The tiny giant of the sea, Ostreococcus's unique adaptations.

Ostreococcus spp. are unicellular organisms with one of the simplest cellular organizations. The sequencing of the genomes of different Ostreococcus species has reinforced this status since Ostreococcus tauri has one most compact nuclear genomes among eukaryotic organisms. Despite this, it has retained a number of genes, setting it apart from other organisms with similar small genomes. Ostreococcus spp. feature a substantial number of selenocysteine-containing proteins, which, due to their higher catalytic activity compared to their selenium-lacking counterparts, may require a reduced quantity of proteins. Notably, O. tauri encodes several ammonium transporter genes, that may provide it with a competitive edge for acquiring nitrogen (N). This characteristic makes it an intriguing model for studying the efficient use of N in eukaryotes. Under conditions of low N availability, O. tauri utilizes N from abundant proteins or amino acids, such as L-arginine, similar to higher plants. However, the presence of a nitric oxide synthase (L-arg substrate) sheds light on a new metabolic pathway for L-arg in algae. The metabolic adaptations of O. tauri to day and night cycles offer valuable insights into carbon and iron metabolic configuration. O. tauri has evolved novel strategies to optimize iron uptake, lacking the classic components of the iron absorption mechanism. Overall, the cellular and genetic characteristics of Ostreococcus contribute to its evolutionary success, making it an excellent model for studying the physiological and genetic aspects of how green algae have adapted to the marine environment. Furthermore, given its potential for lipid accumulation and its marine habitat, it may represent a promising avenue for third-generation biofuels.

Presence of inulin in sunflower (Helianthus annuus L.) grown under high irradiance.

Fructans are important polysaccharides synthesized from sucrose which are present in about 12-15% of angiosperms. Sunflower (Helianthus annuus L.) is considered a non-fructan bearing plant even though its close relative, Helianthus tuberosus, accumulates the inulin type of the polymer in large amounts. Previous work suggested that putative fructan-synthesizing enzymes may be expressed in sunflower, but only very limited amounts of the trisaccharide isokestose were found in stems of plants storing high levels of sucrose due to capitulum removal. The present work is aimed at investigating whether intact sunflower plants may indeed synthesize fructans in any of its parts when grown in conditions that favor sucrose availability. Plants were grown in the field at a low density, resulting in a high light availability and low competition for resources, in comparison with controls (usual crop planting density). Plants were harvested at anthesis. Thinned treatment led to an increase in carbohydrates level especially in the capitulum. Carbohydrates analysis of this tissue in thinned plants revealed, for the first time in this species, the presence of inulin-type fructans. The amount of each member of the series appeared to decline starting from isokestose, being DP = 15 the longest fructan detected. Results suggest that, in sunflower, fructans could be synthesized only when sucrose availability exceeds a high threshold, which may not be attained under usual growing conditions. Given the relationship between fructans and tolerance to abiotic stresses including drought, the present finding opens a new perspective for breeding and management of this crop.

TOR signaling in the green picoalga Ostreococcus tauri.

Target of rapamycin (TOR) is a master regulator that controls growth and metabolism by integrating external and internal signals. Although there was a great progress in the study of TOR in plants and in the model alga Chlamydomonas, scarce data are available in other green algae. Thus, in this work we studied TOR signaling in Ostreococcus tauri, the smallest free-living eukaryote described to date. This picoalga is particularly important because it has a key site at the base of the green lineage and is part of the marine phytoplankton, contributing to global photosynthesis. We investigated OtTOR complex in silico and experimentally, by using first- and second-generation TOR inhibitors, such as rapamycin and PP242. We analyzed the effect of TOR down-regulation on cell growth and on the accumulation of carbon reserves. The results showed that O. tauri responds to TOR inhibitors more similarly to plants than to Chlamydomonas, being PP242 a valuable tool to study this pathway. Besides, Ottor expression analysis revealed that the kinase is dynamically regulated under nutritional stress. Our data indicate that TOR signaling is conserved in O. tauri and we propose this alga as a good and simple model for studying TOR kinase and its regulation.

The tip of the iceberg: emerging roles of TORC1, and its regulatory functions in plant cells.

Target of Rapamycin (TOR) is an evolutionarily conserved protein kinase that plays a central role in coordinating cell growth with light availability, the diurnal cycle, energy availability, and hormonal pathways. TOR Complex 1 (TORC1) controls cell proliferation, growth, metabolism, and defense in plants. Sugar availability is the main signal for activation of TOR in plants, as it also is in mammals and yeast. Specific regulators of the TOR kinase pathway in plants are inorganic compounds in the form of major nutrients in the soils, and light inputs via their impact on autotrophic metabolism. The lack of TOR is embryo-lethal in plants, whilst dysregulation of TOR signaling causes major alterations in growth and development. TOR exerts control as a regulator of protein translation via the action of proteins such as S6K, RPS6, and TAP46. Phytohormones are central players in the downstream systemic physiological TOR effects. TOR has recently been attributed to have roles in the control of DNA methylation, in the abundance of mRNA splicing variants, and in the variety of regulatory lncRNAs and miRNAs. In this review, we summarize recent discoveries in the plant TOR signaling pathway in the context of our current knowledge of mammalian and yeast cells, and highlight the most important gaps in our understanding of plants that need to be addressed in the future.

Target of rapamycin signaling is tightly and differently regulated in the plant response under distinct abiotic stresses.

MAIN CONCLUSION: TOR signaling is finely regulated under diverse abiotic stresses and may be required for the plant response with a different time-course depending on the duration and nature of the stress. Target of rapamycin (TOR) signaling is a central regulator of growth and development in eukaryotic organisms. However, its regulation under stress conditions has not yet been elucidated. In Arabidopsis, we show that TOR transcripts and activity in planta are finely regulated within hours after the onset of salt, osmotic, cold and oxidative stress. The expression of genes encoding the partner proteins of the TOR complex, RAPTOR3G and LST8-1, is also regulated. Besides, the data indicate that TOR activity increases at some time during the adverse condition. Interestingly, in oxidative stress, the major TOR activity increment occurred transiently at the early phase of treatment, while in salt, osmotic and cold stress, it was around 1 day after the unfavorable condition was applied. Those results suggest that the TOR signaling has an important role in the plant response to an exposure to stress. Moreover, basal ROS (H2O2) levels and their modification under abiotic stresses were altered in TOR complex mutants. On the other hand, the root phenotypic analysis of the effects caused by the diverse abiotic stresses on TOR complex mutants revealed that they were differently affected, being in some cases less sensitive, than wild-type plants to long-term unfavorable conditions. Therefore, in this work, we demonstrated that TOR signaling is tightly regulated under abiotic stresses, at transcript and activity level, with different and specific time-course patterns according to the type of abiotic stress in Arabidopsis. Taking our results together, we propose that TOR signaling should be necessary during the plant stress response.

TOR and SnRK1 signaling pathways in plant response to abiotic stresses: Do they always act according to the "yin-yang" model?

Plants are sessile photo-autotrophic organisms continuously exposed to a variety of environmental stresses. Monitoring the sugar level and energy status is essential, since this knowledge allows the integration of external and internal cues required for plant physiological and developmental plasticity. Most abiotic stresses induce severe metabolic alterations and entail a great energy cost, restricting plant growth and producing important crop losses. Therefore, balancing energy requirements with supplies is a major challenge for plants under unfavorable conditions. The conserved kinases target of rapamycin (TOR) and sucrose-non-fermenting-related protein kinase-1 (SnRK1) play central roles during plant growth and development, and in response to environmental stresses; these kinases affect cellular processes and metabolic reprogramming, which has physiological and phenotypic consequences. The "yin-yang" model postulates that TOR and SnRK1 act in opposite ways in the regulation of metabolic-driven processes. In this review, we describe and discuss the current knowledge about the complex and intricate regulation of TOR and SnRK1 under abiotic stresses. We especially focus on the physiological perspective that, under certain circumstances during the plant stress response, the TOR and SnRK1 kinases could be modulated differently from what is postulated by the "yin-yang" concept.

TOR signaling downregulation increases resistance to the cereal killer Fusarium graminearum.

TOR is the master regulator of growth and development that senses energy availability. Biotic stress perturbs metabolic and energy homeostasis, making TOR a good candidate to participate in the plant response. Fusarium graminearum (Fusarium) produces important losses in many crops all over the world. To date, the role of TOR in Fusarium infection has remained unexplored. Here, we show that the resistance to the pathogen increases in different Arabidopsis mutants impaired in TOR complex or in wild-type plants treated with a TOR inhibitor. We conclude that TOR signaling is involved in plant defense against Fusarium.

The riddle of mitochondrial alkaline/neutral invertases: A novel Arabidopsis isoform mainly present in reproductive tissues and involved in root ROS production.

Alkaline/neutral invertases (A/N-Inv), glucosidases that irreversibly hydrolyze sucrose into glucose and fructose, play significant roles in plant growth, development, and stress adaptation. They occur as multiple isoforms located in the cytosol or organelles. In Arabidopsis thaliana, two mitochondrial A/N-Inv genes (A/N-InvA and A/N-InvC) have already been investigated. In this study, we functionally characterized A/N-InvH, a third Arabidopsis gene coding for a mitochondrial-targeted protein. The phenotypic analysis of knockout mutant plants (invh) showed a severely reduced shoot growth, while root development was not affected. The emergence of the first floral bud and the opening of the first flower were the most affected stages, presenting a significant delay. A/N-InvH transcription is markedly active in reproductive tissues. It is also expressed in the elongation and apical meristem root zones. Our results show that A/N-InvH expression is not evident in photosynthetic tissues, despite being of relevance in developmental processes and mitochondrial functional status. NaCl and mannitol treatments increased A/N-InvH expression twofold in the columella root cap. Moreover, the absence of A/N-InvH prevented ROS formation, not only in invh roots of salt- and ABA-treated seedlings but also in invh control roots. We hypothesize that this isoform may take part in the ROS/sugar (sucrose or its hydrolysis products) signaling pathway network, involved in reproductive tissue development, cell elongation, and abiotic stress responses.

Ancient signal for nitrogen status sensing in the green lineage: Functional evidence of CDPK repertoire in Ostreococcus tauri.

Calcium-dependent protein kinases (CDPKs) regulate plant development and many stress signalling pathways through the complex cytosolic [Ca2+] signalling. The genome of Ostreococcus tauri (Ot), a model prasinophyte organism that is on the base of the green lineage, harbours three sequences homologous to those encoding plant CDPKs with the three characteristic conserved domains (protein kinase, autoregulatory/autoinhibitory, and regulatory domain). Phylogenetic and structural analyses revealed that putative OtCDPK proteins are closely related to CDPKs from other Chlorophytes. We functionally characterised the first marine picophytoeukaryote CDPK gene (OtCDPK1) and showed that the expression of the three OtCDPK genes is up-regulated by nitrogen depletion. We conclude that CDPK signalling pathway might have originated early in the green lineage and may play a key role in prasinophytes by sensing macronutrient changes in the marine environment.

Sublethal effects of acaricides and Nosema ceranae infection on immune related gene expression in honeybees.

Nosema ceranae is an obligate intracellular parasite and the etiologic agent of Nosemosis that affects honeybees. Beside the stress caused by this pathogen, honeybee colonies are exposed to pesticides under beekeeper intervention, such as acaricides to control Varroa mites. These compounds can accumulate at high concentrations in apicultural matrices. In this work, the effects of parasitosis/acaricide on genes involved in honeybee immunity and survival were evaluated. Nurse bees were infected with N. ceranae and/or were chronically treated with sublethal doses of coumaphos or tau-fluvalinate, the two most abundant pesticides recorded in productive hives. Our results demonstrate the following: (1) honeybee survival was not affected by any of the treatments; (2) parasite development was not altered by acaricide treatments; (3) coumaphos exposure decreased lysozyme expression; (4) N. ceranae reduced levels of vitellogenin transcripts independently of the presence of acaricides. However, combined effects among stressors on imagoes were not recorded. Sublethal doses of acaricides and their interaction with other ubiquitous parasites in colonies, extending the experimental time, are of particular interest in further research work.

Sunflower: a potential fructan-bearing crop?

Grain filling in sunflower (Helianthus annuus L.) mainly depends on actual photosynthesis, being the contribution of stored reserves in stems (sucrose, hexoses, and starch) rather low. Drought periods during grain filling often reduce yield. Increasing the capacity of stem to store reserves could help to increase grain filling and yield stability in dry years. Fructans improve water uptake in soils at low water potential, and allow the storage of large amount of assimilates per unit tissue volume that can be readily remobilized to grains. Sunflower is a close relative to Jerusalem artichoke (H. tuberosus L.), which accumulates large amounts of fructan (inulin) in tubers and true stems. The reason why sunflower does not accumulate fructans is obscure. Through a bioinformatics analysis of a sunflower transcriptome database, we found sequences that are homologous to dicotyledon and monocotyledon fructan synthesis genes. A HPLC analysis of stem sugar composition revealed the presence of low amounts of 1-kestose, while a drastic enhancement of endogenous sucrose levels by capitulum removal did not promote 1-kestose accumulation. This suggests that the regulation of fructan synthesis in this species may differ from the currently best known model, mainly derived from research on Poaceae, where sucrose acts as both a signaling molecule and substrate, in the induction of fructan synthesis. Thus, sunflower might potentially constitute a fructan-bearing species, which could result in an improvement of its performance as a grain crop. However, a large effort is needed to elucidate how this up to now unsuspected potential could be effectively expressed.

The levels of RAC3 expression are up regulated by TNF in the inflammatory response.

RAC3 is a coactivator of glucocorticoid receptor and nuclear factor-κB (NF-κB) that is usually over-expressed in tumors and which also has important functions in the immune system. We investigated the role of the inflammatory response in the control of RAC3 expression levels in vivo and in vitro. We found that inflammation regulates RAC3 levels. In mice, sub-lethal doses of lipopolysaccharide induce the increase of RAC3 in spleen and the administration of the synthetic anti-inflammatory glucocorticoid dexamethasone has a similar effect. However, the simultaneous treatment with both stimuli is mutually antagonistic. In vitro stimulation of the HEK293 cell line with tumor necrosis factor (TNF), one of the cytokines induced by lipopolysaccharide, also increases the levels of RAC3 mRNA and protein, which correlates with an enhanced transcription dependent on the RAC3 gene promoter. We found that binding of the transcription factor NF-κB to the RAC3 gene promoter could be responsible for these effects. Our results suggest that increase of RAC3 during the inflammatory response could be a molecular mechanism involved in the control of sensitivity to both pro- and anti-inflammatory stimuli in order to maintain the normal healthy course of the immune response.

First evidence of sucrose biosynthesis by single cyanobacterial bimodular proteins.

The net synthesis of sucrose (Suc) is catalysed by the sequential action of Suc-phosphate synthase (SPS) and Suc-phosphate phosphatase (SPP). SPS and SPP from Anabaena sp. PCC 7120 (7120-SPS and 7120-SPP) define minimal catalytic units. Bidomainal SPSs, where both units are fused, occur in plants and cyanobacteria, but they display only SPS activity. Using recombinant proteins that have fused 7120-SPS and 7120-SPP, we demonstrated that they are bifunctional chimeras and that the arrangement 7120-SPS/SPP is the most efficient to catalyse the sequential reactions to yield Suc. Moreover, we present the first evidence of a bidomainal SPS present in the cyanobacterium Synechococcus elongatus PCC 7942 with both, SPS and SPP activity.

Sucrose signaling in plants: a world yet to be explored.

The role of sucrose as a signaling molecule in plants was originally proposed several decades ago. However, recognition of sucrose as a true signal has been largely debated and only recently this role has been fully accepted. The best-studied cases of sucrose signaling involve metabolic processes, such as the induction of fructan or anthocyanin synthesis, but a large volume of scattered information suggests that sucrose signals may control a vast array of developmental processes along the whole life cycle of the plant. Also, wide gaps exist in our current understanding of the intracellular steps that mediate sucrose action. Sucrose concentration in plant tissues tends to be directly related to light intensity, and inversely related to temperature, and accordingly, exogenous sucrose supply often mimics the effect of high light and cold. However, many exceptions to this rule seem to occur due to interactions with other signaling pathways. In conclusion, the sucrose role as a signal molecule in plants is starting to be unveiled and much research is still needed to have a complete map of its significance in plant function.

Nuclear receptor coactivator RAC3 inhibits autophagy.

RAC3 is an oncogene naturally overexpressed in several tumors. Besides its role as coactivator, it can exert several protumoral cytoplasmic actions. Autophagy was found to act either as a tumor suppressor during the early stages of tumor development, or as a protector of the tumor cell in later stages under hypoxic conditions. We found that RAC3 overexpression inhibits autophagy when induced by starvation or rapamycin and involves RAC3 nuclear translocation-dependent and -independent mechanisms. Moreover, hypoxia inhibits the RAC3 gene expression leading to the autophagy process, allowing tumor cells to survive until angiogenesis occurs. The interplay between RAC3, hypoxia, and autophagy could be an important mechanism for tumor progression and a good target for a future anticancer therapy.

Cyclin D1 is a NF-κB corepressor.

NF-κB regulates the expression of Cyclin D1 (CD1), while RAC3 is an NF-κB coactivator that enhances its transcriptional activity. In this work, we investigated the regulatory role of CD1 on NF-κB activity. We found that CD1 inhibits NF-κB transcriptional activity through a corepressor function that can be reverted by over-expressing RAC3. In both, tumoral and non-tumoral cells, the expression pattern of RAC3 and CD1 is regulated by the cell cycle, showing a gap between the maximal expression levels of each protein. The individual increase, by transfection, of either CD1 or RAC3 enhances cell proliferation. However the simultaneous and constitutive over-expression of both proteins has an inhibitory effect. Our results suggest that the relative amounts of CD1 and RAC3, and the timing of expression of these oncogenes could tilt the balance of tumor cell proliferation in response to external signals.

[Rapamycin effect on senescence and autophagy processes in human cell lines]. / Efectos de la rapamicina en los procesos de senescencia y autofagia en líneas celulares humanas.

Autophagy and senescence are both processes that firstly avoid tumor development through the inhibition of proliferation of damaged cells. However, autophagy does not imply cell death, because it is also a mechanism of cell survival under stress conditions. Concerning senescence, although these cells do not proliferate, they produce growth factors that contribute to the proliferative response of other cells. Rapamycin is an immunosupressor used in transplanted patients that inhibits the mTOR transduction signal pathway. This pathway is involved in the control of the energetic and nutritional state of the cell allowing protein synthesis and inhibiting autophagy when it is active. In this paper, the action of rapamycin over these processes was investigated and we found that a low concentration of this drug induces the senescence of a normal cell line, while a higher concentration induces autophagy of a transformed cell line. We have also determined that the oncogen RAC3 inhibits autophagy and that its expression is diminished by rapamycin. Therefore, our results contribute to a better understanding of the molecular mechanisms by which this drug is effective, given the relevance of rapamycin for potential tumor therapy.

Specific role of the cyanobacterial PipX factor in the heterocysts of Anabaena sp. strain PCC 7120.

The PipX factor is a regulatory protein that seems to occur only in cyanobacteria. In the filamentous, heterocyst-forming Anabaena sp. strain PCC 7120, open reading frame (ORF) asr0485, identified as the pipX gene, is expressed mainly under conditions of combined-nitrogen deprivation dependent on the global N regulator NtcA and the heterocyst-specific regulator HetR. Primer extension and 5' rapid amplification of cDNA ends (RACE) analyses detected three transcription start points corresponding to a canonical NtcA-activated promoter (to which direct binding of NtcA was observed), an NtcA- and HetR-dependent promoter, and a consensus-type promoter, the last with putative -35 and -10 determinants. Activation of pipX took place in cells differentiating into heterocysts at intermediate to late stages of the process. Accordingly, disruption of pipX led to impaired diazotrophic growth, reduced nitrogenase activity, and impaired activation of the nitrogenase structural genes. The nitrogenase activity of the mutant was low under oxic conditions, likely resulting from inefficient protection against oxygen. In line with this, the activation of the coxB2A2C2 and coxB3A3C3 operons, encoding heterocyst-specific terminal respiratory oxidases responsible for internal oxygen removal, was deficient in the pipX mutant. Therefore, the Anabaena PipX factor shows a spatiotemporal specificity contributing to normal heterocyst function, including full activation of the nitrogenase structural genes and genes of the nitrogenase-protective features of the heterocyst.

NifB and NifEN protein levels are regulated by ClpX2 under nitrogen fixation conditions in Azotobacter vinelandii.

The major part of biological nitrogen fixation is catalysed by the molybdenum nitrogenase that carries at its active site the iron and molybdenum cofactor (FeMo-co). The nitrogen fixation (nif) genes required for the biosynthesis of FeMo-co are derepressed in the absence of a source of fixed nitrogen. The nifB gene product is remarkable because it assembles NifB-co, a complex cluster proposed to comprise a [6Fe-9S-X] cluster, from simpler [Fe-S] clusters common to other metabolic pathways. NifB-co is a common intermediate of the biosyntheses of the cofactors present in the molybdenum, vanadium and iron nitrogenases. In this work, the expression of the Azotobacter vinelandii nifB gene was uncoupled from its natural nif regulation to show that NifB protein levels are lower in cells growing diazotrophically than in cells growing at the expense of ammonium. A. vinelandii carries a duplicated copy of the ATPase component of the ubiquitous ClpXP protease (ClpX2), which is induced under nitrogen fixing conditions. Inactivation of clpX2 resulted in the accumulation of NifB and NifEN and a defect in diazotrophic growth, especially when iron was in short supply. Mutations in nifE, nifN and nifX or in nifA also affected NifB accumulation, suggesting that NifB susceptibility to degradation might vary during its catalytic cycle.

Sugar signaling of fructan metabolism: New insights on protein phosphatases in sucrose-fed wheat leaves.

Protein phosphatase type 2A (PP2A) activity is required for the sucrose induction of fructan metabolism in wheat leaves, as shown in experiments with the addition of the specific inhibitor okadaic acid (OA) together with sucrose. However, a decrease in total PP2A activity has been found along sucrose treatment. Here we analyze the effect of sucrose feeding to wheat leaves on PP2A activity profiles after Deae-Sephacel and Superose separation, in comparison with those of control leaves. The results show no evidence of changes in PP2A activity profiles as a consequence of sucrose feeding. In all, our data suggest that constitutive levels of PP2A activity may be sufficient for the sucrose-mediated induction of fructan metabolism and that general decrease of PP2A activity produced by long-term treatment with sucrose may be due to a negative feedback regulation.