Chrystalla Antoniou

Effect of drought and rewatering on the cellular status and antioxidant response of Medicago truncatula plants

Effects of water stress on plants have been well-documented. However, the combined responses to drought and rewatering and their underlying mechanisms are relatively unknown. The present study attempts to describe spatiotemporal alterations in the physiology and cellular status of Medicago truncatula tissues that result from and subsequently follow a period of moderate water deficit. Physiological processes and cellular damage levels were monitored in roots and leaves by determining lipid peroxidation levels, as well as nitric oxide and hydrogen peroxide content, further supported by stomatal conductance and chlorophyll fluorescence measurements in leaves. During water stress, cells in both organs displayed increased damage levels and reactive oxygen and nitrogen species content, while leaves showed reduced stomatal conductance. Furthermore, both tissues demonstrated increased proline content. Upon rewatering, plants recovered displaying readings similar to pre-stress control conditions. Furthermore, molecular analysis of antioxidant gene expression by quantitative real-time RT-PCR revealed differential spatiotemporal regulation in a number of genes examined (including catalase, cytosolic ascorbate peroxidase, copper/zinc and iron superoxide dismutase and alternative oxidase). Overall, M. truncatula plants demonstrated increased sensitivity to drought-induced oxidative damage; however, this was reversed following rewatering indicating a great elasticity in the plant’s capacity to cope with free oxygen radicals.

The nitric oxide donor sodium nitroprusside regulates polyamine and proline metabolism in leaves of Medicago truncatula plants.

Nitric oxide (NO), polyamines, and proline have all been suggested to play key roles in a wide spectrum of physiological processes and abiotic stress responses. Although exogenous application of polyamines has been shown to induce NO production, the effect of NO on polyamine biosynthesis has not yet been elucidated. Several reports exist that demonstrate the protective action of sodium nitroprusside (SNP), a widely used NO donor, which acts as a signal molecule in plants responsible for the regulation of the expression of many defense-related enzymes. This study attempted to provide a novel insight into the effects of application of low (100 μΜ) and high (2.5 mM) concentrations of SNP on the biosynthesis of two major abiotic stress response-related metabolites, polyamines and proline, in mature (40 day) and senescing (65 day) Medicago truncatula plants. Physiological data showed that long-term (24 h), higher SNP concentration resulted in decreased photosynthetic rate and stomatal conductance followed by intracellular putrescine and proline accumulation, as a result of an increase in biosynthetic arginine decarboxylase (ADC) and Δ(1) -pyrroline-5-carboxylate synthetase (P5CS) enzymatic activity, respectively. Further analysis of polyamine oxidase (PAO)/diamine oxidase (DAO) polyamine catabolic enzymes indicated that DAO enzymatic activity increased significantly in correlation with putrescine accumulation, whereas PAO activity, involved in spermidine/spermine degradation, increased slightly. Moreover, transcriptional analysis of polyamine and proline metabolism genes (P5CS, P5CR, ADC, SPMS, SPDS, SAMDC, PAO, DAO) further supported the obtained data and revealed a complex SNP concentration-, time-, and developmental stage-dependent mechanism controlling endogenous proline and polyamine metabolite production. This is the first report to provide a global analysis leading to a better understanding of the role of the widely used NO donor SNP in the regulation of key stress-related metabolic pathways.

Melatonin systemically ameliorates drought stress-induced damage in Medicago sativa plants by modulating nitro-oxidative homeostasis and proline metabolism

Recent reports have uncovered the multifunctional role of melatonin in plant physiological responses under optimal and suboptimal environmental conditions. In this study, we explored whether melatonin pretreatment could provoke priming effects in alfalfa (Medicago sativa L.) plants subsequently exposed to prolonged drought stress (7 days), by withholding watering. Results revealed that the rhizospheric application of melatonin (10 μmol L−1) remarkably enhanced the drought tolerance of alfalfa plants, as evidenced by the observed plant tolerant phenotype, as well as by the higher levels of chlorophyll fluorescence and stomatal conductance, compared with nontreated drought‐stressed plants. In addition, lower levels of lipid peroxidation (MDA content) as well as of both H2O2 and NO contents in primed compared with nonprimed stressed plants suggest that melatonin pretreatment resulted in the systemic mitigation of drought‐induced nitro‐oxidative stress. Nitro‐oxidative homeostasis was achieved by melatonin through the regulation of reactive oxygen (SOD, GR, CAT, APX) and nitrogen species (NR, NADHde) metabolic enzymes at the enzymatic and/or transcript level. Moreover, melatonin pretreatment resulted in the limitation of cellular redox disruption through the regulation of the mRNA levels of antioxidant and redox‐related components (ADH, AOX, GST7, GST17), as well via osmoprotection through the regulation of proline homeostasis, at both the enzymatic (P5CS) and gene expression level (P5CS, P5CR). Overall, novel results highlight the importance of melatonin as a promising priming agent for the enhancement of plant tolerance to drought conditions through the regulation of nitro‐oxidative and osmoprotective homeostasis.

Unravelling chemical priming machinery in plants: the role of reactive oxygen-nitrogen-sulfur species in abiotic stress tolerance enhancement.

Abiotic stresses severely limit crop yield and their detrimental effects are aggravated by climate change. Chemical priming is an emerging field in crop stress management. The exogenous application of specific chemical agents before stress events results in tolerance enhancement and reduction of stress impacts on plant physiology and growth. However, the molecular mechanisms underlying the remarkable effects of chemical priming on plant physiology remain to be elucidated. Reactive oxygen, nitrogen and sulfur species (RONSS) are molecules playing a vital role in the stress acclimation of plants. When applied as priming agents, RONSS improve stress tolerance. This review summarizes the recent knowledge on the role of RONSS in cell signalling and gene regulation contributing to abiotic stress tolerance enhancement.

NO loading: Efficiency assessment of five commonly used application methods of sodium nitroprusside in Medicago truncatula plants

Nitric oxide (NO) is a bioactive, diffusible molecule involved in a multitude of physiological and developmental processes in plants, which has been reported to display both antioxidant and pro-oxidant properties in plants. Several reports exist highlighting the protective action of sodium nitroprusside (SNP), an NO donor, which demonstrate its important role as a signal molecule in plants responsible for the expression regulation of antioxidant and other defense enzymes. However, the mode of application of this compound varies greatly between studies. The present study provides a comprehensive efficiency comparison of the most commonly used application methods using 2.5mM SNP on mature (40 day) Medicago truncatula plants. Measurement of NO content in both leaves and roots suggests that vacuum infiltration is the most efficient method for NO donation in leaf tissue, whereas hydroponic application resulted in highest NO content in roots. NO content correlated with activity levels of nitrate reductase (NR; EC 1.7.99.4), a key enzyme involved in the generation of NO in plants and which is known to be regulated by NO itself.

Stress-related phenomena and detoxification mechanisms induced by common pharmaceuticals in alfalfa (Medicago sativa L.) plants

Pharmaceutically active compounds (PhACs) have been recently shown to exert phytotoxic effects. The present study explores the uptake, systemic translocation, and abiotic stress responses and detoxification mechanisms induced by the exposure of alfalfa plants grown in sand under greenhouse conditions to four common, individually applied PhACs (10μgL(-1)) (diclofenac, sulfamethoxazole, trimethoprim, 17a-ethinylestradiol) and their mixture. Stress physiology markers (lipid peroxidation, proline, H2O2 and NO content, antioxidant activity assays) and gene expression levels of key plant detoxification components (including glutathione S-transferases, GST7, GST17; superoxide dismutases, CuZnSOD, FeSOD; proton pump, H(+)-ATP, and cytochrome c oxidase, CytcOx), were evaluated. PhACs were detected in significantly higher concentrations in roots compared with leaves. Stress related effects, manifested via membrane lipid peroxidation and oxidative burst, were local (roots) rather than systemic (leaves), and exacerbated when the tested PhACs were applied in mixture. Systemic accumulation of H2O2 in leaves suggests its involvement in signal transduction and detoxification responses. Increased antioxidant enzymatic activities, as well as upregulated transcript levels of GST7, GST17, H(+)-ATPase and CytcOx, propose their role in the detoxification of the selected PhACs in plants. The current findings provide novel biochemical and molecular evidence highlighting the studied PhACs as an emerging abiotic stress factor, and point the need for further research on wastewater flows under natural agricultural environments.

Developmental stage- and concentration-specific sodium nitroprusside application results in nitrate reductase regulation and the modification of nitrate metabolism in leaves of Medicago truncatula plants

Nitric oxide (NO) is a bioactive molecule involved in numerous biological events that has been reported to display both pro-oxidant and antioxidant properties in plants. Several reports exist which demonstrate the protective action of sodium nitroprusside (SNP), a widely used NO donor, which acts as a signal molecule in plants responsible for the expression regulation of many antioxidant enzymes. This study attempts to provide a novel insight into the effect of application of low (100 μΜ) and high (2.5 mM) concentrations of SNP on the nitrosative status and nitrate metabolism of mature (40 d) and senescing (65 d) Medicago truncatula plants. Higher concentrations of SNP resulted in increased NO content, cellular damage levels and reactive oxygen species (ROS) concentration, further induced in older tissues. Senescing M. truncatula plants demonstrated greater sensitivity to SNP-induced oxidative and nitrosative damage, suggesting a developmental stage-dependent suppression in the plant’s capacity to cope with free oxygen and nitrogen radicals. In addition, measurements of the activity of nitrate reductase (NR), a key enzyme involved in the generation of NO in plants, indicated a differential regulation in a dose and time-dependent manner. Furthermore, expression levels of NO-responsive genes (NR, nitrate/nitrite transporters) involved in nitrogen assimilation and NO production revealed significant induction of NR and nitrate transporter during long-term 2.5 mM SNP application in mature plants and overall gene suppression in senescing plants, supporting the differential nitrosative response of M. truncatula plants treated with different concentrations of SNP.

Interplay between GST and nitric oxide in the early response of soybean (Glycine max L.) plants to salinity stress.

Glutathione-s-transferases (GSTs) and nitric oxide (NO) have both been implicated in the response of plants to salinity stress. However, their interplay and underlying mechanisms are relatively unknown. The present study attempts to provide new insight into the time course effects of NO application on GST biosynthesis regulation in Glycine max L. leaves under salt stress. A 150μM concentration of sodium nitroprusside (SNP), a widely used NO donor, was sprayed on soybean seedlings for two days at 24h intervals, followed by application of 200mM NaCl. The relative water content (RWC), total chlorophyll content (CHL), stomatal conductance (gs), ABA content, malondialdehyde (MDA), hydrogen peroxide content (H2O2), along with GST enzyme and isoenzyme activities and GST1 and GST4 transcript levels were determined at 0h, 6h and 12h after stress imposition. The results indicated that salt treatment alone did not alter MDA, H2O2 or ABA content and stomatal conductance in soybean leaves, most likely due to short-term (6h and 12h) application, although lower RWC and CHL were recorded. SNP treatment alone increased ABA content and reduced stomatal conductance, but did not change RWC, CHL, MDA (except at 12h) and H2O2. However, exogenous SNP application protected soybean leaves from salt stress by increasing RWC, CHL and ABA content, as well as by lowering stomatal conductance in order to maintain water balance. A significant increase in GST activity was recorded under salt stress alone at 6h. Conversely, SNP application lowered GST activity in soybean leaves at 0h and 12h, while it increased at 6h, supported by GST isoenzyme activities. Thus, it could be suggested that exogenous NO application induced GST activity in an ABA-dependent manner, while GST activity could also be induced by salt stress independent of ABA. In addition, SNP pre-treatment in salt-stressed seedlings lowered GST activity at 6h and 12h, in line with the GST isoenzyme expression profile. Finally, GST1 and GST4 transcript levels were significantly induced in both salt-stressed and SNP pre-treated and subsequently stressed samples at 6h and 12h, while a more variable regulation pattern was observed in plants treated only with SNP. Overall, our findings suggest that both NO and salt stress act as potent regulators of GST gene and enzyme expression through both ABA-dependent and independent pathways.

REVIEW ARTICLE Hydrogen sulphide: a versatile tool for the regulation of growth and defence responses in horticultural crops

Summary The improvement of plant growth and productivity under biotic and abiotic stress conditions, as well as the reduction of post-harvest losses, are of paramount significance to meet the increasing demand for food by a growing World population. Hydrogen sulphide (H2S) has recently appeared as a key contributor towards this goal through its bioactive role in the regulation of plant defence responses. In this review, we provide an up-to-date overview of recent literature concerning the biosynthesis and regulation of H2S within plant cells, as well as its involvement in a series of plant physiological processes. More specifically, H2S is actively associated with several horticultural plant growth and developmental processes, tolerance responses following exposure to stress factors, as well as in post-harvest fruit physiology.The H2S-mediated enhancement of tolerance to several abiotic stress factors is highlighted, with particular emphasis on the priming effects of H2S on the anti-oxidant capacity, redox regulation and signalling, and transcriptional regulation of cellular defence components in plants. Moreover,H2S has been reported to be associated with seed germination, increased growth, and root organogenesis, while also enhancing the post-harvest performance and anti-oxidant capacity of fruit.The present review proposes a practical role for H2S in horticultural crop physiology, further supporting the notion that H2S acts as a key signalling molecule in plants, possibly acting by “cross-talk” with other secondary plant cell messengers.

Kresoxim-methyl primes Medicago truncatula plants against abiotic stress factors via altered reactive oxygen and nitrogen species signalling leading to downstream transcriptional and metabolic readjustment

Highlight The fungicide kresoxim-methyl displays novel priming properties against key abiotic stress factors (drought and salinity) by modifying reactive oxygen and nitrogen species signalling, inducing osmoprotection through increased proline biosynthesis and suppressing proteolysis.