Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (21): 4318-4327.doi: 10.3864/j.issn.0578-1752.2014.21.017

• HORTICULTURE • Previous Articles     Next Articles

Expression and Protein Interaction Analysis of Light Responsive bZIP Transcription Factor MdHY5

LI Hui-feng1, WANG Xiao-fei2, RAN Kun1, HE Ping1, WANG Hai-bo1, LI Lin-guang1   

  1. 1Shandong Institute of Pomology, Tai’an 271000, Shandong
    2College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an 271018, Shandong
  • Received:2014-03-31 Revised:2014-06-30 Online:2014-11-01 Published:2014-11-01

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Abstract: 【Objective】Cloning of the key light responsive bZIP (Basic-leucine zipper) transcription factors from Malus domestica Borkh, gene expression and protein interaction analysis, which were determined to identify the function and molecular mechanism of MdHY5.【Method】First, semi-quantitative primers used for detecting expression levels were designed from the apple in order to identify the light-responsive bZIP genes. Differentially screened and identified a light responsive gene named as MdHY5 based on comparative analysis of homologous. Subsequently, the primers were designed to clone the MdHY5 gene. Phylogenetic relationship including the MdHY5 protein with Arabidopsis bZIP proteins was analyzed using MEGA5 and the multiple alignment of bZIP domain was constructed, while the conserved protein domain was predicted, spatio expression analysis was made to detect the MdHY5 in different organs. The expression of MdHY5 in response to various light qualities was detected by semi-quantitative RT-PCR based on promoter prediction analysis, combined with the Arabidopsis【Result】A bZIP transcription factor was isolated in apple through the light response analysis. Phylogenetic relationship showed that MdHY5 is a homolog of Arabidopsis AtHY5. This gene is located on chromosome 12 of apple genome, with the gene number MDP0000586302, MdHY5 gene contains four exons and three introns in genome structure, which is similar to its homolog. The length of the cDNA is 1 112 bp, with 214 bp in the 5′ untranslated region, 403 bp in 3′ untranslated region, and 495 bp of open reading frame, which encode a 164 amino acid residues. MdHY5 contains a leucine zipper structure typical (bZIP domain) in the C-terminal of the protein, with a nuclear localization signal domain (NLS domain) included. Expression analysis showed that MdHY5 is expressed in various tissues, with the highest level in leaves. Promoter analysis showed that multiple light responsive cis-acting element exists in the promoters of MdHY5, such as G-box, GT-1-box, I-box and as so on. Quantitative expression analysis showed that MdHY5 gene could be induced by white, blue and UV light. The prokaryotic expression vector was transformed into BL21 and the fusion protein presented mainly in the supernatant after sonication. Pull down assay showed that MdHY5 could interact with MdCOP1 protein in vitro. 【Conclusion】 MdHY5 is a light responsive bZIP transcription factor and homologous to AtHY5, which is the key transcription factor in photomorphogenesis of Arabidopsis, MdHY5 has similar genome structure and conserved protein domain to AtHY5, and can be interacted with MdCOP1. microarray database. Finally, MdHY5 gene was inserted into the prokaryotic vector pGEX-4T-1, and IPTG was used to induce the fusion protein expression in E.coli BL21. Then the fusion protein was purified and detected by Western blot. The interaction between MdHY5 and MdCOP1 was detected by pull down assay.

Key words: apple, bZIP transcription factor, MdHY5, light responsive, protein interaction

[1]    陈学森, 韩明玉, 苏桂林, 刘凤之, 过国南, 姜远茂, 毛志泉, 彭福田, 束怀瑞. 当今世界苹果产业发展趋势及我国苹果产业优质高效发展意见. 果树学报, 2010, 27(4): 598- 604.
Chen X S, Han M Y, Su G L, Liu F Z, Guo G N, Jiang Y M, Mao Z Q, Peng F T, Shu H R. Discussion on today’s world apple industry trends and the suggestions on sustainable and efficient development of apple industry in China. Journal of Fruit Science, 2010, 27(4): 598-604. (in Chinese)
[2]    Kendrick R E, Kronenberg G H M. Photomorphogenesis in Plants. Springer, 1994.
[3]    Smith H. Phytochromes and light signal perception by plants-an emerging synthesis. Nature, 2000, 407(6804): 585-591.
[4]    Sullivan J A, Shirasu K, Deng X W. The diverse roles of ubiquitin and the 26S proteasome in the life of plants. Nature Reviews Genetics, 2003, 4(12): 948-958.
[5]    Ang L H, Chattopadhyay S, Wei N, Oyama T, Okada K, Batschauer A, Deng X W. Molecular interaction between COP1 and HY5 defines a regulatory switch for light control of Arabidopsis development. Molecular Cell, 1998, 1(2): 213-222.
[6]    Chattopadhyay S, Ang L H, Puente P, Deng X W, Wei N. Arabidopsis bZIP protein HY5 directly interacts with light-responsive promoters in mediating light control of gene expression. The Plant Cell, 1998, 10(5): 673-683.
[7]    Jonassen E M, Sandsmark B A, Lillo C. Unique status of NIA2 in nitrate assimilation: NIA2 expression is promoted by HY5/HYH and inhibited by PIF4. Plant Signaling & Behavior, 2009, 4(11): 1084-1086.
[8]    Chen M, Chory J, Fankhauser C. Light signal transduction in higher plants. Annual Review of Genetics, 2004, 38: 87-117.
[9]    Rizzini L, Favory J J, Cloix C, Faggionato D, O’Hara A, Kaiserli E, Ulm R. Perception of UV-B by the Arabidopsis UVR8 protein. Science, 2011, 332(6025): 103-106.
[10]   Christie J M, Arvai A S, Baxter K J, Heilmann M, Pratt A J, O’Hara A, Getzoff E D. Plant UVR8 photoreceptor senses UV-B by tryptophan- mediated disruption of cross-dimer salt bridges. Science, 2012, 335(6075): 1492-1496.
[11]   Wei N, Deng X W. The COP9 signalosome. Annual Review of Cell and Developmental Biology, 2003, 19(1): 261-286.
[12]   Jakoby M, Weisshaar B, Dröge-Laser W, Vicente-Carbajosa J, Tiedemann J, Kroj T, Parcy F. bZIP transcription factors in Arabidopsis. Trends in Plant Science, 2002, 7(3): 106-111.
[13]   Finkelstein R R, Lynch T J. The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor. The Plant Cell, 2000, 12(4): 599-609.
[14]   Oñate L, Vicente-Carbajosa J, Lara P, D??az I, Carbonero P. Barley BLZ2, a seed-specific bZIP protein that interacts with BLZ1 in vivo and activates transcription from the GCN4-like motif of B-hordein promoters in barley endosperm. Journal of Biological Chemistry, 1999, 274(14): 9175-9182.
[15]   Pelaz S, Ditta G S, Baumann E, Wisman E, Yanofsky M F. B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature, 2000, 405(6783): 200-203.
[16]   田义, 陈可钦, 安秀红, 郝红梅, 刘志, 丛佩华, 郝玉金. 两个苹果乙烯反应因子MdAP2D4与MdAP2D19的序列分析及原核表达. 中国农业科学, 2013, 46(17): 3635-3642.
Tian Y, Chen K Q, An X H, Hao H M, Liu Z, Cong P H, Hao Y J. Sequence analysis and prokaryotic expression of two ethylene responsive factors MdAP2D4 and MdAP2D19. Scientia Agricultura Sinica, 2013, 46(17): 3635-3642.
[17]   Koornneef M, Rolff E, Spruit C J P. Genetic control of light-inhibited hypocotyl elongation in Arabidopsis thaliana (L.) Heynh. Zeitschrift für Pflanzenphysiologie, 1980, 100(2): 147-160.
[18]   Oyama T, Shimura Y, Okada K. The Arabidopsis HY5 gene encodes a bZIP protein that regulates stimulus-induced development of root and hypocotyl. Genes & Development, 1997, 11(22): 2983-2995.
[19]   Ulm R, Baumann A, Oravecz A, Máté Z, Adám E, Oakeley E J, Schäfer E, Nagy F. Genome-wide analysis of gene expression reveals function of the bZIP transcription factor HY5 in the UV-B response of Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(5): 1397-1402.
[20]   Li Y Y, Mao K, Zhao C, Zhao X Y, Zhang, H L, Shu H R, Hao, Y J. MdCOP1 ubiquitin E3 ligases interact with MdMYB1 to regulate light-induced anthocyanin biosynthesis and red fruit coloration in apple. Plant Physiology, 2012, 160(2): 1011-1022.
[21]   Osterlund M T, Hardtke C S, Wei N, Deng X W. Targeted destabilization of HY5 during light-regulated development of Arabidopsis. Nature, 2000, 405(6785): 462-466.
[22]   Holm M, Ma L G, Qu L J, Deng X W. Two interacting bZIP proteins are direct targets of COP1-mediated control of light-dependent gene expression in Arabidopsis. Genes & Development, 2002, 16(10): 1247-1259.
[23]   Saijo Y, Sullivan J A, Wang H, Yang J, Shen Y, Rubio V, Deng X W . The COP1-SPA1 interaction defines a critical step in phytochrome A-mediated regulation of HY5 activity. Genes & Development, 2003, 17(21): 2642-2647.
[24]   Seo H S, Watanabe E, Tokutomi S, Nagatani A, Chua N H. Photoreceptor ubiquitination by COP1 E3 ligase desensitizes phytochrome A signaling. Genes & Development, 2004, 18(6): 617-622.
[25]   Jang I C, Yang J Y, Seo H S, Chua N H. HFR1 is targeted by COP1 E3 ligase for post-translational proteolysis during phytochrome A signaling. Genes & Development, 2005, 19(5): 593-602.
[26]   Yang J, Lin R, Sullivan J, Hoe U, Liu B, Xu L, Wang H. Light regulates COP1-mediated degradation of HFR1, a transcription factor essential for light signaling in Arabidopsis. The Plant Cell, 2005, 17(3): 804-821.
[27]   Hardtke C S, Gohda K, Osterlund M T, Oyama T, Okada K, Deng X W. HY5 stability and activity in Arabidopsis is regulated by phosphorylation in its COP1 binding domain. The EMBO Journal, 2000, 19(18): 4997-5006.
[28]   Duek P D, Elmer M V, van Oosten, V R, Fankhauser C. The degradation of HFR1, a putative bHLH class transcription factor involved in light signaling, is regulated by phosphorylation and requires COP1. Current Biology, 2004, 14(24): 2296-2301.
[29]   Cluis C P, Mouchel C F, Hardtke C S. The Arabidopsis transcription factor HY5 integrates light and hormone signaling pathways. The Plant Journal, 2004, 38(2): 332-347.
[30]   Lee J, He K, Stolc V, Lee H, Figueroa P, Gao Y, Deng X W. Analysis of transcription factor HY5 genomic binding sites revealed its hierarchical role in light regulation of development. The Plant Cell, 2007, 19(3): 731-749.
[31]   Zhang H, He H, Wang X, Wang X, Yang X, Li L, Deng X W. Genome-wide mapping of the HY5-mediated gene networks in Arabidopsis that involve both transcriptional and post-transcriptional regulation. The Plant Journal, 2011, 65(3): 346-358.
[32]   Saijo Y, Sullivan J A, Wang H, Yang J, Shen Y, Rubio V, Deng X W. The COP1-SPA1 interaction defines a critical step in phytochrome A-mediated regulation of HY5 activity. Genes & Development, 2003, 17(21): 2642-2647.
[33]   刘志. 苹果抗轮纹病种质资源和基因筛选以及NO介导的防御反应[D]. 山东泰安: 山东农业大学, 2009.
Liu Z. Studies on screening for apple germplasm and genes resistant to ring rot disease, and NO-mediated defensive [D]. Tai’an, Shandong: Shandong Agricultural University, 2009. (in Chinese)
[34]   Poole R L. The TAIR database. Methods Molecular Biology, 2007, 406: 179-212.
[35]   Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, Chu V T. The genome of the domesticated apple (Malus domestica Borkh.). Nature Genetics, 2010, 42(10): 833-839.
[36]   Solovyev V, Kosarev P, Seledsov I, Vorobye D. Automatic annotation of eukaryotic genes, pseudogenes and promoters. Genome Biology, 2006, 7, S10: 1-12.
[37]   Higo K, Ugawa Y, Iwamoto M, Korenaga T. Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Research, 1999, 27(1): 297-300.
[38]   Gutterson N, Reuber T L. Regulation of disease resistance pathways by AP2/ERF transcription factors. Current Opinion in Plant Biology, 2004, 7(4): 465-471.
[39]   Vandenbussche F, Habricot Y, Condiff A S, Maldiney R, Van der Straeten D, Ahmad M. HY5 is a point of convergence between cryptochrome and cytokinin signalling pathways in Arabidopsis thaliana. The Plant Journal, 2007, 49(3): 428-441.
[40]   Kindgren P, Norén L, López Jde D, Shaikhali J, Strand A. Interplay between Heat Shock Protein 90 and HY5 controls PhANG expression in response to the GUN5 plastid signal. Molecular Plant, 2012, 5(4): 901-913.
[41]   Li Z, Zhang L, Yu Y, Quan R, Zhang Z, Zhang H, Huang R. The ethylene response factor AtERF11 that is transcriptionally modulated by the bZIP transcription factor HY5 is a crucial repressor for ethylene biosynthesis in Arabidopsis. The Plant Journal, 2011, 68(1): 88-99.
[42]   Son G H, Wan J, Kim H J, Nguyen X C, Chung W S, Hong J C, Stacey G. Ethylene-responsive element-binding factor 5, ERF5, is involved in chitin-induced innate immunity response. Molecular Plant-Microbe Interactions, 2012, 25(1): 48-60.
[43]   Gu Y Q, Yang C, Thara V K, Zhou J, Martin G B. Pti4 is induced by ethylene and salicylic acid, and its product is phosphorylated by the Pto kinase. The Plant Cell, 2000, 12(5): 771-785.
[44]   Cheong Y H, Moon B C, Kim J K, Kim C Y, Kim M C, Kim I H, Cho M J. BWMK1, a rice mitogen-activated protein kinase, locates in the nucleus and mediates pathogenesis-related gene expression by activation of a transcription factor. Plant Physiology, 2003, 132(4): 1961-1972.
[45]   Wang P, Du Y, Zhao X, Miao Y, Song C P. The MPK6-ERF6- ROS-responsive cis-acting element7/GCC box complex modulates oxidative gene ranscription and the oxidative response in Arabidopsis. Plant Physiology, 2013, 161(3): 1392-1408.
[46]   Brown R L, Kazan K, McGrath K C, Maclean D J, Manners J M. A role for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of Arabidopsis. Plant Physiology, 2003, 132(2): 1020-1032.
[47]   Fujimoto S Y, Ohta M, Usui A, Shinshi H, Ohme-Takagi M. Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression. The Plant Cell, 2000, 12(3): 393-404.
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