The Essential Role of H2S-ABA Crosstalk in Maize Thermotolerance through the ROS-Scavenging System

Hydrogen sulfide (H2S) and abscisic acid (ABA), as a signaling molecule and stress hormone, their crosstalk-induced thermotolerance in maize seedlings and its underlying mechanism were elusive. In this paper, H2S and ABA crosstalk as well as the underlying mechanism of crosstalk-induced thermotolerance in maize seedlings were investigated. The data show that endogenous levels of H2S and ABA in maize seedlings could be mutually induced by regulating their metabolic enzyme activity and gene expression under non-heat stress (non-HS) and HS conditions. Furthermore, H2S and ABA alone or in combination significantly increase thermotolerance in maize seedlings by improving the survival rate (SR) and mitigating biomembrane damage. Similarly, the activity of the reactive oxygen species (ROS)-scavenging system, including enzymatic antioxidants catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POD), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and superoxide dismutase (SOD), as well as the non-enzymatic antioxidants reduced ascorbic acid (AsA), carotenoids (CAR), flavone (FLA), and total phenols (TP), was enhanced by H2S and ABA alone or in combination in maize seedlings. Conversely, the ROS level (mainly hydrogen peroxide and superoxide radical) was weakened by H2S and ABA alone or in combination in maize seedlings under non-HS and HS conditions. These data imply that the ROS-scavenging system played an essential role in H2S-ABA crosstalk-induced thermotolerance in maize seedlings.


Introduction
Hydrogen sulfide (H 2 S), which is similar to other signaling molecules, has an inversely physiological effect at low (as a signaling molecule) and high (cytotoxic agent) concentrations. Currently, the research on H 2 S was actively turned to its signaling role from toxic gas molecules in plants [1]. Signaling molecules, like H 2 S, can be rapidly synthesized and triggered by signaling pathways when plants are subjected to environment stimuli, and even stress, but they maintain homeostasis in plant cellular and subcellular compartments under normal physiological conditions [2]. H 2 S homeostasis in plant cells is strictly controlled by anabolic and catabolic enzymes located in the various subcellular compartments. These metabolic enzymes include L-/D-cysteine desulfhydrase (L/DCD), O-acetylserine (thiol)lyase A1 (OAS-TL A1), and L-cysteine desulfhydrase 1 (DES1, OAS-TL homolog) in the cytosol; sulfite reductase (SiR) and OAS-TL B in chloroplast; and β-cyanoalanine synthase (CAS), OAS-TL C, and nitrogenase Fe-S cluster (NifS) in mitochondria [3]. These enzymes can be activated or inhibited by a specific metabolism, development, and/or environment signals/stress to trigger H 2 S signaling or maintain H 2 S homeostasis in plants [4]. H 2 S, as a novel gasotransmitter, could increase multiple-stress tolerance in plants [5]. In SR was increased to 78%, 82%, and 85%, respectively, from 52% in the control, especially irrigation with NaHS and ABA in combination was more significant among the treatments ( Figure 1A). Similarly, before and after HS, the electrolyte leakage (EL, denoting biomembrane integrity) and malondialdehyde (MDA, membrane lipid peroxidation) content in the seedlings irrigated with NaHS and ABA alone, or in combination, were determined. The data indicate that NaHS and ABA alone, or in combination, had no significant effect on the electrolyte leakage ( Figure 1B) and MDA content ( Figure 1C) before HS. After HS, NaHS and ABA alone, or in combination, obviously remitted the increase in the electrolyte leakage ( Figure 1B) and MDA content ( Figure 1C) compared with the control, especially NaHS combined with ABA in combination showed a more significant remission, similar to the effect on the survival rate ( Figure 1A). These data suggest that H 2 S and ABA alone, or in combination, could induce thermotolerance in maize seedlings. , and malondialdehyde content (C) in maize seedlings under non-heat stress (non-HS) and HS conditions. The significance analysis between the data was performed as a one-way analysis of variance (ANOVA) and Duncan multiple-range test at the 0.05 level. The data in the figures represent the mean ± standard error (SE, n = 5), the same and different letters indicate insignificant and significant difference, respectively.

ABA Increases Endogenous H2S Level
To further illustrate the H2S-ABA crosstalk, before and after HS, the contents of endogenous H2S and ABA, as well as the activities by their metabolic enzymes in seedlings irrigated with NaHS and ABA alone or in combination, NaHS combined with ST or FLU, as well as ABA combined with PAG, HA, or HT, were determined. The data indicate that, before HS, the endogenous H2S content was significantly increased by NaHS and ABA alone, or in combination, and the combination resulted in a more obvious increase ( Figure   Figure 1. Effect of the irrigation with NaHS and abscisic acid (ABA) alone or in combination on the survival rate (A), electrolyte leakage (B), and malondialdehyde content (C) in maize seedlings under non-heat stress (non-HS) and HS conditions. The significance analysis between the data was performed as a one-way analysis of variance (ANOVA) and Duncan multiple-range test at the 0.05 level. The data in the figures represent the mean ± standard error (SE, n = 5), the same and different letters indicate insignificant and significant difference, respectively.

ABA Increases Endogenous H 2 S Level
To further illustrate the H 2 S-ABA crosstalk, before and after HS, the contents of endogenous H 2 S and ABA, as well as the activities by their metabolic enzymes in seedlings irrigated with NaHS and ABA alone or in combination, NaHS combined with ST or FLU, as well as ABA combined with PAG, HA, or HT, were determined. The data indicate that, before HS, the endogenous H 2 S content was significantly increased by NaHS and ABA alone, or in combination, and the combination resulted in a more obvious increase ( Figure 2). Also, NaHS-induced H 2 S was eliminated by the ABA inhibitors ST and FLU, while the ABA-induced H 2 S was weakened by the H 2 S inhibitor PAG, but eliminated by the H 2 S scavengers HA and HT. Similarly, the activity of LCD, DCD, and OAS-TL in the maize seedlings was significantly enhanced by H 2 S and ABA alone, or in combination ( Figure 3). However, the gene expression of ZmLCD1 and ZmOAS-TL was not markedly up-regulated by NaHS and ABA alone, or in combination, except NaHS up-regulated ZmOAS-TL expression ( Figure 4). scavengers HA and HT. Similarly, the activity of LCD, DCD, and OAS-TL in the maize seedlings was significantly enhanced by H2S and ABA alone, or in combination ( Figure  3). However, the gene expression of ZmLCD1 and ZmOAS-TL was not markedly up-regulated by NaHS and ABA alone, or in combination, except NaHS up-regulated ZmOAS-TL expression ( Figure 4).

Figure 2.
Effect of the irrigation with NaHS and abscisic acid (ABA) alone or in combination (A), NaHS alone or in combination with sodium tungstate (ST) or fluoridone (FLU) (B), and ABA alone or in combination with hydroxylamine (HA) or hypotaurine (HT) (C), on endogenous hydrogen sulfide (H2S) content in maize seedlings under non-heat stress (non-HS) and HS conditions. The significance analysis between the data was performed as a one-way analysis of variance (ANOVA) and Duncan multiple-range test at the 0.05 level. The data in the figures represent mean ± standard error (SE, n = 5), the same and different letters indicate the insignificant and significant difference, respectively.

Figure 2.
Effect of the irrigation with NaHS and abscisic acid (ABA) alone or in combination (A), NaHS alone or in combination with sodium tungstate (ST) or fluoridone (FLU) (B), and ABA alone or in combination with hydroxylamine (HA) or hypotaurine (HT) (C), on endogenous hydrogen sulfide (H 2 S) content in maize seedlings under non-heat stress (non-HS) and HS conditions. The significance analysis between the data was performed as a one-way analysis of variance (ANOVA) and Duncan multiple-range test at the 0.05 level. The data in the figures represent mean ± standard error (SE, n = 5), the same and different letters indicate the insignificant and significant difference, respectively. The significance analysis between the data was performed as a one-way analysis of variance (ANOVA) and Duncan multiple-range test at the 0.05 level. The data in the figures represent mean ± standard error (SE, n = 5), the same and different letters indicate the insignificant and significant difference, respectively.
After HS, the endogenous H2S level in maize seedlings irrigated with NaHS and ABA alone or in combination was significantly increased, especially both in combination were shown to be more effective (Figure 2). Similarly, NaHS-induced H2S was removed by the ABA inhibitors ST and FLU; whereas, ABA-induced H2S was impaired by the H2S inhibitor PAG and scavengers HA and HT, respectively ( Figure 2). Analogously, the LCD, DCD, and OAS-TL activity was markedly increased by H2S and ABA alone or in combination, except ABA alone for OAS-TL ( Figure 3). For gene expression, ZmLCD1 expression was significantly up-regulated by ABA alone or combined with NaHS, but down-regulated by conditions. The significance analysis between the data was performed as a one-way analysis of variance (ANOVA) and Duncan multiple-range test at the 0.05 level. The data in the figures represent mean ± standard error (SE, n = 5), the same and different letters indicate the insignificant and significant difference, respectively.
After HS, the endogenous H 2 S level in maize seedlings irrigated with NaHS and ABA alone or in combination was significantly increased, especially both in combination were shown to be more effective (Figure 2). Similarly, NaHS-induced H 2 S was removed by the ABA inhibitors ST and FLU; whereas, ABA-induced H 2 S was impaired by the H 2 S inhibitor PAG and scavengers HA and HT, respectively ( Figure 2). Analogously, the LCD, DCD, and OAS-TL activity was markedly increased by H 2 S and ABA alone or in combination, except ABA alone for OAS-TL ( Figure 3). For gene expression, ZmLCD1 expression was significantly up-regulated by ABA alone or combined with NaHS, but down-regulated by NaHS alone. Also, ZmOAS-TL expression was markedly up-regulated by ABA alone or combined with NaHS, but NaHS had no significant effect on its expression ( Figure 4). nt. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 NaHS alone. Also, ZmOAS-TL expression was markedly up-regulated by ABA alo combined with NaHS, but NaHS had no significant effect on its expression ( Figure   Figure 4. Effect of the irrigation with NaHS and abscisic acid (ABA) alone or in combination gene expression of LCD1 (A) and OAS-TL (B) in maize seedlings under non-heat stress (no and HS conditions. The significance analysis between the data was performed as a one-way an of variance (ANOVA) and Duncan multiple-range test at the 0.05 level. The data in the figure resent mean ± standard error (SE, n = 5), the same and different letters indicate the insignifica significant difference, respectively.

H2S Increases Endogenous ABA Level
To further study the effect of NaHS on the endogenous ABA level, the content ABA and its metabolic enzyme activity in maize seedlings irrigated with NaHS and alone or in combination, NaHS combined with ST and FLU, and ABA combined PAG, HA, and HT, were measured. Before the HS, irrigation with NaHS, ABA, ST + N FLU + NaHS had no significant effect on the endogenous ABA level, which was obvi increased by the ABA + NaHS, whereas it was significantly weakened by the combin of ABA with PAG, HA, or HT ( Figure 5). Also, the activities of ZEP, NCED, and AA seedling mesocotyls were markedly improved by NaHS and ABA alone, or in com tion ( Figure 6). Similarly, the gene expression of ZmZEP, ZmNCED, and ZmAAO mesocotyls of maize seedlings was observably up-regulated by the irrigation with N and ABA alone, or in combination (Figure 7). HS conditions. The significance analysis between the data was performed as a one-way analysis of variance (ANOVA) and Duncan multiple-range test at the 0.05 level. The data in the figures represent mean ± standard error (SE, n = 5), the same and different letters indicate the insignificant and significant difference, respectively.

H 2 S Increases Endogenous ABA Level
To further study the effect of NaHS on the endogenous ABA level, the content of the ABA and its metabolic enzyme activity in maize seedlings irrigated with NaHS and ABA alone or in combination, NaHS combined with ST and FLU, and ABA combined with PAG, HA, and HT, were measured. Before the HS, irrigation with NaHS, ABA, ST + NaHS, FLU + NaHS had no significant effect on the endogenous ABA level, which was obviously increased by the ABA + NaHS, whereas it was significantly weakened by the combination of ABA with PAG, HA, or HT ( Figure 5). Also, the activities of ZEP, NCED, and AAO in seedling mesocotyls were markedly improved by NaHS and ABA alone, or in combination ( Figure 6). Similarly, the gene expression of ZmZEP, ZmNCED, and ZmAAO in the mesocotyls of maize seedlings was observably up-regulated by the irrigation with NaHS and ABA alone, or in combination ( Figure 7).   ditions. The significance analysis between the data was performed as a one-way analysis of (ANOVA) and Duncan multiple-range test at the 0.05 level. The data in the figures represe ± standard error (SE, n = 5), the same and different letters indicate the insignificant and si difference, respectively.
After HS, the endogenous ABA content in the mesocotyls of the maize seedli signally increased by NaHS and ABA alone or in combination, but impaired by t bination of ABA with PAG, HA, or HT, while the combination of NaHS with ST had no significant effect on the endogenous ABA level ( Figure 5). In addition, the a of the ABA metabolic enzymes (ZEP, NCED, and AAO) were memorably incre Figure 6. Effect of the irrigation with NaHS and abscisic acid (ABA) alone or in combination on the activity of zeaxanthin epoxidase (ZEP) (A), 9-cis epoxycarotenoid dioxygenase (NCED) (B), and abscisic aldehyde oxidase (AAO) (C) in maize seedlings under non-heat stress (non-HS) and HS conditions. The significance analysis between the data was performed as a one-way analysis of variance (ANOVA) and Duncan multiple-range test at the 0.05 level. The data in the figures represent mean ± standard error (SE, n = 5), the same and different letters indicate the insignificant and significant difference, respectively.

Figure 7.
Effect of the irrigation with NaHS and abscisic acid (ABA) alone or in combination on the gene expression of ZEP (A), NCED1 (B), and AAO (C) in maize seedlings under non-heat stress (non-HS) and HS conditions. The significance analysis between the data was performed as a oneway analysis of variance (ANOVA) and Duncan multiple-range test at the 0.05 level. The data in the figures represent mean ± standard error (SE, n = 5), the same and different letters indicate the insignificant and significant difference, respectively.

H2S-ABA Crosstalk Activates ROS-Scavenging System
To further understand the mechanism underlying H2S-ABA crosstalk-induced thermotolerance in maize seedlings, the activity of the ROS-scavenging system was analyzed. Before the HS, the results show that the activities of GR, MDHAR, CAT, and DHAR in maize seedlings were dramatically increased by the irrigation with NaHS and ABA alone or in combination (Figures 8-10), while APX, POD, and SOD activity was only activated by NaHS combined with ABA, but NaHS and ABA alone had no significant effect on the activity of the three enzymes (Figures 8 and 9). Correspondingly, the contents of the carotenoids and flavone in the maize seedlings were observably augmented by NaHS and ABA alone or in combination (Figures 10 and 11), whereas the AsA and total phenol contents were only augmented by the combination of NaHS with ABA, but NaHS and ABA had no significant difference on the contents of the AsA and total phenol in the maize seedlings ( Figures 10 and 11). Similarly, the gene expression of ZmGR1 and ZmAPX1 in the mesocotyls of the maize seedlings was significantly up-regulated by NaHS and ABA alone or in combination (Figures 12 and 13), while ZmCAT1 expression was obviously up- , and AAO (C) in maize seedlings under non-heat stress (non-HS) and HS conditions. The significance analysis between the data was performed as a one-way analysis of variance (ANOVA) and Duncan multiple-range test at the 0.05 level. The data in the figures represent mean ± standard error (SE, n = 5), the same and different letters indicate the insignificant and significant difference, respectively.
After HS, the endogenous ABA content in the mesocotyls of the maize seedlings was signally increased by NaHS and ABA alone or in combination, but impaired by the combination of ABA with PAG, HA, or HT, while the combination of NaHS with ST or FLU had no significant effect on the endogenous ABA level ( Figure 5). In addition, the activities of the ABA metabolic enzymes (ZEP, NCED, and AAO) were memorably increased by the irrigation with NaHS and ABA alone or in combination, especially both in combination was more significant ( Figure 6). Analogously, the expression of ZmZEP, ZmNCED, and ZmAAO in the mesocotyls of the maize seedlings was dramatically up-regulated by the irrigation with NaHS and ABA alone, or in combination (Figure 7).

H 2 S-ABA Crosstalk Activates ROS-Scavenging System
To further understand the mechanism underlying H 2 S-ABA crosstalk-induced thermotolerance in maize seedlings, the activity of the ROS-scavenging system was analyzed. Before the HS, the results show that the activities of GR, MDHAR, CAT, and DHAR in maize seedlings were dramatically increased by the irrigation with NaHS and ABA alone or in combination (Figures 8-10), while APX, POD, and SOD activity was only activated by NaHS combined with ABA, but NaHS and ABA alone had no significant effect on the activity of the three enzymes (Figures 8 and 9). Correspondingly, the contents of the carotenoids and flavone in the maize seedlings were observably augmented by NaHS and ABA alone or in combination (Figures 10 and 11), whereas the AsA and total phenol contents were only augmented by the combination of NaHS with ABA, but NaHS and ABA had no significant difference on the contents of the AsA and total phenol in the maize seedlings (Figures 10 and 11). Similarly, the gene expression of ZmGR1 and ZmAPX1 in the mesocotyls of the maize seedlings was significantly up-regulated by NaHS and ABA alone or in combination (Figures 12 and 13), while ZmCAT 1 expression was obviously up-regulated by the irrigation with NaHS alone or combined with ABA, and up-regulation of ZmSOD 4 expression by NaHS and ABA alone was also observed (Figures 12 and 13), but the significant difference from NaHS and ABA alone or in combination on ZmDHAR and ZmMDHAR expression was not observed, except for the significant effect of both in combination on ZmMDHAR expression (Figures 12 and 13).
regulated by the irrigation with NaHS alone or combined with ABA, and up-regulation of ZmSOD4 expression by NaHS and ABA alone was also observed (Figures 12 and 13), but the significant difference from NaHS and ABA alone or in combination on ZmDHAR and ZmMDHAR expression was not observed, except for the significant effect of both in combination on ZmMDHAR expression (Figures 12 and 13).       After HS, the activities of APX, CAT, POD, and MDHAR in the mes maize seedlings were significantly increased by the irrigation with NaHS an or in combination (Figures 7-9), while a significant difference from NaHS an or in combination on the activity of GR, DHAR, and SOD in maize seedlings (Figures 7-9). Also, the contents of the carotenoids, flavone, and total phenol seedlings were obviously increased by the irrigation with NaHS and AB combination, both in combination increased the AsA content, but NaHS an had no significant effect on the AsA (Figures 9 and 10). Correspondingly, the sion of ZmCAT1, ZmSOD4, ZmGR1, and ZmMDHAR in the maize seedlings w up-regulated by NaHS and ABA alone or in combination (Figures 11 ZmAPX1 and ZmDHAR expression was separately up-regulated by NaHS co ABA and ABA alone, or in combination with NaHS (Figures 11 and 12). and HS conditions. The significance analysis between the data was performed as a one-way analysis of variance (ANOVA) and Duncan multiple-range test at the 0.05 level. The data in the figures represent mean ± standard error (SE, n = 5), the same and different letters indicate the insignificant and significant difference, respectively.
After HS, the activities of APX, CAT, POD, and MDHAR in the mesocotyls of the maize seedlings were significantly increased by the irrigation with NaHS and ABA alone or in combination (Figures 7-9), while a significant difference from NaHS and ABA alone or in combination on the activity of GR, DHAR, and SOD in maize seedlings was not noted (Figures 7-9). Also, the contents of the carotenoids, flavone, and total phenols in the maize seedlings were obviously increased by the irrigation with NaHS and ABA alone or in combination, both in combination increased the AsA content, but NaHS and ABA alone had no significant effect on the AsA (Figures 9 and 10). Correspondingly, the gene expression of ZmCAT1, ZmSOD4, ZmGR1, and ZmMDHAR in the maize seedlings was markedly up-regulated by NaHS and ABA alone or in combination (Figures 11 and 12), and ZmAPX1 and ZmDHAR expression was separately up-regulated by NaHS combined with ABA and ABA alone, or in combination with NaHS (Figures 11 and 12).

H 2 S-ABA Crosstalk Modulates the ROS Level
As mentioned above, H 2 S-ABA crosstalk could activate the activity of the ROSscavenging system in maize seedlings (Figures 8-13). To further explore the effect of H 2 S-ABA crosstalk on the ROS level in maize seedlings, the H 2 O 2 content and O 2 .− generation rate was detected. Before the HS, the data show that the generation rate for O 2 .− in the mesocotyls of the maize seedlings was significantly decreased by the irrigation with NaHS and ABA alone or in combination, but the content of the H 2 O 2 was increased through the irrigations ( Figure 14). After the HS, the generation rate for O 2 .− in the mesocotyls of the maize seedlings was significantly increased, but the HS-induced increase in the generation rate for O 2 .− was weakened by NaHS and ABA alone or in combination ( Figure 14). Also, compared with the control, the H 2 O 2 content in the mesocotyls of the seedlings was maintained at a low level by NaHS and ABA alone, or in combination ( Figure 14).    HS) and HS conditions. The significance analysis between the data was way analysis of variance (ANOVA) and Duncan multiple-range test at the 0.05 figures represent mean ± standard error (SE, n = 5), the same and different lette nificant and significant difference, respectively.

H2S-ABA Crosstalk Modulates the ROS Level
As mentioned above, H2S-ABA crosstalk could activate the activit enging system in maize seedlings (Figures 8-13). To further explore the Figure 13. Effect of the irrigation with NaHS and abscisic acid (ABA) alone or in combination on the gene expression of APX1 (A), DHAR (B), and MDHAR (C) in maize seedlings under non-heat stress (non-HS) and HS conditions. The significance analysis between the data was performed as a one-way analysis of variance (ANOVA) and Duncan multiple-range test at the 0.05 level. The data in the figures represent mean ± standard error (SE, n = 5), the same and different letters indicate the insignificant and significant difference, respectively.

Correlation among Indices
After the correlation analysis, the correlation between H 2 S and its metabolic enzymes (LCD, DCD, and OAS-TL) and ABA and its metabolic enzymes (ZEP, NCED, and AAO) is shown in Table 1. The data showed that H 2 S was positively correlated with ABA, ZEP, NCED, and AAO, as well as with ABA, and AAO, and NCED reached significant levels. Table 1. Correlation analysis between H 2 S and its metabolic enzymes and ABA and its metabolic enzymes. In the table, r represents the correlation coefficient (positive and negative numbers denote positive and negative correlation, respectively), the asterisk (*) and double asterisks (**) indicate significant (p < 0.05) and very significant difference (p < 0.01), respectively.   In addition, Table 3 implies that a very significant positive correlation (p < 0.01) was observed between SR and POD, CAT, GR, APX, and DHAR, while a significant positive correlation (p < 0.05) existed between SR and MDHAR.  Table 4. Correlation analysis between the survival rate and antioxidants. In the table, r represents the correlation coefficient (positive and negative numbers denote positive and negative correlation, respectively), the asterisk (*) and double asterisks (**) indicate significant (p < 0.05) and very significant difference (p < 0.01), respectively.

Discussion
H 2 S, as a novel signaling molecule, and ABA, as a stress hormone, can exert their physiological functions in an independent or dependent (antagonistic or synergistic) manner in plants [16,19,25]. However, in the development of thermotolerance in plants, whether and how H 2 S interacts with ABA remains elusive. In this paper, using maize seedlings as the materials, we found that H 2 S interacted with ABA by mutually modulating their endogenous levels via metabolic enzyme activity and corresponding gene expression (Figures 2-6), and the ROS-scavenging system played an essential role in H 2 S-ABA crosstalk-induced thermotolerance in maize seedlings (Figures 7-13).
Generally, the crosstalk among signaling molecules maybe achieve by chemical reaction among the signaling molecules, by modulating the metabolic enzymes, by competing with common target proteins, and/or by regulating some nodes in the signaling pathways, and so on [26,27]. Therefore, some signaling pathways, through signaling crosstalk, can be strengthened, or weakened, and can even trigger a novel signal pathway [26,27]. In this paper, irrigation with NaHS up-regulated the gene expression of the ABA metabolic enzymes (ZmZEP, ZmNCED1, and ZmAAO) in the maize seedlings, which in turn increased the level of endogenous ABA under both non-HS and HS conditions (Figures 6 and 7). Also, NaHS-induced ABA was weakened by the ABA inhibitors ST and FLU, respectively (Figures 6 and 7), indicating that H 2 S could trigger ABA signaling by activating its metabolic pathways. Similarly, root irrigation with ABA up-regulated the gene expression of the H 2 S metabolic enzymes (ZmLCD1 and ZmOAS-TL) in the maize seedlings, followed by increasing the level of endogenous H 2 S under both non-HS and HS conditions (Figures 2  and 3). In addition, ABA-induced H 2 S was impaired by the H 2 S inhibitor PAG and scavengers HA and HT, respectively (Figures 2 and 3), indicating that ABA could trigger H 2 S signaling by modulating its metabolic enzymes. Also, Pearson correlation analysis showed that H 2 S and its metabolic enzymes LCD, DCD, and OAS-TL were positive correlation with ABA and its metabolic enzymes ZEP, NCED, and AAO, and their correlation reached a significant (for ZEP and AAO) and very significant (for NCED) difference (Table 1). Similarly, in wheat and rice plants, H 2 S regulated the formation of thermotolerance by interacting with ABA, melatonin, ETH, and NO [28][29][30]. These results suggest that the crosstalk between H 2 S and ABA occurred in the development of plant thermotolerance by mutually modulating the metabolic enzymes.
In addition, numerous studies show that H 2 S and ABA alone or in combination could increase the thermotolerance in plants, but their detailed mechanisms were not completely clear [20,31,32]. In this paper, the thermotolerance in maize seedlings was significantly improved by increasing the survival rate and decreasing the electrolyte leakage and MDA accumulation after the seedlings were irrigated with NaHS and ABA alone or in combination, particularly in combination ( Figure 1). Also, Pearson correlation analysis indicated that the survival rate was positively correlated with tissue viability, negatively correlated with electrolyte leakage and MDA accumulation, and their correlation reached a significant difference ( Table 2). These results further support the fact that H 2 S-ABA crosstalk induces thermotolerance in maize seedlings.
Oxidative stress is the major heat injury in plants due to the excessive accumulation of ROS under HS conditions [23,[33][34][35]. Correspondingly, the acquirement of thermotolerance in plants and the ROS-scavenging system goes hand in hand [36][37][38][39][40][41]. In this paper, HS promoted the generation rate of O 2 .− in the mesocotyls of the maize seedlings ( Figure 14), indicating HS-triggered oxidative stress. Interestingly, HS-promoted O 2 .− production was significantly impaired by NaHS and ABA alone or in combination, especially in combination ( Figure 14). To further understand the mechanisms underlying H 2 S-ABA crosstalk-induced thermotolerance and the mitigative effect of NaHS and ABA alone or in combination on oxidative stress in maize seedlings, the ROS-scavenging system activity was analyzed. Before the HS, the enhanced ROS-scavenging system activity by NaHS and ABA alone or in combination laid the foundation for the acquirement of subsequent thermotolerance in the maize seedlings (Figures 8-13). After irrigation with NaHS and ABA alone or in combination, the activity of the ROS-scavenging enzymes, such as CAT, APX, POD, and MDHAR, as well as the content of the non-enzymatic antioxidants, such as AsA, carotenoids, flavone, and total phenols, in the maize seedlings were significantly enhanced, particularly when irrigating with NaHS in combination with ABA (Figures 8-13). Correspondingly, the gene expression of the ROS-scavenging enzymes in the mesocotyls of the maize seedlings, such as ZmCAT1, ZmAPX, ZmSOD4, and ZmMDHAR, was markedly up-regulated by the irrigation with NaHS and ABA alone or in combination, especially in combination (Figures 8-13).
In HS situations, the ROS-scavenging system plays a key role in the development of thermotolerance in plants [42][43][44][45][46][47]. As mentioned above, before HS, the enhanced ROSscavenging system by H 2 S and ABA laid the foundation for the formation of the subsequent thermotolerance (Figures 8-13). Analogously, under HS conditions, compared with the control, the ROS-scavenging enzymes (i.e., CAT, APX, POD, and MDHAR) activities and the non-enzymatic antioxidants (i.e., AsA, carotenoids, flavone, and total phenols) contents in the mesocotyls of the maize seedlings were significantly enhanced by the irrigation with NaHS and ABA alone or in combination, especially the combination showed more physiological effects (Figures 8-10). Accordingly, the expression of the ROS-scavenging enzyme genes (i.e., ZmCAT1, ZmAPX, ZmSOD4, and ZmMDHAR) in the mesocotyls of the maize seedlings was obviously up-regulated by NaHS and ABA alone or in combination, particularly NaHS combined with ABA had the most significantly molecular effect among the irrigations (Figures 12 and 13). In addition, Pearson correlation analysis suggested that the survival rate was positively related to the activity of the ROS-scavenging enzymes, POD, CAT, SOD, GR, APX, DHAR, and MDHAR, and their correlation showed a significant (for MDHAR) and very significant (for POD, CAT, GR, APX, and DHAR) level (Table 3).
Similarly, the survival rate and the ROS-scavengers AsA, flavone, total phenols, and carotenoids had a positive correlation, while a negative correlation for the survival rate and O 2 .− was observed (Table 4). These indicate that the ROS-scavenging system in maize seedlings could be enhanced by the irrigation with NaHS and ABA alone or in combination under HS conditions, which in turn reduced the accumulation of ROS (Figure 14), thus mitigating the oxidative stress induced by HS and improving the thermotolerance in maize seedlings ( Figure 1). As discussed above, H 2 S initiated the formation of thermotolerance by interplaying with ABA, melatonin, ETH, and NO in wheat and rice plants via antioxidants, redox homeostasis, osmolytes, and photosynthetic metabolism [28][29][30], further supporting the essential role of H 2 S-ABA crosstalk in plant thermotolerance development by the ROS-scavenging system.

Measurement of Thermotolerance Parameters
To study the effect of H 2 S-ABA crosstalk on thermotolerance in maize seedlings, after HS and recovery, the survival rate (SR), tissue viability (TV), biomembrane peroxidation, and electrolyte leakage (EL) of the seedlings irrigated with NaHS and ABA alone or in combination were separately measured, according to the methods reported by Wang et al. [53]. The survival rate and electrolyte leakage were expressed in %, while the biomembrane peroxidation was expressed as nmol g −1 fresh weight (FW), respectively.

Analysis of H 2 S Content and Its Metabolic Enzymes
To investigate the effect of ABA on H 2 S content and its metabolic enzyme activity, the seedlings were irrigated with NaHS alone or combined with the ABA inhibitors ST and FLU, as well as ABA alone or combined with the H 2 S scavenger HT and the inhibitors HA and PAG. After irrigation and HS, the H 2 S content and activity of LCD, DCD, and OAS-TL in seedling mesocotyls were analyzed, as per the procedures by Ye et al. [54]. The H 2 S content was expressed in nmol g −1 FW, whereas the LCD, DCD, and OAS-TL activities were indicated as nmol min −1 mg −1 protein. The content of the soluble proteins in the seedling mesocotyls was analyzed using the Bradford method [55], using bovine serum albumin as the standard sample.

Determination of ABA Content and Its Metabolic Enzymes
Similarly, to explore the effect of H 2 S on the ABA content and its metabolic enzyme activity, the seedlings were irrigated with NaHS alone or combined with the ABA inhibitors ST and FLU, as well as ABA alone or combined with the H 2 S scavenger HT and the inhibitors HA and PAG. After irrigation and HS, the ABA content and activity of ZEP, NCED, and OAA in seedling mesocotyls were determined, as per the procedures in the instruction book for the kits. The ABA content was expressed in µg g −1 FW, whereas the ZEP, NCED, and OAA activities were expressed in nmol min −1 mg −1 protein.

Determination of ROS
To estimate the effect of H 2 S-ABA crosstalk on the ROS level, after irrigation and HS, the generation rate for the superoxide radical (O 2 .− ) and hydrogen peroxide (H 2 O 2 ) content in seedlings irrigated with NaHS and ABA alone or in combination was determined according to the titanium sulfate method [53] and Na,3 -[1-[(phenylamino)-carbonyl]-3, 4-tetrazolium] (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) method [52]. The O 2 .− production and H 2 O 2 content were calculated using the millimolar extinction coefficient at 21.6 and 0.28 mM −1 cm −1 , and expressed as nmol min −1 g −1 FW and nmol g −1 FW, respectively.

Quantification of Gene Expression
To further explore the effect of H 2 S-ABA crosstalk on the gene expression of the H 2 Sand ABA-metabolic enzymes and the enzymatic ROS-scavenging system, after irrigation and HS, the genes ZmLCD1, ZmOAS-TL, ZmZEP, ZmNCED, ZmOAA, ZmCAT1, ZmSOD4, ZmGR1, ZmAPX1, ZmDHAR, and ZmMDHAR in the seedling mesocotyls were quantified by qRT-PCR (using Zea mays beta-5 tubulin (ZmTUB) as an internal reference), and the relative expression level was calculated using 2 −∆∆CT [57]. For the gene primers refer to Table S1.

Statistical Analysis
The experiments were a random design and the significance analysis between the data was performed as a one-way analysis of variance (ANOVA) and Duncan multiple-range test at the 0.05 level. The data in the figures represent mean ± standard error (SE, n = 5), and the same and different letters indicate the insignificant and significant difference, respectively. Also, to further estimate the correlation among the parameters, the correlation between H 2 S and its metabolic enzymes and ABA and its metabolic enzymes, as well as SR and the thermotolerance index, antioxidant enzymes, non-enzymatic antioxidant, and ROS was analyzed using Pearson correlation in SigmaPlot 25 software. In the tables, r represents the correlation coefficient (positive and negative numbers denote positive and negative correlation, respectively), and the asterisk (*) and double asterisks (**) indicate significant (p < 0.05) and very significant difference (p < 0.01), respectively.

Conclusions
In sum, the crosstalk between H 2 S and ABA in maize seedlings before and after HS was found to modulate the activity and gene expression of metabolic enzymes related to H 2 S (LCD, DCD, and OAS-TL, as well as ZmLCD1 and ZmOAS-TL) and ABA (ZEP, NCED, and AAO, as well as ZmZEP, ZmNCED, and ZmAAO) biosynthesis. The H 2 S-ABA crosstalk induced the thermotolerance in maize seedlings by improving the survival rate (SR) and tissue viability (TV), as well as relieving electrolyte leakage (EL) and MDA accumulation.
Also, the ROS-scavenging system was enhanced by NaHS and ABA alone or in combination via activation of the enzymatic system (i.e., CAT, APX, GR, POD, SOD, DHAR, and MDHAR) and the non-enzymatic system (i.e., AsA, CAR, FLA, and TP), as well as the up-regulation of the gene expression of the corresponding enzymes (i.e., ZmCAT1, ZmSOD4, ZmGR1, ZmAPX1, ZmDHAR, and ZmMDHAR) in maize seedlings before and after HS. These data indicate the essential role of the ROS-scavenging system in H 2 S-ABA crosstalk-induced thermotolerance in maize seedlings, which lays the foundation for breeding a heat-resilient crop variety, and are very important for maize production.