Unearthing The Alleviatory Mechanisms of Hydrogen Sulde in Aluminum Toxicity in Rice

Hydrogen sulde (H 2 S) improves aluminum (Al) resistance in rice; however, the underlying molecular mechanism remains unclear. In the present study, treatment with 30-μM Al signicantly inhibited rice root growth and increased the total Al content and apoplastic and cytoplasm Al concentration in the rice roots. However, pretreatment with NaHS (H 2 S donor) reversed these negative effects. Transcriptomics and physiological experiments conrmed that H 2 S increased the ATP, sucrose, glutathione, and ascorbic acid contents, which was accompanied by decreased O 2·- and H 2 O 2 contents, to alleviate Al toxicity. H 2 S signicantly inhibited ethylene emissions in the rice and then inhibited pectin synthesis and increased the pectin methylation degree to reduce cell wall Al deposition. The phytohormones indole-3-acetic and brassinolide were also involved in the alleviation of Al toxicity by H 2 S. In addition, other pathways of material and energy metabolism, secondary metabolism, cell wall components, signal transduction, and transcriptional and translational pathways in the rice roots were also regulated by H 2 S under Al toxicity conditions. These ndings improve our understanding of how H 2 S affects rice responses to Al toxicity, which will facilitate further studies on crop safety.


Introduction
Hydrogen sul de (H 2 S) is an important endogenous gasotransmitter that maintains a dynamic equilibrium with Lcysteine desulfhydrase, D-cysteine desulfhydrase, sulphite reductase, cyanoalanine synthase, cysteine synthase, and Oacetyl-l-serine(thiol)lyase in plants (Banerjee et al., 2018;Li, 2013;Sirko et al., 2004;Tai and Cook, 2000). Although a high H 2 S concentration is harmful to plant growth, the appropriate H 2 S concentrations acts as signaling molecule to regulate plant development and the response to environmental stress (Yamasaki and Cohen, 2016). For example, H 2 S induces protein persul dation to protect plants from oxidative damage (Filipovic, 2015), maintains a higher K + /Na + ratio to ameliorate salt stress (Wang et al., 2012), reduces peroxidation damage to promote wheat seed germination under drought stress (Zhang et al., 2010c), and reduces electrolyte leakage in tobacco (Nicotiana tabacum L.) suspensioncultured cells to alleviate heat stress (Li et al., 2012b).  Zhang et al., 2008). The application of NaHS signi cantly enhanced the AsA-GSH cycle to alleviate As toxicity in pea seedlings (Singh et al., 2015), promoted the protoplast sequestering of Cd in Arabidopsis (Guan et al., 2018), boosted photosynthesis to alleviate Ni toxicity in rice, and reduced peroxidation damage to alleviate Cr, Cu, and Zn toxicity (Rizwan et al., 2019). About 30%-50% of arable land in the world is acidic and has limited crop production capacity due to Al toxicity (He et al., 2012). alleviates Al toxicity has mainly focused on the physiological level, while the molecular mechanisms remain unclear.
Recently, transcriptomics has been used to explore Al response genes in plants; for instance, the possible transporter genes involved in Al resistance in buckwheat leaves were found following treatment with short-term moderate Al stress (Yokosho et al., 2014). In the present study, seedlings of the rice cultivar "Kasalath" were pretreated with or without the H 2 S donor NaHS under Al toxicity conditions for one day, and the roots were collected for genome-wide transcriptome and physiological analysis.

Materials And Methods
The seeds of the indica rice "Kasalath" were soaked in distilled water for 24 h and then transferred to 0.5-mM CaCl 2 solution (pH 5.6) at 30°C under total darkness. The rice seedlings were treated with or without 2 μM of the H 2 S donor NaHS in 0.5-mM CaCl 2 solution (pH 5.6) until the rice roots reached about 1 cm. After 8 h of treatment, the solution was discarded, and the roots were treated with or without 30-μM Al (AlCl 3 ·6H 2 O) in 0.5-mM CaCl 2 solution (pH 4.5). The treatments were set as CK (without NaHS and Al treatment), H 2 S (only treated with NaHS), Al (only treated with Al), and Al+H 2 S (treated with NaHS and Al). After 24 h of treatment, the fresh roots were collected and frozen in liquid nitrogen immediately. The root lengths were measured before and after Al treatment.

Measurement of Al content in rice
The fresh roots were collected and dried in the oven to the point where the weight no longer changed. The dry roots (0.1 g) were digested in 2 mL of HNO 3 :HClO 4 (v:v, 4:1) at 120°C and then diluted to 50 mL with ultrapure water to measure the total Al content (Shen et al., 2002). The rice root tips (1 cm) were then placed into 1.5-mL ultra-free MC tubes (Millipore, Billerica, MA, USA) and stored at −80°C for 1 d, following which they were heated at 25°C to break the cells. The residues after centrifugation were used to extract an apoplastic solution with 1 mL of 2-M HCl. The Al concentrations were measured by inductively coupled plasma mass spectrometry (ICP-MS).
The cytoplasmic Al content was indicated by the intensity of the green uorescence after staining with morin. One centimeter of the rice root tip was collected and stained in 0.01% morin for 30 min at room temperature, and the extra dye was washed off using deionized water. Images were captured using a uorescence microscope (LEICA DM2500) (Li et al., 2016).

Transcriptome library construction, sequencing, and bioinformatics analysis
The total RNA was extracted by TRIzol reagent (Invitrogen, Germany) according to the manufacturer's instructions. The RNA, which met the experimental requirements, was enriched by Oligo(dT) beads and reverse transcribed to cDNA immediately after fragmenting into short fragments. The synthesis of second-strand cDNA, puri cation of cDNA fragments, ampli cation of cDNA, and sequencing by Illumina HiSeq TM 2500 were completed by Gene Denovo Biotechnology Co. (Guangzhou, China) according to Yang et al. (2021). Reads were mapped to the ribosome RNA (rRNA) database by Bowtie2 (Langmead and Salzberg, 2012). TopHat2 was used to map the rRNA removed reads (version 2.0.3.12) (Kim et al., 2013a). Unmapped reads were then re-aligned with Bowtie2 and split into smaller segments (Trapnell et al., 2010). The reconstruction of transcripts was carried out with the Cu inks software (Trapnell et al., 2012), which together with TopHat2 allowed for the identi cation of new genes and new splice variants of known genes.
The RSEM software was used to quantify gene abundances (Li and Dewey, 2011), and the FPKM (Fragments Per Kilobase of transcript per Million mapped reads) method was used to normalize the gene expression levels. To identify differentially expressed genes (DEGs) across samples or groups, the edgeR package (http://www.rproject.org/) was used.
We identi ed genes with a fold change log 2 (fc) ≥ 1 and P < 0.05 in a comparison as a signi cant DEG. The Gene Ontology (GO) annotation (https://www.ebi.ac.uk/GOA/) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (https://www.kegg.jp/kegg/pathway.html) were used to analyze gene functions and enrichment.

Measurement of sucrose and ATP content
Fresh rice roots (0.1 g) were ground with 1 mL of 0.83-M cold perchloric acid (Sigma Aldrich, St. Louis, USA) on ice, and the supernatant was adjusted to a neutral pH with 3-M KOH. The BacTiter-Glo Microbial Cell Viability Assay (Promega, Madison, USA) was used to determine the ATP concentration in a white opaque-walled 96-well microplate (Greiner Bio-One, Frickenhausen, Germany) (Köpnick et al., 2018).
The rice roots were dried in the oven and ground into powder. The sucrose was extracted with 80% ethanol and measured using the resorcinol hydrochloric acid method (Li et al., 2012a).

Measurement of pectin demethylesteri cation degree
The extraction method of pectin and the concentration of pectin and methanol in the solution were in accordance with a previous study. The degree of pectin demethylesteri cation was calculated as (1−methanol concentration/pectin concentration) × 100% (Zhu et al., 2020).

Phytohormone measurement
Ethylene emissions: about 2 g of fresh roots were incubated in 20-mL glass vials sealed with a silica gel plug at 30°C for 2 h to collect the ethylene released from the roots. Then, 10 mL of incubated gas was injected into a gas chromatograph The ascorbic acid was extracted in 15% metaphosphoric acid from 0.1 g of fresh rice roots and measured using a highperformance liquid chromatography (HPLC; Nanospace SI-1, Shiseido, Japan) system equipped with a PDA Detector The content of O 2 ·was indicated by the intensity of red uorescence after staining with dihydroethidium according to the manufacturer's instructions (S0063, Beyotime, China). The 1-cm rice root tips were collected and stained in 1-μM dihydroethidium for 30 min at room temperature, and the extra dye was washed off using deionized water. The uorescence was captured at an emission wavelength of 300 nm and an excitation wavelength of 535 nm.  Table S1. The relative expression of selected genes was calculated according to a previous study (Livak and Schmittgen, 2001).

Western blot
The total protein was extracted, and the Bradford method was used to determine the protein content (Bradford, 1976).

Statistical analysis
All experiments in the present study were performed with three independent biological replicates. A one-way ANOVA was used to analyze the data, and a post hoc Tukey's test was used to compare the mean values at P < 0.05 (SPSS 13.0). The R program (version 3.0.0; R Development Core Team) with the vegan package was used to perform multivariate analyses.

Effect of H 2 S on rice growth under Al toxicity conditions
The Al treatment signi cantly inhibited the elongation of the rice roots, ampli ed the total Al content in the rice roots and shoots, and increased the Al concentration in the rice apoplast and cytoplasm ( Fig. 1). However, pretreatment with NaHS (H 2 S donor) signi cantly reversed the inhibition of root growth induced by Al toxicity and also decreased the total Al content and the Al concentration in the rice apoplast and cytoplasm under Al conditions ( Fig. 1), indicating that H 2 S is involved in alleviating Al toxicity in rice.

Identi cation of DEGs by transcriptome sequencing
The results of the clean reads, Pearson's correlation coe cient analysis, principal components analysis (PCA), and hierarchical clustering analysis for each sample are provided in the supplementary material (Table S1- The Venn diagram indicated that 385, 584, and 236 genes were only differentially expressed in the Al-S/Al, Al/CK, and S/CK groups, 27 DEGs co-existed in the Al-S/Al and S/CK groups, 98 DEGs co-existed in the Al/CK and Al-S/Al groups, 103 DEGs co-existed in the Al/CK and S/CK groups, and 26 DEGs co-existed in three groups (Fig. 2B).
The GO classi cation in the biological process indicated that the top three DEGs belonged to metabolic process, cellular process, and single-organism process. For the molecular function category, the top three genes belonged to binding fractions, catalytic activity, and nucleic acid binding transcription factor activity. In addition, cellular component analysis found that the locations of the top three genes were the membrane, cell, and cell part (Fig. 3A).
The KEGG analysis also showed that most of the DEGs were involved in metabolism, such as carbohydrate metabolism, energy metabolism, and biosynthesis of other secondary metabolism. The other pathways included genetic information processing, environmental information processing, and cellular processes (Fig. 3B).
H 2 S improved energy production and the antioxidant system in rice The Al treatment signi cantly decreased the sucrose content, ATP content, and the protein abundance of ATP synthase in the rice roots compared with CK. However, pretreatment with NaHS signi cantly reversed these negative effects induced by Al toxicity (Fig. 4).
The Al treatment also affected the antioxidant system in the rice roots. GSH and AsA, which are involved in alleviating peroxidation damage, were induced by Al toxicity and further increased after applying NaHS to the rice roots. In addition, the O 2 ·and H 2 O 2 content, indicated by the intensity of the red and green uorescence, signi cantly increased under Al toxicity conditions and decreased following pretreatment with NaHS under Al toxicity conditions (Fig. 4). The protein abundance of APXs (sAPX and pAPX) decreased under Al toxicity conditions and increased following pretreatment with NaHS under Al toxicity (Fig. 4), suggesting that H 2 S reduced the peroxidation stress induced by Al toxicity.  5). A single application of the ethylene synthesis inhibitor AVG alone or in combination with NaHS signi cantly improved rice root growth, decreased the Al content in the rice roots and cell walls, and decreased the pectin content and pectin methylation degree; however, the application of the ethylene synthesis precursor ACC had an opposite tendency and even negated the positive role of NaHS in alleviating Al toxicity (Fig. 5).
In addition, the content of IAA and BL in the rice roots decreased under Al toxicity conditions and increased after pretreatment with NaHS under Al toxicity, and the exogenous application of IAA and BL both decreased the rice root Al content (Fig. S4).         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 signi cantly increased (Fig. 4 I), further con rming that H 2 S alleviates peroxidation damage by reducing peroxides.  Table 1). In addition, a previous study found that ethylene stimulates pectin synthesis in rice, and thus there is a hypothesis that H 2 S 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 signi cantly 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 esteri cation, whereas the ethylene synthesis precursor ACC had an opposite tendency and even negated the positive role of H 2 S in alleviating Al toxicity once applied with NaHS (Fig. 5). ). 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 con rmed that H 2 S cross-talks with IAA and BL to alleviate Al toxicity in rice.

H 2 S 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).  H 2 S 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 Cd 2+ from the roots to shoots . In the present study, the expression of heavy-metal-associated domain-containing protein (No. 59) signi cantly decreased in the Al+S/Al set. This was concurrent with a decreasing shoot-root-Al-content ratio (Fig. 1E), indicating that H 2 S not only decreased the Al uptake but also inhibited its transportation from the roots to the shoots.

Conclusion
As show in Fig. 9, H 2 S improved energy production, reduced peroxidation damage, and decreased Al binding in the cell membrane to alleviate Al toxicity. In addition, H 2 S inhibited ethylene emissions to reduce pectin content and improved the pectin methylation degree to decrease cell wall Al deposition. The phytohormones IAA and BL were also found to be

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Availability of supporting data
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any quali ed researcher.

Competing interests
The authors declare that they have no competing interests.  There were three independent biological replicates for the transcriptomics analysis. The differentially expressed genes that changed more than log 2 (fc) ≥ 1 and passed Student's t-test (P ≤ 0.05) were de ned as signi cantly different. The Al stress concentration in the solution was 30 μM, the NaHS concentration was 2 μM. CK: without Al and NaHS; Al: single Al treatment; S: single NaHS treatment; Al+S: both Al and NaHS treatment. NC: no change.