Persulfidation protects from oxidative stress under nonphotorespiratory conditions in Arabidopsis

Persulfidation protects from oxidative stress under nonphotorespiratory conditions in Arabidopsis Margarita Garcıa-Calderon 1 , Thibaut Vignane 2 , Milos R. Filipovic 2 , M. Teresa Ruiz 3 , Luis C. Romero 3 , Antonio J. Marquez 1 , Cecilia Gotor 3 and Angeles Aroca 1,3 1 Departamento de Bioquımica Vegetal y Biologıa Molecular, Universidad de Sevilla, Prof. Garcıa Gonzalez 1, 41012 Sevilla, Spain; 2 Leibniz Institute for Analytical Sciences, ISAS e.V., 44227 Dortmund, Germany; 3 Instituto de Bioquımica Vegetal y Fotosıntesis (Universidad de Sevilla, Consejo Superior de Investigaciones Cientıficas), Americo Vespucio 49, 41092 Sevilla, Spain Authors for correspondence: Angeles Aroca Email: [email protected] Cecilia Gotor Email: [email protected] Received: 21 November 2022 Accepted: 18 February 2023 New Phytologist (2023) 238: 1431–1445 doi: 10.1111/nph.18838 Key words: Arabidopsis, high CO 2 , hydrogen sulfide, persulfidation, photorespiration, reactive oxygen species, stomatal movement. Summary Hydrogen sulfide is a signaling molecule in plants that regulates essential biological processes through protein persulfidation. However, little is known about sulfide-mediated regulation in relation to photorespiration. Here, we performed label-free quantitative proteomic analysis and observed a high impact on protein persulfidation levels when plants grown under nonphotorespiratory conditions were transferred to air, with 98.7% of the identified proteins being more persulfidated under suppressed photorespiration. Interestingly, a higher level of reactive oxygen species (ROS) was detected under nonphotorespiratory conditions. Analysis of the effect of sulfide on aspects associated with non- or photorespiratory growth conditions has demonstrated that it protects plants grown under suppressed photorespiration. Thus, sulfide amends the imbalance of carbon/nitrogen and restores ATP levels to concentrations like those of air-grown plants; balances the high level of ROS in plants under nonphotorespiratory conditions to reach a cellular redox state similar to that in air-grown plants; and regulates stomatal closure, to decrease the high guard cell ROS levels and induce stomatal aperture. In this way, sulfide signals the CO 2 -dependent stomata movement, in the opposite direction of the established abscisic acid-dependent movement. Our findings suggest that the high persulfidation level under suppressed photorespiration reveals an essential role of sulfide signaling under these conditions. Introduction Photorespiration has been frequently considered a wasteful pathway that competes with the Calvin cycle because of the lack of specificity of RuBisCO, which also oxygenates RuBP, wasting some of the energy produced by photosynthesis. This process is light dependent and tends to be higher at high temperatures, low CO 2 concentrations, and high concentrations of O 2 (Sharkey, 1988). It is well known that high CO 2 suppresses photorespiration (Somerville & Ogren, 1979;Marquez et al., 2005), and engineered plants with metabolic bypasses to avoid photorespiration show enhanced productivity (Kebeish et al., 2007). However, this process plays an important role in plant carbon metabolism, since many photorespiratory mutants grow weakly under normal CO 2 conditions (Timm & Bauwe, 2013), and, in fact, it is becoming clear that photorespiration protects plants from certain stresses, conferring protection of photosynthesis from photoinhibition, and can alleviate oxidative stress under several conditions, such as salinity, altered CO 2 concentrations, drought, or heat/chilling stress (Voss et al., 2013; Dusenge et al., 2019). Therefore, in the last decade, photorespiration has been described as an important stress response in green tissues to prevent the accumulation of reactive oxygen species (ROS). Photorespiration consumes NADPH and ATP generated in photosynthesis, reducing the capacity of the Calvin–Benson cycle, which can be beneficial during drought stress or high-light conditions where an imbalance between photosynthetic electron transport rates and the use of its products can be generated, which could lead to an increase in excitation pressure, photoinhibition, and ROS formation. Photorespiration can help alleviate this acceptor limitation and ROS damage by consuming reducing equivalents (Takahashi & Badger, 2011). However, photorespiration is also a source of H 2 O 2 that is possibly involved in signal transduction, resulting in modulation of gene expression. Consequently, recent investigations indicate that photorespiration plays a major role in the adjustment of redox homeostasis (Foyer & Noctor, 2020). The crosstalk of redox signaling between the chloroplast and mitochondria has been largely discussed, highlighting the role of photorespiration in this crosstalk to adapting plant cells to environmental changes (Voss et al., 2013). Furthermore, photorespiration has been intimately related to plant nitrogen cycling, since transamination is required to produce glycine, and ammonium is produced during serine formation and must be efficiently Ó2023 The Authors New Phytologist Ó2023 New Phytologist Foundation New Phytologist (2023) 238: 1431–1445 1431 www.newphytologist.com This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Research reassimilated (Marquez et al., 2005;Perez-Delgado et al., 2016). Photorespiration has been associated with increased nitrogen uptake capacity (Bloom et al., 2012,2014). Elevated CO 2 concentrations, and consequently reduced photorespiration, have been shown to reduce nitrate assimilation, also threatening food quality by depleting crop protein concentrations (Bloom, 2009). Therefore, future atmospheric CO 2 concentrations predicted for 2100 point to major implications for plant physiology (Taub, 2010), including higher rates of photosynthesis and therefore, increased plant growth (Dusenge et al., 2019), stomatal closure and decreased water use, but lowered tissue concentrations of nitrogen and protein (Taub & Wang, 2008). Consequently, the main concern is how rising CO 2 over the next century will probably affect both agricultural production and food quality. In addition, plants with dysfunctional photorespiration show enhanced susceptibility to pathogen attack (Rojas et al., 2012) and lower tolerance to abiotic stresses (Voss et al., 2013). In plants, the concept of sulfide has recently changed from a toxic molecule to a highly important signaling molecule, which has been proved to regulate a wide range of cellular processes. Sulfide may react with metal centers of metalloproteins and may quench directly free radicals acting as a reducing agent, but the posttranslational modification of proteins named persulfidation is the main mechanism proposed for sulfide regulation (Aroca et al., 2018,2020; Filipovic et al., 2018). Persulfidation of a large number of proteins involved in the regulation of important biological processes has been previously demonstrated (Aroca et al., 2015,2017). Among these proteins, several have been found to participate in photorespiration (Aroca et al., 2017). Previous research demonstrated that almost all photorespiratory enzymes can be modified by oxidative posttranslational protein modifications (PTMs) in their cysteine residues, suggesting that they are potential targets for redox regulation (Keech et al., 2017). However, to our knowledge, little is known about the possible involvement of sulfide-mediated regulation associated with photorespiration. Therefore, further understanding is needed to unveil how redox modifications affect the regulation of photorespiration to cope with the climatic change. In this study, we show the existence of important differences in PTMs, such as persulfidation and sulfenylation, in plants growing under photorespiratory or nonphotorespiratory conditions, increasing CO 2 to a final concentration of 0.7% (v/v) (Murray et al., 1987). In addition, changes in many other biological parameters of these plants resulting from sulfide addition are described. The results obtained shed light on new aspects of sulfide signaling related to important issues, such as the nutritional quality of the plants, stomatal aperture, and ROS balance. Materials and Methods Plant materials and treatments Arabidopsis thaliana (Linnaeus) Heynhold Col-0 wild-type plants were grown at constant humidity of 70% with 16 h : 8 h, light (120 lmol m 2 s 1 ) : dark, 20°C:18°C. Plants were grown for 25 d under nonphotorespiratory conditions by automatically injecting CO 2 to a final concentration of 0.7% (v/v), and a batch of plants was harvested (high CO 2 treatment –nonphotorespiratory conditions, NPC). Then, other batches of plants were transferred for 1, 2, 3, and 6 d from high CO 2 (0.7% CO 2 v/v) to normal CO 2 conditions (0.04% CO 2 v/v) and then harvested for further analysis (1, 2, 3, and 6 d active-photorespiratory conditions, APC). For the sulfide treatment, plants were irrigated and sprayed with 200 lM NaHS every 3 d at least five times before harvesting the NPC samples and transferring to APC conditions. Control plants were treated with water. Protein persulfidation for mass spectrometry analysis A total of 500 mg of Arabidopsis leaves from plants growing under NPC and APC for 3 d were subjected to the dimedone switch method to identify persulfidated proteins by LC–MS/MS analysis, as described (Aroca et al., 2021a,2022). The final protein samples were digested with a 1 : 20 ratio of trypsin (V5111; Promega). The peptides were lyophilized and redissolved in 0.1% TFA before being analyzed by high-resolution LC–MS/MS using an Ultimate 3000 Nano Ultra High-Pressure Chromatography (UPLC) system (Thermo Fisher Scientific, Erlangen, Germany) coupled with an Orbitrap Fusion TM Lumos TM Tribrid TM Mass Spectrometer via an EASY-spray (Thermo Fisher Scientific). Peptide separation was carried out with an Acclaim TM Pep- Map TM 100 C18 column (Thermo Fisher Scientific) using a 60 min gradient (0 min, 7% B; 19 min, 10% B; 56 min, 20% B; 74 min, 30% B; B: 84% acetonitrile, 0.1% formic acid) at a flow rate of 250 nl min 1 . An Orbitrap Fusion TM Lumos TM Tribrid TM mass spectrometer was operated in DDA mode, and MS1 survey scans were acquired from m/z 300 to 1800 at a resolution of 120 000 using Orbitrap mode. The most intense ions were isolated for 3 s with a 1 m/z window and then fragmented by highenergy collision-induced dissociation with a normalized collision energy of 28%, considering a dynamic exclusion of 20 s. MS/MS spectra were recorded using Orbitrap mode with a resolution of 30 000. Data evaluation was performed with PEAKS XPRO software using 15 ppm for precursor mass tolerance, 0.5 Da for fragment mass tolerance, specific tryptic digest, and a maximum of three missed cleavages. Sulfenic acid-reactive 3-(2,4-dioxocyclohexyl) propyl (DCP) (+168.08 Da) on cysteine residues and nitro- 2,1,3-benzoxadiazole (NBF) (+163.00 Da) on cysteine, lysine, and arginine residues were added as variable modifications. Both peptide-spectrum matching and proteins were filtered at 1% false discovery rate. The data were normalized using the EIGENMS R package (Karpievitch et al., 2009,2015; R Core Team, 2021), and statistical tests were performed using PERSEUS (Tyanova et al., 2016). Gene ontology (GO) term enrichment was performed using PANTHER SLIM GO (Mi et al., 2021) and mapped in semantic space using REVIGO (Supek et al., 2011). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed using DAVID (Huang et al., 2009; Sherman et al., 2022). New Phytologist (2023) 238: 1431–1445 www.newphytologist.com Ó2023 The Authors New Phytologist Ó2023 New Phytologist Foundation Research New Phytologist 1432 14698137, 2023, 4, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.18838 by Universidad De Sevilla, Wiley Online Library on [19/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License In-gel persulfidation and sulfenylation The in-gel persulfidation and sulfenylation detection was performed in 500 mg of Arabidopsis leaves from plants growing under NPC and APC for 1, 2, 3, and 6 d, following the previously described dimedone switch detection methods (Zivanovic et al., 2019; Aroca et al., 2022). For persulfidation, NBF-Cl was used to react with all Cys residues and amino groups, giving a characteristic fluorescence (k ex =488 nm; Bernal-Perez et al., 2012; representing the loading control), and the persulfide adducts were selectively labeled with Daz-2/Cy5-alkyne (640 nm). Sulfenylated proteins were detected by labeling with DCP-Bio1 and visualized with Alexa Fluor TM 488 streptavidin as previously described (Zivanovic et al., 2019). Sulfenylation levels were calculated using b-tubulin assayed by western blot (As10680; Agrisera, V€ann€as, Sweden; dilution 1 : 5000) as a loading control. The fluorescence intensity of the bands measured in an Ettan DIGE imager (GE Healthcare, Chicago, IL, USA) was analyzed with IMAGEJ software, and the Cy5 : NBF and Alexa 488 : Tubulin ratios were calculated for the quantification of persulfidation and sulfenylation, respectively. Quantification of H 2 O 2 The determination of H 2 O 2 in Arabidopsis leaves was performed on 50 mg of Arabidopsis leaves from plants growing under NPC and APC for 1, 2, 3, and 6 d, treated or not treated with 200 lM NaHS, using the Amplex ® Red hydrogen peroxide/peroxidase assay kit (Thermo Fisher, Waltham, MA, USA) following the manufacturer’s instructions. The fluorescence was measured using a Varioskan plate reader (Thermo Fisher) using 560 nm excitation and 590 nm emission filters. The results are presented as the mean values SDs of six different biological replicates. ATP quantification ATP quantification was performed in 50 mg of Arabidopsis leaves from plants growing under NPC and APC for 3 d, treated or not with 200 lM NaHS, using an ATP Determination Kit (Thermo Fisher) based on the luciferin-luciferase bioluminescence reaction following the manufacturer’s instructions. The luminescence readings for the assay were recorded on a Varioskan plate reader (Thermo Fisher). The results are presented as the mean values SDs of six different biological replicates. Determination of soluble sugars and starch The soluble sugar concentration was determined by anion exchange chromatography in leaves from 25-d-old Arabidopsis plants growing under NPC and then transferred to APC for 3 d, treated or not with 200 lM NaHS, according to a previously described method (Ortiz-Marchena et al., 2014b). The samples were applied to a Dionex HPLC system equipped with a Carbo- Pac PA10 column and precolumn. Detection was carried out by amperometry, and the chromatograms were analyzed using CHROMELEON software (v.7.0; Thermo Fisher Scientific). The results are presented as the mean values SDs of four different biological replicates and three technical replicates. Starch was also determined in the same Arabidopsis leaves as previously described (Ortiz-Marchena et al., 2014a). Determination of amino acids The total amino acid content was determined in 100 mg of Arabidopsis leaves from plants growing under NPC and APC for 3 d, treated or not treated with 200 lM NaHS, following the same previous method (Jurado-Flores et al., 2021). Quantification of H 2 S Sulfide quantification was performed by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) using 100 mg of Arabidopsis leaves from plants growing under NPC and APC for 3 d, as previously described using monobromobimane derivatization (Tan et al., 2017). A standard curve of Na 2 S concentrations was generated from 2.5 to 100 lM and the concentration of H 2 S in the samples was calculated based on the standard curve. The results are presented as the mean values SD of five different biological replicates. Stomatal aperture and ROS quantification in stoma To measure stomatal aperture, leaves from 25-d-old Arabidopsis plants growing under NPC and APC for 3 d, treated with or without 200 lM NaHS, were fixed in 4% formaldehyde for 15 min to avoid interference with stomatal movement during manipulation. Then, after washing, epidermal peels were obtained from fixed leaves and kept in MES until microscopy visualization. The width apertures of a total of c. 150 stomata were measured from each experiment and for each treatment. The level of ROS in leaves was monitored using CM- H 2 DCFDA (Thermo Fisher) in plants treated with or without 200 M NaHS in plants growing under NPC and APC for 3 d. The abaxial epidermis from leaves was incubated with 10 lM CM-H 2 DCFDA dissolved in 10 mM MES, pH 6.5, for 15 min. After incubation, the epidermal peels were washed with MES buffer and fixed with 10% methanol for 5 min. The fluorescence was observed using a confocal laser scanning microscope (Olympus, Tokyo, Japan; excitation 488 nm, emission 510 nm). The average fluorescence of 100 stomata was quantified from the captured images of three different replicates and at least five different plants from each treatment and experiment by using the integrated density (sum of all pixels in the ROI, using the same area) in IMAGEJ software (https://imagej.nih.gov/ij/). Enzymatic activities Serine hydroxymethyltransferase (SHMT) activity was determined in 300 mg of leaves from 25-d-old Arabidopsis plants growing under NPC and APC for 3 d, treated or not with 200 lM NaHS, by following the production of glycine from serine, measured by HPLC as previously described (Liu et al., 2019). Ó2023 The Authors New Phytologist Ó2023 New Phytologist Foundation New Phytologist (2023) 238: 1431–1445 www.newphytologist.com New Phytologist Research 1433 14698137, 2023, 4, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.18838 by Universidad De Sevilla, Wiley Online Library on [19/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Hydroxypyruvate reductase (HPR) activity was measured in 150 mg of leaves from 25-d-old Arabidopsis plants growing under NPC and APC for 3 d, treated or not with 200 lM NaHS, using a previously described method (Chistoserdova & Lidstrom, 1991). Glycolate oxidase (GOX) activity was measured in 100 mg of leaves from 25-d-old Arabidopsis plants growing under NPC and APC for 3 d, treated or not with 200 lM NaHS, using an existing method (Kaundal et al., 2012). Glutamine synthetase (GS) activity was determined using the biosynthetic enzyme assay as previously described (Garcıa- Calderon et al., 2012;Perez-Delgado et al., 2013). Catalase (CAT) activity was measured in 100 mg of leaves from 25-d-old Arabidopsis plants growing under NPC and APC for 3 d, treated or not with 200 lM NaHS, following the decomposition of H 2 O 2 as described previously (Aebi, 1984). All results of enzymatic activities are presented as the mean values SDs of three different biological replicates. Results Identification and quantitative comparison of persulfidated proteins by LC–MS/MS analysis In recent publications, we reported the presence of persulfidated modified cysteine residues in several proteins involved in photorespiration and associated pathways identified from Arabidopsis leaf extracts by the modified biotin switch method (Aroca et al., 2015) and the tag switch method (Aroca et al., 2017). To assess the role of persulfidation and other targets involved in these pathways, we used a label-free quantitative approach combined with a dimedonebased method to identify protein persulfidation in leaves of 25-d- old Arabidopsis plants growing under NPC compared with plants adapted to air (APC) for 3 d. Protein samples from three biological replicates (independent pools) were isolated and subjected to the dimedone-switch method (Zivanovic et al., 2019;Aroca et al., 2021a,2022) to label persulfidated proteins (Supporting Information Fig. S1). The proteins eluted from streptavidin beads were digested, and the peptide solutions were analyzed by liquid chromatography tandem mass spectrometry. The biological triplicates and treatments showed peptide correlation ≥95% (Fig. S2). Although the most desired way to quantify the changes in protein persulfidation would be normalization to actual protein levels determined by global proteome screening, this approach is almost never done in the field of posttranslational modifications detection. There are several technical challenges that have made this solution difficult to apply globally. Because of relatively low abundance and highly reactive chemical properties, persulfidated proteins must be enriched before mass spectrometric analysis making it impossible to detect all and the same targets in separated total proteome screening, even with extensive fractionation. In addition, using different protocols for sample preparation and quantification (one for total proteome and the other for persulfidome) will undoubtedly affect the MS results. To minimize the effect of total protein changes, after label-free quantification, the data were normalized using the EIGENMS R package (Karpievitch et al., 2009,2015; R Core Team, 2021), which aims to preserve the original differences between treatment groups while removing the bias imposed by the datasets themselves (Fig. S3). Labelfree quantitative persulfidome analysis led to the identification of a total of 963 proteins differentially persulfidated in response to an elevated concentration of CO 2 in at least two out of three biological replicates, with a threshold of 0.05 (P<0.05) (Fig. 1a; Dataset S1). In addition, persulfidation of 119 proteins was found to decrease below the detection limit under APC treatment, while only 14 proteins were found to be exclusively persulfidated in APC (Dataset S2). Therefore, the total number of proteins differentially persulfidated was 1096. Quantification of persulfidation levels in all 1096 identified proteins revealed that 98.7% of the total targets were more persulfidated under suppressed photorespiration (NPC), while only 1.3% of proteins showed more persulfidation in samples adapted to photorespiration in normal air conditions (Datasets S1,S2). The differential changes in protein persulfidation between plants grown under NPC and APC are represented in a heatmap (Figs 1b,S4). In the heatmap, the color indicates the level of persulfidation at individual protein targets per replicate, with the red and blue colors representing the highest and lowest levels of persulfidation, respectively (Fig. 1b). There is a drastic effect on the persulfidation level when plants grown under nonphotorespiratory conditions are transferred to air. Almost all proteins are quantitatively more persulfidated under NPC than when plants have active photorespiration (Fig. S4), which correlates with an increase in H 2 S content in NPC in comparison with APC (Table S1). A selection of proteins involved in the sucrose–starch pathway, mitochondrial electron transport, redox and photorespiration, according to the MAPMAN classification (Thimm et al., 2004; Klie & Nikoloski, 2012), has been highlighted to demonstrate the impact of persulfidation on the regulation of these pathways depending on the photorespiratory conditions (Fig. 1b). Gene ontology enrichment analysis was performed on 963 proteins found to significantly change using the PANTHER SLIM-GO classification (Fig. 1c; Dataset S3). The analysis revealed an enrichment of GO terms grouped around protein intracellular transport, translation-related processes, processes involved in cellular division, misfolding control, and cellular stress response, among others. Furthermore, metabolic pathways such as sugar metabolism, cellular respiration, and energy conversion were also among the significantly enriched GO terms (Fig. 1c; Dataset S3). Similar results were obtained upon analyzing the same proteins for KEGG pathway enrichment (Fig. 1d; Dataset S4). Enrichment of different amino acid metabolism pathways, as well as general carbon metabolism pathways, including the tricarboxylic acid (TCA), pyruvate metabolism, glycolysis/gluconeogenesis, and pentosaphosphate pathways, was noticeable. Carbon fixation had the strongest fold of enrichment. Interestingly, glutathione, cysteine, and methionine metabolism, related to H 2 S production, were also significantly enriched. In-gel detection of persulfidation and sulfenylation levels Hydrogen peroxide signaling is related to posttranslational modification protein S-sulfenylation (P-SOH), and the crosstalk New Phytologist (2023) 238: 1431–1445 www.newphytologist.com Ó2023 The Authors New Phytologist Ó2023 New Phytologist Foundation Research New Phytologist 1434 14698137, 2023, 4, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.18838 by Universidad De Sevilla, Wiley Online Library on [19/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Fig. 1 Comparison of persulfidated proteins identified in Arabidopsis plants grown under nonphotorespiratory conditions (NPC) and adapted for 3 d to active photorespiratory conditions (APC). (a) Volcano plot of log 2 -transformed ratios of protein persulfidation (fold change) against the log 10 of Pvalues (Welch-test) for the observed changes. (b) Heatmap analysis of changes in persulfidation levels of proteins identified by mass spectrometry in Arabidopsis plants under NPC and APC. (c) Gene Ontology (GO) term enrichment (PANTHER SLIM-GO) of the 963 proteins that significantly change between APC and NPC groups, plotted using REVIGO. The size of the bubbles is indicative of the number of proteins annotated with that GO term; bubbles are color coded according to significance. (d) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Changes in persulfidation with a P-value of <0.05 were selected for analysis. Ó2023 The Authors New Phytologist Ó2023 New Phytologist Foundation New Phytologist (2023) 238: 1431–1445 www.newphytologist.com New Phytologist Research 1435 14698137, 2023, 4, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.18838 by Universidad De Sevilla, Wiley Online Library on [19/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License between H 2 S and H 2 O 2 signaling is well known (Zivanovic et al., 2019; Aroca et al., 2021b;Wanget al., 2021). To elucidate the crosstalk between these two signaling molecules, the levels of persulfidation (Fig. 2a) and sulfenylation (Figs 2b,S5) were analyzed in-gel during the transition of plants growing under NPC to the acclimation to photorespiration in a normal air atmosphere (APC) after 1, 2, 3, and 6 d. A drastic change in persulfidation and sulfenylation levels was observed during the transition from NPC to APC (Fig. 2c). The persulfidation level was higher under NPC than under APC, in agreement with the results of the quantitative proteomic analysis (NPC vs APC3). Sulfenylation levels revealed a high rate in plants under NPC, suggesting that the H 2 O 2 level must be high in that condition. During the transition to photorespiration, plants showed a drastic decrease in sulfenylation (Fig. 2c). After 3 d under normal air conditions, the plants showed a new increase in PSOH levels, which increased to a maximum at 6 d after acclimation to air. The kinetics of intracellular sulfenylation and persulfidation during the transition from non- to photorespiration in Arabidopsis showed opposing waves in a phaseshifted manner, as previously observed in other publications where the effect of sulfide has been demonstrated to lead to better resistance to oxidative stress (Zivanovic et al., 2019). Effect of sulfide on the whole-leaf H 2 O 2 content under nonphotorespiratory conditions Photorespiration can alter the cellular redox balance, producing and consuming both reducing and oxidizing equivalents. We found that plants grown under NPC showed a higher concentration of H 2 O 2 than those adapted to APC (Fig. 3). In addition, it was observed that the H 2 O 2 concentration sharply decreased during the first days of transition to photorespiration adaptation. On the other hand, the concentration of H 2 O 2 was much lower in the plants treated with sulfide under NPC, showing the same level during the transition to active photorespiration (Fig. 3). Therefore, these results suggest that the effect of sulfide in plants during NPC is to further increase the protection against redox species, modulating the level of H 2 O 2 . Effects of sulfide on carbon and nitrogen metabolism and ATP storage It is well known that primary carbon metabolism is dependent on nitrogen assimilation, which requires high energy and carbon skeletons that are mainly obtained from the TCA cycle. Therefore, the C : N balance is narrowly related, and an imbalance of any of these nutrients may involve a catastrophic outcome in the photosynthetic organism. Plants grown under NPC showed a significantly higher accumulation of soluble sugars and starch than plants adapted to photorespiratory conditions (Fig. 4a,b); nevertheless, they showed lower concentrations of total amino acids (Fig. 4c) when grown without photorespiration, confirming previous results found by other authors (Zhu et al., 2016; Wang et al., 2019). Consistent with previous results, the Gly : Ser ratio was decreased in plants under NPC compared with those adapted to photorespiration (Fig. 4d). To investigate the sulfide regulation of plants under both growth conditions at the metabolic level, plants were exogenously treated with NaHS and their carbon and nitrogen Cy5NBF Alexa488 Tub (a) (b) (c) NPC APC1 APC2 APC3 APC6 NPC APC1 APC2 APC3 APC6 0 1 2 3 4 5 6 7 0.0 0.5 1.0 1.5 2.0 2.5 NPC APC1 APC2 APC3 APC6 P-SOH levels (Alexa488/Tub) P-SSH levels (Cy5 : NBF) SSH SOH a a b d A B C C D c Fig. 2 In-gel protein persulfidation and sulfenylation detection of plants during the transition from nonphotorespiratory conditions (NPC) to active photorespiratory conditions for 1 (APC1), 2 (APC2), 3 (APC3), and 6 (APC6) d. (a) In-gel detection of protein persulfidation (PSSH) levels. Fire pseudo-coloring was used to visualize Cy5 signal. Green coloring corresponds to the total protein load (nitro-2,1,3- benzoxadiazole (NBF)-protein adducts). Ratio of Cy5 : NBF signals is used for quantification (n=3). (b) In-gel detection of protein sulfenylation (PSOH) levels (labeled with DCP-Bio1 and visualized with streptavidin-488) (n=3). b-Tubulin was used as a loading control. (c) Quantification of PSSH and PSOH changes (n=3). Values are presented as a mean SD and different letters indicate statistically significant differences (ANOVA, Tukey test, P<0.05). New Phytologist (2023) 238: 1431–1445 www.newphytologist.com Ó2023 The Authors New Phytologist Ó2023 New Phytologist Foundation Research New Phytologist 1436 14698137, 2023, 4, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.18838 by Universidad De Sevilla, Wiley Online Library on [19/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License metabolism was compared with untreated plants. Under NPC, sulfide-treated plants showed significantly less soluble sugars and starch but an increase in total amino acids, suggesting a balanced C : N metabolism similar to that found in untreated plants adapted to photorespiration (Fig. 4a–c). The Gly : Ser ratio was also significantly higher in plants treated with sulfide, even higher than that found in untreated plants adapted to photorespiration. The effect of sulfide on the ATP concentration was also significant (Fig. 4e). Plants growing under NPC showed a decrease in ATP accumulation compared to those growing in normal air, supporting previous publications (Li et al., 2013). However, plants treated with sulfide under NPC showed significantly higher accumulation of ATP than the untreated plants, reaching similar concentrations as those plants adapted to normal air conditions (Fig. 4e). These findings appear to suggest that the exogenous sulfide treatment ameliorates the imbalanced C, N, and energy metabolisms observed in nonphotorespiratory conditions grown plants. Sulfide regulates stomatal movement by regulating stomatal ROS Stomata movement is sensitive to various environmental factors, and it is well known that high levels of CO 2 trigger stomata closure; this closure requires an increase in ROS in guard cells. The stomatal ROS in Arabidopsis leaves grown under NPC and plants adapted to photorespiration for 3 d was quantified by fluorescence microscopy using the H 2 DCFDA probe, which is a cell-permeant indicator for ROS that detects hydrogen peroxide, peroxyl radicals, and peroxynitrite anions (Dikalov & Harrison, 2014). Our results indicate that the stomata showed higher accumulation of ROS under NPC, as previously described (Ma & Bai, 2021), than in plants acclimated to photorespiration, which showed a decrease in fluorescence in the stomata (Fig. 5). Interestingly, plants grown under high-CO 2 and treated with sulfide showed a significant decrease in ROS concentration in comparison with untreated plants, reaching concentrations more similar to those of plants adapted to normal air. In addition, when plants grown under photorespiratory conditions were treated with sulfide, the level of ROS detected was the same as that observed in treated plants under nonphotorespiratory conditions. These results demonstrate that sulfide was able to regulate the ROS burst in stomata while growing without photorespiration (Fig. 5a,b). Furthermore, the stomatal width was measured under the microscope, and the results corroborate with previous data (Ma & Bai, 2021), demonstrating that stomata were mainly closed under high CO 2 and open wide in air (Fig. 6a,b) and that under APC sulfide regulates the stomata closing as described previously (Shen et al., 2020; Zhang et al., 2020). However, plants treated with sulfide under suppressed photorespiration showed more opened stomata than untreated plants. Therefore, the results indicate that the opening and closing of stomata in NPC conditions is regulated by sulfide, possibly by regulation of the ROS levels in guard cells. Therefore, the in situ ROS fluorescence determinations are quite well in accordance with previous results in crude extracts shown in Fig. 3. Furthermore, several targets for persulfidation involved in stomatal movement and abscisic acid (ABA) signaling gene ontologies were found in plants grown under NPC (Table S2), similar to the stomatal proteins already known to be involved in guard cell ABA signaling under photorespiratory conditions (Shen et al., 2020; Zhang et al., 2020; Aroca et al., 2021b), thus demonstrating the contribution of sulfide in the regulation of stomatal movement under NPC growth conditions. Enzymatic activities Previous data indicate different aspects of the positive effects of sulfide treatments under nonphotorespiratory conditions in Arabidopsis. We have also examined if there is any influence of sulfide on photorespiratory enzymes activities under conditions where these enzymes are operative. Several photorespiratory enzymes were selected to analyze the possible changes taking place under active photorespiration (Fig. 7). The results indicate that there was a common pattern of behavior for most of the enzymes assayed and demonstrated that sulfide regulates photorespiration enhancing the activity of several enzymes under photorespiratory active conditions. In fact, sulfide produced a substantial increase on GS and HPR enzyme activities reaching the double respecting to the untreated plants. A lesser increase but significant was found in the GOX and CAT activities (40% and 10% increase, respectively), and a slight increase in activity, but not significant, was observed for SHMT. Discussion Whether photorespiration may be a wasteful or useful process is still a matter of intense debate. Traditionally, it was considered as a wasteful process because of the loss of fixed CO 2 due to the oxygenation of RUBISCO at the normal atmospheric CO 2 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 H2O2(μg mg–1 FW) Control NaHS a b c c b A AB AA B * * NPC APC1 APC2 APC3 APC6 Fig. 3 Impact of sulfide on the H 2 O 2 content in Arabidopsis plants grown under non-photorespiratory conditions (NPC) and adapted for 1 (APC1), 2 (APC2), 3 (APC3), and 6 (APC6) d to active photorespiratory conditions (n=6). Bars represent means SD, different letters indicate statistically significant differences among samples and asterisks indicate statistically significant differences between treatments (ANOVA, Tukey test: *, P<0.05). Ó2023 The Authors New Phytologist Ó2023 New Phytologist Foundation New Phytologist (2023) 238: 1431–1445 www.newphytologist.com New Phytologist Research 1437 14698137, 2023, 4, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.18838 by Universidad De Sevilla, Wiley Online Library on [19/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License concentration. In recent years, it is becoming clear that photorespiration safeguards photosynthesis and connects with a number of subcellular processes (Timm, 2020). In fact, an emerging concept has been established for photorespiration as an important part of the stress responses to prevent ROS accumulation and to mitigate the oxidative stress under abiotic stress (Voss et al., 2013). Different environmental factors affecting the rate of photorespiration could interfere with the intracellular ROS levels by increasing or decreasing ROS production (Foyer et al., 2009). The present paper shows new evidence for a role for sulfide in the protection of Arabidopsis plants against ROS accumulation produced as a consequence of the lack of photorespiration. The growth of plants under high CO 2 leads to inhibition of the photorespiratory pathway, which has a variety of secondary effects on the physiology of plants. The availability of an elevated concentration of CO 2 enables plants to grow faster due to increased photosynthetic activity, which seems to be the perfect situation to increase biomass of harvestable crops (Long et al., 2006; Ainsworth, 2008). Nevertheless, inhibition of photorespiration also leads to changes in the chemical composition of plants, such as an increase in sugar and starch concentrations and a decrease in nitrogen assimilation (Ainsworth, 2008; Bloom et al., 2010), which are likely to affect both agricultural production and food quality (Taub, 2010). Interestingly, our results reveal other important effects on plants under conditions of suppression of photorespiration, and also the level of protein persulfidation in plants was quantitatively induced (Fig. 1). Furthermore, we found that sulfide regulates several pathways through the persulfidation of specific targets under suppressed photorespiration, including mitochondrial 0 100 200 300 400 500 600 700 Control NaHS Control NaHS High CO2 Air [ATP] (nmol g–1 FW) a bb b 0 0.5 1 1.5 2 2.5 3 CNaHSCNaHS High CO2 Air Starch (mg mg–1 FW) b a cc (a) (b) (c) 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 ControlNaHSControlNaHS High CO2 Air Rao Gly : Ser aa c b 0 100 200 300 400 500 600 700 800 900 1000 Control NaHS Control NaHS High CO2 Air Amino acids (nmol g–1 FW) ac d b (d) (e) 0.0 0.5 1.0 1.5 2.0 2.5 CNaHSCNaHS High CO2 Air Soluble sugars (μmol g–1 FW) aa bb NPC APC NPC APC NPC APC NPC APC NPC APC Control NaHS Control NaHS Control NaHS Control NaHS Fig. 4 Effect of sulfide in carbon and nitrogen metabolisms and ATP storage in Arabidopsis plants grown under nonphotorespiratory conditions (NPC) and adapted for 3 d to active photorespiratory conditions (APC). (a) Determination of total soluble sugars (n=4). (b) Determination of total starch (n=4). (c) Total amino acids content (n=5). (d) Ratio Gly : Ser. (e) Quantification of ATP concentration (n=6). Bars represent means SD, and different letters indicate statistically significant differences (ANOVA, Tukey test: P<0.05). New Phytologist (2023) 238: 1431–1445 www.newphytologist.com Ó2023 The Authors New Phytologist Ó2023 New Phytologist Foundation Research New Phytologist 1438 14698137, 2023, 4, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.18838 by Universidad De Sevilla, Wiley Online Library on [19/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License electron transport, carbon metabolism, and redox status. Previous studies in plants have described the wide-range effect of sulfide, which regulates numerous physiological processes by increasing or decreasing the persulfidation level of certain targets (Aroca et al., 2017,2018,2020), but the huge increase in the persulfidation level in most of the proteins detected under suppressed photorespiration is astounding, undoubtedly suggesting an essential role of sulfide signaling under these conditions. Interestingly, the growth of Arabidopsis plants in high CO 2 conditions to suppress photorespiration is a common practice since the early work of Somerville & Ogren (1979), and it has been used also for other plant species (Murray et al., 1987; Lacuesta et al., 1992; Marquez et al., 2005;Perez-Delgado et al., 2013,2016). However, plants in these conditions have never shown any apparent external phenotype. In our opinion, this reinforce the main conclusion established in our work that the high extent of protein persulfidation detected is very efficient to protect the plants against the lack of photorespiration. (a) (b) lortnoc_CPN NPC_NaHS lortnoc_CPA APC_NaHS Fig. 5 Sulfide decreases reactive oxygen species (ROS) in guard cells in Arabidopsis. (a) Confocal microscopy images of the guard cells showing ROS levels indicated by CM- H 2 DCFDA fluorescent probe, in response to sulfide treatment under nonphotorespiratory conditions (NPC) and active photorespiratory conditions (APC). (b) Quantification of the intensity of pixels fluorescence (IntDent) from CM-H 2 DCFDA. Box plots show the average (red lines), and 25–75% percentiles of at least 100 randomly selected stomata from three different replicates and at least five different plants from each treatment. Whiskers and cross symbols representing the outlier and 1–99% percentiles, respectively. Different letters indicate statistically significant differences (ANOVA, Fisher’s least significant difference test: P<0.05). Ó2023 The Authors New Phytologist Ó2023 New Phytologist Foundation New Phytologist (2023) 238: 1431–1445 www.newphytologist.com New Phytologist Research 1439 14698137, 2023, 4, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.18838 by Universidad De Sevilla, Wiley Online Library on [19/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License