Skip to main content
SpringerLink
Log in

Isolation and functional characterization of two novel seed-specific promoters from sunflower (Helianthus annuus L.)

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

The promoter region of two sunflower (Helianthus annuus L. HA89 genotype) seed specifically expressed genes, coding for an oleate desaturase (HaFAD2-1) and a lipid transfer protein (HaAP10), were cloned and in silico characterized. The isolated fragments are 867 and 964 bp long, respectively, and contain several seed-specific motifs, such as AACA motif, ACGT element, E-Boxes, SEF binding sites and GCN4 motif. Functional analysis of these promoters in transgenic Arabidopsis plants was investigated after fusing them with the β-glucuronidase (GUS) reporter gene. None of the promoters triggered GUS activity in any vegetative tissue, with the exception of early seedling cotyledons. HaFAD2-1 and HaAP10 promoters were tested along seed development from globular stage to mature seeds. GUS staining was restricted to embryonic tissue and quantitative fluorometric assays showed high activity values at the later stages of development. In this work we demonstrate that HaFAD2-1 promoter is as strong as 35S promoter even though it is a tissue-specific promoter and its activity derived just from the embryo, thus confirming that it can be considered a strong highly specific seed promoter useful for biotechnology applications.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Allen RD, Nessler CL, Thomas TL (1985) Developmental expression of sunflower 11S storage protein genes. Plant Mol Biol 5:165–173

    Article  CAS  Google Scholar 

  • Allen RD, Bernier F, Lessard PA, Beachy RN (1989) Nuclear factors interact with a soybean beta-conglycinin enhancer. Plant Cell 1:623–631

    Article  CAS  PubMed  Google Scholar 

  • Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, Görlach J (2001) Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13:1499–1510

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Cercos M, Gomez-Cadenas A, Ho T-HD (1999) Hormonal regulation of a cysteine proteinase gene, EPB-1, in barley aleurone layers: cis- and trans-acting elements involved in the co-ordinated gene expression regulated by gibberellins and abscisic acid. Plant J 19:107–118

    Article  CAS  PubMed  Google Scholar 

  • Chandrasekharan MB, Bishop KJ, Hall TC (2003) Module-specific regulation of the beta-phaseolin promoter during embryogenesis. Plant J 33:853–866

    Article  CAS  PubMed  Google Scholar 

  • Chung K-J, Hwang S-K, Hahn B-S, Kim K-H, Kim J-B, Kim Y-H, Yang J-S, Ha S-H (2008) Authentic seed-specific activity of the Perilla oleosin 19 gene promoter in transgenic Arabidopsis. Plant Cell Rep 27:29–37

    Article  CAS  PubMed  Google Scholar 

  • Clough S, Bent A (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  CAS  PubMed  Google Scholar 

  • D’Aoust M-A, Nguyen-Quoc B, Le V-Q, Yelle S (1999) Upstream regulatory regions from the maize Sh1 promoter confer tissue-specific expression of the â-glucuronidase gene in tomato. Plant Cell Rep 18:803–808

    Article  Google Scholar 

  • Edqvist J, Farbos I (2002) Characterization of germination-specific lipid transfer proteins from Euphorbia lagascae. Planta 215:41–50

    Article  CAS  PubMed  Google Scholar 

  • Forde B, Heyworth A, Pywell J, Kreis M (1985) Nucleotide sequence of a B1 hordein gene and the identification of possible upstream regulatory elements in endosperm storage protein genes from barley, wheat and maize. Nucleic Acids Res 13:7327–7339

    Article  CAS  PubMed  Google Scholar 

  • Fujiwara T, Nambara E, Yamagishi K, Goto DB, Naito S (2002) Storage proteins. In: Biologist ASoP (ed) The Arabidopsis book. Rockville, MD, pp 1–12

    Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Hsieh TH, Lee JT, Charng YY, Chan MT (2002) Tomato plants ectopically expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress. Plant Physiol 130:618–626

    Article  CAS  PubMed  Google Scholar 

  • Huang AHC (1996) Oleosins and oil bodies in seeds and other organs. Plant Physiol 110:1055–1061

    Article  CAS  PubMed  Google Scholar 

  • Hwang Y-S, Yang D, McCullar C, Wu L, Chen L, Pham P, Nandi S, Huang N (2002) Analysis of the rice endosperm-specific globulin promoter in transformed rice cells. Plant Cell Rep 20:842–847

    Article  CAS  Google Scholar 

  • Jefferson R, Bevan M, Kavanagh T (1987) The use of the Escherichia coli beta-glucuronidase as a gene fusion marker for studies of gene expression in higher plants. Biochem Soc Trans 15:17–18

    CAS  PubMed  Google Scholar 

  • Jordano J, Almoguera C, Thomas TL (1989) A sunflower helianthinin gene upstream sequence ensemble contains an enhancer and sites of nuclear protein interaction. Plant Cell 1:855–866

    Article  CAS  PubMed  Google Scholar 

  • Kader JC (1996) Lipid-transfer proteins in plants. Annu Rev Plant Physiol Plant Mol Biol 47:627–654

    Article  CAS  PubMed  Google Scholar 

  • Kader JC (1997) Lipid-transfer proteins: a puzzling family of plant proteins. Trends Plant Sci 2:66–70

    Article  Google Scholar 

  • Karimi M, Depicker A, Hilson P (2007) Recombinational cloning with plant gateway vectors. Plant Physiol 145:1144–1154

    Article  CAS  PubMed  Google Scholar 

  • Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol 17:287–291

    Article  CAS  PubMed  Google Scholar 

  • Kasuga M, Miura S, Shinozaki K, Yamaguchi-Shinozaki K (2004) A combination of the Arabidopsis DREB1A gene and stress-inducible rd29A promoter improved drought- and low-temperature stress tolerance in tobacco by gene transfer. Plant Cell Physiol 45:346–350

    Article  CAS  PubMed  Google Scholar 

  • Kaushik V, Yadav MK, Bhatla SC (2009) Temporal and spatial analysis of lipid accumulation, oleosin expression and fatty acid partitioning during seed development in sunflower (Helianthus annuus L.). Acta Physiol Plant

  • Kim SY, Chung H-J, Thomas TL (1997) Isolation of a novel class of bZIP transcription factors that interact with ABA-responsive and embryo-specification elements in the Dc3 promoter using a modified yeast one-hybrid system. Plant J 11:1237–1251

    Article  CAS  PubMed  Google Scholar 

  • Kim M, Kim H, Shin J, Chung C-H, Ohlrogge J, Suh M (2006) Seed-specific expression of sesame microsomal oleic acid desaturase is controlled by combinatorial properties between negative cis-regulatory elements in the SeFAD2 promoter and enhancers in the 5′-UTR intron. Mol Gen Genomics 276:351–368

    Article  CAS  Google Scholar 

  • Kridl J, McCarter D, Rose R, Scherer D, Knutzon D, Radke S, Knauf V (1991) Isolation and characterization of an expressed napin gene from Brassica rapa. Seed Sci Res 1:209–219

    Article  CAS  Google Scholar 

  • Lee JT, Prasad V, Yang PT, Wu JF, Ho THD, Charng YY, Chan MT (2003) Expression of Arabidopsis CBF1 regulated by an ABA/stress promoter in transgenic tomato confers stress tolerance without affecting yield. Plant Cell Environ 26:1181–1190

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Lewi D, Hopp H, Escandón A (2006) Sunflower (Helianthus annuus L.). In: Wang K (ed) Agrobacterium protocols. Humana Press, Clifton, pp 291–297

    Chapter  Google Scholar 

  • Liu Q, Brubaker CL, Green AG, Marshall DR, Sharp PJ, Singh SP (2001) Evolution of the FAD2-1 fatty acid desaturase 5′ UTR intron and the molecular systematics of Gossypium (Malvaceae). Am J Bot 88:92–102

    Article  CAS  PubMed  Google Scholar 

  • Martınez-Rivas JM, Sperling P, Lühs W, Heinz E (2001) Spatial and temporal regulation of three different microsomal oleate desaturase genes (FAD2) from normal-type and high-oleic varieties of sunflower (Helianthus annuus L.). Mol Breed 8:159–168

    Article  Google Scholar 

  • Mazhar H, Quayle R, Fido RJ, Stobart AK, Napier JA, Shewry PR (1998) Synthesis of storage reserves in developing seeds of sunflower. Phytochemistry 48:429–432

    Article  CAS  Google Scholar 

  • Morita A, Umemura T, Kuroyanagi M, Futsuhara Y, Perata P, Yamaguchi J (1998) Functional dissection of a sugar-repressed K-amylase gene (RAmy1A) promoter in rice embryos. FEBS Lett 423:81–85

    Article  CAS  PubMed  Google Scholar 

  • Muller M, Knudsen S (1993) The nitrogen response of a barley C-hordein promoter is controlled by positive and negative regulation of the GCN4 and endosperm box. Plant J 4:343–355

    Article  CAS  PubMed  Google Scholar 

  • Nunberg AN, Li Z, Bogue MA, Vivekananda J, Reddy AS, Thomas TL (1994) Developmental and hormonal regulation of sunflower helianthinin genes: proximal promoter sequences confer regionalized seed expression. Plant Cell 6:473–486

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Ogawa M, Hanada A, Yamauchi Y, Kuwahara A, Kamiya Y, Yamaguchi S (2003) Gibberellin biosynthesis and response during Arabidopsis seed germination. Plant Cell 15:1591–1604

    Article  CAS  PubMed  Google Scholar 

  • Okuley J, Lightner J, Feldmann K, Yadav N, Lark E, Browse J (1994) Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell 6:147–158

    Article  CAS  PubMed  Google Scholar 

  • Pagnussat LA, Lombardo C, Regente M, Pinedo M, Martin M, de la Canal L (2009) Unexpected localization of a lipid transfer protein in germinating sunflower seeds. J Plant Physiol 166:797–806

    Article  CAS  PubMed  Google Scholar 

  • Paniego N, Heinz R, Hopp H (2006) Sunflower. In: Kole C (ed) Genome mapping and molecular breeding. Springer, Heidelberg, pp 153–178

    Google Scholar 

  • Potrykus I (2001) Golden rice and beyond. Plant Physiol 125:1157–1161

    Article  CAS  PubMed  Google Scholar 

  • Prieto-Dapena P, Almoguera C, Rojas A, Jordano J (1999) Seed-specific expression patterns and regulation by ABI3 of an unusual late embryogenesis-abundant gene in sunflower. Plant Mol Biol 39:615–627

    Article  CAS  PubMed  Google Scholar 

  • Radonic LM, Zimmermann JM, Zavallo D, López N, López Bilbao M (2008) Rooting in Km selective media as efficient in vitro selection method for sunflower genetic transformation. Electron J Biotechnol 9:315–319

    Google Scholar 

  • Rasmussen TB, Donaldson IA (2006) Investigation of the endosperm-specific sucrose synthase promoter from rice using transient expression of reporter genes in guar seed tissue. Plant Cell Rep 25:1035–1042

    Article  CAS  PubMed  Google Scholar 

  • Regente M, de la Canal L (2003) A cDNA encoding a putative lipid transfer protein expressed in sunflower seeds. J Plant Physiol 160:201–203

    Article  CAS  PubMed  Google Scholar 

  • Rombauts S, Dehais P, Montagu MV, Rouze P (2002) PlantCARE, a plant cis-acting regulatory element database. Nucleic Acids Res 27:295–296

    Article  Google Scholar 

  • Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers (http://frodo.wi.mit.edu/). In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386

  • Salzman RA, Fujita T, Zhu-Salzman K, Hasegawa PM, Bressan RA (1999) An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates. Plant Mol Biol Rep 17:11–17

    Article  CAS  Google Scholar 

  • Schneiter AA, Miller JR (1981) Description of sunflower growth stages. Crop Sci 21:901–904

    Article  Google Scholar 

  • Schuppert GF, Tang S, Slabaugh MB, Knapp SJ (2006) The sunflower high-oleic mutant Ol carries variable tandem repeats of FAD2–1, a seed-specific oleoyl-phosphatidyl choline desaturase. Mol Breed 17:241–256

    Article  CAS  Google Scholar 

  • Senkoylu N, Dale N (2006) Nutritional evaluation of a high-oil sunflower meal in broiler starter diets. J Appl Poult Res 15:40–47

    CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Stangeland B, Salehian Z (2002) An improved clearing method for GUS assay in Arabidopsis endosperm and seed. Plant Mol Biol 20:107–114

    Article  Google Scholar 

  • Tzen J, Cao Y, Laurent P, Ratnayake C, Huang A (1993) Lipids, proteins, and structure of seed oil bodies from diverse species. Plant Physiol 101:267–276

    CAS  PubMed  Google Scholar 

  • Vos E, Cunnane S (2003) α-Linolenic acid, linoleic acid, coronary artery disease, overall mortality. Am J Clin Nutr 77:521–522

    CAS  PubMed  Google Scholar 

  • Wu C, Washida H, Onodera Y, Harada K, Takaiwa F (2000) Quantitative nature of the prolamin-box, ACGT and AACA motifs in a rice glutelin gene promoter: minimal cis-element requirements for endosperm-specific gene expression. Plant J 23:415–421

    Article  CAS  PubMed  Google Scholar 

  • Yoshihara T, Washida H, Takaiwa F (1996) A 45-bp proximal region containing AACA and GCN4 motif is sufficient to confer endosperm-specific expression of the rice storage protein glutelin gene. GluA-3 FEBS Letters 383:213–218

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by grants of CONICET (PIP 5788), ANPCyT (PICTO ASAGIR Nº08-13164) and INTA (PE AEBIO4461, PE AEGR3426). We thank Lic. Laura Radonic (IB-INTA) for kindly facilitation of the plasmid 35S::GUS. We thank Valeria Beracochea and Teresa Cabrera (IB-INTA) for expert technical assistance. We acknowledge Guillermo Maroniche, Natalia Almasia, Corina Fusari and Florencia del Viso for critically reading of the manuscript and helpful discussions and Dr. Veronica Lia for her assistance in statistical analysis. DZ holds a CONICET fellowship; MLB, HEH and RH are independent researchers at INTA, HEH is researcher of the CIC and Professor at the University of Buenos Aires, RH is researcher of CONICET.

Author information

Authors and Affiliations

  1. Instituto de Biotecnología, CNIA, Instituto Nacional de Tecnología Agropecuaria (INTA)-Castelar, Los Reseros y Nicolas Repeto, 1686, Hurlingham, Buenos Aires Province, Argentina

    Diego Zavallo, Marisa Lopez Bilbao, H. Esteban Hopp & Ruth Heinz

  2. Facultad de Ciencias Exactas y Naturales, Ciudad Universitaria, Universidad de Buenos Aires, 1428, Buenos Aires, Argentina

    H. Esteban Hopp & Ruth Heinz

Authors
  1. Diego Zavallo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marisa Lopez Bilbao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. H. Esteban Hopp
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ruth Heinz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ruth Heinz.

Additional information

Communicated by M. Jordan.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zavallo, D., Lopez Bilbao, M., Hopp, H.E. et al. Isolation and functional characterization of two novel seed-specific promoters from sunflower (Helianthus annuus L.). Plant Cell Rep 29, 239–248 (2010). https://doi.org/10.1007/s00299-010-0816-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-010-0816-x

Keywords

Search

Navigation