Protoplast-Esculin Assay as a New Method to Assay Plant Sucrose Transporters: Characterization of AtSUC6 and AtSUC7 Sucrose Uptake Activity in Arabidopsis Col-0 Ecotype.

The best characterized function of sucrose transporters of the SUC family in plants is the uptake of sucrose into the phloem for long-distance transport of photoassimilates. This important step is usually performed by one specific SUC in every species. However, plants possess small families of several different SUCs which are less well understood. Here, we report on the characterization of AtSUC6 and AtSUC7, two members of the SUC family in . Heterologous expression in yeast () revealed that AtSUC6 is a high-affinity H-symporter that mediates the uptake of sucrose and maltose across the plasma membrane at exceptionally low pH values. Reporter gene analyses revealed a strong expression of in reproductive tissues, where the protein product might contribute to sugar uptake into pollen tubes and synergid cells. A knockout of did not interfere with vegetative development or reproduction, which points toward physiological redundancy of AtSUC6 with other sugar transporters. Reporter gene analyses showed that is expressed in roots and pollen tubes and that this sink specific expression of is regulated by intragenic regions. Transport activity of AtSUC7 could not be analyzed in baker's yeast or oocytes because the protein was not correctly targeted to the plasma membrane in both heterologous expression systems. Therefore, a novel approach to analyze sucrose transporters was developed. Plasma membrane localized SUCs including AtSUC6 and also sucrose specific SWEETs were able to mediate transport of the fluorescent sucrose analog esculin in transformed mesophyll protoplasts. In contrast, AtSUC7 is not able to mediate esculin transport across the plasma membrane which implicates that might be a non-functional pseudogene. The novel protoplast assay provides a useful tool for the quick and quantitative analysis of sucrose transporters in an expression system.