Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (16): 3234-3249.doi: 10.3864/j.issn.0578-1752.2024.16.011

• HORTICULTURE • Previous Articles     Next Articles

Genome-Wide Identification of GST Gene Family and Functional Analysis of the BcGSTF6 Gene Related to Anthocyanin in Pak Choi

YIN YuQin(), XU HuanHuan, TANG LiPing, WANG XinYa, HU ChunMei, HOU XiLin, LI Ying()   

  1. College of Horticulture, Nanjing Agricultural University/National Key Laboratory of Crop Genetics and Germplasm Innovation and Utilization/Engineering Research Center for Horticultural Crop Germplasm Innovation and Utilization of the Ministry of Education, Nanjing 210000
  • Received:2024-01-25 Accepted:2024-04-15 Online:2024-08-16 Published:2024-08-27
  • Contact: LI Ying

RichHTML

39

PDF

1940

Abstract:

【Background】Anthocyanin is one of the important nutritional components in purple pak choi (Brassica campestris ssp. chinensis), and the glutathione S-transferase (GST) plays an important role in anthocyanin transport. 【Objective】This study aimed to identify the genes encoding GST in pak choi and to elucidate the role of BcGSTF6 in anthocyanin transport through transcriptome and molecular biology methods, which would help to elucidate the molecular basis of anthocyanin accumulation in pak choi. 【Method】Using bioinformatics methods to identify members of the GST gene family and to analyze their positions and motifs on chromosomes. The transcriptome analysis was performed in purple and green pak choi to screen genes involving in anthocyanin transport. To further validate the functions of BcGSTF6 and BcTT19, the expression pattern analysis, subcellular localization, and heterologous expression of BcGSTF6 and BcTT19 in the Arabidopsis tt19 mutant were conducted. By comparing the amino acid sequences of BcGSTF6 and BcTT19 with anthocyanin transporters in other species, the differences between BcGSTF6 and others were analyzed. In vitro binding experiments and gene silencing experiments of the BcGSTF6 protein with cyanidin (Cya) demonstrated whether BcGSTF6 played a crucial role in anthocyanin transport and accumulation in pak choi.【Result】In total, 83 members of the GST gene family were identified. They were divided into 8 subfamilies, and they were distributed on all 10 chromosomes of the pak choi. The transcriptome analysis results showed that BcGSTF6 and BcTT19 may be important genes involved in anthocyanin transport in pak choi. Furthermore, qRT-PCR analysis revealed that both BcGSTF6 and BcTT19 were highly expressed during the anthocyanin accumulation; subcellular localization results indicated that both BcGSTF6 and BcTT19 were located in the endoplasmic reticulum (ER) and tonoplast; multiple comparisons of amino acid sequences revealed high homology between BcTT19 and other anthocyanin transport proteins in other species, while BcGSTF6 showed significant differences in the conserved domain; the overexpression of BcGSTF6 and BcTT19 in the Arabidopsis tt19 mutant rescued the phenotype that seedlings could accumulate anthocyanin, but the brown color of the seed coats was not rescued. These results suggested that both BcGSTF6 and BcTT19 were involved in anthocyanin transport but could not transport proanthocyanidins (PAs); In vitro binding assay showed that BcGSTF6 protein could increase the solubility of Cya at room temperature; gene silencing of BcGSTF6 in pak choi not only resulted a significant decrease in anthocyanin content in the leaves, but also led to a decrease in the expression of genes related to anthocyanin synthesis and regulation. These results demonstrated that BcGSTF6 was involved in anthocyanin transport and accumulation in pak choi, and played an important role in the coloring process of pak choi leaves. 【Conclusion】 This study identified the GST gene family and further elucidated the functions of BcTT19 and BcGSTF6 in anthocyanin transport in pak choi. This experiment elucidated the partial mechanism of BcGSTs regulation of anthocyanin transport and accumulation in pak choi.

Key words: pak choi (Brassica campestris ssp. chinensis), anthocyanins, glutathione S-transferase, BcGSTF6, transport

Fig. 1

Member identification and classification of BcGST in pak choi A: The phylogenetic tree of GST genes in pak choi; B: Chromosomal mapping analysis of GST genes in pak choi; C: Analysis of GST protein motifs in pak choi"

Fig. 2

The expression patterns of BcGSTF6 and BcTT19 A：Differential expression heatmap of BcGST in ‘HG072’ and ‘HP072’; B: The total anthocyanin content of ‘HG072’and ‘HP072’, and the expression levels of BraC10g001530, BraC10g024210 and BraC08g036270 in ‘HG072’ and ‘HP072’; C: S1-S8 during the development process of ‘G125’ leaves, total anthocyanin content in leaves, and expression levels of BcGSTF6 and BcTT19. The scale is 2 cm. Different lowercase letters indicate significant difference (P<0.05). The same as below"

Fig. 3

Protein sequence alignment and subcellular localization analysis of BcGSTF6 and BcTT19 A: Protein sequence alignment of BcGSTF6 and BcTT19 with other species; B: Subcellular localization of BcGSTF6 and BcTT19"

Fig. 4

Transformation of Arabidopsis mutant tt19 by BcGSTF6 and BcTT19 A:7-day-old Arabidopsis seedlings were grown on MS medium containing 5% sucrose, and the scale is 2 mm; B: Images of Arabidopsis seeds, and the scale is 1 mm; C: Relative expression levels of BcGSTF6 and BcTT19 in transgenic Arabidopsis"

Fig. 5

In vitro binding of BcGSTF6 protein to Cya A: Denatured protein gel electrophoresis; B: UPLC detection of the absorbance of Cya in BcGSTF6 protein and empty protein solution, * indicate the absorbance after being placed at room temperature for 2.5 minutes"

Fig. 6

BcGSTF6 silencing inhibits anthocyanin accumulation in pak choi A: Phenotypes of ‘G125’ after silencing BcGSTF6, and PTY-Empty was used as a control; B: The total anthocyanin content of silenced plants and control plants; C: The content of Cya in both silenced and control plants were measured by UPLC; D: The gene expression levels of BcGSTF6 in both silenced and control plants; E: Relative expression levels of anthocyanin synthesis pathway genes in both silenced and control plants. LODX: Leucoanthocyanidin dioxygenase; UF3GT: UDP-glucose flavonoid-3-O-glucosyltransferase; F3H: Flavanone 3-hydroxylase; CHI: Chalcone isomerase; F3′H: Flavonoid 3'-hydroxylase; DFR: Dihydroflavonol-4-reductase"

[1]
POURCEL L, ROUTABOUL J M, CHEYNIER V, LEPINIEC L, DEBEAUJON I. Flavonoid oxidation in plants: From biochemical properties to physiological functions. Trends in Plant Science, 2007, 12(1): 29-36.

doi: 10.1016/j.tplants.2006.11.006 pmid: 17161643
[2]
LI P, LI Y J, ZHANG F J, ZHANG G Z, JIANG X Y, YU H M, HOU B K. The Arabidopsis UDP-glycosyltransferases UGT79B2 and UGT79B3, contribute to cold, salt and drought stress tolerance via modulating anthocyanin accumulation. The Plant Journal, 2017, 89(1): 85-103.
[3]
XIE P, SHI J Y, TANG S Y, CHEN C X, KHAN A, ZHANG F X, XIONG Y, LI C, HE W, WANG G D, LEI F M, WU Y R, XIE Q. Control of bird feeding behavior by Tannin1 through modulating the biosynthesis of polyphenols and fatty acid-derived volatiles in Sorghum. Molecular Plant, 2019, 12(10): 1315-1324.
[4]
NAKABAYASHI R, YONEKURA-SAKAKIBARA K, URANO K, SUZUKI M, YAMADA Y, NISHIZAWA T, MATSUDA F, KOJIMA M, SAKAKIBARA H, SHINOZAKI K, MICHAEL A J, TOHGE T, YAMAZAKI M, SAITO K. Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids. The Plant Journal, 2014, 77(3): 367-379.
[5]
FAN X P, FAN B H, WANG Y X, YANG W C. Anthocyanin accumulation enhanced in Lc-transgenic cotton under light and increased resistance to bollworm. Plant Biotechnology Reports, 2016, 10(1): 1-11.
[6]
BISHAYEE A, MBIMBA T, THOPPIL R J, HÁZNAGY-RADNAI E, SIPOS P, DARVESH A S, FOLKESSON H G, HOHMANN J. Anthocyanin-rich black currant (Ribes nigrum L.) extract affords chemoprevention against diethylnitrosamine-induced hepatocellular carcinogenesis in rats. The Journal of Nutritional Biochemistry, 2011, 22(11): 1035-1046.
[7]
SINGLETARY K W, JUNG K J, GIUSTI M. Anthocyanin-rich grape extract blocks breast cell DNA damage. Journal of Medicinal Food, 2007, 10(2): 244-251.

doi: 10.1089/jmf.2006.258 pmid: 17651059
[8]
FILIPIAK K, HIDALGO M, SILVAN J M, FABRE B, CARBAJO R J, PINEDA-LUCENA A, RAMOS A, DE PASCUAL-TERESA B, DE PASCUAL-TERESA S. Dietary Gallic acid and anthocyanin cytotoxicity on human fibrosarcoma HT1080 cells. A study on the mode of action. Food & Function, 2014, 5(2): 381-389.
[9]
ZHAO J, DIXON R A. The ‘ins’ and ‘outs’ of flavonoid transport. Trends in Plant Science, 2010, 15(2): 72-80.
[10]
MUELLER L A, GOODMAN C D, SILADY R A, WALBOT V. AN9, a Petunia glutathione S-transferase required for anthocyanin sequestration, is a flavonoid-binding protein. Plant Physiology, 2000, 123(4): 1561-1570.
[11]
GOMEZ C, TERRIER N, TORREGROSA L, VIALET S, FOURNIER- LEVEL A, VERRIÈS C, SOUQUET J M, MAZAURIC J P, KLEIN M, CHEYNIER V, AGEORGES A. Grapevine MATE-type proteins act as vacuolar H+-dependent acylated anthocyanin transporters. Plant Physiology, 2009, 150(1): 402-415.

doi: 10.1104/pp.109.135624 pmid: 19297587
[12]
REA P A. Plant ATP-binding cassette transporters. Annual Review of Plant Biology, 2007, 58: 347-375.

pmid: 17263663
[13]
ZHANG H B, WANG L, DEROLES S, BENNETT R, DAVIES K. New insight into the structures and formation of anthocyanic vacuolar inclusions in flower petals. BMC Plant Biology, 2006, 6: 29.

pmid: 17173704
[14]
MARRS K A, ALFENITO M R, LLOYD A M, WALBOT V. A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2. Nature, 1995, 375: 397-400.
[15]
SUN Y, LI H, HUANG J R. Arabidopsis TT19 functions as a carrier to transport anthocyanin from the cytosol to tonoplasts. Molecular Plant, 2012, 5(2): 387-400.
[16]
PÉREZ-DÍAZ R, MADRID-ESPINOZA J, SALINAS-CORNEJO J, GONZÁLEZ-VILLANUEVA E, RUIZ-LARA S. Differential roles for VviGST1, VviGST3, and VviGST4 in proanthocyanidin and anthocyanin transport in Vitis vinifera. Frontiers in Plant Science, 2016, 7: 1166.
[17]
CONN S, CURTIN C, BÉZIER A, FRANCO C, ZHANG W. Purification, molecular cloning, and characterization of glutathione S-transferases (GSTs) from pigmented Vitis vinifera L. cell suspension cultures as putative anthocyanin transport proteins. Journal of Experimental Botany, 2008, 59(13): 3621-3634.
[18]
LIEBERHERR D, WAGNER U, DUBUIS P H, MÉTRAUX J P, MAUCH F. The rapid induction of glutathione S-transferases AtGSTF2 and AtGSTF6 by avirulent Pseudomonas syringae is the result of combined salicylic acid and ethylene signaling. Plant and Cell Physiology, 2003, 44(7): 750-757.
[19]
TANG L P, XIAO D, YIN Y Q, WANG H B, WANG J J, LIU T K, HOU X L, LI Y. Comparative transcriptome analysis of purple and green non-heading Chinese cabbage and function analyses of BcTT8 gene. Genes, 2022, 13(6): 988.
[20]
LI Y, LIU G F, MA L M, LIU T K, ZHANG C W, XIAO D, ZHENG H K, CHEN F, HOU X L. A chromosome-level reference genome of non-heading Chinese cabbage [Brassica campestris (syn. Brassica rapa) ssp. Chinensis]. Horticulture Research, 2020, 7: 212.
[21]
CHEN C J, WU Y, LI J W, WANG X, ZENG Z H, XU J, LIU Y L, FENG J T, CHEN H, HE Y H, XIA R. TBtools-II: A “one for all, all for one” bioinformatics platform for biological big-data mining. Molecular Plant, 2023, 16(11): 1733-1742.
[22]
CLOUGH S J, BENT A F. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal, 1998, 16(6): 735-743.
[23]
JIANG S H, CHEN M, HE N B, CHEN X L, WANG N, SUN Q G, ZHANG T L, XU H F, FANG H C, WANG Y C, ZHANG Z Y, WU S J, CHEN X S. MdGSTF6, activated by MdMYB1, plays an essential role in anthocyanin accumulation in apple. Horticulture Research, 2019, 6: 40.

doi: 10.1038/s41438-019-0118-6 pmid: 30854214
[24]
ISLAM S, RAHMAN I A, ISLAM T, GHOSH A. Genome-wide identification and expression analysis of glutathione S-transferase gene family in tomato: Gaining an insight to their physiological and stress-specific roles. PLoS ONE, 2017, 12(11): e0187504.
[25]
HAN L L, ZOU H Z, ZHOU L, WANG Y. Transcriptome-based identification and expression analysis of the glutathione S-transferase (GST) family in tree peony reveals a likely role in anthocyanin transport. Horticultural Plant Journal, 2022, 8(6): 787-802.
[26]
KOU M, LIU Y J, LI Z Y, ZHANG Y G, TANG W, YAN H, WANG X, CHEN X G, SU Z X, ARISHA M H, LI Q, MA D F. A novel glutathione S-transferase gene from sweetpotato, IbGSTF4, is involved in anthocyanin sequestration. Plant Physiology and Biochemistry, 2019, 135: 395-403.
[27]
LUO H F, DAI C, LI Y P, FENG J, LIU Z C, KANG C Y. Reduced Anthocyanins in Petioles codes for a GST anthocyanin transporter that is essential for the foliage and fruit coloration in strawberry. Journal of Experimental Botany, 2018, 69(10): 2595-2608.

doi: 10.1093/jxb/ery096 pmid: 29538703
[28]
CHAI Q C, WANG X L, GAO M W, ZHAO X C, CHEN Y, ZHANG C, JIANG H, WANG J B, WANG Y C, ZHENG M N, BALTAEVICH A M, ZHAO J, ZHAO J S. A glutathione S- transferase GhTT19 determines flower petal pigmentation via regulating anthocyanin accumulation in cotton. Plant Biotechnology Journal, 2023, 21(2): 433-448.
[29]
ZHAO Y, DONG W Q, ZHU Y C, ALLAN A C, KUI L W, XU C J. PpGST1, an anthocyanin-related glutathione S-transferase gene, is essential for fruit coloration in peach. Plant Biotechnology Journal, 2020, 18(5): 1284-1295.
[30]
KITAMURA S, SHIKAZONO N, TANAKA A. TRANSPARENT TESTA 19 is involved in the accumulation of both anthocyanins and proanthocyanidins in Arabidopsis. The Plant Journal, 2004, 37(1): 104-114.
[31]
HAN L L, ZHOU L, ZOU H Z, YUAN M, WANG Y. PsGSTF3, an anthocyanin-related glutathione S-transferase gene, is essential for petal coloration in tree peony. International Journal of Molecular Sciences, 2022, 23(3): 1423.
[32]
HU B, ZHAO J T, LAI B, QIN Y H, WANG H C, HU G B. LcGST4 is an anthocyanin-related glutathione S-transferase gene in Litchi chinensis Sonn. Plant Cell Reports, 2016, 35(4): 831-843.
[33]
DUAN A Q, DENG Y J, TAN S S, LIU S S, LIU H, XU Z S, SHU S, XIONG A S. DcGST1, encoding a glutathione S-transferase activated by DcMYB7, is the main contributor to anthocyanin pigmentation in purple carrot. The Plant Journal, 2024, 117(4): 1069-1083.
[34]
SHAO D N, LI Y J, ZHU Q H, ZHANG X Y, LIU F, XUE F, SUN J. GhGSTF12, a glutathione S-transferase gene, is essential for anthocyanin accumulation in cotton (Gossypium hirsutum L.). Plant Science, 2021, 305: 110827.
[35]
LIU Y F, QI Y W, ZHANG A L, WU H X, LIU Z D, REN X L. Molecular cloning and functional characterization of AcGST1, an anthocyanin-related glutathione S-transferase gene in kiwifruit (Actinidia chinensis). Plant Molecular Biology, 2019, 100(4): 451-465.
[36]
WANGWATTANA B, KOYAMA Y, NISHIYAMA Y, KITAYAMA M, YAMAZAKI M, SAITO K. Characterization of PAP1-upregulated glutathione S-transferase genes in Arabidopsis thaliana. Plant Biotechnology, 2008, 25(2): 191-196.
[37]
OSMANI S A, BAK S, MØLLER B L. Substrate specificity of plant UDP-dependent glycosyltransferases predicted from crystal structures and homology modeling. Phytochemistry, 2009, 70(3): 325-347.

doi: 10.1016/j.phytochem.2008.12.009 pmid: 19217634
[38]
CUI Y M, FAN J W, LU C F, REN J S, QI F T, HUANG H, DAI S L. ScGST3 and multiple R2R3-MYB transcription factors function in anthocyanin accumulation in Senecio cruentus. Plant Science, 2021, 313: 111094.
[1] FENG WeiQing, NI YuanQian, FEI Teng, LI YouMei, XIE ZhaoSen. Differences in Vascular Bundle Morphological Structure, Distribution, and Water Transport Function in Grape Fruits of Different Shapes [J]. Scientia Agricultura Sinica, 2026, 59(1): 161-178.
[2] TANG Yu, LEI BiXin, WANG ChuanWei, YAN XuanTao, WANG Hao, ZHENG Jie, ZHANG WenJing, MA ShangYu, HUANG ZhengLai, FAN YongHui. Response Mechanism of Anthocyanin Accumulation in Colored Wheat to Post-Anthesis High Temperature Stress [J]. Scientia Agricultura Sinica, 2025, 58(6): 1083-1101.
[3] GUO AoLin, LIN JunXuan, LAI GongTi, HE LiYuan, CHE JianMei, PAN Ruo, YANG FangXue, HUANG YuJi, CHEN GuiXin, LAI ChengChun. Effect of VdF3′5′H2 Overexpression on the Accumulation of Anthocyanin Composition in Spine Grape Cells [J]. Scientia Agricultura Sinica, 2025, 58(4): 802-818.
[4] ZHUANG LiHua, LUO Lei, ZHAO ChunFang, WANG JiZhong, ZHANG YaDong, HE Lei. Identification and Gene Mapping of Rice Grain Shape Mutant sgd13 [J]. Scientia Agricultura Sinica, 2025, 58(24): 5097-5109.
[5] HUANG HongMei, WANG SiQi, YANG QingChuan, GUO ChangHong, WANG Xue. Phosphate Transporter MsPT5 Regulates Phosphate Uptake and Utilization in Alfalfa [J]. Scientia Agricultura Sinica, 2025, 58(21): 4544-4556.
[6] WANG HuiLing, ZHANG YingYing, YAN AiLing, WANG XiaoYue, LIU ZhenHua, REN JianCheng, XU HaiYing, SUN Lei. Multi-Omics Analysis Reveals the Changes of Monoterpenes and Anthocyanins Accumulation During Veraison in Red Muscat-Type Grape [J]. Scientia Agricultura Sinica, 2025, 58(13): 2645-2662.
[7] LI YongFei, LI ZhanKui, ZHANG ZhanSheng, CHEN YongWei, KANG JianHong, WU HongLiang. Effects of Postponing Nitrogen Fertilizer Application on Flag Leaf Physiological Characteristics and Yield of Spring Wheat Under High Temperature Stress [J]. Scientia Agricultura Sinica, 2024, 57(8): 1455-1468.
[8] LUO LiDan, CHEN JiaMing, AN Qi, LIU Lei, SUN QinZhe, LIU Huan, WANG SenShan, SONG LiWen. Effects of Extreme High Temperature on Trehalose Content and Trehalose Transporter Gene in Tetranychus truncatus [J]. Scientia Agricultura Sinica, 2024, 57(6): 1091-1101.
[9] ZHAO YiYan, GUO HongFang, LIU WeiMin, ZHAO XiaoMing, ZHANG JianZhen. Effects of Apolipophorin on Ovarian Development and Lipid Deposition in Locusta migratoria [J]. Scientia Agricultura Sinica, 2024, 57(4): 711-720.
[10] SHI DeYang, LI YanHong, WANG FeiFei, XIA DeJun, JIAO YanLin, SUN NiNa, ZHAO Jian. Regulation Effects of Line-Spacing Expansion and Row-Spacing Shrinkage on Dry Matter and Nutrient Accumulation and Transport of Summer Maize Under High Plant Density [J]. Scientia Agricultura Sinica, 2024, 57(23): 4658-4672.
[11] LIU YiHan, MU QingShan, HE Xiang, CHEN Min, HU Jin, GUAN YaJing. Study on the Involvement of OsFWL3 in the Regulation of Metal Ion Transport and Accumulation in Rice [J]. Scientia Agricultura Sinica, 2024, 57(21): 4161-4174.
[12] WEI XiaoDong, SONG XueMei, WANG Ning, ZHAO QingYong, ZHU Zhen, CHEN Tao, ZHAO Ling, WANG CaiLin, ZHANG YaDong. Distribution Characteristics of Photosynthetic Products of Nanjing Series of Super Rice During Filling Stage [J]. Scientia Agricultura Sinica, 2024, 57(12): 2309-2321.
[13] WANG Yu, SONG YiFan, ZHANG Rong, MU HaiMeng, SUN LiFang, FU KaiXia, WU ZiJun, HUANG QingQing, XU YingMing, LI GeZi, WANG YongHua, GUO TianCai. Effects of Soil Application of Passivating Agent and Compound Microbial Fertilizer on Cadmium Accumulation in Winter Wheat [J]. Scientia Agricultura Sinica, 2024, 57(1): 126-141.
[14] ZHANG PengYun, CHEN Min, LIU MingXing, ZHOU Hong, LIN HuiXing, FAN HongJie. Development and Application of Indirect ELISA Kits for Antibody Detection of Haemophilus parasuis [J]. Scientia Agricultura Sinica, 2023, 56(8): 1606-1614.
[15] AN Yong, QIN ShiZhen, SHI ZhaoGuo, GONG LiYuan, ZHANG Shuai, JI Feng. Influences of Phosphorus Level in Diet of Parent Pigeons on Biochemical Index, Untargeted Metabolomics Profile of Serum, and Gene Expression of Phosphate Transporters in Squabs [J]. Scientia Agricultura Sinica, 2023, 56(23): 4772-4788.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd