Nitroblue Tetrazolium Staining Research Articles

The isoleucic acid triad: distinct impacts on plant defense, root growth, and formation of reactive oxygen species.

Isoleucic acid (ILA), a branched-chain amino acid-related 2-hydroxycarboxylic acid, occurs ubiquitously in plants. It enhances pathogen resistance and inhibits root growth of Arabidopsis. The salicylic acid (SA) glucosyltransferase UGT76B1 is able to conjugate ILA. Here, we investigate the role of ILA in planta in Arabidopsis and reveal a triad of distinct responses to this small molecule. ILA synergistically co-operates with SA to activate SA-responsive gene expression and resistance in a UGT76B1-dependent manner in agreement with the observed competitive ILA-dependent repression of SA glucosylation by UGT76B1. However, ILA also shows an SA-independent stress response. Nitroblue tetrazolium staining and pharmacological experiments indicate that ILA induces superoxide formation of the wild type and of an SA-deficient (NahG sid2) line. In contrast, the inhibitory effect of ILA on root growth is independent of both SA and superoxide induction. These effects of ILA are specific and distinct from its isomeric compound leucic acid and from the amino acid isoleucine. Leucic acid and isoleucine do not induce expression of defense marker genes or superoxide production, whereas both compounds inhibit root growth. All three responses to ILA are also observed in Brassica napus.

Manganese Superoxide Dismutase Activity Assay in the Yeast Saccharomyces cerevisiae.

Superoxide dismutases (SODs) act as a primary defence against reactive oxygen species (ROS) by converting superoxide anion radicals (O2 -) into molecular oxygen (O2) and hydrogen peroxide (H2O2). Members of this enzyme family include CuZnSODs, MnSODs, FeSODs, and NiSODs, depending on the nature of the cofactor that is required for proper activity. Most eukaryotes, including yeast, possess CuZnSOD and MnSOD. This protocol aims at assessing the activity of the yeast Saccharomyces cerevisiae MnSOD Sod2p from cellular extracts using nitroblue tetrazolium staining. This method can be used to estimate the cellular bioavailability of Mn2+ as well as to evaluate the redox state of the cell.

The effect of Trichoderma biofertilizer on the quality of flowering Chinese cabbage and the soil environment

In the present study, four Trichoderma species were isolated and made into a biofertilizer. Following treatment with the biofertilizer for 30 days, the germination rate (↑22.5%), height (↑24.4%), fresh weight (↑41.7%), and yield (↑37.4%) of flowering Chinese cabbage increased markedly compared with those of the control. In addition, the contents of soluble sugar (2.044%), soluble protein (5.938 %), and chlorophyll (2.803 mg/g) were higher in flowering Chinese cabbage treated with the biofertilizer for 30 days, compared to the control (1.7.4%, 5.611% and 2.639 mg/g respectively); however, the content of nitric nitrogen, with toxicological consequences for human health, was lower (↓23.6%), indicating that Trichoderma could also improve the quality of flowering Chinese cabbage. After Evans blue and Nitro blue tetrazolium staining, the leaves of flowering Chinese cabbage treated with the biofertilizer showed a smaller blue area than in the control, suggesting that the biofertilizer enhanced the tolerance of flowering Chinese cabbage to environment stresses. Furthermore, the biofertilizer contributed to the increasing of soil enzyme activity at 30th days, including urease (↑25.1%), phosphatase (↑13.1%), and catalase (↑14.0%), providing more inorganic N and P to the soil and reducing the harm done to flowering Chinese cabbage by reactive oxygen species. Taken together, the results showed the Trichoderma biofertilizer enhanced the nutrient uptake and tolerance environment stresses, further improving the quality and production of flowering Chinese cabbage.

AMELIORATION WITH TITANIUM DIOXIDE NANOPARTICLE FOR REGULATION OF OXIDATIVE STRESS IN MAIZE (Zea mays L.)

In the present experiment, Zea mays L. was evaluated for its C4 modality through oxidative stress responses. Moreover, under variable concentrations (0, 50, 100µM) of hydrogen peroxide, the impacts were variable and modulated with titanium dioxide nanoparticle (TiO2-NP). Plants scored with elevated concentrations of H2O2 and superoxide (O2.-) maximum by 1.31 & 1.52-fold respectively over 0µM H2O2. TiO2-NP recovered those maximums with 8.69 & 7.89% as compared to control plants. The effects of H2O2 were moderated with malondialdehyde and carbonyl content by peak values 7.45 & 5.91% under TiO2-NP treatment respectively. A depletion in proline content recorded under H2O2 stress but recovered through highest 1.23-fold with TiO2-NP application. TiO2-NP recorded NADP-ME activities up-regulated in concentration gradient of H2O2. Oxidative exposure was also documented by in-vivo detection of H2O2 and O2.- by 3,3′-diaminobenzidine and nitroblue tetrazolium staining. For the anti-oxidative cascades superoxide dismutase activity had induced by 1.42-fold under H2O2 when pre-treated with TiO2-NP. However, glutathione reductase activity was differentially modulated under H2O2 through interaction with TiO2-NP. Activity of guaiacol peroxidase was significantly up-regulated by 1.07-fold when TiO2-NP applied. The inductions of oxidative stress recorded more evident when a distinct polymorphism for GPX protein resolved variations in band intensities, but not in number. Activity of catalase experience a down-regulated trend all through the H2O2 exposure and had significant sensitivity to TiO2-NP. The results depict the nature and reactivity of C4 modules under oxidative stress that would be the selection pressures and that might be modulated with TiO2-NP application.

Hydrophobin HFBII-4 from Trichoderma asperellum induces antifungal resistance in poplar.

Herein, the class II hydrophobin gene HFBII-4 was cloned from the biocontrol agent Trichoderma asperellum ACCC30536 and recombinant rHFBII-4 was expressed in Pichia pastoris GS115. Treatment of Populus davidiana × P. alba var. pyramidalis (PdPap poplar) with rHFBII-4 altered the expression levels of genes in the auxin, salicylic acid (SA), and jasmonic acid (JA) signal transduction pathways. Polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) enzyme activities were induced with rHFBII-4. Evans Blue and nitro blue tetrazolium (NBT) staining indicated that cell membrane permeability and reactive oxygen species were lower in the leaves of plants treated with rHFBII-4. The chlorophyll content was higher than that of control at 2-5days after treatment. Furthermore, poplar seedlings were inoculated with Alternaria alternata, disease symptoms were observed. The diseased area was smaller in leaves induced with rHFBII-4 compared with control. In summary, rHFBII-4 enhances resistance to A. alternata.

The association between iron, calcium, and oxidative stress in seminal plasma and sperm quality.

The present study aimed to determine the level of iron and calcium in the seminal plasma of men with different fertility potentials and to examine its relationship with oxidative stress. Seventy-nine sub-fertile patients with asthenoteratozoospermia (AT), n 27; teratoleucozoospermia (TL), n 20; teratozoospermia (Terato), n 32; and 29 healthy donors were included. The ability of spermatozoa to produce reactive oxygen species (ROS) was evaluated by using nitroblue tetrazolium (NBT) staining. The lipid peroxidation end product, malondialdehyde (MDA), and the trace element levels (iron and calcium) were measured spectrophotometrically. Iron and calcium concentrations in seminal plasma of the patient groups were significantly more elevated than the normal group. Nevertheless, both calcium and iron showed strong negative correlations with the total sperm motility and normal sperm morphology, but only iron was positively and significantly associated with multiple anomalies index and seminal leucocyte concentration. On the other hand, the rates of MDA and ROS production in semen were significantly higher in the three abnormal groups than in controls. These two oxidative stress biomarkers were significantly associated with the percentage of atypical forms in semen. However, only semen ROS level was significantly associated with the decreased sperm motility and the sperm leucocytes concentration. Meanwhile, there are positive correlations between seminal iron and calcium content and the studied oxidative stress biomarkers. Oxidative stress and trace element excess are implicated in low sperm quality. Iron and calcium might be the mediators of the effects of oxidative damage and induces lipid peroxidation.

An insight into the mechanism of antifungal activity of biogenic nanoparticles than their chemical counterparts

Herein, we describe the enhanced antifungal activity of silver nanoparticles biosynthesized by cell free filtrate of Trichoderma viride (MTCC 5661) in comparison to chemically synthesized silver nanoparticles (CSNP) of similar shape and size. Biosynthesized silver nanoparticles (BSNP) enhanced the reduction in dry weight by 20 and 48.8% of fungal pathogens Fusarium oxysporum and Alternaria brassicicola respectively in comparison to their chemical counterparts (CSNP). Nitroblue tetrazolium and Propidium iodide staining demonstrated the higher generation of superoxide radicals lead to higher death in BSNP treated fungus in comparison to CSNP. Scanning electron microscopy of A. brassicicola revealed the osmotic imbalance and membrane disintegrity to be major cause for fungal cell death after treatment with BSNP. To gain an insight into the mechanistic aspect of enhanced fungal cell death after treatment of BSNP in comparison to CSNP, stress responses and real time PCR analysis was carried out with A. brassicicola. It revealed that generation of ROS, downregulation of antioxidant machinery and oxidative enzymes, disruption of osmotic balance and cellular integrity, and loss of virulence are the mechanisms employed by BSNP which establishes them as superior antifungal agent than their chemical counterparts. With increasing drug resistance and ubiquitous presence of fungal pathogens in plant kingdom, BSNP bears the candidature for new generation of antifungal agent.

Identification and characterization of proteins involved in stolon adhesion in the highly invasive fouling ascidian Ciona robusta

Adhesive ascidians have caused serious biofouling problems and huge economic losses in marine ecosystems. However, adhesion mechanisms, particularly on functional proteins involved in ascidian adhesion, remain largely unexplored. Here, we identified 26 representative stolon proteins from the highly invasive fouling ascidian Ciona robusta using the proteomics approach. The uncharacterized stolon proteins were rich in adhesion-related conserved domains. Real-time quantitative PCR further revealed specific expressions of these uncharacterized protein genes in stolon tissue, suggesting their potential roles in stolon adhesion.> A recombinant vWFA domain-containing uncharacterized protein, ascidian stolon protein 1 (ASP-1), was successfully expressed in a baculovirus-insect cell system and purified in vitro. Coating experiment showed that tyrosinase-modified ASP-1 could absorb to glass and organic glass stronger than unmodified ASP-1, while only modified ASP-1 could absorb to aluminum foil. Quartz crystal microbalance analysis also showed the increase in absorption ability of ASP-1 after modification. In addition, abundant 3,4-l-dihydroxyphenylalanine (DOPA) in modified protein was detected by nitroblue tetrazolium staining. These results suggest that ASP-1 be involved in ascidian DOPA-dependent and material-selective adhesion. Overall, this study provides insight into molecular mechanisms of C. robusta stolon adhesion, and findings here are expected to be conductive to develop strategies against biofouling caused by ascidians.

MicroRNA-183 improve myocardial damager via NF-kb pathway: In vitro and in vivo study.

The aim of this study is to evaluate the effects of microRNA-183 (miRNA-183) in myocardial damager. In the cell experiment, the H9C2 cell was divided into three groups: NC group, Model group, and miRNA group. The cell apoptosis and relative proteins' expressions were measured by flow cytometry and Western blot assay. The in vivo study of the rats was divided into three groups: Sham group, Model group, and miRNA group. The pathology, Infarct size, cell apoptosis, and relative protein expressions were evaluated by hematoxylin and eosin staining, nitro blue tetrazolium staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and immunohistochemistry. Compared with NC groups, the in vitro and in vivo study showed that cell apoptosis rate of miRNA groups was significantly suppressed (P < 0.05). The pathology and infarct size of miRNA group were significantly improved compared with the NC group. Meanwhile, the relative proteins expression (nuclear factor-κB [NF-kb], caspase-3, Bax, and Bcl-2) of miRNA groups were significantly different compared with those of NC groups ( P < 0.05). In vitro and in vivo study revealed that miRNA-183 improves myocardial damager through the NF-κb pathway.

Investigation on the origin of sperm morphological defects: oxidative attacks, chromatin immaturity, and DNA fragmentation.

DNA fragmentation can be deleterious on spermatozoon morphology but the pathogenesis of teratozoospermia associated with DNA breaks is not fully understood, even if oxidative attacks and defects in chromatin maturation are hypothesized. Therefore, this study is one of the first to clarify on the underlying hypothesizes behind such observations. The objectives of our study were to assess the role of oxidative attacks in DNA damage pathogenesis in ejaculated spermatozoa from patients with isolated teratozoospermia. We aimed to assess the correlation of DNA breaks with morphologically abnormal spermatozoa, as well as ROS level and impairment chromatin condensation. A total of 90 patients were divided into two groups, men with isolated teratozoospermia (n = 60) and men with normal semen parameters (n = 30) as controls. DNA fragmentation was evaluated by TUNEL assay; chromatin immaturity was studied using acridine orange and toluidine blue staining. We evaluated the ability of spermatozoa to produce reactive oxygen species with nitro blue tetrazolium staining. Patient with teratozoospermia when compared to fertile men showed significantly higher rates of semen ROS production, sperm hypocondensated chromatin, denaturated DNA, and fragmented DNA. All these parameters were positively correlated with abnormal sperm morphology. The studied DNA integrity markers were also correlated with ROS production. Fragmented DNA is the main pathway leading to morphology defects in the sperm. In fact, impaired chromatin compaction may induce DNA breaks and free radicals, which can break the DNA backbone indirectly, by reducing protamination and disulphide bond formation, as oxidative attack appears to be the major cause of poor semen morphology.

A novel gene of Kalanchoe daigremontiana confers plant drought resistance

Kalanchoe (K.) daigremontiana is important for studying asexual reproduction under different environmental conditions. Here, we describe a novel KdNOVEL41 (KdN41) gene that may confer drought resistance and could thereby affect K. daigremontiana development. The detected subcellular localization of a KdN41/Yellow Fluorescent Protein (YFP) fusion protein was in the nucleus and cell membrane. Drought, salt, and heat stress treatment in tobacco plants containing the KdN41 gene promoter driving β-glucuronidase (GUS) gene transcription revealed that only drought stress triggered strong GUS staining in the vascular tissues. Overexpression (OE) of the KdN41 gene conferred improved drought resistance in tobacco plants compared to wild-type and transformed with empty vector plants by inducing higher antioxidant enzyme activities, decreasing cell membrane damage, increasing abscisic acid (ABA) content, causing reinforced drought resistance related gene expression profiles. The 3,3′-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining results also showed less relative oxygen species (ROS) content in KdN41-overexpressing tobacco leaf during drought stress. Surprisingly, by re-watering after drought stress, KdN41-overexpressing tobacco showed earlier flowering. Overall, the KdN41 gene plays roles in ROS scavenging and osmotic damage reduction to improve tobacco drought resistance, which may increase our understanding of the molecular network involved in developmental manipulation under drought stress in K. daigremontiana.

Night Light-Adaptation Strategies for Photosynthetic Apparatus in Yellow-Poplar (Liriodendron tulipifera L.) Exposed to Artificial Night Lighting

Plants can undergo external fluctuations in the natural light and dark cycle. The photosynthetic apparatus needs to operate in an appropriate manner to fluctuating environmental factors, especially in light. Yellow-poplar seedlings were exposed to nighttime artificial high-pressure sodium (HPS) lighting to evaluate night light-adaptation strategies for photosynthetic apparatus fitness relative to pigment contents, photosystem II photochemistry, photosynthetic parameters, histochemical analysis of reactive oxygen species, and plant biomass. As a result, seedlings exhibited dynamic changes including the enhancement of accessory pigments, the reduction of photosystem II photochemistry, increased stomatal limitation, downregulation of photosynthesis, and the decreased aboveground and belowground biomass under artificial night lighting. Histochemical analysis with 3,3′-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining indicates the accumulation of in situ superoxide radicals (O2−) and hydrogen peroxide (H2O2) in leaves exposed to the lowest level of artificial night lighting compared to control. Moreover, these leaves exposed to artificial night lighting had a lower nighttime respiration rate. These results indicated that HPS lighting during the night may act as a major factor as repressors of the fitness of photosynthesis and growth patterns, via a modification of the photosynthetic light harvesting apparatus.

Methods to Quantify Biotic-Induced Stress in Plants.

Plant pathogens such as fungi, oomycetes, viruses and bacteria infect important crops and account for significant economic losses worldwide. Therefore, it is critical to gain insights into plant-pathogen interactions at the cellular and molecular level. The outcome of the interaction between plants and pathogens greatly differs depending on the species, strains and cultivars involved as well as environmental factors, yet typically results in stress for the plant, the pathogen or both. These biotic-induced stresses can be monitored using a wide range of techniques, of which some of the most commonly used techniques are outlined in this chapter. One widely observed feature of biotic stress in plants is the generation of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide (O2-). We describe the quantification of hydrogen peroxide by 3,3'-diaminobenzidine (DAB) staining and luminol-based assays, and of superoxide by nitroblue tetrazolium (NBT) staining. Other techniques detailed here include measurement of callose deposition by aniline blue staining, evaluation of cell death by trypan blue staining and analysis of the loss of membrane integrity by monitoring electrolyte leakage.

Effect of β glucan dietary levels on immune response and hematology of channel catfish Ictalurus punctatus juveniles

Biological response modifiers such as pro and prebiotics have been recently used in aquaculture showing abilities as immunostimulants, enhancing resistance to different fungal, bacterial and parasitic pathogens and tolerance to environmental stress. This experiment evaluated three different concentrations (0.05, 0.1 and 0.5 %) of β-1,3/1,6 glucans on nonspecific immune parameters of channel catfish ( Ictalurus punctatus ). Results showed that intake supplementation with 0.05 % of β-1,3/1,6 glucans provides a greater immunostimulation over the fourth treatment week. Significant higher differences ( p < 0.05) were obtained on parameters related to phagocytic activity on blood cells, also other assays showed more reactive cells to nitroblue tetrazolium (NbT) stain on spleen and head kidney analysis. We conclude that the use of 0.05 % of β-1,3/1,6 glucans added to a commercial diet improves nonspecific immune system of channel catfish ( I. punctatus ).

Partial alleviation of oxidative stress induced by gamma irradiation in Vigna radiata by polyamine treatment

Purpose: Environmental changes generate free radicals and reactive oxygen species (ROS) resulting in abiotic stress in plants. This causes alterations in germination, morphology, growth and development ultimately leading to yield loss. Gamma irradiation was used to experimentally induce oxidative damage in an important pulse crop Vigna radiata (L.) Wilczek or mung bean. Our research was aimed towards augmentation of oxidative stress tolerance through treatment with a group of aliphatic amines known as polyamines.Materials and methods: We used sub-lethal doses of gamma irradiation to generate oxidative damage which was evaluated using Nitro blue tetrazolium (NBT) staining, total antioxidant activity, 1, 1-Diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assay, proline content and lipid peroxidation. Changes in internal free polyamines and messenger ribonucleic acid (mRNA) expression of key rate-limiting S-adenosylmethionine decarboxylase (SAMDC) enzyme in polyamine biosynthetic pathway was studied using real-time polymerase chain reaction (PCR).Results: We observed increased oxidative damage with higher irradiation dose which was partially alleviated by putrescine treatment. Internal levels of putrescine and spermidine increased with 1 mM (50 and 100 Gy) and 2 mM putrescine treatment. Expression of SAMDC also increased with putrescine treatment.Conclusion: This study shows that treatment with putrescine can partially alleviate oxidative damage caused by gamma rays.

Nitro Blue Tetrazolium (NBT) Staining as an Assessment of Thermal Penetration

Medical devices utilizing hyperthermic tissue injury are increasingly being used in surgical procedures. It is clinically essential to accurately assess the extent of thermal tissue damage these devices have on tissues. Histological stains such as hematoxylin and eosin (H&amp;E) and trichrome staining provide a generic tissue staining, of nucleic acids and proteins. As a result of its generality, the application of these stains in the assessment of tissue injury and viability is similarly limited. In contrast, Nitro Blue Tetrazolium (NBT) staining is requires functional cellular proteins and energy production; as such, this stain has the potential to more accurately establish viability and the extent of tissue injury. Here, we describe a validated methodology for NBT staining to assess the extent of thermal tissue damage/injury using the ex vivo porcine model. Following hyperthermic treatment, the tissues are maintained at room temperature for at least two but not longer than four hours and are then frozen at −80°C. The treatment sites are subsequently dissected and embedded in Optimal Cutting Temperature (OCT) Compound on a liquid nitrogen freezing stage. NBT staining is performed on tissue sections cut between 10–12 microns thick. Histologically, the thermally damaged/injured tissue is quantified by assessing the extent of NBT‐negative (yellow‐tan in color) staining within the treatment zone. In contrast, the thermally unaffected tissue stains NBT‐positive (purple‐blue) due to its preservation of diaphorase protein function and maintenance of energy production. Our results validate NBT staining, as an effective assessment of thermal tissue injury, in several different tissues including muscle, liver, stomach and colon.

Gametophyte Development Needs Mitochondrial Coproporphyrinogen III Oxidase Function.

Tetrapyrrole biosynthesis is one of the most essential metabolic pathways in almost all organisms. Coproporphyrinogen III oxidase (CPO) catalyzes the conversion of coproporphyrinogen III into protoporphyrinogen IX in this pathway. Here, we report that mutation in the Arabidopsis (Arabidopsis thaliana) CPO-coding gene At5g63290 (AtHEMN1) adversely affects silique length, ovule number, and seed set. Athemn1 mutant alleles were transmitted via both male and female gametes, but homozygous mutants were never recovered. Plants carrying Athemn1 mutant alleles showed defects in gametophyte development, including nonviable pollen and embryo sacs with unfused polar nuclei. Improper differentiation of the central cell led to defects in endosperm development. Consequently, embryo development was arrested at the globular stage. The mutant phenotype was completely rescued by transgenic expression of AtHEMN1 Promoter and transcript analyses indicated that AtHEMN1 is expressed mainly in floral tissues and developing seeds. AtHEMN1-green fluorescent protein fusion protein was found targeted to mitochondria. Loss of AtHEMN1 function increased coproporphyrinogen III level and reduced protoporphyrinogen IX level, suggesting the impairment of tetrapyrrole biosynthesis. Blockage of tetrapyrrole biosynthesis in the AtHEMN1 mutant led to increased reactive oxygen species (ROS) accumulation in anthers and embryo sacs, as evidenced by nitroblue tetrazolium staining. Our results suggest that the accumulated ROS disrupts mitochondrial function by altering their membrane polarity in floral tissues. This study highlights the role of mitochondrial ROS homeostasis in gametophyte and seed development and sheds new light on tetrapyrrole/heme biosynthesis in plant mitochondria.

Defensive responses in Capsicum annuum (L) plants, induced due to the feeding by different larval instars of Spodoptera litura (F)

A study was conducted to demonstrate the induced defence responses of chilli plants against herbivory by different larval instars of the tobacco cutworm, Spodoptera litura (F) at different intervals of time (0, 24, 48 and 72 h) after feeding. The rate of various antioxidative enzymes such as peroxidase, catalase (CAT), superoxide and polyphenol oxidases generated in chilli, Capsicum annuum (L) plant leaves due to damage caused by different larval stages of S. litura, and the primary and secondary metabolite contents were quantified. Also, lipid peroxidation content in plant leaves was measured by a malondialdehyde method, while the photosynthetic pigment concentrations were estimated spectrophotometrically. Early instar larval feeding caused an enhanced production of reactive oxygen species (ROS) compared with the older instars. Not much variation occurred in the primary metabolite content of plants fed upon by herbivores, and normal chilly plants. However, a considerable increase in the activities of CAT and superoxide dismutase was recorded after 24 h of insect feeding. Hydrogen peroxide accumulation was higher in plants fed by 2nd instar larvae when estimated after 0 h (immediately after 2 h of feeding was completed), whereas no hydrogen peroxide accumulation was observed due to the feeding by 5th instar larvae using the 3,3′-diaminobenzidine (DAB) staining method. Nitro blue tetrazolium staining for the location of superoxide ions revealed the immediate accumulation of superoxide ions at the damaged site due to the feeding by all the tested instar larvae of S. litura. Among the different larval stages tested, 2nd and 3rd instar feeding led to more superoxide radical accumulation as an indication of ROS generated as a counteraction to herbivory.

Characterization and fine-mapping of a novel premature leaf senescence mutant yellow leaf and dwarf 1 in rice

Leaves are the main organs in which photosynthates are produced. Leaf senescence facilitates the translocation of photosynthates and nutrients from source to sink, which is important for plant development and especially for crop yield. However, the molecular mechanism of leaf senescence is unknown. Here, we identified a mutant, yellow leaf and dwarf 1 (yld1), which exhibited decreased plant height and premature leaf senescence. Nitroblue tetrazolium and diamiobenzidine staining analyses revealed that the concentrations of reactive oxygen species were higher in yld1 leaves than in wild type leaves. The photosynthetic pigment contents were significantly decreased in yld1. The yld1 chloroplasts had collapsed and were filled with abnormal starch granules. Combining bulk segregant and MutMap gene mapping approaches, the mutation responsible for the yld1 phenotype was mapped to a 7.3 Mb centromeric region, and three non-synonymous single nucleotide polymorphisms located in three novel genes were identified in this region. The expression patterns of the three candidate genes indicated that LOC_Os06g29380 had the most potential for functional verification. Plant hormone measurements showed that salicylic acid was highly accumulated in yld1 leaves when compared with wild type leaves, and yld1 was more sensitive to salicylic acid than wild type. This work lays the foundation for understanding the molecular regulatory mechanism of leaf senescence, and may reveal new connections among the molecular pathways related to leaf senescence, starch metabolism and salicylic acid signaling.

EFFECT OF NESFATIN-1 ON ISCHAEMIA-REPERFUSION INJURY IN ISOLATED HEART OF ALBINO RATS

Nesfatin-1 is a hypothalamic neuropeptide which is involved in control of food intake and glucose homeostasis. Lower plasma level of Nesfatin-1 in patients with acute myocardial infarction (AMI) was reported. Objective: This study was designed to explore possible effect of Nesfatin-1 on ischemia-reperfusion injury in isolated heart of adult male albino rats and to explain the possible involved mechanisms, in a trial to clarify Nesfatin-1 expected cardioprotective effect. Design: This study was carried out on eighteen adult male albino rats which were divided equally (n=6) into 3 groups: Group I (ischemia-reperfusion I/R group); hearts were stabilized then subjected to (I/R) protocol, Group II (Nesfatin-1 pre-conditioning group); Nesfatin-1 was infused for 20 minutes before hearts were subjected to ischemia and Group III (Nesfatin-1 post-conditioning group); Nesfatin-1 was infused for 20 minutes at the beginning of 120 minutes of reperfusion. Cardiac performance indicators as left ventricular pressure (LVP), +max (LV dp/dt), -max (LVdP/dt)+, in addition to heart rate were recorded. Lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), superoxide dismutase (SOD) and C-reactive protein (CRP) were measured in the collected perfusate and cardiac Malondialdehyde (MDA) was measured. Finally, Nitro blue tetrazolium stain was used to detect the necrotic tissue percentage to the whole left ventricular mass. Results: In group III (post conditioning group), there was a significant increase of the studied cardiac parameters compared to group I (I/R). Nesfatin-1 significantly increased LVP, +max (dp/dt), -max (dp/dt) and HR in comparison with I/R group. This was associated with a significant decrease in LDH and CK-MB levels, a significant increase in SOD level and a significant decrease in MDA and CRP levels. Moreover, Nesfatin-1 caused a significant decrease in percentage of necrotic tissue to the whole left ventricular mass. Regarding group II, no significant changes were detected in all parameters except for significant increase in SOD level and significant decrease in CRP level. Conclusion: Nesfatin-1 protects against ischemia/reperfusion injury in vitro through its antioxidant and anti-inflammatory properties by limiting the infarction area, only if given as a post conditioning factor after I/R. Those results open the way to include Nesfatin-1 among the strategies for management of cardiac infarction during reperfusion. Key words: Nesfatin-1, cardiac ischemia-reperfusion, oxidative stress, inflammation.