Unstressed Conditions Research Articles

Reciprocal interplay between OTULIN–LUBAC determines genotoxic and inflammatory NF-κB signal responses

Targeting nuclear factor-kappa B (NF-κB) represents a highly viable strategy against chemoresistance in cancers as well as cell death. Ubiquitination, including linear ubiquitination mediated by the linear ubiquitin chain assembly complex (LUBAC), is emerging as a crucial mechanism of overactivated NF-κB signaling. Ovarian tumor family deubiquitinase OTULIN is the only linear linkage-specific deubiquitinase; however, the molecular mechanisms of how it counteracts LUBAC-mediated NF-κB activation have been largely unknown. Here, we identify Lys64/66 of OTULIN for linear ubiquitination facilitated in a LUBAC-dependent manner as a necessary event required for OTULIN-LUBAC interaction under unstressed conditions, which becomes deubiquitinated by OTULIN itself in response to genotoxic stress. Furthermore, this self-deubiquitination of OTULIN occurs intermolecularly, mediated by OTULIN dimerization, resulting in the subsequent dissociation of OTULIN from the LUBAC complex and NF-κB overactivation. Oxidative stress induces OTULIN dimerization via cysteine-mediated covalent disulfide bonds. Our study reveals that the status of the physical interaction between OTULIN and LUBAC is a crucial determining factor for the genotoxic NF-κB signaling, as measured by cell survival and proliferation, while OTULIN loss of function resulting from its dimerization and deubiquitination leads to a dissociation of OTULIN from the LUBAC complex. Of note, similar molecular mechanisms apply to the inflammatory NF-κB signaling in response to tumor necrosis factor α. Hence, a fuller understanding of the detailed molecular mechanisms underlying the disruption of the OTULIN-LUBAC interaction will be instrumental for developing future therapeutic strategies against cancer chemoresistance and necroptotic processes pertinent to numerous human diseases.

Bio-Stimulating Effect of Natural Polysaccharides from Lobularia maritima on Durum Wheat Seedlings: Improved Plant Growth, Salt Stress Tolerance by Modulating Biochemical Responses and Ion Homeostasis.

Bioactivities of polysaccharides derived from halophyte plants have gained attention in recent years. The use of biostimulants in agriculture is an innovative method of dealing with environmental stressors affecting plant growth and development. Here, we investigated the use of natural polysaccharides derived from the halophyte plant Lobularia maritima (PSLm) as a biostimulant in durum wheat seedlings under salt stress. Treatment with polysaccharide extract (0.5, 1, and 2 mg/mL PSLm) stimulated in vitro wheat growth, including germination, shoot length, root length, and fresh weight. PSLm at 2 mg/mL provided tolerance to plants against NaCl stress with improved membrane stability and low electrolyte leakage, increased antioxidant activities (catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD)), enhanced leaf chlorophyll fluorescence, proline, and total sugar contents, decreased lipid peroxidation (MDA), and reactive oxygen species (H2O2) levels, and coordinated the efflux and compartmentation of intracellular ions. The expression profile analyses of ten stress-related genes (NHX1, HKT1.4, SOS1, SOD, CAT, GA20-ox1, GA3-ox1, NRT1.1, NRT2.1, and GS) using RT-qPCR revealed the induction of several key genes in durum wheat growing in media supplemented with PSLm extract, even in unstressed conditions that could be related to the observed tolerance. This study revealed that PSLm extract contributes to salt tolerance in durum wheat seedlings, thereby enhancing their reactive oxygen species scavenging ability, and provided evidence for exploring PSLm as a plant biostimulant for sustainable and organic agriculture.

TORC1 regulates actin dynamics to control proteasome homeostasis

The conserved protein kinase complex TORC1 is a central regulator of protein homeostasis. TORC1 is active under nutrient replete, unstressed conditions and promotes bulk protein synthesis, cell growth, and proliferation. Many stresses inactivate TORC1, including nutrient starvation. When TORC1 is inactivated, bulk protein synthesis is inhibited, and stress-responsive protein synthesis activated. Degradation of damaged or unwanted proteins is increased through autophagy activation and enhanced proteasome assembly. The 26S proteasome is composed of the 20S core particle (CP), capped with one or two 19S regulatory particles (RP). Following TORC1 inhibition, the Mpk1 kinase is activated. Mpk1 facilitates increased translation of proteasome regulatory particle assembly chaperones (RPACs), leading to increased proteasome assembly. The mechanisms underlying the RPAC translation increase remain unclear, however. Here, we show that actin depolarisation upon TORC1 inhibition causes RPAC mRNAs to associate more with actin patch structures. Increasing this interaction enhances translation further. We identified Ede1 as a protein which binds translating RPAC mRNA and show that Ede1 is required to increase RPAC mRNA association with actin patches, RPAC translation and proteasome assembly upon stress.

An invasive weed-associated bacteria confers enhanced heat stress tolerance in wheat

Global temperatures are expected to increase due to climate change, and heat stress is one of the major limiting factors affecting future agriculture. To identify plant-associated microorganisms which can promote heat stress tolerance in wheat, we have screened several bacteria isolated from etiolated seedlings of the invasive noxious weed Parthenium hysterophorus. One isolate designated as Ph-04 was found to confer enhanced heat stress tolerance in wheat. The 16S rRNA gene sequence analysis showed that Ph-04 isolate shared highest sequence identity with Bacillus paramycoides species of the Bacillus cereus group. Ph-04 treated wheat seeds exhibited enhanced germination, longer coleoptile, radicle and seminal root length than control seedlings when grown in the dark at optimum and high temperatures. Similarly, under autotrophic conditions, Ph-04 treated plants also exhibited enhanced heat stress tolerance with a significant increase in membrane integrity and significantly reduced levels of H2O2 under heat stress compared to control plants. This observed heat stress tolerance is associated with constitutively higher basal levels of proline, and activity of antioxidant enzymes, catalase (CAT) and ascorbate peroxidase (APX) in Ph-04 treated plants grown under unstressed conditions with further increase under heat stress conditions compared to controls. Plant recovery after heat stress also showed that the Ph-04 treated plants exhibited significantly less damage in terms of survival percentage and exhibited better morphological and physiological characteristics compared to control plants. The study proves that invasive weeds can harbour potentially beneficial microorganisms, which can be transferred to non-native crop (host) plants to improve climate resilience characteristics.

BLM Sumoylation Is Required for Replication Stability and Normal Fork Velocity During DNA Replication.

BLM is sumoylated in response to replication stress. We have studied the role of BLM sumoylation in physiologically normal and replication-stressed conditions by expressing in BLM-deficient cells a BLM with SUMO acceptor-site mutations, which we refer to as SUMO-mutant BLM cells. SUMO-mutant BLM cells exhibited multiple defects in both stressed and unstressed DNA replication conditions, including, in hydroxyurea-treated cells, reduced fork restart and increased fork collapse and, in untreated cells, slower fork velocity and increased fork instability as assayed by track-length asymmetry. We further showed by fluorescence recovery after photobleaching that SUMO-mutant BLM protein was less dynamic than normal BLM and comprised a higher immobile fraction at collapsed replication forks. BLM sumoylation has previously been linked to the recruitment of RAD51 to stressed forks in hydroxyurea-treated cells. An important unresolved question is whether the failure to efficiently recruit RAD51 is the explanation for replication stress in untreated SUMO-mutant BLM cells.

Effects of Visual Environment on Students Adjustment to Stress

The impact of stress on visual landscape perception was assessed in a photo-based survey. The survey was first performed when the student participants were expected to be stressed just prior to an important examination. The same students were asked to respond to the same questionnaire a month after the examination when they were expected to have a lower level of stress. Then respondents answered some daily activities, personal study habits, and feelings before an exam. They also provided ratings of how much a selection of environmental factors generally influence their ability to study and their academic success. In the main perceptual survey reactions to a selection of 22 landscape scenes photos were reported by ratings (1–5) of the extent to which each of six emotions was associated with each scene. Differences in emotions ratings for the represented landscapes during high-stress and low-stress periods were analyzed by multiple comparison and Pearson correlational methods using the SPSS 17.00 package. Stress tests confirmed higher stress in the first versus second survey and perceptual ratings showed significant statistical differences in emotion ratings between landscape scenes, as well as both main effects and interactions between high stress and low stress conditions. Scene ratings for each emotion were strongly positively correlated between high stress and lower stress conditions. At the same time, respondents generally gave slightly higher ratings for positive emotions -excited, relaxing, happiness-when in the high stress condition and moderately higher ratings for negative emotions -stressed, irritating, scary-, compared to their ratings when tested later under lower stress conditions. This study indicated that stress conditions affect perception, and stressed conditions gave higher emotionality overall than the unstressed condition. In general, in both stressed and unstressed conditions, the students gave the highest scores (>3.4) to convenience and the lowest score (<2) to scary. The main limitations of this study are the large number of environmental factors that influence people's perception. The strongest determiner of emotion ratings was the landscape scenes themselves. Inspection of outliers in the scatter plots and multiple comparisons articulating higher order interactions with stress conditions revealed clear differences in the patterns of emotions ratings, especially for scenes representing water surfaces, open green spaces, and seasonal plant scenes.

Caloric restriction stress causes epigenetic effects evidenced by longevity in Caenorhabditis elegans

Caenorhabditis elegans are members of the nematode family and have been used as model organisms of epigenetic studies for many years. C. elegans have a relatively short life cycle; the worms grow from an egg to an adult within the span of about 3-4 days and an overall lifespan of about 2-3 weeks. The worms are very easy to maintain, requiring for survival only nematode growth medium (NGM) agar plates that are kept at a temperature of about 20-25 °C and contain E. coli for food. For this study, which replicates aspects of a study reported by Kishimoto et al. (2017), C. elegans was used to explore the epigenetic effects of stress on longevity. The animals in this study that were subjected to the environmental stressor starvation showed an increase in overall lifespan based on the statistical analysis of a Kaplan-Meier Survivorship test. This increased survival rate was also found to be passed to at least three subsequent generations who were grown under unstressed conditions. These results showcase the hormetic epigenetic effects of starvation in C. elegans.

The role of cycloastragenol at the intersection of NRF2/ARE, telomerase, and proteasome activity

Aging is well-characterized by the gradual decline of cellular functionality. As redox balance, proteostasis, and telomerase systems have been found to be associated with aging and age-related diseases, targeting these systems with small compounds has been considered a promising therapeutic approach. Cycloastragenol (CA), a small molecule telomerase activator obtained from Astragalus species, has been reported to positively affect several age-related pathophysiologies, but the mechanisms underlying CA activity have yet to be reported. Here, we presented that CA increased NRF2 nuclear localization and activity leading to upregulation of cytoprotective enzymes and attenuation of oxidative stress-induced ROS levels. Furthermore, CA-mediated induction of telomerase activity was found to be regulated by NRF2. CA not only increased the expression of hTERT but also its nuclear localization via upregulating the Hsp90-chaperon complex. In addition to modulating nuclear hTERT levels at unstressed conditions, CA alleviated oxidative stress-induced mitochondrial hTERT levels while increasing nuclear hTERT levels. Concomitantly, H2O2-induced mitochondrial ROS level was found to be significantly decreased by CA administration. Our data also revealed that CA strongly enhanced proteasome activity and assembly. More importantly, the proteasome activator effect of CA is dependent on the induction of telomerase activity, which is mediated by NRF2 system. In conclusion, our results not only revealed the cross-talk among NRF2, telomerase, and proteasome systems but also that CA functions at the intersection of these three major aging-related cellular pathways.

Plant Host Traits Mediated by Foliar Fungal Symbionts and Secondary Metabolites.

Fungal symbionts living inside plant leaves ("endophytes") can vary from beneficial to parasitic, but the mechanisms by which the fungi affect the plant host phenotype remain poorly understood. Chemical interactions are likely the proximal mechanism of interaction between foliar endophytes and the plant, as individual fungal strains are often exploited for their diverse secondary metabolite production. Here, we go beyond single strains to examine commonalities in how 16 fungal endophytes shift plant phenotypic traits such as growth and physiology, and how those relate to plant metabolomics profiles. We inoculated individual fungi on switchgrass, Panicum virgatum L. This created a limited range of plant growth and physiology (2-370% of fungus-free controls on average), but effects of most fungi overlapped, indicating functional similarities in unstressed conditions. Overall plant metabolomics profiles included almost 2000 metabolites, which were broadly correlated with plant traits across all the fungal treatments. Terpenoid-rich samples were associated with larger, more physiologically active plants and phenolic-rich samples were associated with smaller, less active plants. Only 47 metabolites were enriched in plants inoculated with fungi relative to fungus-free controls, and of these, Lasso regression identified 12 metabolites that explained from 14 to 43% of plant trait variation. Fungal long-chain fatty acids and sterol precursors were positively associated with plant photosynthesis, conductance, and shoot biomass, but negatively associated with survival. The phytohormone gibberellin, in contrast, was negatively associated with plant physiology and biomass. These results can inform ongoing efforts to develop metabolites as crop management tools, either by direct application or via breeding, by identifying how associations with more beneficial components of the microbiome may be affected.

APEX2-Mediated Proximity Labeling Resolves the DDIT4-Interacting Proteome.

DNA damage-inducible transcript 4 (DDIT4) is a ubiquitous protein whose expression is transiently increased in response to various stressors. Chronic expression has been linked to various pathologies, including neurodegeneration, inflammation, and cancer. DDIT4 is best recognized for repressing mTORC1, an essential protein complex activated by nutrients and hormones. Accordingly, DDIT4 regulates metabolism, oxidative stress, hypoxic survival, and apoptosis. Despite these well-defined biological functions, little is known about its interacting partners and their unique molecular functions. Here, fusing an enhanced ascorbate peroxidase 2 (APEX2) biotin-labeling enzyme to DDIT4 combined with mass spectrometry, the proteins in the immediate vicinity of DDIT4 in either unstressed or acute stress conditions were identified in situ. The context-dependent interacting proteomes were quantitatively but not functionally distinct. DDIT4 had twice the number of interaction partners during acute stress compared to unstressed conditions, and while the two protein lists had minimal overlap in terms of identity, the proteins’ molecular function and classification were essentially identical. Moonlighting keratins and ribosomal proteins dominated the proteomes in both unstressed and stressed conditions, with many of their members having established non-canonical and indispensable roles during stress. Multiple keratins regulate mTORC1 signaling via the recruitment of 14-3-3 proteins, whereas ribosomal proteins control translation, cell cycle progression, DNA repair, and death by sequestering critical proteins. In summary, two potentially distinct mechanisms of DDIT4 molecular function have been identified, paving the way for additional research to confirm and consolidate these findings.

Med13 Plays a Dual Role in Promoting P‐Body Assembly

In response to stress, cells rapidly adjust gene expression, translation, and protein levels to mitigate damage and initiate repair pathways. The Cdk8 Kinase Module (CKM) is a highly conserved, detachable unit of the Mediator complex that plays a vital role in regulating transcription and communicating stress signals from the nucleus to other organelles. Previously, our laboratory has shown that the scaffold protein within the CKM, Med13, is exported from the nucleus and degraded via a selective autophagy pathway following nitrogen starvation. In periods of starvation, cells attenuate translation and biosynthetic pathways to reserve nutrient pools. To rapidly adapt to starvation, cytosolic ribonucleoprotein (RNP) granules such as processing bodies (P‐bodies) tightly regulate mRNA accessibility and translation. Here we show that Med13 plays a dual role in promoting P‐body assembly. In physiological conditions, Med13 functions as a positive transcriptional regulator of P‐body formation as the deletion of MED13 significantly decreases the quantity of P‐bodies. In addition, during stress Med13 co‐localizes with the P‐body marker, Edc3, suggesting that Med13 may also play a scaffolding role within these membrane‐less organelles. Taken together, these data demonstrate the duality of Med13 as a transcriptional regulator of P‐bodies in unstressed conditions and a scaffolding component during stress. The nutrient sensing kinase, Ksp1, plays an important role in mediating P‐body localization of Med13 and other mRNA binding proteins following nitrogen starvation. These data demonstrate a novel cytosolic role for Med13 and expand the known proteome of P‐bodies.

Brief Developmental Exposure to Fluoxetine Causes Life-Long Alteration of the Brain Transcriptome in Zebrafish.

Fluoxetine (FLX) and other selective serotonin reuptake inhibitors are widely used to treat depressive disorders during pregnancy. Early-life exposure to FLX is known to disrupt the normal function of the stress axis in humans, rodents, and teleosts. We used a zebrafish line with a cortisol-inducible fluorescent transgene to study the effects of developmental daily exposure to FLX (54 µg/L) on the transcriptomic profile of brain tissues in exposed larvae and later as 6-month-old adults. High throughput RNA sequencing was conducted on brain tissues in unstressed and stressed conditions. Long-lasting effects of FLX were observed in telencephalon (Tel) and hypothalamus (Hyp) of adult zebrafish with 1927 and 5055 genes significantly (≥1.2 fold-change, false-discovery p-value < 0.05) dysregulated in unstressed condition, respectively. Similar findings were observed in Hyp with 1245 and 723 genes being significantly dysregulated in stressed adults, respectively. Differentially expressed genes converted to Homo sapiens orthologues were used for Ingenuity Pathway Analysis. The results showed alteration of pathways involved in neuroendocrine signaling, cholesterol metabolism and synaptogenesis. Enriched networks included lipid metabolism, molecular transport, and nervous system development. Analysis of putative upstream transcription regulators showed potential dysregulation of clocka and nr3c1 which control circadian rhythm, stress response, cholesterol metabolism and histone modifications. Several genes involved in epigenetic regulation were also affected by FLX, including dnmt3a, adarb1, adarb2, hdac4, hdac5, hdac8, and atf2. We report life-long disruptive effects of FLX on pathways associated with neuroendocrine signaling, stress response and the circadian rhythm, and all of which are implicated in the development of depressive disorders in humans. Our results raise concern for the persistent endocrine-disrupting potential of brief antidepressant exposure during embryonic development.

On the Estimation of Potential Evaporation Under Wet and Dry Conditions

AbstractPotential evaporation (EP) is an important concept that has been extensively used in hydrology, climate, agriculture and many other relevant fields. However, EP estimates using conventional approaches generally do not conform with the underlying idea of EP, since meteorological forcing variables observed under real conditions are not necessarily equivalent to those over a hypothetical surface with an unlimited water supply. Here, we estimate EP using a recently developed ocean surface evaporation model (i.e., the maximum evaporation model) that explicitly acknowledges the inter‐dependence between evaporation, surface temperature (Ts) and radiation such that is able to recover radiation and Ts to a hypothetical wet surface. We first test the maximum evaporation model over land by validating its evaporation estimates with evaporation observations under unstressed conditions at 86 flux sites and found an overall good performance. We then apply the maximum evaporation model to the entire terrestrial surfaces under both wet and dry conditions to estimate EP. The mean annual (1979–2019) global land EP from the maximum evaporation model (EP_max) is 1,272 mm yr−1, which is 11.2% higher than that estimated using the widely adopted Priestley‐Taylor model (EP_PT). The difference between EP_max and EP_PT is negligible in humid regions or under wet conditions but becomes increasingly larger as the surface moisture availability decreases. This difference is primarily caused by increased net radiation (Rn) when restoring the dry surfaces to hypothetical wet surfaces, despite a lower Ts obtained under hypothetical wet conditions in the maximum evaporation model.

Enhancing Irrigation Salinity Stress Tolerance and Increasing Yield in Tomato Using a Precision Engineered Protein Hydrolysate and Ascophyllum nodosum-Derived Biostimulant

Most vegetable crops are salt sensitive, growing inadequately in salinised soils due to the accumulation of toxic ions from prolonged irrigation regimes. Plant biostimulants are a potential tool that can be used to counteract salinity stress and increase crop yield. The aim of this study was to investigate the ability of the proprietary protein hydrolysate and Ascophyllum nodosum-derived biostimulant PSI-475 to activate salinity stress tolerance responses in plants. After characterising PSI-475 composition, initial biostimulant activity screening was performed using Arabidopsis thaliana. PSI-475 stimulated primary root growth (+5.5–20.0%) and photosynthetic pigments content (18.8–63.0%) under unstressed and salinity stressed conditions. Subsequently, PSI-475 was assessed by foliar application on tomato plants (cv. Micro-Tom) that received a saline irrigation water program, which caused a significant decrease in fruit yield (−37.5%). Stressed plants treated with PSI-475 increased this parameter by 31.8% versus the stressed control. Experimental data suggest that PSI-475 can alleviate the negative effects of saline irrigation by improving osmotic adjustment and ion homeostasis markers. PSI-475 was also able to provide significant yield benefits in unstressed plants (+16.9%) that were associated with improved leaf biochemical markers. The data presented support the use of this precision biostimulant to target the negative effects of salinity stress from irrigation.

POTENTIAL OF COW DUNG AMENDMENT TO ENHANCE LINDANE BIODEGRADATION IN CONTAMINATED AGRICULTURAL SOIL

Efficacy of cow dung (CD) amendment as potential bioremediation tool for cleanup of lindane contaminated agricultural soil was investigated in microcosms simulated in the laboratory. Microcosm CDS contaminated with 5 mg kg-1 of lindane was fortified with 200 g of CD while CTS1 was similar to CDS except that it received no CD amendment. Setup CTS2 contained uncontaminated soil only and was designed to evaluate the physico-chemical and biological dynamics of the soil under unstressed conditions. The trends observed for the dynamics of the physico-chemistry were much more pronounced in setups CDS and CTS1 when compared with CTS2. This was relatively significant between days 5 and 15 which incidentally were periods of striking microbial population changes, whereas, that of CTS2 remained relatively stable. In spite of the initial population drop at the onset of study (though less pronounced in CDS than CTS1), over 18% lindane degradation occurred in system CDS as against 9% in CTS1. Although overall degradation rates in both soils presented no statistical significant differences (P &lt; 0.05), CDS effected a higher level of degradation. Data presented in this investigation suggest that CD is a potential stimulant of relevant organisms with lindane metabolic functions in the test soil especially at early stages of study. Therefore, utilization of organic wastes in bioremediation of contaminated soil is of great potential that should be exploited. This will help reduce the amount of waste destined for landfill, thus reducing landfill gas emissions. It will also provide a cheaper source of organic supplement for cleanup purposes with the ultimate aim of reducing the cost of bioremediation.

Trade-offs in Root and Shoot Growth in Forage Pea [Pisum sativum (L.) arvense] with Foliar Applications of Synthetic Elicitor DPMP (2,4-Dichloro-6-{(E)-[(3-Methoxyphenyl) Imino] Methyl} Phenol) and SA (Salicylic Acid)

Background: Climate change, abiotic and biotic stress pressure are forcing breeders and farmers to find alternative ways to improve and extend food production. Even though there are multiple ways to cope with stress conditions, alleviation of the stress by enhancing plant responses is one of the cheapest, environmentally safest and most direct ways. This study aimed to evaluate the effects of two common plant defense elicitors, 2,4-dichloro-6-{(E)-[(3-methoxyphenyl) imino] methyl} phenol (DPMP) and salicylic acid (SA) on plant growth and seedling vigor with forage pea [Pisum sativum (L.) arvense] as a model plant. Methods: Two different chemicals, 100 µM SA and 10 µM DPMP were evaluated in response to Polyethylene glycol 8000 (PEG) treatment in a semi-hydroponic growth system. Root architecture and shoot growth parameters were evaluated. The experiment was designed according to completely randomized design with three replications and ten plants per replication. Result: The effects of SA and DPMP foliar applications were significant on tap, lateral and total root lengths, number of lateral roots and root fresh weight. For most of the traits, SA and DPMP did not inhibit plant growth compared to control under treated and untreated conditions. Average lateral root length (aLatRL) was the noteworthy trait with significantly higher values in DPMP + unstressed conditions. Plants sprayed with DPMP had significantly higher (5.57 cm plant-1) aLatRL values compared to SA (4.53 cm plant-1) and control (3.01 cm plant-1). The results of the current study suggest that SA and DPMP foliar spraying can be beneficial to reduce the effects of abiotic stresses at optimal doses defined for each species. DPMP can be a candidate as a sustainable pesticide alternative and growth-enhancing agent, similar to SA.

Regulatory role of LexA in modulating photosynthetic redox poise and cadmium stress tolerance in the cyanobacterium, Anabaena sp. PCC7120

Strategies developed by organisms to overcome disruption in redox poise of photosynthetic electron transport chain (pETC) are important for its survival under abiotic stress. The process needs to be tightly regulated for optimal functioning. While the redox poising processes are well known in cyanobacteria, understanding of their regulatory network is lacking. Since LexA is one of the known global regulators of stress response in the cyanobacterium Anabaena sp. PCC7120, its role in pETC redox poising was investigated using cadmium (Cd) as an abiotic stressor to disrupt photosynthesis. Assessment of the photosynthetic responses of recombinant Anabaena strains, An lexA + (LexA-overexpressing) and AnpAM (vector control), under unstressed and Cd-stressed conditions using transmission electron microscopy (TEM) and chlorophyll a fluorescence, indicated that some pETC redox poising responses, including PSII photodamage, energy dissipation, PSI photoprotection, and NDH-mediated cyclic electron flow were decreased in An lexA + under unstressed conditions. Disturbance in pETC redox poise during Cd stress observed in Anabaena was accentuated upon overexpression of LexA. The decreased photodamage of PSII and increased photoinhibition of PSI in An lexA + in the presence or absence of Cd stress, correlated well with the changes in pETC complexes observed in blue native (BN)-PAGE and the regulation of over 70 of the 90 pETC component genes by LexA demonstrated through transcript, electromobility shift assay (EMSA), and bioinformatics studies. In a nutshell, LexA has been identified as one of the regulators involved in the streamlining of pETC redox poising responses under normal growth and during abiotic stress through transcriptional regulation of some of the redox-controlled pETC component genes. • Abiotic stresses disrupt photosynthetic redox poise, triggering poising responses. • Poising responses are known in cyanobacteria, but their regulatory network is not. • LexA overexpression in Anabaena reduced these responses, lowering stress tolerance. • In-silico , qRT-PCR and EMSA showed LexA regulation on over 70 photosynthetic genes. • LexA was shown to modulate these responses by regulating some photosynthetic genes.

In Situ Peroxidase Labeling Followed by Mass-Spectrometry Reveals TIA1 Interactome.

Simple SummaryT-cell intracellular antigen 1 (TIA1) is a DNA/RNA-binding protein best known for its different roles in RNA metabolism. Currently, little is known about the interacting protein partners of TIA1 in control and stress conditions that could shed light on its multiple context-specific molecular functions. Proximity labeling is a technique in which a labeling enzyme, here APEX2, that is fused to the protein of interest, in this case TIA1, marks the protein’s interacting network in living cells, allowing for subsequent ex vivo analysis using protein identification methods such as mass spectrometry. Hereby, combining these two techniques, it was revealed that the TIA1 interactome has very distinct protein partners in control and unstressed cells and that these partners are involved in not only previously identified processes such as splicing, nucleocytoplasmic transport, and different levels of protein translation control, but also in novel ones such as DNA double-strand break repair and mitochondrial metabolism. Overall, these findings provide a more precise definition of TIA1’s function in cells and pave the way to dissect its role in each of these processes.TIA1 is a broadly expressed DNA/RNA binding protein that regulates multiple aspects of RNA metabolism. It is best known for its role in stress granule assembly during the cellular stress response. Three RNA recognition motifs mediate TIA1 functions along with a prion-like domain that supports multivalent protein-protein interactions that are yet poorly characterized. Here, by fusing the enhanced ascorbate peroxidase 2 (APEX2) biotin-labeling enzyme to TIA1 combined with mass spectrometry, the proteins in the immediate vicinity of TIA1 were defined in situ. Eighty-six and 203 protein partners, mostly associated with ribonucleoprotein complexes, were identified in unstressed control and acute stress conditions, respectively. Remarkably, the repertoire of TIA1 protein partners was highly dissimilar between the two cellular states. Under unstressed control conditions, the biological processes associated with the TIA1 interactome were enriched for cytosolic ontologies related to mRNA metabolism, such as translation initiation, nucleocytoplasmic transport, and RNA catabolism, while the protein identities were primarily represented by RNA binding proteins, ribosomal subunits, and eicosanoid regulators. Under acute stress, TIA1-labeled partners displayed a broader subcellular distribution that included the chromosomes and mitochondria. The enriched biological processes included splicing, translation, and protein synthesis regulation, while the molecular function of the proteins was enriched for RNA binding activity, ribosomal subunits, DNA double-strand break repair, and amide metabolism. Altogether, these data highlight the TIA1 spatial environment with its different partners in diverse cellular states and pave the way to dissect TIA1 role in these processes.

Reactive feedback control and adaptation to perturbed speech timing in stressed and unstressed syllables

This study examines speakers’ reaction to focally applied temporal real-time auditory feedback perturbation in a word-initial unstressed syllable (Unstressed condition) and a similar word-medial stressed syllable (Stressed condition) in a three-syllabic word. Speakers compensate locally in both conditions for the perturbed syllable’s nucleus (V; compressed by the perturbation) but not for the complex onsets (CC; stretched by the perturbation). The perturbation of the first, unstressed syllable causes a global slowing down of all segments following the perturbation (syllable two and three), while the perturbation in the Stressed condition elicits local adjustments only in the perturbed (second) syllable. When viewed in a larger prosodic context, the timing strategy in the Unstressed condition indicates that speakers aim to keep relative durations within the word constant when the word-initial onset is auditorily stretched, leading to a compensatory pattern for both CC and V in word-proportional durations. In the Stressed condition, increasing the stressed vowel’s duration seems to be of the highest priority, causing all other segments to take up a shorter portion within the word. Adaptation effects of the stressed vowel indicate a durational representation on the segment level. Further adaptation effects additionally suggest a representation of timing/coordination in larger prosodic units. Complementary investigation of aperiodicity, spectral skewness, and intensity (RMS) indicates that spectral properties can change along with compensatorily increased duration.

Measurement of Macromolecular Crowding in Rhodobacter sphaeroides under Different Growth Conditions.

ABSTRACTThe bacterial cytoplasm is a very crowded environment, and changes in crowding are thought to have an impact on cellular processes including protein folding, molecular diffusion and complex formation. Previous studies on the effects of crowding have generally compared cellular activity after imposition of stress. In response to different light intensities, in unstressed conditions, Rhodobacter sphaeroides changes the number of 50-nm intracytoplasmic membrane (ICM) vesicles, with the number varying from a few to over a thousand per cell. In this work, the effects of crowding induced by ICM vesicles in photoheterotrophic R. sphaeroides were investigated using a fluorescence resonance energy transfer (FRET) sensor and photoactivated localization microscopy (PALM). In low light grown cells where the cytoplasm has large numbers of ICM vesicles, the FRET probe adopts a more condensed conformation, resulting in higher FRET ratio readouts compared to high light cells with fewer ICM vesicles. The apparent diffusion coefficients of different sized proteins, PAmCherry, PAmCherry-CheY6, and L1-PAmCherry, measured via PALM showed that diffusion of protein molecules >27 kDa decreased as the number of ICM vesicles increased. In low light R. sphaeroides where the crowding level is high, protein molecules were found to diffuse more slowly than in aerobic and high light cells. This suggests that some physiological activities might show different kinetics in bacterial species whose intracellular membrane organization can change with growth conditions.