Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (16): 3127-3141.doi: 10.3864/j.issn.0578-1752.2024.16.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Function Analysis of bHLH93 Transcription Factor in Tartary Buckwheat in Response to Aluminum Stress

LIU Tong1,2(), WANG ZhiRong2, LI Wei2, LIU Yang2, WANG XiangRu2, LAI DiLi2, HE YuQi2, ZHANG KaiXuan2, ZHAO ZhenJun1(), ZHOU MeiLiang2()   

  1. 1 College of Life Science, Yantai University, Yantai 264000, Shandong
    2 Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2024-02-09 Accepted:2024-05-08 Online:2024-08-16 Published:2024-08-27
  • Contact: ZHAO ZhenJun, ZHOU MeiLiang

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Abstract:

【Objective】Buckwheat is an important cereal and economic crop. Compared with other crops, buckwheat has strong aluminum tolerance. A transcription factor FtbHLH93 in response to aluminum stress was identified in transcriptome data of aluminum treatment. Exploring the function of FtbHLH93 will provide ideas and clues for solving the problem of aluminum toxicity in acidic soil and molecular breeding of new varieties of buckwheat with aluminum tolerant, and provide theoretical basis for the molecular mechanism of tolerance aluminum in buckwheat.【Method】 The cDNA of Pinku1 was used as a template to clone FtbHLH93. qRT-PCR was used to detect the expression of FTbHLH93 in different tissues of Tartary buckwheat and at different time points after aluminum treatment. Yeast system was used to identify the transcriptional activation activity. The localization of intracellular expression was determined by subcellular localization. The flavonoid content of the overexpressed materials was examined, and SOD and POD activities were measured under untreated and Al-treated conditions. The differentially expressed genes were analyzed by transcriptome analysis, potential downstream target genes were screened, and their promoters were predicted. The dual luciferase reporter gene assay was used to verify the results.【Result】The coding region of FtbHLH93 transcription factor was 573 bp in length, encoding 190 amino acid residues. The predicted molecular weight of FtbHLH93 was 21.759 kDa, and its isoelectric point was 8.64. qRT-PCR results showed that FtbHLH93 was highly expressed in roots. The expression level of FtbHLH93 is highest at 24 h after aluminum treatment. FtbHLH93 is localized in the nucleus without self-activating activity. Overexpression of FtbHLH93 in Tartary buckwheat hairy roots enhanced aluminum tolerance, and the activities of SOD and POD were significantly higher than those of the control group. The detection results of flavonoid metabolites in the overexpressed FtbHLH93 hairy roots showed that the contents of rutin, catechin, and fireworks were significantly higher than those of the control group. GO enrichment analysis showed that it was related to metal ion transport and cadmium and manganese ion entries, and KEGG enrichment analysis showed that it was related to ABC transporter. Three genes responsive to aluminum stress may be downstream target genes of FtbHLH93, and co-expression analysis showed that two of the candidate downstream target genes had a similar expression pattern to FtbHLH93.【Conclusion】FtbHLH93 transcription factor may alleviate aluminum toxicity by promoting the accumulation of flavonoids and the increase of SOD and POD activities. FtbHLH93 may act as an upstream regulator to regulate the expression of FtPinG0100930100.01, FtPinG0303102000.01 and FtPinG0403996200.01.

Key words: tartary buckwheat, aluminum, bHLH, overexpression, transcriptome, Dual-luciferase

Fig. 1

Cloning and bioinformatics analysis of FtbHLH93 in Tartary buckwheat A: Buckwheat FtbHLH93 clone; B: Gene structure of Tartary buckwheat FtbHLH93; C: Secondary structure of Tartary buckwheat FtbHLH93 protein; D: Tertiary structure of Tartary buckwheat FtbHLH93 protein"

Fig. 2

Expression pattern, yeast autoactivation, and subcellular localization of the FtbHLH93 A: Differential expression of FtbHLH93 in different tissues; B: Differential expression of FtbHLH93 induced by aluminum at different times; C: Yeast autoactivation of FtbHLH93; D: Subcellular localization of FtbHLH93; **: Significant difference at 0.01 level; ***: Significant difference at 0.001 level; ****: Significant difference at 0.0001 level. The same as below"

Fig. 3

Identification of positive hairy roots A: Identification of FtbHLH93 overexpression hairy root DNA level; M: DNA Marker; -: Negative control (Tartary buckwheat DNA as template); +: Positive control (FtbHLH93-1307 plasmid as template); 1: FtbHLH93 overexpressed hairy root strain 4; 2: FtbHLH93 overexpressed hairy root line 12; 3: FtbHLH93 overexpressed hairy root line 19; B: Identification of FtbHLH93 overexpression hairy root RNA levels"

Fig. 4

Analysis of hairy root system function and physiological indexes under aluminum stress A: Phenotype of FtbHLH93 overexpressing strain 19 after aluminum treatment; 1: Empty A4 hairy roots cultured in MS liquid medium; 2: Empty A4 hairy roots cultured in MS liquid medium with 100 µmol·L-1 aluminum chloride; 3: FtbHLH93 overexpression line 19 hairy roots cultured in MS liquid medium; 4: FtbHLH93 overexpression line 19 hairy roots cultured in MS liquid medium supplemented with 100 µmol·L-1 aluminum chloride; B: Fresh and dry weight of FtbHLH93 overexpressing strain 19; C: SOD and POD activity determination of FtbHLH93 overexpressing hairy roots; D: FtbHLH93 overexpression of rutin, catechin, kaempferol-3-O-rutinoside content in hairy roots. Different lowercase letters show significant differences(P<0.05); ns: The difference was not significant; *: Significant difference at the 0.05 level. The same as below"

Fig. 5

Differential expression analysis and GO and KEGG enrichment analysis of FtbHLH93 A: All differentially expressed genes in FtbHLH93-vs-CK; B: The top 20 items of KEGG enrichment analysis of all differentially expressed genes in FtbHLH93-vs-CK; C: Top 30 items of GO enrichment analysis of differentially expressed genes down-regulated by FtbHLH93-vs-CK; D: Top 30 entries from GO enrichment analysis of all differentially expressed genes in FtbHLH93-vs-CK"

Table 1

Significantly differentially expressed genes related to metal ions"

基因ID Gene ID 表达水平Expression level GO条目 GO term
FtPinG0707878600.01 下调 Down 铁离子结合，酸还原双加氧酶[需要铁]活性
Iron-binding, acid-reducing dioxygenase [iron required] activity
FtPinG0303292100.01 上调 Up 金属离子结合Metal ion binding
FtPinG0303287700.01 上调 Up 金属离子结合Metal ion binding
FtPinG0201816600.01 上调 Up 金属离子结合，负调控对缺水的反应
Metal ion binding and negative regulation of water deficiency response

Table 2

Candidate downstream target genes of FtbHLH93"

基因ID
Gene ID		 表达水平Expression
level		 基因命名
Gene symbol		 注释
Description		 GO条目
GO term		 表达集群
Express
cluster		 FC值
log2 (FoldChange)		 P值
P-value
FtPinG0100930100.01 下调
Down		 ABCB25 ABC转运蛋白B家族成员25
A member of the ABC transporter B family 25		 铝离子跨膜转运活性Aluminium ion transmembrane transport activity 14 -1.045611368 0.002076521
FtPinG0303102000.01 上调
Up		 ALMT4 铝活化苹果酸转运蛋白4
Aluminum activates malate transporter 4		 离子通道活性
Ion channel activity		 13 2.282465572 2.69E-10
FtPinG0403996200.01 上调
Up		 PLDGAMMA1 磷脂酶Dγ1
Phospholipase Dγ1		 响应铝离子，钙离子结合
In response to aluminum ions, calcium ions binding		 13 1.168803299 3.45E-08

Fig. 6

Co-expression analysis of FtbHLH93 (A), promoter prediction of downstream target genes (B, C, D), and Dual Luciferase reporter assay (E)"

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