An ABA-responsive bZIP protein, OsBZ8, mediates sugar repression of a-amylase gene expression Yi-Ching Lee a , Chung-An Lu b , Jose Casaretto c and Su-May Yu b,* a Graduate Institute of Life Sciences, National Defense University, and Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115, Taiwan, Republic of China b Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan 115, Republic of China c Department of Biology, Washington University, St. Louis, MO 63130, USA *Corresponding author, e-mail: sumay@ccvax.sinica.edu.tw Received 26 December 2002; revised 14 February 2003 Expression of some a-amylase genes in cereals is suppressed by sugars and activated by sugar starvation. A 100-bp sugar response sequence (SRS) identified in the promoter of a rice a-amylase gene, aAmy3, contains three essential motifs: the GC box, the G box, and the TATCCA element. To study the mechanism of sugar regulation of aAmy3 transcription, an ABA-responsive bZIP protein, OsBZ8, which binds specifi- cally to the G box in aAmy3 SRS was characterized and function analysed. In sucrose-starved rice suspension cells and embryos, decline in OsBZ8 mRNA levels coincided with the induction of aAmy3 mRNA accumulation. In vivo gain- and loss-of-function studies by transient expression assays in rice embryos revealed that OsBZ8 suppresses SRS activity through the G box and overrides the activity of an activator, OsMYBS1, which binds to the TATCCA element. Gel mobi- lity shift assays revealed that OsBZ8 binds specifically to the G box in vitro. These studies suggest that OsBZ8 is a sup- pressor responsible for sugar repression of aAmy3 expression, and OsMYBS1 is responsible for sugar starvation induced expression of aAmy3. Introduction Sugar production through photosynthesis is the most fundamental activity in plant life. The processes of sugar production, transport, utilization and storage are tightly associated with cellular activities, tissue and organ development, and responses to environment. Genes involved in these processes are highly regulated by sugars. In general, sugar favours the expression of enzymes in connection with biosynthesis, utilization, and storage of reserves (including starch, lipid and pro- tein), while repressing the expression of enzymes involved in photosynthesis and reserve mobilization (Koch 1996). Recent studies with Arabidopsis have revealed intimate cross-talks between sugars, plant hor- mones, environmental stimuli and metabolic signalling pathways through the formation of an interconnected web (Sheen et al. 1999, Smeekens 2000, Coruzzi and Zhou 2001, Gazzarrini and McCourt 2001). As sugars play a central role in metabolism, as well as regulating many developmental and physiological processes in plants, it is conceivable that sugar affects, either directly or indirectly, each pathway in the web. Despite the importance of soluble sugars in regulating these funda- mental processes, our understanding regarding the mechanisms and components involved in sugar regula- tion of gene expression in plants remains very limited. The expression of a-amylase genes in cereals is induced by both gibberellin (GA) and sugar starvation. Sugar regulation of a-amylase gene expression has been used as a model system for studying the molecular mechan- isms that mediate sugar repression in plants (Yu 1999a, b). Expression of a-amylase genes in the embryos of germinating rice and barley seeds (Yu et al. 1996, Perata et al. 1997) and in cultured rice suspension cells (Yu et al. 1991, 1992) is activated by sugar starvation and repressed by sugar provision. Sugar repression of a-amylase gene expression involves control of both transcription PHYSIOLOGIA PLANTARUM 119: 78–86. 2003 Copyright # Physiologia Plantarum 2003 Printed in Denmark – all rights reserved ISSN 1399-3054 Abbreviations – GA, gibberellin; SRS, sugar response sequence. 78 Physiol. Plant. 119, 2003