Abstract
Rice storage protein glutelin genes are coordinately regulated during seed development. A previous 5′ deletion analysis using transgenic tobacco revealed that the minimum 5′ region necessary for endosperm specificity was within −245 bp of the transcription start site, and included the AACA and GCN4 motifs that are highly conserved in the 5′-flanking regions of all glutelin genes. In this paper, the sequence elements essential for endosperm-specific expression are characterized in stable transgenic tobacco plants by both loss-of-function and gain-of-function experiments using this minimum promoter. Base substitution analysis shows that the proximal AACA motif between −73 and −61, and the GCN4 motif between −165 and −158 act as critical elements. An ACGT motif between −81 and −75, and Skn-I-like elements between −173 and −169 also play important roles in controlling the seed-specific expression. When the distal region between −245 and −145 containing the AACA and the GCN4 motifs or the proximal region between −113 and −46 containing the ACGT and AACA motifs is fused to a truncated promoter (−90 to +9) of the CaMV 35S gene fused to the β-glucuronidase (GUS) reporter gene, high levels of seed-specific expression are observed in these fusions, thereby indicating that either pair of motifs is sufficient to confer seed expression in these fusions. However, when substituted for by the CaMV 35S core promoter (−46 to +1), seed expression is abolished, suggesting that the sequence between −90 and −46 of the CaMV 35S promoter containing G-box-like motif (as-1 element) is required for such specific expression in addition to AACA and GCN4 motifs. Therefore, we conclude that at least three cis-regulatory elements, the AACA motif, GCN4 motif and ACGT motif, are necessary to mediate endosperm expression of the GluB-1 glutelin gene.
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References
Albani D, Hammond-Kosack MC, Smith C, Conlan S, Colot V, Holdsworth M, Bevan MW: The wheat transcriptional activator SPA: a seed-specific bZIP protein that recognizes the GCN4-like motif in the bifactorial endosperm box of prolamin genes. Plant Cell 9: 171–184 (1997).
Benfey PN, Chua NH: The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants. Science 250: 959–966 (1990).
Blackwell TK, Bowerman B, Priess JR, Weintraub H: Formation of a monomeric DNA binding domain by Skn-1 bZIP and homeodomain elements. Science 266: 621–628 (1994).
Bradford MM: Arapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976).
Chen ZL, Pan NS, Beachy RN: A DNA sequence element that confers seed-specific enhancement to a constitutive promoter. EMBO J 7: 297–302 (1988).
de Freitas FA, Yunes a JA, da Silva MJ, Arruda P, Leite A: Structural characterization and promoter activity analysis of the-kafirin gene from sorghum. Mol Gen Genet 245: 177–186 (1994).
Goldberg RB, de Paiva G, Yadegari R: Plant embryogenesis: zygote to seed. Science 266: 605–614 (1994).
Hammond-Kosack MC, Holdsworth MJ, Bevan MW: In vivo footprinting of a low molecular weight glutenin gene (LMWG-1D1) in wheat endosperm. EMBO J 12: 545–554 (1993).
Holdsworth MJ, Munoz-Blanco J, Hammond-Kosack M, Colot V, Schuch W, Bevan MW: The maize transcription factor Opaque-2 activates a wheat glutenin promoter in plant and yeast cells. Plant Mol Biol 29: 711–720 (1995).
Jefferson RA: Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387–405 (1987).
Kim SY, Wu R: Multiple protein factors bind to a rice glutelin promoter region. Nucl Acids Res 18: 6845–6852 (1990).
Kosugi S, Ohashi Y, Nakajima K, Arai Y: An improved assay forα-glucuronidase in transformed cells: methanol almost completely suppresses a putative endogenousα-glucuronidase activity. Plant Sci 70: 133–140 (1990).
Lam H, Benfey PN, Gilmartin PM, Fang RX, Chua NH: Sitespecific mutations alter in vitro factor binding and change promoter expression patterns in transgenic plants. Proc Natl Acad Sci USA 86: 7890–7894 (1989).
Liu ZB, Ulmasov T, Shi X, Hagen G, Guilfoyle TJ: Soybean GH3 promoter contains multiple auxin-inducible elements. Plant Cell 6: 645–657 (1994).
Marzabal P, Busk PK, LudevidMD, Torrent M: The bifactorial endosperm box of-zein gene: characterization and fuction of the Pb3 and GZM cis-acting elements. Plant J 16: 41–52 (1998).
Mikaelian I, Sergent A: A general and fast method to generate multiple site directed mutations. Nucl Acids Res 20: 376 (1992).
Morton RL, Quiggin D, Higgins TJV: Regulation of seed storage protein gene expression. In: Kigel J, Galili G <nt>(ed)</nt> Seed Development and Germination, pp. 103–138. Marcel Dekker, New York (1995).
Müller M, Knudsen S: 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 (1993).
Okita TW, Hwang YS, Huilo J, Kim WT, Aryan, AP, Larsen R, Krishnan HB: Structure and expression of the rice glutelin multigene family. J Biol Chem 264: 12573–12581 (1989).
Ottoboni LM, Leite A, Yunes JA, Targon ML, de Souza Filho GA, Arruda P: Sequence analysis of 22 kDa-like alphacoixin genes and their comparison with homologous zein and kafirin genes reveals highly conserved protein structure and regulatory elements. Plant Mol Biol 21: 765–778 (1993).
de Pater S, Pham K, Chua NH, Memelink J, Kijne J: A 22-bp fragment of the pea lectin promoter containing essential TGAC-like motifs confers seed-specific gene expression. Plant Cell 5: 877–886 (1993).
de Pater S, Katagiri F, Kijne J, Chua NH: bZIP proteins bind to a palindromic sequence without an ACGT core located in a seed-specific element of pea lectin promoter. Plant J 6: 133–140 (1994).
Quayle T, Feix G: Functional analysis of the-300 region of maize zein genes. Mol Gen Genet 231: 369–374 (1992).
Rogers JC, Lanahan MB, Rogers SW: The cis-acting gibberellin response complex in high-pIα-amylase gene promoters. Plant Physiol 105: 151–158 (1994).
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Shewry PR, Napier JA, Tatham AS: Seed storage proteins: structure and biosynthesis. Plant Cell 7: 945–956 (1995).
Shotwell MA, Boyer SK, Chesnut RC, Larkins BA: Analysis of seed storage protein genes of oats. J Biol Chem 265: 9552–9558 (1990).
Szabados L, Ratet P, Grunenberg B, de Bruijn FJ: Functional analysis of the Sesbanla rostrata leghemoglobin glb3 gene 50-upstream region in transgenic Lotus corniculatus and Nicotiana tabacum plants. Plant Cell 2: 973–986 (1990).
Takaiwa F, Kikuchi S, Oono K: A rice glutelin family: major type of glutelin mRNAs can be divided into two classes. Mol Gen Genet 208: 15–22 (1987).
Takaiwa F, Ebinuma H, Kikuchi S, Oono K: Nucleotide sequence of a rice glutelin gene. FEBS Lett 206: 33–35 (1987).
Takaiwa F, Oono K: Genomic DNA sequences of two new genes for new storage protein glutelin in rice. Jpn J Genet 66: 161–171 (1990).
Takaiwa F, Oono K, Wing D, Kato A: Sequence of three members and expression of a new major subfamily of glutelin genes from rice. Plant Mol Biol 17: 875–885 (1991).
Takaiwa F, Oono K, Kato A: Analysis of the 50-flanking region responsible for the endosperm-specific expression of a rice glutelin chimeric gene in transgenic tobacco. Plant Mol Biol 16: 49–58 (1991).
Takaiwa F, Yamanouchi U, Yoshihara T, Washida H, Tanabe F, Kato A, Yamada K: Characterization of common cisregulatory elements responsible for the endosperm-specific expression of members of the rice glutelin multigene family. Plant Mol Biol 30: 1207–1221 (1996).
Thomas TL: Gene expression during plant embryogenesis and germination: an overview. Plant Cell 5: 1401–1410 (1993).
Ueda T, Wang Z, Pham N, Messing J: Identification of a transcriptional activator-binding element in the 27-kilodalton zein promoter, theα300 element. Mol Cell Biol: 14: 4350–4359 (1994).
Wu CY, Suzuki A, Washida H, Takaiwa F: The GCN4 motif in rice glutelin gene is essential for endosperm-specific gene expression and is activated by Opaque-2 in transgenic plants. Plant J 14: 673–683 (1998).
Yoshihara T, Washida H, Takaiwa F: 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 Lett 383: 213–218 (1996).
Yunes JA, Neto GC, da Silva MJ, Leite A, Ottoboni LMM. Arrud P: The transcriptional activator Opaque2 recognizes two different target sequences in the 22-kD-likeα-prolamin genes. Plant Cell 6: 237–249 (1994)
Zheng Z, Kawagoe Y, Xiao S, Li Z, Okita T, Hau TL, Lin A, Murai N: 50 distal and proximal cis-acting regulatory elements are required for developmental control of a rice seed storage protein gene. Plant J 4: 357–366 (1993).
Zhou X, Fan Y-L: The endosperm-specific expression of a rice prolamin chimeric gene in transgenic tobacco plants. Transgen Res 2: 141–146 (1993).
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Washida, H., Wu, CY., Suzuki, A. et al. Identification of cis-regulatory elements required for endosperm expression of the rice storage protein glutelin gene GluB-1. Plant Mol Biol 40, 1–12 (1999). https://doi.org/10.1023/A:1026459229671
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DOI: https://doi.org/10.1023/A:1026459229671