H2S regulated material metabolism to alleviate Al toxicity
The Al stress significantly affected material metabolism in the plants, and exogenously applied signaling material alleviated the Al toxicity by regulating plant material metabolism. In our study, pretreatment with the H2S donor NaHS induced a series of changes to material metabolism genes, including carbon metabolism (Nos. 1, 2), protein metabolism (Nos. 3, 4, 5), lipid metabolism (Nos. 6, 7, 8, 9, 10), energy production (Nos. 26, 27), and secondary metabolism (Nos. 56, 57, 58) (Table 1). Sucrose is responsible for energy supply and also acts as a signaling molecule in plants (Kühn and Grof, 2010). Producing enough sucrose and transporting sucrose to the right site is pivotal for plants to resist stress. A previous study demonstrated that sucrose transporters are involved in orchestrating sucrose allocation both intracellularly and at the whole plant level (Kühn and Grof 2010). In our study, the gene expression of the sucrose transporter (No. 2, Table 1) significantly decreased in the Al/CK set and increased in the Al+S/Al set, and this was accompanied by an increased sucrose content when the rice roots were pretreated with NaHS under Al toxicity conditions (Fig. 4B), suggesting that H2S not only stimulates the production of sucrose but is also involved in regulating sucrose allocation to resist Al toxicity. ATP is the direct energy source for plant development, and most of the ATP in cells is produced in the mitochondria. Thus, maintaining the integrity and normal functioning of the mitochondria is important for producing enough energy in organisms (Harris and Das 1991). The GCN5-like protein 1 is responsible for the regulation of mitochondrial biogenesis and mitophagy (Scott et al. 2014). Kinesin superfamily proteins play an important role in mitochondrial and lysosomal dispersion. In our study, the expression of GCN5-like 1 domain containing protein (No. 26) and kinesin heavy chain (No. 27) genes significantly increased in the Al+S/Al set (Table 1), and it was also accompanied by increased ATP content and ATP synthase abundance in the rice roots once pretreated with NaHS under Al toxicity conditions (Figs. 4 A and I). This indicated that H2S maintained sufficient ATP in the rice roots to help the rice resist Al toxicity.
F-box proteins are responsible for catalyzing protein ubiquitination and maintaining the balance between protein synthesis and degradation (Boycheva et al., 2015); therefore, the increased expression of cyclin-like F-box domain containing protein (No. 3) in the Al+S/Al set in our study (Table 1) indicated that H2S regulated the protein synthesis under Al toxicity. Previous studies have demonstrated that the carboxyl in the cell wall and phosphate in the plasma membrane are the main sites that bind with Al3+ and aggravate Al toxicity in plants (Kochian et al., 2005). Non-specific lipid transfer proteins (nsLTPs) are located in the plant cell wall and respond to the transfer of phospholipids (Thoma et al., 1993; Zhang et al., 2019). 3-Ketoacyl-coenzyme A synthase (KCS) and β-ketoacyl-CoA synthase are involved in the synthesis of long chain fatty acids in plants, including sphingolipids and phospholipids (Kim et al., 2013b; Li et al., 2018). In the present study, the expression of nonspecific lipid-transfer protein 3 (Nos. 6–8), 3-ketoacyl-CoA synthase (No. 9), and beta-ketoacyl-CoA synthase (No. 10) significantly decreased in the Al+S/Al set (Table 1), indicating that H2S might decrease the transfer and synthesis of phospholipids to inhibit Al accumulation and then alleviate Al toxicity in rice.
Flavonoids are secondary metabolites present in higher plants that play an important role in plant development and abiotic and biotic stress resistance (Mo et al., 1992; Stapleton and Walbot, 1994). Flavanone 3-hydroxylase catalyzes the formation of dihydroflavonols from flavanones, therefore providing precursors for many classes of flavonoid compounds (Pelletier and Shirley, 1996). MYBL2 is a negative regulator of flavonoid synthesis in Arabidopsis (Dubos et al., 2008), and OsWD40 family genes are co-expressed with MYB factors to initiate their diverse functions (Ouyang et al., 2012). In the present study, the expression of flavanone 3-hydroxylase 3 (No. 56) significantly increased; however, the expression of both MYBL2 (No. 57) and OsWD40-77 (No. 58) significantly decreased in the Al+S/Al set (Table 1), indicating that H2S increased flavonoid synthesis to improve rice Al toxicity resistance.
H2S alleviates Al toxicity by reducing cell wall deposition
Cell walls are the main site for Al deposition, and the removal of Al from the cell wall significantly improves plant growth. Pectin and hemicellulose are the major Al deposition sites in plant cell walls; for example, about 76% of Al in the tobacco cell wall is in the pectin (Chang et al., 1999) and most of cell wall Al in Arabidopsis roots is in the hemicellulose fraction (Yang et al., 2011). In our study, five DEGs (Nos. 11–15) associated with polysaccharide synthesis and construction modification were identified (Table 1). Among them, pectinesterase inhibitor is involved in blocking the process of demethylation in plants and thus reduces the negative charges in pectin in plants to decrease the binding of Al3+ (Van Beusichem et al., 1988). Glycoside hydrolase family proteins are involved in hemicellulose breakdown (Langston et al., 2011). Xylanase is responsible for catalyzing the degradation of the hemicellulose main composition material xylan and the glycoside hydrolase family 18-member xylanase inhibitor protein I (XIP-I), which are responsible for inhibiting the activity of xylanase (Anne et al., 2005; Jensen et al., 2018). Arabinogalactan-proteins (AGPs) participate in cell wall polysaccharide synthesis; for example, the mutant of agp30 in Arabidopsis exhibited significantly decreased pectin content (Hengel and Roberts, 2003). In the present study, the expression of the pectinesterase inhibitor domain containing protein (No. 11) and glycoside hydrolase (No. 12) increased, while the expression of glycoside hydrolase family 18 (No. 13) and arabinogalactan protein 1 and 2 (Nos. 14, 15) decreased, following the application of NaHS under Al toxicity conditions (Table 1), which indicated that H2S reduced the cell wall polysaccharide content to reduce cell wall Al deposition. Furthermore, we also found that two wall-associated kinase (WAK) genes (Nos. 18, 19), which are related to cell growth (Kim and Guerinot, 2007; Walker and Connolly, 2008), significantly increased after the application of NaHS under Al toxicity conditions (Table 1). This implies that H2S improved the expression of the WAK gene to improve rice root growth under Al toxicity conditions.
H2S alleviates the peroxidation damage induced by Al toxicity
Al toxicity significantly increased the H2O2 and O2·- content in the rice roots (Figs. 4 E-G), suggesting that Al induced peroxidation damage in rice. To remove the excess oxidizing substances, the content of the most important antioxidants in rice roots, namely GSH and AsA (Jones, 2002), both significantly increased after NaHS application (Figs 4 C and D). In addition, enzymes belonging to the antioxidant system were also identified in the present study. Among them, germins and germin-like proteins (GLPs) possess the enzyme functions of superoxide dismutase (SOD) to produce H2O2 and play a pivotal role in plant development and defense, such as by improving blast disease resistance in rice (Manosalva et al., 2009). Thioredoxins are major cellular protein disulfide reductases that play a role in H2O2 removal (Chae et al., 1999). Peroxisomes are simple organelles present in most organisms and play an important role in ROS metabolism (Corpas et al., 2017). Ascorbate oxidase (AO) is responsible for converting ascorbate to monodehydroascorbate and reducing oxygen to water at the same time (Stevens et al., 2017). In our study, the expression of germin-like proteins (Nos. 28–34), thioredoxin domain 2 containing protein (No. 35), peroxisomal biogenesis factor 19 (No. 36), and L-ascorbate oxidase precursor (No. 39) all increased in the Al+S/Al set (Table1). However, the expression of benzothiadiazole-induced protein (No. 37), which participates in inhibiting CAT and APX to increase H2O2 content in plants (Iriti et al., 2003), decreased (Table 1). In addition, although the protein content of CAT did not change before or after the application of NaHS, the protein contents of sAPX and tAPX both significantly increased (Fig. 4 I), further confirming that H2S alleviates peroxidation damage by reducing peroxides.
The phytohormones involved in H2S alleviation of Al toxicity
In addition, H2S often interacts with other signaling molecules to regulate the plant response to various stresses (Jin and Pei, 2015), such as nitric oxide, salicylic acid, abscisic acid, and Ca2+ (Fang et al., 2014; Li et al., 2015; Peng et al., 2016; Jin et al., 2013; Wang et al., 2018). In our study, we also identified some genes involved in the regulation of phytohormone synthesis, metabolism, and signal transduction. Ethylene emissions are induced by Al toxicity and aggravate Al toxicity in Lotus japonicus L. and Arabidopsis (Sun et al., 2007; Sun et al., 2010). The ACC oxidase is responsible for the final step of ethylene production, which catalyzes 1-aminocyclopropane carboxylic acid (ACC) to ethylene (Kende, 1993). GDSL lipase-like 1 regulates systemic resistance and is required for ethylene signaling in plants (Kwon et al., 2009). In the present study, ethylene emissions and the related gene ACC oxidase (No. 23) decreased in the Al+S/Al set and therefore decreased the expression of lipase and GDSL domain-containing protein (Nos. 24, 25) (Table 1). In addition, a previous study found that ethylene stimulates pectin synthesis in rice, and thus there is a hypothesis that H2S decreases ethylene synthesis to reduce the pectin content, which then reduces the Al content in rice roots. In the present study, the application of the ethylene synthesis inhibitor AVG alone or together with NaHS significantly increased rice root growth, decreased cell wall Al and total Al content in the rice roots, and inhibited the degree of pectin synthesis and demethylation esterification, whereas the ethylene synthesis precursor ACC had an opposite tendency and even negated the positive role of H2S in alleviating Al toxicity once applied with NaHS (Fig. 5).
Furthermore, the expression of some genes related to IAA (Nos. 21, 40–42) and BL (No. 43) also increased after pretreatment with H2S under Al toxicity conditions, including tryptophan decarboxylase, which is responsible for catalyzing the first step of IAA synthesis that transfers tryptophan to tryptamine (Majerus et al. 2009) (Table 1). The small auxin-up RNA (SAUR) genes are early auxin-responsive genes that play a role in auxin-mediated cell elongation (Longnecker and Welch, 1990). Brassinosteroid insensitive 1 (BRI1)-Associated Kinase I (BAK1) is one of the key components in the brassinosteroid signal transduction pathway and is involved in regulating rice growth and development (Huang et al., 2012; Lei et al., 2014). In addition, the content of IAA and BL both increased in the rice roots following pretreatment with NaHS under Al toxicity conditions and was accompanied by a decrease in the Al content in the rice root tips after exogenous application of IAA and BL under Al toxicity (Fig. S4). This further confirmed that H2S cross-talks with IAA and BL to alleviate Al toxicity in rice.
H2S regulated transcriptional and translational pathways to alleviate Al toxicity
Transcriptional and translational pathways that control the expression of stress-responsive genes are pivotal for the plant response to various stresses (Romheld and Marschner, 1986). Eukaryotic translation initiation factor 2 is involved in the initiation of polypeptide chain synthesis (Bienfait et al., 1985). Toll-interleukin-1 receptor domain (TIR) is associated with the response of growth factors in plants (Guerinot and Yi, 1994; Mori, 1999). Apetala2 (AP2) transcription factor genes belong to the ethylene response factor (ERF) subfamily (Jofuku et al., 1994; Ohme-Takagi and Shinshi, 1995). AP2/ERF family transcription factors, including the subfamily of dehydration-responsive element, play an important role in seed development (Boutilier et al., 2002) and response to environmental stress (Zhu et al., 2010). In the present study, the expression of eukaryotic translation initiation factor 2 (No. 48), toll-interleukin receptor domain containing protein (No. 49), AP2/ERF transcription factor (Nos. 50, 51), dehydration-responsive element (Nos. 52, 53), ethylene-responsive transcription factor 2 (No. 54), and ERF domain containing protein (No. 55) all significantly increased in the Al+S/Al set (Table 1), indicating that H2S stimulates ethylene response transcription factors to increase Al resistance ability.
H2S inhibited the transport of Al from the roots to the shoots by mediating genes related to ion uptake and transport
Heavy metal-associated (HMA) proteins are involved in heavy metal uptake and internal transportation in plants; for example, OsHMA2 localizes to root pericycle cells and is associated with the transportation of heavy metal Cd2+ from the roots to shoots (Yamaji et al., 2013). In the present study, the expression of heavy-metal-associated domain-containing protein (No. 59) significantly decreased in the Al+S/Al set. This was concurrent with a decreasing shoot–root-Al-content ratio (Fig. 1E), indicating that H2S not only decreased the Al uptake but also inhibited its transportation from the roots to the shoots.