Isolation of ripening rice ( Oryza sativa L.) aleurone protoplasts for uptake studies

Abstract

The aleurone layer of the developing rice ( Oryza sativa L.) caryopsis is the first filial tissue in a position to take up sucrose released into the endosperm apoplast by the maternal nucellus. Adhesion of the aleurone layer to the nucellus during ripening makes it difficult to characterize sucrose transport by the aleurone layer. As an alternative, aleurone protoplasts were isolated enzymatically in a two-step process in which the cells were released from the caryopsis before the protoplasts were isolated. The procedure prevented the formation of maternal and giant (fused) aleurone protoplasts. Low protoplast yield (10% of the number of cells) was correlated with the low viability of the isolated cells. To test the utility of the protoplast system, sucrose uptake by the protoplasts was investigated using the silicone oil centrifugation technique. The concentration-dependence of the influx was biphasic and could be fitted by a function that depended on three parameters to describe the saturable and non-saturable components of the influx. These results demonstrate that it is possible to isolate protoplasts rapidly from the aleurone layer in high enough yield for protoplast uptake studies and are consistent with the aleurone layer having a saturable and a non-saturable component of sucrose influx.