Influence of ATP-Binding Cassette Transporters in Root Exudation of Phytoalexins, Signals, and in Disease Resistance

Abstract

The roots of plants secrete compounds as a way to exchange information with organisms living in the soil. Here, we report the involvement of seven root-expressed ATP-binding cassette (ABC) transporters corresponding to both full and half-size molecules (Atabcg36, Atabcg37, Atabcc5, Atabcf1, Atabcf3, Atnap5, and Atath10) in root exudation processes using Arabidopsis thaliana. Root exuded phytochemicals were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS), and it was determined that some of the root exudates from the corresponding ABC transporter mutants were significantly different compared to the wild type. For example, Atabcg37 and Atabcc5 secreted higher levels of the phytoalexin camalexin, and Atabcg36 secreted higher levels of organic acids, specifically salicylic acid (SA). Furthermore, we analyzed the root tissue metabolites of these seven ABC transporter mutants and found that the levels of SA, quercetin, and kaempferol glucosides were higher in Atabcg36, which was correlated with higher expression levels of defense genes in the root tissues compared with the wild type. We did not observe significant changes in the root exudates of the half-size transporters except for Atabcf1 that showed lower levels of few organic acids. In summary, full-size transporters are involved in root secretion of phytochemicals.

Keywords: ABC transporters; defense proteins; disease resistance; phytoalexin; root exudates; salicylic acid.

Figure 1

Phenotypic characterization of ABC transporter T-DNA KO mutants used in this study. (A) Organ-specific expression of ABC transporters in wild type roots, leaves, and flowers. (B) RT-PCR assays of the ABC transporter mutants used in this study. (C) Characterization of root phenotype by growing wild type and mutants in MS agar medium supplemented with 0.5 and 1% sucrose. Number of lateral roots was measured when the seedlings were 14 days old. (D) Characterization of root phenotype by growing wild type and mutants in MS agar medium supplemented with 0.5 and 1% sucrose. Root length was measured when the seedlings were 14 days old. cDNA was prepared from respective tissues. Total RNA and RT-PCR was performed using gene-specific primers. The lower panel is the actin control for the cDNA of wild type root, leaf, and flower. R, root; L, leaf; F, flower. The results represent experiments repeated two times with three replicates each.

Figure 2

Bray–Curtis ordination analyses with Sorenson distance of the root exudates of wild type and ABC transporter mutants constructed from HPLC-MS data obtained from root exudates profiles.

Figure 3

Bray–Curtis ordination analyses with Sorenson distance of the root exudates of wild type and ABC transporter mutants constructed from GC-MS data obtained from root exudates profiles.

Figure 4

Bray–Curtis ordination analyses with Sorenson distance of the root tissue metabolites of wild type and ABC transporter mutants constructed from HPLC-MS data obtained from root tissue metabolites profiles.

Figure 5

Total salicylic acid concentrations of the root tissues of wild type and Atabcg36 mutant lines (Atabcg36-1 and Atabcg36-2). *Indicates the values are significant at p-value below 0.05 compared to wild type. Values represented are the mean of three biological replicates.

Figure 6

Gene expression analyses of wild type (Wt) and Atabcg36 by RT-PCR assays. (A) RT-PCR assay of defense genes expressions in wild type and Atabcg36 root tissues. (B) RT-PCR assay of ABC transporters gene expressions in wild type and Atabcg36 root tissues. *Indicates the gene expression of AtABCB1 is higher in Atabcg36 compared to wild type. The results represent experiments repeated two times with three replicates each.

Figure A1

Root exudates profile of 21-day-old wild type (Col-0) and ABC transporter mutant plants analyzed by HPLC-MS at wavelength 280 nm. Arrows indicate the peaks present or absent in respective mutants. The numbers indicate the positive ESIMS of the peaks. The chromatogram represent experiments repeated two times with three replicates each.

Figure A2

Graphs illustrating the representative phenolic compounds that show different levels in the ABC transporter mutants compared to wild type analyzed by GC-MS. *Indicates the values are statistically significant (p < 0.05) compared with wild-type (Col-0) line (n = 8).

Figure A3

Graphs illustrating the representative sugars that show different levels in the ABC transporter mutants compared to wild type analyzed by GC-MS. *Indicates the values are statistically significant (p < 0.05) compared with wild-type (Col-0) line (n = 8).

Figure A4

Mass trace of compound camalexin (positive ESIMS 201) in the root exudates of wild type (Wt) and ABC transporter mutants used in this study overlayed with authentic camalexin.