Proline accumulation and antioxidant response are crucial for citrus tolerance to UV-B light-induced stress.

Plants face a wide range of biotic and abiotic stress conditions, which are further intensified by climate change. Among these stressors, increased irradiation in terms of intensity and wavelength range can lead to detrimental effects, such as chlorophyll degradation, destruction of the PSII reaction center, generation of ROS, alterations to plant metabolism, and even plant death. Here, we investigated the responses of two citrus genotypes, Citrus macrophylla (CM), and Troyer citrange (TC) to UV-B light-induced stress, by growing plants of both genotypes under control and UV-B stress conditions for 5 days to evaluate their tolerance mechanisms. TC seedlings had higher sensitivity to UV-B light than CM seedlings, as they showed more damage and increased levels of oxidative harm (indicated by the accumulation of MDA). In contrast, CM seedlings exhibited specific adaptive mechanisms, including accumulation of higher levels of proline under stressful conditions, and enhanced antioxidant capacity, as evidenced by increased ascorbate peroxidase activity and upregulation of the CsAPX2 gene. Phytohormone accumulation patterns were similar in both genotypes, with a decrease in ABA content in response to UV-B light. Furthermore, expression of genes involved in light perception and response was specifically affected in the tolerant CM seedlings, which exhibited higher expression of CsHYH/CsHY5 and CsRUP1-2 genes. These findings underscore the importance of the antioxidant system in citrus plants subjected to UV-B light-induced stress and suggest that CsHYH/CsHY5 and CsRUP1-2 could be considered genes associated with tolerance to such challenging conditions.

Quest for a stable Cu-ligand complex with a high catalytic activity to produce ROS.

Metal ion-catalysed overproduction of reactive oxygen species (ROS) are believed to contribute significantly to oxidative stress and be involved in several biological processes, from immune defence to development of diseases. Among the essential metal ions, copper is one of the most efficient catalysts in ROS production in the presence of O2 and a physiological reducing agent such as ascorbate. To control this chemistry, Cu ions are tightly coordinated to biomolecules. Free or loosely bound Cu ions are generally avoided to prevent their toxicity. In the present report, we aim to find stable Cu-ligand complexes (Cu-L) that can catalyse efficiently the production of ROS in presence of ascorbate under aerobic conditions. Thermodynamic stability would be needed to avoid dissociation in biological environment and high ROS catalysis is of interest for applications as in antimicrobial or anticancer agents. A series of Cu complexes with the well-known tripodal and tetradentate ligands containing a central amine linked to three pyridyl-alkyl arms of different lengths were investigated. The two of them with mixed armlength showed higher catalytic activity in oxidation of ascorbate and subsequent ROS production than Cu salts in buffer, which is an unprecedented result. Despite these high catalytic activities, no increased antimicrobial activity towards E. coli or cytotoxicity against eukaryotic AGS cells in culture related to Cu-L based ROS production could be observed. The potential reasons for discrepancy between in vitro and in cell data will be discussed.

Influence of brassinosteroid and silicon on growth, antioxidant enzymes, and metal uptake of leafy vegetables under wastewater irrigation.

Vegetable cultivation under sewage irrigation is a common practice mostly in developing countries due to a lack of freshwater. Long-term usage provokes heavy metals accumulation in soil and ultimately hinders the growth and physiology of crop plants and deteriorates the quality of food. A study was performed to investigate the role of brassinosteroid (BRs) and silicon (Si) on lettuce, spinach, and cabbage under lead (Pb) and cadmium (Cd) contaminated sewage water. The experiment comprises three treatments (control, BRs, and Si) applied under a completely randomized design (CRD) in a growth chamber. BRs and Si application resulted in the highest increase of growth, physiology, and antioxidant enzyme activities when applied under canal water followed by distilled water and sewage water. However, BRs and Si increased the above-determined attributes under the sewage water by reducing the Pb and Cd uptake as compared to the control. It's concluded that sewerage water adversely affected the growth and development of vegetables by increasing Pb and Cd, and foliar spray of Si and BRs could have great potential to mitigate the adverse effects of heavy metals and improve the growth. The long-term alleviating effect of BRs and Si will be evaluated in the field conditions at different ecological zones.

Comparative investigation on chemical and green synthesized titanium dioxide nanoparticles against chromium (VI) stress eliciting differential physiological, biochemical, and cellular attributes in Helianthus annuus L.

Nanotechnology is a new scientific area that promotes unique concepts to comprehend the optimal mechanics of nanoparticles (NPs) in plants under heavy metal stress. The present investigation focuses on effects of synthetic and green synthesized titanium dioxide nanoparticles (TiO2 NPs and gTiO2 NPs) against Cr(VI). Green TiO2 NPs have been produced from plant leaf extract (Ricinus communis L.). Synthesis was confirmed employing an array of optical spectroscopic and electron microscopic techniques. Chromium strongly accelerated H2O2 and MDA productions by 227 % and 266 % at highest chromium concentration (60 mg/kg of soil), respectively, and also caused DNA damage, and decline in photosynthesis. Additionally, anomalies were observed in stomatal cells with gradual increment in chromium concentrations. Conversely, foliar applications of TiO2 NPs and gTiO2 NPs considerably mitigated chromium stress. Sunflower plants treated with modest amounts of green TiO2 NPs had significantly better growth index compared to chemically synthesized ones. Principal component analysis highlighted the variations among photosynthetic attributes, oxidative stress markers, and antioxidant defense systems. Notably, gTiO2 supplementation to the Cr(VI) strained plants minimized PC3 production which is a rare report so far. Conclusively, gTiO2 NPs have been identified to be promising nano-based nutrition resource for farming applications.

Electrochemical Detection of Total Antioxidant Capacity (TAC) in Plant Tissues from Different Origins.

Total antioxidant capacity (TAC) is a reliable indicator of antioxidant content in animal and plant samples. The different experimental approaches available allow the determination of TAC using, as a reference, diverse compounds with recognized antioxidant capacities such as Trolox, ascorbic acid, gallic acid, or melatonin. A new portable device, named BRS (BQC redox system), is now commercially available that, through an electrochemical approach, allows the determination of TAC in a simple, fast, reproducible, and robust way. In this chapter, using this portable device, a comparative analysis of the TAC is assayed in different red, citrus, and Solanaceae fruits, several Allium species, and organs of different plant species, including Arabidopsis thaliana. The obtained results demonstrate the versatility of the BRS portable device.

Measurements of Ascorbate and Dehydroascorbate in Plants Using High-Performance Liquid Chromatography.

The current concepts emphasize the fundamental role of reactive oxygen species (ROS) as signaling molecules that coordinate defense mechanisms, cell death, and the growth and development processes in plants. However, due to the inherent reactivity of ROS, achieving precise control over their levels within plant cells, both spatially and temporally, becomes important to effectively harness the potential of ROS signaling while concurrently minimizing the risk of oxidative damage. Ascorbate is an exceptional antioxidant and contributes to the antioxidant defense system in plants. Its role is further reinforced by the presence of ascorbate peroxidases and enzymes responsible for recycling ascorbate from its oxidized forms. Ascorbate metabolism plays a pivotal role in averting oxidative damage and facilitates meticulous regulation of ROS signal availability. This chapter outlines the preferred protocol for the measurement of ascorbate.

Detection of Ascorbate Peroxidase (APX) Activity in Plant Tissues: Using Non-denaturing PAGE and Spectrophotometric Assay.

At the cellular level, the generation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), due to different abiotic or biotic stress, causes oxidative stress that induces an imbalance in the metabolism. Among the different H2O2-scavenging enzymatic antioxidants, ascorbate peroxidase (APX) is a heme-peroxidase that plays an important role in the ascorbate-glutathione pathway using ascorbate to reduce H2O2 to water. Using non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with a spectrophotometric assay for APX activity, the protocol allows identifying diverse APX isozymes present in different organs and plant species.

Genetics aspect of vitamin C (Ascorbic Acid) biosynthesis and signaling pathways in fruits and vegetables crops.

Vitamin C, also known as ascorbic acid, is an essential nutrient that plays a critical role in many physiological processes in plants and animals. In humans, vitamin C is an antioxidant, reducing agent, and cofactor in diverse chemical processes. The established role of vitamin C as an antioxidant in plants is well recognized. It neutralizes reactive oxygen species (ROS) that can cause damage to cells. Also, it plays an important role in recycling other antioxidants, such as vitamin E, which helps maintain the overall balance of the plant's antioxidant system. However, unlike plants, humans cannot synthesize ascorbic acid or vitamin C in their bodies due to the absence of an enzyme called gulonolactone oxidase. This is why humans need to obtain vitamin C through their diet. Different fruits and vegetables contain varying levels of vitamin C. The biosynthesis of vitamin C in plants occurs primarily in the chloroplasts and the endoplasmic reticulum (ER). The biosynthesis of vitamin C is a complex process regulated by various factors such as light, temperature, and plant hormones. Recent research has identified several key genes that regulate vitamin C biosynthesis, including the GLDH and GLDH genes. The expression of these genes is known to be regulated by various factors such as light, temperature, and plant hormones. Recent studies highlight vitamin C's crucial role in regulating plant stress response pathways, encompassing drought, high salinity, and oxidative stress. The key enzymes in vitamin C biosynthesis are L-galactose dehydrogenase (GLDH) and L-galactono-1, 4-lactone dehydrogenase (GLDH). Genetic studies reveal key genes like GLDH and GLDH in Vitamin C biosynthesis, offering potential for crop improvement. Genetic variations influence nutritional content through their impact on vitamin C levels. Investigating the roles of genes in stress responses provides insights for developing resilient techniques in crop growth. Some fruits and vegetables, such as oranges, lemons, and grapefruits, along with strawberries and kiwi, are rich in vitamin C. Guava. Papaya provides a boost of vitamin C and dietary fiber. At the same time, red and yellow bell peppers, broccoli, pineapple, mangoes, and kale are additional sources of this essential nutrient, promoting overall health. In this review, we will discuss a brief history of Vitamin C and its signaling and biosynthesis pathway and summarize the regulation of its content in various fruits and vegetables.

Characterization of cinnamate 4-hydroxylase (CYP73A) and p-coumaroyl 3'-hydroxylase (CYP98A) from Leucojum aestivum, a source of Amaryllidaceae alkaloids.

Biosynthesis of Amaryllidaceae alkaloids (AA) starts with the condensation of tyramine with 3,4-dihydroxybenzaldehyde. The latter derives from the phenylpropanoid pathway that involves modifications of trans-cinnamic acid, p-coumaric acid, caffeic acid, and possibly 4-hydroxybenzaldehyde, all potentially catalyzed by hydroxylase enzymes. Leveraging bioinformatics, molecular biology techniques, and cell biology tools, this research identifies and characterizes key enzymes from the phenylpropanoid pathway in Leucojum aestivum. Notably, we focused our work on trans-cinnamate 4-hydroxylase (LaeC4H) and p-coumaroyl shikimate/quinate 3'-hydroxylase (LaeC3'H), two key cytochrome P450 enzymes, and on the ascorbate peroxidase/4-coumarate 3-hydroxylase (LaeAPX/C3H). Although LaeAPX/C3H consumed p-coumaric acid, it did not result in the production of caffeic acid. Yeasts expressing LaeC4H converted trans-cinnamate to p-coumaric acid, whereas LaeC3'H catalyzed specifically the 3-hydroxylation of p-coumaroyl shikimate, rather than of free p-coumaric acid or 4-hydroxybenzaldehyde. In vivo assays conducted in planta in this study provided further evidence for the contribution of these enzymes to the phenylpropanoid pathway. Both enzymes demonstrated typical endoplasmic reticulum membrane localization in Nicotiana benthamiana adding spatial context to their functions. Tissue-specific gene expression analysis revealed roots as hotspots for phenylpropanoid-related transcripts and bulbs as hubs for AA biosynthetic genes, aligning with the highest AAs concentration. This investigation adds valuable insights into the phenylpropanoid pathway within Amaryllidaceae, laying the foundation for the development of sustainable production platforms for AAs and other bioactive compounds with diverse applications.

Temporal Changes in Biochemical Responses to Salt Stress in Three Salicornia Species.

Halophytes adapt to salinity using different biochemical response mechanisms. Temporal measurements of biochemical parameters over a period of exposure to salinity may clarify the patterns and kinetics of stress responses in halophytes. This study aimed to evaluate short-term temporal changes in shoot biomass and several biochemical variables, including the contents of photosynthetic pigments, ions (Na+, K+, Ca2+, and Mg2+), osmolytes (proline and glycine betaine), oxidative stress markers (H2O2 and malondialdehyde), and antioxidant enzymes (superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase) activities of three halophytic Salicornia species (S. persica, S. europaea, and S. bigelovii) in response to non-saline, moderate (300 mM NaCl), and high (500 mM NaCl) salinity treatments at three sampling times. Salicornia plants showed maximum shoot biomass under moderate salinity conditions. The results indicated that high Na+ accumulation in the shoots, coupled with the relative retention of K+ and Ca2+ under salt stress conditions, contributed significantly to ionic and osmotic balance and salinity tolerance in the tested Salicornia species. Glycine betaine accumulation, both constitutive and salt-induced, also seems to play a crucial role in osmotic adjustment in Salicornia plants subjected to salinity treatments. Salicornia species possess an efficient antioxidant enzyme system that largely relies on the ascorbate peroxidase and peroxidase activities to partly counteract salt-induced oxidative stress. The results also revealed that S. persica exhibited higher salinity tolerance than S. europaea and S. bigelovii, as shown by better plant growth under moderate and high salinity. This higher tolerance was associated with higher peroxidase activities and increased glycine betaine and proline accumulation in S. persica. Taking all the data together, this study allowed the identification of the biochemical mechanisms contributing significantly to salinity tolerance of Salicornia through the maintenance of ion and osmotic homeostasis and protection against oxidative stress.

A Module for Analyzing Interactomes via APEX-MS Integrated into PatternLab for Proteomics.

Proximity labeling techniques, such as APEX-MS, provide valuable insights into proximal interactome mapping; however, the verification of biotinylated peptides is not straightforward. With this as motivation, we present a new module integrated into PatternLab for proteomics to enable APEX-MS data interpretation by targeting diagnostic fragment ions associated with APEX modifications. We reanalyzed a previously published APEX-MS data set and report a significant number of biotinylated peptides and, consequently, a confident set of proximal proteins. As the module is part of the widely adopted PatternLab for proteomics software suite, it offers users a comprehensive, easy, and integrated solution for data analysis. Given the broad utility of the APEX-MS technique in various biological contexts, we anticipate that our module will be a valuable asset to researchers, facilitating and enhancing interactome studies. PatternLab's APEX, including a usage protocol, is available at http://patternlabforproteomics.org/apex.

Ascorbate-glutathione cycle in wheat and rice seedlings under anoxia and subsequent reaeration.

The most important part of the plant antioxidant system is the ascorbate-glutathione cycle (AGC), the activity of which is observed upon exposure to a range of stressors, including lack of O2, and oxidative stress occurring immediately after the restoration of oxygen access, hereafter termed reaeration or post-anoxia. The operation of the AGC (enzymes and low-molecular components) in wheat (Triticum aestivum, cv. Leningradka, non-resistant to hypoxia) and rice (Oryza sativa, cv. Liman, resistant) seedlings after 24 h anoxia and 1 h or 24 h reaeration was studied. Significant accumulation of oxidized forms of ascorbate and glutathione was revealed in the non-resistant plant (wheat) after 24 h of anoxia and reaeration, indicating the development of oxidative stress. In the resistant plant (rice), reduced forms of these antioxidants prevailed both in normoxia and under stress, which may indicate their intensive reduction. In wheat, the activities of ascorbate peroxidase and dehydroascorbate reductase in shoots, and monodehydroascorbate reductase and glutathione reductase in roots decreased under anoxia and reaeration. The activity of antioxidant enzymes was maintained in rice under lack of oxygen (ascorbate peroxidase, glutathione reductase) and increased during post-anoxia (AGC reductases). Anoxia stimulated accumulation of mRNA of the organellar ascorbate peroxidase genes OsAPX3, OsAPX5 in shoots, and OsAPX3-5 and OsAPX7 in roots. At post-anoxia, the contribution of the OsAPX1 and OsAPX2 genes encoding the cytosolic forms of the enzyme increased in the whole plant, and so did that of the OsAPX8 gene for the plastid form of the enzyme. The accumulation of mRNA of the genes OsMDAR2 and OsMDAR4 encoding peroxisomal and cytosolic monodehydroascorbate reductase as well as the OsGR2 and OsGR3 for cytosolic and organellar glutathione reductase was activated during reaeration in shoots and roots. In most cases, O2 deficiency activated the genes encoding the peroxisomal, plastid, and mitochondrial forms of the enzymes, and upon reaeration, an enhanced activity of the genes encoding the cytoplasmic forms was observed. Taken together, the inactivation of AGC enzymes was revealed in wheat seedlings during anoxia and subsequent reaeration, which disrupted the effective operation of the cycle and triggered the accumulation of oxidized forms of ascorbate and glutathione. In rice, anoxia led to the maintenance of the activity of AGC enzymes, and reaeration stimulated it, including at the level of gene expression, which ensured the effective operation of AGC.

Magnetic Resonance Spectroscopy (MRS) in Alzheimer's Disease.

MRS is a noninvasive technique to measure different metabolites in the brain. Changes in the levels of certain metabolites can be used as surrogate markers for Alzheimer's disease. They can potentially be used for diagnosis, prediction of prognosis, or even assessing response to treatment.There are different techniques for MRS acquisitions including STimulated Echo Acquisition Mode (STEAM) and Point Resolved Spectroscopy (PRESS). In terms of localization, single or multi-voxel methods can be used. Based on current data: 1. NAA, marker of neuronal integrity and viability, reduces in AD with longitudinal changes over the time as the disease progresses. There are data claiming that reduction of NAA is associated with tau accumulation, early neurodegenerative processes, and cognitive decline. Therefore, it can be used as a stage biomarker for AD to assess the severity of the disease. With advancement of disease modifying therapies, there is a potential role for NAA in the future to be used as a marker of response to treatment. 2. mI, marker of glial cell proliferation and activation, is associated with AB pathology and has early changes in the course of the disease. The NAA/mI ratio can be predictive of AD development with high specificity and can be utilized in the clinical setting to stratify cases for further evaluation with PET for potential treatments. 3. The changes in the level of other metabolites such as Chol, Glu, Gln, and GABA are controversial because of the lack of standardization of MRS techniques, current technical limitations, and possible region specific changes. 4. Ultrahigh field MRS and more advanced techniques can overcome many of these limitations and enable us to measure more metabolites with higher accuracy. 5. Standardization of MRS techniques, validation of metabolites' changes against PET using PET-guided technique, and longitudinal follow-ups to investigate the temporal changes of the metabolites in relation to other biomarkers and cognition will be crucial to confirm the utility of MRS as a potential noninvasive biomarker for AD.

Evolutionary insights into strategy shifts for the safe and effective accumulation of ascorbate in plants.

Plants accumulate high concentrations of ascorbate, commonly in their leaves, as a redox buffer. While ascorbate levels have increased during plant evolution, the mechanisms behind this phenomenon are unclear. Moreover, has the increase in ascorbate concentration been achieved without imposing any detrimental effects on the plants? In this review, we focus on potential transitions in two regulatory mechanisms related to ascorbate biosynthesis and the availability of cellular dehydroascorbate (DHA) during plant evolution. The first transition might be that the trigger for the transcriptional induction of VTC2, which encodes the rate-limiting enzyme in ascorbate biosynthesis, has shifted from oxidative stress (in green algae) to light/photosynthesis (in land plants), probably enabling the continuous accumulation of ascorbate under illumination. This could serve as a preventive system against the unpredictable occurrence of oxidative stress. The second transition might be that DHA-degrading enzymes, which protect cells from the highly reactive DHA in green algae and mosses, have been lost in ferns or flowering plants. Instead, flowering plants may have increased glutathione concentrations to reinforce the DHA reduction capacity, possibly allowing ascorbate accumulation and avoiding the toxicity of DHA. These potential transitions may have contributed to strategies for plants' safe and effective accumulation of ascorbate.

Enzymes Involved in Antioxidant and Detoxification Processes Present Changes in the Expression Levels of Their Coding Genes under the Stress Caused by the Presence of Antimony in Tomato.

Currently, there is an increasing presence of heavy metals and metalloids in soils and water due to anthropogenic activities. However, the biggest problem caused by this increase is the difficulty in recycling these elements and their high permanence in soils. There are plants with great capacity to assimilate these elements or make them less accessible to other organisms. We analyzed the behavior of Solanum lycopersicum L., a crop with great agronomic interest, under the stress caused by antimony (Sb). We evaluated the antioxidant response throughout different exposure times to the metalloid. Our results showed that the enzymes involved in the AsA-GSH cycle show changes in their expression level under the stress caused by Sb but could not find a relationship between the NITROSOGLUTATHIONE REDUCTASE (GSNOR) expression data and nitric oxide (NO) content in tomato roots exposed to Sb. We hypothesize that a better understanding of how these enzymes work could be key to develop more tolerant varieties to this kind of abiotic stress and could explain a greater or lesser phytoremediation capacity. Moreover, we deepened our knowledge about Glutathione S-transferase (GST) and Glutathione Reductase (GR) due to their involvement in the elimination of the xenobiotic component.

Function of the ascorbate peroxidase (APX) in fruits and their modulation by reactive species.

Ascorbate peroxidase (APX) is one of the enzymes of the ascorbate-glutathione cycle and is the key enzyme that breaks down H2O2 with the aid of ascorbate as an electron source, although other enzymes also break down H2O2 such as catalase, peroxiredoxins, among others. APX is present in all photosynthetic eukaryotes from algae to higher plants and at the cellular level, it is localized in all subcellular compartments where H2O2 is generated, including apoplast, cytosol, plastids, mitochondria, and peroxisomes, either in soluble form or attached to the organelle membranes. The APX activity can be modulated by various post-translational modifications (PTMs) including tyrosine nitration, S-nitrosation, persulfidation, and S-sulfenylation among others. This allows the connection of the H2O2 metabolism with other relevant signaling molecules such as NO and H2S thus building a complex coordination system. In both climacteric and non-climacteric fruits, APX plays a key role during the ripening process as well as during postharvest, since it participates in the regulation of both H2O2 and ascorbate levels affecting fruit quality. Currently, the exogenous application of molecules such as NO, H2S, H2O2, and more recently melatonin has been seen as a new alternative to maintain and extend the shelf life and quality of the fruits because these molecules can modulate APX activity as well as other antioxidant systems. Therefore, these molecules are being considered new biotechnological tools to improve crop quality in the horticultural industry.

Boron Reduced Copper Excess-Induced Oxidative Damage in Citrus sinensis by Modulating Reactive Oxygen Species and Methylglyoxal Formation and Their Detoxification Systems.

Citrus is mainly cultivated in acid soil with low boron (B) and high copper (Cu). In this study, Citrus sinensis seedlings were submitted to 0.5 (control) or 350 µM Cu (Cu excess or Cu exposure) and 2.5, 10, or 25 µM B for 24 weeks. Thereafter, H2O2 production rate (HPR), superoxide production rate (SAPR), malondialdehyde, methylglyoxal, and reactive oxygen species (ROS) and methylglyoxal detoxification systems were measured in leaves and roots in order to test the hypothesis that B addition mitigated Cu excess-induced oxidative damage in leaves and roots by reducing the Cu excess-induced formation and accumulation of ROS and MG and by counteracting the impairments of Cu excess on ROS and methylglyoxal detoxification systems. Cu and B treatments displayed an interactive influence on ROS and methylglyoxal formation and their detoxification systems. Cu excess increased the HPR, SAPR, methylglyoxal level, and malondialdehyde level by 10.9% (54.3%), 38.9% (31.4%), 50.3% (24.9%), and 312.4% (585.4%), respectively, in leaves (roots) of 2.5 µM B-treated seedlings, while it only increased the malondialdehyde level by 48.5% (97.8%) in leaves (roots) of 25 µM B-treated seedlings. Additionally, B addition counteracted the impairments of Cu excess on antioxidant enzymes, ascorbate-glutathione cycle, sulfur metabolism-related enzymes, sulfur-containing compounds, and methylglyoxal detoxification system, thereby protecting the leaves and roots of Cu-exposed seedlings against oxidative damage via the coordinated actions of ROS and methylglyoxal removal systems. Our findings corroborated the hypothesis that B addition alleviated Cu excess-induced oxidative damage in leaves and roots by decreasing the Cu excess-induced formation and accumulation of ROS and MG and by lessening the impairments of Cu excess on their detoxification systems. Further analysis indicated that the pathways involved in the B-induced amelioration of oxidative stress caused by Cu excess differed between leaves and roots.

Complex Formation between the Transcription Factor WRKY53 and Antioxidative Enzymes Leads to Reciprocal Inhibition.

The transcription factor WRKY53 of the model plant Arabidopsis thaliana is an important regulator of leaf senescence. Its expression, activity and degradation are tightly controlled by various mechanisms and feedback loops. Hydrogen peroxide is one of the inducing agents for WRKY53 expression, and a long-lasting intracellular increase in H2O2 content accompanies the upregulation of WRKY53 at the onset of leaf senescence. We have identified different antioxidative enzymes, including catalases (CATs), superoxide dismutases (SODs) and ascorbate peroxidases (APXs), as protein interaction partners of WRKY53 in a WRKY53-pulldown experiment at different developmental stages. The interaction of WRKY53 with these enzymes was confirmed in vivo by bimolecular fluorescence complementation assays (BiFC) in Arabidopsis protoplasts and transiently transformed tobacco leaves. The interaction with WRKY53 inhibited the activity of the enzyme isoforms CAT2, CAT3, APX1, Cu/ZuSOD1 and FeSOD1 (and vice versa), while the function of WRKY53 as a transcription factor was also inhibited by these complex formations. Other WRKY factors like WRKY18 or WRKY25 had no or only mild inhibitory effects on the enzyme activities, indicating that WRKY53 has a central position in this crosstalk. Taken together, we identified a new additional and unexpected feedback regulation between H2O2, the antioxidative enzymes and the transcription factor WRKY53.

Pharmacologic Ascorbate Radiosensitizes Pancreatic Cancer but Radioprotects Normal Tissue: The Role of Oxidative Stress-Induced Lipid Peroxidation.

The toxicity of ionizing radiation limits its effectiveness in the treatment of pancreatic ductal adenocarcinoma. Pharmacologic ascorbate (P-AscH-) has been shown to radiosensitize pancreatic cancer cells while simultaneously radioprotecting normal cells. We hypothesize that P-AscH- protects the small intestine while radiosensitizing pancreatic cancer cells partially through an oxidative stress mechanism. Duodenal samples from pancreaticoduodenectomy specimens of patients who underwent radio-chemotherapy ± P-AscH- and mouse tumor and jejunal samples treated with radiation ± P-AscH- were evaluated. Pancreatic cancer and non-tumorigenic cells were treated with radiation ± P-AscH- to assess lipid peroxidation. To determine the mechanism, pancreatic cancer cells were treated with selenomethionine or RSL3, an inhibitor of glutathione peroxidase 4 (GPx4). Radiation-induced decreases in villi length and increases in 4-HNE immunofluorescence were reversed with P-AscH- in human duodenum. In vivo, radiation-induced decreases in villi length and increased collagen deposition were reversed in P-AscH--treated jejunal samples. P-AscH- and radiation increased BODIPY oxidation in pancreatic cancer cells but not in non-tumorigenic cells. Selenomethionine increased GPx4 protein and activity in pancreatic cancer and reversed P-AscH--induced toxicity and lipid peroxidation. RSL3 treatment inhibited GPx4 activity and increased lipid peroxidation. Differences in oxidative stress may play a role in radioprotecting normal cells while radiosensitizing pancreatic cancer cells when treated with P-AscH-.

High-dose ascorbate exerts anti-tumor activities and improves inhibitory effect of carboplatin through the pro-oxidant function pathway in uterine serous carcinoma cell lines.

OBJECTIVE: Uterine serous carcinoma is a highly aggressive non-endometrioid subtype of endometrial cancer with poor survival rates overall, creating a strong need for new therapeutic strategies to improve outcomes. High-dose ascorbate (vitamin C) has been shown to inhibit cell proliferation and tumor growth in multiple preclinical models and has shown promising anti-tumor activity in combination with chemotherapy, with a favorable safety profile. We aimed to study the anti-tumor effects of ascorbate and its synergistic effect with carboplatin on uterine serous carcinoma cells. METHODS: Cell proliferation was evaluated by MTT and colony formation assays in ARK1, ARK2 and SPEC2 cells. Cellular stress, antioxidant ability, cleaved caspase 3 activity and adhesion were measured by ELISA assays. Cell cycle was detected by Cellometer. Invasion was measured using a wound healing assay. Changes in protein expression were determined by Western immunoblotting. RESULTS: High-dose ascorbate significantly inhibited cell proliferation, caused cell cycle arrest, induced cellular stress, and apoptosis, increased DNA damage, and suppressed cell invasion in ARK1 and SPEC2 cells. Treatment of both cells with 1 mM N-acetylcysteine reversed ascorbate-induced apoptosis and inhibition of cell proliferation. The combination of ascorbate and carboplatin produced significant synergistic effects in inhibiting cell proliferation and invasion, inducing cellular stress, causing DNA damage, and enhancing cleaved caspase 3 levels compared to each compound alone in both cells. CONCLUSIONS: Ascorbate has potent antitumor activity and acts synergistically with carboplatin through its pro-oxidant effects. Clinical trials of ascorbate combined with carboplatin as adjuvant treatment of uterine serous carcinoma are worth exploring.