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A novel cis-acting element, ESP, contributes to high-level endosperm-specific expression in an oat globulin promoter

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Abstract

To examine the genetic controls of endosperm (ES) specificity, several cereal seed storage protein (SSP) promoters were isolated and studied using a transient expression analysis system. An oat globulin promoter (AsGlo1) capable of driving strong ES-specific expression in barley and wheat was identified. Progressive 5′ deletions and cis element mutations demonstrated that the mechanism of specificity in the AsGlo1 promoter was distinct from that observed in glutelin and prolamin promoters. A novel interrupted palindromic sequence, ACATGTCATCATGT, was required for ES specificity and substantially contributed to expression strength of the␣AsGlo1 promoter. This sequence was termed the endosperm specificity palindrome (ESP) element. The GCN4 element, which has previously been shown to be required for ES specificity in cereal SSP promoters, had a quantitative role but was not required for tissue specificity. The 960-bp AsGlo1 promoter and a 251-bp deletion containing the ESP element also drove ES-specific expression in stably transformed barley. Reporter gene protein accumulated at very high levels (10% of total soluble protein) in ES tissues of plants transformed with an AsGlo1:GFP construct. Expression strength and tissue specificity were maintained over five transgenic generations. These attributes make the AsGlo1 promoter an ideal promoter for biotechnology applications. In conjunction with previous findings, our data demonstrate that there is more than one genetically distinct mechanism by which ES specificity can be achieved in cereal SSP promoters, and also suggest that there is redundancy between transcriptional and post-transcriptional tissue specificity mechanisms in cereal globulin genes.

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Abbreviations

ABA:

abscisic acid

Act1 :

rice actin gene

AsGlo1 :

oat 12S globulin gene

bZIP:

basic leucine zipper protein

DAP:

days after pollination

ES:

endosperm

ESP:

endosperm specificity palindrome

GA:

gibberellin

GFP:

green fluorescent protein

GluB-1 :

rice B1 glutelin gene

Glu-B1 :

wheat high-molecular weight glutenin gene

GP:

Hordeum vulgare cv. Golden Promise

GUSPlus :

modified Staphylococcus sp. β-glucuronidase

Hor2-4 :

barley B1 hordein gene

Hor3−1 :

barley D hordein gene

SSP:

seed storage protein

sUTR:

synthetic untranslated leader

TC:

tissue culture

TEP:

total extractable protein

TSP:

total soluble protein

XYN:

xylanase

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Acknowledgments

This research was supported by an Australian Government Grains Research and Development Corporation grant (to CEV). The authors wish to thank Stephen Mudge (CSIRO Plant Industry, Brisbane) and Diana Buzas (ARC Centre for Integrative Legume Research, The University of Queensland, Brisbane) for technical advice, Adrian Elliott (Bureau of Sugar Experimental Stations, Brisbane, Australia) for donation of partially purified s65tGFP, Danial Bishop (CSIRO Plant Industry, Canberra) for assistance in generating transgenic barley and Joan Vickers (The University of Southern Queensland, Toowoomba) for useful comments during manuscript preparation.

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Authors and Affiliations

  1. CSIRO Plant Industry, 306 Carmody Rd, St Lucia, Brisbane, 4067, Australia

    Claudia E. Vickers & Gangping Xue

  2. ARC Centre of Excellence for Integrative Legume Research, The University of Queensland, St Lucia, 4072, Australia

    Claudia E. Vickers & Peter M. Gresshoff

  3. Department of Biological Sciences, The University of Essex, Wivenhoe Park, Colchester, UK

    Claudia E. Vickers

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  1. Claudia E. Vickers
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  2. Gangping Xue
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Correspondence to Claudia E. Vickers.

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Accession numbers: AY294282, AY795082, AY293730, AY795083

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Vickers, C.E., Xue, G. & Gresshoff, P.M. A novel cis-acting element, ESP, contributes to high-level endosperm-specific expression in an oat globulin promoter. Plant Mol Biol 62, 195–214 (2006). https://doi.org/10.1007/s11103-006-9014-1

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