Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (16): 3082-3092.doi: 10.3864/j.issn.0578-1752.2022.16.002


The Functional Analysis of High Mobility Group MsHMG-Y Involved in Flowering Regulation in Medicago sativa L.

ZHANG YunXiu(),JIANG Xu,WEI ChunXue,JIANG XueQian,LU DongYu,LONG RuiCai,YANG QingChuan,WANG Zhen,KANG JunMei()   

  1. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193
  • Received:2022-03-16 Accepted:2022-05-05 Online:2022-08-16 Published:2022-08-11
  • Contact: JunMei KANG;


【Objective】Flowering is an important signal indicating the transformation from vegetative growth to reproductive growth and has a significant effect on plant biomass. Alfalfa is one of the upmost forage crops worldwide, its yield and quality are closely related to flowering time. The optimum harvest time for alfalfa is during the early flowering stage, which could give the highest yield and the best quality. In the current study, an alfalfa flowering related gene, Medicago sativa High Mobility Group Y (MsHMG-Y), was cloned. The gene structure and expression pattern of MsHMG-Y were studied. Function of MsHMG-Y in alfalfa flowering regulation was analyzed. This work could provide theoretical support for mechanism study underlying flowering regulation. 【Method】MsHMG-Y was cloned by homology cloning strategy and the amino acid sequence was analyzed by multiple sequence alignment. The phylogenetic tree was also constructed. qRT-PCR analysis was used to detect the expression level of MsHMG-Y in different tissues and different flowering stages. The expression pattern of MsHMG-Y under light, gibberellin (GA3), salicylic acid (SA) or methyl jasmonate (MeJA) treatment were analyzed. The phenotype of MsHMG-Y-overexpressing alfalfa was analyzed, and the expression levels of flowering activators and suppressors were also analyzed. 【Result】Phylogenetic analysis showed that MsHMG-Y has the closest relationship with MtHMG-Y in Medicago truncatula. Spatial expression pattern analysis showed that MsHMG-Y was expressed in flowers, stems and leaves, with the highest expression level in flowers and the lowest expression level in leaves in both paternal and maternal alfalfa. In paternal alfalfa with early flowering phenotype, the expression level of MsHMG-Y was the highest at early flowering stage. The highest expression level of MsHMG-Y was detected at flower bud differentiation stage in maternal alfalfa with late flower phenotype. Photoperiod analysis showed that MsHMG-Y was down-regulated after 16-hour light treatment. After 28 hours of light treatment, the expression level of MsHMG-Y was continuously lower than that in the control group, indicating that MsHMG-Y was down-regulated after light treatment. After 50 μmol·L-1 GA3, 100 μmol·L-1 SA or 100 μmol·L-1 MeJA treatment, the expression level of MsHMG-Y was up-regulated compared with the mock treatment. In detail, the expression level of MsHMG-Y was the highest at 1 h under GA3 treatment, which was 3.5 folds higher than control. Under SA treatment, the expression level of MsHMG-Y was the highest at 6 h, which was 24 folds higher than the mock treatment. The expression level of MsHMG-Y was the highest at 3 h under MeJA treatment, which was 11 folds higher than the control. These results indicated that the expression of MsHMG-Y was inducible by the above three hormones. MsHMG-Y-overexpressing alfalfa has late flowering phenotype. The expression levels of flowering activator genes were down-regulated in MsHMG-Y-overexpressing alfalfa, while the expression levels of flowering inhibitor genes were up-regulated. Among these genes, expression of flowering activator genes MsPHYA, MsGI and MsFTa1 was significantly down-regulated by 4.9 folds, 3.9 folds and 2.8 folds respectively, and the expression level of flowering inhibitor genes MsTEM and MsSVP was increased by 2.5 folds and 1.9 folds, respectively. 【Conclusion】The expression of MsHMG-Y is inducible by photoperiod and exogenous hormone treatment, including GA3, SA and MeJA. Overexpression of MsHMG-Y in alfalfa resulted in delayed flowering time. MsHMG-Y plays an important role in regulatory mechanism underlying late flowering in alfalfa.

Key words: alfalfa, High Mobility Group-Y, flowering regulation, expression pattern

Fig. 1

Analysis of multiple sequences The black line and bold letters A-D represent the HMG-Y conserved domain, and square represents the HMG-Y functional domain"

Fig. 2

Phylogenetic analysis of MsHMG-Y GLYMA: Glycine max; Ms: Medicago sativa; MTR: Medicago truncatula; At: Arabidopsis thaliana; Os: Oyza sativa; Zm: Zea mays; Traes: Triticum aestium; POPTR: Poplar tree. The value at the bifurcation is the positive value of bootstrap correction"

Table 1

Predictive analysis of promoter acting elements"

Cis acting element
Sequence (5′-3′)
TCA-element CCATCTTTTT 烟草Nicotiana tabacum 1 参与水杨酸反应的顺式作用元件
Cis-acting element involved in salicylic acid responsiveness
TGACG-motif TGACG 大麦Hordeum vulgare 1 参与茉莉酸甲酯反应的顺式作用元件
Cis-acting regulatory element involved in the MeJA-responsiveness
CGTCA-motif CGTCA 大麦Hordeum vulgare 1 参与茉莉酸甲酯反应的顺式作用元件
Cis-acting regulatory element involved in the MeJA-responsiveness
GT1-motif GGTTAA 拟南芥Arabidopsis thaliana 1 光响应元件 Light responsive element
TCT-motif TCTTAC 拟南芥Arabidopsis thaliana 1 光响应元件 A light responsive element
G-box CACGAC 玉米Zea mays 1 光响应顺式作用元件
Cis-acting regulatory element involved in light responsiveness

Fig. 3

Tissue specificity and expression analysis of MSHMG-Y in different developmental stages of parental flowers A: Tissue specific expression; B: Differential expression in three stages of parental flowering. P: Paternal parent, M: Maternal parent. Different letters represented the significant difference. The same as below"

Fig. 4

Expression analysis of MSHMG-Y treated by light and exogenous hormone A: Light treatment; B: GA3 treatment; C: SA treatment; D: MeJA treatment. CK: Control, GA3: Gibberellin 3, SA: Salicylic acid, MeJA: Methyl jasmonate"

Fig. 5

Phenotypic identification and identification of transgenic Alfalfa A: Phenotypic identification of transgenic Alfalfa; B: Expression level of MSHMG-Y in transgenic Alfalfa using qRT-PCR"

Fig. 6

Expression analysis of flowering related genes in transgenic Alfalfa A: Flowering promoting genes; B: Flower inhibiting genes"

[1] JUNG C, MULLER A E. Flowering time control and applications in plant breeding. Trends in Plant Science, 2009, 14(10): 563-573.
doi: 10.1016/j.tplants.2009.07.005
[2] FORNARA F, MONTAIGU A, COUPIAND G. SnapShot: Control of flowering in Arabidopsis. Cell, 2010, 141(3): 550, 550.e1-2.
doi: 10.1016/j.cell.2010.04.024
[3] KINOSHITA A, RICHTER R. Genetic and molecular basis of floral induction in Arabidopsis thaliana. Journal of Experimental Botany, 2020, 71(9): 2490-2504.
doi: 10.1093/jxb/eraa057
[4] AUNG B, GRUBER M Y, AMYOT L, OMARI K, BERTRAND A, HANNOUFA A. MicroRNA156 as a promising tool for alfalfa improvement. Plant Biotechnology Journal, 2015, 13(6): 779-790.
doi: 10.1111/pbi.12308
[5] BIANCHI M E, BELTRAME M, PANOESSA G. Specific recognition of cruciform DNA by nuclear protein HMG1. Science, 1989, 243: 1056-1059.
doi: 10.1126/science.2922595
[6] CLAUS P, SCHULZE E, WISNIEWSKI J R. Insect proteins homologous to mammalian high mobility group proteins I/Y (HMG I/Y). Characterization and binding to linear and four-way junction DNA. Journal of Biological Chemistry, 1994, 269: 33042-33048.
doi: 10.1016/S0021-9258(20)30095-8
[7] JACOBSEN K, LAURSEN N B, JENSEN E O, MARCKER A, POULSEN C, MARCKER K A. HMG I-like proteins from leaf and nodule nuclei interact with different AT motifs in soybean nodulin promoters. The Plant Cell, 1990, 2: 85-94.
[8] PEDERSEN T J, ARWOOD L J, SPIER S, GUILTINAN M J, THOMPSON W F. High mobility group chromosomal proteins bind to AT-rich tracts flanking plant genes. Plant Molecular Biology, 1991, 16: 95-104.
doi: 10.1007/BF00017920
[9] NIETO-SOTELO J, ICHIDA A, QUAIL P H. PF1: An A-T hook-containing DNA binding protein from rice that interacts with a functionally defined d(AT)-rich element in the oat phytochrome A3 gene promoter. The Plant Cell, 1994, 6: 287-301.
[10] PWEE K H, WEBSTER C I, GRAY J C. HMG protein binding to an A/T-rich positive regulatory region of the pea plastocyanin gene promoter. Plant Molecular Biology, 1994, 26: 1907-1920.
doi: 10.1007/BF00019502
[11] YAMAMOTO S, MINAMIKAWA T. Two genes for the high mobility group protein HMG-Y are present in the genome of Canavalia gladiata D.C. Plant Molecular Biology, 1997, 33: 537-544.
doi: 10.1023/A:1005791728975
[12] MALLIK R, PRASAD P, KUNDU A, SACHDEV S, CHAUDHURI S. Identification of genome-wide targets and DNA recognition sequence of the Arabidopsis HMG-box protein AtHMGB15 during cold stress response. Biochimica et Biophysica Acta, 2020, 1863(12): 194644.
[13] GUPTA R, WEBSTER C I, WALKER A R, GRAY J C. Chromosomal location and expression of the single-copy gene encoding high- mobility-group protein HMG-I/Y in Arabidopsis thaliana. Plant Molecular Biology, 1997, 34(3): 529-536.
doi: 10.1023/A:1005828430861
[14] SAWA S, WATANABE K, GOTO K, LIU Y G, SHIBATA D, KANAYA E, MORITA E H, OKADA K. FILAMENTOUS FLOWER, a meristem and organ identity gene of Arabidopsis, encodes a protein with a zinc finger and HMG-related domains. Genes & Development, 1999, 13(9): 1079-1088.
doi: 10.1101/gad.13.9.1079
[15] GALVAO V C, COLLANI S, HORRER D, SCHMID M. Gibberellic acid signaling is required for ambient temperature-mediated induction of flowering in Arabidopsis thaliana. The Plant Journal, 2015, 84(5): 949-962.
doi: 10.1111/tpj.13051
[16] MARTINEZ-GARCIA J F, QUAI P H. The HMG-I/Y protein PF1 stimulates binding of the transcriptional activator GT-2 to the PHYA gene promoter. The Plant Journal, 1999, 18(2): 173-183.
doi: 10.1046/j.1365-313X.1999.00440.x
[17] XU M, XU Z, LIU B, KONG F, TSUOKURA Y, WATANABE S, XIA Z, HARADA K, KANAZAWA A, YAMADA T, ABE J. Genetic variation in four maturity genes affects photoperiod insensitivity and PHYA-regulated post-flowering responses of soybean. BMC Plant Biology, 2013, 13: 91.
doi: 10.1186/1471-2229-13-91
[18] LONG R, ZHANG F, ZHANG Z, LI M, CHEN L, WANG X, LIU W, ZHANG T, YU L X, HE F, JIANG X, YANG X, YANG C, WANG Z, KANG J, YANG Q. Assembly of chromosome-scale and allele-aware autotetraploid genome of the Chinese alfalfa cultivar Zhongmu-4 and identification of SNP loci associated with 27 agronomic traits. Genomics Proteomics Bioinformatics, 2022.
[19] TAMURA K, PETERSON D, PETERSON N, STECHER G, NEI M, KUMAR S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 2011, 28(10): 2731-2739.
doi: 10.1093/molbev/msr121
[20] WANG K. Preface. Agrobacterium protocols. Methods in Molecular Biology, 2015, 1224: vii-viii.
[21] BLUMEL M, DALLY N, JUNG C. Flowering time regulation in crops-what did we learn from Arabidopsis? Current Opinion in Biotechnology, 2015, 32: 121-129.
doi: 10.1016/j.copbio.2014.11.023
[22] 雍伟东, 种康, 许智宏, 谭克辉, 朱至清. 高等植物开花时间决定的基因调控研究. 科学通报, 2000, 45(5): 455-466.
YONG W D, ZHONG K, XU Z H, TAN K H, ZHU Z Q. Gene regulation of flowering time determination in higher plants. Chinese Science Bulletin, 2000, 45(5): 455-466. (in Chinese)
[23] WANG L F. Physiological and molecular responses to variation of light intensity in rubber tree (Hevea brasiliensis Muell Arg). PLoS ONE, 2014, 9(2): e89514.
doi: 10.1371/journal.pone.0089514
[24] CHO L H, YOON J, AN G. The control of flowering time by environmental factors. The Plant Journal, 2017, 90(4): 708-719.
doi: 10.1111/tpj.13461
[25] BAO S, HUA C, SHEN L, YU H. New insights into gibberellin signaling in regulating flowering in Arabidopsis. Journal of Integrative Plant Biology, 2020, 62(1): 118-131.
doi: 10.1111/jipb.12892
[26] KHAN F S, GAN Z M, LI E Q, REN M K, HU C G, ZHANG J Z. Transcriptomic and physiological analysis reveals interplay between salicylic acid and drought stress in citrus tree floral initiation. Plant Molecular Biology, 2021, 255(1): 24.
[27] LI L, ZHAO Y, MCCAIG B C, WINGERD B A, WANG J, WHALON M E, PICHERSKY E, HOWE G A. The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development. The Plant Cell, 2004, 16(1): 126-143.
doi: 10.1105/tpc.017954
[28] 龚建英, 王华新, 汪小玉, 梁萍, 覃荣, 唐遒冥, 陈宝玲, 苏莉花. 不同外源激素对绿莹石斛萌芽及开花的影响. 广西林业科学, 2021, 50(5): 551-556.
GONG J Y, WANG H X, WANG X Y, LIANG P, QIN R, TANG Q M, CHEN B L, SU L H. Effects of Different exogenous hormones on germination and flowering of Dendrobium greenum. Guangxi Forestry Science, 2021, 50(5): 551-556. (in Chinese)
[29] LORENZO C D, GARCIA-GAGLIARDI P, ANTONIETTI M S, SANCHEZ-LAMAS M, MANCINI E, DEZAR C A, VAZQUEZ M, WATSON G, YANOVSKY M J, CERDAN P D. Improvement of alfalfa forage quality and management through the down- regulation of MsFTa1. Plant Biotechnology Journal, 2020, 18(4): 944-954.
doi: 10.1111/pbi.13258
[30] 杨凤玺, 徐庆全, 朱根发. 不同植物激素处理对建兰开花的影响. 中国观赏园艺研究进展, 2015: 447-451.
YANG F X, XU Q Q, ZHU G F. Effects of different plant hormones on flowering of Cymbidium chinensis. Advances in Ornamental Horticulture of China, 2015: 447-451. (in Chinese)
[31] JAUDAL M, WEN J, MYSORE K S, PUTTERILL J. Medicago PHYA promotes flowering, primary stem elongation and expression of flowering time genes in long days. BMC Plant Biology, 2020, 20: 329.
doi: 10.1186/s12870-020-02540-y
[32] PARK D H, SOMERS D E, KIM Y S, CHOY Y H, LIM H K, SOH M S, KIM H J, KAY S A, NAM H G. Control of circadian rhythms and photoperiodic flowering by the Arabidopsis GIGANTEA gene. Science, 1999, 285: 1579-1582.
doi: 10.1126/science.285.5433.1579
[33] HARTMANN U, HOHMANN S, NETTESHEIM K, WISMAN E, AEDLER H, HUIJSER P. Molecular cloning of SVP: A negative regulator of the floral transition in Arabidopsis. The Plant Journal, 2000, 21: 351-360.
doi: 10.1046/j.1365-313x.2000.00682.x
[34] FUKAZAWA J, OHASHI Y, TAKAHASHI R, NAKAI K, TAKAHASHI Y. DELLA degradation by gibberellin promotes flowering via GAF1-TPR-dependent repression of floral repressors in Arabidopsis. The Plant Cell, 2021, 33(7): 2258-2272.
doi: 10.1093/plcell/koab102
[1] SU Qian,DU WenXuan,MA Lin,XIA YaYing,LI Xue,QI Zhi,PANG YongZhen. Cloning and Functional Analyses of MsCIPK2 in Medicago sativa [J]. Scientia Agricultura Sinica, 2022, 55(19): 3697-3709.
[2] MA Lin,WEN HongYu,WANG XueMin,GAO HongWen,PANG YongZhen. Cloning and Function Analysis of MsMAX2 Gene in Alfalfa (Medicago sativa L.) [J]. Scientia Agricultura Sinica, 2021, 54(19): 4061-4069.
[3] XU HuanHuan,LI Yi,GAO Wei,WANG YongQin,LIU LeCheng. Cloning and Identification of γ-Glutamyl Transpeptidase AcGGT Gene from Onion (Allium cepa) [J]. Scientia Agricultura Sinica, 2021, 54(19): 4169-4178.
[4] SHI GuoLiang,WU Qiang,YANG NianWan,HUANG Cong,LIU WanXue,QIAN WanQiang,WAN FangHao. Gene Cloning, Expression Pattern and Molecular Characterization of Chitin Deacetylase 2 in Cydia pomonella [J]. Scientia Agricultura Sinica, 2021, 54(10): 2105-2117.
[5] ZeMin LI,Chen ZHANG,ChongYu ZHANG,GuiGuo ZHANG. The Relationship Between Nutrients and Biological Yield of Different Varieties of Alfalfa [J]. Scientia Agricultura Sinica, 2020, 53(6): 1269-1277.
[6] XING QiKai,LI LingXian,CAO Yang,ZHANG Wei,PENG JunBo,YAN JiYe,LI XingHong. Prediction and Analysis of Candidate Secreted Proteins from the Genome of Lasiodiplodia theobromae [J]. Scientia Agricultura Sinica, 2020, 53(24): 5027-5038.
[7] KANG JunMei,ZHANG QiaoYan,JIANG Xu,WANG Zhen,ZHANG TieJun,LONG RuiCai,CUI HuiTing,YANG QingChuan. Cloning MsSQE1 from Alfalfa and Functional Analysis in Saponin Synthesis [J]. Scientia Agricultura Sinica, 2020, 53(2): 247-260.
[8] JIANG Xu,CUI HuiTing,WANG Zhen,ZHANG TieJun,LONG RuiCai,YANG QingChuan,KANG JunMei. Cloning and Function Analysis of MsNST in Lignin and Cellulose Biosynthesis Pathway from Alfalfa [J]. Scientia Agricultura Sinica, 2020, 53(18): 3818-3832.
[9] HAO ShuLin,CHEN HongWei,LIAO FangLi,LI Li,LIU ChangYan,LIU LiangJun,WAN ZhengHuang,SHA AiHua. Analysis of F-Box Gene Family Based on Salt-Stressed Transcriptome Sequencing in Vicia faba L. [J]. Scientia Agricultura Sinica, 2020, 53(17): 3443-3454.
[10] LIU JiaoJiao,WANG XueMin,MA Lin,CUI MiaoMiao,CAO XiaoYu,ZHAO Wei. Isolation, Identification, and Response to Abiotic Stress of MsWRKY42 Gene from Medicago sativa L. [J]. Scientia Agricultura Sinica, 2020, 53(17): 3455-3466.
[11] GONG Hao,YANG Liu,LI DanDan,LIU GuoFu,XIAO ZhiXin,WU QingYing,CUI GuoWen. Response of Alfalfa Production and Quality to Fertilization and Cutting Frequency and Benefit Analysis in Mollisol Agricultural Area in Cold Region [J]. Scientia Agricultura Sinica, 2020, 53(13): 2657-2667.
[12] XIAO ZhiXin,WANG Yang,LIU GuoFu,GONG Hao,LI DanDan,GONG Lin,BAI ZhenJian,CUI GuoWen. Effects of Fertilizing Time in Early Spring on Alfalfa (Medicago sativa) Production Performance and Nutritional Quality in Mollisol Area in Cold Region [J]. Scientia Agricultura Sinica, 2020, 53(13): 2668-2677.
[13] XIAO LuoDan, TANG Lei, WANG WeiDong, GAO YueFang, HUANG YiFan, MENG Yang, YANG YaJun, XIAO Bin. Cloning and Functional Analysis of CsWRKYIIcs Transcription Factors in Tea Plant [J]. Scientia Agricultura Sinica, 2020, 53(12): 2460-2476.
[14] XiaoDong LI,YiShun SHANG,ShiGe LI,GuangJi CHEN,ChengJiang PEI,Fang SUN,XianQin XIONG. The Mechanism of Ectopic Expression of Brassica juncea Multidrug and Toxic Compound Extrusion (BjMATE) to Enhance the Resistance to Acid and Aluminum Stress in Alfalfa [J]. Scientia Agricultura Sinica, 2020, 53(1): 18-28.
[15] JunBo PENG,XingHong LI,Wei ZHANG,Ying ZHOU,JinBao HUANG,JiYe YAN. Pathogenicity and Gene Expression Pattern of the Exocrine Protein LtGH61A of Grape Canker Fungus [J]. Scientia Agricultura Sinica, 2019, 52(24): 4518-4526.
Full text



No Suggested Reading articles found!