Identification of HMA Gene Family and Cadmium Transport Function of SlHMA1 in Tomato

doi:10.13560/j.cnki.biotech.bull.1985.2023-0896

Abstract

Abstract:

【Objective】 The heavy metal ATPase（HMA）gene family is extensively involved in the absorption and transport of metal elements in plants. Therefore, this study is aimed to systematically identify the members of the tomato HMA gene family and their characteristics, as well as to investigate their functions in response to cadmium stress.【Method】 Tomato HMA gene family members were identified through bioinformatics, and their systematic evolution tree, protein physicochemical properties, gene structure, cis-regulatory elements, and gene expression patterns, etc., were analyzed. Additionally, yeast functional complementation experiments were conducted to validate the Cd²⁺ transport activity of SlHMA1.【Result】 Eight SlHMAs were identified in the tomato genome, categorized into 2 subgroups. Remarkable differences in gene structure were observed among SlHMAs genes and their homologs in Arabidopsis and rice. Notably, the promoter regions of SlHMAs members were found to harbor numerous cis-regulatory elements associated with stress responses, and RT-qPCR analysis also indicated that most SlHMAs genes showed tissue-specific responses to cadmium stress. Furthermore, yeast functional complementation experiments provided conclusive evidence for Cd²⁺ transport activity of SlHMA1, while evolutionary analysis indicated the widespread presence of HMA1 in the plant kingdom, with the motif DKTGT related to ATP hydrolysis activity within HMA1 also being highly conserved across the plant kingdom.【Conclusion】 The eight SlHMAs present typical characteristics of the HMA gene family while also having functional diversity. SlHMAs, along with their conserved amino acid motif DKTGT, are closely associated with metal ion transport and cadmium stress response, offering significant potential for application in the breeding of low cadmium-accumulating crop varieties.

Key words: tomato, metal transporter, heavy metal ATPase, cadmium, SlHMA1

ZHAO Yao, WEN Lang, LUO Shao-dan, LI Zi-xing, LIU Chao-chao. Identification of HMA Gene Family and Cadmium Transport Function of SlHMA1 in Tomato[J]. Biotechnology Bulletin, 2024, 40(2): 212-222.

Figures/Tables 10

References 34

[1]	陈玉鹏, 梁东丽, 刘中华, 等. 大棚蔬菜土壤重金属污染及其控制的研究进展与展望[J]. 农业环境科学学报, 2018, 37(1): 9-17.
	Chen YP, Liang DL, Liu ZH, et al. Analysis of present situation and control of heavy metal pollution in vegetable greenhouse soils[J]. J Agro-Environ Sci, 2018, 37(1): 9-17.
[2]	Hu WY, Huang B, Tian K, et al. Heavy metals in intensive greenhouse vegetable production systems along Yellow Sea of China: Levels, transfer and health risk[J]. Chemosphere, 2017, 167: 82-90. doi: S0045-6535(16)31323-6 pmid: 27710846
[3]	Sun JT, Pan LL, Li ZH, et al. Comparison of greenhouse and open field cultivations across China: Soil characteristics, contamination and microbial diversity[J]. Environ Pollut, 2018, 243: 1509-1516. doi: S0269-7491(18)32285-1 pmid: 30292159
[4]	Tao JY, Lu LL. Advances in genes-encoding transporters for cadmium uptake, translocation, and accumulation in plants[J]. Toxics, 2022, 10(8): 411. doi: 10.3390/toxics10080411 URL
[5]	Yang Z, Yang F, Liu JL, et al. Heavy metal transporters: Functional mechanisms, regulation, and application in phytoremediation[J]. Sci Total Environ, 2022, 809: 151099. doi: 10.1016/j.scitotenv.2021.151099 URL
[6]	Feki K, Tounsi S, Mrabet M, et al. Recent advances in physiological and molecular mechanisms of heavy metal accumulation in plants[J]. Environ Sci Pollut Res, 2021, 28(46): 64967-64986. doi: 10.1007/s11356-021-16805-y
[7]	Ajeesh Krishna TP, Maharajan T, Victor Roch G, et al. Structure, function, regulation and phylogenetic relationship of ZIP family transporters of plants[J]. Front Plant Sci, 2020, 11: 662. doi: 10.3389/fpls.2020.00662 pmid: 32536933
[8]	Fu S, Lu YS, Zhang X, et al. The ABC transporter ABCG36 is required for cadmium tolerance in rice[J]. J Exp Bot, 2019, 70(20): 5909-5918. doi: 10.1093/jxb/erz335 pmid: 31328224
[9]	Zhang J, Zhang M, Song HY, et al. A novel plasma membrane-based NRAMP transporter contributes to Cd and Zn hyperaccumulation in Sedum alfredii Hance[J]. Environ Exp Bot, 2020, 176: 104121. doi: 10.1016/j.envexpbot.2020.104121 URL
[10]	Naz M, Dai ZC, Tariq M, et al. CHAPTER16-Role of NRAMP transporters for Fe, mineral uptake, and accumulation in rice and other plants[M]//Aftab T, Hakeem K. Metals Metalloids Soil Plant Water Systems. Amsterdam: Elsevier, 2022: 331-348.
[11]	刘元峰, 李素贞, 郭晋杰, 等. 植物YSL家族基因研究进展[J]. 生物技术通报, 2017, 33(9): 1-9. doi: 10.13560/j.cnki.biotech.bull.1985.2017-0375
	Liu YF, Li SZ, Guo JJ, et al. Research progress on YSL transporters gene family[J]. Biotechnol Bull, 2017, 33(9): 1-9.
[12]	Das N, Bhattacharya S, Maiti MK. Enhanced cadmium accumulation and tolerance in transgenic tobacco overexpressing rice metal tolerance protein gene OsMTP1 is promising for phytoremediation[J]. Plant Physiol Biochem, 2016, 105: 297-309. doi: 10.1016/j.plaphy.2016.04.049 URL
[13]	Huang XY, Deng FL, Yamaji N, et al. A heavy metal P-type ATPase OsHMA4 prevents copper accumulation in rice grain[J]. Nat Commun, 2016, 7(1): 12138. doi: 10.1038/ncomms12138
[14]	Tang B, Luo MJ, Zhang YX, et al. Natural variations in the P-type ATPase heavy metal transporter gene ZmHMA3 control cadmium accumulation in maize grains[J]. J Exp Bot, 2021, 72(18): 6230-6246. doi: 10.1093/jxb/erab254 pmid: 34235535
[15]	Cobbett CS, Hussain D, Haydon MJ. Structural and functional relationships between type 1B heavy metal-transporting P-type ATPases in Arabidopsis[J]. New Phytol, 2003, 159(2): 315-321. doi: 10.1046/j.1469-8137.2003.00785.x URL
[16]	Argüello JM, Eren E, González-Guerrero M. The structure and function of heavy metal transport P1B-ATPases[J]. Biometals, 2007, 20(3): 233-248. doi: 10.1007/s10534-006-9055-6 URL
[17]	Leonhardt N, Cun P, Richaud P, et al. Zn/Cd/Co/Pb P1b-ATPases in plants, physiological roles and biological interest[M]// Gupta DK, Sandalio LM. Metal Toxicity in Plants: Perception, Signaling and Remediation. Berlin, Heidelberg: Springer Berlin Heidelberg, 2011: 227-248.
[18]	E ZG, Li TT, Chen C, et al. Genome-wide survey and expression analysis of P1B-ATPases in rice, maize and sorghum[J]. Rice Sci, 2018, 25(4): 208-217. doi: 10.1016/j.rsci.2018.06.004 URL
[19]	Ma YT, Wei N, Wang QX, et al. Genome-wide identification and characterization of the heavy metal ATPase(HMA)gene family in Medicago truncatula under copper stress[J]. Int J Biol Macromol, 2021, 193: 893-902. doi: 10.1016/j.ijbiomac.2021.10.197 URL
[20]	Zhang CR, Yang QH, Zhang XQ, et al. Genome-wide identification of the HMA gene family and expression analysis under Cd stress in barley[J]. Plants, 2021, 10(9): 1849. doi: 10.3390/plants10091849 URL
[21]	黄治皓, 刘婷婷, 等. 芥菜HMA家族基因鉴定及其在镉胁迫下的表达分析[J]. 园艺学报, 2023, 50(6): 1230-1242. doi: 10.16420/j.issn.0513-353x.2022-0509
	Huang ZH, Liu TT, et al. Identification and expression analysis of HMA gene family in Brassica juncea under cadmium stress[J]. Acta Hortic Sin, 2023, 50(6): 1230-1242.
[22]	Lu CN, Zhang LX, Tang Z, et al. Producing cadmium-free Indica rice by overexpressing OsHMA3[J]. Environ Int, 2019, 126: 619-626. doi: 10.1016/j.envint.2019.03.004 URL
[23]	Hosmani PS, Flores-Gonzalez M, van de Geest H, et al. An improved de novo assembly and annotation of the tomato reference genome using single-molecule sequencing, Hi-C proximity ligation and optical maps[J]. bioRxiv, 2019: 767764.
[24]	Fang XL, Wang L, Deng XJ, et al. Genome-wide characterization of soybean P1B-ATPases gene family provides functional implications in cadmium responses[J]. BMC Genomics, 2016, 17(1): 376. doi: 10.1186/s12864-016-2730-2 URL
[25]	Li NN, Xiao H, Sun JJ, et al. Genome-wide analysis and expression profiling of the HMA gene family in Brassica napus under cd stress[J]. Plant Soil, 2018, 426(1): 365-381. doi: 10.1007/s11104-018-3637-2
[26]	Xu GX, Guo CC, Shan HY, et al. Divergence of duplicate genes in exon-intron structure[J]. P Natl Acad Sci USA, 2012, 109(4): 1187-1192. doi: 10.1073/pnas.1109047109 URL
[27]	Castillo-Davis CI, Mekhedov SL, Hartl DL, et al. Selection for short introns in highly expressed genes[J]. Nat Genet, 2002, 31(4): 415-418. doi: 10.1038/ng940 pmid: 12134150
[28]	Heyn P, Kalinka AT, et al. Introns and gene expression: Cellular constraints, transcriptional regulation, and evolutionary consequences[J]. BioEssays, 2015, 37(2): 148-154. doi: 10.1002/bies.201400138 pmid: 25400101
[29]	Morel Ml, Crouzet Jrm, Gravot A, et al. AtHMA3, a P1B-ATPase allowing Cd/Zn/Co/Pb vacuolar storage in Arabidopsis[J]. Plant Physiol, 2008, 149(2): 894-904. doi: 10.1104/pp.108.130294 URL
[30]	Chen YY, Chao ZF, Jin M, et al. A heavy metal transporter gene ZmHMA3a promises safe agricultural production on cadmium-polluted arable land[J]. J Genet Genomics, 2023, 50(2): 130-134. doi: 10.1016/j.jgg.2022.08.003 URL
[31]	Liu H, Zhao HX, Wu LH, et al. Heavy metal ATPase 3(HMA3)confers cadmium hypertolerance on the cadmium/zinc hyperaccumulator Sedum plumbizincicola[J]. New Phytol, 2017, 215(2): 687-698. doi: 10.1111/nph.2017.215.issue-2 URL
[32]	Wahinya FW, Yamazaki K, Jing Z, et al. Sorghum ionomics reveals the functional SbHMA3a allele that limits excess cadmium accumulation in grains[J]. Plant Cell Physiol, 2022, 63(5): 713-728. doi: 10.1093/pcp/pcac035 URL
[33]	Seigneurin-Berny D, Gravot A, Auroy P, et al. HMA1, a new Cu-ATPase of the chloroplast envelope, is essential for growth under adverse light conditions[J]. J Biol Chem, 2006, 281(5): 2882-2892. doi: 10.1074/jbc.M508333200 pmid: 16282320
[34]	Zhao HX, Wang LS, Zhao FJ, et al. SpHMA1 is a chloro plast cadmium exporter protecting photochemical reactions in the Cd hyperaccumulator Sedum plumbizincicola[J]. Plant, Cell Environ, 2019, 42(4): 1112-1124. doi: 10.1111/pce.v42.4 URL

基因Gene	正向引物Forward primer（5'-3'）	反向引物 Reverse primer（5'-3'）
SlHMA1	CAGGAGGCCTTATAATGGTAGG	TAGTACATCAGCAACAGCTACC
SlHMA3	GTGTTCTGACTCAACAGACAAC	TTTCCACATGTCATTGAGCTTG
SlHMA4	GATGGCGTCGTTATTAATGGTC	ACAGTTCCACCAATGACCTTAT
SlHMA5.1	ATTCATCATTGGACGACGTTTC	AGCTGGTTCTTAACACCGAATA
SlHMA5.2	GCTTATGGATTTAGCCCCAAAG	TCGTTCTTTTGTATCAATCGGC
SlHMA6	TAGTCAGTGGAGATTTAGGGGA	CAATGATCTGATCTCCGACTGA
SlHMA7	CTGCAGAATACGTGTTGATGAG	GGGATGGAAATCACATTGTACG
SlHMA8	ATTCTCTCTCTCACACGATCAC	CAATAGCTGACTAGTACGACGT
SlActin	CCATTCTCCGTCTTGACTTGG	TCTTTCCTAATATCCACGTCAC

基因Gene	正向引物Forward primer（5'-3'）	反向引物 Reverse primer（5'-3'）
SlHMA1	CAGGAGGCCTTATAATGGTAGG	TAGTACATCAGCAACAGCTACC
SlHMA3	GTGTTCTGACTCAACAGACAAC	TTTCCACATGTCATTGAGCTTG
SlHMA4	GATGGCGTCGTTATTAATGGTC	ACAGTTCCACCAATGACCTTAT
SlHMA5.1	ATTCATCATTGGACGACGTTTC	AGCTGGTTCTTAACACCGAATA
SlHMA5.2	GCTTATGGATTTAGCCCCAAAG	TCGTTCTTTTGTATCAATCGGC
SlHMA6	TAGTCAGTGGAGATTTAGGGGA	CAATGATCTGATCTCCGACTGA
SlHMA7	CTGCAGAATACGTGTTGATGAG	GGGATGGAAATCACATTGTACG
SlHMA8	ATTCTCTCTCTCACACGATCAC	CAATAGCTGACTAGTACGACGT
SlActin	CCATTCTCCGTCTTGACTTGG	TCTTTCCTAATATCCACGTCAC

基因ID Gene ID	基因名称 Gene name	氨基酸数目 No. of amino acid	分子量 Molecular weight/kD	等电点 pI	稳定性 Instability index	脂肪系数 Aliphatic index	亲水性 GRAVY	亚细胞定位 Subcellular localization	跨膜结构域 TM
Solyc02g092920.2	SlHMA1	821	89.08	8.44	34.91	100.4	0.13	叶绿体 Chloroplast	0
Solyc07g009130.2	SlHMA3	1 302	140.44	6.94	45.63	80.46	-0.231	细胞质膜 Plasma membrane	8
Solyc11g062100.1	SlHMA4	698	76.04	5.68	36.13	104.44	0.227	细胞质膜 Plasma membrane	7
Solyc08g080870.2	SlHMA5.1	954	103.58	5.58	39.69	102.69	0.139	细胞质膜 Plasma membrane	6
Solyc08g080890.2	SlHMA5.2	890	96.58	5.80	32.38	103.96	0.200	细胞质膜 Plasma membrane	8
Solyc01g105160.4	SlHMA6	964	101.94	8.30	33.79	97.68	0.092	叶绿体 Chloroplast	0
Solyc02g068490.3	SlHMA7	1 015	108.59	5.16	30.72	106.97	0.324	细胞质膜 Plasma membrane	10
Solyc08g061610.2	SlHMA8	894	95.20	5.94	38.01	103.66	0.118	叶绿体 Chloroplast	0

基因ID Gene ID	基因名称 Gene name	氨基酸数目 No. of amino acid	分子量 Molecular weight/kD	等电点 pI	稳定性 Instability index	脂肪系数 Aliphatic index	亲水性 GRAVY	亚细胞定位 Subcellular localization	跨膜结构域 TM
Solyc02g092920.2	SlHMA1	821	89.08	8.44	34.91	100.4	0.13	叶绿体 Chloroplast	0
Solyc07g009130.2	SlHMA3	1 302	140.44	6.94	45.63	80.46	-0.231	细胞质膜 Plasma membrane	8
Solyc11g062100.1	SlHMA4	698	76.04	5.68	36.13	104.44	0.227	细胞质膜 Plasma membrane	7
Solyc08g080870.2	SlHMA5.1	954	103.58	5.58	39.69	102.69	0.139	细胞质膜 Plasma membrane	6
Solyc08g080890.2	SlHMA5.2	890	96.58	5.80	32.38	103.96	0.200	细胞质膜 Plasma membrane	8
Solyc01g105160.4	SlHMA6	964	101.94	8.30	33.79	97.68	0.092	叶绿体 Chloroplast	0
Solyc02g068490.3	SlHMA7	1 015	108.59	5.16	30.72	106.97	0.324	细胞质膜 Plasma membrane	10
Solyc08g061610.2	SlHMA8	894	95.20	5.94	38.01	103.66	0.118	叶绿体 Chloroplast	0