Differential regulation of starch synthesis gene expression during amyloplast development in cultured tobacco BY-2 cells

Transfer of stationary-phase cultured tobacco (Nicotiana tabacum L.) BY-2 cells into auxin-depleted culture medium induces amyloplast formation. Measurements and microscopic observations of starch content revealed that amyloplast formation begins 18 to 24 h after transfer to amyloplast-inducing medium, and involves morphological changes in plastids and the enlargement of starch granules. To investigate the timing and requirement for de novo protein synthesis in amyloplast development and the enhancement of starch synthesis gene expression, we added cycloheximide to cells grown in amyloplast-inducing media at various times and monitored the changes in plastid morphology, starch accumulation, and the mRNA levels of starch synthesis genes. Cycloheximide immediately inhibited both cell growth and amyloplast development. RNA gel-blot analyses revealed that cycloheximide treatment reduced the accumulation of mRNA for the ADP-Glc pyrophosphorylase small subunit (AgpS) and granule-bound starch synthase (GBSS) mRNA levels, but did not affect the starch branching enzyme (SBE) mRNA levels. These results suggest that (i) the expression of at least one isoform of SBE is regulated differently from the expression of AgpS and GBSS at the transcriptional or post-transcriptional level, and (ii) at least one de novo synthesized protein is required in order to control the expression of AgpS and GBSS