In higher eukaryotes regulation of gene expression is controlled at the transcriptional, post transcriptional, translational level or a combination of all the above. Such control of gene expression is a sophisticated strategy for energy utilization. Primary signal molecules or elicitors are perceived external or internal to the cell membrane. Thus in response to various cues, the plant cell interacts and responds for survival. Perception of signal molecules is followed by the induction of a signal transduction cascade eventually leading to the interaction of protein molecules (transcription factors) with specific sequences (cis-elements) located within the promoter region of genes. Promoters of genes can be isolated and studied to identify cis-elements and transcription factors that control the expression of genes. In this study, the promoters of the bifunctional α-amylase and subtilisin inhibitor gene (asi), and the pathogenesis related gene (prb-1) both from barley, were investigated.
The bifunctional α-amylase and subtilisin inhibitor protein (ASI) is an important component of the barley (Hordeum vulgare L.) grain, comprising up to 0.5% of total seed protein [Munck, 1985 #6086; Jarrett, 1997 #164]. Although the physiological function of this protein is not clear, it is known that it selectively inhibits the high pl-group of α-amylases [Mundy, 1983 #2872; Weselake, 1983 #2875] in addition to the bacterial serine protease, subtilisin [Mundy, 1983 #2872; Yoshikawa, 1976 #2873]. The levels of ASI protein and mRNA in the endosperm and aleurone layers is developmentally regulated and is thought to be under control of abscisic acid (ABA) and gibberellic acid (GA) [Leah, 1989 #2416; Mundy, 1984 #2876]. These two hormones are also involved in regulating the expression of the high pl-group of α-amylases, where the hormonal control is antagonistic to that of the ASI protein [Mundy, 1986 #2465; Rogers, 1985 #2877].
In barley, the prb-l gene is one of at least five-members of the pathogenesis related type-1 (PR-1) gene family. Northern-blot analysis was used to demonstrate that transcript homologous to the prb-1 gene is induced in response to biotic agents such as barley powdery mildew fungus (Erysiphe graminis) and abiotic agents, such as chemicals like salicylic acid (SA), 2,4- dichloro isonicotinic acid (INA), and ultra violet (UV) light [Mouradov, 1994 #5296]. Based on this data, it was likely that the promoter of the prb-l gene is inducible by the agents outlined above. The 1033 bp promoter of the asi gene was isolated and investigated by a transient reporter-gene expression assay to demonstrate hormonal and tissue-specific expression. Rice was transformed with the 1033-asi promoter linked to a reporter gene to investigate 1033-asi promoter-directed temporal, tissue-specific and developmental control of gene expression. Specifically:
• The 1033-asi promoter was shown to confer aleurone-specific gene expression.
• The 1033-asi promoter was isolated and a number of sequences were identified which were known to play a part in ABA and GA mediated induction of other genes.
• The 1033-asi promoter was demonstrated to not be regulated in the mature aleurone tissue by the hormones ABA and GA.
Likewise, a 1239 bp promoter of the prb-1 gene was investigated In transgenic rice plants or by transient reporter-gene expression assays that demonstrated the following results:
• The 1239-prb-1 promoter not induced in barley, wheat and rice by the chemicals (INA) and benzothiadiazole (BTH).
• The 1239-prb-1 promoter is not induced in barley and wheat by the barley and wheat powdery mildew fungus respectively.
• In leaves of transgenic rice plants the 1239-prb-1 promoter is however induced locally around necrotic-like lesions of unknown causes.
• The 1239-prb-1 promoter directs constitutive expression of gfp in terminal cells of trichomes for unknown reasons.
Based on the above data, it is possible to conclude that the 1033-asi promoter could be used to express transgenes in the developing endosperm of barley and possibly other cereals. The 1239-prb-1 promoter could be used to express transgenes locally around the site of necrotic-like lesions.