Induced genetic diversity through mutagenesis in wheat gene pool and significant use of SCoT markers to underpin key agronomic traits.

BACKGROUND: This research explores the efficacy of mutagenesis, specifically using sodium azide (SA) and hydrazine hydrate (HZ) treatments, to introduce genetic diversity and enhance traits in three wheat (Triticum aestivum L.) genotypes. The experiment entails subjecting the seeds to different doses of SA and HZ and cultivating them in the field for two consecutive generations: M1 (first generation) and M2 (second generation). We then employed selective breeding techniques with Start Codon Targeted (SCoT) markers to select traits within the wheat gene pool. Also, the correlation between SCoT markers and specific agronomic traits provides insights into the genetic mechanisms underlying mutagenesis-induced changes in wheat. RESULTS: In the study, eleven genotypes were derived from parent varieties Sids1, Sids12, and Giza 168, and eight mutant genotypes were selected from the M1 generation and further cultivated to establish the M2 generation. The results revealed that various morphological and agronomical characteristics, such as plant height, spikes per plant, spike length, spikelet per spike, grains per spikelet, and 100-grain weight, showed increases in different genotypes from M1 to M2. SCoT markers were employed to assess genetic diversity among the eleven genotypes. The bioinformatics analysis identified a correlation between SCoT markers and the transcription factors ABSCISIC ACID INSENSITIVE3 (ABI3) and VIVIPAROUS1 (VP1), crucial for plant development, growth, and stress adaptation. A comprehensive examination of genetic distance and the function identification of gene-associated SCoT markers may provide valuable insights into the mechanisms by which SA and HZ act as mutagens, enhancing wheat agronomic qualities. CONCLUSIONS: This study demonstrates the effective use of SA and HZ treatments to induce gene diversity through mutagenesis in the wheat gene pool, resulting in the enhancement of agronomic traits, as revealed by SCoT markers. The significant improvements in morphological and agronomical characteristics highlight the potential of mutagenesis techniques for crop improvement. These findings offer valuable information for breeders to develop effective breeding programs to enhance wheat quality and resilience through increased genetic diversity.

A New Family of High Oxidation State Antiperovskite Nitrides: La3MN5 (M=Cr, Mn and Mo).

Three new nitrides La3MN5 (M=Cr, Mn, and Mo) have been synthesized using a high pressure azide route. These are the first examples of ternary Cs3CoCl5-type nitrides, and show that this (MN4)NLa3 antiperovskite structure type may be used to stabilise high oxidation-state transition metals in tetrahedral molecular [MN4]n- nitridometallate anions. Magnetic measurements confirm that Cr and Mo are in the M6+ state, but the M=Mn phase has an anomalously small paramagnetic moment and large cell volume. Neutron powder diffraction data are fitted using an anion-excess La3MnN5.30 model (space group I4/mcm, a=6.81587(9) Šand c=11.22664(18) Šat 200 K) in which Mn is close to the +7 state. Excess-anion incorporation into Cs3CoCl5-type materials has not been previously reported, and this or other substitution mechanisms may enable many other high oxidation state transition metal nitrides to be prepared.

Inhibition of peroxidases and oxidoreductases is crucial for avoiding false-positive reactions in the localization of reactive oxygen species in intact barley root tips.

MAIN CONCLUSION: NBT and HE may be efficiently used for the detection of superoxide, while DCDHF-DA and DHR123 for the detection of peroxynitrite in intact barley root tips, only if PRXs and oxidoreductases are inhibited to avoid false-positive reactions. Strong peroxidase (PRX) and oxidoreductase activities were observed in the barley root tips that were markedly inhibited by NaN3. Rapid and strong nitro-blue tetrazolium chloride (NBT) reduction is associated mainly with the vital functions of root cells but not with superoxide formation. In turn, the inhibition of root surface redox activity by NaN3 strongly reduced the formation of formazan, but its slight accumulation, observed in the root elongation zone, was a result of NADPH oxidase-mediated apoplastic superoxide formation. A longer staining time period with NBT was required for the detection of antimycin A-mediated superoxide formation inside the cells. This antimycin A-induced superoxide was clearly detectable by hydroethidine (HE) after the inhibition of PRXs by NaN3, and it was restricted into the root transition zone. TEMPOL, a superoxide scavenger, strongly inhibited both NBT reduction and HE oxidation in the presence of NaN3. Similarly, the DCDHF-DA and DHR123 oxidation was markedly reduced after the inhibition of apoplastic PRXs by NaN3 and was detectable mainly in the root transition zone. This fluorescence signal was not influenced by the application of pyruvate but was strongly reduced by urea, a peroxynitrite scavenger. The presented results suggest that if the root PRXs and oxidoreductases are inhibited, both NBT and HE detect mainly superoxide, whereas both DCDHF-DA and DHR123 may be efficiently used for the detection of peroxynitrite in intact barley root tips. The inhibition of PRXs and oxidoreductases is crucial for avoiding false-positive reactions in the localization of reactive oxygen species in the intact barley root tip.

Mutagenic effects of sodium azide on in vitro mutagenesis, polymorphism and genomic instability in wheat (Triticum aestivum L.).

INTRODUCTION: Breeding studies are commonly conducted to develop new cultivars with high yield levels and improved quality traits. Chemically-induced mutations are used to create genetic variations in wheat genomes. Various physical and chemical mutagens are used to increase frequency of mutations and facilitate the selection processes. Sodium azide (SA) is largely employed to induce mutations of the genes regulating essential traits. Such mutations may also elucidate gene functions of the mutant phenotypes. Present experiments were conducted to investigate potential use of conventional chemical mutagenesis technique through SA for mature embryo culture in wheat. METHODS AND RESULTS: Sodium azide mutagenesis was experimented with 4 treatment durations (1, 2, 3 and 4 h) and 5 treatment concentrations (0, 1, 2, 3 and 4 mM). Mature embryos were subjected to experimental treatments to detect optimum doses of mutagenesis and to estimate polymorphism and genomic instability. Primarily, 50% reduction in number of regenerated plants as compared to the control (LD50) was adopted as the optimum dose. Based on LD50 criterion, the optimum value was achieved at 1 h duration of 4 mM SA concentration. Afterwards, inter-primer binding site markers were applied to investigate polymorphism and genomic instability in the regenerated plants. CONCLUSIONS: Present findings revealed that efficiency of chemical mutagenesis could be improved through the use of molecular technology and such mutations may assist plant breeders in developing high-yield cultivars.

Mitochondrial Inhibition by Sodium Azide Induces Assembly of eIF2α Phosphorylation-Independent Stress Granules in Mammalian Cells.

Mitochondrial stress is involved in many pathological conditions and triggers the integrated stress response (ISR). The ISR is initiated by phosphorylation of the eukaryotic translation initiation factor (eIF) 2α and results in global inhibition of protein synthesis, while the production of specific proteins important for the stress response and recovery is favored. The stalled translation preinitiation complexes phase-separate together with local RNA binding proteins into cytoplasmic stress granules (SG), which are important for regulation of cell signaling and survival under stress conditions. Here we found that mitochondrial inhibition by sodium azide (NaN3) in mammalian cells leads to translational inhibition and formation of SGs, as previously shown in yeast. Although mammalian NaN3-induced SGs are very small, they still contain the canonical SG proteins Caprin 1, eIF4A, eIF4E, eIF4G and eIF3B. Similar to FCCP and oligomycine, other mitochodrial stressors that cause SG formation, NaN3-induced SGs are formed by an eIF2α phosphorylation-independent mechanisms. Finally, we discovered that as shown for arsenite (ASN), but unlike FCCP or heatshock stress, Thioredoxin 1 (Trx1) is required for formation of NaN3-induced SGs.

Acid-Switchable Synthesis of Trifluoromethylated Triazoles and Isoxazoles via Reaction of CF3-Ynones with NaN3: DFT Study of the Reaction Mechanism.

A detailed study of the reaction of CF3-ynones with NaN3 was performed. It was found that the reaction permits the selective synthesis of either 4-trifluoroacetyltriazoles or 5-CF3-isoxazoles. The chemoselectivity of the reaction was switchable via acid catalysis. The reaction of CF3-ynones with NaN3 in EtOH produced high yields of 4-trifluoroacetyltriazoles. In contrast, the formation of 5-CF3-isoxazoles was observed under catalysis by acids. This acid-switchable procedure can be performed at sub-gram scale. The possible reaction mechanism was supported by DFT calculations. The synthetic utility of the prepared 4-trifluoroacetyltriazoles was demonstrated.

Elucidation of the RNA-granule inducing sodium azide stress response through transcriptome analysis.

Sodium azide is a commonly used cytochrome oxidase inhibitor that leads to translation repression and RNA granule assembly. The global changes in mRNA abundance in response to this stressor are unknown. RGG-motif proteins Scd6 and Sbp1 are translation-repressors and decapping-activators that localize to and affect the assembly of RNA granules in response to sodium azide stress. Transcriptome-wide effects of these proteins remain unknown. To address this, we have sequenced transcriptome of the: a) wild type strain under unstressed and sodium azide stress, b) Δscd6 and Δsbp1 strains under unstressed and sodium azide stress. Transcriptome analysis identified altered abundance of many transcripts belonging to stress-responsive pathways which were further validated by qRT-PCR results. Abundance of several transcripts was altered in Δscd6/Δsbp1 under normal conditions and upon stress. Overall, this study provides critical insights into transcriptome changes in response to sodium azide stress and the role of RGG-motif proteins in these changes.

Eosin Y as a high-efficient photooxidase mimic for colorimetric detection of sodium azide.

The reported fluorescent dye-based artificial light-responsive oxidase mimics are suffering from their low catalytic efficiency. To overcome the limitation, we report the photooxidase-mimicking activity of Eosin Y which can catalyze the oxidation of various chromogenic substrates such as 3,3',5,5'-tetramethylbenzydine (TMB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 3,3'-diaminobenzidine (DAB), and o-phenylenediamine (OPD) by dissolved oxygen. The photooxidase-like activity of Eosin Y is highly efficient for TMB substrate, and its catalytic efficiency is higher than that of the reported fluorescein (130 fold) and 9-mesityl-10-methylacridinium ion (7.7-fold) mimetic photooxidase. Moreover, the photosensitized Eosin Y-TMB chromogenic system is utilized for colorimetric detection of highly toxic and explosive sodium azide (NaN3) in a linear range from 5 to 500 µM with a limit of detection of 3.5 µM. The resulting colorimetric assay is selective and applied to determine NaN3 in real lake water samples.

Evaluation of neuroprotective activity of digoxin and semisynthetic derivatives against partial chemical ischemia.

Recently, cardiotonic steroids (CTS) have been shown to lead to the activation of Na,K-ATPase at low concentrations in brain, promoting neuroprotection against ischemia. We report here the results of the use of digoxin and its semisynthetic derivatives BD-14, BD-15, and BD-16 against partial chemical ischemic induction followed by reperfusion in murine neuroblastoma cells neuro-2a (N2a). For chemical ischemic induction, sodium azide (5 mM) was used for 5 hours, and then reperfusion was induced for 24 hours. Na,K-ATPase activity and protein levels were analyzed in membrane preparation of N2a cells pretreated with the compounds (150 nM), in the controls and in induced chemical ischemia. In the Na,K-ATPase activity and protein levels assays, the steroids digoxin and BD-15 demonstrated a capacity to modulate the activity of the enzyme directly, increasing its levels of expression and activity. Oxidative parameters, such as superoxide dismutase (SOD) activity, lipid peroxidation (thiobarbituric acid reactive substance), glutathione peroxidase (GPx), glutathione (GSH) levels, hydrogen peroxide content, and the amount of free radicals (reactive oxygen species) during induced chemical ischemia were also evaluated. Regarding the redox state, lipid peroxidation, hydrogen peroxide content, and GPx activity, we have observed an increase in the chemical ischemic group, and a reduction in the groups treated with CTS. SOD activity increased in all treated groups when compared to control and GSH levels decreased when treated with sodium azide and did not change with CTS treatments. Regarding the lipid profile, we saw a decrease in the content of phospholipids and cholesterol in the chemical ischemic group, and an increase in the groups treated with CTS. In conclusion, the compounds used in this study demonstrate promising results, since they appear to promote neuroprotection in cells exposed to chemical ischemia.