Chemico-Proteomics Reveal the Enhancement of Salt Tolerance in an Invasive Plant Species via H2S Signaling.

H2S is a small molecule known to have multiple signaling roles in animals. Recently, evidence shows that H2S also has signaling functions in plants; however, the role of H2S in invasive plants is unknown. Spartina alterniflora is a typical invasive species growing along the beaches of southern China. A physiological comparison proves that S. alterniflora is highly tolerant to salinity stress compared with the native species Cyperus malaccensis. To decipher the mechanism that enables S. alterniflora to withstand salinity stress, a chemico-proteomics analysis was performed to examine the salt stress response of the two species; an inhibitor experiment was additionally designed to investigate H2S signaling on salinity tolerance in S. alterniflora. A total of 86 proteins belonging to nine categories were identified and differentially expressed in S. alterniflora exposed to salt stress. Moreover, the expression level of enzymes responsible for the H2S biosynthesis was markedly upregulated, indicating the potential role of H2S signaling in the plant's response to salt stress. The results suggested that salt triggered l-CD enzyme activity and induced the production of H2S, therefore upregulating expression of the antioxidants ascorbate peroxidase, superoxide dismutase, and S-nitrosoglutathione reductase, which mitigates damage from reactive nitrogen species. Additionally, H2S reduced the potassium efflux, thereby sustaining intracellular sodium/potassium ion homeostasis and enhancing S. alterniflora salt tolerance. These findings indicate that H2S plays an important role in the adaptation of S. alterniflora to saline environments, which provides greater insight into the function of H2S signaling in the adaptation of an invasive plant species.

Introduction

Although the small gas molecule hydrogen sulfide (H2S) has long since been thought to be toxic, evidence shows that H2S is the third endogenous gaseous transmitter besides nitric oxide (NO) and carbon monoxide.[1,2] H2S functions in a majority of physiological processes in animals, including neuronal excitability, vasorelaxation, anti-inflammatory response, smooth muscle relaxation, and blood pressure regulation.[3−5] Recently, the positive effect of H2S in improving the ability of plants to adapt to multiple environmental stimuli has also been reported.[6,7] In addition, H2S acts as a critical signal in response to abiotic stress, modulating processes such as stomatal closure, iron availability, leaf senescence, osmotic stress, heat shock, and hypoxia stress.[8−13] Cystathionine β-synthase (CBS, EC4.2.1.22) and cystathionine γ-lyase (CSE, EC 4.4.1.1) are mainly used by mammalian cells for the synthesis of H2S. CBS hydrolyses l-cysteine to l-serine and CSE hydrolyses l-serine to H2S, pyruvate, and ammonia.[14] In plants, H2S generation is mainly related with the pyridoxal 5′-phosphate (PLP)-dependent d/l-cysteine desulfhydrases (d/l-CDs) enzymes. Meanwhile, another PLP-dependent enzyme (β-cyanoalanine synthase) could convert cysteine and cyanide to H2S and β-cyanoalanine.[15] However, H2S is consumed by the cysteine synthesis complex in the synthesis of l-cys from O-acetyl serine which is catalyzed by O-acetylserine-thiol-lyase.[16]Arabidopsis DES1 encodes the cytosolic l-CDes, which modulates endogenous H2S levels in the cytosol.[17] Abscisic acid (ABA), which triggers stomatal closure, induces a DES1 expression in Arabidopsis guard cells. ABA treatment could not close the stomata in isolated epidermal strips of the des1 mutant, and this defect was restored because of exogenous H2S addition.[18] These data demonstrate the essential function of H2S in response to the environmental stress in the plant.

The cross-communication between H2S and other small molecular signals, such as NO and reactive oxygen species (ROS), has been thoroughly investigated in plants exposed to biotic or abiotic stress. For example, Arabidopsis DES1 is involved in ABA-dependent NO production and stomatal closure, and NO acts downstream of H2S in the ABA-induced stomatal closure.[18] H2S treatment also improves the tolerance of rice to salt stress or mercury toxicity.[19,20] Salt stress can induce l- activity and raise the production of endogenous H2S in plant Medicago sativa. Furthermore, exogenous H2S treatment was shown to minimize the salt toxicity effects by modulating the activities of antioxidant enzymes, maintaining K+/Na+ homeostasis, and preventing K+-efflux triggered by NaCl.[21] H2S also alleviates heat stress in maize, arsenate toxicity in pea, and cadmium toxicity in Brassica through the regulation of the ascorbate–glutathione cycle and ROS signaling.[21] However, the mechanism by which H2S interacts with other signals to modulate plant adaptation to environmental stress is yet to be elucidated. The H2S signal increases the activity of plasma membrane NADPH oxidase, which generates hydrogen peroxide (H2O2) to control stomatal closure.[22]

Spartina alterniflora is a perennial rhizomatous grass which is a native species in the Atlantic and Gulf coasts of North America. It is considered to be an important “environmental engineer” in terms of silting land, repairing wetlands, and fixing carbon dioxide. However, since S. alterniflora was introduced to South China in the 1970’s, it has become established as an invasive species due to its rapid growth, changing the biodiversity of the region, and disrupting the mangrove ecosystem.[23−28] The biomass of S. alterniflora could be five times greater than those of native species such as Suaeda salsa in the coastal region of the Yangtze River Basin.[29] Although S. alterniflora has the potential to increase the soil organic carbon content and primary productivity (as well as improving the carbon sequestration capability of the flora), as a pioneer plant, it has successfully adapted to environmental stresses including flooding, high salinity, and sediment burial. However, there is a dearth of literature in deciphering the mechanism by which S. alterniflora has adapted to the saline environment.

Mass spectrometry-based chemical proteomics approaches have been used in several aspects of small molecular drug research, including identifying drug targets, verifying drug to target interaction affinity, and selectivity profiling. It has also been used to profile enzyme activity at a systemic level across different samples, becoming an integrated research engine that removes the hurdle of identifying protein targets of biologically active small molecules with unknown modes of action.[30−32]

In this study, the physiological and chemico-proteomics comparisons focused on the role of H2S in salt tolerance were studied in order to systemically understand the adaptation of S. alterniflora to salt stress, as compared with the native species Cyperus malaccensis in southern China. Therefore, an inhibitor experiment was designed to explain the effects of H2S signaling on salinity tolerance in S. alterniflora. Our study therefore demonstrated that H2S is a novel signal mediating tolerance to salinity stress in the invasive plant S. alterniflora, providing important clues to control this species and curb its encroachment on native plant species.

Results

Physiological Response of S. alterniflora and C. malaccensis Induced by Salt Treatment and the Salt-Related Dynamic Protein Profiling in S. alterniflora

To decipher the mechanism underlying S. alterniflora tolerance to NaCl stress, the responses of the invading S. alterniflora and the domestic C. malaccensis to different concentrations of salt were researched. A salt concentration of 100 mM reduced the Fv/Fm ratio and increased ion leakage in C. malaccensis, while a salt concentration of over 300 mM only somewhat decreased the Fv/Fm ratio and increased the ion leakage in S. alterniflora (Figure S1); when the salt concentration exceeded 500 Mm, the damage inflicted by salt toxicity was more severe, and possibly caused side effects such as unhealthy growth, extremely wilted leaves, and so forth. Thus, a salt concentration of 300 mM was selected to challenge S. alterniflora in subsequent experiments, in which S. alterniflora plants were exposed for 1, 3, and 7 days. As the control, the native species C. malaccensis was simultaneously treated with 300 mM NaCl. The leaf photosynthesis capability, ion leakage, and malondialdehyde (MDA) content, important criteria to estimate the leaf damage degree, were measured. As shown in Figure A–C, NaCl treatment for different periods did not markedly affect Fv/Fm, leaf ion leakage and MDA degree in S. alterniflora, but markedly reduced Fv/Fm, and increased leaf ion leakage and MDA content in the native species C. malaccensis after 3 or 5 days of NaCl treatment. These data support the notion that S. alterniflora has a higher tolerance for NaCl stress than does C. malaccensis.

Figure 1

Effects of salt stress on the leaf photosynthesis capability and electrolyte leakage. S. alterniflora and C. malaccensis were collected and cultivated in a greenhouse for 7 days, and then treated with 300 mM NaCl. At the indicated time points, the average Fv/Fm values (A), electrolyte leakage (B), and MDA content (C) were determined using whole leaves. Data represent mean ± SE of three replicate experiments. Means denoted by different letters show significant differences at p < 0.05 according to Tukey’s test.

In order to further explore the mechanism underlying salt stress response in S. alterniflora, the comparative proteomics approach was used to monitor the protein profile in S. alterniflora after 1, 3, and 5 days of 300 mM NaCl stress. A total of 86 proteins were successfully identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)/TOF and changed significantly in response to salt stress (p < 0.05) compared with the control (Figures A,B and 3A). These identified proteins were divided into nine and seven groups based on the annotated and putative biological functions, respectively (Figure B,C and Table S1). Most of these proteins were metabolism- and energy-related proteins, followed by defense proteins and transcription factors. Proteins associated with epigenetics, protein stability, and hormone metabolism were also detected (Figure B). In addition, we found that proteins involved in the H2S metabolism, such as protein spot 65 (putative d-cysteine desulfhydrase 2) and protein spot 79 (cystathionine γ-lyase/cysteine synthase), and proteins involved in autophagy, such as spot 44 (autophagy protein 5) and spot 50 (cysteine protease ATG4), were regulated differentially after salt stress (Table S1), suggesting the critical role of H2S during the response to NaCl stress in S. alterniflora.

Figure 2

Changes in protein spots derived from the leaves of S. alterniflora after treatment with 300 M NaCl. Plant samples were collected at different time points [0 day (control) and 1, 3, and 5 days] and 1 mg of total protein was extracted and loaded onto gels. (A) Representative BR-20-stained 2-D gel of the total leaf protein at 0 days showing protein spot position. (B–G) Enlarged windows from panel (A) showing spot changes from samples collected after different periods of salt treatment.

Figure 3

Protein species expression profiles from the leaves of S. alterniflora after exposure to salt stress. Hierarchical clustering (A), functional classification, and (B) Venn diagram analysis of protein species expression profiles in S. alterniflora leaf samples obtained after different periods of salt stress treatment. The hierarchical cluster analysis was conducted using Cluster 3.0 and Treeview software (http://bonsai.ims.u-tokyo.ac.jp/?mdehoon/software/cluster). The different colors correspond to the log-transformed values of the protein species change-fold ratio shown in the bar at the bottom of the figure (C). Data represent mean ± SE of three replicate experiments.

Comparing the Chemico-Proteomics Characteristics in S. alterniflora and C. malaccensis Induced by Salt Treatment

H2S Biosynthesis and the Main Responsible Enzyme (l-CD) Activity

H2S is a signal in the plant defense response.[9] Proteomics data in this study showed that some proteins involved in the H2S biosynthesis coordinately accumulated after NaCl treatment (Table S1), indicating the potential role of the H2S signal in S. alterniflora tolerance to salt stress. Thus, the endogenous H2S content in the leaves of S. alterniflora was measured and found that NaCl gradually induced the generation of H2S, peaking after 5 days of NaCl treatment. NaCl treatment also induced H2S generation in the leaves of C. malaccensis, but to a lesser extent than in S. alterniflora (Figure ). Consistent with H2S generation, the enzyme activity of l-CD was also higher in the leaves of S. alterniflora than in those of C. malaccensis after NaCl treatment (Figure ). These data show that NaCl treatment induced higher levels of H2S generation in S. alterniflora than in C. malaccensis, and indicate the potential role of H2S in enhancing S. alterniflora tolerance to salt stress.

Figure 4

Comparing the generation of H2S in S. alterniflora and C. malaccensis induced by salinity. S. alterniflora and C. malaccensis plants were treated with 300 mM NaCl. At the indicated time points, the leaves of S. alterniflora and C. malaccensis plants were collected, the H2S content (upper) and l-CD activity (bottom) were measured. Data represent mean ± SE of three replicate experiments.

H2O2 and O2– Generation and the Activities of Related Antioxidant Enzymes [Ascorbate Peroxidase (APX) and Superoxide Dismutase (SOD)]

Plants have evolved protective mechanisms such as the antioxidant enzyme system to sustain the cytosolic redox balance.[33−35] In this study, a series of antioxidant enzymes, such as spot 54 (thioredoxin reductase NTRC), spot 57 (glutathione synthetase), spot 77 (cysteine synthase), spot 84 (SOD), spot 85 (APX), and spot 87 (glutathione S-transferase 1), were found and their differential regulation after salt stress indicated the possible role of antioxidant enzymes in the response to salt stress in S. alterniflora. To investigate the functions of these antioxidant enzymes, in situ H2O2 and O2– generation by histochemical diaminobenzidine (DAB) staining (for detecting H2O2, dark brown color) and nitro-blue tetrazolium (NBT) staining (for detecting O2–, purple blue color) were first monitored. The leaves of C. malaccensis plants exhibited more purple blue and dark brown staining after NaCl treatment than those of S. alterniflora (Figure A), indicating that the H2O2 and O2– contents of the C. malaccensis leaves were higher (Figure B,C). H2O2 and O2– changes suggested that more ROS accumulated in the C. malaccensis leaves than in the S. alterniflora leaves following exposure to salt stress. Besides, the activities of antioxidant enzymes, including APX and SOD, gradually increased after salt treatment was also found in this study (Figure A,B). These results indicate that these antioxidant enzymes have a protective role by scavenging ROS after exposure to salt stress.

Figure 5

Comparing the generation of H2O2 and O2– in S. alterniflora and C. malaccensis induced by salinity. S. alterniflora and C. malaccensis plants were treated with 300 mM NaCl for 7 days. At the indicated time points, leaves were collected for H2O2 staining [(A), left panel] by DAB, or O2– staining (a, right panel) by NBT. The H2O2 content (B) and O2– content (C) were measured. Data represent mean ± SE of three replicate experiments.

Figure 6

Comparing the activities of antioxidant proteins in S. alterniflora and C. malaccensis induced by salinity. S. alterniflora and C. malaccensis were treated with 300 mM NaCl for 7 days. At the indicated time points, S. alterniflora and C. malaccensis leaves were collected and APX activity (A), SOD activity (B), SNO content (C), and GSNOR activity (D) were measured. Data represent mean ± SE of three replicate experiments. Means denoted by different letters show significant differences at p < 0.05 according to Tukey’s test.

S-Nitrosothiols (SNO) Content and the Related S-Nitrosoglutathione Reductase (GSNOR) Activity

Besides H2O2, NO also acts as a signal that regulates the cellular redox balance. NO-derived RNS, such as peroxynitrite (ONOO–), dinitrogen trioxide (N2O3), and nitrogen dioxide (NO2–), reacts with cellular GSH to form GSNO, which then transfers the NO group to cellular protein thiols to form the longer-lived SNOs through protein S-nitrosylation.[36,37] A previous study showed that the metabolism of GSNO could be catalyzed by the evolutionarily conserved GSNOR. The proteomic data also showed that the accumulation of GSNOR (spot 69) was upregulated by salt stress (Figure A and Table S1), indicating a potential role of GSNOR in controlling SNOs in S. alterniflora after exposure to salt stress. Here, the level of SNOs in S. alterniflora and C. malaccensis after exposure to salt stress was compared, and the result suggested that salt stress increased SNO accumulation in both S. alterniflora and C. malaccensis, with the SNO level in S. alterniflora being markedly lower than that in C. malaccensis (Figure C). Consistent with the proteomic data, the enzyme activity of GSNOR was upregulated in S. alterniflora after exposure to salt stress. While such an increase in activity was also observed in C. malaccensis after exposure to salt stress, the increase was not as striking as in C. malaccensis (Figure D). These data suggest that GSNOR catalyzes the SNO metabolism in plants after exposure to abiotic stress.

Na+/K+ Ratio and K+ Efflux

Cellular ion homeostasis is impaired in plants exposed to salt stress, and the cytosol of cells of salt-tolerant plants typically sustain high levels of K+ and low levels of Na+ under salt stress.[38,39] Plasma membrane H+-ATPase and Na+/H+ antiporter play key roles in maintaining cellular Na+/K+ homeostasis.[40,41] In this study, proteomics data showed that the accumulation of protein spot 16 encoding H+-ATPase and spot 111 encoding V-type proton ATPase subunit G1 were increased, indicating that they function in sustaining ion homeostasis in S. alterniflora after exposure to salt stress. To investigate cellular Na+/K+ homeostasis following exposure to salt stress, Na+ content, K+ content, and Na+/K+ ratio in the leaves of S. alterniflora and C. malaccensis plants were then examined under salt stress. Salt stress increased the concentration of Na+ and decreased K+, resulting in a sharp increase in the Na+/K+ ratio in the C. malaccensis leaves (Figure A). The increase in Na+ percentage and Na+/K+ ratio and decrease in K+ percentage were not as obvious in S. alterniflora (Figure A), possibly due to the increased PM H+-ATPase activity.

Figure 7

Comparing the ion homeostasis and potassium efflux in S. alterniflora and C. malaccensis induced by salinity. S. alterniflora and C. malaccensis plants were treated with 300 mM NaCl for 7 days. The leaves were collected at the indicated time points, and the Na+ and K+ contents and Na+/K+ ratio were measured (A). Time-course analysis of the transient net K+ efflux (B) was performed using the noninjuring technique after the addition of salt. The average K extrusion during 20 min of NaCl stress was also determined (C). Data represent mean ± SE of three replicate experiments. Means denoted by different letters show significant differences at p < 0.05 according to Tukey’s test.

To further characterize how NaCl modulates K+ homeostasis in S. alterniflora and C. malaccensis upon salt stress, noninvasive microtest technology (NMT) was used to detect dynamic changes in K+ in the S. alterniflora and C. malaccensis roots after exposure to salt stress. NaCl stress led to a large K+ efflux in C. malaccensis roots but not in S. alterniflora roots (Figure B,C), suggesting that the lower K+ efflux in S. alterniflora contributes to its salt tolerance.

H2S Signal Functions in Salt Tolerance in S. alterniflora

Through the chemico-proteomics comparison between invasive plant S. alterniflora and native plant C. malaccensis induced by salinity stress, we found that salt stress induced higher levels of H2S and the activity of the responsible enzyme (l-CD), activities of the activated antioxidant enzymes (APX and SOD), as well as of the enzyme GSNOR in S. alterniflora. To further understand the potential role of H2S in sustaining S. alterniflora tolerance to salt stress, S. alterniflora was pretreated with a H2S inhibitor and then exposed to salt stress. dl-propargylglycine (PAG) inhibits l-DES activity and hypotaurine (HT) scavenges H2S. In this experiment, NaHS was used as the exogenous H2S donor. Pretreatment of S. alterniflora with different concentrations of PAG or HT reduced the salt-induced H2S accumulation (Figure S2), and this reduction was more obvious at high concentrations of PAG (2 mM) and HT (100 μM). Thus, these concentrations were used to investigate the role of H2S signaling in mediating the salt stress response in S. alterniflora.

First, the redox status of S. alterniflora under salt stress with or without PAG or HT pretreatment were compared. As shown in Figure A, PAG or HT treatment aggravated salt-induced ROS accumulation and suppressed salt-induced antioxidant APX or SOD enzyme activities. These findings agreed with a previous study[35] and suggest that salt-induced H2S is the signal that activates the antioxidant enzyme activity to reduce ROS accumulation during salt stress. Similarly, a NMT analysis showed that PAG or HT pretreatment increased the K+ efflux, resulting in an increase in the Na+/K+ ratio compared with plants not subjected to a PAG or HT pretreatment (Figure B). This suggests that suppressing the H2S signal affected Na+/K+ homeostasis in S. alterniflora under salt stress. PAG or HT pretreatment significantly reduced the Fv/Fm ratio and increased the MDA content in S. alterniflora leaves after 3 days of exposure to salt stress (Figure C), in contrast to samples lacking PAG or HT pretreatment. These data suggest that H2S mediates tolerance to salt stress in S. alterniflora.

Figure 8

H2S signal and GSNOR activity modulated ROS and RNS generation, antioxidant enzyme activities, ion homeostasis, and leaf viability in S. alterniflora after exposure to salt stress. Seven-day-old S. alterniflora plants were treated with 300 mM NaCl with or without the H2S biosynthesis inhibitor PAG (2 mM) or scavenger HT (100 μM). After 3 days of treatment, the accumulation of H2O2 and O2–, the activities of APX and SOD, the generation of SNOs, and GSNOR activity were measured (A). The effect of HT and DA on the net K+ efflux was monitored by the noninjuring technique after the addition of salt (B). The inset shows the mean efflux rates 0–20 min after treatment. Furthermore, the effects of PAG and HT on the MDA content and Fv/Fm ratio were evaluated in S. alterniflora leaves (C). Data represent mean ± SE of three replicate experiments. Means denoted by different letters show significant differences at p < 0.05 according to Tukey’s test.

Pretreatment with PAG or HT also aggravated NaCl-induced SNO content and reduced GSNOR activity in S. alterniflora (Figure S3), suggesting that salt-induced H2S also regulates the NO metabolism and GSNOR activity. To determine the cross-talk between H2S and GSNOR in S. alterniflora after exposure to salt stress, S. alterniflora was pretreated with the GSNOR inhibitor dodecanoic acid (DA) and it was found that DA pretreatment increased salt-induced MDA degree and reduced salt-induced Fv/Fm (Figure S4), suggesting that GSNOR activity modulates salt tolerance in S. alterniflora. However, the DA treatment did not obviously affect salt-induced H2S generation (Figure S5). These data indicate that H2S acts upstream of the NO signal to enhance S. alterniflora tolerance to salt stress. H2S can buffer the overaccumulation of NO that may cause plant damage by leveraging GSNOR activity, which serves as a protective shelter for S. alterniflora under a high salinity environment.

Discussion

In China, S. alterniflora is regarded as an invasive species based on its aggressive growth capability and tolerance to salt stress. However, the mechanism underlying salt stress tolerance in S. alterniflora was hitherto unknown. In this study, S. alterniflora was more tolerant to salt stress than C. malaccensis was, and further proteomic analysis showed that a series of proteins associated with sugar signaling, such as spot 14 (sucrose synthase), spot 30 (soluble starch synthase 1), spot 42 (β-amylase), and spot 43 (α-amylase isozyme 2A), were differentially upregulated, suggesting that sugar signaling may mediate the salt stress response in S. alterniflora. In addition to providing energy, sugar has been reported to act as a signal mediating many physiological processes, such as iron deficiency and plant lifespan.[42,43] It is possible that the sugar signal regulates intracellular osmotic levels. Other plant hormone-response-related proteins, such as spot 49 (ninja-family protein 8), spot 62 (gibberellin 20 oxidase), spot 63 (S-adenosylmethionine synthase), and spot 109 (auxin-responsive protein SAUR40) were also differentially regulated after exposure to salt stress; several hormone-responsive transcriptional factors, such as spot 23 (transcription factor MYC2), spot 25 (ETHYLENE INSENSITIVE 3-like 3 protein), spot 95 (ethylene-responsive transcription factor 13), and spot 97 (GRF1-interacting factor 3) presented differential accumulation, suggesting the possible role of jasmonic acid, gibberellin, and auxin signaling in response to salt stress in S. alterniflora. Furthermore, salt stress modulated protein stability through the protein ubiquitination system, as proteins associated with this system, including spot 13 (ATP-dependent zinc metalloprotease FTSH 9), spot 21 (BTB family PROTEIN 1), spot 80 (ubiquitin-conjugating enzyme E2), spot 86 (proteasome subunit alpha type-2), spot 104 (probable E3 ubiquitin-protein ligase XERICO), and spot 117 (ubiquitin-40S ribosomal protein S27a), were markedly modulated after exposure to salt stress. Proteins associated with cell autophagy, such as spot 44 (autophagy protein 5) and spot 50 (cysteine protease ATG4), were also reduced by salt stress, possibly through the H2S signal. A previous study demonstrated that H2S is the signal that represses cell autophagy in Arabidopsis.[44,45] The proteomic data also showed that proteins related to splicing or epigenetic modification components, such as spot 2 (isoform 5 of nuclear poly(A) polymerase), spot 5 [poly (ADP-ribose) polymerase], spot 6 (SWI/SNF complex subunit SWI3C), spot 20 (probable DNA helicase MCM8), spot 27 (U4/U6 small nuclear ribonucleoprotein PRP4-like protein), and spot 76 (PHD finger protein ALFIN-LIKE), were also differentially expressed after exposure to salt stress, indicating their role in modulating S. alterniflora tolerance to salt at the splicing or epigenetic level. These proteomics data suggest that S. alterniflora uses multiple strategies to enhance its tolerance to salt stress.

Among these differentially expressed proteins, antioxidant enzymes and defense proteins presented high expression after exposure to salt stress, suggesting that they enhance the S. alterniflora tolerance to salt stress. In agreement with this notion, salt treatment efficiently increased the activities of antioxidant enzymes in the leaves, which scavenge excess H2O2 and O2–, thus limiting ROS damage, while salt stress induced the rapid accumulation of ROS because of the reduced activities of antioxidant enzymes. Compared with S. alterniflora, the Fv/Fm ratio of C. malaccensis was also lower. These data suggest that S. alterniflora efficiently promoted antioxidant enzyme activity, and thereby enhanced the plant’s ability to adapt to salt stress.

NO also plays a central role in regulating various physiological processes, such as flowering, stomatal closure, germination, root development, gravitropism, and the response to abiotic and biotic stresses.[36−52] However, NO is a short-lived signal that reacts with the antioxidant glutathione to form GSNO, which then transfers its NO group to the cys thiol to form SNOs. This process, called protein S-nitrosylation, is a key redox-based post-translational modification in plants.[53] Cellular SNO levels are also regulated by GSNOR. This enzyme function is conserved among bacteria, plants, and animals and plays an important role in plant de-nitrosylation.[54,55] As demonstrated before, GSNOR efficiently removes excess SNOs to mitigate chilling damage to poplar or salt stress to alga.[44] Here, the proteomics data showed that salt stress induced the accumulation of GNSOR, indicating its role in sustaining the RNS status in S. alterniflora in response to salt stress. Consistent with this, salt stress in this study also increased the activity of the GSNOR enzyme, which would limit damage to the leaves by removing excess SNOs. The finding that exposure to salt stress caused SNOs to accumulate and resulted in lower levels of GSNOR indicates that these enzymes mediate tolerance to saline stress in S. alterniflora.

While a previous study demonstrated the critical role of H2S in plant adaptation to environmental stress, its role in invasive plant adaptation was unknown.[56] Here, the proteomic data revealed that several proteins associated with the H2S metabolism were upregulated after the plants were exposed to salt stress. Further analysis showed that salt stress induced the activity of l-CD and the generation of H2S in S. alterniflora, and removing endogenous H2S accumulation by pretreatment with a H2S scavenger resulted in the salt stress causing more damage to the leaves, as indicated by the increased ion leakage and MDA content and reduced Fv/Fm ratio. These data suggest that H2S enhances the tolerance to salt stress in S. alterniflora. Compared with C. malaccensis, the rates of K+ efflux were lower in S. alterniflora. This lower efflux of K+ would result in higher levels of cytosolic K+ and maintain the Na+/K+ balance in S. alterniflora. In agreement with this, suppressing H2S generation increased the K+ efflux and the Na+/K+ ratio, and reduced salt-induced antioxidant enzyme activity to increase ROS accumulation in the leaves, which possibly caused the lower Fv/Fm ratio in the leaves. Furthermore, H2S-mediated salt-induced GSNOR activity reduced SNO damage in S. alterniflora after exposure to salt stress, and suppressing H2S generation reduced salt-induced GSNOR activity increases SNO generation. This finding is in agreement with the results of a previous study[31] and supports the conclusion that H2S enhances S. alterniflora tolerance to salt stress through increasing GSNOR activity.

Conclusions

In this study, physiological and chemico-proteomics approaches were applied to explore the mechanism underlying salt stress tolerance in the invasive plant species S. alterniflora compared with the native species C. malaccensis. Additionally, the effects of H2S signaling on S. alterniflora were also determined through an inhibitor experiment. The results demonstrated that H2S signaling plays a central role in enhancing S. alterniflora tolerance to salt stress. Salinity stress triggers l-CD enzyme activity to rapidly induce H2S generation in S. alterniflora; the H2S signal activates antioxidant enzymes (APX and SOD), thereby limiting ROS accumulation, and also activates GSNOR, thereby reducing RNS damage. H2S also improves S. alterniflora tolerance to salinity stress by reducing the K+ efflux and thereby maintaining an intracellular Na+/K+ homeostasis. Thus, the findings provide insights into the H2S mechanism underlying salt stress tolerance in the invasive plant species S. alterniflora and show that H2S has an important role in this process by modulating the ROS and RNS status of the stressed cells. The findings also provide clues as to how to control this species and curb its encroachment on the native plant species by reducing H2S signaling.

Materials and Methods

Material and Salt Treatment

S. alterniflora was collected in the Shanyutan wetland in the Min River Estuary, Southeast China (119°34′12″–119°40′40″ W; 26°00′36″–26°03′42″ N; ca. 3120 ha) (Figure S6). In this region, S. alterniflora invaded the native species C. malaccensis that dominated the marsh in 2004. C. malaccensis occupies from the intertidal area to near the bank.[57] In this experiment, the collected plants were about 25 cm in height and then transplanted into 4 L pots filled with 1 L sandy soil and irrigated with 3 L of one-fourth strength Hoagland’s nutrient solution, pH 6.5. The Hoagland’s nutrient solution was changed every three days. The plants were grown under greenhouse conditions with an average air temperature of 25/18 °C (day/night), an air relative humidity ranging from 65 to 90%, and an average irradiance of 300/500 μmol m–2 s–1 for 7 days, and then were subjected to salt treatment. We chose a 7 day growth of plants because the roots, stems, and leaves of both plants were in a withered state after 7 days. For the salt or chemical inhibitor treatment, sodium chloride (NaCl) was dissolved in the Hoagland’s nutrient solution at the indicated concentrations (100, 300, 500, and 700 mM) and used to irrigate the sandy soil. For the chemical inhibitor treatment, PAG and HT were dissolved in 1% dimethyl sulfoxide at 1 M as the stock solution which was then diluted into the indicated concentration with the Hoagland’s solution before being used to irrigate the plants. The treated leaves were collected at various time points for further analysis. Three to five replicates were conducted and each replicate comprised six or ten individual plants. Samples not exposed to salt or chemical treatment were used as the controls.

Analysis of Chlorophyll Fluorescence

A pulse-amplitude modulated chlorophyll fluorometer (Heinz Walz GmbH, Effeltrich, Germany) was used to measure chlorophyll fluorescence.[58] After adapting the leaves in darkness for 30 min, the maximum quantum yield of PSII (Fv/Fm) was obtained by analyzing the entire leaf. Maximal fluorescence (Fm) and minimum fluorescence (Fo) were obtained using a 0.8 s pulsed light at 4000 μmol s–1 m–2 and the weak measuring pulses, respectively.

H2O2 and O2– Detection and Quantification

As reported, in situ detection of H2O2 and O2– was tested with some modifications.[59] Three leaf discs exposed to different salt treatments were punched out and vacuum infiltrated with 10 mL of 1 mg mL–1 of DAB solution for 2 h. After infiltration, using boiling ethanol (95%), the leaves were cleaned for 30 min and H2O2 was detected using DAB staining. The accumulation of O2– in leaves after different periods of treatment was determined using 10–2 M NBT reduction. The leaf discs were then vacuum infiltrated for 2 h using 10 mL of NBT and cleaned for 30 min using boiling ethanol (95%).

H2O2 content was determined based on the method described by Hu et al.[60] The leaves (1 g) after different treatments were ground into powder using liquid nitrogen and then homogenized with 5 mL of 0.2 M HClO4 at 4 °C. After keeping the extract on ice for 5 min and then centrifuging at 10,000g for 10 min at 4 °C, the supernatant obtained was stored in a refrigerator at −70 °C until further analysis. At room temperature for 1 h, a total of 100 μL of the supernatant was added to 1 mL of reaction buffer containing 0.25 mM (NH4)2SO4, 0.25 mM FeSO4, 1.25 M xylenol orange, 25 mM H2SO4, and 1 mM sorbitol. H2O2 levels were calculated via measuring absorbance at 560 nm by reference to standards.

Mixing 1 mL of the supernatant, 1 mL of 50 mM potassium phosphate buffer (pH 7.0), 10 mM hydroxylamine hydrochloride, 2 mL of 7 mM α-naphthyl and 2 mL of 17 mM sulphanilic acid, and then measuring the absorbance of the pink phase at 530 nm. The O2– content was determined by comparison with a standard curve according to the method previously described by Hu et al.[60]

H2S Measurements

The leaf tissue collected under different treatment conditions was ground into fine powder in liquid nitrogen, and after vortexing for 1 min in distilled water, the content of H2S was recorded by a micro sulfide ion electrode (LIS-146AGSCM; Lazar Research Lab. Inc., Los Angeles, CA, USA) at 25 °C.[61] Each measurement is repeated thrice.

Measurement of Lipid Peroxidation

Lipid peroxidation was measured using the thiobarbituric acid-reacting substance method with MDA as a standard.[62]

Relative Ion Leakage Measurement

Relative ion leakage was determined as reported.[58] The fresh leaf sample (0.5 g) and 10 mL of deionized water were kept in a petri dish at 25 °C for 2 h. The first conductivity of the bathing solution was tested after incubation (C1). The second conductivity was determined after boiling the bathing solution continuously for 15 min (C2). The relative ion leakage was calculated as: C1/C2 × 100.

APX and SOD Enzyme Activity Analysis

Leaf samples (1 g), 5 mL of extraction medium containing 50 mM Tris-HCl (pH 7.8), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.2% (v/v) Triton X-100, 2 mM ascorbate, and 1 mM phenylmethylsulfonyl fluoride were homogenized. APX activity was determined by the decreasing of the absorbance of a reaction medium containing 50 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid-NaOH (pH 7.6), 0.25 mM ascorbate, 0.1 mM H2O2, and 10–40 μL of extract under 290 nm. The SOD enzyme activity was calculated by the 50% inhibition of the photochemical reduction of NBT.

SNO Content and GSNOR Measurement

The total SNO content was measured using the gas-phase chemiluminescence method based on the NO release from the reductive decomposition of nitrogen species in an iodine/triiodide mixture. A nitric oxide monitor 410 (2B Technologies, Boulder, CO, USA) was used to determine the released NO content.

GSNOR activity was tested at 25 °C via recording the reduced nicotinamide adenine dinucleotide (NADH) oxidation at 340 nm as reported.[30] Samples exposed to different treatments were collected and quickly homogenized in liquid nitrogen, using extraction buffer (20 mM Tris-HCl (pH 8.0), 0.2 mM NADH, and 0.5 mM EDTA) at 4 °C. After centrifuging the homogenates at 3000g for 10 min at 4 °C, the supernatants were then collected for GSNOR enzyme activity assays. Nanomoles NADH consumed per minute per milligram of protein (e340 6.22 mM–1 cm–1) represented the GSNOR activity.

l-Cysteine Desulfhydrases Enzyme Activity Measurement

The activities of l-CDs were measured as reported.[63] The leaf tissue (1 g) exposed to different salt treatments was collected and ground to a fine powder using liquid nitrogen. The sample was vortexed after adding 1.5 mL of cold extraction buffer [20 mM Tris-HCl (pH 8.0), 0.1% (w/v) dithiothreitol and 0.2% (w/v) sodium ascorbate]. The homogenate was centrifuged at 13,000g for 15 min at 4 °C and the resulting supernatant was collected for analysis of the activities of l-CDs enzyme.

Protein Extraction and 2D Electrophoresis

Protein extraction and 2D separation were performed according to the method previously described by Yang et al. and Ma et al.[64,65]

In-Gel Digestion

We excised manually the protein spots which significantly changed in expression from colloidal CBB-stained 2DE gels, and then performed protein digestion with trypsin based on the method described by Yang et al. and Ma et al.[64,65]

MALDI-TOF/TOF Analysis and Database Search

The lyophilized peptide samples were completely dissolved in 0.1% trifluoroacetic acid. A MALDI-TOF/TOF mass spectrometer 4800-plus proteomics analyzer (Applied Biosystems; Massachusetts, USA) was used for the MS analyses.[64] (see the details for above mentioned measurements in Supporting Information Text).