Glutathione: a key modulator of plant defence and metabolism through multiple mechanisms.

Redox reactions are fundamental to energy conversion in living cells, and also determine and tune responses to the environment. Within this context, the tripeptide glutathione plays numerous roles. As an important antioxidant, glutathione confers redox stability on the cell and also acts an interface between signalling pathways and metabolic reactions that fuel growth and development. It also contributes to the assembly of cell components, biosynthesis of sulphur-containing metabolites, inactivation of potentially deleterious compounds, and control of hormonal signalling intensity. The multiplicity of these roles probably explains why glutathione status has been implicated in influencing plant responses to many different conditions. In particular, there is now a considerable body of evidence that glutathione is a crucial player in governing the outcome of biotic stresses. This review provides an overview of glutathione synthesis, transport, degradation, and redox turnover in plants. It examines the expression of genes associated with these processes during pathogen challenge and related conditions, and considers the diversity of mechanisms by which glutathione can influence protein function and gene expression.

Translatome analysis of an NB-LRR immune response identifies important contributors to plant immunity in Arabidopsis.

An important branch of plant immunity involves the recognition of pathogens by nucleotide-binding, leucine-rich repeat (NB-LRR) proteins. However, signaling events downstream of NB-LRR activation are poorly understood. We have analysed the Arabidopsis translatome using ribosome affinity purification and RNA sequencing. Our results show that the translational status of hundreds of transcripts is differentially affected upon activation of the NB-LRR protein RPM1, showing an overall pattern of a switch away from growth-related activities to defense. Among these is the central translational regulator and growth promoter, Target of Rapamycin (TOR) kinase. Suppression of TOR expression leads to increased resistance to pathogens while overexpression of TOR results in increased susceptibility, indicating an important role for translational control in the switch from growth to defense. Furthermore, we show that several additional genes whose mRNAs are translationally regulated, including BIG, CCT2, and CIPK5, are required for both NB-LRR-mediated and basal plant innate immunity, identifying novel actors in plant defense.

NB-LRR signaling induces translational repression of viral transcripts and the formation of RNA processing bodies through mechanisms differing from those activated by UV stress and RNAi.

Plant NB-LRR proteins confer resistance to multiple pathogens, including viruses. Although the recognition of viruses by NB-LRR proteins is highly specific, previous studies have suggested that NB-LRR activation results in a response that targets all viruses in the infected cell. Using an inducible system to activate NB-LRR defenses, we find that NB-LRR signaling does not result in the degradation of viral transcripts, but rather prevents them from associating with ribosomes and translating their genetic material. This indicates that defense against viruses involves the repression of viral RNA translation. This repression is specific to viral transcripts and does not involve a global shutdown of host cell translation. As a consequence of the repression of viral RNA translation, NB-LRR responses induce a dramatic increase in the biogenesis of RNA processing bodies (PBs). We demonstrate that other pathways that induce translational repression, such as UV irradiation and RNAi, also induce PBs. However, by investigating the phosphorylation status of eIF2α and by using suppressors of RNAi we show that the mechanisms leading to PB induction by NB-LRR signaling are different from these stimuli, thus defining a distinct type of translational control and anti-viral mechanism in plants.

Quantitative Measurements of Biochemical and Molecular Markers of Oxidative Stress Signaling and Responses.

Increases in cellular oxidation are a part of most plant responses to challenging conditions and are commonly described as oxidative stress. While this phenomenon is closely related to the accumulation of reactive oxygen species, these latter compounds can be difficult to measure. Complementary measurements to assess cellular redox state are, therefore, very useful in studies of plant responses to stress. Here, we detail protocols for three complementary approaches that can be used to assess the intensity of oxidative stress. These involve quantification of marker transcripts, assays of the extractable activities of major antioxidative enzymes, and measurement of antioxidant buffers. We confirm experimentally that the data obtained by such approaches can provide reliable information on the intensity of oxidative stress.

Metabolite modification in oxidative stress responses: A case study of two defense hormones.

Studies of the Arabidopsis cat2 mutant lacking the major leaf isoform of catalase have allowed the potential impact of intracellular H2O2 on plant function to be studied. Here, we report a robust analysis of modified gene expression associated with key families involved in metabolite modification in cat2. Through a combined transcriptomic and metabolomic analysis focused on the salicylic acid (SA) and jasmonic acid (JA) pathways, we report key features of the metabolic signatures linked to oxidative stress-induced signaling via these defence hormones and discuss the enzymes that are likely to be involved in determining these features. We provide evidence that specific UDP-glycosyl transferases contribute to the glucosylation of SA that accumulates as a result of oxidative stress in cat2. Glycosides of dihydroxybenzoic acids that accumulate alongside SA in cat2 are identified and, based on the expression of candidate genes, likely routes for their production are discussed. We also report that enhanced intracellular H2O2 triggers induction of genes encoding different enzymes that can metabolize JA. Integrated analysis of metabolite and transcript profiles suggests that a gene network involving specific hydrolases, hydroxylases, and sulfotransferases functions to limit accumulation of the most active jasmonates during oxidative stress.

Towards plant resistance to viruses using protein-only RNase P.

Plant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5' maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases.

Accidental venous and dural puncture during epidural analgesia in obese parturients (BMI > 40 kg/m2): three different body positions during insertion.

STUDY OBJECTIVE: To assess the frequency of blood vessel punctures in morbidly obese parturients [body mass index (BMI) > 40 kg/m(2)] during epidural catheterization, in three different body positions. DESIGN: Prospective, randomized study. SETTING: Delivery room of a university-affiliated hospital. PATIENTS: 347 obese parturients (BMI > 40 kg/m(2)) undergoing continuous epidural analgesia during labor. INTERVENTIONS: Patients were randomized to undergo epidural catheterization in the sitting, lateral recumbent horizontal, or lateral recumbent head-down positions. MEASUREMENTS AND MAIN RESULTS: A lower frequency of epidural venous cannulation was noted when this procedure was performed in the lateral recumbent head-down position (4.8%) than in the lateral recumbent horizontal (11.6%) or sitting position (18.3%) (P = 0.001). Frequency of accidental subarachnoid puncture did not differ significantly (2.5%, 2.6%, and 3.7%), respectively. CONCLUSION: Adoption of the lateral recumbent head-down position for the performance of lumbar epidural blockade in labor at term reduces the frequency of lumbar epidural venous puncture in obese parturients (BMI > 40 kg/m(2)).

Total cell associated electrolyte homeostasis in rat spinal cord cells following apparently irreversible injury.

BACKGROUND: We investigated total electrolyte homeostasis in spinal cord cells of rats subjected to irreversible spinal cord trauma. MATERIAL/METHODS: Forty-two rats underwent total transection of spinal cord (Group 1); chemical neurolysis by 10% lidocaine overdose (Group 2); sham "injury" (Group 3). Spinal cords were isolated 24 h, 72 h or 7 days following injury. Total cellular Ca, Mg, Na and K were measured in the spinal cord thoracic or lumbar parts using an atomic absorption spectrometer. RESULTS: Group 1: A significant Ca, Mg, Na, and K efflux was observed in thoracic and lumbar parts 24 h following transection. By 72 h, a significant re-entrance of Ca was evident. By 168 h, an influx of all electrolytes was demonstrable, sometimes reaching concentrations above the pre-trauma levels. Group 2: Following 24 h, Na, K, Ca, and Mg concentrations dropped significantly both in thoracic and lumbar parts. By 72 h, the electrolyte re-entrance was evident in the thoracic, but not the lumbar part. By 168 h, Na, K, Ca, and Mg influx was observed both in thoracic and lumbar parts, the concentration approaching pre-trauma levels. Group 3: No changes in electrolyte content were observed in spinal cords of sham-operated animals. CONCLUSIONS: Following massive, apparently irreversible injury of the spinal cord, some restorative processes do take place at the cellular level. Subsequent supernormal accumulation of intracellular electrolytes, especially Ca, might eventually contribute to a secondary injury. Should this be the case, pharmacotherapeutic intervention might prove beneficial.

The lateral recumbent head-down position decreases the incidence of epidural venous puncture during catheter insertion in obese parturients.

PURPOSE: The unintentional and unrecognized cannulation of an extradural vein is a potentially serious complication of an epidural anesthetic. The present study was undertaken to assess the incidence of blood vessel puncture related to epidural catheterization in three different body positions, in a cohort of morbidly obese parturients, following the completion of a similar study published in 2001 from which such parturients were excluded. METHODS: The study was conducted in 450 (three groups of 150) morbidly obese, obstetric patients undergoing continuous epidural analgesia during labour. Epidural catheterization was performed on patients randomized to the sitting, lateral recumbent horizontal, or lateral recumbent head-down position. RESULTS: There was a lower incidence of vessel cannulation when this procedure was performed in the lateral recumbent head-down position [1.3%; body mass index (BMI): 37.0] than in the lateral recumbent horizontal [12.9%; BMI: 38.0] and in the sitting position [12.0%; BMI: 38.0]. The incidence of accidental subarachnoid puncture was 2%, 1.3% and 2% respectively, in these same positions. CONCLUSION: Adoption of the lateral recumbent head-down position for the performance of lumbar epidural blockade, in labour at term, reduces the incidence of lumbar epidural venous puncture in these obese parturients.