Transcriptome analysis reveals insight into molecular hydrogen-induced cadmium tolerance in alfalfa: the prominent role of sulfur and (homo)glutathione metabolism

Abstract

Background: Hydrogen gas (H₂) is hypothesised to play a role in plants that are coping with stresses by regulating signal transduction and gene expression. Although the beneficial role of H₂ in plant tolerance to cadmium (Cd) has been investigated previously, the corresponding mechanism has not been elucidated. In this report, the transcriptomes of alfalfa seedling roots under Cd and/or hydrogen-rich water (HRW) treatment were first analysed. Then, the sulfur metabolism pathways were focused on and further investigated by pharmacological and genetic approaches.

Results: A total of 1968 differentially expressed genes (DEGs) in alfalfa seedling roots under Cd and/or HRW treatment were identified by RNA-Seq. The DEGs were classified into many clusters, including glutathione (GSH) metabolism, oxidative stress, and ATP-binding cassette (ABC) transporters. The results validated by RT-qPCR showed that the levels of relevant genes involved in sulfur metabolism were enhanced by HRW under Cd treatment, especially the genes involved in (homo)glutathione metabolism. Additional experiments carried out with a glutathione synthesis inhibitor and Arabidopsis thaliana cad2-1 mutant plants suggested the prominent role of glutathione in HRW-induced Cd tolerance. These results were in accordance with the effects of HRW on the contents of (homo)glutathione and (homo)phytochelatins and in alleviating oxidative stress under Cd stress. In addition, the HRW-induced alleviation of Cd toxicity might also be caused by a decrease in available Cd in seedling roots, achieved through ABC transporter-mediated secretion.

Conclusions: Taken together, the results of our study indicate that H₂ regulated the expression of genes relevant to sulfur and glutathione metabolism and enhanced glutathione metabolism which resulted in Cd tolerance by activating antioxidation and Cd chelation. These results may help to elucidate the mechanism governing H₂-induced Cd tolerance in alfalfa.

Keywords: (homo)glutathione; Cadmium; Medicago sativa; Molecular hydrogen; RNA-Seq.

Fig. 1

Differential abundance of transcripts involved in H₂-mediated cadmium (Cd) resistance. Transcriptome of alfalfa seedling roots treated with 100 μM Cd for 12 h, with or without hydrogen-rich water (HRW) pretreatment for 12 h. a Summary of significantly upregulated and downregulated transcripts in the three experimental groups. b Heatmap showing the increasing or decreasing expression of all the differentially abundant transcripts identified in the three groups. c Venn diagram of the differential abundance of transcripts identified in three groups, and the number in brackets indicates the counts of transcripts that exhibited opposite regulatory tendencies

Fig. 2

Clusters of unigenes in the M. sativa transcriptome. a Gene Ontology (GO) terms. The 10 most significantly enriched terms in the level 4 Gene Ontology hierarchy. Information on the percentage and number of involved proteins in a term are shown on the left and right y-axes. b Clusters of enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are arranged into the metabolism subcategories, and the number of involved proteins in a specific pathway and corresponding P value are shown on the right side of column

Fig. 3

Transcriptional changes in genes identified by RNA-Seq and RT-qPCR involved in sulfur and glutathione metabolism in alfalfa seedling roots. Arrows represent enzymatic reactions. Coloured bars near the arrows indicate the relative expression of the corresponding homologous genes in different treatments. Transcripts detected by RT-qPCR are presented relative to the control samples (Con → Con), with the expression being normalized to two internal reference genes in each sample. Abbreviations: APS, adenosine-phosphosulfate; γ-EC, γ-glutamylcysteine; GSH, reduced glutathione; GSSG, oxidized glutathione; PCs, phytochelatins; hGSH, reduced homoglutathione; hGSSGh, oxidized homoglutathione; hPCs, homophytochelatins; OAS, O-acetyl serine; SAM, S-adenosyl-methionine. [1], high affinity sulfate transporter type 1 (Mtr_3g073780 homologue); [2], ATP sulfurylase (Mtr_1g102550 homologue); [3], 5′-adenylylsulfate reductase (Mtr_2g023540 homologue); [4], sulfite reductase [ferredoxin] protein (Mtr_4g077190 homologue); [5], O-acetylserine(thiol)lyase (Mtr_5g006340 homologue); [6], glutamate-cysteine ligase B (Mtr_8g098350 homologue); [7], glutathione synthetase (Mtr_7g113890 homologue); [8], phytochelatin synthase (Mtr_7g097190 homologue); [9], homoglutathione synthetase (Mtr_7g113880 homologue); [10], glutathione S-transferase (Mtr_2g070070 homologue); [11], glutathione reductase (Mtr_6g033515 homologue); [12], NADP-dependent isocitrate dehydrogenase (Mtr_2g062840 homologue); [13], decarboxylating-like 6-phosphogluconate dehydrogenase (Mtr_7g017900 homologue); [14], glucose-6-phosphate 1-dehydrogenase (Mtr_7g111760 homologue); [15],serine acetyltransferase (Mtr_3g058410 homologue); [16], cystathionine γ-synthase (Mtr_7g011230 homologue); [17], cystathionine beta-lyase (Mtr_1g064320 homologue); [18], homocysteine S-methyltransferase (Mtr_1g103290 homologue); [19], S-adenosylmethionine synthase (Mtr_2g046710 homologue); [20], nicotianamine synthase (Mtr_1g084050 homologue)

Fig. 4

Effects of H₂, _L-buthionine-S,R-sulfoximine (BSO), and reduced glutathione (GSH) on the Cd-induced inhibition of alfalfa seedling growth and fresh weight (a), root elongation (b), and thiobarbituric acid-reactive substance (TBARS) content (c) in alfalfa roots. Five-day-old seedlings were pretreated with or without HRW, 500 μM BSO, and 1 mM GSH individually or in combination for 12 h followed by another 72 h (a and b) and 24 h (c) treatment with 100 μM CdCl₂. Data in the top and bottom of part (a) indicate the fresh weight of 30 seedlings above ground and underground, respectively. Bar = 2 cm. Values are the mean ± SE of three independent experiments with at least three replicates for each. Bars with different letters indicate significant differences (P < 0.05) according to Duncan’s multiple range test

Fig. 5

Effects of H₂, BSO, GSH, and Cd on lipid peroxidation (a), loss of plasma membrane integrity (b), localization of reactive oxygen species (ROS) (c), and the activity of peroxidase (POD) (d), superoxide dismutase (SOD) (e), ascorbate peroxidase (APX) (f), and catalase (CAT) (g) in alfalfa seedling roots. Five-day-old seedlings were pretreated with or without HRW, 500 μM BSO, and 1 mM GSH individually or in combination for 12 h followed by another 24 h treatment with 100 μM CdCl₂. Bar = 1 mm. Values are the mean ± SE of three independent experiments with at least three replicates for each. Bars with different letters indicate significant differences (P < 0.05) according to Duncan’s multiple range test

Fig. 6

Effects of H₂, BSO, GSH, and Cd on the mass spectra of cysteine (b), γ-glutamylcysteine (γ-EC) (c), GSH (d), homoglutathione (hGSH) (e), PC2 (f), hPC2 (g), PC3 (h), and hPC3 (i) with MBBR in alfalfa seedling roots. Five-day-old seedlings were pretreated with or without HRW, 500 μM BSO, and 1 mM GSH individually or in combination for 12 h followed by another 12-h treatment with 100 μM CdCl₂. a Mass spectra of biothiols in the m/z ranges. ND, not detected. Values are the mean ± SE of three independent experiments with two replicates for each. Bars with different letters indicate significant differences (P < 0.05) according to Duncan’s multiple range test

Fig. 7

Content of Cd and the transcripts of ABC transporter genes in alfalfa seedling roots. Five-day-old seedlings were pretreated with or without HRW for 12 h followed by another 12 h (b-f) or 72 h (a) treatment with 100 μM CdCl₂. The Cd content in alfalfa seedling roots was determined after 72 h of Cd exposure (a). Relative expression of the ABC transporter gene homologues of Mtr_1g086080 (b), Mtr_4g077930 (c), Mtr_4g124040 (d), Mtr_6g008800 (e), and Mtr_6g008820 (f) was detected by RT-qPCR. The sample without chemicals was the control (Con). Expression levels of corresponding genes are presented relative to the control samples and were normalized to the expression of two internal reference genes in each sample. Values are the mean ± SE of three independent experiments with at least three replicates for each. Bars with different letters indicate significant differences (P < 0.05) according to Duncan’s multiple range test