Amyloplast formation in cultured tobacco BY-2 cells requires a high cytokinin content.

When cytokinin-autonomous tobacco BY-2 cell cultures are transferred into 2,4-dichlorophenoxyacetic acid (2,4-D)-deprived medium, amyloplast development is initiated. Using this in vitro amyloplast-inducing system, the role of cytokinins in amyloplast formation was investigated. We show that addition of lovastatin, an inhibitor of mevalonate synthesis, to amyloplast-inducing medium reduced starch accumulation. Microscopic observation also revealed that lovastatin treatment decreased starch deposition; however, the overall morphologies of cells and plastids were less affected than control cell cultures. In addition, lovastatin lowered the transcription level of the ADP-glucose pyrophosphorylase small subunit (AgpS) gene. Application of mevalonate or zeatin dramatically restored the decrease in starch deposition, and restored AgpS mRNA accumulation. Moreover, addition of other molecules with cytokinin activity, such as adenine- and phenylurea-type compounds, restored starch accumulation and AgpS transcript levels, whereas other isopentenyl pyrophosphate-derived phytohormones did not. Liquid chromatography-mass spectrometry/mass spectrometry quantification of endogenous cytokinins revealed that endogenous cytokinins increased when BY-2 cells were transferred into 2,4-D-deprived medium from conventional medium containing 2,4-D. In addition, lovastatin treatment decreased endogenous cytokinins to some extent when cultured under 2,4-D-deprived conditions. Our results suggest that both 2,4-D deprivation and an increase in endogenous cytokinins have important roles in accelerating the changes in plastid morphology, starch accumulation, and AgpS gene expression.