Construction of AtLHT1 probe for its transport substrate screening based on FRET principle

Objective  In order to improve the efficiency of guided-pesticides screening and accelerate the research and development of molecular design, synthesis and screening of guided-pesticides, a fluorescence resonance energy transfer (FRET) probe based on Arabidopsis thaliana amino acid transporter AtLHT1 was constructed as guided-pesticide screening platform in this study.

Method  The sandwich molecular probe of CFP-LHT1-YFP was constructed and expressed in prokaryotic cell of Escherichia coli BL21(DE3) and BY4741 yeast cell, respectively, then identified and purified. The change of FRET efficiency was detected with fluorescence microplate reader and laser scanning confocal microscope.

Result  The CFP-AtLHT1-YFP fusion protein probe was purified and mixed with the tested amino acids and glyphosate, respectively. The addition of the tested amino acids and glyphosate resulted in significant changes in the FRET ratio of the probe protein. Glyphosate treatment resulted in the D_{535 nm}/D_{480 nm} increase of 7% to 12%. The similar tendency was also observed in the treatments of positive ligands using glycine and glutamic acid, but no obvious change was observed in the treatment of negative ligand using arginine. The FRET ratio showed substrate concentration dependence. At 20 min after the addition of 1, 5 and 30 mmol/L glycine, the increase of D_{535 nm}/D_{480 nm} were 3%, 1% and 13% respectively. FRET ratio increased with the increase of subatrate concentration in treatments with positive ligand, while the changes were not regular in treatments with negative ligand. Significant changes in FRET efficiency were also detected in individual yeast cells after treatment with amino acids or glyphosate using the photobleaching method. Changes in FRET efficiency were more pronounced with the addition of positive controls of glycine and glutamic acid, with 30% FRET efficiency for glycine and 26% for glutamic acid. The addition of glyphosate resulted in 26% FRER efficiency which was close to that of glutamic acid. The blank control containing PBS buffer showed no significant change in FRET efficiency.

Conclusion  The AtLHT1-FRET probe can bind to the neutral amino acid glycine and the acidic amino acid glutamic acid, but not to the basic amino acid arginine. The FRET efficiency varies among different substrates with some concentration dependence. It is demonstrated that glyphosate can be transported by the amino acid transporter protein AtLHT1.