Original Contribution
Glutathione half-cell reduction potential: A universal stress marker and modulator of programmed cell death?

https://doi.org/10.1016/j.freeradbiomed.2006.02.013Get rights and content

Abstract

The elucidation of factors that contribute to cell viability loss is presently compromised by the lack of a universal measure that quantifies “stress.” We have investigated mechanisms of viability loss in plant seeds to find a reliable marker of stress response. Oxidative damage has previously been correlated with degenerative processes and death, but how exactly this contributes to viability loss is unknown. We show in four species subjected to ageing or desiccation that seed viability decreased by 50% when the half-cell reduction potential of glutathione (EGSSG/2GSH), a major cellular antioxidant and redox buffer, increased to −180 to −160 mV. We then conducted a metaanalysis of data representative of 13 plant and fungal orders to show that plant stress generally becomes lethal when EGSSG/2GSH exceeds −160 mV. We put forward that this change in EGSSG/2GSH is part of the signaling cascade that initiates programmed cell death (PCD), finally causing internucleosomal DNA fragmentation in the final, or execution phase, of PCD. EGSSG/2GSH is therefore a universal marker of plant cell viability and allows us to predict whether a seed will live, germinate, and produce a new plant, or if it will die.

Introduction

Ageing phenomena and programmed cell death (PCD) are typically studied in human or animal cells [1], [2] rather than in plants. However, the seeds of higher plants represent excellent models for the study of ageing because viability loss can be easily induced experimentally. “Orthodox” seeds [3] are desiccation tolerant and can be stored in the dry state. “Recalcitrant” seeds [3] are desiccation sensitive. Lethal damage, as judged by germination tests, can be induced by artificial ageing in orthodox, and by drying in recalcitrant seeds. The dynamics of viability loss in the dry state [4], [5] and underlying biophysical features [6] have previously received much attention. Weakening of the glassy state of the cytoplasm, combined with hydrolysis of sugars and the unleashing of an array of oxidative processes [4], [7], results in damage to lipids, proteins and DNA [6], [8], [9]. In particular the damage to macro-molecules strongly points at oxidative stress as the culprit [10], [11].

Reactive oxygen species (ROS) are produced as by-products of normal metabolism and they also play important roles in signaling [8], [9]. In “healthy” tissue, control of ROS is supported by ROS-processing enzymes such as superoxide dismutase, catalase, and peroxidases [10], [12] and by molecular antioxidants that donate electrons to scavenge ROS [11], [13], [14], [15]. Lipid-soluble, membrane-bound antioxidants include tocopherols and carotenes. The major water-soluble, cytoplasmatic molecular antioxidants are the sulfur-containing tripeptide glutathione (γ-glutamyl-cysteinyl-glycine; GSH) and ascorbate. Glutathione is also the most abundant low-molecular-weight thiol in plants. Its numerous functions in plants include the long-distance transport of reduced sulfur, stress signaling, and sequestering of heavy metals and xenobiotics [15], [16], [17], [18]. Most importantly, however, it is the major cellular thiol antioxidant and redox buffer [15], [19].

Oxidative stress [20] arises as a consequence of imbalances between prooxidative and antioxidative processes; re-reduction of antioxidants fails and the loss of the mechanisms that control ROS results in an increase in ROS-provoked deteriorative processes, ageing, and eventually death. Others have shown that ROS are involved in PCD signaling [8], [9] and that PCD is associated with changes in the intracellular “redox environment” [19], but the precise nature of signals that initiate PCD, often referred to as “cell death triggers,” is still under debate [21]. In addition, our knowledge of biochemical pathways that contribute to viability loss in plants is still limited, as is the availability of reliable stress markers.

Numerous attempts have been made to use antioxidants as stress markers [22]. Using antioxidant concentrations alone has limitations, because they often show a Gaussian response to stress [23], [24], with an initial increase followed by a breakdown, making the interpretation of a single measurement ambiguous. Correlations of the redox state of antioxidants with the physiological state of plant tissue are more satisfactory [23], [24], [25], [26]. In particular the ratios between the two components of the glutathione disulfide/glutathione (GSSG/2GSH) and the dehydroascorbate/ascorbate redox couples [15] are frequently used as markers of plant stress. However, the redox state of a concentration-dependent redox couple can be defined more accurately by its half-cell reduction potential and its reducing capacity [19]. For example, the redox state of the GSSG/2GSH couple can be defined by its half-cell reduction potential (EGSSG/2GSH) and GSH concentration (e.g., −180 mV; 3 mM).

A recently proposed correlation between EGSSG/2GSH and the physiological state of human cells [19] encouraged us to investigate the precise relationship between the viability of plant tissue and EGSSG/2GSH. After establishing a negative sigmoidal correlation between viability and EGSSG/2GSH in three orthodox seed systems subjected to ageing, and one recalcitrant seed system subjected to desiccation, we validated the use of EGSSG/2GSH as a viability marker on a wide range of plant species across 13 plant and fungal orders, including plant cells, tissues, and organs subjected to a variety of stress treatments. We then further hypothesized that the observed increase in EGSSG/2GSH is one of the “death triggers” that initiate PCD. We will present evidence that in ageing seeds DNA is cleaved into intranucleosomal fragments, which is a classic marker of PCD [1], and finally suggest a model of biochemical pathways that shows the possible involvement of EGSSG/2GSH in PCD.

Section snippets

Seed material, ageing, and germination treatments, and sampling

We used commercially available seeds of Pisum sativum L. Alaska Early, Lathyrus pratensis L., and Cytisus scoparius L. Seeds of Quercus robur L. were collected from the grounds of the Royal Botanic Gardens, Kew, Wakehurst Place. To remove physical dormancy in L. pratensis and C. scoparius, seed coats were chipped before experimental treatments; P. sativum seeds do not require this treatment as they do not display dormancy. All orthodox seeds were equilibrated for approximately 5 weeks in

Viability loss in model orthodox and recalcitrant seeds coincides with loss and oxidation of glutathione

We first investigated the concentrations of GSH and GSSG in an orthodox crop seed, Pisum sativum (garden pea), aged at 12 and 13% moisture content (MC; g water per 100 g seed). In general agreement with species-specific “viability constants” for seed ageing [5], a decrease in MC of 1% approximately doubled the life span (Figs. 1A and B), but the pattern of GSH conversion to GSSG was almost identical (Figs. 1A and B). A loss of 50% viability coincided with a 50% loss of total glutathione

Glutathione is a major, probably ancient, redox buffer and its half-cell reduction potential is a universal marker of viability

The major finding of this paper is that EGSSG/2GSH is a universal marker of plant viability. EGSSG/2GSH describes the redox state of the GSSG/GSH couple much more accurately than %GSSG (Fig. 2) and it is a superior marker of viability. In agreement with reports on human cells [19], EGSSG/2GSH shifts toward more positive values as plant tissue loses viability (Fig. 3). A shift of EGSSG/2GSH to the zone between −180 and −160 mV appears to trigger irreversible, deleterious changes (Fig. 2, Fig. 3)

Acknowledgments

We thank L. Castle, D.J. Osborne, C.H. Foyer, A. Lawen, and P.R. Crane for valuable comments on an early draft of the manuscript, K. Müller for help with the desiccation experiments on Quercus robur seeds, C.B. Wood and E. York for providing germination data for Uapaca kirkiana and Elephantorrhiza elephantina, respectively, and Defra for financial support (Grant CSA 6343). The Millennium Seed Bank Project is supported by the Millennium Commission, The Wellcome Trust, Orange Plc., and Defra. The

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