths. The antiparasitic activities of racemic ABZSO and its (+)-(R)- and (-)-(S) enantiomers against T. solium cysts were evaluated in vitro. Parasites were collected from naturally infected pigs, cultured, and exposed to the racemic mixture or to each enantiomer (range, 10 to 500 ng/ml) or to praziquantel as a reference drug. The activity of each compound against cysts was assayed by measuring the ability to evaginate and inhibition of alkaline phosphatase (AP) and parasite antigen release. (+)-(R)-ABZSO was significantly more active than (-)-(S)-ABZSO in suppressing the release of AP and antigen into the supernatant in a dose- and time-dependent manner, indicating that most of the activity of ABZSO resides in the (+)-(R) enantiomer. Use of this enantiomer alone may lead to increased efficacy and/or less toxicity compared to albendazole.

Fish as models to study liver and blood lipid-related effects of fibrates and statins and screen new hypolipidemic drugs.

Fibrates and statins lead worldwide prescriptions of lipid-lowering drugs, whose consumption is increasing considerably due to the growing incidence of dyslipidemias, particularly in high-income areas. Consequently, these chemicals are frequently found in aquatic environments, usually closer to highly urbanized and populated areas, reaching the water systems primarily through waste-water treatment plant (WWTP) effluents. Despite that, the knowledge regarding the effects caused by fibrates and statins in fish, namely in liver lipid metabolism and blood-related parameters, is still very limited. There is yet no standardized fish model for testing the effects of those drugs. However, experimental evidence suggests that the mechanisms of action (MoA) of fibrates and statins are fairly similar to those observed in humans, which makes these aquatic organisms viable alternatives for toxicological and mechanistic studies. This graphical review serves as a state point regarding the potential use of fish as a model for the study of hypolipidemic compounds, addressing (I) the current state of aquatic pollution caused by statins and fibrates, (II) the experimental designs used in the literature to assess effects on fish, (III) the liver metabolism and blood effects caused by exposure to fibrates and statins, as well as (IV) the MoA of both drugs. It further focuses on the current and future benefits of establishing a standardized fish model(s) for testing hypolipidemic drugs.

Integrated climate, ecological and socioeconomic scenarios for the whale watching sector.

Unprecedented human induced changes to the climate system have already contributed to a variety of observed impacts to both ecosystems and populations. Decision-makers demand impact assessments at the regional-to-local scale to be able to plan and define effective climate action measures. Integrated socio-ecological assessments that properly consider system uncertainties require the use of prospective scenarios that project potential climate impacts, while accounting for sectoral exposure and adaptive capacity. Here we provide an integrated assessment of climate change to the whale watching sector by: 1) extending the European Shared Socio-economic Pathways (Eur-SSPs) and developing four whale watching SSP narratives (WW-SSPs) and 2) characterize each key element comprised in the WW-SSPs for the time period 2025-2055. We applied this approach in a case study for the Macaronesia region where we developed scenarios which integrate the socio-economic (WW-SSPs), climate (RCPs) and ecological (species' thermal suitability responses) dimensions of whale watching. These scenarios were used by local stakeholders to identify the level of preparedness of the whale watching sector. When confronted with scenarios that combine this ecological dimension with projected climate changes and the four different socioeconomic narratives, stakeholders assessed the whale watching sector in Macaronesia as being somewhat prepared for a Sustainable World and a Fossil Fuel Development World, but somewhat unprepared for a Rivalry World. No consensus was reached regarding the sector's preparedness level under an Inequality World scenario. Our study demonstrates the importance of considering multiple dimensions when assessing the potential challenges posed by climate change and provides a needed resource to help the whale watching sector in Macaronesia, and elsewhere, in its effort to devise efficient climate action policies and strategies.

A novel expert-driven methodology to develop thermal response curves and project habitat thermal suitability for cetaceans under a changing climate.

Over the last decades, global warming has contributed to changes in marine species composition, abundance and distribution, in response to changes in oceanographic conditions such as temperature, acidification, and deoxygenation. Experimentally derived thermal limits, which are known to be related to observed latitudinal ranges, have been used to assess variations in species distribution patterns. However, such experiments cannot be undertaken on free-swimming large marine predators with wide-range distribution, like cetaceans. An alternative approach is to elicit expert's knowledge to derive species' thermal suitability and assess their thermal responses, something that has never been tested in these taxa. We developed and applied a methodology based on expert-derived thermal suitability curves and projected future responses for several species under different climate scenarios. We tested this approach with ten cetacean species currently present in the biogeographic area of Macaronesia (North Atlantic) under Representative Concentration Pathways 2.6, 4.5 and 8.5, until 2050. Overall, increases in annual thermal suitability were found for Balaenoptera edeni, Globicephala macrorhynchus, Mesoplodon densirostris, Physeter macrocephalus, Stenella frontalis, Tursiops truncatus and Ziphius cavirostris. Conversely, our results indicated a decline in thermal suitability for B. physalus, Delphinus delphis, and Grampus griseus. Our study reveals potential responses in cetaceans' thermal suitability, and potentially in other highly mobile and large predators, and it tests this method's applicability, which is a novel application for this purpose and group of species. It aims to be a cost-efficient tool to support conservation managers and practitioners.

Seven zinc-finger transcription factors are novel regulators of the stress responsive gene OsDREB1B.

Plants have evolved several mechanisms in order to cope with adverse environmental conditions. The transcription factors (TFs) belonging to the DREB1/CBF subfamily have been described as major regulators of the plant responses to different abiotic stresses. This study focused on the rice gene OsDREB1B, initially described as highly and specifically induced by cold. However, here it is shown that OsDREB1B is not only induced by low temperatures, but also by drought and mechanical stress. In order to identify novel TFs that bind to its promoter, a yeast one-hybrid system was used to screen a cold-induced cDNA expression library. Thereby seven novel Zn-finger TFs were identified that bind to the promoter of OsDREB1B. Among them, there were four Zn-finger homeodomain (ZF-HD) and three C(2)H(2)-type Zn-finger TFs. Gene expression studies showed that these TFs are differentially regulated at transcriptional level by different abiotic stress conditions, which is illustrative of the crosstalk between stress signalling pathways. Protein-protein interaction studies revealed the formation of homo- and heterodimers among the ZF-HD TFs identified, but not for the C(2)H(2)-type. Using a transactivation assay in Arabidopsis protoplasts, all the TFs identified repressed the expression of the reporter gene, driven by the promoter of OsDREB1B. This assay also showed that the dimerization observed between the ZF-HD TFs may play a role on their transactivation activity. The results here presented suggest a prominent role of Zn-finger TFs in the regulation of OsDREB1B.