Cloning and characterization of a trypsin inhibitor cDNA from amaranth (Amaranthus hypochondriacus) seeds

We previously isolated and sequenced the major trypsin inhibitor from Amara nthus 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 indu ction 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 re sidue (Ser-41), the polypeptide agrees with the amino acid sequence previou sly reported, plus 7 more residues at the N-terminus. These N-terminal resi dues 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 stem s of plants growing under normal conditions. The kinetics of accumulation of the AmTI-mRNA, protein, and inhibitory acti vity during seed development and imbibition was determined. AmTI-mRNA accum ulation reached a maximum at 14 days after anthesis (daa) and then graduall y decreased, being barely detectable 36 daa. The AmTI protein accumulation followed the same profile as the inhibitory activity, both were delayed wit h respect to the mRNA. The maximum level was observed 22 daa, and then grad ually decreased until a steady state was reached as seed maturation proceed ed. Upon imbibition, a gradual decrease in AmTI protein and inhibitory acti vity was shown; however, an AmTI transcript was detected 24 h after imbibit ion. In contrast to representative members of the potato I family, this inh ibitor was not inducible by wounding of leaves.