Current approaches to measure nitric oxide in plants.

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.

Nitric oxide accelerates germination via the regulation of respiration in chickpea.

Seed germination is crucial for the plant life cycle. We investigated the role of nitric oxide (NO) in two chickpea varieties that differ in germination capacity: Kabuli, which has a low rate of germination and germinates slowly, and Desi, which shows improved germination properties. Desi produced more NO than Kabuli and had lower respiratory rates. As a result of the high respiration rates, Kabuli had higher levels of reactive oxygen species (ROS). Treatment with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) reduced respiration in Kabuli and decreased ROS levels, resulting in accelerated germination rates. These findings suggest that NO plays a key role in the germination of Kabuli. SNAP increased the levels of transcripts encoding enzymes involved in carbohydrate metabolism and the cell cycle. Moreover, the levels of amino acids and organic acids were increased in Kabuli as a result of SNAP treatment. 1H-nuclear magnetic resonance analysis revealed that Kabuli has a higher capacity for glucose oxidation than Desi. An observed SNAP-induced increase in 13C incorporation into soluble alanine may result from enhanced oxidation of exogenous [13C]glucose via glycolysis and the pentose phosphate pathway. A homozygous hybrid that originated from a recombinant inbred line population of a cross between Desi and Kabuli germinated faster and had increased NO levels and a reduced accumulation of ROS compared with Kabuli. Taken together, these findings demonstrate the importance of NO in chickpea germination via the control of respiration and ROS accumulation.

Nitrate nutrition influences multiple factors in order to increase energy efficiency under hypoxia in Arabidopsis.

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.

Characterization of plant growth-promoting, antifungal, and enzymatic properties of beneficial bacterial strains associated with pulses rhizosphere from Bundelkhand region of India.

The present study was conducted to characterize the native plant growth-promoting rhizobacteria (PGPRs) from the pulse rhizosphere of the Bundelkhand region of India. Twenty-four bacterial isolates belonging to nineteen species (B. amyloliquefaciens, B. subtilis, B. tequilensis, B. safensis, B. haynesii, E. soli, E. cloacae, A. calcoaceticus, B. valezensis, S. macrescens, P. aeruginosa, P. fluorescens, P. guariconensis, B. megaterium, C. lapagei, P. putida, K. aerogenes, B. cereus, and B. altitudinis) were categorized and evaluated for their plant growth-promoting potential, antifungal properties, and enzymatic activities to identify the most potential strain for commercialization and wider application in pulse crops. Phylogenetic identification was done on the basis of 16 s rRNA analysis. Among the 24 isolates, 12 bacterial strains were gram positive, and 12 were gram negative. Among the tested 24 isolates, IIPRAJCP-6 (Bacillus amyloliquefaciens), IIPRDSCP-1 (Bacillus subtilis), IIPRDSCP-10 (Bacillus tequilensis), IIPRRLUCP-5 (Bacillus safensis), IIPRCDCP-2 (Bacillus subtilis), IIPRAMCP-1 (Bacillus safensis), IIPRMKCP-10 (Bacillus haynesii), IIPRANPP-3 (Bacillus amyloliquefaciens), IIPRKAPP-5 (Enterobacter soli), IIPRAJCP-2 (Enterobacter cloacae), IIPRDSCP-11 (Acinetobacter calcoaceticus), IIPRDSCP-9 (Bacillus valezensis), IIPRMKCP-3 (Seratia macrescens), IIPRMKCP-1 (Pseudomonas aeruginosa), IIPRCKPP-3 (Pseudomonas fluorescens), IIPRMKCP-9 (Pseudomonas guariconensis), IIPRMKCP-8 (Bacillus megatirium), IIPRMWCP-9 (Cedecea lapagei), IIPRKUCP-10 (Pseudomonas putida), IIPRAMCP-4 (Klebsiella aerogenes), IIPRCKPP-7 (Enterobacter cloacae), IIPRAMCP-5 (Bacillus cereus), IIPRSHEP-6 (Bacillus subtilis), IIPRRSBa89 (Bacillus altitudinis) bacterial isolates, IIPRMKCP-9, IIPRAJCP-6, IIPRMKCP-10, IIPRAMCP-5, IIPRSHEP-6, and IIPRMKCP-3 showed the maximum antagonistic activity against Fusarium oxysporum f. sp. ciceris (FOC), Fusarium oxysporum f. sp. lentis (FOL), and Fusarium udum (FU) causing wilt disease of chickpea, lentil, and pigeonpea, respectively, and maximum plant growth-promoting enzyme (phosphatase), plant growth hormone (IAA), and siderophore production show promising results under greenhouse conditions. This study is the first report of bacterial diversity in the pulse-growing region of India.

Rehashing Our Insight of Seaweeds as a Potential Source of Foods, Nutraceuticals, and Pharmaceuticals.

In a few Southeast Asian nations, seaweeds have been a staple of the cuisine since prehistoric times. Seaweeds are currently becoming more and more popular around the world due to their superior nutritional value and medicinal properties. This is because of rising seaweed production on a global scale and substantial research on their composition and bioactivities over the past 20 years. By reviewing several articles in the literature, this review aimed to provide comprehensive information about the primary and secondary metabolites and various classes of bioactive compounds, such as polysaccharides, polyphenols, proteins, and essential fatty acids, along with their bioactivities, in a single article. This review also highlights the potential of seaweeds in the development of nutraceuticals, with a particular focus on their ability to enhance human health and overall well-being. In addition, we discuss the challenges and potential opportunities associated with the advancement of pharmaceuticals and nutraceuticals derived from seaweeds, as well as their incorporation into different industrial sectors. Furthermore, we find that many bioactive constituents found in seaweeds have demonstrated potential in terms of different therapeutic attributes, including antioxidative, anti-inflammatory, anticancer, and other properties. In conclusion, seaweed-based bioactive compounds have a huge potential to play an important role in the food, nutraceutical, and pharmaceutical sectors. However, future research should pay more attention to developing efficient techniques for the extraction and purification of compounds as well as their toxicity analysis, clinical efficacy, mode of action, and interactions with regular diets.

Isolation and Measurement of Respiration and Structural Studies of Purified Mitochondria from Heterotrophic Plant Tissues.

Mitochondria are the power houses of eukaryotic cells. These organelles contain various oxidoreductase complexes. Electron transfer from different reducing equivalents channeled via these complexes drives proton translocation across the inner mitochondrial membrane, leading to ATP generation. Plant mitochondria contain alternative NAD(P)H dehydrogenases, alternative oxidase, and uncoupling protein, and TCA cycle enzymes are located in their matrix. Apart from ATP production, mitochondria are also involved in synthesis of vitamins and cofactors and participate in fatty acid, nucleotide, photorespiratory, and antioxidant metabolism. Recent emerging evidence suggests that mitochondria play a role in redox signaling and generation of reactive oxygen and nitrogen species. For mitochondrial studies, it is essential to isolate physiologically active mitochondria with good structural integrity. In this article, we explain a detailed procedure for isolation of mitochondria from various heterotrophic tissues, such as germinating chickpea seeds, potato tubers, and cauliflower florets. This procedure requires discontinuous Percoll gradient centrifugation and can give a good yield of mitochondria, in the range of 4 to 8 mg per 50 g tissue with active respiratory capacity. After MitoTracker staining, isolated mitochondria can be visualized by using a confocal microscope. The structure of mitochondria can be monitored by scanning electron microscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of mitochondria from germinating chickpea seeds, potato tubers, and cauliflower florets Basic Protocol 2: Quantification of mitochondrial protein concentration by Bradford assay Basic Protocol 3: Quantification of mitochondrial respiration using single-channel free-radical analyzer Basic Protocol 4: Staining of mitochondria and confocal imaging Basic Protocol 5: Visualization of isolated mitochondria under scanning electron microscope.

Plants Metabolome Study: Emerging Tools and Techniques.

Metabolomics is now considered a wide-ranging, sensitive and practical approach to acquire useful information on the composition of a metabolite pool present in any organism, including plants. Investigating metabolomic regulation in plants is essential to understand their adaptation, acclimation and defense responses to environmental stresses through the production of numerous metabolites. Moreover, metabolomics can be easily applied for the phenotyping of plants; and thus, it has great potential to be used in genome editing programs to develop superior next-generation crops. This review describes the recent analytical tools and techniques available to study plants metabolome, along with their significance of sample preparation using targeted and non-targeted methods. Advanced analytical tools, like gas chromatography-mass spectrometry (GC-MS), liquid chromatography mass-spectroscopy (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) matrix-assisted laser desorption/ionization (MALDI), ion mobility spectrometry (IMS) and nuclear magnetic resonance (NMR) have speed up precise metabolic profiling in plants. Further, we provide a complete overview of bioinformatics tools and plant metabolome database that can be utilized to advance our knowledge to plant biology.

Senescent Hepatocytes in Decompensated Liver Show Reduced UPRMT and Its Key Player, CLPP, Attenuates Senescence In Vitro.

BACKGROUND AND AIMS: Non-dividing hepatocytes in end-stage liver disease indicates permanent growth arrest similar to senescence. Identifying senescence in vivo is often challenging and mechanisms inhibiting senescence are poorly understood. In lower organisms mitochondrial unfolded protein response (UPRMT) helps in increasing longevity; however, its role in senescence and liver disease is poorly understood. Aim of this study was to identify hepatocyte senescence and the role of UPRMT in cryptogenic cirrhosis. METHODS: Doxorubicin was used to induce senescence in non-neoplastic hepatocytes (PH5CH8) and hepatoma cells (HepG2 and Huh7). Senescence-associated markers and unfolded protein response was evaluated by fluorescence microscopy, immunoblotting and gene expression. Explants/biopsies from normal, fibrosis, compensated and decompensated cirrhosis without any known etiology were examined for presence of senescence and UPRMT by immunohistochemistry and gene expression. RESULTS: Accumulation of senescent hepatocytes in cryptogenic cirrhosis was associated with reduced proliferation, increased expression of γH2AX and p21, together with loss of LaminB1. Dysfunctional mitochondria and compromised UPRMT were key features of senescent hepatocytes both in vitro and also in decompensated cirrhosis. Intriguingly, compensated cirrhotic liver mounted strong UPRMT, with high levels of mitochondrial protease, CLPP. Overexpression of CLPP inhibited senescence in vitro, by reducing mitochondrial ROS and altering oxygen consumption. CONCLUSIONS: Our results implicate a role of hepatocyte senescence in cryptogenic cirrhosis together with a crucial role of UPRMT in preventing hepatocyte senescence. A compromised UPRMT may shift the fate of cirrhotic liver toward decompensation by exaggerating hepatocyte senescence. Restoring CLPP levels at least in cell culture appears as a promising strategy in mitohormesis, thereby, preventing senescence and possibly improving hepatocyte function.

Variations in macrophage migration inhibitory factor gene are not associated with visceral leishmaniasis in India.

BACKGROUND: The host genetic factors play important role in determining the outcome of visceral leishmaniasis (VL). Macrophage migration inhibitory factor (MIF) is an important host cytokine, which is a key regulator of innate immune system. Genetic variants in MIF gene have been found to be associated with several inflammatory and infectious diseases. Role of MIF is well documented in leishmaniasis diseases, including Indian visceral leishmaniasis, where elevated level of serum MIF has been associated with VL phenotypes. However, there was no genetic study to correlate MIF variants in VL, therefore, we aimed to study the possible association of three reported MIF gene variants -794 CATT, -173G > C and non-coding RNA gene LOC284889 in Indian VL phenotype. METHODS: Study subjects comprised of 214 VL patients along with ethnically and demographically matched 220 controls from VL endemic regions of Bihar state in India. RESULTS: We found no significant difference between cases and controls in allelic, genotypic and haplotype frequency of the markers analysed [-794 CATT repeats (χ2=0.86; p=0.35; OR=0.85; 95% CI=0.61-1.19); -173 G>C polymorphism (χ2=1.11; p=0.29; OR=0.83; 95% CI=0.59-1.16); and LOC284889 (χ2=0.78; p=0.37; OR=0.86; 95% CI=0.61-1.20)]. CONCLUSION: Since we did not find any significant differences between case and control groups, we conclude that sequencing of complete MIF gene and extensive study on innate and adaptive immunity genes may help in identifying genetic variations that are associated with VL susceptibility/resistance among Indians.

Isolation of Physiologically Active and Intact Mitochondria from Chickpea.

Chickpea is an important leguminous crop that belongs to Fabaceae family, highly valued for its nutritious seeds. Seeds contain reserve food for the developing embryos. Mitochondria are crucial organelle for generation of chemical energy in the form of ATP which is required for achieving metabolically active state; therefore, investigating mitochondrial function and respiration rate is crucial for exploring various metabolic and physio-biochemical changes that occur during seed germination. Here we describe a method for isolation of mitochondria from germinating seeds of two chickpea varieties, i.e., Desi and Kabuli. Structure of Mitotracker-stained isolated mitochondria was observed by confocal microscopy and respiration rate was measured using an oxygen microsensor.

Measurement of Respiration and Internal Oxygen in Germinating Cicer arietinum L. Seeds Using Optic Microsensor.

Internal oxygen concentrations vary in different tissues depending on tissue size, developmental stage, and their location. Respiratory rate of tissue also determines internal oxygen levels. For studying various signaling pathways it is essential to establish a correlation between respiration and internal oxygen. Seed germination is associated with increase in respiration which can dictate the internal oxygen and subsequent production of reactive oxygen species. Using optic oxygen microsensor we made an attempt to measure respiratory rate and internal oxygen. We found that microsensor is able to sense internal oxygen and it is also possible to measure oxygen levels in a close vial that contains seeds. Step-by-step protocol is described here along with illustration.

In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance.

Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas.

Physio-Biochemical Composition and Untargeted Metabolomics of Cumin (Cuminum cyminum L.) Make It Promising Functional Food and Help in Mitigating Salinity Stress.

Cumin is an annual, aromatic, herbaceous, medicinal, spice plant, most widely used as a food additive and flavoring agent in different cuisines. The study is intended to comprehensively analyse physiological parameters, biochemical composition and metabolites under salinity stress. Seed germination index, rate of seed emergence, rate of seed germination, mean germination time, plant biomass, total chlorophyll and carotenoid contents decreased concomitantly with salinity. In contrast, total antioxidant activity, H2O2, proline and MDA contents increased concurrently with stress treatments. Total phenolic and flavonoid contents were decreased initially about 1.4-fold at 50 mM, and thereafter increased about 1.2-fold at 100 mM NaCl stress. Relative water content remained unchanged up to 50 mM NaCl stress, and thereafter decreased significantly. About 2.8-fold electrolyte leakage was found in 50 mM, which increases further 4-fold at 100 mM NaCl stress. Saturated fatty acids (FAs) increased gradually with salinity, whereas unsaturation index and degree of unsaturation change arbitrarily along with the percent quantity of unsaturated FAs. Total lipid and fatty acid composition were significantly influenced by salinity stress. A total of 45 differentially expressed metabolites were identified, including luteolin, salvianolic acid, kaempferol and quercetin, which are phenolic, flavonoid or alkaloids in nature and contain antioxidant activities. Additionally, metabolites with bioactivity such as anticancerous (docetaxel) and antimicrobial (megalomicin) properties were also identified. The study evidenced that plant shoots are a rich source of metabolites, essential amino acids, phenolic compounds and fatty acids, which unveil the medicinal potential of this plant, and also provide useful insight about metabolic responses under salinity stress.

An efficient method for Agrobacterium-mediated genetic transformation and plant regeneration in cumin (Cuminum cyminum L.).

Cumin is an annual herbaceous medicinally important plant having diverse applications. An efficient and reproducible method of Agrobacterium-mediated genetic transformation was herein established for the first time. A direct regeneration method without callus induction was optimised using embryos as explant material in Gamborg's B5 medium supplemented with 0.5-µM 6-benzyladenine and 2.0-µM α-naphthalene acetic acid. About 1,020 embryos (a mean of 255 embryos per batch) were used for the optimisation of transformation conditions. These conditions were an Agrobacterium cell suspension of 0.6 OD600, a co-cultivation time of 72 h, 300-µM acetosyringone and wounding of explants using a razor blade. Pre-cultured elongated embryos were treated using optimised conditions. About 720 embryos (a mean of 180 embryos per batch) were used for transformation and 95 % embryos showed transient ß-glucuronidase expression after co-cultivation. Putative transformed embryos were cultured on B5 medium for shoot proliferation and 21 regenerated plants were obtained after selection and allowed to root. T0 plantlets showed ß-glucuronidase expression and gene integration was confirmed via PCR amplification of 0.96 and 1.28 kb fragments of the hygromycin-phosphotransferase II and ß-glucuronidase genes, respectively. In this study, a transformation efficiency of 1.5 % was demonstrated and a total of 11 transgenic plants were obtained at the hardening stage, however, only four plants acclimatised during hardening. Gene copy number was analysed by Southern blot analysis of hardened plants and single-copy gene integration was observed. This is the first successful attempt of Agrobacterium-mediated genetic transformation of cumin.