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Purpose of Review The olive tree (Olea europaea L.) has featured as a significant part of medicinal history, used to treat a variety of ailments within folk medicine. The Mediterranean diet, which is rich in olive products, is testament to Olea europaeas positive effects on health, associated with reduced incidences of cancer and cardiovascular disease. This review aims to summarise the current literature regarding the therapeutic potential of Olea europaea products in cancer, detailing the possible compounds responsible for its chemotherapeutic effects. Recent Findings Much of the existing research has focused on the use of cell culture models of disease, demonstrating Olea europaea extracts, and specific compounds within these extracts, have efficacy in a range of in vitro and in vivo cancer models. The source of Olea europaeas cytotoxicity is yet to be fully defined; however, compounds such as oleuropein and verbascoside have independent cytotoxic effects on animal models of cancer. Summary Initial results from animal models are promising but need to be translated to a clinical setting. Treatments utilising these compounds are likely to be well tolerated and represent a promising direction for future research.

Although silver nanoparticles (AgNPs) appear to be promising for certain medical/pharmaceutical applications, they present significant disadvantages when it comes to environmental applications due to the need for recovery of the metal (Ag) which is considered hazardous for the environment. The present study examines the antimicrobial properties and mechanism of action of immobilized (on Al2O3) silver nanoparticles’ (1 wt% Ag-iNPs) regarding the treatment of E. coli microbial solutions. Antimicrobial experiments were conducted in semi-batch mode using a three-phase continuous flow stirred tank reactor. Every treated sample was taken from the outlet of the reactor for the time intervals of 0, 5 and 25 min. To ensure that the bactericidal property of Ag-iNPs is not attributed to the dissolution of surface silver (as free Ag+), a suitable Ag+ scavenger was used as already described in our earlier studies. Regarding the molecular analysis performed under this study, the regulation of key enzymes involved in bactericidal activity of E. coli, was examined, after their treatment either with immobilized silver nanoparticles (Ag-iNPs) or AgNO3. Specifically, 50 mL sample for each case was centrifuged (10 min at 10,000 rpm) and the pellet (≈109 cells) was immediately subjected to total RNA extraction. For real-time RT-qPCR analyses, 1 μg of total RNA was converted into cDNA. It was found that PldA gene, which encodes outer membrane’s phospholipase A (OMPLA), was up-regulated after 5 and 25 min of treatment with Ag-iNPs. OMPLA’s activation appears to be the initial step of Ag-iNPs bactericidal mechanism that ultimately leads to the creation of holes on the outer membrane (OM), irreversibly disturbing the cells’ respiration cycle. In addition, after treatment with Ag-iNPs, Blue copper oxidase CueO (encoded by cueO gene) was found to be over-produced and appears to play a key-role in the oxidative transfer of silver from the surface of Ag-iNPs to the cell. This leads to the Ag-induced displacement of copper from its native protein sites such as CusS/CusR, a fact that causes the release of labile copper and ROS inside the cell. In addition, TEM analysis of bacteria treated with Ag-iNPs revealed severe morphological changes (“holes”) on bacterial outer membrane, indicating that there is an intermediate step in the proposed antimicrobial mechanism which takes place on the surface of Ag-iNPs.

Lycopene is a naturally occurring chemical compound that gives fruits and vegetables a red/orange color. As an intermediate metabolite of β-carotene biosynthesis, lycopene represents one of the major carotenoids. This work studies the gene expression pattern of lycopene biosynthesis/catabolism transcripts and the accumulation pattern of lycopene/β-carotene/chlorophyll contents at three developmental stages and three tissues in five widely consumed, commercial tomato cultivars (cvs. ‘Cherry Ninolino F1’, ‘Elpida F1’, ‘Daphne F1’, ‘Eliseo Plum F1’, and ‘Oxheart’). These contents were correlated with in vitro antioxidant activity. Higher levels of lycopene were observed in the peel than other tissues and in the fully ripe stage, with ‘Oxheart’ displaying the highest content. Differential regulation of lycopene biosynthetic genes (SlZDS and SlCRTISO) was demonstrated in all cultivars. Interestingly, a gene implicated in lycopene catabolism (SlLCYB) demonstrated a general induction in the tested tissues. High lycopene content correlates with significant induction of both SlZDS and SlCRTISO in ‘Oxheart’. Significantly increased antioxidant activity was observed in the peel of ‘Cherry Ninolino F1’ and ‘Elpida F1’ cultivars compared with other tested tissues across all cultivars. The exploration of lycopene activity in different cultivars and the consumption of ripe tomatoes including the peel could be useful for the protection of human health.

While imparting gastronomic novelty and sensory delight, microgreens also constitute rudimentary leafy greens packed with nutrients and phytochemicals. As such, they comprise an upcoming class of functional foods. However, apart from bioactive secondary metabolites, microgreens also accumulate antinutritive agents such as nitrate, especially under conducive protected cultivation conditions. The current work examined nutrient deprivation before harvest (DBH), applied by replacing nutrient solution with osmotic water for six and twelve days, as a strategy for reducing microgreen nitrate levels in different species (lettuce, mustard, and rocket). The three species were sown on a peat-based substrate, cultivated in a controlled climate chamber, and harvested 18 days after sowing, when the first two true leaves emerged. DBH impact on major constituents of the secondary metabolome, mineral content, colorimetric, and yield traits was appraised. Nitrate and mineral content were determined through ion chromatography, phenolic composition through UHPLC-Q-Orbitrap HRMS, and carotenoid composition through HPLC-DAD. Nutrient deprivation was effective in reducing nitrate content, however, effective treatment duration differed between species and decline was more precipitous in nitrate hyperaccumulating species such as rocket. Quercetin and kaempferol glycosides were the flavonol glycosides most abundant in brassicaceous microgreens, whereas lettuce microgreens were steeped in caffeoyl quinic acid. DBH interacted with species as it increased the total phenolic content of lettuce, decreased that of rocket, but did not affect mustard. Further research to link changes in phenolic composition to the sensory and in vivo bioactive profile of microgreens is warranted. Notably, brief (≤6 days) DBH can be applied across species with moderate or no impact on the phenolic, carotenoid, and mineral composition of microgreens. Brief DBH applications also have limited impact on microgreens’ yield and colorimetric traits hence on the commercial value of the product. They can therefore be applied for reducing microgreen nitrate levels without significantly impacting key secondary metabolic constituents and their potential bioactive role.

Six critical stages corresponding to major morphophysiological events in carob fruit ripening were defined, and changes in the primary and secondary metabolome and in vitro antioxidant capacity were examined in two genotypes collected at low (15 m) and high (510 m) altitudes from genetically identified and georeferenced trees. Soluble carbohydrates were analyzed by HPLC-RI, macro-minerals by ion chromatography coupled to conductivity detection and polyphenols by UHPLC-Q-Orbitrap-HRMS. spectroscopy facilitated assays for condensed tannins and in vitro free-radical scavenging capacity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power (FRAP). The fruit respiration rate and moisture content declined sharply during the transition from the breaker to green pedicel stage. Sugar accumulation spiked at the onset of fruit coloration and culminated at 498.7 &plusmn, 8.4 mg g&minus, 1 dry weight (dw) in the late ripe stage, while the ratio of reducing sugars to sucrose decreased from 3.45 &plusmn, 0.32 to 0.41 &plusmn, 0.02. The total phenolic compounds and condensed tannins declined with ripening, particularly during the transition from the breaker to green pedicel stage. Eighteen polyphenols were identified and quantitated, with catechins and hydrolyzable tannins being dominant until the onset of fruit coloration. The transition to the green pedicel stage signaled a precipitous decline (90.9%) in catechins, hydrolyzable tannins (60.2%) and flavonol glycosides (52.1%) concomitant to the rise in gallic acid, which was putatively fueled by the enzymatic hydrolysis of gallotannins in immature fruit. Catechins, hydrolyzable tannins and flavone glycosides were more abundant at higher altitudes and gallic acid at lower altitudes. An antioxidant capacity was also favored by higher elevations and declined with ripening, particularly after the breaker stage. Correlations with FRAP and DPPH assays were significant for the total phenolic content, condensed tannins, catechins and hydrolyzable tannins. The highest correlation factors were obtained for epigallocatechin-gallate (r = 0.920 and r = 0.900, p &lt, 0.01). Although the sharp drop in hydrolyzable and nonhydrolyzable tannins and catechins compromised the in vitro antioxidant capacity at physiological maturity, it also reduced the astringency and configured a palatable organoleptic fruit profile. These changes unraveled significant episodes in the ripening-related secondary metabolism of the carob fruit. They further highlighted the value of immature carob as a potent source of gallotannins, with putative in vivo anti-inflammatory action, and of catechins beneficial in preventing and protecting against diseases caused by oxidative stress.

Foliar application of a vegetal-derived protein hydrolysate as a biostimulant was assessed for possible interaction with the ripening of diploid watermelon grafted onto interspecific hybrid rootstock. Assessment encompassed crop performance, fruit morphometric and sensory quality traits, soluble carbohydrates, macrominerals, and bioactive composition at 10, 20, 30, 40, and 50 days post anthesis (dpa). The biostimulant effect on yield components was confounded by the vigorous rootstock effect. Pulp firmness declined precipitously with cell enlargement from 10 to 30 dpa, and the biostimulant phyto-hormonal potential on firmness and rind thickness was masked by grafting. Pulp colorimetry was determined solely by ripening and peaked at 40 dpa. The biostimulant effect reduced lycopene content by 8% compared to the control. Total sugars coevolved with soluble solids content, peaked at 30 dpa, and then stabilized. Fructose and glucose prevailed during rapid fruit growth from 10 to 30 dpa and sucrose prevailed at advanced ripeness between 40–50 dpa, whereas acidity peaked at 20 dpa and then decreased. Potassium, which was the most abundant micromineral, peaked before full ripeness at 30 dpa. The biostimulant effect on the watermelon fruit ripening process is not granted, at least regarding the conditions this study was carried out under. The absence of biostimulant effect might relate to rootstock vigorousness, the grafted watermelon physiology, or the type of biostimulant used.

Rising temperature and solar radiation drive the mobilization and depletion of crown-deposited metabolites harnessed for asparagus spear regeneration during the spring harvest season. We presently examined how successive same-season harvests impact the sensory, nutritive and bioactive composition of select green asparagus genotypes. Soluble carbohydrates were analyzed by HPLC-RI, organic acids and polyphenols by HPLC-DAD and metals by ion chromatography. Higher sugars and lower acids accentuated sweetness and lower polyphenols contributed to reduced astringency at the beginning of the harvest season. This trend was reversed as the season advanced and spear sensory quality was compromised by declining sugars and rising acids; however, functional quality improved as antioxidant capacity increased along with the concentrations of quercetin-3-O-rutinoside (rutin) and ascorbic acid. The compositional changes presently described were uniform across all genotypes examined and thus contribute toward our understanding of seasonal variation in the sensory and functional quality of this acclaimed health-promoting product.

Graphical abstract, Highlight • Τhe response of different Medicago truncatula genotypes to salt stress was examined. • Systems biology revealed tissue-specific metabolic and transcriptional signatures. • RFO metabolites linked with tolerance were identified. • Several genes belonging to the TIR-NBS-LRR class were linked with hyper-sensitivity., Salt stress is an important factor limiting plant productivity by affecting plant physiology and metabolism. To explore salt tolerance adaptive mechanisms in the model legume Medicago truncatula, we used three genotypes with differential salt-sensitivity: TN6.18 (highly sensitive), Jemalong A17 (moderately sensitive), and TN1.11 (tolerant). Cellular damage was monitored in roots and leaves 48 h after 200 mM NaCl treatment by measuring lipid peroxidation, nitric oxide, and hydrogen peroxide contents, further supported by leaf stomatal conductance and chlorophyll readings. The salt-tolerant genotype TN1.11 displayed the lowest level of oxidative damage, in contrast to the salt sensitive TN6.18, which showed the highest responses. Metabolite profiling was employed to explore the differential genotype-related responses to stress at the molecular level. The metabolic data in the salt tolerant TN1.11 roots revealed an accumulation of metabolites related to the raffinose pathway. To further investigate the sensitivity to salinity, global transcriptomic profiling using microarray analysis was carried out on the salt-stressed sensitive genotypes. In TN6.18, the transcriptomic analysis identified a lower expression of many genes related to stress signalling, not previously linked to salinity, and corresponding to the TIR-NBS-LRR gene class. Overall, this global approach contributes to gaining significant new insights into the complexity of stress adaptive mechanisms and to the identification of potential targets for crop improvement.

Trigonella foenum-graecum (fenugreek) is a leguminous plant that produces secondary metabolites frequently used in medicinal and food preparation. Here, the role of an arbuscular mycorrhizal (AM) fungal inoculum, alone or in combination with methyl jasmonate (MeJA) chemical treatment, was evaluated on the production of trigonelline and diosgenin in fenugreek plants grown under mild water limitation. After inoculation, fenugreek plants were subjected to several combined treatments, i.e. well-watered (NS) and water-stressed (WS), both treated with different MeJA concentrations (0, 50, 100, 200 mL/L). At the end of the experiment, in addition to trigonelline and diosgenin content, an array of plant growth parameters (i.e. fruit, stem and root dry weights, fruit number) and biochemical stress markers (i.e. proline, nitric oxide, hydrogen peroxide, MDA) were examined. Results mainly showed a significant increase in production of the considered secondary metabolites, although they are differentially influenced by the individual factors (Stress, SYM, MeJA) and their interactions. SYM treatment was more effective on the production of trigonelline, independently from growth conditions, while diosgenin biosynthesis was influenced by the presence of root symbioses only in combination with MeJA treatment. A differential impact, depending on the applied treatment (inoculum, MeJA treatments, water deficit), on plant growth and on production of the considered bioactive molecules was revealed, providing new information on the effect of biological and chemical priming treatments.

Land plants are continuously exposed to multiple abiotic stress factors like drought, heat, and salinity. Nitric oxide (NO) and hydrogen sulfide (H2S) are two well-examined signaling molecules that act as priming agents, regulating the response of plants to stressful conditions. Several chemical donors exist that provide plants with NO and H2S separately. NOSH is a remarkable novel donor as it can donate NO and H2S simultaneously to plants, while NOSH-aspirin additionally provides the pharmaceutical molecule acetylsalicylic acid. The current study aimed to investigate the potential synergistic effect of these molecules in drought-stressed Medicago sativa L. plants by following a pharmacological approach. Plants were initially pre-treated with both donors (NOSH and NOSH-aspirin) via foliar spraying, and were then subsequently exposed to a moderate water deficit while NO and H2S inhibitors (cPTIO and HA, respectively) were also employed. Phenotypic and physiological data showed that pre-treatment with NOSH synthetic compounds induced acclimation to subsequent drought stress and improved the recovery following rewatering. This was accompanied by modified reactive-oxygen and nitrogen-species signaling and metabolism, as well as attenuation of cellular damage, as evidenced by altered lipid peroxidation and proline accumulation levels. Furthermore, real-time RT-qPCR analysis revealed the differential regulation of multiple defense-related transcripts, including antioxidant enzymes. Overall, the present study proposed a novel role for NOSH compounds as efficient plant priming agents against environmental constraints through the coordinated regulation of multiple defense components, thus opening new horizons in the field of chemical priming research toward the use of target-selected compounds for stress tolerance enhancement.

Environmental stress is increasingly wearing down crop productivity. As a consequence, a major priority of plant research is to get deeper insights on tolerance mechanisms, providing solutions with the generation of stress-tolerant cultivars. To this goal, a common target of genetic modification is the polyamine signaling pathway. Its association with a protective role against stressful stimuli is widely acknowledged; however, the nature of this function is highly complex. In consequence, this review aims to present up-to-date evidence in regard with metabolic and physiological role of polyamines, protecting plants during severe stressful events (such as drought and soil salinization). The most recent evidence from stress physiology research highlight polyamines as key players in signaling responses involved in central metabolism, sugar and lipid homeostasis, maintenance, and induction of antioxidant capacity as well as osmotic regulation. Nevertheless, a number of questions remain open, such as the extent of their roles and whether they represent hub metabolic molecules. Recent advances on polyamine metabolism are therefore summarized in relation to salt and drought stress tolerance and its possible implication on the generation of tolerant crops. This review highlights recent findings related to polyamine protective role during drought and salt stress. A clear synergy is established between these amine compounds, ABA, and reactive oxygen and nitrogen species. However, the involvement of these amine compounds on stress physiology goes far beyond the modulation of nitro-oxidative homeostasis and ABA signaling, with an increasing body of evidence demonstrating that polyamines are emerging metabolic hubs of plant stress signaling.

Microgreens constitute novel gastronomic ingredients that combine visual, kinesthetic and bioactive qualities. The definition of the optimal developmental stage for harvesting microgreens remains fluid. Their superior phytochemical content against mature leaves underpins the current hypothesis of significant changes in compositional profile during the brief interval of ontogeny from the appearance of the first (S1) to the second true leaf (S2). Microgreens of four brassicaceous genotypes (Komatsuna, Mibuna, Mizuna and Pak Choi) grown under controlled conditions and harvested at S1 and S2 were appraised for fresh and dry yield traits. They were further analyzed for macro- and micromineral content using inductively coupled plasma optical emission spectrometry (ICP-OES), carotenoid content using high-performance liquid chromatography with a diode-array detector (HPLC-DAD), volatile organic compounds using solid-phase microextraction followed by gas chromatography-mass spectrometry (SPME-GC/MS), anthocyanins and polyphenols using liquid chromatography-high resolution-tandem mass spectrometry (LC-MS/MS) with Orbitrap technology and for chlorophyll and ascorbate concentrations, well as antioxidant capacity by spectrophotometry. Analysis of compositional profiles revealed genotype as the principal source of variation for all constituents. The response of mineral and phytochemical composition and of antioxidant capacity to the growth stage was limited and largely genotype-dependent. It is, therefore, questionable whether delaying harvest from S1 to S2 would significantly improve the bioactive value of microgreens while the cost-benefit analysis for this decision must be genotype-specific. Finally, the lower-yielding genotypes (Mizuna and Pak Choi) registered higher relative increase in fresh yield between S1 and S2, compared to the faster-growing and higher-yielding genotypes. Although the optimal harvest stage for specific genotypes must be determined considering the increase in yield against reduction in crop turnover, harvesting at S2 seems advisable for the lower-yielding genotypes.

Salinity constitutes one of the most important causes leading to severe reduction in plant yield. Several reports correlate the accumulation of polyamines in plants with tolerance to abiotic stress cues. The present study examined three Medicago truncatula genotypes with differing sensitivities to salinity (TN1.11, tolerant; Jemalong A17, moderately sensitive; TN6.18, sensitive), with the aim of examining the genotype-specific involvement of the polyamine metabolic pathway in plant response to salinity. The study was carried out with leaves harvested 48 h after watering plants with 200 mM NaCl. A comprehensive profile of free polyamines was determined using high performance liquid chromatography. All genotypes showed spermidine and spermine as the most abundant polyamines under control conditions. In salinity conditions, spermine levels increased at the expense of putrescine and spermidine, indicating a drift of polyamine metabolism towards the synthesis of increasing polycationic forms as a stress response. The increasing balance between high and low polycationic forms was clearly diminished in the salt-sensitive genotype TN6.18, showing a clear correlation with its sensitive phenotype. The polyamine metabolic profile was then supported by molecular evidence through the examination of polyamine metabolism transcript levels by RT-qPCR. General suppression of genes that are involved upstream in the PA biosynthetic pathway was determined. Contrarily, an induction in the expression of genes involved in the biosynthesis of spermine and spermidine was observed, in agreement with the metabolic analysis. A significant induction in diamino oxidase expression, involved in the catabolism of putrescine, was specifically found in the sensitive genotype ΤΝ6.18, indicating a distinct metabolic response to stress. Present findings highlight the involvement of polyamines in the defense response of Medicago genotypes showing sensitivity to salt stress.

Reactive oxygen, nitrogen and sulfur species are key signalling molecules involved in multiple physiological processes that can be examined in qualitative and quantitative manners. Here, we describe simple spectrophotometric assays that allow the quantification of hydrogen peroxide, nitrite-derived nitric oxide and hydrogen sulphide from plant tissues.

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 H2 O2 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.

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 150 μM concentration of sodium nitroprusside (SNP), a widely used NO donor, was sprayed on soybean seedlings for two days at 24 h intervals, followed by application of 200 mM NaCl. The relative water content (RWC), total chlorophyll content (CHL), stomatal conductance ( g s ), ABA content, malondialdehyde (MDA), hydrogen peroxide content (H 2 O 2 ), along with GST enzyme and isoenzyme activities and GST1 and GST4 transcript levels were determined at 0 h, 6 h and 12 h after stress imposition. The results indicated that salt treatment alone did not alter MDA, H 2 O 2 or ABA content and stomatal conductance in soybean leaves, most likely due to short-term (6 h and 12 h) 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 12 h) and H 2 O 2. 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 6 h. Conversely, SNP application lowered GST activity in soybean leaves at 0 h and 12 h, while it increased at 6 h, 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 6 h and 12 h, 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 6 h and 12 h, 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.

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.

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 2.5 mM 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.

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 and nitrogen radicals.

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.

Terrestrial plants are constantly exposed to multiple abiotic stress factors such as drought, salinity and heat. Nitric oxide (NO) and hydrogen sulfide (H 2 S) are two well-documented signaling molecules, which act as priming agents, regulating the response of plants under stress condition. Several donors exist which provide plants with NO and H 2 S separately. NOSH is a remarkable novel donor as it can provide plants simultaneously with NO and H 2 S, while NOSH– aspirin additionally provides the pharmaceutical molecule acetylsalicylic acid. The present study attempts to investigate the synergistic effect of these molecules in drought-stressed Medicago truncatula L. plants. Plants were initially pre-treated with both donors (NOSH and NOSH-aspirin) by vacuum infiltration, and were then subsequently exposed to moderate water deficit. Preliminary experiments examined the extent of cellular damage in leaves by determining lipid peroxidation (MDA content) and hydrogen peroxide (H 2 O 2 ) levels. Interestingly, drought-stressed plants pre-treated with either one of these compounds demonstrated significantly lower oxidative damage levels compared with non-primed stressed plants, with lowest cellular damage levels being recorded in NOSH-aspirin-treated plants. In addition, real-time RT-PCR analysis is currently underway, examining the expression of several defense-related genes including enzymatic and non-enzymatic antioxidants. Our results propose a novel role for NOSH-aspirin as a plant priming agent against abiotic stress factors, while further experiments are planned which will attempt to delve deeper in the modus operandi of this hybrid compound.