A spatiotemporal analysis of enzymatic activities associated with carbon metabolism in wild‐type and mutant embryos of Arabidopsis using in situ histochemistry

Abstract

Arabidopsis as a molecular genetic model offers many advantages for the study of seed development, but these do not extend to biochemical and enzymatic studies, which are often compromised by the limited amount of material available from the small developing embryos. A set of assays based on the coupling of an enzymatic reaction to the reduction of NAD, NADP or FAD, and subsequent reduction and precipitation of a tetrazolium salt, have been adapted to investigate 18 enzyme activities associated with carbon metabolism in developing Arabidopsis embryos. The use of organelle-specific marker enzymes demonstrates the utility of the method for detection of activities in mitochondria, plastids and peroxisomes as well as the cytosol. The temporal staining patterns obtained allow classification of the activities into three main categories based on whether they peak in the early, intermediate or late stages of maturation. An interesting switch from ATP to pyrophosphate consuming pathways occurs at the onset of the maturation phase, which involves key steps in primary carbon metabolism such as phosphofructokinase. This spatiotemporal characterization of carbon metabolism has also been applied to various mutants disrupted in embryo development including gnom (gn), acetyl-CoA carboxylase1 (acc1), schlepperless (slp), and wrinkled1 (wri1). The data obtained demonstrate that the extent to which carbon metabolism is affected in mutants is not necessarily correlated to the severity of the mutation considered. Through the advanced characterization of trehalose-6-P synthase1 (tps1) embryos, this approach finally provides new insight into the regulatory role played by trehalose metabolism in embryo development.