Contrasting effects of high‐intensity photosynthetically active radiation on two bloom‐forming dinoflagellates

While light limitation can inhibit bloom formation in dinoflagellates, the potential for high‐intensity photosynthetically active radiation (PAR) to inhibit blooms by causing stress or damage has not been well‐studied. We measured the effects of high‐intensity PAR on the bloom‐forming dinoflagellates Alexandrium fundyense and Heterocapsa rotundata. Various physiological parameters (photosynthetic efficiency F_v/F_m, cell permeability, dimethylsulfoniopropionate [DMSP], cell volume, and chlorophyll‐a content) were measured before and after exposure to high‐intensity natural sunlight in short‐term light stress experiments. In addition, photosynthesis‐irradiance (P‐E) responses were compared for cells grown at different light levels to assess the capacity for photophysiological acclimation in each species. Experiments revealed distinct species‐specific responses to high PAR. While high light decreased F_v/F_m in both species, A. fundyense showed little additional evidence of light stress in short‐term experiments, although increased membrane permeability and intracellular DMSP indicated a response to handling. P‐E responses further indicated a high light‐adapted species with Chl‐a inversely proportional to growth irradiance and no evidence of photoinhibition; reduced maximum per‐cell photosynthesis rates suggest a trade‐off between photoprotection and C fixation in high light‐acclimated cells. Heterocapsa rotundata cells, in contrast, swelled in response to high light and sometimes lysed in short‐term experiments, releasing DMSP. P‐E responses confirmed a low light‐adapted species with high photosynthetic efficiencies associated with trade‐offs in the form of substantial photoinhibition and a lack of plasticity in Chl‐a content. These contrasting responses illustrate that high light constrains dinoflagellate community composition through species‐specific stress effects, with consequences for bloom formation and ecological interactions within the plankton.     

The octadecaneuropeptide ODN prevents 6‐hydroxydopamine‐induced apoptosis of cerebellar granule neurons through a PKC‐MAPK‐dependent pathway

Oxidative stress, induced by various neurodegenerative diseases, initiates a cascade of events leading to apoptosis, and thus plays a critical role in neuronal injury. In this study, we have investigated the potential neuroprotective effect of the octadecaneuropeptide (ODN) on 6‐hydroxydopamine (6‐OHDA)‐induced oxidative stress and apoptosis in cerebellar granule neurons (CGN). ODN, which is produced by astrocytes, is an endogenous ligand for both central‐type benzodiazepine receptors (CBR) and a metabotropic receptor. Incubation of neurons with subnanomolar concentrations of ODN (10^?18 to 10^?12 M) inhibited 6‐OHDA‐evoked cell death in a concentration‐dependent manner. The effect of ODN on neuronal survival was abrogated by the metabotropic receptor antagonist, cyclo_1–8[DLeu⁵]OP, but not by a CBR antagonist. ODN stimulated polyphosphoinositide turnover and ERK phosphorylation in CGN. The protective effect of ODN against 6‐OHDA toxicity involved the phospholipase C/ERK MAPK transduction cascade. 6‐OHDA treatment induced an accumulation of reactive oxygen species, an increase of the expression of the pro‐apoptotic gene Bax, a drop of the mitochondrial membrane potential and a stimulation of caspase‐3 activity. Exposure of 6‐OHDA‐treated cells to ODN blocked all the deleterious effects of the toxin. Taken together, these data demonstrate for the first time that ODN is a neuroprotective agent that prevents 6‐OHDA‐induced oxidative stress and apoptotic cell death.     

Arbuscular mycorrhiza influences carbon‐use efficiency and grain yield of wheat grown under pre‐ and post‐anthesis salinity stress

• Soil salinity severely affects and constrains crop production worldwide. Salinity causes osmotic and ionic stress, inhibiting gas exchange and photosynthesis, ultimately impairing plant growth and development. Arbuscular mycorrhiza (AM) have been shown to maintain light and carbon use efficiency under stress, possibly providing a tool to improve salinity tolerance of the host plants. Thus, it was hypothesized that AM will contribute to improved growth and yield under stress conditions.
• Wheat plants (Triticum aestivum L.) were grown with (AMF+) or without (AMF?) arbuscular mycorrhizal fungi (AMF) inoculation. Plants were subjected to salinity stress (200 mm NaCl) either at pre‐ or post‐anthesis or at both stages. Growth and yield components, leaf chlorophyll content as well as gas exchange parameters and AMF colonization were analysed.
• AM plants exhibited a higher rate of net photosynthesis and stomatal conductance and lower intrinsic water use efficiency. Furthermore, AM wheat plants subjected to salinity stress at both pre‐anthesis and post‐anthesis maintained higher grain yield than non‐AM salinity‐stressed plants.
• These results suggest that AMF inoculation mitigates the negative effects of salinity stress by influencing carbon use efficiency and maintaining higher grain yield under stress.

     

The oxalyl‐CoA synthetase‐regulated oxalate and its distinct effects on resistance to bacterial blight and aluminium toxicity in rice

• Oxalic acid is widely distributed in biological systems and known to play functional roles in plants. The gene AAE3 was recently identified to encode an oxalyl‐CoA synthetase (OCS) in Arabidopsis that catalyses the conversion of oxalate and CoA into oxalyl‐CoA. It will be particularly important to characterise the homologous gene in rice since rice is not only a monocotyledonous model plant, but also a staple food crop.
• Various enzymatic and biological methods have been used to characterise the homologous gene.
• We first defined that AAE3 in the rice genome (OsAAE3) also encodes an OCS enzyme. Its K_m for oxalate is 1.73 ± 0.12 mm , and V_m is 6824.9 ± 410.29 U·min^?1·mg protein^?1. Chemical modification and site‐directed mutagenesis analyses identified thiols as the active site residues for rice OCS catalysis, suggesting that the enzyme might be regulated by redox state. Subcellular localisation assay showed that the enzyme is located in the cytosol and predominantly distributed in leaf epidermal cells. As expected, oxalate levels increased when OCS was suppressed in RNAi transgenic plants. More interestingly, OCS‐suppressed plants were more susceptible to bacterial blight but more resistant to Al toxicity.
• The results demonstrate that the OsAAE3‐encoded protein also acts as an OCS in rice, and may play different roles in coping with stresses. These molecular, enzymatic and functional data provide first‐hand information to further clarify the function and mechanism of OCS in rice plants.

     

AMPK promotes survival of c‐Myc‐positive melanoma cells by suppressing oxidative stress

Although c‐Myc is essential for melanocyte development, its role in cutaneous melanoma, the most aggressive skin cancer, is only partly understood. Here we used the Nras^Q61KINK4a^?/? mouse melanoma model to show that c‐Myc is essential for tumor initiation, maintenance, and metastasis. c‐Myc‐expressing melanoma cells were preferentially found at metastatic sites, correlated with increased tumor aggressiveness and high tumor initiation potential. Abrogation of c‐Myc caused apoptosis in primary murine and human melanoma cells. Mechanistically, c‐Myc‐positive melanoma cells activated and became dependent on the metabolic energy sensor AMP‐activated protein kinase (AMPK), a metabolic checkpoint kinase that plays an important role in energy and redox homeostasis under stress conditions. AMPK pathway inhibition caused apoptosis of c‐Myc‐expressing melanoma cells, while AMPK activation protected against cell death of c‐Myc‐depleted melanoma cells through suppression of oxidative stress. Furthermore, TCGA database analysis of early‐stage human melanoma samples revealed an inverse correlation between C‐MYC and patient survival, suggesting that C‐MYC expression levels could serve as a prognostic marker for early‐stage disease.     

Climatic and biotic extreme events moderate long‐term responses of above‐ and belowground sub‐Arctic heathland communities to climate change

Climate change impacts are not uniform across the Arctic region because interacting factors causes large variations in local ecosystem change. Extreme climatic events and population cycles of herbivores occur simultaneously against a background of gradual climate warming trends and can redirect ecosystem change along routes that are difficult to predict. Here, we present the results from sub‐Arctic heath vegetation and its belowground micro‐arthropod community in response to the two main drivers of vegetation damage in this region: extreme winter warming events and subsequent outbreaks of the defoliating autumnal moth caterpillar (Epirrita autumnata). Evergreen dwarf shrub biomass decreased (30%) following extreme winter warming events and again by moth caterpillar grazing. Deciduous shrubs that were previously exposed to an extreme winter warming event were not affected by the moth caterpillar grazing, while those that were not exposed to warming events (control plots) showed reduced (23%) biomass from grazing. Cryptogam cover increased irrespective of grazing or winter warming events. Micro‐arthropods declined (46%) following winter warming but did not respond to changes in plant community. Extreme winter warming and caterpillar grazing suppressed the CO₂ fluxes of the ecosystem. Evergreen dwarf shrubs are disadvantaged in a future sub‐Arctic with more stochastic climatic and biotic events. Given that summer warming may further benefit deciduous over evergreen shrubs, event and trend climate change may both act against evergreen shrubs and the ecosystem functions they provide. This is of particular concern given that Arctic heath vegetation is typically dominated by evergreen shrubs. Other components of the vegetation showed variable responses to abiotic and biotic events, and their interaction indicates that sub‐Arctic vegetation response to multiple pressures is not easy to predict from single‐factor responses. Therefore, while biotic and climatic events may have clear impacts, more work is needed to understand their net effect on Arctic ecosystems.     

Two‐step d‐ononitol epimerization pathway in Medicago truncatula

Methylated inositol, d ‐pinitol (3‐O‐methyl‐d ‐chiro‐inositol), is a common constituent in legumes. It is synthesized from myo‐inositol in two reactions: the first reaction, catalyzed by myo‐inositol‐O‐methyltransferase (IMT), consists of a transfer of a methyl group from S‐adenosylmethionine to myo‐inositol with the formation of d ‐ononitol, while the second reaction, catalyzed by d ‐ononitol epimerase (OEP), involves epimerization of d ‐ononitol to d ‐pinitol. To identify the genes involved in d ‐pinitol biosynthesis in a model legume Medicago truncatula, we conducted a BLAST search on its genome using soybean IMT cDNA as a query and found putative IMT (MtIMT) gene. Subsequent co‐expression analysis performed on publicly available microarray data revealed two potential OEP genes: MtOEPA, encoding an aldo‐keto reductase and MtOEPB, encoding a short‐chain dehydrogenase. cDNAs of all three genes were cloned and expressed as recombinant proteins in E. coli. In vitro assays confirmed that putative MtIMT enzyme catalyzes methylation of myo‐inositol to d ‐ononitol and showed that MtOEPA enzyme has NAD⁺‐dependent d ‐ononitol dehydrogenase activity, while MtOEPB enzyme has NADP⁺‐dependent d ‐pinitol dehydrogenase activity. Both enzymes are required for epimerization of d ‐ononitol to d ‐pinitol, which occurs in the presence of NAD⁺ and NADPH. Introduction of MtIMT, MtOEPA, and MtOEPB genes into tobacco plants resulted in production of d ‐ononitol and d ‐pinitol in transformants. As this two‐step pathway of d ‐ononitol epimerization is coupled with a transfer of reducing equivalents from NADPH to NAD⁺, we speculate that one of the functions of this pathway might be regeneration of NADP⁺ during drought stress.     

Overexpression of the leucine‐rich receptor‐like kinase gene LRK2 increases drought tolerance and tiller number in rice

Drought represents a key limiting factor of global crop distribution. Receptor‐like kinases play major roles in plant development and defence responses against stresses such as drought. In this study, LRK2, which encodes a leucine‐rich receptor‐like kinase, was cloned and characterized and found to be localized on the plasma membrane in rice. Promoter–GUS analysis revealed strong expression in tiller buds, roots, nodes and anthers. Transgenic plants overexpressing LRK2 exhibited enhanced tolerance to drought stress due to an increased number of lateral roots compared with the wild type at the vegetative stage. Moreover, ectopic expression of LRK2 seedlings resulted in increased tiller development. Yeast two‐hybrid screening and bimolecular fluorescence complementation (BiFC) indicated a possible interaction between LRK2 and elongation factor 1 alpha (OsEF1A) in vitro. These results suggest that LRK2 functions as a positive regulator of the drought stress response and tiller development via increased branch development in rice. These findings will aid our understanding of branch regulation in other grasses and support improvements in rice genetics.     

Protection from cigarette smoke‐induced vascular injury by recombinant human relaxin‐2 (serelaxin)

Smoking is regarded as a major risk factor for the development of cardiovascular diseases (CVD). This study investigates whether serelaxin (RLX, recombinant human relaxin‐2) endowed with promising therapeutic properties in CVD, can be credited of a protective effect against cigarette smoke (CS)‐induced vascular damage and dysfunction. Guinea pigs exposed daily to CS for 8 weeks were treated with vehicle or RLX, delivered by osmotic pumps at daily doses of 1 or 10 μg. Controls were non‐smoking animals. Other studies were performed on primary guinea pig aortic endothelial (GPAE) cells, challenged with CS extracts (CSE) in the absence and presence of 100 ng/ml (17 nmol/l) RLX. In aortic specimens from CS‐exposed guinea pigs, both the contractile and the relaxant responses to phenylephrine and acetylcholine, respectively, were significantly reduced in amplitude and delayed, in keeping with the observed adverse remodelling of the aortic wall, endothelial injury and endothelial nitric oxide synthase (eNOS) down‐regulation. RLX at both doses maintained the aortic contractile and relaxant responses to a control‐like pattern and counteracted aortic wall remodelling and endothelial derangement. The experiments with GPAE cells showed that CSE significantly decreased cell viability and eNOS expression and promoted apoptosis by sparkling oxygen free radical‐related cytotoxicity, while RLX counterbalanced the adverse effects of CSE. These findings demonstrate that RLX is capable of counteracting CS‐mediated vascular damage and dysfunction by reducing oxidative stress, thus adding a tile to the growing mosaic of the beneficial effects of RLX in CVD.     

The diversion of 2‐C‐methyl‐d‐erythritol‐2,4‐cyclodiphosphate from the 2‐C‐methyl‐d‐erythritol 4‐phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana

2‐C‐Methyl‐d ‐erythritol‐2,4‐cyclodiphosphate (MEcDP) is an intermediate of the plastid‐localized 2‐C‐methyl‐d ‐erythritol‐4‐phosphate (MEP) pathway which supplies isoprenoid precursors for photosynthetic pigments, redox co‐factor side chains, plant volatiles, and phytohormones. The Arabidopsis hds‐3 mutant, defective in the 1‐hydroxy‐2‐methyl‐2‐(E)‐butenyl‐4‐diphosphate synthase step of the MEP pathway, accumulates its substrate MEcDP as well as the free tetraol 2‐C‐methyl‐d ‐erythritol (ME) and glucosylated ME metabolites, a metabolic diversion also occurring in wild type plants. MEcDP dephosphorylation to the free tetraol precedes glucosylation, a process which likely takes place in the cytosol. Other MEP pathway intermediates were not affected in hds‐3. Isotopic labeling, dark treatment, and inhibitor studies indicate that a second pool of MEcDP metabolically isolated from the main pathway is the source of a signal which activates salicylic acid induced defense responses before its conversion to hemiterpene glycosides. The hds‐3 mutant also showed enhanced resistance to the phloem‐feeding aphid Brevicoryne brassicae due to its constitutively activated defense response. However, this MEcDP‐mediated defense response is developmentally dependent and is repressed in emerging seedlings. MEcDP and ME exogenously applied to adult leaves mimics many of the gene induction effects seen in the hds‐3 mutant. In conclusion, we have identified a metabolic shunt from the central MEP pathway that diverts MEcDP to hemiterpene glycosides via ME, a process linked to balancing plant responses to biotic stress.     

Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway

Kallistatin, a plasma protein, protects against vascular and organ injury. This study is aimed to investigate the role and mechanism of kallistatin in endothelial senescence. Kallistatin inhibited H₂O₂‐induced senescence in human endothelial cells, as indicated by reduced senescence‐associated‐β‐galactosidase activity, p16^INK4a and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked H₂O₂‐induced superoxide formation, NADPH oxidase levels and VCAM‐1, ICAM‐1, IL‐6 and miR‐34a synthesis. Kallistatin reversed H₂O₂‐mediated inhibition of endothelial nitric oxide synthase (eNOS), SIRT1, catalase and superoxide dismutase (SOD)‐2 expression, and kallistatin alone stimulated the synthesis of these antioxidant enzymes. Moreover, kallistatin's anti‐senescence and anti‐oxidant effects were attributed to SIRT1‐mediated eNOS pathway. Kallistatin, via interaction with tyrosine kinase, up‐regulated Let‐7g, whereas Let‐7g inhibitor abolished kallistatin's effects on miR‐34a and SIRT1/eNOS synthesis, leading to inhibition of senescence, oxidative stress and inflammation. Furthermore, lung endothelial cells isolated from endothelium‐specific kallistatin knockout mice displayed marked reduction in mouse kallistatin levels. Kallistatin deficiency in mouse endothelial cells exacerbated senescence, oxidative stress and inflammation compared to wild‐type mouse endothelial cells, and H₂O₂ treatment further magnified these effects. Kallistatin deficiency caused marked reduction in Let‐7g, SIRT1, eNOS, catalase and SOD‐1 mRNA levels, and elevated miR‐34a synthesis in mouse endothelial cells. These findings indicate that endogenous kallistatin through novel mechanisms protects against endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway.     

State‐of‐the‐art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids

CRISPR/Cas9 technology has revolutionized biology. This prokaryotic defense system against foreign DNA has been repurposed for genome editing in a broad range of cell tissues and organisms. Trypanosomatids are flagellated protozoa belonging to the order Kinetoplastida. Some of its most representative members cause important human diseases affecting millions of people worldwide, such as Chagas disease, sleeping sickness and different forms of leishmaniases. Trypanosomatid infections represent an enormous burden for public health and there are no effective treatments for most of the diseases they cause. Since the emergence of the CRISPR/Cas9 technology, the genetic manipulation of these parasites has notably improved. As a consequence, genome editing is now playing a key role in the functional study of proteins, in the characterization of metabolic pathways, in the validation of alternative targets for antiparasitic interventions, and in the study of parasite biology and pathogenesis. In this work we review the different strategies that have been used to adapt the CRISPR/Cas9 system to Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., as well as the research progress achieved using these approaches. Thereby, we will present the state‐of‐the‐art molecular tools available for genome editing in trypanosomatids to finally point out the future perspectives in the field.     

MicroRNA‐183‐5p is stress‐inducible and protects neurons against cell death in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the death of motor neurons. A fundamental pathogenesis of ALS is the prolonged cell stress in neurons, which is caused by either accumulation of protein aggregates or reactive oxygen species. However, the mechanistic link between stress sensing and cell death is unsettled. Here, we identify that miR‐183‐5p, a neuron‐enriched miRNA, couples stress sensing and cell death programming in ALS. miR‐183‐5p is immediately induced by hydrogen peroxide, tunicamycin or TNF‐α in neurons. The overexpression of miR‐183‐5p increases neuron survival under stress conditions, whereas its knockdown causes neuron death. miR‐183‐5p coordinates apoptosis and necroptosis pathways by directly targeting PDCD4 and RIPK3, and thus protects neurons against cell death under stress conditions. The consistent reduction of miR‐183‐5p in ALS patients and mouse models enhances the notion that miR‐183‐5p is a central regulator of motor neuron survival under stress conditions. Our study supplements current understanding of the mechanistic link between cell stress and death/survival, and provides novel targets for clinical interventions of ALS.