Changes in Antioxidant Defence System in Durum Wheat under Hyperosmotic Stress: A Concise Overview

Durum wheat is one of the most commonly cultivated species in the world and represents a key commodity for many areas worldwide, as its grain is used for production of many foods, such as pasta, bread, couscous, and bourghul. Durum wheat grain has a relevant role in the human diet, providing carbohydrates, proteins, lipids, fibres, vitamins, and minerals, as well as highly valued bioactive compounds contributing to a healthy diet. Durum wheat is largely cultivated in the Mediterranean basin, where it is mainly grown under rain-fed conditions, thus currently undergoing drought stress, as well as soil salinity, which can hamper yield potential and influence the qualitative characteristics of grain. When plants suffer drought and/or salinity stress, a condition known as hyperosmotic stress is established at cellular level. This leads to the accumulation of ROS thus generating in turn an oxidative stress condition, which can ultimately result in the impairment of cellular integrity and functionality. To counteract oxidative damage due to excessive ROS production under stress, plants have evolved a complex array of both enzymatic and non-enzymatic antioxidant mechanisms, working jointly and synergically for maintenance of ROS homeostasis. Enhancement of antioxidant defence system has been demonstrated as an adaptive mechanism associated to an increased tolerance to hyperosmotic stress. In the light of these considerations, this review provides a concise overview on recent advancements regarding the role of the ascorbate-glutathione cycle and the main antioxidant enzymes (superoxide dismutase, catalase, and peroxidases) in durum wheat response to drought and salt stresses that are expected to become more and more frequent due to the ongoing climate changes.

Hyperosmotic stress induces epithelial-mesenchymal transition through rearrangements of focal adhesions in tubular epithelial cells

Epithelial-mesenchymal transition (EMT) of tubular epithelial cells is a hallmark of renal tubulointerstitial fibrosis and is associated with chronic renal injury as well as acute renal injury. As one of the incidences and risk factors for acute renal injury, increasing the osmolality in the proximal tubular fluid by administration of intravenous mannitol has been reported, but the detailed mechanisms remain unclear. Hyperosmotic conditions caused by mannitol in the tubular tissue may generate not only osmotic but also mechanical stresses, which are known to be able to induce EMT in epithelial cells, thereby contributing to renal injury. Herein, we investigate the effect of hyperosmolarity on EMT in tubular epithelial cells. Normal rat kidney (NRK)-52E cells were exposed to mannitol-induced hyperosmotic stress. Consequently, the hyperosmotic stress led to a reduced expression of the epithelial marker E-cadherin and an enhanced expression of the mesenchymal marker, α-smooth muscle actin (α-SMA), which indicates an initiation of EMT in NKR-52E cells. The hyperosmotic condition also induced time-dependent disassembly and rearrangements of focal adhesions (FAs) concomitant with changes in actin cytoskeleton. Moreover, prevention of FAs rearrangements by cotreatment with Y-27632, a Rho-associated protein kinase inhibitor, could abolish the effects of hyperosmotic mannitol treatment, thus attenuating the expression of α-SMA to the level in nontreated cells. These results suggest that hyperosmotic stress may induce EMT through FAs rearrangement in proximal tubular epithelial cells.

First Responders to Hyperosmotic Stress in Murine Astrocytes: Connexin 43 Gap Junctions Are Subject to an Immediate Ultrastructural Reorganization

In a short-term model of hyperosmotic stress, primary murine astrocytes were stimulated with a hyperosmolar sucrose solution for five minutes. Astrocytic gap junctions, which are mainly composed of Connexin (Cx) 43, displayed immediate ultrastructural changes, demonstrated by freeze–fracture replica immunogold labeling: their area, perimeter, and distance of intramembrane particles increased, whereas particle numbers per area decreased. Ultrastructural changes were, however, not accompanied by changes in Cx43 mRNA expression. In contrast, transcription of the gap junction regulator zonula occludens (ZO) protein 1 significantly increased, whereas its protein expression was unaffected. Phosphorylation of Serine (S) 368 of the Cx43 C–terminus has previously been associated with gap junction disassembly and reduction in gap junction communication. Hyperosmolar sucrose treatment led to enhanced phosphorylation of Cx43S368 and was accompanied by inhibition of gap junctional intercellular communication, demonstrated by a scrape loading-dye transfer assay. Taken together, Cx43 gap junctions are fast reacting elements in response to hyperosmolar challenges and can therefore be considered as one of the first responders to hyperosmolarity. In this process, phosphorylation of Cx43S368 was associated with disassembly of gap junctions and inhibition of their function. Thus, modulation of the gap junction assembly might represent a target in the treatment of brain edema or trauma.

Both Clathrin-Mediated and Membrane Microdomain-Associated Endocytosis Contribute to the Cellular Adaptation to Hyperosmotic Stress in Arabidopsis

As sessile organisms, plants must directly deal with an often complex and adverse environment in which hyperosmotic stress is one of the most serious abiotic factors, challenging cellular physiology and integrity. The plasma membrane (PM) is the hydrophobic barrier between the inside and outside environments of cells and is considered a central compartment in cellular adaptation to diverse stress conditions through dynamic PM remodeling. Endocytosis is a powerful method for rapid remodeling of the PM. In animal cells, different endocytic pathways are activated in response to osmotic stress, while only a few reports are related to the endocytosis response pathway and involve a mechanism in plant cells upon hyperosmotic stress. In this study, using different endocytosis inhibitors, the microdomain-specific dye di-4-ANEPPDHQ, variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), and confocal microscopy, we discovered that internalized Clathrin Light Chain-Green Fluorescent Protein (CLC-GFP) increased under hyperosmotic conditions, accompanied by decreased fluorescence intensity of CLC-GFP at the PM. CLC-GFP tended to have higher diffusion coefficients and a fraction of CLC-GFP molecules underwent slower diffusion upon hyperosmotic stress. Meanwhile, an increased motion range of CLC-GFP was found under hyperosmotic treatment compared with the control. In addition, the order of the PM decreased, but the order of the endosome increased when cells were in hyperosmotic conditions. Hence, our results demonstrated that clathrin-mediated endocytosis and membrane microdomain-associated endocytosis both participate in the adaptation to hyperosmotic stress. These findings will help to further understand the role and the regulatory mechanism involved in plant endocytosis in helping plants adapt to osmotic stress.

Hyperosmotic Stress Induces a Specific Pattern for Stress Granule Formation in Human-Induced Pluripotent Stem Cells

Stress granules (SGs) are assemblies of selective messenger RNAs (mRNAs), translation factors, and RNA-binding proteins in small untranslated messenger ribonucleoprotein (mRNP) complexes in the cytoplasm. Evidence indicates that different types of cells have shown different mechanisms to respond to stress and the formation of SGs. In the present work, we investigated how human-induced pluripotent stem cells (hiPSCs/IMR90-1) overcome hyperosmotic stress compared to a cell line that does not harbor pluripotent characteristics (SH-SY5Y cell line). Gradient concentrations of NaCl showed a different pattern of SG formation between hiPSCs/IMR90-1 and the nonpluripotent cell line SH-SY5Y. Other pluripotent stem cell lines (hiPSCs/CRTD5 and hESCs/H9 (human embryonic stem cell line)) as well as nonpluripotent cell lines (BHK-21 and MCF-7) were used to confirm this phenomenon. Moreover, the formation of hyperosmotic SGs in hiPSCs/IMR90-1 was independent of eIF2α phosphorylation and was associated with low apoptosis levels. In addition, a comprehensive proteomics analysis was performed to identify proteins involved in regulating this specific pattern of hyperosmotic SG formation in hiPSCs/IMR90-1. We found possible implications of microtubule organization on the response to hyperosmotic stress in hiPSCs/IMR90-1. We have also unveiled a reduced expression of tubulin that may protect cells against hyperosmolarity stress while inhibiting SG formation without affecting stem cell self-renewal and pluripotency. Our observations may provide a possible cellular mechanism to better understand SG dynamics in pluripotent stem cells.

m6A Modified Short RNA Fragments Inhibit Cytoplasmic TLS/FUS Aggregation Induced by Hyperosmotic Stress

Translocated in LipoSarcoma/Fused in Sarcoma (TLS/FUS) is a nuclear RNA binding protein whose mutations cause amyotrophic lateral sclerosis. TLS/FUS undergoes LLPS and forms membraneless particles with other proteins and nucleic acids. Interaction with RNA alters conformation of TLS/FUS, which affects binding with proteins, but the effect of m6A RNA modification on the TLS/FUS–RNA interaction remains elusive. Here, we investigated the binding specificity of TLS/FUS to m6A RNA fragments by RNA pull down assay, and elucidated that both wild type and ALS-related TLS/FUS mutants strongly bound to m6A modified RNAs. TLS/FUS formed cytoplasmic foci by treating hyperosmotic stress, but the cells transfected with m6A-modified RNAs had a smaller number of foci. Moreover, m6A-modified RNA transfection resulted in the cells obtaining higher resistance to the stress. In summary, we propose TLS/FUS as a novel candidate of m6A recognition protein, and m6A-modified RNA fragments diffuse cytoplasmic TLS/FUS foci and thereby enhance cell viability.

Cat1 Regulate ROS Metabolism and Microsclerotia Formation in the Dimorphic Fungus Nomuraea Rileyi

Catalases are the most important enzymes in the metabolism of reactive oxygen species (ROS), as they convert hydrogen peroxide (H2O2) into water and molecular oxygen. They are also involved in virulence, and in oxidative, heat, hyperosmotic stress and UV-B radiation responses in some entomopathogenic fungi. In this study, the Cat1 gene from Nomuraea rileyi was cloned and its function was studied by gene deletion. The NrCat1 deletion mutant (ΔNrCat1) was generated using the split-marker method. No significant differences in colony growth or dimorphic switching of ΔNrCat1 were observed under regular culture conditions, whereas oxidative stress inhibited colony growth and the yeast-hyphal transition. In contrast, there was no significant difference in tolerance to hyperosmotic stress between ΔNrCat1 and wild type (WT) strains. In the ΔNrCat1 strain, microsclerotia (MS) formation time of the ΔNrCat1 was delayed, and MS size was less uniform than in the WT. MS yield was decreased by 76% in the ΔNrCat1 strain compared to the WT strain. Furthermore, virulence was attenuated in the ΔNrCat1 strain. Gene expression analysis showed that NrCat2, NrCat4, and NrAox are up-regulated to compensate for NrCat1 deletion. Thus, the NrCat1 gene in N. rileyi appears to be involved in essential functions, including H2O2 metabolism, MS formation, and virulence.

Formation of Non-Nucleoplasmic Proteasome Foci during the Late Stage of Hyperosmotic Stress

Cellular stress induces the formation of membraneless protein condensates in both the nucleus and cytoplasm. The nucleocytoplasmic transport of proteins mainly occurs through nuclear pore complexes (NPCs), whose efficiency is affected by various stress conditions. Here, we report that hyperosmotic stress compartmentalizes nuclear 26S proteasomes into dense nuclear foci, independent of signaling cascades. Most of the proteasome foci were detected between the condensed chromatin mass and inner nuclear membrane. The proteasome-positive puncta were not colocalized with other types of nuclear bodies and were reversibly dispersed when cells were returned to the isotonic medium. The structural integrity of 26S proteasomes in the nucleus was slightly affected under the hyperosmotic condition. We also found that these insulator-body-like proteasome foci were possibly formed through disrupted nucleus-to-cytosol transport, which was mediated by the sequestration of NPC components into osmostress-responding stress granules. These data suggest that phase separation in both the nucleus and cytosol may be a major cell survival mechanism during hyperosmotic stress conditions.