Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes

Abstract

Background: We have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. We utilized the full-length cDNA databases to generate a normalized library consisting of 239 full-length Arabidopsis thaliana transporter cDNAs. The genes were arranged into a 96-well format and optimized for expression in Xenopus oocytes by cloning each coding sequence into a Xenopus expression vector.

Results: Injection of 96 in vitro transcribed cRNAs from the library in pools of columns and rows into oocytes and subsequent screening for glucose uptake activity identified three glucose transporters. One of these, AtSTP13, had not previously been experimentally characterized.

Conclusion: Expression of the library in Xenopus oocytes, combined with uptake assays, has great potential in assignment of plant transporter function and for identifying membrane transporters for the many plant metabolites where a transporter has not yet been identified.

Figure 1

Screening for glucose uptake activity in Xenopus oocytes expressing pools of cRNA of Arabidopsis transporter cDNAs. cRNA of 96 genes transcribed with native plant UTRs were pooled in columns and rows and screened for glucose uptake in Xenopus oocytes. High glucose uptake was measured in column 5 and row C, which suggests that the marked well contains a glucose transporter.

Figure 2

Confirmation of glucose uptake activity by AtSTP13. cRNA of AtSTP13 representing the single clone C5 was injected into nine oocytes. Measurement of glucose uptake by the oocytes was compared to oocytes injected with row C and column 5, respectively.

Figure 3

Effect of different UTRs on gene expression in Xenopus oocytes. CDSs of AtSTP1, AtSTP4, AtSTP13, AtSUCI, and AtSUC2 were expressed with their native plant UTRs or with Xenopus-specific β-globin gene UTRs. Water injected oocytes functioned as negative controls for endogenous glucose or sucrose uptake by oocytes. Assays were performed with 15 μM radio labelled glucose or sucrose (~100 μCi/μmol).

Figure 4

Screening for glucose uptake in oocytes expressingpools of cRNA of Arabidopsis transporter cDNAs optimized for Xenopus expression. cRNAs of 96 genes transcribed with Xenopus UTRs were pooled in columns and rows and screened for glucose uptake in oocytes. High glucose uptake was measured in row A, C and E and column 4, 5 and 7, which suggests that the marked wells, contain glucose transporters.