Plant Molecular Biology

, Volume 88, Issue 4–5, pp 415–428 | Cite as

Functional architecture of two exclusively late stage pollen-specific promoters in rice (Oryza sativa L.)

  • Shuo Yan
  • Zhongni Wang
  • Yuan Liu
  • Wei Li
  • Feng Wu
  • Xuelei Lin
  • Zheng Meng


Late stage pollen-specific promoters are important tools in crop molecular breeding. Several such promoters, and their functional motifs, have been well characterized in dicotyledonous plants such as tomato and tobacco. However, knowledge about the functional architecture of such promoters is limited in the monocotyledonous plant rice. Here, pollen-late-stage-promoter 1 (PLP1) and pollen-late-stage-promoter 2 (PLP2) were characterized using a stable transformation system in rice. Histochemical staining showed that the two promoters exclusively drive GUS expression in late-stage pollen grains in rice. 5′ deletion analysis revealed that four regions, including the −1159 to −720 and the −352 to −156 regions of PLP1 and the −740 to −557 and the −557 to −339 regions of PLP2, are important in maintaining the activity and specificity of these promoters. Motif mutation analysis indicated that ‘AGAAA’ and ‘CAAT’ motifs in the −740 to −557 region of PLP2 act as enhancers in the promoter. Gain of function experiments indicated that the novel TA-rich motif ‘TACATAA’ and ‘TATTCAT’ in the core region of the PLP1 and PLP2 promoters is necessary, but not sufficient, for pollen-specific expression in rice. Our results provide evidence that the enhancer motif ‘AGAAA’ is conserved in the pollen-specific promoters of both monocots and eudicots, but that some functional architecture characteristics are different.


Pollen-specific promoter Rice cis-Element Functional architecture Stable transformation 



We thank Dr. Hualiang Liu and Dr. Wenjing Li for their technical assistance with the GUS activity analysis. This work was supported by grants from The National Natural Science Foundation of China (Grant 31270280) and The Ministry of Science and Technology of China (Grant 2011CB100405).

Supplementary material

11103_2015_331_MOESM1_ESM.pdf (123 kb)
Supplementary material 1 (PDF 122 kb)
11103_2015_331_MOESM2_ESM.pdf (203 kb)
Supplementary material 2 (PDF 202 kb)
11103_2015_331_MOESM3_ESM.pdf (217 kb)
Supplementary material 3 (PDF 216 kb)
11103_2015_331_MOESM4_ESM.pdf (29 kb)
Supplementary material 4 (PDF 28 kb)


  1. Bate N, Twell D (1998) Functional architecture of a late pollen promoter: pollen-specific transcription is developmentally regulated by multiple stage-specific and co-dependent activator elements. Plant Mol Biol 37:859–869PubMedCrossRefGoogle Scholar
  2. Benfey PN, Ren L, Chua NH (1989) The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns. EMBO J 8:2195–2202PubMedCentralPubMedGoogle Scholar
  3. Boschen S (2009) Hybrid regimes of knowledge? Challenges for constructing scientific evidence in the context of the GMO-debate. Environ Sci Pollut R 16:508–520CrossRefGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  5. Bruce WB, Christensen AH, Klein T, Fromm M, Quail PH (1989) Photoregulation of a phytochrome gene promoter from oat transferred into rice by particle bombardment. Proc Natl Acad Sci U S A 86:9692–9696PubMedCentralPubMedCrossRefGoogle Scholar
  6. Cai M, Wei J, Li X, Xu C, Wang S (2007) A rice promoter containing both novel positive and negative cis-elements for regulation of green tissue-specific gene expression in transgenic plants. Plant Biotechnol J 5:664–674PubMedCrossRefGoogle Scholar
  7. Chapman GP (1987) The tapetum. Int Rev Cytol 107:111–125CrossRefGoogle Scholar
  8. Chen L, Tu ZM, Hussain J, Cong L, Yan YJ, Jin L, Yang GX, He GY (2010) Isolation and heterologous transformation analysis of a pollen-specific promoter from wheat (Triticum aestivum L.). Mol Biol Rep 37:737–744PubMedCrossRefGoogle Scholar
  9. Chen L, Miao YJ, Wang C, Su PP, Li TH, Wang R, Hao XL, Yang GX, He GY, Gao CB (2012) Characterization of a novel pollen-specific promoter from wheat (Triticum Aestivum L.). Plant Mol. Biol. Rep. 30:1426–1432CrossRefGoogle Scholar
  10. Cheon BY, Kim HJ, Oh KH, Bahn SC, Ahn JH, Choi JW, Ok SH, Bae JM, Shin JS (2004) Overexpression of human erythropoietin (EPO) affects plant morphologies: retarded vegetative growth in tobacco and male sterility in tobacco and Arabidopsis. Transgenic Res 13:541–549PubMedCrossRefGoogle Scholar
  11. 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 5:213–218PubMedCrossRefGoogle Scholar
  12. Christensen AH, Sharrock RA, Quail PH (1992) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol 18:675–689PubMedCrossRefGoogle Scholar
  13. Cockburn A (2002) Assuring the safety of genetically modified (GM) foods: the importance of an holistic, integrative approach. J Biotechnol 98:79–106PubMedCrossRefGoogle Scholar
  14. Denning GL, Mew TW (1998) China and IRRI: Improving China’s rice productivity in the 21st century. International Rice Research Institute, ManilaGoogle Scholar
  15. Fang RX, Nagy F, Sivasubramaniam S, Chua NH (1989) Multiple cis regulatory elements for maximal expression of the cauliflower mosaic virus 35S promoter in transgenic plants. Plant Cell 1:141–150PubMedCentralPubMedCrossRefGoogle Scholar
  16. Filichkin SA, Leonard JM, Monteros A, Liu PP, Nonogaki H (2004) A novel endo-beta-mannanase gene in tomato LeMAN5 is associated with anther and pollen development. Plant Physiol 134:1080–1087PubMedCentralPubMedCrossRefGoogle Scholar
  17. Frewer LJ, van der Lans IA, Fischer ARH, Reinders MJ, Menozzi D, Zhang XY, van den Berg I, Zimmermann KL (2013) Public perceptions of agri-food applications of genetic modification—a systematic review and meta-analysis. Trends Food Sci. Tech 30:142–152CrossRefGoogle Scholar
  18. Gardner N, Felsheim R, Smith AG (2009) Production of male- and female-sterile plants through reproductive tissue ablation. J Plant Physiol 166:871–881PubMedCrossRefGoogle Scholar
  19. Glover J, Grelon M, Craig S, Chaudhury A, Dennis E (1998) Cloning and characterization of MS5 from Arabidopsis: a gene critical in male meiosis. Plant J 15:345–356PubMedCrossRefGoogle Scholar
  20. Hamilton DA, Schwarz YH, Mascarenhas JP (1998) A monocot pollen-specific promoter contains separable pollen-specific and quantitative elements. Plant Mol Biol 38:663–669PubMedCrossRefGoogle Scholar
  21. Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282PubMedCrossRefGoogle Scholar
  22. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27:297–300PubMedCentralPubMedCrossRefGoogle Scholar
  23. Hsu SW, Liu MC, Zen KC, Wang CS (2014) Identification of the tapetum/microspore-specific promoter of the pathogenesis-related 10 gene and its regulation in the anther of Lilium longiflorum. Plant Sci 215:124–133PubMedCrossRefGoogle Scholar
  24. Huang ZY, Gan ZS, He YS, Li YH, Liu XD, Mu H (2011) Functional analysis of a rice late pollen-abundant UDP-glucose pyrophosphorylase (OsUgp2) promoter. Mol Biol Rep 38:4291–4302PubMedCrossRefGoogle Scholar
  25. Jefferson RA (1987) Assaying chimeric genes in plant: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405CrossRefGoogle Scholar
  26. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedCentralPubMedGoogle Scholar
  27. Khurana R, Kapoor S, Tyagi AK (2012) Anthology of anther/pollen-specific promoters and transcription factors. Crit Rev Plant Sci 31:359–390CrossRefGoogle Scholar
  28. Khush GS (2005) What it will take to feed 5.0 billion rice consumers in 2030. Plant Mol Biol 59:1–6PubMedCrossRefGoogle Scholar
  29. Konagaya K, Ando S, Kamachi S, Tsuda M, Tabei Y (2008) Efficient production of genetically engineered, male-sterile Arabidopsisthaliana using anther-specific promoters and genes derived from Brassica oleracea and B. rapa. Plant Cell Rep 27:1741–1754PubMedCrossRefGoogle Scholar
  30. Kurek I, Stoger E, Dulberger R, Christou P, Breiman A (2002) Overexpression of the wheat FK506-binding protein 73 (FKBP73) and the heat-induced wheat FKBP77 in transgenic wheat reveals different functions of the two isoforms. Transgenic Res 11:373–379PubMedCrossRefGoogle Scholar
  31. Kuriakose B, Arun V, Gnanamanickam SS, Thomas G (2009) Tissue-specific expression in transgenic rice and Arabidopsis thaliana plants of GUS gene driven by the 5′ regulatory sequences of an anther specific rice gene YY2. Plant Sci 177:390–397CrossRefGoogle Scholar
  32. Lee YH, Chung KH, Kim HU, Jin YM, Kim HI, Park BS (2003) Induction of male sterile cabbage using a tapetum-specific promoter from Brassica campestris L. ssp pekinensis. Plant Cell Rep 22:268–273PubMedCrossRefGoogle Scholar
  33. Li SQ, Yang DC, Zhu YG (2007) Characterization and use of male sterility in hybrid rice breeding. J Integr Plant Biol 49:791–804CrossRefGoogle Scholar
  34. Liu Y, Cui S, Wu F, Yan S, Lin X, Du X, Chong K, Schilling S, Theissen G, Meng Z (2013) Functional conservation of MIKC*-type MADS box genes in Arabidopsis and rice pollen maturation. Plant Cell 25:1288–1303PubMedCentralPubMedCrossRefGoogle Scholar
  35. Luo H, Lee JY, Hu Q, Nelson-Vasilchik K, Eitas TK, Lickwar C, Kausch AP, Chandlee JM, Hodges TK (2006) RTS, a rice anther-specific gene is required for male fertility and its promoter sequence directs tissue-specific gene expression in different plant species. Plant Mol Biol 62:397–408PubMedCrossRefGoogle Scholar
  36. Mariani C, Debeuckeleer M, Truettner J, Leemans J, Goldberg RB (1990) Induction of male-sterility in plants by a chimeric ribonuclease gene. Nature 347:737–741CrossRefGoogle Scholar
  37. Mariani C, Gossele V, Debeuckeleer M, Deblock M, Goldberg RB, Degreef W, Leemans J (1992) A chimeric ribonuclease-inhibitor gene restores fertility to male sterile plants. Nature 357:384–387CrossRefGoogle Scholar
  38. Mascarenhas JP (1990) Gene activity during pollen development. Annu Rev Plant Phys 41:317–338CrossRefGoogle Scholar
  39. Matzke MA, Mette MF, Matzke AJ (2000) Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Mol Biol 43:401–415PubMedCrossRefGoogle Scholar
  40. Mcelroy D, Brettell RIS (1994) Foreign gene-expression in transgenic cereals. Trends Biotechnol 12:62–68CrossRefGoogle Scholar
  41. Mcelroy D, Zhang WG, Cao J, Wu R (1990) Isolation of an efficient actin promoter for use in rice transformation. Plant Cell 2:163–171PubMedCentralPubMedCrossRefGoogle Scholar
  42. Park JI, Hakozaki H, Endo M, Takada Y, Ito H, Uchida M, Okabe T, Watanabe M (2006) Molecular characterization of mature pollen-specific genes encoding novel small cysteine-rich proteins in rice (Oryza sativa L.). Plant Cell Rep 25:466–474PubMedCrossRefGoogle Scholar
  43. Paul W, Hodge R, Smartt S, Draper J, Scott R (1992) The isolation and characterisation of the tapetum-specific Arabidopsis thaliana A9 gene. Plant Mol Biol 19:611–622PubMedCrossRefGoogle Scholar
  44. Rogers HJ, Bate N, Combe J, Sullivan J, Sweetman J, Swan C, Lonsdale DM, Twell D (2001) Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10. Plant Mol Biol 45:577–585PubMedCrossRefGoogle Scholar
  45. Schrauwen JA, de Groot PF, van Herpen MM, van der Lee T, Reynen WH, Weterings KA, Wullems GJ (1990) Stage-related expression of mRNAs during pollen development in lily and tobacco. Planta 182:298–304PubMedCrossRefGoogle Scholar
  46. Shirsat A, Wilford N, Croy R, Boulter D (1989) Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco. Mol Gen Genet 215:326–331PubMedCrossRefGoogle Scholar
  47. Sinha NR, Williams RE, Hake S (1993) Overexpression of the maize homeo box gene, KNOTTED-1, causes a switch from determinate to indeterminate cell fates. Genes Dev 7:787–795PubMedCrossRefGoogle Scholar
  48. Swapna L, Khurana R, Kumar SV, Tyagi AK, Rao KV (2011) Pollen-specific expression of Oryza sativa Indica pollen allergen gene (OSIPA) promoter in rice and Arabidopsis transgenic systems. Mol Biotechnol 48:49–59PubMedCrossRefGoogle Scholar
  49. Toki S, Hara N, Ono K, Onodera H, Tagiri A, Oka S, Tanaka H (2006) Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant J 47:969–976PubMedCrossRefGoogle Scholar
  50. Twell D, Klein TM, Fromm ME, Mccormick S (1989) Transient expression of chimeric genes delivered into pollen by microprojectile bombardment. Plant Physiol 91:1270–1274PubMedCentralPubMedCrossRefGoogle Scholar
  51. Twell D, Yamaguchi J, Wing RA, Ushiba J, Mccormick S (1991) Promoter analysis of genes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements. Gene Dev 5:496–507PubMedCrossRefGoogle Scholar
  52. Willing RP, Bashe D, Mascarenhas JP (1988) An analysis of the quantity and diversity of messenger RNAs from pollen and shoots of Zea mays. Theor Appl Genet 75:751–753CrossRefGoogle Scholar
  53. Zhang DB, Wilson ZA (2009) Stamen specification and anther development in rice. Chinese Sci Bull 54:2342–2353CrossRefGoogle Scholar
  54. Zhang WG, Mcelroy D, Wu R (1991) Analysis of rice act1 5′ region activity in transgenic rice plants. Plant Cell 3:1155–1165PubMedCentralPubMedGoogle Scholar
  55. Zhou P, Yang F, Yu JJ, Ao GM, Zhao Q (2010) Several cis-elements including a palindrome involved in pollen-specific activity of SBgLR promoter. Plant Cell Rep 29:503–511PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Shuo Yan
    • 1
    • 2
  • Zhongni Wang
    • 1
    • 2
  • Yuan Liu
    • 1
  • Wei Li
    • 3
  • Feng Wu
    • 1
  • Xuelei Lin
    • 1
  • Zheng Meng
    • 1
  1. 1.Key Laboratory of Plant Molecular Physiology, Institute of BotanyChinese Academy of SciencesBeijingChina
  2. 2.University of Chinese Academy of SciencesBeijingChina
  3. 3.College of Life ScienceNortheast Forestry UniversityHarbinChina

Personalised recommendations