Symbiosis between cyanobacteria and plants: from molecular studies to agronomic applications.

Nitrogen-fixing cyanobacteria from the order Nostocales are able to establish symbiotic relationships with diverse plant species. They are promiscuous symbionts, as the same strain of cyanobacterium is able to form symbiotic biological nitrogen-fixing relationships with different plants species. This review will focus on the different types of cyanobacterial-plant associations, both endophytic and epiphytic, and provide insights from a structural viewpoint, as well as our current understanding of the mechanisms involved in the symbiotic crosstalk. In all these symbioses, the benefit for the plant is clear; it obtains from the cyanobacterium fixed nitrogen and other bioactive compounds, such as phytohormones, polysaccharides, siderophores, or vitamins, leading to enhanced plant growth and productivity. Additionally, there is increasing use of different cyanobacterial species as bio-inoculants for biological nitrogen fixation to improve soil fertility and crop production, thus providing an eco-friendly, alternative, and sustainable approach to reduce the over-reliance on synthetic chemical fertilizers.

Sulfide promotes tolerance to drought through protein persulfidation in Arabidopsis.

Hydrogen sulfide (H2S) is a signaling molecule that regulates essential plant processes. In this study, the role of H2S during drought was analysed, focusing on the underlying mechanism. Pretreatments with H2S before imposing drought on plants substantially improved the characteristic stressed phenotypes under drought and decreased the levels of typical biochemical stress markers such as anthocyanin, proline, and hydrogen peroxide. H2S also regulated drought-responsive genes and amino acid metabolism, and repressed drought-induced bulk autophagy and protein ubiquitination, demonstrating the protective effects of H2S pretreatment. Quantitative proteomic analysis identified 887 significantly different persulfidated proteins between control and drought stress plants. Bioinformatic analyses of the proteins more persulfidated in drought revealed that the most enriched biological processes were cellular response to oxidative stress and hydrogen peroxide catabolism. Protein degradation, abiotic stress responses, and the phenylpropanoid pathway were also highlighted, suggesting the importance of persulfidation in coping with drought-induced stress. Our findings emphasize the role of H2S as a promoter of enhanced tolerance to drought, enabling plants to respond more rapidly and efficiently. Furthermore, the main role of protein persulfidation in alleviating reactive oxygen species accumulation and balancing redox homeostasis under drought stress is highlighted.

Proteome Dynamics of Persulfidation in Leaf Tissue under Light/Dark Conditions and Carbon Deprivation.

Hydrogen sulfide (H2S) acts as a signaling molecule in plants, bacteria, and mammals, regulating various physiological and pathological processes. The molecular mechanism by which hydrogen sulfide exerts its action involves the posttranslational modification of cysteine residues to form a persulfidated thiol motif. This research aimed to study the regulation of protein persulfidation. We used a label-free quantitative approach to measure the protein persulfidation profile in leaves under different growth conditions such as light regimen and carbon deprivation. The proteomic analysis identified a total of 4599 differentially persulfidated proteins, of which 1115 were differentially persulfidated between light and dark conditions. The 544 proteins that were more persulfidated in the dark were analyzed, and showed significant enrichment in functions and pathways related to protein folding and processing in the endoplasmic reticulum. Under light conditions, the persulfidation profile changed, and the number of differentially persulfidated proteins increased up to 913, with the proteasome and ubiquitin-dependent and ubiquitin-independent catabolic processes being the most-affected biological processes. Under carbon starvation conditions, a cluster of 1405 proteins was affected by a reduction in their persulfidation, being involved in metabolic processes that provide primary metabolites to essential energy pathways and including enzymes involved in sulfur assimilation and sulfide production.

Detection of protein persulfidation in plants by the dimedone switch method.

Hydrogen sulfide (H2S) is a well-known signaling molecule in both animals and plants, endogenously produced by cells, and involved in a wide variety of biological functions. In plants, H2S regulates a wide range of essential aspects of plant life, including plant responses to numerous stresses and physiological processes as important as abscisic acid (ABA)-dependent stomatal movement, photosynthesis, and autophagy. The best studied molecular mechanism responsible of sulfide signaling is protein persulfidation, a post-translational modification of cysteine residues, where a thiol group (P-SH) is transformed into a persulfide group (P-SSH). In this way, persulfidation has emerged as a new type of cellular redox mechanism that can regulate protein structure and function and interest in this modification has increased exponentially. However, the identification and the development of detection methods have been challenging. Nevertheless, on the basis of the chemical differences between the thiol and the persulfide groups, different methods have been implemented. In plants, different high-throughput proteomic analyzes have been performed using a tag-switch method where in the first step all thiols and persulfides are blocked and then in the second step persulfides are selectively labeled using a specific nucleophile. This chapter outlines a new method, previously described in mammals, that has been applied to detect persulfidation in plants and is based on the same chemical premise but consists of chemoselective persulfide labeling with dimedone-based probes. Here, we provide a detailed workflow of this method that includes procedures for the determination of the persulfidation level of a protein extract visualized and quantified by fluorescence on the gel on one side, and on the other, the labeling and purification of persulfidated proteins for identification by mass spectrometry.

Label-Free Quantitative Proteomic Analysis of Nitrogen Starvation in Arabidopsis Root Reveals New Aspects of H2S Signaling by Protein Persulfidation.

Hydrogen sulfide (H2S)-mediated signaling pathways regulate many physiological and pathophysiological processes in mammalian and plant systems. The molecular mechanism by which hydrogen sulfide exerts its action involves the posttranslational modification of cysteine residues to form a persulfidated thiol motif. We developed a comparative and label-free quantitative proteomic analysis approach for the detection of endogenous persulfidated proteins in N-starved Arabidopsis thaliana roots by using the tag-switch method. In this work, we identified 5214 unique proteins from root tissue that were persulfidated, 1674 of which were quantitatively analyzed and found to show altered persulfidation levels in vivo under N deprivation. These proteins represented almost 13% of the entire annotated proteome in Arabidopsis. Bioinformatic analysis revealed that persulfidated proteins were involved in a wide range of biological functions, regulating important processes such as primary metabolism, plant responses to stresses, growth and development, RNA translation and protein degradation. Quantitative mass spectrometry analysis allowed us to obtain a comprehensive view of hydrogen sulfide signaling via changes in the persulfidation levels of key protein targets involved in ubiquitin-dependent protein degradation and autophagy, among others.

Exploring the Functional Relationship between y-Type Thioredoxins and 2-Cys Peroxiredoxins in Arabidopsis Chloroplasts.

Thioredoxins (Trxs) are small, ubiquitous enzymes that catalyze disulphide-dithiol interchange in target enzymes. The large set of chloroplast Trxs, including f, m, x and y subtypes, use reducing equivalents fueled by photoreduced ferredoxin (Fdx) for fine-tuning photosynthetic performance and metabolism through the control of the activity of redox-sensitive proteins. Although biochemical analyses suggested functional diversity of chloroplast Trxs, genetic studies have established that deficiency in a particular Trx subtype has subtle phenotypic effects, leading to the proposal that the Trx isoforms are functionally redundant. In addition, chloroplasts contain an NADPH-dependent Trx reductase with a joint Trx domain, termed NTRC. Interestingly, Arabidopsis mutants combining the deficiencies of x- or f-type Trxs and NTRC display very severe growth inhibition phenotypes, which are partially rescued by decreased levels of 2-Cys peroxiredoxins (Prxs). These findings indicate that the reducing capacity of Trxs f and x is modulated by the redox balance of 2-Cys Prxs, which is controlled by NTRC. In this study, we explored whether NTRC acts as a master regulator of the pool of chloroplast Trxs by analyzing its functional relationship with Trxs y. While Trx y interacts with 2-Cys Prxs in vitro and in planta, the analysis of Arabidopsis mutants devoid of NTRC and Trxs y suggests that Trxs y have only a minor effect, if any, on the redox state of 2-Cys Prxs.

Abscisic Acid-Triggered Persulfidation of the Cys Protease ATG4 Mediates Regulation of Autophagy by Sulfide.

Hydrogen sulfide is a signaling molecule that regulates essential processes in plants, such as autophagy. In Arabidopsis (Arabidopsis thaliana), hydrogen sulfide negatively regulates autophagy independently of reactive oxygen species via an unknown mechanism. Comparative and quantitative proteomic analysis was used to detect abscisic acid-triggered persulfidation that reveals a main role in the control of autophagy mediated by the autophagy-related (ATG) Cys protease AtATG4a. This protease undergoes specific persulfidation of Cys170 that is a part of the characteristic catalytic Cys-His-Asp triad of Cys proteases. Regulation of the ATG4 activity by persulfidation was tested in a heterologous assay using the Chlamydomonas reinhardtii CrATG8 protein as a substrate. Sulfide significantly and reversibly inactivates AtATG4a. The biological significance of the reversible inhibition of the ATG4 by sulfide is supported by the results obtained in Arabidopsis leaves under basal and autophagy-activating conditions. A significant increase in the overall ATG4 proteolytic activity in Arabidopsis was detected under nitrogen starvation and osmotic stress and can be inhibited by sulfide. Therefore, the data strongly suggest that the negative regulation of autophagy by sulfide is mediated by specific persulfidation of the ATG4 protease.

Signaling by hydrogen sulfide and cyanide through post-translational modification.

Two cysteine metabolism-related molecules, hydrogen sulfide and hydrogen cyanide, which are considered toxic, have now been considered as signaling molecules. Hydrogen sulfide is produced in chloroplasts through the activity of sulfite reductase and in the cytosol and mitochondria by the action of sulfide-generating enzymes, and regulates/affects essential plant processes such as plant adaptation, development, photosynthesis, autophagy, and stomatal movement, where interplay with other signaling molecules occurs. The mechanism of action of sulfide, which modifies protein cysteine thiols to form persulfides, is related to its chemical features. This post-translational modification, called persulfidation, could play a protective role for thiols against oxidative damage. Hydrogen cyanide is produced during the biosynthesis of ethylene and camalexin in non-cyanogenic plants, and is detoxified by the action of sulfur-related enzymes. Cyanide functions include the breaking of seed dormancy, modifying the plant responses to biotic stress, and inhibition of root hair elongation. The mode of action of cyanide is under investigation, although it has recently been demonstrated to perform post-translational modification of protein cysteine thiols to form thiocyanate, a process called S-cyanylation. Therefore, the signaling roles of sulfide and most probably of cyanide are performed through the modification of specific cysteine residues, altering protein functions.