Plant Molecular Biology 41: 15–23, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Cloning and characterization of a trypsin inhibitor cDNA from amaranth
(Amaranthus hypochondriacus) seeds
, Alejandro Blanco-Labra
, Glenda Guti
, Alfredo Herrera-Estrella
and June Simpson
Unidad de Biotecnolog´ıa e Ingenier´ıa Gen´etica de Plantas, Centro de Investigaci´on y de Estudios Avanzados
del IPN, Apdo postal 629, C.P. 36500, Irapuato, Guanajuato, M´exico (
author for correspondence);
Ciencias Agr´ıcolas, Universidad de Guanajuato, Apdo postal 311, C.P. 36500 and Irapuato, Guanajuato, M´exico
Received 12 February 1999; accepted in revised form 9 June 1999
Key words: Amaranthus hypochondriacus), gene expression, trypsin inhibitor
We previously isolated and sequenced the major trypsin inhibitor from Amaranthus hypochondriacus seeds. This
amaranth trypsin inhibitor (AmTI) is a 69 amino acid protein with high homology to members of the potato-1
inhibitor family. This paper describes the cloning and expression of a cDNA encoding this trypsin inhibitor in
various vegetative tissues of the amaranth plant during seed development and imbibition, and investigates the
possible induction of AmTI expression by wounding.
We obtained a 393 bp cDNA sequence with an open reading frame corresponding to a polypeptide with
76 amino acid residues. With the exception of one residue (Ser-41), the polypeptide agrees with the amino acid
sequence previously reported, plus 7 more residues at the N-terminus. These N-terminal residues are thought to be
part of the signal used for intracellular sorting.
The organ speciﬁcity of AmTI gene expression was investigated by northern analysis, showing that mRNA
corresponding to AmTI genes was present in stems of plants growing under normal conditions.
The kinetics of accumulation of the AmTI-mRNA, protein, and inhibitory activity during seed development
and imbibition was determined. AmTI-mRNA accumulation reached a maximum at 14 days after anthesis (daa)
and then gradually decreased, being barely detectable 36 daa. The AmTI protein accumulation followed the same
proﬁle as the inhibitory activity, both were delayed with respect to the mRNA. The maximum level was observed
22 daa, and then gradually decreased until a steady state was reached as seed maturation proceeded. Upon imbi-
bition, a gradual decrease in AmTI protein and inhibitory activity was shown; however, an AmTI transcript was
detected 24 h after imbibition. In contrast to representative members of the potato I family, this inhibitor was not
inducible by wounding of leaves.
Proteinase inhibitors have long been proposed to play
an important role as defense proteins in plants against
the attack of insects (Ryan, 1990). This has been
demonstrated by the transfer of proteinase inhibitor
genes from different sources to several plants of eco-
The nucleotide sequence data reported will appear in the
EMBL and GenBank Nucleotide Sequence Databases under the
accession number AJ132473.
nomic interest, resulting in transgenic plants more
resistant to predation (Boulter, 1993; Altpeter et al.,
1999). However, another important and less under-
stood role of these inhibitors is related to the regulation
of the activity of endogenous proteases, particularly
during the stage of protein deposition in storage tis-
sues (Baumgartner and Chrispeels, 1977). In addition,
inhibitorproteinsthemselves have also been suggested
to act as storage proteins (Richardson, 1991).