Activities Of Reactive Oxygen Species Scavenging Enzymes Research Articles

α-Tocopherol Long-Chain Metabolite α-T-13'-COOH Exhibits Biphasic Effects on Cell Viability, Induces ROS-Dependent DNA Damage, and Modulates Redox Status in Murine RAW264.7 Macrophages.

The α-tocopherol long-chain metabolite α-tocopherol-13'-hydroxy-chromanol (α-T-13'-COOH) is a proposed regulatory intermediate of endogenous vitamin E metabolism. Effects of α-T-13'-COOH on cell viability and adaptive stress response are not well understood. The present study aims to investigate the concentration-dependent effects of α-T-13'-COOH on cellular redox homeostasis, genotoxicity, and cytotoxicity in murine RAW264.7 macrophages as a model system. Murine RAW264.7 macrophages are exposed to various dosages of α-T-13'-COOH to determine its regulatory effects on reactive oxygen species (ROS) production, DNA damage, expression of stress-related markers, and the activity of ROS scavenging enzymes including superoxide dismutases, catalase, and glutathione-S-transferases. The impact on cell viability is assessed by analyzing cell proliferation, cell cycle arrest, and cell apoptosis. α-T-13'-COOH influences ROS production and induces DNA damage in a dose-dependent manner. The metabolite modulates the activity of ROS-scavenging enzymes, with significant changes observed in the activities of antioxidant enzymes. A biphasic response affecting cell viability is noted: sub-micromolar doses of α-T-13'-COOH promote cell proliferation and enhance DNA synthesis, whereas supraphysiological doses lead to DNA damage and cytotoxicity. It hypothesizes an adaptive stress response, characterized by upregulation of ROS detoxification mechanisms, enhanced cell cycle arrest, and increased apoptosis, indicating a correlation with oxidative stress and subsequent cellular damage.

Regulation of plum fruit cracking process during cold storage period by treatment of preharvest chitosan

Cracking fruit is a serious quality change phenomenon in the storage and transportation of postharvest plum fruit. To reduce the cracking rate of plum fruit during cold storage, the 'Guofeng No.7′ plum was sprayed with chitosan with 1.5% concentration 30d from preharvest. The postharvest plum fruit was stored at (0 ± 0.5) °C and (85–90) % relative humidity. The results showed that chitosan treatment could reduce the reactive oxygen species (ROS) level of plum fruit, effectively delaying the fruit cracking. Compared with the control, chitosan treated fruits inhibited the increase of free radical content such as O2−· and H2O2 and delayed the increase of membrane permeability and the accumulation of MDA (from 71.9 mmol/g to 53.5 mmol/g). It also regulates lipoxygenase (LOX), peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) (enzymatic activity from 34.15 g/min to 42.21 g/min) and ascorbate peroxidase (APX) activity and gene expression during fruit cracking to maintain membrane integrity and intracellular compartmentalization, thereby increasing the crack tolerance of plum fruit during cold storage. These results indicate that preharvest chitosan treatment can inhibit the metabolism of active oxygen species, increase the activity of ROS scavenging enzymes and gene expression, and delay the postharvest cracking of plum fruit.

ROS Detection and Quantification in Plant-Nematode Interactions.

Reactive oxygen species (ROS) accumulation is one of the earliest hallmarks upon successful pathogen recognition in plants. H2O2 is considered the most important ROS in plant defense considering its relatively high stability and capacity to cross long distances in the plant. However, ROS also play roles in cell development and could hence facilitate nematode feeding site development. Several methods to analyze the cellular redox state exist, among which ROS detection and quantification and the evaluation of ROS scavenging enzyme activity (peroxidase activity, catalase activity, etc.). Here, we describe DAB staining, which is used to detect and localize ROS in planta upon an external trigger. Furthermore, ROS quantification using the FOX assay is described. Both methods have been used extensively in research and yield repeatable results in various plants.

Effects of Reactive Oxygen Levels on Chilling Injury and Storability in 21 Apricot Varieties from Different Production Areas in China.

The key factors for resistance to chilling injury in apricot fruits were obtained by analyzing the low-temperature storage characteristics of 21 varieties of apricot fruits in the main producing areas of China. Twenty-one varieties of apricots from different production areas in China were stored at 0 °C for 50 d and then shelved at 25 °C. The storage quality, chilling injury, reactive oxygen species (ROS), antioxidant ability, and contents of bioactive substances of the apricots were measured and analyzed. The results showed that the 21 varieties of apricot fruits could be divided into two categories according to tolerance during low-temperature storage, where there was chilling tolerance and lack of chilling tolerance. Eleven varieties of apricots, of which Xiangbai and Yunbai are representative, suffered from severe chilling injury after cold storage and shelf life. After 50 d of storage at 0 °C, the levels of superoxide anions and hydrogen peroxide accumulated in the 11 varieties of apricots with a lack of chilling tolerance during storage were significantly higher than those in the remaining 10 varieties of apricots with chilling tolerance. In addition, the activities of ROS scavenging enzymes, represented by superoxide dismutase, catalase and peroxidase, were significantly decreased in 11 varieties of apricots with a lack of chilling tolerance during storage. The contents of bioactive substances with ROS scavenging ability, represented by ascorbic acid, total phenols, carotenoids, and total flavonoids, also significantly decreased. The 10 varieties of apricots, of which Akeximixi and Suanmao are representative, were less affected by chilling injury because the production and removal of ROS were maintained at normal levels, avoiding the damaging effects of ROS accumulation in the fruit. In addition, the 10 apricot varieties with chilling tolerance during storage had higher sugar and acid contents after harvest. This could supply energy for physiological metabolism during cold storage and provide carbon skeletons for secondary metabolism, thus enhancing the chilling tolerance of the fruits. Based on the results of cluster analysis combined with the geographical distribution of the 21 fruit varieties, it was found that apricot varieties with chilling tolerance during storage were all from the northwestern region of China where diurnal temperature differences and rapid climate changes occur. In conclusion, maintaining the balance of ROS production and removal in apricots during cold storage is a key factor to enhance the storage tolerance of apricots. Moreover, apricots with higher initial glycolic acid and bioactive substance contents are less susceptible to chilling injury.

ROS accumulation-induced tapetal PCD timing changes leads to microspore abortion in cotton CMS lines

BackgroundCytoplasmic male sterility (CMS) is the basis of heterosis exploitation. CMS has been used to hybrid production in cotton, but its molecular mechanism remains unclear. CMS is associated with advanced or delayed tapetal programmed cell death (PCD), and reactive oxygen species (ROS) may mediate this process. In this study, we obtained Jin A and Yamian A, two CMS lines with different cytoplasmic sources.ResultsCompared with maintainer Jin B, Jin A anthers showed advanced tapetal PCD with DNA fragmentation, producing excessive ROS which accumulated around the cell membrane, intercellular space and mitochondrial membrane. The activities of peroxidase (POD) and catalase (CAT) enzymes which can scavenge ROS were significantly decreased. However, Yamian A tapetal PCD was delayed with lower ROS content, and the activities of superoxide dismutase (SOD) and POD were higher than its maintainer. These differences in ROS scavenging enzyme activities may be caused by isoenzyme gene expressions. In addition, we found the excess ROS generated in Jin A mitochondria and ROS overflow from complex III might be the source in parallel with the reduction of ATP content.ConclusionROS accumulation or abrogation were mainly caused by the joint action of ROS generation and scavenging enzyme activities transformation, which led to the abnormal progression of tapetal PCD, affected the development of microspores, and eventually contributed to male sterility. In Jin A, tapetal PCD in advance might be caused by mitochondrial ROS overproduction, accompanied by energy deficiency. The above studies will provide new insights into the cotton CMS and guide the follow-up research ideas.

Salinity effects on the activities of ROS scavenging enzymes in leaves of two sweet potato clones

AbstractSweet potato production, particularly in coastal areas is often prone to salinity. Salt‐tolerant clones will be needed to maintain production, but to date, little is known about salt tolerance traits in sweet potato. Salt stress may result in excessive uptake of unwanted ions into plant tissues leading to the formation of reactive oxygen species (ROS), which in turn may destroy membranes and reduce photosynthesis and growth. Antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), glutathione reductase (GR) and ascorbate peroxidase (APX) scavenge ROS and early changes in the activities of such enzymes could be used to identify salinity tolerant genotypes. Therefore, cuttings of two contrasting cultivars of sweet potato, BARI SP 8 (tolerant) and BARI SP 4 (sensitive) were greenhouse‐cultivated in nutrient solution for 21 days and then exposed to 100 mmol NaCl for 7 days. Three, five and seven days after salt application the youngest leaves were sampled individually and enzyme activities, potassium (K) and sodium (Na) concentrations, and SPAD (as a proxy for chlorophyll content) were determined. In both varieties leaf growth was not affected by salinity and young leaves grown under salinity had higher SPAD values than older leaves. Na concentration increased over time, particularly in earlier and in older leaves, whereas K was reduced in younger leaves. In general, enzyme activities were strongly affected by leaf age and leaf position. SOD and APX showed varietal but no salinity effects, CAT increased under salinity in both varieties, whereas POX was strongly reduced and GR was strongly increased under salinity in BARI SP 8 with no effect in BARI SP 4. Enzyme activities were not correlated to leaf Na, neither in relation to leaf age, nor leaf number or duration of salt stress in both varieties. However, varietal differences were observed regarding leaf K. Activities of SOD were highly positive and of CAT highly negatively correlated with leaf K under salinity in BARI SP 8 but not in BARI SP 4, whereas activities of GR and POX were strongly positively correlated with leaf K in BARI SP 4 under salinity but not in BARI SP 8. We conclude that potassium may have a strong regulating role on leaf stress levels and therefore on the activities of antioxidant enzymes. Varieties may differ in their tolerance strategy and we have shown that salinity does not generally increase levels of ROS‐scavenging enzymes in sweet potato leaves under salt stress. Confounding factors such as leaf age and leaf position as well as maintaining high leaf level K concentrations need to be considered when evaluating metabolic traits for salinity tolerance traits.

Exogenous melatonin alleviates apple replant disease by regulating rhizosphere soil microbial community structure and nitrogen metabolism

Apple replant disease (ARD) is a common soil-borne disease afflicting apple plants. Melatonin is a broad-spectrum oxygen scavenger that plays a key role in alleviating stress-induced damage in plants. In this study, we aimed to determine whether adding melatonin to replant soil can promote plant growth by improving the rhizosphere soil environment and nitrogen metabolism. In replant soil, chlorophyll synthesis was blocked, reactive oxygen species (ROS) accumulated in large quantities, and membrane lipid peroxidation was aggravated; this eventually resulted in slow plant growth. However, the application of 200 μM exogenous melatonin enhanced the tolerance of plants to ARD by up-regulating the expression of antioxidant enzyme-related genes and increasing ROS scavenging enzyme activity. Exogenous melatonin also increased the absorption and utilization of 15N by increasing the expression of nitrogen absorption genes and the activity of nitrogen metabolism enzymes. Exogenous melatonin enhanced the soil microbial environment by promoting soil enzyme activity and bacterial richness and decreasing the abundance of several harmful fungi in rhizosphere soil. Mantel test results showed that soil properties (except for AP) and growth indexes were positively correlated with the rate of 15N absorption and utilization. Spearman correlation analysis showed that the above factors were closely related to the richness and diversity of bacteria and fungi, indicating that the composition of microbial communities might play a key role in mediating change in the soil environment and thus affect nutrient absorption and growth. These findings provide new insights into how melatonin enhances ARD tolerance.

OsLPXC negatively regulates tolerance to cold stress via modulating oxidative stress, antioxidant defense and JA accumulation in rice.

Exposure of crops to low temperature (LT) during emerging and reproductive stages influences their growth and development. In this study, we have isolated a cold induced, nucleus-localized lipid A gene from rice named OsLPXC, which encodes a protein of 321amino acids. Knockout of OsLPXC resulted in enhance sensitivity to LT stress in rice, with increased accumulation of reactive oxygen species (ROS), malondialdehyde and electrolyte leakage, while expression and activities of antioxidant enzymes were significantly suppressed. The accumulation of chlorophyll content and net photosynthetic rate of knockout plants were also decreased compared with WT under LT stress. The functional analysis of differentially expressed genes (DEGs), showed that numerous genes associated with antioxidant defense, photosynthesis, cold signaling were solely expressed and downregulated in oslpxc plants compared with WT under LT. The accumulation of methyl jasmonate (MeJA) in leave and several DEGs related to the jasmonate biosynthesis pathway were significantly downregulated in OsLPXC knockout plants, which showed differential levels of MeJA regulation in WT and knockout plants in response to cold stress. These results indicated that OsLPXC positively regulates cold tolerance in rice via stabilizing the expression and activities of ROS scavenging enzymes, photosynthetic apparatus, cold signaling genes, and jasmonate biosynthesis.

TaBAS1 encoding a typical 2-Cys peroxiredoxin enhances salt tolerance in wheat.

Efficient antioxidant enzymatic system contributes to salt tolerance of plants via avoiding ROS over-accumulation. Peroxiredoxins are crucial components of the reactive oxygen species (ROS) scavenging machinery in plant cells, but whether they offer salt tolerance with potential for germplasm improvement has not been well addressed in wheat. In this work, we confirmed the role of a wheat 2-Cys peroxiredoxin gene TaBAS1 that was identified through the proteomic analysis. TaBAS1 overexpression enhanced the salt tolerance of wheat at both germination and seedling stages. TaBAS1 overexpression enhanced the tolerance to oxidative stress, promoted the activities of ROS scavenging enzymes, and reduced ROS accumulation under salt stress. TaBAS1 overexpression promoted the activity of ROS production associated NADPH oxidase, and the inhibition of NADPH oxidase activity abolished the role of TaBAS1 in salt and oxidative tolerance. Moreover, the inhibition of NADPH-thioredoxin reductase C activity erased the performance of TaBAS1 in the tolerance to salt and oxidative stress. The ectopic expression of TaBAS1 in Arabidopsis exhibited the same performance, showing the conserved role of 2-Cys peroxiredoxins in salt tolerance in plants. TaBAS1 overexpression enhanced the grain yield of wheat under salt stress but not the control condition, not imposing the trade-offs between yield and tolerance. Thus, TaBAS1 could be used for molecular breeding of wheat with superior salt tolerance.

Astragaloside IV, as a potential anticancer agent.

Cancer is a global intractable disease, and its morbidity and mortality are increasing year by year in developing countries. Surgery and chemotherapy are often used to treat cancer, but they result in unsatisfactory outcomes, such as severe side effects and drug resistance. With the accelerated modernization of traditional Chinese medicine (TCM), an increasing body of evidence has shown that several TCM components have significant anticancer activities. Astragaloside IV (AS-IV) is considered the main active ingredient of the dried root of Astragalus membranaceus. AS-IV exhibits various pharmacological effects, such as anti-inflammatory, hypoglycemic, antifibrotic, and anticancer activities. AS-IV possesses a wide range of activities, such as the modulation of reactive oxygen species-scavenging enzyme activities, participation in cell cycle arrest, induction of apoptosis and autophagy, and suppression of cancer cell proliferation, invasiveness, and metastasis. These effects are involved in the inhibition of different malignant tumors, such as lung, liver, breast, and gastric cancers. This article reviews the bioavailability, anticancer activity, and mechanism of AS-IV and provides suggestions for further research of this TCM.

Molecular, biochemical, and comparative genome analysis of a rhizobacterial strain Klebsiella Sp. KBG6.2 imparting salt stress tolerance to Oryza sativa L.

One-third of global agricultural land is affected by salinity, thereby limiting the, productivity and sustainability of the cropping ecosystem. Rice is a globally important, economical crop but is largely affected by soil salinity , leading to loss of standing crop. Plant growth-promoting rhizobacteria (PGPR) can be a potential solution to this problem by improving soil fertility, tolerance to stress and ultimately enhancing crop yield. However, the usage of PGPR in alleviating stress is limited and requires further investigation. In the current work, a PGPR strain, namely, Klebsiella sp. KBG6.2 was isolated from the rice rhizosphere of salt-affected agricultural fields in Odisha, India., The strain was found to be imparting salt tolerance to rice seedlings grown up to 200 mM of sodium chloride. The rice seedlings when treated with sodium chloride and coinoculated with Klebsiella sp. KBG6.2 showed better germination percentage, biomass, shoot, root length, chlorophyll content, and reducing sugar accumulation. The PGPR treatment also led to a reduction in reactive oxygen species (ROS) production, as evidenced by reduced ROS scavenging enzyme (catalase, superoxide dismutase , and peroxidase) activities and lower accumulation of the corresponding mRNA as, verified by real time PCR analysis. This was further supported by the reduced, accumulation of malondialdehyde which is an indicator of stress-induced oxidative damage. The genome sequence analysis of the Klebsiella sp. KBG6.2 strain revealed, it to contain the required genes responsible for colonization and acclimation during stress conditions. The successful root colonization was further verified by scanning electron microscopy. Further, the obtained PGPR strain could provide saline stress tolerance to rice and serve as bioinoculant for sustainable agriculture in salt-affected areas. • Isolation and characterization of a potential PGPR strain named Klebsiella sp. KBG6.2. • The PGPR strain Klebsiella sp. KBG6.2 could be used to impart saline stress tolerance to rice. • A successful strain in maintaining cellular reactive oxygen species homeostasis.

IbTLD modulates reactive oxygen species scavenging and DNA protection to confer salinity stress tolerance in tobacco

Plants accumulate reactive oxygen species (ROS) that may damage the cells under prolonged stress conditions. Reduction of the excessive ROS production can alleviate oxidative damage and enhance the survival rates under stress. TLDc–containing protein (TLD) was reported to confer tolerance to oxidative stress, but the regulatory mechanism of TLD remains unclear. In this study, we ectopically overexpressed the Ipomoea batatas TLDc gene (IbTLD) in tobacco and characterized its functions. RNA-sequencing analysis and Gene Ontology term enrichment analysis revealed that IbTLD up-regulates auxin-responsive genes in response to oxidative stress. Under salinity stress, the IbTLD transgenic lines showed higher germination rates, chlorophyll contents, and root lengths than wild type (W38). In addition, the IbTLD transgenic lines showed higher expression of ROS scavenging genes, nudix hydrolases, ROS scavenging enzyme activity, and lesser DNA damage compared to W38 under salinity stress. Therefore, our results suggest that IbTLD activates the expression of ROS scavenging genes and confers tolerance to salinity stress in planta.

ALL-TRANS RETINOIC ACID ALLEVIATES ARSENIC-INDUCED ENDOCRINE DISRUPTION IN SWISS ALBINO MICE

Exposure to endocrine-disrupting chemicals (EDCs), such as arsenic, leads to severe health problems and surreptitiously accentuates stressful conditions in humans. Additionally, arsenic-induced endocrine stress leads to severe disturbances in glucose metabolism, mostly by disrupting the structure-function adroitness of the adrenal, thyroid and pancreas. Mechanistically, elevated levels of reactive oxygen species (ROS) generated by arsenic accentuate perturbation of cellular redox balance and eventually cell death. All-trans retinoic acid (ATRA), an active metabolite of vitamin A, is known for its anti-oxidant properties. Therefore, ATRA was used as a protection against arsenic-induced deteriorations of physiological conditions in mice. The present study reveals arsenic induced ROS generation in the adrenal and thyroid glands, accompanied by a decline in the activities of ROS scavenging enzymes, leading to disruption of the architecture of the tissues and induction of apoptosis therein, culminating in an imbalance in the hormonal secretions from the respective tissues. Structural damage to the pancreas accompanied with severe imbalances in glucose metabolism and associated biochemical parameters like glucose, pancreatic amylase and liver glycogen were also noted. Treatment with ATRA could efciently reverse the deleterious effects induced by arsenic. Hence, ATRA can be used as an efcient nutraceutical which can lead to attenuation of endocrine stress induced by arsenic.

HPR1 Is Required for High Light Intensity Induced Photorespiration in Arabidopsis thaliana.

High light intensity as one of the stresses could lead to generation of large amounts of reactive oxygen species (ROS) in plants, resulting in severe plant growth retardation. The photorespiration metabolism plays an important role in producing and removing a variety of ROS, maintaining the dynamic balance of the redox reaction, and preventing photoinhibition. Arabidopsis hydroxypyruvate reductase 1 (HPR1) is a primary metabolic enzyme in the photorespiration cycle. However, the role of HPR1 in plants response to high light is not clear. Here, we found that the expression of HPR1 could be induced by high light intensity. The growth and photosynthetic capacity of hpr1 mutants are seriously affected under high light intensity. The absence of HPR1 suppresses the rates of photorepair of Photosystem II (PSII), aggravates the production of ROS, and accelerates photorespiration rates. Moreover, the activity of ROS scavenging enzymes in the hpr1 mutants is significantly higher. These results indicate that HPR1 is involved in plant response to high light intensity and is essential for maintaining the dynamic balance of ROS and photorespiration.

Pretreatment with high oxygen controlled atmosphere enhanced fresh‐cut white mushroom (Agaricus bisporus) quality via activating wounding stress defenses

High oxygen treatment has been proven to be effective in fresh-cut white mushroom preservation, however, the preservation effect and possible mechanisms in high oxygen controlled atmosphere pretreatment (HOCAP) on wounding stress are incompletely understood. In this study, based on the time chosen of HOCAP research, whole white mushrooms treated with 3h HOCAP (80% O2 + 20% CO2 ) and the wounding resistant responses of their slices were mainly investigated through phenylpropane pathway, reactive oxygen species (ROS) scavenging system, and ascorbate-glutathione (AsA-GSH) cycle. Results showed that 3h HOCAP can induce the production of hydrogen peroxide (H2 O2 ) and superoxide anion (O2 -• ) in the early stage, as well as the NADPH oxidase activity. Enzymes and endogenous antioxidants involved in ROS scavenging were enhanced by HOCAP during the whole storage. Besides, HOCAP maintained high level of phenylalanine ammonia-lyase (PAL) activity, enhanced the content of total phenolic and lignin, accelerated the AsA-GSH cycle. The results demonstrated that HOCAP induced defense responses by increasing the ROS in the early stage which stimulated the activities of ROS scavenging enzymes, along with the capability of increasing for wounding stress defense and resistance. This study provides a theoretical pretreatment technology for fresh-cut white mushroom preservation. © 2021 Society of Chemical Industry.

Pt-Fe3O4, Pd-Fe3O4, and Au-Fe3O4 Nanoheterodimers and Their Efficacy as Radiosensitizers in Cancer Therapy.

The X-radiation enhancing effect of caffeic acid-functionalized Au-Fe3O4, Pt-Fe3O4, and Pd-Fe3O4 nanoheterodimers (NHDs) on 2D and 3D breast tumor (MCF-7) and healthy breast epithelial (MCF-10A) cells was comprehensively examined by performing cell viability, reactive oxygen species (ROS) detection, enzyme activity, and clonogenic assays. Intracellular NHDs were observed to cause DNA fragmentation and to enhance superoxide and hydroxyl radical formation in MCF-7 cells when exposed to a single dose of 1 Gy. MCF-7-derived multicellular tumor spheroids (MCF-7 MCTS) and MCF-10A-derived multicellular spheroids (MCF-10A MCS) were incubated with the NHDs and irradiated with five daily doses of 2 Gy. The Au-Fe3O4 and Pt-Fe3O4 NHD-loaded MCF-7 MCTS significantly decreased in volume after being exposed to fractionated irradiation, whereas intracellular Au-Fe3O4 and Pt-Fe3O4 NHDs promoted the growth of the irradiated MCF-10A MCS. Moreover, Au-Fe3O4 and Pt-Fe3O4 NHDs were shown to impede ROS formation and inhibit DNA strand breakage in the noncancerous MCF-10A cells. On the other hand, the Pd-Fe3O4 NHDs boosted X-radiation-induced damage of MCF-10 A cells, which was ascribed to impairment of the ROS scavenging enzyme activities. In summary, caffeic acid-functionalized Au-Fe3O4 and Pt-Fe3O4 NHDs performed as excellent X-radiation enhancing agents in breast tumor cells, while they protect healthy breast epithelial cells against X-radiation.

OsNAC109 regulates senescence, growth and development by altering the expression of senescence- and phytohormone-associated genes in rice

Key messageWe demonstrate that OsNAC109 regulates senescence, growth and development via binding to the cis-element CNTCSSNNSCAVG and altering the expression of multiple senescence- and hormone-associated genes in rice.The NAC family is one of the largest transcripton factor families in plants and plays an essential role in plant development, leaf senescence and responses to biotic/abiotic stresses through modulating the expression of numerous genes. Here, we isolated and characterized a novel yellow leaf 3 (yl3) mutant exhibiting arrested-growth, increased accumulation of reactive oxygen species (ROS), decreased level of soluble proteins, increased level of malondialdehyde (MDA), reduced activities of ROS scavenging enzymes, altered expression of photosynthesis and senescence/hormone-associated genes. The yellow leaf and arrested-growth trait was controlled by a single recessive gene located to chromosome 9. A single nucleotide substitution was detected in the mutant allele leading to premature termination of its coding protein. Genetic complementation could rescue the mutant phenotype while the YL3 knockout lines displayed similar phenotype to WT. YL3 was expressed in all tissues tested and predicted to encode a transcriptional factor OsNAC109 which localizes to the nucleus. It was confirmed that OsNAC109 could directly regulate the expression of OsNAP, OsNYC3, OsEATB, OsAMTR1, OsZFP185, OsMPS and OsGA2ox3 by targeting to the highly conserved cis-element CNTCSSNNSCAVG except OsSAMS1. Our results demonstrated that OsNAC109 is essential to rice leaf senescence, growth and development through regulating the expression of senescence- and phytohormone-associated genes in rice.

Olive leaf extract impairs mitochondria by pro-oxidant activity in MDA-MB-231 and OVCAR-3 cancer cells

Breast and ovarian cancers are the leading and fifth reason for tumor death among females, respectively. Recently, many studies demonstrated antiproliferative activities of natural aliments in cancer. In this study, we investigated the antitumor potential of Olive Leaf Extract (OLE) in triple-negative breast and ovarian cancer cells.A HPLC/DAD analysis on OLE has been performed to assess the total polyphenolics and other secondary metabolites content. HCEpiC, MDA-MB-231, and OVCAR-3 cell lines were used. MTS, Cytofluorimetric, Western Blot analysis were performed to analyze cell viability, cell proliferation, apoptosis, and oxidative stress. Fluorimetric and IncuCyte® analyses were carried out to evaluate apoptosis and mitochondrial function.We confirmed that OLE, containing a quantity of oleuropein of 87 % of the total extract, shows anti-proliferative and pro-apoptotic activity on MDA-MB-231 cells. For the first time, our results indicate that OLE inhibits OVCAR-3 cell viability inducing cell cycle arrest, and it also increases apoptotic cell death up-regulating the protein level of cleaved-PARP and caspase 9. Moreover, our data show that OLE treatment causes a significant decrease in mitochondrial functionality, paralleled by a reduction of mitochondrial membrane potential. Interestingly, OLE increased the level of intracellular and mitochondrial reactive oxygen species (ROS) together with a decreased activity of ROS scavenging enzymes, confirming oxidative stress in both models. Our data demonstrate that mitochondrial ROS generation represented the primary mechanism of OLE antitumor activity, as pretreatment with antioxidant N-acetylcysteine prevented OLE-induced cell cycle arrest and apoptosis.

Responses of AG1 and AG2 QTL introgression lines and seed pre-treatment on growth and physiological processes during anaerobic germination of rice under flooding

Rice seeds germinating in flooded soils encounter hypoxia or even anoxia leading to poor seed germination and crop establishment. Introgression of AG1 and AG2 QTLs associated with tolerance of flooding during germination, together with seed pre-treatment via hydro-priming or presoaking can enhance germination and seedling growth in anaerobic soils. This study assessed the performance of elite lines incorporating AG1, AG2 and their combination when directly seeded in flooded soils using dry seeds. The QTLs were in the background of two popular varieties PSB Rc82 and Ciherang-Sub1, evaluated along with the donors Kho Hlan On (AG1) and Ma-Zhan Red (AG2) and recipient parents PSB Rc82 and Ciherang-Sub1. In one set of experiments conducted in the greenhouse, seedling emergence, growth, and carbohydrate mobilization from seeds were assessed. Metabolites associated with reactive oxygen species (ROS) scavenging including malondialdehyde (MDA) as a measure of lipid peroxidation, ascorbate, total phenolic concentration (TPC), and activities of ROS scavenging enzymes were quantified in seeds germinating under control (saturated) and flooded (10 cm) soils. In another set of experiments conducted in a natural field with 3–5 cm flooding depths, control and pretreated seeds of Ciherang-Sub1 introgression lines and checks were used. Flooding reduced seedling emergence of all genotypes, though emergence of AG1 + AG2 introgression lines was greater than the other AG lines. Soluble sugars increased, while starch concentration decreased gradually under flooding especially in the tolerant checks and in AG1 + AG2 introgression lines. Less lipid peroxidation and higher α-amylase activity, higher ascorbate (RAsA) and TPC were observed in the tolerant checks and in the AG1 + AG2 introgression lines. Lipid peroxidation correlated negatively with ascorbate, TPC, and with ROS scavengers. Seed hydro-priming or pre-soaking increased emergence by 7–10% over that of dry seeds. Introgression of AG2 and AG1 + AG2 QTLs with seed pretreatment showed 101–153% higher emergence over dry seeds of intolerant genotypes in the field. Lines carrying AG1 + AG2 QTLs showed higher α-amylase activity, leading to rapid starch degradation and increase in soluble sugars, ascorbate, and TPC, together leading to higher germination and seedling growth in flooded soils. Seed hydro-priming or pre-soaking for 24 h also improved traits associated with flooding tolerance. Combining tolerance with seed management could therefore, improve crop establishment in flooded soils and encourage large-scale adoption of direct seeded rice system.

Rice (Oryza sativa) Receptor for Activated C Kinase1B (OsRACK1B) Regulates Chlorophyll Catabolism and NADPH oxidase‐dependent H2O2 Signaling Pathways during Salinity Stress

The Receptor for Activated C Kinase1B (RACK1B) is a WD‐40 type scaffold protein that regulates diverse environmental stress signal transduction pathways. Arabidopsis RACK1A has been reported to interact with various proteins in salt stress and Light‐Harvesting Complex (LHC) pathways. However, the mechanism of how RACK1 contributes to the photosystem and chlorophyll metabolism in stress conditions remains elusive. Using T‐DNA‐mediated activation tagging transgenic rice (Oryza sativa L.) lines, we show that leaves from rice RACK1B gene (OsRACK1B) gain‐of‐function (OsRACK1B‐OX) plants exhibit the stay‐green phenotype by stabilizing chlorophyll under salinity stress. In contrast, leaves from down‐regulated OsRACK1B (OsRACK1B‐UX) plants display an accelerated yellowing phenotype accompanied by chlorophyll degradation. qRT‐PCR analysis revealed that genes encoding chlorophyll catabolic enzymes (CCEs) are differentially expressed in both OsRACK1B‐OX and OsRACK1B‐UX rice plants. In addition to CCEs, STAY‐GREEN (SGR) is a key component that forms the SGR‐CCE complex in senescing chloroplasts, which causes LHCII complex instability. Transcript and protein profiling revealed a significant upregulation of OsSGR in RACK1B‐UX plants than that in RACK1B‐OX plants during salt stress. The BiFC assay demonstrated that OsRACK1B interacts with OsSGR in planta in both cytoplasm and the nucleus. Our findings suggest that overexpression of OsRACK1B negatively regulates chlorophyll degradation, delays salinity‐induced senescence and OsRACK1B functions in this process, at least in part, by antagonizing the function of OsSGR through a direct interaction. Interestingly, histochemical staining revealed a global increase of hydrogen peroxide (H2O2) in plants overexpressing OsRACK1B compared to that of the wild‐type and the trend can be reversed by pre‐treatment leaves with diphenylidonium (DPI), a NADPH oxidase (NOX) inhibitor. In gel‐activity of NOX and Reactive Oxygen Species (ROS) scavenging enzymes revealed that OsRACK1B regulates NOX and ROS scavenging enzyme activity in a salt stress dependent manner indicating a complex interplay and a novel regulatory module involving OsRACK1, NOX dependent H2O2 signaling, and a balanced ROS scavenging enzyme activity. BiFC analysis demonstrated that RACK1 also interacts with N‐terminal region of NOX at the plasma membrane. We hypothesize that elevated level of H2O2 acts as a signaling molecule to counteract salt stress and chlorophyll catabolism in OsRACK1B transgenic leaves under salinity stress. Taken together, these results provide important insights into the molecular mechanisms of chlorophyll catabolism, salinity‐induced senescence which can be useful in circumventing the effect of salt on photosynthesis and in reducing yield penalty of important cereal crops, like rice, in global climate change conditions.Support or Funding InformationThis work was partially supported by National Science Foundation (NSF), USA grant to Howard University (Grant number MCB 0542312); Dept. of Biology and Graduate School of the Howard University.Proposed working model of how OsRACK1B delays chlorophyll breakdown through regulation of Stay‐Green (SGR) protein and Rboh/NADPH‐Oxidase‐dependent H2O2 signaling to cope with high salinity stress.OsRACK1B localizes in the nucleus and the cytoplasm in rice. During abiotic stresses such as high salinity, OsRACK1B directly or indirectly represses the transcripts of chlorophyll catabolic genes and activates defense‐related genes in the nucleus. After synthesized in the cytosol, OsRACK1B shuttles to the chloroplast where it binds to the Stay‐Green (SGR) protein and limits the availability of SGR to form SGR‐CCE‐LHCII multi‐protein complex, a prerequisite for chlorophyll breakdown. Simultaneously, OsRACK1B interacts with the N‐terminal region of Rboh/NADPH oxidase in the cytoplasm and regulates the NADPH oxidase‐dependent H2O2 production via the action of CuZn‐SOD resulting in a ROS dependent systemic wave inside the cell. At a certain level, H2O2 can act as a signaling molecule to generate a positive feedback loop for chloroplast homeostasis and may trigger retrograde signaling to regulate nuclear gene expression to protect the chloroplast and enhance stress tolerance of the plant. High salinity stress also induces stress phytohormone abscisic acid (ABA). OsRACK1B can also protect the photosystem complex through negative regulation of ABA signaling pathway. Solid and dotted lines indicate actual and hypothetical regulations of the signaling effect, respectively.Figure 1