Subcellular glutathione homeostasis and characterisation of glutathione transport across the plasma membrane in Arabidopsis thaliana

Challenging environmental conditions are known to alter glutathione homeostasis, notably by inducing the accumulation of oxidized glutathione, an effect that may be influential in the perception or transduction of stress signals. The tripeptide glutathione (reduced form: GSH; oxidized form: GSSG) is a key player in maintaining cellular thiol-redox homeostasis. In Arabidopsis, two proteins are responsible for GSH biosynthesis, GSH1 and GSH2. GSH1 is exclusively localized in plastids, while GSH2 is localized in both plastids and cytosol. Thus, GSH synthesis is restricted to plastids and cytosol but there is a requirement for glutathione also in other subcellular compartments with the need for intracellular GSH transport. In addition, there is good indication pointing at long-distance transport of glutathione between different organs. The latter implies the need for transport of glutathione across the plasma membrane which is poorly understood. Null mutants of GSH1 are embryo lethal, while disruption of GSH2 is seedling lethal. In several genetic screens, different gsh1 mutants with defects in GSH biosynthesis have been identified. However, the effect of these mutations at the subcellular level was still elusive. In this study, the effect of GSH1 mutations on subcellular glutathione distribution was analysed in an allelic series of gsh1 mutants and wild-type plants. Fluorescent labelling of GSH with monochlorobimane (MCB) and HPLC measurements showed that the total amount of glutathione was affected by the respective mutations. Furthermore, the relative glutathione redox potential (EGSH) in different subcellular compartments measured with redox-sensitive fluorescent protein2 (roGFP2), showed that these mutations differentially affect the subcellular glutathione pool. The data indicate that mutations in GSH1 have a stronger effect on cytosolic and plastidial glutathione homeostasis than on mitochondrial glutathione homeostasis. Furthermore, crossing of bir6 (a mutant with diminished glutathione turnover) with the severely GSH deficient mutants, zir1 (a mutant The glutathione concentration is limited by the γ-glutamyl cycle, which is based on glutathione synthesis, degradation and transport. While the γ-glutamyl cycle was suggested as a classical pathway involved in glutathione transport across the plasma membrane there is also evidence for cytosolic glutathione degradation. Under sulfur deficiency, intense MCB fluorescence in γ-glutamyl-cyclotransferase (GGCT) null mutants and diminished MCB fluorescence in wild-type suggests that GGCTs specifically degrade GSH in the cytosol. While glutathione transport studies in yeast (Saccharomyces cerevisiae) have led to identification of Hgt1p as a high affinity glutathione transporter, genes encoding glutathione-specific transporters in the plasma membrane of plants remain largely unknown. To investigate the transport of glutathione across the plasma membrane, the severely glutathione-deficient Arabidopsis mutant rml1 was analysed using the roGFP2, which are able to monitor the local EGSH. Changes in the fluorescence ratio of roGFP2 expressed in the cytosol of rml1 with external supply of GSH, in combination with inhibitor studies revealed a highly efficient secondary active uptake of GSH across the plasma membrane. Furthermore, reduction of roGFP2 was only seen with GSH, but not with individual amino acids or GSSG. Additionally, a generated opt4rml1 double mutant further proved that the oligopeptide transporter 4 (OPT4) reported earlier is not the only GSH transporter in Arabidopsis but is rather complemented by a yet unknown high affinity transporter. These results have major implications for our understanding of the glutathione homeostasis in plants, with a particular focus on subcellular compartmentation, degradation, functionality and transport

The Effects of Osmosis and Thermo-Priming on Salinity Stress Tolerance in Vigna radiata L.

A plant’s response to osmotic stress is a complex phenomenon that causes many abnormal symptoms due to limitations in growth and development or even the loss of yield. The current research aimed to analyze the agronomical, physiological, and biochemical mechanisms accompanying the acquisition of salt resistance in the Vigna radiata L. variety ‘Ramzan’ using seed osmo- and thermopriming in the presence of PEG-4000 and 4 °C under induced salinity stresses of 100 and 150 mM NaCl. Seeds were collected from CCRI, Nowshera, and sowing was undertaken in triplicate at the Department of Botany, Peshawar University, during the 2018–2019 growing season. Rhizospheric soil pH (6.0), E.C (2.41 ds/m), field capacity, and moisture content level were estimated in the present study. We observed from the estimated results that the agronomic characteristics, i.e., shoot fresh weight and shoot dry weight in T9 (4oC + 150 mM NaCl), root fresh weight and root dry weight in T4 (PEG + 100 mM NaCl), shoot moisture content in T5 (PEG + 100 mM NaCl), and root moisture content in T6 (PEG + 150 mM NaCl) were the highest, followed by the lowest in T1 (both shoot and root fresh weights) and T2 (shoot and root dry weights). Similarly, the shoot moisture content was the maximum in T5 and the minimum in T6, and root moisture was the highest in T6. We observed from the estimated results that agronomical parameters including dry masses (T4, T6, T4), leaf area index, germination index, leaf area, total biomass, seed vigor index under treatment T9, and relative water content and water use efficiency during T5 and T6 were the highest. Plant physiological traits such as proline, SOD enhanced by T1, carotenoids in treatment T2, and chlorophyll and protein levels were the highest under treatment T4, whereas sugar and POD were highest under treatments T7 and T8. The principal component analysis enclosed 63.75% of the total variation among all biological components. These estimated results confirmed the positive resistance by Vigna radiata during osmopriming (PEG) and thermopriming (4 °C) on most of the features with great tolerance under a low-saline treatment such as T4 (PEG), T5 (PEG + 100 mM NaCl), T7 (4 °C), and T8 (4 °C + 100 mM NaCl), while it was susceptible in the case of T6 (PEG + 150 mM NaCl) and T9 (4 °C + 150 mM NaCl) to high salt application. We found that the constraining impact of several priming techniques improved low salinity, which was regarded as economically inexpensive and initiated numerous metabolic processes in plants, hence decreasing germination time. The current study will have major applications for combatting the salinity problem induced by climate change in Pakistan

The Effects of Osmosis and Thermo-Priming on Salinity Stress Tolerance in Vigna radiata L.

A plant’s response to osmotic stress is a complex phenomenon that causes many abnormal symptoms due to limitations in growth and development or even the loss of yield. The current research aimed to analyze the agronomical, physiological, and biochemical mechanisms accompanying the acquisition of salt resistance in the Vigna radiata L. variety ‘Ramzan’ using seed osmo- and thermopriming in the presence of PEG-4000 and 4 °C under induced salinity stresses of 100 and 150 mM NaCl. Seeds were collected from CCRI, Nowshera, and sowing was undertaken in triplicate at the Department of Botany, Peshawar University, during the 2018–2019 growing season. Rhizospheric soil pH (6.0), E.C (2.41 ds/m), field capacity, and moisture content level were estimated in the present study. We observed from the estimated results that the agronomic characteristics, i.e., shoot fresh weight and shoot dry weight in T9 (4oC + 150 mM NaCl), root fresh weight and root dry weight in T4 (PEG + 100 mM NaCl), shoot moisture content in T5 (PEG + 100 mM NaCl), and root moisture content in T6 (PEG + 150 mM NaCl) were the highest, followed by the lowest in T1 (both shoot and root fresh weights) and T2 (shoot and root dry weights). Similarly, the shoot moisture content was the maximum in T5 and the minimum in T6, and root moisture was the highest in T6. We observed from the estimated results that agronomical parameters including dry masses (T4, T6, T4), leaf area index, germination index, leaf area, total biomass, seed vigor index under treatment T9, and relative water content and water use efficiency during T5 and T6 were the highest. Plant physiological traits such as proline, SOD enhanced by T1, carotenoids in treatment T2, and chlorophyll and protein levels were the highest under treatment T4, whereas sugar and POD were highest under treatments T7 and T8. The principal component analysis enclosed 63.75% of the total variation among all biological components. These estimated results confirmed the positive resistance by Vigna radiata during osmopriming (PEG) and thermopriming (4 °C) on most of the features with great tolerance under a low-saline treatment such as T4 (PEG), T5 (PEG + 100 mM NaCl), T7 (4 °C), and T8 (4 °C + 100 mM NaCl), while it was susceptible in the case of T6 (PEG + 150 mM NaCl) and T9 (4 °C + 150 mM NaCl) to high salt application. We found that the constraining impact of several priming techniques improved low salinity, which was regarded as economically inexpensive and initiated numerous metabolic processes in plants, hence decreasing germination time. The current study will have major applications for combatting the salinity problem induced by climate change in Pakistan

Arabidopsis glutathione reductase 2 is indispensable in plastids, while mitochondrial glutathione is safeguarded by additional reduction and transport systems

A highly negative glutathione redox potential (EGSH ) is maintained in the cytosol, plastids and mitochondria of plant cells to support fundamental processes, including antioxidant defence, redox regulation and iron-sulfur cluster biogenesis. Out of two glutathione reductase (GR) proteins in Arabidopsis, GR2 is predicted to be dual-targeted to plastids and mitochondria, but its differential roles in these organelles remain unclear. We dissected the role of GR2 in organelle glutathione redox homeostasis and plant development using a combination of genetic complementation and stacked mutants, biochemical activity studies, immunogold labelling and in vivo biosensing. Our data demonstrate that GR2 is dual-targeted to plastids and mitochondria, but embryo lethality of gr2 null mutants is caused specifically in plastids. Whereas lack of mitochondrial GR2 leads to a partially oxidised glutathione pool in the matrix, the ATP-binding cassette (ABC) transporter ATM3 and the mitochondrial thioredoxin system provide functional backup and maintain plant viability. We identify GR2 as essential in the plastid stroma, where it counters GSSG accumulation and developmental arrest. By contrast a functional triad of GR2, ATM3 and the thioredoxin system in the mitochondria provides resilience to excessive glutathione oxidation

The Effects of Osmosis and Thermo-Priming on Salinity Stress Tolerance in Vigna radiata L.

A plant's response to osmotic stress is a complex phenomenon that causes many abnormal symptoms due to limitations in growth and development or even the loss of yield. The current research aimed to analyze the agronomical, physiological, and biochemical mechanisms accompanying the acquisition of salt resistance in the Vigna radiata L. variety 'Ramzan' using seed osmo- and thermopriming in the presence of PEG-4000 and 4 degrees C under induced salinity stresses of 100 and 150 mM NaCl. Seeds were collected from CCRI, Nowshera, and sowing was undertaken in triplicate at the Department of Botany, Peshawar University, during the 2018-2019 growing season. Rhizospheric soil pH (6.0), E.C (2.41 ds/m), field capacity, and moisture content level were estimated in the present study. We observed from the estimated results that the agronomic characteristics, i.e., shoot fresh weight and shoot dry weight in T9 (4oC + 150 mM NaCl), root fresh weight and root dry weight in T4 (PEG + 100 mM NaCl), shoot moisture content in T5 (PEG + 100 mM NaCl), and root moisture content in T6 (PEG + 150 mM NaCl) were the highest, followed by the lowest in T1 (both shoot and root fresh weights) and T2 (shoot and root dry weights). Similarly, the shoot moisture content was the maximum in T5 and the minimum in T6, and root moisture was the highest in T6. We observed from the estimated results that agronomical parameters including dry masses (T4, T6, T4), leaf area index, germination index, leaf area, total biomass, seed vigor index under treatment T9, and relative water content and water use efficiency during T5 and T6 were the highest. Plant physiological traits such as proline, SOD enhanced by T1, carotenoids in treatment T2, and chlorophyll and protein levels were the highest under treatment T4, whereas sugar and POD were highest under treatments T7 and T8. The principal component analysis enclosed 63.75% of the total variation among all biological components. These estimated results confirmed the positive resistance by Vigna radiata during osmopriming (PEG) and thermopriming (4 degrees C) on most of the features with great tolerance under a low-saline treatment such as T4 (PEG), T5 (PEG + 100 mM NaCl), T7 (4 degrees C), and T8 (4 degrees C + 100 mM NaCl), while it was susceptible in the case of T6 (PEG + 150 mM NaCl) and T9 (4 degrees C + 150 mM NaCl) to high salt application. We found that the constraining impact of several priming techniques improved low salinity, which was regarded as economically inexpensive and initiated numerous metabolic processes in plants, hence decreasing germination time. The current study will have major applications for combatting the salinity problem induced by climate change in Pakistan.Peer reviewe

Perceptions of the learning environment, learning preferences, and approaches to studying among medical students in Pakistan

Students at a Pakistani medical college were surveyed using instruments taken from Western research. The students who rated their courses positively tended to adopt an organised approach in their learning and studying. The students who perceived that their assessment and workload were inappropriate preferred a transmissive approach to teaching and adopted a surface approach in their learning and studying. The students who preferred a student-centred approach to teaching tended to adopt a deep approach in their learning and studying. This confirms the idea, well established in Western research, that there exists a strong relationship between students’ perceptions of their courses and the approaches that they adopt on those courses. However, the incorporation of problem-based learning in the medical curriculum had not led to any enhancement of their perceptions and preferences, nor had it led to an unambiguous improvement in their approaches to studying. This is attributed to the hybrid nature of their programmes, in which problem-based activities were combined with more conventional forms of teaching and assessment, and to the anxiety and stress which seem to be common among students at medical schools in Pakistan