Genetic Manipulation of Reactive Oxygen Species (ROS) Homeostasis Utilizing CRISPR/Cas9-Based Gene Editing in Rice.

Reactive oxygen species (ROS) are now recognized as key signals in plant stress responses. Adverse environmental conditions can either promote ROS production or downregulate antioxidative enzymes, leading to the alteration of redox homeostasis and activation of ROS-linked stress signaling. To uncover their signaling mechanisms and to characterize related components, genetic modification of ROS homeostasis is a central approach. CRISPR/Cas9-based genome editing system has become a powerful tool for gene mutation in a variety of organisms, including plants. Within this chapter, we describe a method that can be applied to manipulate ROS homeostasis in rice (Oryza sativa L.) utilizing CRISPR/Cas9 technology. Step-by-step protocols including the design and construction of Cas9/sgRNA, agrobacterium-mediated transformation, and mutation characterization are described. Application of this system in editing a rice catalase gene CatC, a key antioxidative enzyme in controlling ROS homeostasis, is also presented.

Analyzing the Function of Catalase and the Ascorbate-Glutathione Pathway in H2O2 Processing: Insights from an Experimentally Constrained Kinetic Model.

SIGNIFICANCE: Plant stress involves redox signaling linked to reactive oxygen species such as hydrogen peroxide (H2O2), which can be generated at high rates in photosynthetic cells. The systems that process H2O2 include catalase (CAT) and the ascorbate-glutathione pathway, but interactions between them remain unclear. Modeling can aid interpretation and pinpoint areas for investigation. Recent Advances: Based on emerging data and concepts, we introduce a new experimentally constrained kinetic model to analyze interactions between H2O2, CAT, ascorbate, glutathione, and NADPH. The sensitivity points required for accurate simulation of experimental observations are analyzed, and the implications for H2O2-linked redox signaling are discussed. CRITICAL ISSUES: We discuss several implications of the modeled results, in particular the following. (i) CAT and ascorbate peroxidase can share the load in H2O2 processing even in optimal conditions. (ii) Intracellular H2O2 concentrations more than the low µM range may rarely occur. (iii) Ascorbate redox turnover is largely independent of glutathione until ascorbate peroxidation exceeds a certain value. (iv) NADPH availability may determine glutathione redox status through its influence on monodehydroascorbate reduction. (v) The sensitivity of glutathione status to oxidative stress emphasizes its potential suitability as a sensor of increased H2O2. FUTURE DIRECTIONS: Important future questions include the roles of other antioxidative systems in interacting with CAT and the ascorbate-glutathione pathway as well as the nature and significance of processes that achieve redox exchange between different subcellular compartments. Progress in these areas is likely to be favored by integrating kinetic modeling analyses into experimentally based programs, allowing each approach to inform the other.

Glutathione oxidation in response to intracellular H2O2: Key but overlapping roles for dehydroascorbate reductases.

Glutathione is a pivotal molecule in oxidative stress, during which it is potentially oxidized by several pathways linked to H2O2 detoxification. We have investigated the response and functional importance of 3 potential routes for glutathione oxidation pathways mediated by glutathione S-transferases (GST), glutaredoxin-dependent peroxiredoxins (PRXII), and dehydroascorbate reductases (DHAR) in Arabidopsis during oxidative stress. Loss-of-function gstU8, gstU24, gstF8, prxIIE and prxIIF mutants as well as double gstU8 gstU24, gstU8 gstF8, gstU24 gstF8, prxIIE prxIIF mutants were obtained. No mutant lines showed marked changes in their phenotype and glutathione profiles in comparison to the wild-type plants in either optimal conditions or oxidative stress triggered by catalase inhibition. By contrast, multiple loss of DHAR functions markedly decreased glutathione oxidation triggered by catalase deficiency. To assess whether this effect was mediated directly by loss of DHAR enzyme activity, or more indirectly by upregulation of other enzymes involved in glutathione and ascorbate recycling, we measured expression of glutathione reductase (GR) and expression and activity of monodehydroascorbate reductases (MDHAR). No evidence was obtained that either GRs or MDHARs were upregulated in plants lacking DHAR function. Hence, interplay between different DHARs appears to be necessary to couple ascorbate and glutathione pools and to allow glutathione-related signaling during enhanced H2O2 metabolism.

Cytosolic and Chloroplastic DHARs Cooperate in Oxidative Stress-Driven Activation of the Salicylic Acid Pathway.

The complexity of plant antioxidative systems gives rise to many unresolved questions. One relates to the functional importance of dehydroascorbate reductases (DHARs) in interactions between ascorbate and glutathione. To investigate this issue, we produced a complete set of loss-of-function mutants for the three annotated Arabidopsis (Arabidopsis thaliana) DHARs. The combined loss of DHAR1 and DHAR3 expression decreased extractable activity to very low levels but had little effect on phenotype or ascorbate and glutathione pools in standard conditions. An analysis of the subcellular localization of the DHARs in Arabidopsis lines stably transformed with GFP fusion proteins revealed that DHAR1 and DHAR2 are cytosolic while DHAR3 is chloroplastic, with no evidence for peroxisomal or mitochondrial localizations. When the mutations were introduced into an oxidative stress genetic background (cat2), the dhar1 dhar2 combination decreased glutathione oxidation and inhibited cat2-triggered induction of the salicylic acid pathway. These effects were reversed in cat2 dhar1 dhar2 dhar3 complemented with any of the three DHARs. The data suggest that (1) DHAR can be decreased to negligible levels without marked effects on ascorbate pools, (2) the cytosolic isoforms are particularly important in coupling intracellular hydrogen peroxide metabolism to glutathione oxidation, and (3) DHAR-dependent glutathione oxidation influences redox-driven salicylic acid accumulation.

Missing links in understanding redox signaling via thiol/disulfide modulation: how is glutathione oxidized in plants?

Glutathione is a small redox-active molecule existing in two main stable forms: the thiol (GSH) and the disulphide (GSSG). In plants growing in optimal conditions, the GSH:GSSG ratio is high in most cell compartments. Challenging environmental conditions are known to alter this ratio, notably by inducing the accumulation of GSSG, an effect that may be influential in the perception or transduction of stress signals. Despite the potential importance of glutathione status in redox signaling, the reactions responsible for the oxidation of GSH to GSSG have not been clearly identified. Most attention has focused on the ascorbate-glutathione pathway, but several other candidate pathways may couple the availability of oxidants such as H2O2 to changes in glutathione and thus impact on signaling pathways through regulation of protein thiol-disulfide status. We provide an overview of the main candidate pathways and discuss the available biochemical, transcriptomic, and genetic evidence relating to each. Our analysis emphasizes how much is still to be elucidated on this question, which is likely important for a full understanding of how stress-related redox regulation might impinge on phytohormone-related and other signaling pathways in plants.