Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

The rice OsLTP6 gene promoter directs anther-specific expression by a combination of positive and negative regulatory elements

  • 971 Accesses

  • 20 Citations

Abstract

Characterization of tissue-specific plant gene promoters will benefit genetic improvement in crops. Here, we isolated a novel rice anther-specific plant lipid transfer protein (OsLTP6) gene through high through-put expressional profiling. The promoter of OsLTP6 was introduced to the upstream of the uidA gene, which encodes β-glucuronidase (GUS), and transformed into rice plants for functional analysis. Histochemical and fluorometric GUS assay showed that GUS was specifically expressed in the anthers and pollens in OsLTP6 promoter::uidA transgenic plants. Transverse section of the rice anther further indicated that the OsLTP6 promoter directed the reporter gene specifically expressed in anther tapetum. To identify regulatory elements within OsLTP6 promoter region, four progressive deletions of the OsLTP6 promoter were constructed. The results indicated that the OsLTP6 promoter achieved anther-specific expression through a combination of positive and negative regulatory elements. A 26-bp motif upstream of TATA box was a key transcriptional activator for OsLTP6 gene. CAAT box and GTGA box were the putative motifs to increase the transcription level to full expression. Two negative regulatory elements were also found in two distinct regions within this promoter. They repressed the expression in leaf and stem, respectively. These results revealed the regulating complexity of anther-specific expression.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Abbreviations

4-MU:

4-Methylumbelliferone

CaMV:

Cauliflower mosaic virus

GUS:

β-Glucuronidase

MMC:

Microspore mother cell

MUG:

4-Methylumbelliferyl β-d-glucuronide

nsLTP:

Non-specific lipid transfer protein

OsLTP6 :

Oryza sativa lipid transfer protein 6

TA29 promoter:

Tobacco anther-specific gene 29 promoter

TSS:

Transcriptional start site

X-gluc:

5-Bromo-4-chloro-3-indoyl-β-d-glucuronide solution

St:

Anther developmental stage

References

  1. Aya K, Ueguchi-Tanaka M, Kondo M, Hamada K, Yano K, Nishimura M, Matsuoka M (2009) Gibberellin modulates anther development in rice via the transcriptional regulation of GAMYB. Plant Cell 21:1453–1472

  2. Beyer P, Al-Babili S, Ye X, Lucca P, Schaub P, Welsch R, Potrykus I (2002) Golden rice: introducing the β-carotene biosynthesis pathway into rice endosperm by genetic engineering to defeat vitamin A deficiency. J Nutr 132:506S–510S

  3. Bhattacharyya J, Chowdhury AH, Ray S, Jha JK, Das S, Gayen S, Chakraborty A, Mitra J, Maiti MK, Basu A, Sen SK (2012) Native polyubiquitin promoter of rice provides increased constitutive expression in stable transgenic rice plants. Plant Cell Rep 31:271–279

  4. Blein JP, Coutos-Thevenot P, Marion D, Ponchet M (2002) From elicitins to lipid-transfer proteins: a new insight in cell signalling involved in plant defence mechanisms. Trends Plant Sci 7:293–296

  5. Boutrot F, Chantret N, Gautier MF (2008) Genome-wide analysis of the rice and Arabidopsis non-specific lipid transfer protein (nsLtp) gene families and identification of wheat nsLtp genes by EST data mining. BMC Genom 9:86

  6. 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 72:248–254

  7. Chen H, Tang W, Xu C, Li X, Lin Y, Zhang Q (2005) Transgenic indica rice plants harboring a synthetic cry2A* gene of Bacillus thuringiensis exhibit enhanced resistance against lepidopteran rice pests. Theor Appl Genet 111:1330–1337

  8. Cheng HC, Cheng PT, Peng P, Lyu PC, Sun YJ (2004) Lipid binding in rice nonspecific lipid transfer protein-1 complexes from Oryza sativa. Prot Sci 13:2304–2315

  9. 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 18:675–689

  10. Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: a sequence logo generator. Genom Res 14:1188–1190

  11. Daniell H (2002) Molecular strategies for gene containment in transgenic crops. Nat Biotechnol 20:581–586

  12. Engelke T, Hirsche J, Roitsch T (2010) Anther-specific carbohydrate supply and restoration of metabolically engineered male sterility. J Exp Bot 61:2693–2706

  13. Engelke T, Hirsche J, Roitsch T (2011) Metabolically engineered male sterility in rapeseed (Brassica napus L.). Theor Appl Genet 122:163–174

  14. Garcia-Garrido JM, Menossi M, Puigdomenech P, Martinez-Izquierdo JA, Delseny M (1998) Characterization of a gene encoding an abscisic acid-inducible type-2 lipid transfer protein from rice. FEBS Lett 428:193–199

  15. Gincel E, Simorre JP, Caille A, Marion D, Ptak M, Vovelle F (1994) Three-dimensional structure in solution of a wheat lipid-transfer protein from multidimensional 1H-NMR data. A new folding for lipid carriers. Eur J Biochem 226:413–422

  16. Gomez MD, Beltran JP, Canas LA (2004) The pea END1 promoter drives anther-specific gene expression in different plant species. Planta 219:967–981

  17. Guiderdoni E, Cordero MJ, Vignols F, Garcia-Garrido JM, Lescot M, Tharreau D, Meynard D, Ferriere N, Notteghem JL, Delseny M (2002) Inducibility by pathogen attack and developmental regulation of the rice Ltp1 gene. Plant Mol Biol 49:683–699

  18. Gupta V, Khurana R, Tyagi AK (2007) Promoters of two anther-specific genes confer organ-specific gene expression in a stage-specific manner in transgenic systems. Plant Cell Rep 26:1919–1931

  19. Halpin C (2005) Gene stacking in transgenic plants––the challenge for 21st century plant biotechnology. Plant Biotechnol J 3:141–155

  20. He C, Lin Z, McElroy D, Wu R (2009) Identification of a rice actin2 gene regulatory region for high-level expression of transgenes in monocots. Plant Biotechnol J 7:227–239

  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 6:271–282

  22. High SM, Cohen MB, Shu QY, Altosaar I (2004) Achieving successful deployment of Bt rice. Trends Plant Sci 9:286–292

  23. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucl Acid Res 27:297–300

  24. Hirsche J, Engelke T, Voller D, Gotz M, Roitsch T (2009) Interspecies compatibility of the anther specific cell wall invertase promoters from Arabidopsis to tobacco for generating male sterile plants. Theor Appl Genet 118:235–245

  25. Hoh F, Pons JL, Gautier MF, de Lamotte F, Dumas C (2005) Structure of a liganded type 2 non-specific lipid-transfer protein from wheat to the molecular basis of lipid binding. Acta Crystallogr D Biol Crystallogr 61:397–406

  26. Huang N, Wu LY, Nandi S, Bowman E, Huang JM, Sutliff T, Rodriguez RL (2001) The tissue-specific activity of a rice β-glucanase promoter (Gns9) is used to select rice transformants. Plant Sci 161:589–595

  27. Huang MD, Wei FJ, Wu CC, Hsing YI, Huang AH (2009) Analyses of advanced rice anther transcriptomes reveal global tapetum secretory functions and potential proteins for lipid exine formation. Plant Physiol 149:694–707

  28. Jang IC, Choi WB, Lee KH, Song SI, Nahm BH, Kim JK (2002) High-level and ubiquitous expression of the rice cytochrome c gene OsCc1 and its promoter activity in transgenic plants provides a useful promoter for trans genesis of monocots. Plant Physiol 129:1473–1481

  29. Jefferson RA (1987) Assay chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405

  30. Jeon JS, Chung YY, Lee S, Yi GH, Oh BG, An G (1999) Isolation and characterization of an anther-specific gene, RA8, from rice (Oryza sativa L.). Plant Mol Biol 39:35–44

  31. Jose-Estanyol M, Gomis-Ruth FX, Puigdomenech P (2004) The eight-cysteine motif, a versatile structure in plant proteins. Plant Physiol Biochem 42:355–365

  32. Jung KH, Han MJ, Lee YS, Kim YW, Hwang IW, Kim MJ, Kim YK, Nahm BH, An GH (2005) Rice Undeveloped Tapetum1 is a major regulator of early tapetum development. Plant Cell 17:2705–2722

  33. Jung KH, Han MJ, Lee DY, Lee YS, Schreiber L, Franke R, Faust A, Yephremov A, Saedler H, Kim YW, Hwang I, An G (2006) Wax-deficient anther1 is involved in cuticle and wax production in rice anther walls and is required for pollen development. Plant Cell 18:3015–3032

  34. Kaneko M, Inukai Y, Ueguchi-Tanaka M, Itoh H, Izawa T, Kobayashi Y, Hattori T, Miyao A, Hirochika H, Ashikari M, Matsuoka M (2004) Loss-of-function mutations of the rice GAMYB gene impair alpha-amylase expression in aleurone and flower development. Plant Cell 16:33–44

  35. Kim TH, Park JH, Kim MC, Cho SH (2008) Cutin monomer induces expression of the rice OsLTP5 lipid transfer protein gene. J Plant Physiol 165:345–349

  36. Konagaya K, Ando S, Kamachi S, Tsuda M, Tabei Y (2008) Efficient production of genetically engineered, male-sterile Arabidopsis thaliana using anther-specific promoters and genes derived from Brassica oleracea and B. rapa. Plant Cell Rep 27:1741–1754

  37. Lee SB, Go YS, Bae HJ, Park JH, Cho SH, Cho HJ, Lee DS, Park OK, Hwang I, Suh MC (2009) Disruption of glycosylphosphatidylinositol-anchored lipid transfer protein gene altered cuticular lipid composition, increased plasto globules, and enhanced susceptibility to infection by the fungal pathogen Alternaria brassicicola. Plant Physiol 150:42–54

  38. Lerche MH, Poulsen FM (1998) Solution structure of barley lipid transfer protein complexed with palmitate. Two different binding modes of palmitate in the homologous maize and barley nonspecific lipid transfer proteins. Prot Sci 7:2490–2498

  39. Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouze P, Rombauts S (2002) Plant CARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucl Acid Res 30:325–327

  40. Li N, Zhang DS, Liu HS, Yin CS, Li XX, Liang WQ, Yuan Z, Xu B, Chu HW, Wang J, Wen TQ, Huang H, Luo D, Ma H, Zhang DB (2006) The rice tapetum degeneration retardation gene is required for tapetum degradation and anther development. Plant Cell 18:2999–3014

  41. Li H, Pinot F, Sauveplane V, Werck-Reichhart D, Diehl P, Schreiber L, Franke R, Zhang P, Chen L, Gao Y, Liang W, Zhang D (2010) Cytochrome P450 family member CYP704B2 catalyzes the omega-hydroxylation of fatty acids and is required for anther cutin biosynthesis and pollen exine formation in rice. Plant Cell 22:173–190

  42. Li H, Yuan Z, Vizcay-Barrena G, Yang CY, Liang WQ, Zong J, Wilson ZA, Zhang DB (2011) PERSISTENT TAPETAL CELL1 encodes a PHD-finger protein that is required for tapetal cell death and pollen development in rice. Plant Physiol 156:615–630

  43. Liu X, Lu T, Yu S, Li Y, Huang Y, Huang T, Zhang L, Zhu J, Zhao Q, Fan D, Mu J, Shangguan Y, Feng Q, Guan J, Ying K, Zhang Y, Lin Z, Sun Z, Qian Q, Lu Y, Han B (2007) A collection of 10,096 indica rice full-length cDNAs reveals highly expressed sequence divergence between Oryza sativa indica and japonica subspecies. Plant Mol Biol 65:403–415

  44. 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 62:397–408

  45. Maldonado AM, Doerner P, Dixon RA, Lamb CJ, Cameron RK (2002) A putative lipid transfer protein involved in systemic resistance signalling in Arabidopsis. Nature 419:399–403

  46. Mariani C, De Beuckeleer M, Truettner J, Leemans J, Goldberg RB (1990) Induction of male sterility in plants by a chimeric ribonuclease gene. Nature 347:737–741

  47. Mariani C, Gossele V, De Beuckeleer M, De Block M, Goldberg RB, De Greef W, Leemans J (1992) A chimeric ribonuclease-inhibitor gene restores fertility to male-sterile plants. Nature 357:384–387

  48. McElroy D, Zhang W, Cao J, Wu R (1990) Isolation of an efficient actin promoter for use in rice transformation. Plant Cell 2:163–171

  49. McElroy D, Blowers AD, Jenes B, Wu R (1991) Construction of expression vectors based on the rice actin 1 (Act1) 5′ region for use in monocot transformation. Mol Gen Genet 231:150–160

  50. Medina M, Roque E, Pineda B, Canas L, Rodriguez-Concepcion M, Beltran JP, Gomez-Mena C (2013) Early anther ablation triggers parthenocarpic fruit development in tomato. Plant Biotechnol J. doi:10.1111/pbi.12069

  51. Niu NN, Liang WQ, Yang XJ, Jin WL, Wilson ZA, Hu JP, Zhang DB (2013) EAT1 promotes tapetal cell death by regulating aspartic proteases during male reproductive development in rice. Nat Commun 4:1445. doi:10.1038/ncomms2396

  52. Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812

  53. Paine JA, Shipton CA, Chaggar S, Howells RM, Kennedy MJ, Vernon G, Wright SY, Hinchliffe E, Adams JL, Silverstone AL, Drake R (2005) Improving the nutritional value of golden rice through increased pro-vitamin A content. Nat Biotechnol 23:482–487

  54. 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 25:466–474

  55. Park SH, Yi N, Kim YS, Jeong MH, Bang SW, Choi YD, Kim JK (2010) Analysis of five novel putative constitutive gene promoters in transgenic rice plants. J Exp Bot 61:2459–2467

  56. 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 19:611–622

  57. 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 45:577–585

  58. Roque E, Gomez MD, Ellul P, Wallbraun M, Madueno F, Beltran JP, Canas LA (2007) The PsEND1 promoter: a novel tool to produce genetically engineered male-sterile plants by early anther ablation. Plant Cell Rep 26:313–325

  59. Samuel D, Liu YJ, Cheng CS, Lyu PC (2002) Solution structure of plant nonspecific lipid transfer protein-2 from rice (Oryza sativa). J Biol Chem 277:35267–35273

  60. Sarowar S, Kim YJ, Kim KD, Hwang BK, Ok SH, Shin JS (2009) Overexpression of lipid transfer protein (LTP) genes enhances resistance to plant pathogens and LTP functions in long-distance systemic signaling in tobacco. Plant Cell Rep 28:419–427

  61. Shelton AM, Zhao JZ, Roush RT (2002) Economic, ecological, food safety, and social consequences of the deployment of bt transgenic plants. Annu Rev Entomol 47:845–881

  62. Shi J, Tan HX, Yu XH, Liu YY, Liang WQ, Ranathunge K, Franke RB, Schreiber L, Wang YJ, Kai GY, Shanklin J, Ma H, Zhang DB (2011) Defective Pollen Wall is required for anther and microspore development in rice and encodes a fatty acyl carrier protein reductase. Plant Cell 23:2225–2246

  63. Stalberg K, Ellerstom M, Ezcurra I, Ablov S, Rask L (1996) Disruption of an overlapping E-box/ABRE motif abolished high transcription of the napA storage-protein promoter in transgenic Brassica napus seeds. Planta 199:515–519

  64. Thoma S, Hecht U, Kippers A, Botella J, De Vries S, Somerville C (1994) Tissue-specific expression of a gene encoding a cell wall-localized lipid transfer protein from Arabidopsis. Plant Physiol 105:35–45

  65. Wang L, Xie WB, Chen Y, Tang WJ, Yang JY, Ye RJ, Liu L, Lin YJ, Xu CG, Xiao JH, Zhang QF (2010) A dynamic gene expression atlas covering the entire life cycle of rice. Plant J 61:752–766

  66. Ye X, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000) Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287:303–305

  67. Yin T, Wu H, Zhang S, Lu H, Zhang L, Xu Y, Chen D, Liu J (2009) Two negative cis-regulatory regions involved in fruit-specific promoter activity from watermelon (Citrullus vulgaris S.). J Exp Bot 60:169–185

  68. Yokoi S, Tsuehiya T, Toriyama K, Hinata K (1997) Tapetum specific expression of the Osg6B promoter-β-glucuronidase gene in transgenic rice. Plant Cell Rep 16:363–367

  69. Zhang D, Wilson Z (2009) Stamen specification and anther development in rice. Chinese Sci Bull 54:2342–2353

  70. Zhang D, Liang W, Yin C, Zong J, Gu F (2010) OsC6, encoding a lipid transfer protein, is required for postmeiotic anther development in rice. Plant Physiol 154:149–162

Download references

Acknowledgments

The authors are grateful to Yiwen Yan and Tingting Lu for their technical help in this research. This work was supported by the grants from the National Natural Science Foundation of China (Grant No. 30771159, 30871319).

Author information

Correspondence to Bin Han.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Liu, X., Shangguan, Y., Zhu, J. et al. The rice OsLTP6 gene promoter directs anther-specific expression by a combination of positive and negative regulatory elements. Planta 238, 845–857 (2013). https://doi.org/10.1007/s00425-013-1934-9

Download citation

Keywords

  • Anther-specific
  • Lipid transfer protein
  • Oryza
  • OsLTP6 promoter