New Insights on the Role of ß-Cyanoalanine Synthase CAS-C1 in Root Hair Elongation through Single-Cell Proteomics.

(1) Background: Root hairs are specialized structures involved in water and plant nutrient uptake. They elongate from epidermal cells following a complex developmental program. ß-cyanoalanine synthase (CAS), which is mainly involved in hydrogen cyanide (HCN) detoxification in Arabidopsis thaliana, plays a role in root hair elongation, as evidenced by the fact that cas-c1 mutants show a severe defect in root hair shape. In addition to root hairs, CAS C1 is expressed in the quiescent center and meristem. (2) Methods: To identify its role in root hair formation, we conducted single-cell proteomics analysis by isolating root hair cells using Fluorescence-activated Cell Sorting (FACS) from wild-type and cas-c1 mutants. We also analyzed the presence of S-cyanylation, a protein post-translational modification (PTM) mediated by HCN and affecting cysteine residues and protein activity in proteins of wild type and cas-c1 mutants. (3) Results and Conclusions: We have found that the cas-c1 mutation has no visible effect on quiescent center or meristem root tissue, in both control and nutrient-deprivation conditions. We have identified more than 3900 proteins in root hairs and we have found that several proteins involved in root hair development, related to the receptor kinase FERONIA signaling and DNA methylation, are modified by S-cyanylation.

Mutation in Arabidopsis ß-cyanoalanine synthase overcomes NADPH oxidase action in response to pathogens.

Plant responses to pathogens comprise a complex process, implying a plethora of signals and reactions. Among them, endogenous production of hydrogen cyanide (HCN) has been shown to induce resistance in Arabidopsis to the hemibiotrophic bacterium Pseudomonas syringae pv. tomato (Pst) DC3000. ß-cyanoalanine synthase (CAS-C1) is responsible for the detoxification of HCN in Arabidopsis mitochondria. Here, we show that green fluorescent protein-tagged CAS-C1 is transiently reduced in leaves infected with an avirulent strain of Pst during early interactions and increased in leaves infected with a virulent strain of Pst, supporting previous transcriptional data. Genetic crosses show that mutation in CAS-C1 in Arabidopsis resembles the action of the NADPH oxidase RbohD independently of reactive oxygen species production and that the accumulation of salicylic acid is required for HCN-stimulated resistance to Pst. Finally, we show that the cas-c1 mutation acts on the salicylic acid-dependent response to pathogens by mechanisms other than protein ubiquitination or the increase of monomerization and entry to the nucleus of NPR1, the central regulator of the salicylic acid-mediated response. Considering these results, we propose new mechanisms for modulation of the immune response by HCN.

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.

HCN Regulates Cellular Processes through Posttranslational Modification of Proteins by S-cyanylation.

Hydrogen cyanide (HCN) is coproduced with ethylene in plant cells and is primarily enzymatically detoxified by the mitochondrial ß-CYANOALANINE SYNTHASE (CAS-C1). Permanent or transient depletion of CAS-C1 activity in Arabidopsis (Arabidopsis thaliana) results in physiological alterations in the plant that suggest that HCN acts as a gasotransmitter molecule. Label-free quantitative proteomic analysis of mitochondrially enriched samples isolated from the wild type and cas-c1 mutant revealed significant changes in protein content, identifying 451 proteins that are absent or less abundant in cas-c1 and 353 proteins that are only present or more abundant in cas-c1 Gene ontology classification of these proteins identified proteomic changes that explain the root hairless phenotype and the altered immune response observed in the cas-c1 mutant. The mechanism of action of cyanide as a signaling molecule was addressed using two proteomic approaches aimed at identifying the S-cyanylation of Cys as a posttranslational modification of proteins. Both the 2-imino-thiazolidine chemical method and the direct untargeted analysis of proteins using liquid chromatography-tandem mass spectrometry identified a set of 163 proteins susceptible to S-cyanylation that included SEDOHEPTULOSE 1,7-BISPHOSPHATASE (SBPase), the PEPTIDYL-PROLYL CIS-TRANS ISOMERASE 20-3 (CYP20-3), and ENOLASE2 (ENO2). In vitro analysis of these enzymes showed that S-cyanylation of SBPase Cys74, CYP20-3 Cys259, and ENO2 Cys346 residues affected their enzymatic activity. Gene Ontology classification and protein-protein interaction cluster analysis showed that S-cyanylation is involved in the regulation of primary metabolic pathways, such as glycolysis, and the Calvin and S-adenosyl-Met cycles.

Role of mitochondrial cyanide detoxification in Arabidopsis root hair development.

In non-cyanogenic plants, cyanide is produced during ethylene biosynthesis and is mainly detoxified by the ß-cyanoalanine synthase CAS-C1. Arabidopsis plants lacking CAS-C1 show abnormal root hairs, which stop growing at early stages. Root hair elongates by polarized cell expansion at the tip, and we have observed that CAS-C1-driven GFP fluorescence locates in mitochondria and accumulates in root hair tips during root hair elongation. Genetic crosses have been performed between cas-c1 plants and scn1-1 mutants, defective in the SCN1 protein that regulates the NADPH oxidase RHD2/AtrbohC, and between cas-c1 and rhd2-1, defective in the NADPH oxidase necessary for the generation of ROS and the Ca2+ gradient necessary for root hair elongation. The phenotypic and molecular analysis of these crosses indicates that cas-c1 is hypostatic to scn1-1 and epistatic to rhd2-1. Furthermore, the action of cyanide in root hair development is independent of ROS and of direct NADPH oxidase inhibition by cyanide.

ß-Cyanoalanine Synthase Action in Root Hair Elongation is Exerted at Early Steps of the Root Hair Elongation Pathway and is Independent of Direct Cyanide Inactivation of NADPH Oxidase.

In Arabidopsis thaliana, cyanide is produced concomitantly with ethylene biosynthesis and is mainly detoxified by the ß-cyanoalanine synthase CAS-C1. In roots, CAS-C1 activity is essential to maintain a low level of cyanide for proper root hair development. Root hair elongation relies on polarized cell expansion at the growing tip, and we have observed that CAS-C1 locates in mitochondria and accumulates in root hair tips during root hair elongation, as shown by observing the fluorescence in plants transformed with the translational construct ProC1:CASC1-GFP, containing the complete CAS-C1 gene fused to green fluorescent protein (GFP). Mutants in the SUPERCENTIPEDE (SCN1) gene, that regulate the NADPH oxidase gene ROOT HAIR DEFECTIVE 2 (RHD2)/AtrbohC, are affected at the very early steps of the development of root hair that do not elongate and do not show a preferential localization of the GFP accumulation in the tips of the root hair primordia. Root hairs of mutants in CAS-C1 or RHD2/AtrbohC, whose protein product catalyzes the generation of ROS and the Ca2+ gradient, start to grow out correctly, but they do not elongate. Genetic crosses between the cas-c1 mutant and scn1 or rhd2 mutants were performed, and the detailed phenotypic and molecular characterization of the double mutants demonstrates that scn1 mutation is epistatic to cas-c1 and cas-c1 is epistatic to rhd2 mutation, indicating that CAS-C1 acts in early steps of the root hair development process. Moreover, our results show that the role of CAS-C1 in root hair elongation is independent of H2O2 production and of a direct NADPH oxidase inhibition by cyanide.