Fig. 1.

(A) Expression of the HvPap-1 gene in aleurone layers after 48 h of incubation in the presence or absence of 1 μM GA, determined by qRT-PCR. Values are expressed as relative mRNA levels of barley protease and standardized using the barley Actin2 mRNA content. (B) Nucleotide and deduced amino acid sequences of the cDNA encoding the cathepsin F-like HvPap-1 peptidase. The dark and light grey boxes represent the signal peptide and the propeptide sequences, respectively. The active site residues C¹⁶⁶, H³⁰⁸, and N³³⁵, characteristic of C1A CysProts, are boxed. The conserved motif EX₃RX₃FX₂NX₃AX₃Q specific for cathepsin F-like proteases is also boxed. The GXN/TXFXD and the GCNGG like-motifs, common to all CysProts, are double underlined. The consensus QVVGG sequence present in cystatins and responsible for the protease–inhibitor interaction is marked by a dotted box. Cysteine residues are circled and the stop codon is indicated by an asterisk. The predicted N-glycosylation motif NYS is underlined by a dotted line. The horizontal double arrowhead indicates the amino acid sequence used for specific antibody production (HvPap-1-IgG). (C) Ribbon plots of HvPap-1 and human cathepsin-F. The three-dimensional structure of HvPap-1 was predicted using the automated SWISS-MODEL program with the known human cathepsin-F structure as template (1M6D). The figure was prepared with RasMol 2.7. The active site residues of HvPap-1 are coloured in green. (This figure is available in colour at JXB online.)