Significantly less is known about regulation
Significantly less is known about regulation of phytocystatin gene expression. A genomic clone corresponding to the barley cystatin Icy gene has been characterized (Gaddour et al., 2001) and the function of its promoter region has been analysed by Martínez et al. (2005b). Bioinformatic analysis of the potential cis-regulatory elements in the promotor region revealed numerous elements, among which are three CCTTTT/A pyrimidine boxes recognized by the DOF transcription factors and nine C/TAAC R2-R3 MYB-binding motifs. The expression of the Icy gene in the barley aleurone layer and the presence of DOF-binding motifs in the promotor of the Icy gene suggest that its expression might be regulated by the two barley DOF factors, BPBF and SAD (Mena et al., 1998, Isabel-LaMoneda et al., 2003). Martínez et al. (2005b) showed that Icy transcripts were abundantly expressed in aleurone WIKI4 at 8h of imbibition, but their expression decreased until it was almost undetectable at 24h. Furthermore, in isolated aleurone layers incubated with GA3, the level of the Icy transcript was also markedly decreased. Martínez et al. (2005b) stated that the Icy expression pattern in the barley aleurone layer indicated that the two factors might compete for binding to the same cis-motifs. BPBF behaves as a strong transcriptional repressor of Icy, whereas SAD acts as an activator of this gene. Moreover, BPBF strongly counteracts the SAD-mediated trans-activation of the Icy gene promoter when the two factors are present in equimolar amounts. The repressor activity of BPBF might also operate indirectly through its interaction with other transcription factors that are putatively associated with the Icy promoter, such as GAMYB. Isabel-LaMoneda et al. (2003) demonstrated that SAD and BPBF both interact with the MYB R2R3 transcriptional activator of several hydrolase genes in barley aleurone cells during germination (Gubler et al., 1999, Isabel-LaMoneda et al., 2003) and with endosperm protein genes during seed development (Diaz et al., 2002). These data suggest that the two DOF factors, SAD and BPBF, are an important part of the transcription combinatorial complex mediated by the hormones GA and ABA and are responsible for fine-tuning the expression of genes encoding inhibitors of hydrolases that trigger the mobilization of storage materials that provide nutrients for seedling growth (Martínez et al., 2005b). Upstream analysis of the Arabidopsis cystatin (AtCYS6) promoter region also revealed several putative cis-elements (Hwang et al., 2009). Among them are the ABA-responsive element (ABRE), which is involved in abscisic-acid responsiveness (Washio, 2003), and pyrimidine boxes (P-box; CTTTT) (Huang et al., 1990). The ABRE motif and the P-box motif have been identified as binding sites for ABRE-binding proteins (AREBs) (Lopez-Molina and Chua, 2000) and DOF transcription factors (Yanagisawa, 2002), respectively. Hwang et al. (2009) observed that the levels of AtCYS6 expression were decreased by treatment with GA, whereas AtCYS6 expression persisted at a high level after ABA treatment. These results are consistent with the results of Martínez et al. (2003b), who showed that ABA represses the steady-state levels of cathepsin B-like CP gene expression and induces phytocystatin gene (Icy) expression. Therefore, the putative AREBs in the AtCYS6 promoter may be responsible for the ABA-mediated control of AtCYS6 expression (Hwang et al., 2009). These studies suggest that, like AtCYS6 and Icy, the expression of other phytocystatin genes might be regulated by transcriptional regulators that are activated in response to ABA and GA signalling. Analyses of upstream promotor sequences of cysteine proteases and phytocystatins and interaction between transcription factors are the initial step to understanding the regulatory mechanisms that control their gene expression. Analyses of the expression levels of TFs and their targets confirm this interaction and indicate that cysteine proteases and their inhibitors are transcriptionally regulated. In B. distachyon seeds, transcripts of two factors: BdGamyb and BdDof24 are induced by GA in aleurone cells during germination and reach the highest expression level just before the gene encoding cathepsin B-like protease (BdCathB) (González-Calle et al., 2014). In turn, ABA and paclobutrazol (a GA biosynthesis inhibitor) down regulated the expression of BdGamyb and its orthologs in barley and rice as well as the gene encoding cathepsin B-like protease (BdCathB) (Gubler et al., 1999, Sutoh and Yamauchi, 2003). Moreover, transient expression experiments have shown that BPBF strongly decreased transcription of the native promotor of a barley cystatin gene (Icy) but SAD activated this system in vivo (Martínez et al., 2005b). Finally, mutants of transcription factor VP1 (a key component of ABA signaling) in maize and Arabidopsis, are characterized by inability of seeds to initiate dormancy leading to their premature germination in the ear (Abraham et al., 2016, Gubler et al., 2005). These numerous results indicate that the success of seed development and germination depends on their responses to GA or ABA, which activate the expression of many genes encoding firstly TFs and next their targets such as proteolytic enzymes and their inhibitors.