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Nitric oxide (NO) is a signaling molecule which controls a variety of biological functions and plays important roles in plant physiology. NO is involved in the majority of plant responses to biotic and abiotic stress, either indirectly through gene activation or interaction with hormones and reactive oxygen species (ROS), or directly as a result of altering enzyme activities primarily through S-nitrosylation. Protein S-nitrosylation is a redox-based post-translational modification that involves covalent attaching of a NO molecule to a reactive cysteine thiol of a target protein. This modification performs many significant physiological functions of NO. However, S-nitrosylation is a conserved evolutionary mechanism that regulates various facts of cellular signaling such as hormone signaling and responses to pathogens. It is challenging to find S-nitrosylated proteins since the S-NO bond is labile and therefore, methods to identify these modified proteins becomes difficult under different pathological conditions. Biotin switch method is well known in identifying S-nitrosylated proteins. S-nitrosylation of proteins plays important role in regulating different cellular processes like signal transduction, autophagy, SUMOylation and also impacts pathogen’s virulence. However, much about S-nitrosylation is known due to abiotic stress. This short review highlights S-nitrosylation in response to biotic disturbances.

Plant tissues, particularly roots, can be subjected to periods of hypoxia due to environmental circumstances. Plants have developed various adaptations in response to hypoxic stress and these have been described extensively. Less well-appreciated is the body of evidence demonstrating that scavenging of nitric oxide (NO) and the reduction of nitrate/nitrite regulate important mechanisms that contribute to tolerance to hypoxia. Although ethylene controls hyponasty and aerenchyma formation, NO production apparently regulates hypoxic ethylene biosynthesis. In the hypoxic mitochondrion, cytochrome c oxidase, which is a major source of NO, also is inhibited by NO, thereby reducing the respiratory rate and enhancing local oxygen concentrations. Nitrite can maintain ATP generation under hypoxia by coupling its reduction to the translocation of protons from the inner side of mitochondria and generating an electrochemical gradient. This reaction can be further coupled to a reaction whereby nonsymbiotic haemoglobin oxidizes NO to nitrate. In addition to these functions, nitrite has been reported to influence mitochondrial structure and supercomplex formation, as well as playing a role in oxygen sensing via the N-end rule pathway. These studies establish that nitrite and NO perform multiple functions during plant hypoxia and suggest that further research into the underlying mechanisms is warranted.

Nitric oxide (NO) is now established as an important signalling molecule in plants where it influences growth, development, and responses to stress. Despite extensive research, the most appropriate methods to measure and localize these signalling radicals are debated and still need investigation. Many confounding factors such as the presence of other reactive intermediates, scavenging enzymes, and compartmentation influence how accurately each can be measured. Further, these signalling radicals have short half-lives ranging from seconds to minutes based on the cellular redox condition. Hence, it is necessary to use sensitive and specific methods in order to understand the contribution of each signalling molecule to various biological processes. In this review, we summarize the current knowledge on NO measurement in plant samples, via various methods. We also discuss advantages, limitations, and wider applications of each method.

Abstract Background and Aims Nitrogen (N) levels vary between ecosystems, while the form of available N has a substantial impact on growth, development and perception of stress. Plants have the capacity to assimilate N in the form of either nitrate (NO3–) or ammonium (NH4+). Recent studies revealed that NO3– nutrition increases nitric oxide (NO) levels under hypoxia. When oxygen availability changes, plants need to generate energy to protect themselves against hypoxia-induced damage. As the effects of NO3– or NH4+ nutrition on energy production remain unresolved, this study was conducted to investigate the role of N source on group VII transcription factors, fermentative genes, energy metabolism and respiration under normoxic and hypoxic conditions. Methods We used Arabidopsis plants grown on Hoagland medium with either NO3– or NH4+ as a source of N and exposed to 0.8 % oxygen environment. In both roots and seedlings, we investigated the phytoglobin–nitric oxide cycle and the pathways of fermentation and respiration; furthermore, NO levels were tested using a combination of techniques including diaminofluorescein fluorescence, the gas phase Griess reagent assay, respiration by using an oxygen sensor and gene expression analysis by real-time quantitative reverse transcription–PCR methods. Key Results Under NO3– nutrition, hypoxic stress leads to increases in nitrate reductase activity, NO production, class 1 phytoglobin transcript abundance and metphytoglobin reductase activity. In contrast, none of these processes responded to hypoxia under NH4+ nutrition. Under NO3– nutrition, a decreased total respiratory rate and increased alternative oxidase capacity and expression were observed during hypoxia. Data correlated with decreased reactive oxygen species and lipid peroxidation levels. Moreover, increased fermentation and NAD+ recycling as well as increased ATP production concomitant with the increased expression of transcription factor genes HRE1, HRE2, RAP2.2 and RAP2.12 were observed during hypoxia under NO3– nutrition. Conclusions The results of this study collectively indicate that nitrate nutrition influences multiple factors in order to increase energy efficiency under hypoxia.

Citronella oil is the main product of Java citronella grass (Cymbopogon winterianus Jowitt) rich in geraniol and citronellol, widely used in mosquito repellents and perfumeries. The age of the plant plays a key role in oil composition and its yield such that young leaves have lesser oil content than the mature leaves. Also, a remarkable difference between fresh and dried leaves regarding oil yield is observed. The various methods of extracting essential oils from citronella grass with respect to yield (%) were studied. Average percent yield in the manual extraction and hydro-distillation procedure was 0.8 and 1 % respectively, which was better as compared to steam distilled oil (0.7 %). The chromatographic analysis of essential oils with respect to standards geraniol and citronellol were studied by high performance thin layer chromatography (HPTLC) with n-hexane and ethyl acetate (3:2) as mobile phase followed by its separation on plates. The developed plates showed geraniol, citronellol and citronellal as major bands. The analysis of all extracted oil samples by means of electrospray ionization-mass spectrometry (ESI-MS) in the positive ion mode showed rapid mass fingerprints of constituents present in the samples according to the observed mass of standards. Furthermore, the analysis of vibrational spectra was accomplished with Fourier transform infra-red spectroscopy (FTIR) specifying all the functional groups as major peaks confirming all of them as monoterpene alcohols with conjugated double bonds. Thus, HPTLC, ESI-MS and FTIR studies evidenced that the two essential oil components were majorly present in the methanol extract suggesting methanol as a good extractant in the manual extraction process.

Nitrogen (N) is essential for growth, development and defense but, how low N effects defense and the role of Trichoderma in enhancing defense under low nitrate is not known. Low nitrate fed Arabidopsis plants displayed reduced growth and compromised LAR & SAR response when infected with avirulent and virulent Pseudomonas syringae DC3000. These responses were enhanced in the presence of Trichoderma. The mechanism of increased LAR and SAR mediated by Trichoderma involve increased N uptake and enhanced protein levels via modulation of nitrate transporter genes. nrt2.1 mutant is compromised in LAR and SAR response suggesting a link between enhanced N transport and defense. Enhanced N uptake was mediated by Trichoderma elicited nitric oxide (NO). Low NO producing nia1,2 mutant and nsHb+ over expressing lines were unable to induce nitrate transporters and compromised defense in presence of Trichoderma under low N suggesting a signaling role of Trichoderma elicited NO. Trichoderma also induced SA and defense gene expression under low N. SA deficient NahG and npr1 mutants were compromised in LAR and SAR response mediated by Trichoderma. The mechanism of enhanced plant defense under low N mediated by Trichoderma involve NO, ROS, SA production and induction of NRT and SAR marker genes.

Nitric oxide (NO) is a free radical molecule that plays an important role in hypoxic stress. We studied the impact of hypoxia-induced NO production on the expression of genes and production of metabolites involved in carbon, nitrogen and antioxidant metabolism using wild type (WT) and non-symbiotic haemoglobin-overexpressing (Hb+) and nitrate reductase double mutant (nia1,2) and application of NO scavenger cPTIO of Arabidopsis. We found that imposing hypoxia leads to the increase of NO and reactive oxygen species (ROS) levels in WT, while the reduced levels of NO and higher levels of ROS were observed in roots of Hb+ and nia1,2 mutant. Expression of the genes encoding group VII ERFs and the enzymes involved in fermentative pathways, activities of these enzymes and metabolite levels were highly induced in WT suggesting that NO plays a role in the induction of fermentation. Several genes and metabolites involved in the TCA cycle were induced in WT in comparison to Hb+ and nia mutant line suggesting that NO can accelerate TCA cycle to regenerate reducing equivalents under hypoxia. Interestingly, we found that the genes and metabolites involved in the ascorbate-glutathione cycle were modulated by NO under hypoxia. The alternative oxidase gene (AOX1A) was induced under hypoxia in WT due to increased levels of NO rather than ROS. Overall these findings suggest that NO increases expression of the genes of carbon, nitrogen and antioxidant metabolism to improve plant survival under hypoxia.