Functional analysis of the 5′ regulatory region of the maize GALACTINOL SYNTHASE2 gene

Maize (Zea mays) GALACTINOL SYNTHASE (GolS) is a key enzyme in the raffinose biosynthetic pathway. We have previously characterized the maize GolS2 (ZmGolS2) gene as heat shock induced in maize germinating seeds and cultured cells. Here we report the identification, isolation and characterization of the 1.5Kb, 5' regulatory region of the ZmGolS2 gene. The 1.5kb fragment and its deletions were tested for promoter activity by their regulation of the Renilla (Renilla reniformis) luciferase reporter gene expression in maize protoplasts cultured at either 25°C or 42°C for 24h. The expression of a constitutively expressed firefly (Photinus ssp.) luciferase gene in the same vector was used as a reference. One heat shock element (HSE) was identified by comparing the promoter activity of each fragment under normal and heat shock conditions. Deletion or triplication of this HSE motif, abolished or enhanced the heat shock response of the ZmGolS2 promoter, respectively. This HSE motif is specifically bound by proteins in the nuclear extracts of heat shock stressed, but not unstressed maize cells as confirmed by DNA-EMSA. This work helps to understand the regulatory mechanism of the ZmGolS2 gene under stress conditions.