Plant Molecular Biology 34: 331–338, 1997.
1997 Kluwer Academic Publishers. Printed in Belgium.
Characterization of a barley gene coding for an
-amylase inhibitor subunit
(CMd protein) and analysis of its promoter in transgenic tobacco plants and
in maize kernels by microprojectile bombardment
, Marie-Franc¸oise Gautier
, Marcelo Menossi
e A. Martinez-Izquierdo
and Philippe Joudrier
e de Biochimie et Biologie Mol
eculaire des C
eales, INRA, 2 Place Viala, 34060 Montpellier C
author for correspondence);
Departamento de Gen
etica Molecular, CID-CSIC, Jordi Girona 18–26,
08034 Barcelona, Spain;
J. Grosset and R. Alary contributed equally to the research presentedin this paper and
are both considered to be ﬁrst author.
Received 16 October 1996; accepted in revised form 13 February 1997
Key words: Act 1,
-amylase inhibitor, Hordeum vulgare, CM protein, promoter, tissue-speciﬁc expression
A gene coding for a barley CMd protein was isolated from a genomic library using a cDNA probe encoding the
wheat CM3 protein. Promotersequence analysis reveals motifs found in genes speciﬁcally expressed in endosperm
and aleurone cells, as well as TATA and other putative functional boxes. 720 bp of the Hv85.1 CMd protein gene
promoter,when fused to a gus coding region, were unable to direct GUS activity in the seeds of transgenic tobacco
plants. In contrast, the same construction delivered into immature maize kernels by microprojectile bombardment
was able to direct expression of GUS in the outermost cell layers of maize endosperm in both a tissue-speciﬁc and
a developmentally determined manner.
CM proteins were originally found in cereal endo-
sperm and belong to the trypsin/
family . They were initially characterized by their
solubility in a mixture of chloroform and methanol
and therefore named CM proteins . The
inhibitorshavebeen classiﬁed into monomeric,dimer-
ic and tetrameric forms on the basis of their degree of
aggregation. Of the ﬁve major CM proteins present in
barley, CMc and CMe are trypsin inhibitors and CMa
is an inhibitor of insect
-amylases [1, 30]. Although
no in vitro
-amylase inhibitory activity individually,
they do show increased in vitro inhibitory activity to
-amylases when they are combined with CMa
to form a tetrameric protein in a CMa/CMb/CMd ratio
of 1:1:2 [5, 34].
The nucleotide sequences data reported will appear in the
EMBL, GenBank and DDBJ Nucleotide Sequence Databases under
the accession numbers X69911 (Hv85.1) and U47641 (Hv57).
Although the in planta biological role of the CM
proteins is as yet unknown, they are thought to
be involved in the plant defense mechanism against
insects; in vitro experiments have shown that CM pro-
teins are active against insect
-amylases [16, 36].
More recently, transgenic tobacco plants carrying the
barleyCMe or the wheat monomeric
itor coding sequence have been found to be lethal to
lepidopterous larvae in leaf disk assays .
Wheat CM proteins and other
-amylase or trypsin
inhibitorsare reduced by the NADP/thioredoxinh sys-
tem in physiological conditions compatible with cell
life . Thus thioredoxincould control the biological
activity of CM proteins by changing their redox status.
Anotheraspectconcernsthe role of CM proteins in the
technological quality of wheat products like pasta [12,
21]. After reduction by the thioredoxinsystemandthen
modiﬁcation of their sulfhydryl status, these cysteine-
rich proteins may interact, via SH/SS exchange reac-
Article: Plan3719 , GR: 201001908, Pips nr. 135301 BIO2KAP
plan3719.tex; 12/05/1997; 7:21; v.7; p.1