Comparative Transcriptome Analysis of Cadmium Stress Response Induced by Exogenous Sulfur in Tartary Buckwheat

doi:10.13560/j.cnki.biotech.bull.1985.2022-0951

Abstract

Abstract:

Excessive Cd（cadmium）content is the main heavy metal pollution to tartary buckwheat（Fagopyrum tararicum）. To investigate the molecular mechanism of adding exogenous S on the Cd content of tartary buckwheat, differentially expressed genes in the tartary buckwheat leaves and their functions were analyzed via comparative transcriptome while adding S（100 mg/kg（NH₄）₂SO₄）on tartary buckwheat root and Cd（5 mg/kg CdCl₂）treatment. A total of 3 93 983 500 clean sequences were obtained and 19 875 expressed genes were identified, and 1 760 differentially expressed genes were obtained under different treatments. The analysis of these differentially expressed genes by GO annotation and KEGG pathway demonstrated that the exogenous S enhanced glutathione pathway, inhibited oxidoreduction process, induced the responses to salicylic acid, abscisic acid response and auxin response pathway to Cd stress. Transcriptome data identified that tartary buckwheat FtbHLH68 was specifically induced by Cd+S treatment, and the Arabidopsis thaliana’s homologous gene mutant Atbhlh68 presented a deficient phenotype under Cd+S treatment, and changes in chlorophyll content, net photosynthesis, GR activity and GSH activity in Atbhlh68 did not respond to Cd+S treatment. The results revealed the transcriptional level regulation process of tartary buckwheat in response to Cd stress, and clarified the molecular mechanism of exogenous addition of S enhancing plant resistances to Cd stress, which provides theoretical basis and target for application of exogenous S in Cd resistance of tartary buckwheat.

Key words: cadmium stress, exogenous sulfur, tartary buckwheat, comparative transcriptome, verification of gene function, oxidoreduction pathway, abscisic acid, auxin

LIU Hui, LU Yang, YE Xi-miao, ZHOU Shuai, LI Jun, TANG Jian-bo, CHEN En-fa. Comparative Transcriptome Analysis of Cadmium Stress Response Induced by Exogenous Sulfur in Tartary Buckwheat[J]. Biotechnology Bulletin, 2023, 39(5): 177-191.

Figures/Tables 8

Table 1 Primers for RT-qPCR

Gene	Primer-F（5'-3'）	Primer-R（5'-3'）
FtPinG0001636600.01	CAAGCGCTTTCCAAGATCGG	GCACTCATGACTTCGTTCGC
FtPinG0007992900.01	TGTTGTGTGTCCCACTCTGT	TTGGCTTGAGTCCCATGCTC
FtPinG0004475100.01	GGCTTGATATTGCTGCGGTG	TTGCCAGCTCCTGCTTAGAC
FtPinG0002599400.01	CACTTCTCCTCGCTAACGCT	CAATGGGTGAGTGGCGTAGT
FtPinG0005827500.01	GTTCGGGAAGAAGTACGGGG	CCAGCTAACCCAACCTAGCC
FtPinG0000987900.01	AAGTCCGTGGAGCAACTGAG	TGTGACTTCCTCCATGACGC
FtPinG0002633300.01	AAGACCGCAGCGTACTGAAA	TCTCAAAGGCGCACAGCTTA
FtPinG0008441500.01	GCCTTGGAGTGTCGACAAGA	TCCCTAACCCGAATGCACAC
FtPinG0001390600.01	GCTGGCGAGGAATAATTGCG	GCGCAATCCTTGCACTTGAA
FtPinG0007214300.01	AGGCATCACCAACATGGGAG	CTTGTGTGCTGAAAGTGCCC
FtPinG0002002200.01	TCACGAGGCTGGTGTTTTGA	ATCTAGTGGCAACCGCGTAG
FtPinG0000414800.01	GGGGAGAGCTCCATGTTGTG	TCCATGGCCGAATTGGTGAA

Table 2 Statistical table of sample analysis results

样品 Sample	原始读取序列 Raw reads/bp	干净读取序列总数 Total clean reads/bp	总序列中质量值大于30的碱基数的比例 Clean Q30 bases rate/%	比对到参考基因组序列比例 Mapping rate/%	检测到表达的基因数目 Total gene
CK1	41 740 092	40 412 240	91.84	67.42	19 224
CK2	46 423 990	45 073 886	92.23	68.31	19 362
CK3	45 942 460	43 884 754	92.46	64.74	18 854
Cd1	41 384 496	40 064 950	92.9	92.22	19 109
Cd2	43 464 826	42 209 070	92.14	81.49	19 593
Cd3	46 953 848	45 429 048	92.14	85.88	19 560
Cd+S1	45 970 716	44 405 584	92.48	78.95	19 875
Cd+S2	48 106 694	46 437 132	92.12	83.07	19 593
Cd+S3	48 272 452	46 066 836	92.27	78.48	19 614

Fig. 1 Transcriptome analysis of Cd treatment and Cd+S treatment in Qianku 4's leaves A: Transcriptome heat map in Cd treatment and Cd+S treatment. B: Statistics of the number of differentially expressed genes in Cd treatment and Cd+S treatment. C: Wayne diagram of differentially expressed genes under Cd treatment and Cd+S treatment

Fig. 2 GO analysis and KEGG analysis of differentially expressed genes under Cd treatment A-C: GO analysis of up-regulated genes under Cd treatment. A: Biological processes. B: Cellular components. C: Molecular function. D: KEGG analysis of up-regulated gene under Cd treatment. E-G: GO analysis of down-regulated genes under Cd treatment. E: Biological processes. F: Cell components. G: Molecular function. H: KEGG analysis of down-regulated gene under Cd treatment

Fig. 3 GO analysis and KEGG analysis of differentially expressed genes under Cd+S treatment A-C: GO analysis of up regulated genes under Cd+S treatment. A: Biological processes. B: Cellular components. C: Molecular function. D: KEGG analysis of up regulated gene under Cd+S treatment. E-G: GO analysis of down regulated genes under Cd+S treatment. E: Biological processes. F: Cell components. G: Molecular function. H: KEGG analysis of down regulated gene under Cd+S treatment

Fig. 4 GO analysis and KEGG analysis of differentially expressed genes in CD+S treatment compared with CD treatment A-C: GO analysis of up-regulated genes under Cd+S treatment. A: Biological processes. B: Cellular components. C: Molecular function. D: KEGG analysis of up-regulated gene under Cd+S treatment. E-G: GO analysis of down-regulated genes under Cd+S treatment. E: Biological processes. F: Cell components. G: Molecular function. H: KEGG analysis of down-regulated gene under Cd+S treatment

Fig. 5 Effects of exogenous S addition on transcriptional levels in different pathways of Cd treatment A: Response to cadmium ion. B: Oxidation-reduction process. C: Glutaredoxin activity. D: Response to salicylic acid. E: Response to abscisic acid. F: Response to auxin.G: Real-time quantitative PCR validation of several selected genes response to Cd stress and exogenous sulfur

Fig. 6 Functional verification of tartary buckwheat FtbHLH68 gene A: Analysis of FtbHLH68 gene expression in tartary buckwheat. B-C: Phenotype (B) and green leaves rate (C) of wild-type Col-0 and bhlh68 mutants under Cd stress and Cd+S treatment. D-E: Root length phenotype (D) and statistical results (E) of wild-type Col-0 and bhlh68 mutants under Cd stress and Cd+S treatment. F-I: Chlorophyll content (F), net photosynthesis (G), GR activity (H) and GSH activity (I) of wild-type Col-0 and bhlh68 mutants under Cd stress and Cd+S treatment. * indicate significant difference at 0.05 level

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