The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (β-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (β-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

The wheat bZip transcription factor TaABF1 mediates both abscisic acid (ABA)-induced and ABA-suppressed gene expression. As levels of TaABF1 protein do not change in response to ABA, and TaABF1 is in a phosphorylated state in vivo, we investigated whether TaABF1 could be regulated at the post-translational level. In bombarded aleurone cells, a TaABF1 protein carrying phosphomimetic mutations (serine to aspartate) at four sites (S36D, S37D, S113D, S115D) was three to five times more potent than wild-type TaABF1 in activating HVA1, an ABA-responsive gene. The phosphomimetic mutations also increased the ability of TaABF1 to downregulate the ABA-suppressed gene Amy32b. These findings strongly suggest that phosphorylation at these sites increases the transcriptional regulatory activity of TaABF1. In contrast to the activation observed by the quadruple serine to aspartate mutation, a single S113D mutation completely eliminated the ability of TaABF1 to upregulate HVA1 or downregulate Amy32b. Thus phosphorylation of TaABF1 can either stimulate or inhibit the activity of TaABF1 in regulating downstream genes, depending on the site and pattern of phosphorylation. Mutation of S318 and S322 (in the bZIP domain) eliminated the ability of TaABF1 to activate HVA1, but had no effect on the ability of TaABF1 to downregulate Amy32b, suggesting that TaABF1 represses Amy32b expression through a mechanism other than direct DNA binding. An important step towards understanding how ABA and gibberellin (GA) signals are integrated through TaABF1 phosphorylation to regulate downstream gene expression is to clarify the effects of those hormones on the expression of specific genes. In contrast to some other ABA-induced genes, we found that HVA1 induction by ABA or TaABF1 is not inhibited by GA.