Journal of Experimental Botany, Vol. 54, No. 391, pp. 2251-2263,
October 1, 2003
© 2003 Oxford University Press
Differential gene expression for suicide-substrate serine proteinase inhibitors (serpins) in vegetative and grain tissues of barley
Received 21 February 2003; Accepted 14 June 2003
1 Biochemistry and Nutrition, BioCentrum, Building 224, Technical University of Denmark, DK-2800 Lyngby, Denmark
2 Department of Crop Science, Swedish University of Agricultural Sciences, PO Box 44, SE-23053 Alnarp, Sweden
3 Plant Research Department, Building 301, Risø National Laboratory, Frederiksborgvej 399, PO Box 49, DK-4000 Roskilde, Denmark
* Present address and to whom correspondence should be sent: Department of Biological Sciences, Building E8A, Macquarie University NSW 2109, Australia. Fax: +61 2 98508245. E-mail: troberts{at}els.mq.edu.au
Abbreviations: BSZ, barley serpin (protein Z family); dpa, days post-anthesis; rBSZ, recombinant barley serpin (protein Z family); RT-PCR, reverse-transcription polymerase chain reaction.
Proteins of the serpin superfamily (
43 kDa) from mature cereal grains are in vitro suicide-substrate inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the ‘standard-mechanism’ serine proteinase inhibitors (<25 kDa), the biological functions of plant serpins are unknown. Expression studies of genes encoding members of three subfamilies of serpins (BSZx, BSZ4 and BSZ7) in developing grain and vegetative tissues of barley (Hordeum vulgare L.) showed that transcripts encoding BSZx, which inhibits distinct proteinases at overlapping reactive centres in vitro, were ubiquitous at low levels, but the protein could not be detected. EST analysis showed that expression of genes for serpins with BSZx-type reactive centres in vegetative tissues is widespread in the plant kingdom, suggesting a common regulatory function. For BSZ4 and BSZ7, expression at the protein level was highest in the maturing grain (
15 d post-anthesis), where these serpins were localized by immunomicroscopy to the central and peripheral starchy endosperm, subaleurone, and (at lower levels) to the aleurone. Serpins were also localized to the meristem and vascular tissues of roots, and to the phloem of coleoptiles and leaves. The identification of BSZ4 in vegetative tissues by western blotting was confirmed for the roots by purification and amino acid sequencing, and for the leaves by in vitro reactive-centre loop cleavage studies. Plant serpins are likely to use their irreversible inhibitory mechanism in the inhibition of exogenous proteinases capable of breaking down seed storage proteins, and in the defence of specific cell types in vegetative tissues.
Key words: Barley, Hordeum vulgare, gene expression, plant defence, protease inhibitor, serine proteinase inhibitor, serpin.
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