Autophagy is a highly conserved process that degrades damaged macromolecules and organelles. Unlike animals, only scant information is available regarding nitric oxide (NO)-induced autophagy in plants. Such lack of information prompted us to study the roles of the NO donors’ nitrate, nitrite, and sodium nitroprusside in this catabolic process in wheat roots. Furthermore, spermine, a polyamine that is found in all eukaryotic cells, was also tested as a physiological NO donor. Here, we show that in wheat roots, NO donors and spermine can trigger autophagy, with NO and reactive oxygen species (ROS) playing signaling roles based on the visualization of autophagosomes, analyses of the levels of NO, ROS, mitochondrial activity, and the expression of autophagic (ATG) genes. Treatment with nitrite and nitroprusside causes an energy deficit, a typical prerequisite of autophagy, which is indicated by a fall in mitochondrial potential, and the activity of mitochondrial complexes. On the contrary, spermine sustains energy metabolism by upregulating the activity of appropriate genes, including those that encode glyceraldehyde 3-phosphate dehydrogenase GAPDH and SNF1-related protein kinase 1 SnRK1. Taken together, our data suggest that one of the key roles for NO in plants may be to trigger autophagy via diverse mechanisms, thus facilitating the removal of oxidized and damaged cellular constituencies.

Autophagy is a highly conserved catabolic process in eukaryotic cells. Reactive nitrogen species play roles as inductors and signaling molecules of autophagy. A key mechanism of NO-mediated signaling is S-nitrosylation, a post-translational modification (PTM) of proteins at cysteine residues. In the present work, we analyzed the patterns of protein S-nitrosylation during the induction of autophagy in Triticum aestivum roots. The accumulation of S-nitrosylated proteins in the cells during autophagy induced with KNO₂ and antimycin A was visualized using monoclonal antibodies with a Western blot analysis, and proteins were identified using a standard bottom-up proteomics approach. Protein S-nitrosylation is a labile and reversible PTM, and therefore the SNO group can be lost during experimental procedures. A subsequent bioinformatic analysis using predictive algorithms and protein-ligand docking showed that identified proteins possess hypothetical S-nitrosylation sites. Analyzing protein–protein interaction networks enabled us to discover the targets that can directly interact with autophagic proteins, and those that can interact with them indirectly via key multifunctional regulatory proteins. In this study, we show that S-nitrosylation is a key mechanism of NO-mediated regulation of autophagy in wheat roots. A combination of in silico predictive algorithms with a mass spectrometry analysis provides a targeted approach for the identification of S-nitrosylated proteins.

Plant dehydration-responsive element binding (DREB) transcription factors (TFs) play important roles during stress tolerance by regulating the expression of numerous genes involved in stresses. DREB TFs have been extensively studied in a variety of angiosperms and bryophytes. To date, no information on the identification and characterization of DREB TFs in Dicranum scoparium has been reported. In this study, a new DBF1 gene from D. scoparium was identified by cloning and sequencing. Analysis of the conserved domain and physicochemical properties revealed that DsDBF1 protein has a classic AP2 domain encoding a 238 amino acid polypeptide with a molecular mass of 26 kDa and a pI of 5.98. Subcellular prediction suggested that DsDBF1 is a nuclear and cytoplasmic protein. Phylogenetic analysis showed that DsDBF1 belongs to group A-5 DREBs. Expression analysis by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) revealed that DsDBF1 was significantly upregulated in response to abiotic stresses such as desiccation/rehydration, exposure to paraquat, CdCl₂, high and freezing temperatures. Taken together, our data suggest that DsDBF1 could be a promising gene candidate to improve stress tolerance in crop plants, and the characterization of TFs of a stress tolerant moss such as D. scoparium provides a better understanding of plant adaptation mechanisms.

Mitochondria play a key role in providing energy to cells. These organelles are constantly undergoing dynamic processes of fusion and fission that change in stressful conditions. The role of mitochondrial fusion in wheat root cells was studied using Mdivi-1, an inhibitor of the mitochondrial fragmentation protein Drp1. The effect of the inhibitor was studied on mitochondrial dynamics in the roots of wheat seedlings subjected to a wounding stress, simulated by excision. Treatment of the stressed roots with the inhibitor increased the size of the mitochondria, enhanced their functional activity, and elevated their membrane potentials. Mitochondrial fusion was accompanied by a decrease in ROS formation and associated cell damage. Exposure to Mdivi-1 also upregulated genes encoding the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and an energy sensor AMP-dependent protein sucrose non-fermenting-related kinase (SnRK1), suggesting that mitochondrial fusion is associated with a general activation of energy metabolism. Controlling mitochondrial fusion rates could change the physiology of wheat plants by altering the energy status of the cell and helping to mitigate the effects of stress.

Autophagy is a highly conserved catabolic process in eukaryotic cells. Reactive nitrogen species play roles as inductors and signaling molecules of autophagy. A key mechanism of NO-mediated signaling is S-nitrosylation, a posttranslational modification (PTM) of proteins at cysteine resi-dues. In the present work we analyzed the patterns of protein S-nitrosylation during the induc-tion of autophagy in Triticum aestivum roots. The accumulation of S-nitrosylated proteins in the cells during autophagy induced by KNO2 and antimycin A was visualized using monoclonal an-tibodies by Western blot analysis, and proteins were identified using a standard bottom-up pro-teomics approach. Protein S-nitrosylation is a labile and reversible PTM, and therefore SNO group can be lost during experimental procedures. Subsequent bioinformatic analysis using predictive algorithms and protein-ligand docking showed that identified proteins possess hypothetical S-nitrosylation sites. Analyzing protein-protein interaction networks enabled us to discover the targets that can directly interact with autophagic proteins, and those that can interact with them indirectly via key multifunctional regulatory proteins. In this study, we show that S-nitrosylation is a key mechanism of NO-mediated regulation of autophagy in wheat roots. A combination of in silico predictive algorithms with a mass spectrometry analysis provides a targeted approach for the identification of S-nitrosylated proteins.