To better understand the antagonistic relationship between p

To better understand the antagonistic relationship between proteases and phytocystatins and to elucidate the regulatory mechanisms that control their expression, the upstream promoter sequences of their genes have been analysed. The analyses revealed that both protease and phytocystatin genes contain several interesting cis-elements that are responsive to the phytohormones involved in development and germination, i.e., ABA and GAs (Fig. 4). Many hydrolytic enzymes that are synthesised in response to GAs in barley aleurone cells, which include high and low pI α-amylases, α-glucosidases, proteases and nucleases are secreted and they digest the reserve materials stored in the endosperm (Gubler et al., 1999). Functional analysis of the promoters of high and low pI α-amylase genes of barley, wheat and rice revealed a conserved cis-acting response complex (GARC), which very often contains three sequence motifs, the TAACAAA box or GA-responsive DL-Dithiothreitol mg (GARE), the pyrimidine box CCTTTT and the TATCCAC box, which are necessary for a full response to GA (Gubler and Jacobsen, 1992, Jacobsen et al., 1995, Lanahan et al., 1992). The promoters of other GA-responsive aleurone genes, such as the cathepsin B-like gene (Cejudo et al., 1992, Gubler and Jacobsen, 1992) and L-like cysteine protease genes (Cercos et al., 1999, Mikkonen et al., 1996) have cis-elements similar to those found in the α-amylase gene promoters (Isabel-LaMoneda et al., 2003). However, the sequences and positions of these cis-elements in each of the promoters differ, which explains the diverse spatial and temporal patterns of expression of these genes upon seed germination (Isabel-LaMoneda et al., 2003). Comparing the expression patterns of GA-regulated genes indicated that the early-response genes encode signal transduction proteins, such as calmodulin (Schuurink et al., 1996), Ca+2 ATPase (Chen et al., 1997) and transcription factors, whereas the late-response genes encode hydrolases and nucleases (Banik et al., 1997Taiz and Starks, 1977). The first transcription factor (TF) known to interact with one of the cis-acting GAREs is GAMYB protein, the expression of which is induced by GA in the aleurone cells of barley (HvGAMYB) (Gubler and Jacobsen, 1992). GAMYB is an important regulator of endosperm functions during seed development and germination (Yan et al., 2014). The binding of this TF to the GARE activates the expression of a number of hydrolase genes required for the mobilization of storage materials during seed germination (Gubler et al., 1995, Gubler et al., 1999). HvGAMYB-responsive promoters are present in the genes encoding low- and high-pI α- amylases, the EII (1-3, 1-4-β)-glucanase, the cathepsin B-like protease and EP-B cysteine protease (Cejudo et al., 1992, Cercos et al., 1999, Gubler et al., 1995, Gubler et al., 1999). Additionally, GAMYB has been shown to be involved in GA-mediated PCD in the aleurone cells of both cereals and Arabidopsis (Alonso-Peral et al., 2010, Guo and Ho, 2008). The GAMYB of rice seeds (OsGAMYB) also induces the expression of a different set of gibberellin-responsive genes in the aleurone layer, which indicates that other TFs may be required for the endosperm- specific gibberellin response (Tsuji et al., 2006). The rice gamyb mutant and Arabidopsis triple mutant myb33myb65myb101 show defects in GA-induced respones in the aleurone layer of the endosperm (Alonso-Peral et al., 2010, Kaneko et al., 2004, Yan et al., 2014).