K. Toyofuku and J. YAMAGUCHI

      BioScience Center and Graduate School of Bioagricultural Sciences, 
      Nagoya University, Chikusa-ku, Nagoya, 464-8601 Japan

     The induction of a-amylase by gibberellins (GAs) in cereal grains and the counteractive role of abscisic acid (ABA) on the same process represents a classical model system for studying the mode of action of GA. Besides the aleurone layers, the scutellum also plays an important role in the production of a-amylase. The rice a-amylase gene RAmy3D, which is expressed in the scutellar epithelium, is not induced by GA, but down-regulated by sugars. Sugars act not only as an important source of energy and carbon skeletons, but also as signaling molecules involved in developmental processes in plants. In this study, we report on the change of endogenous ABA content and the relationships between ABA and sugar signaling in the sugar-starved rice embryos.

     We already reported that the G and GATA motifs as consensus regulatory cis-acting sequences are important for the sugar repression of the RAmy3D gene (Toyofuku et a!. 1998). G motif sequence resides in the promoters of many genes that are switched on in response to diverse stimulatory pathways (i.e. light, anaerobiosis, p-coumaric acid and

phytohormone such as ABA). Transcription of RAmy3D gene was promoted by sugar deletion in callus-forming embryos of rice. ABA content in callus-forming rice embryos was determined by radioimmunoassay using a highly specific monoclonal antibody described by Vernieri et a!. (1989) (Table 1). Increase in ABA content in the embryos corresponding to the sugar starvation was observed, indicating that ABA might be a intermediate signal for the sugar repression process.

     To analyze the effect of ABA on sugar repression of RAmy3D gene, the embryos were treated with exogenously applied ABA and fluridone as an inhibitor of ABA biosynthesis. We performed Northern analysis using Rabl6A cDNA clone which is known to be ABA-inducible gene. Exogenous ABA treatment resulted in an increased mRNA level of the Rabl6A gene, but remarkably reduced in the presence of fluridone (data not shown). However, addition of exogenous ABA and fluridone to the embryos showed no effect on the sugar repression of RAmy3D gene by Northern analysis using RAmy3D cDNA probe (Fig. 1A). To analyze the promoter activity of RAmy3D gene in the embryos, we performed co-delivery transformation experiment of RAmy3D-GUS and 35S-LUC genes with particle-bombardment (described by Toyofuku et a!. 1998). Sugar repression of RAmy3D promoter activity was also unaffected in the presence of ABA and fluridone (Fig. 1B). These results indicate that increase in endogenous ABA level does not affect the promotion of RAmy3D gene transcription.

     Sugar starvation treatment did result in gain of endogenous ABA level in callus-forming rice embryos (Table 1), but did not in barley embryos (Perata et al. 1997), although expression of a-amylase genes in both embryos is promoted by sugar depletion. The increase in ABA content in rice embryos, therefore, might be due to the effect of additives in the medium during callus-formation. 2,4-dichlorophenoxyacetic acid (2,4-D), artificial auxin, seems to be potent inducer for ABA biosynthesis. The biosynthesis of ABA in plants is usually promoted by some environmental changes such as drought and/or Salinity stress. Gain of ABA level in the embryos under the sugar starvation reported in this paper may be due to a sort of stress triggered by sugar starvation.

     Recently it has been suggested that hexokinases take part in the sugar repression. Regulatory mechanism(s) of sugar repression of RAmy3D gene transcription is of interested to clarify how plants sense sugars.

contents of callus-forming embryos of rice

a) ABA content was quantified by using monoclonal antibody. b) 90mM glucose was added to MS medium
 

 

References
Vernieri P., P. Perata, D. Armellini, M. Bugnoli, R. Presentini, R. Lorenzi, N. Ceccarelli, A. Alpi and F Tognoni,
             1989. Solid phase radioimmunoassay for the quantitation of abscisic acid in plant crude extracts using 
             a new monoclonal antibody. J. Plant Physiol. 134: 441-446.