Plant Cell Reports

, Volume 38, Issue 8, pp 899–914 | Cite as

Identification of sugar response complex in the metallothionein OsMT2b gene promoter for enhancement of foreign protein production in transgenic rice

  • Chia-Yu Chang
  • Kuo-Wei Lee
  • Chung-Shen Wu
  • Yu-Hsing Huang
  • Ho-Chun Chang
  • Chien-Lung Chen
  • Chen-Tung Li
  • Min-Jeng Li
  • Chung-Fu Chang
  • Peng-Wen ChenEmail author
Original Article


Key message

A 146-bp sugar response complex MTSRC is identified in the promoter of rice metallothionein OsMT2b gene conferring high-level expression of luciferase reporter gene and bioactive recombinant haFGF in transgenic rice.


A rice subfamily type 2 plant metallothionein (pMT) gene, OsMT2b, encoding a reactive oxygen species (ROS) scavenger protein, has been previously shown to exhibit the most abundant gene expression in young rice seedling. Expression of OsMT2b was found to be regulated negatively by ethylene and hydrogen peroxide in rice stem node under flooding stress, but little is known about its response to sugar depletion. In this study, transient expression assay and transgenic approach were employed to characterize the regulation of the OsMT2b gene expression in rice. We found that the expression of OsMT2b gene is induced by sugar starvation in both rice suspension cells and germinated embryos. Deletion analysis and functional assay of the OsMT2b promoter revealed that the 5′-flanking region of the OsMT2b between nucleotides − 351 and − 121, which contains the sugar response complex (− 266 to − 121, designated MTSRC) is responsible for high-level promoter activity under sugar starvation. It was also found that MTSRC significantly enhances the Act1 promoter activity in transgenic rice cells and seedlings. The modified Act1 promoter, Act1-MTSRC, was used to produce the recombinant human acidic fibroblast growth factor (haFGF) in rice cells. Our result shows that the bioactive recombinant haFGF is stably produced in transformed rice cell culture and yields are up to 2% of total medium proteins. Our studies reveal that MTSRC serves as a strong transcriptional activator and the Act1-MTSRC promoter can be applicable in establishing an efficient expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings.


Rice metallothionein OsMT2b promoter Act1 promoter Transcriptional activator Sugar response complex (SRC) Human acidic fibroblast growth factor (haFGF) 



We thank Dr. Carol PeiYin Wu for critical review of the manuscript. This work was supported by grants from the Ministry of Science and Technology (NSC96-2311-B-415-003-MY3 and 105A11) of Taiwan.

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest.

Supplementary material

299_2019_2411_MOESM1_ESM.pptx (59 kb)
OsMT2b promoter sequence in Japonica rice variety Lomello (GenBank accession no. AF048750). The nucleotides are numbered relative to the transcription start site (+1) of OsMT2b gene. The consensus sequences of G box (5’-TACGTG-3’) and the TA box (5’-TATCCA-3’), and putative TATA box are highlighted and underlined (PPTX 59 kb)
299_2019_2411_MOESM2_ESM.pptx (105 kb)
The nucleotide sequence of the T-DNA region of pAAct1-MTSRC::ShaFGF. The T-DNA containsCaMV35S::hpt and Act1-MTSRC::ShaFGF chimeric genes. Nucleotide and deduced amino acid sequence of theαAct1-MTSRC chimeric promoter and the codon optimized αAmy3 SP-haFGF gene are shown. The three copies of MTSRC and the signal peptide (SP) of αAmy3 gene are indicated (PPTX 105 kb)
299_2019_2411_MOESM3_ESM.pptx (1.7 mb)
The level of OsMT2b mRNA significantly decreased in hypoxic embryos during seed germination and seedling growth in rice. Seeds were germinated and grown in air (Air) or under hypoxic conditions (Hypoxia, 1% O2) with an oxygen-controlled system (Model 101, Prosperous Instrument Co., Ltd.) at 28 ℃ in the dark for up to 3 days. Total RNA was purified from aerobic and hypoxic embryos at indicated time points and RNA gel blot was performed using the OsMT2b specific cDNA probe (PPTX 1754 kb)
299_2019_2411_MOESM4_ESM.pptx (53 kb)
The MTSRC confers higher promoter activity than αAmy3 SRC in rice embryo. a Schematic representation of constructs for αAmy3 SRC and MTSRC promoter analysis. b Transient expression assay of luciferase activity in rice embryos. Rice embryos were transfected with plasmids pαAmy3SRC::Luc, pMTSRC::Luc, or p35mA::Luc by particle bombardment. Transfected embryos were incubated at 28 ℃ for 24 hours in glucose-containing (+ Glu, open bar) or glucose-free (-Glu, filled bar) cultured medium. X indicates fold induction by sugar starvation. Error bars indicate the standard deviations of data collected from three replicate experiments for each construct. The plasmid p35mA::Luc was used as a negative control in these assays for sugar-starvation responses (PPTX 53 kb)
299_2019_2411_MOESM5_ESM.pptx (52 kb)
The MTSRC significantly enhances the Act1 promoter activity and confers higher promoter activity than αAmy3 SRC in rice embryo. a Schematic representation of constructs for Act1, Act1-αAmy3SRC, and Act1-MTSRC promoter activity analysis. b Transient luciferase activity assay in rice embryos. Rice embryos were transfected with plasmids pAct1::Luc, pAct1-αAmy3SRC::Luc, or pAct1-MTSRC::Luc by particle bombardment. Transfected embryos were incubated at 28 ℃ for 24 hours in glucose-containing (+ Glu, open bar) or glucose-free (-Glu, filled bar) cultured medium. X indicates fold induction by sugar starvation. Error bars indicate the standard deviations of luciferase activity readings from three replicate experiments for each construct. Significant differences between the wild type and modified Act1 promoters are at P ≤ 0.01 (t-test) (PPTX 52 kb)


  1. Benfey PN, Ren L, Chua N-H (1989) The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns. The EMBO Journal 8(8):2195Google Scholar
  2. Burgess WH (1991) Structure-function studies of acidic fibroblast growth factor. Ann N Y Acad Sci 638(1):89–97Google Scholar
  3. Chen PW, Fang LW, Lin JW, Tsay HS, Wu HK, Chen LJ (1997) Isolation of cDNA clones for genes that are specifically expressed in the rice embryo. Bot Bull Acad Sin 38:13–20Google Scholar
  4. Chen PW, Lu CA, Yu TS, Tseng TH, Wang CS, Yu SM (2002) Rice alpha-amylase transcriptional enhancers direct multiple mode regulation of promoters in transgenic rice. J Biol Chem 277(16):13641–13649Google Scholar
  5. Chen PW, Chiang CM, Tseng TH, Yu SM (2006) Interaction between rice MYBGA and the gibberellin response element controls tissue-specific sugar sensitivity of alpha-amylase genes. Plant Cell 18(9):2326–2340Google Scholar
  6. Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5(3):213–218Google Scholar
  7. Davies JP, Reddy V, Liu XL, Reddy AS, Ainley WM, Thompson M, Sastry-Dent L, Cao Z, Connell J, Gonzalez DO, Wagner DR (2014) Identification and use of the sugarcane bacilliform virus enhancer in transgenic maize. BMC Plant Biol 14(1):1–12. Google Scholar
  8. Emamverdian A, Ding Y, Mokhberdoran F, Xie Y (2015) Heavy metal stress and some mechanisms of plant defense response. Sci World J 2015:18. Google Scholar
  9. Fantoni A, Bill RM, Gustafsson L, Hedfalk K (2007) Improved yields of full-length functional human FGF1 can be achieved using the methylotrophic yeast Pichia pastoris. Protein Expr Purif 52(1):31–39Google Scholar
  10. Freisinger E (2011) Structural features specific to plant metallothioneins. J Biol Inorg Chem 16(7):1035–1045. Google Scholar
  11. Gospodarowicz D (1974) Localisation of a fibroblast growth factor and its effect alone and with hydrocortisone on 3T3 cell growth. Nature 249(5453):123Google Scholar
  12. Ha J-H, Kim H-N, Moon K-B, Jeon J-H, Jung D-H, Kim S-J, Mason HS, Shin S-Y, Kim H-S, Park K-M (2017) Recombinant human acidic fibroblast growth factor (aFGF) expressed in nicotiana benthamiana potentially inhibits skin photoaging. Planta Med 83(10):862–869Google Scholar
  13. Hassinen VH, Tervahauta AI, Schat H, Karenlampi SO (2011) Plant metallothioneins–metal chelators with ROS scavenging activity? Plant Biol (Stuttg) 13(2):225–232. Google Scholar
  14. Ho SL, Tong WF, Yu SM (2000) Multiple mode regulation of a cysteine proteinase gene expression in rice. Plant Physiol 122(1):57–66Google Scholar
  15. Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168(3):1291–1301Google Scholar
  16. Hsieh HM, Liu WK, Huang PC (1995) A novel stress-inducible metallothionein-like gene from rice. Plant Mol Biol 28(3):381–389Google Scholar
  17. Hsieh HM, Liu WK, Chang A, Huang PC (1996) RNA expression patterns of a type 2 metallothionein-like gene from rice. Plant Mol Biol 32(3):525–529Google Scholar
  18. Hsing Y-I, Chern C-G, Fan M-J, Lu P-C, Chen K-T, Lo S-F, Sun P-K, Ho S-L, Lee K-W, Wang Y-C (2007) A rice gene activation/knockout mutant resource for high throughput functional genomics. Plant Mol Biol 63(3):351–364Google Scholar
  19. Jeong DH, An S, Kang HG, Moon S, Han JJ, Park S, Lee HS, An K, An G (2002) T-DNA insertional mutagenesis for activation tagging in rice. Plant Physiol. Google Scholar
  20. Koch KE, Ying Z, Wu Y, Avigne WT (2000) Multiple paths of sugar-sensing and a sugar/oxygen overlap for genes of sucrose and ethanol metabolism. J Exp Bot 51(suppl 1):417–427Google Scholar
  21. Kumar G, Kushwaha HR, Panjabi-Sabharwal V, Kumari S, Joshi R, Karan R, Mittal S, Pareek SL, Pareek A (2012) Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-P confers multiple abiotic stress tolerance in tobacco via ROS scavenging. BMC Plant Biol 12(1):107Google Scholar
  22. Last D, Brettell R, Chamberlain D, Chaudhury A, Larkin P, Marsh E, Peacock W, Dennis E (1991) pEmu: an improved promoter for gene expression in cereal cells. Theor Appl Genet 81(5):581–588Google Scholar
  23. Lee KW, Chen PW, Lu CA, Chen S, Ho TH, Yu SM (2009) Coordinated responses to oxygen and sugar deficiency allow rice seedlings to tolerate flooding. Sci Signal 2(91):ra61. Google Scholar
  24. Lee KW, Chen PW, Yu SM (2014) Metabolic adaptation to sugar/O2 deficiency for anaerobic germination and seedling growth in rice. Plant Cell Environ 37(10):2234–2244. Google Scholar
  25. Leszczyszyn OI, Imam HT, Blindauer CA (2013) Diversity and distribution of plant metallothioneins: a review of structure, properties and functions. Metallomics 5(9):1146–1169. Google Scholar
  26. Liu J, Shi X, Qian M, Zheng L, Lian C, Xia Y, Shen Z (2015) Copper-induced hydrogen peroxide upregulation of a metallothionein gene, OsMT2c, from Oryza sativa L. confers copper tolerance in Arabidopsis thaliana. J Hazard Mater 294:99–108Google Scholar
  27. Loreti E, Yamaguchi J, Alpi A, Perata P (2003) Sugar modulation of alpha-amylase genes under anoxia. Ann Bot (Lond) 91:143–148Google Scholar
  28. Lu CA, Lim EK, Yu SM (1998) Sugar response sequence in the promoter of a rice alpha-amylase gene serves as a transcriptional enhancer. J Biol Chem 273(17):10120–10131Google Scholar
  29. Lu C-A, T-hD Ho, Ho S-L, Yu S-M (2002) Three novel MYB proteins with one DNA binding repeat mediate sugar and hormone regulation of α-amylase gene expression. Plant Cell 14(8):1963–1980Google Scholar
  30. Lu CA, Lin CC, Lee KW, Chen JL, Huang LF, Ho SL, Liu HJ, Hsing YI, Yu SM (2007) The SnRK1A protein kinase plays a key role in sugar signaling during germination and seedling growth of rice. Plant Cell 19(8):2484–2499.Google Scholar
  31. Matsumura H, Nirasawa S, Terauchi R (1999) Technical advance: transcript profiling in rice (Oryza sativa L.) seedlings using serial analysis of gene expression (SAGE). Plant J 20(6):719–726Google Scholar
  32. McElroy D, Brettell RIS (1994) Foreign gene expression in transgenic cereals. Trends Biotechnol 12:62–68Google Scholar
  33. Morkunas I, Bednarski W (2008) Fusarium oxysporum-induced oxidative stress and antioxidative defenses of yellow lupine embryo axes with different sugar levels. J Plant Physiol 165(3):262–277Google Scholar
  34. Morkunas I, Gmerek J (2007) The possible involvement of peroxidase in defense of yellow lupine embryo axes against Fusarium oxysporum. J Plant Physiol 164(2):185–194Google Scholar
  35. Olive MR, Walker JC, Singh K, Dennis ES, Peacock WJ (1990) Functional properties of the anaerobic responsive element of the maize Adh1 gene. Plant Mol Biol 15(4):593–604Google Scholar
  36. Ornitz DM, Itoh N (2015) The fibroblast growth factor signaling pathway. Wiley Interdis Rev Dev Biol 4(3):215–266Google Scholar
  37. Perata P, Matsukura C, Vernieri P, Yamaguchi J (1997) Sugar repression of a gibberellin-dependent signaling pathway in barley embryos. Plant Cell 9(12):2197–2208Google Scholar
  38. Schledzewski K, Mendel RR (1994) Quantitative transient gene expression: comparison of the promoters for maize polyubiquitin1, rice actin1, maize-derived Emu and CaMV 35S in cells of barley, maize and tobacco. Transgenic Res 3:249–255Google Scholar
  39. Sheen J (1990) Metabolic repression of transcription in higher plants. Plant Cell 2(10):1027–1038Google Scholar
  40. Sheu JJ, Yu TS, Tong WF, Yu SM (1996) Carbohydrate starvation stimulates differential expression of rice alpha-amylase genes that is modulated through complicated transcriptional and posttranscriptional processes. J Biol Chem 271(43):26998–27004Google Scholar
  41. Singer SD, Cox KD, Liu Z (2010) Both the constitutive Cauliflower Mosaic Virus 35S and tissue-specific AGAMOUS enhancers activate transcription autonomously in Arabidopsis thaliana. Plant Mol Biol 74(3):293–305Google Scholar
  42. Singer SD, Cox KD, Liu Z (2011) Enhancer–promoter interference and its prevention in transgenic plants. Plant Cell Rep 30(5):723–731Google Scholar
  43. Smeekens S, Hellmann HA (2014) Sugar sensing and signaling in plants. Front Plant Sci 5:113Google Scholar
  44. Steffens B, Sauter M (2009) Epidermal cell death in rice is confined to cells with a distinct molecular identity and is mediated by ethylene and H2O2 through an autoamplified signal pathway. Plant Cell 21(1):184–196.Google Scholar
  45. Stoykova P, Radkova M, Stoeva-Popova P, Atanasov N, Chassovnikarova T, Wang X, Iantcheva A, Vlahova M, Atanassov A (2011) Expression of the human acidic fibroblast growth factor in transgenic tomato and safety assessment of transgenic lines. Biotechnol Biotechnol Equip 25(1):2187–2196Google Scholar
  46. Tan Y, Wang KY, Wang N, Li G, Liu D (2014) Ectopic expression of human acidic fibroblast growth factor 1 in the medicinal plant, Salvia miltiorrhiza, accelerates the healing of burn wounds. BMC Biotechnol 14(1):74Google Scholar
  47. Umemura T, Perata P, Futsuhara Y, Yamaguchi J (1998) Sugar sensing and alpha-amylase gene repression in rice embryos. Planta 204(4):420–428Google Scholar
  48. Vichai V, Kirtikara K (2006) Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 1(3):1112–1116Google Scholar
  49. Walpurgis K, Thomas A, Laussmann T, Horta L, Metzger S, Schänzer W, Thevis M (2011) Identification of fibroblast growth factor 1 (FGF-1) in a black market product. Drug Test Anal 3(11–12):791–797Google Scholar
  50. Wang F, Wang R, Wang Y, Zhao P, Xia Q (2015) Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons. Scientific Reports 5:16323Google Scholar
  51. Wong HL, Sakamoto T, Kawasaki T, Umemura K, Shimamoto K (2004) Down-regulation of metallothionein, a reactive oxygen scavenger, by the small GTPase OsRac1 in rice. Plant Physiol 135(3):1447–1456. Google Scholar
  52. Wu X, Kamei K, Sato H, S-i Sato, Takano R, Ichida M, Mori H, Hara S (2001) High-level expression of human acidic fibroblast growth factor and basic fibroblast growth factor in silkworm (Bombyx mori L.) using recombinant baculovirus. Protein Express Purif 21(1):192–200Google Scholar
  53. Wu CS, Chen DY, Chang CF, Li MJ, Hung KY, Chen LJ, Chen PW (2014a) The promoter and the 5′-untranslated region of rice metallothionein OsMT2b gene are capable of directing high-level gene expression in germinated rice embryos. Plant Cell Rep 33(5):793–806. Google Scholar
  54. Wu CS, Kuo WT, Chang CY, Kuo JY, Tsai YT, Yu SM, Wu HT, Chen PW (2014b) The modified rice alphaAmy8 promoter confers high-level foreign gene expression in a novel hypoxia-inducible expression system in transgenic rice seedlings. Plant Mol Biol 85(1–2):147–161. Google Scholar
  55. Yang Y, Singer SD, Liu Z (2010) Two similar but distinct second intron fragments from tobacco AGAMOUS homologs confer identical floral organ-specific expression sufficient for generating complete sterility in plants. Planta 231(5):1159–1169Google Scholar
  56. Yang J, Guan L, Guo Y, Du L, Wang F, Wang Y, Zhen L, Wang Q, Zou D, Chen W (2015) Expression of biologically recombinant human acidic fibroblast growth factor in Arabidopsis thaliana seeds via oleosin fusion technology. Gene 566(1):89–94Google Scholar
  57. Yu S-M (1999) Cellular and genetic responses of plants to sugar starvation. Plant Physiol 121(3):687–693Google Scholar
  58. Yu SM, Kuo YH, Sheu G, Sheu YJ, Liu LF (1991) Metabolic derepression of alpha-amylase gene expression in suspension-cultured cells of rice. J Biol Chem 266(31):21131–21137Google Scholar
  59. Yu S-M, Lee Y-C, Fang S-C, Chan M-T, Hwa S-F, Liu L-F (1996) Sugars act as signal molecules and osmotica to regulate the expression of α-amylase genes and metabolic activities in germinating cereal grains. Plant Mol Biol 30(6):1277–1289Google Scholar
  60. Yuan J, Chen D, Ren Y, Zhang X, Zhao J (2008) Characteristic and expression analysis of a metallothionein gene, OsMT2b, down-regulated by cytokinin suggests functions in root development and seed embryo germination of rice. Plant Physiol 146(4):1637–1650. Google Scholar
  61. Yun Y-R, Won JE, Jeon E, Lee S, Kang W, Jo H, Jang J-H, Shin US, Kim H-W (2010) Fibroblast growth factors: biology, function, and application for tissue regeneration. J Tissue Eng 1(1):218142Google Scholar
  62. Zakrzewska M, Marcinkowska E, Wiedlocha A (2008) FGF-1: from biology through engineering to potential medical applications. Crit Rev Clin Lab Sci 45(1):91–135Google Scholar
  63. Zazo M, Lozano RM, Ortega S, Varela J, Diaz-Orejas R, Ramirez JM, Giménez-Gallego G (1992) High-level synthesis in Escherichia coli of shortened and full-length human acidic fibroblast growth factor and purification in a form stable in aqueous solutions. Gene 113(2):231–238Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BioAgricultural SciencesNational Chiayi UniversityChiayiTaiwan
  2. 2.PRIT Biotech Co., Ltd.ChunanTaiwan

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