Altered profile of secondary metabolites in the root exudates of Arabidopsis ATP-binding cassette transporter mutants

Abstract

Following recent indirect evidence suggesting a role for ATP-binding cassette (ABC) transporters in root exudation of phytochemicals, we identified 25 ABC transporter genes highly expressed in the root cells most likely to be involved in secretion processes. Of these 25 genes, we also selected six full-length ABC transporters and a half-size transporter for in-depth molecular and biochemical analyses. We compared the exuded root phytochemical profiles of these seven ABC transporter mutants to those of the wild type. There were three nonpolar phytochemicals missing in various ABC transporter mutants compared to the wild type when the samples were analyzed by high-performance liquid chromatography-mass spectrometry. These data suggest that more than one ABC transporter can be involved in the secretion of a given phytochemical and that a transporter can be involved in the secretion of more than one secondary metabolite. The primary and secondary metabolites present in the root exudates of the mutants were also analyzed by gas chromatography-mass spectrometry, which allowed for the identification of groups of compounds differentially found in some of the mutants compared to the wild type. For instance, the mutant Atpdr6 secreted a lower level of organic acids and Atmrp2 secreted a higher level of amino acids as compared to the wild type. We conclude that the release of phytochemicals by roots is partially controlled by ABC transporters.

Figure 1.

RT-PCR assays of the ABC transporter mutants used in this study. cDNA was prepared from 1.5 μg of root tissue total RNA with superscript reverse transcriptase (Invitrogen) and RT-PCR was performed using gene-specific primers. Below is the actin control for the cDNA of mutants and the wild type. The corresponding Salk line mutants for these ABC transporters are listed in Supplemental Table S3. The primers used for this RT-PCR are listed in Supplemental Table S5. Wt, Wild type; M, mutant lines. [See online article for color version of this figure.]

Figure 2.

Profile shows the M_r trace of compound 3 (647) in the root exudates of the wild type (Col-0), ABC transporter mutant Atpgp4-1 (pgp4-1), and AtPGP4 overexpresser line in the homozygote Atpgp4-1 mutant background (Atpgp4OX-pgp4-1) after 3 d of continuous root secretion (details in “Materials and Methods”). Arrows indicate the absence of peak. Asterisks indicate the presence of peak. The number indicates the peak number. The results represent experiments repeated two times with three replicates each. [See online article for color version of this figure.]

Figure 3.

PCA of the root exudate profile of ABC transporter mutants compared to wild type analyzed by GC-MS. Col-0, black circles; Atpdr6, white rectangles; Atpdr2, black rectangles; Atmrp2, asterisks; Atath6, white diamonds; Atpgp4-1, X.