ABCG1 contributes to suberin formation in Arabidopsis thaliana roots

Abstract

Diffusion barriers enable plant survival under fluctuating environmental conditions. They control internal water potential and protect against biotic or abiotic stress factors. How these protective molecules are deposited to the extracellular environment is poorly understood. We here examined the role of the Arabidopsis ABC half-size transporter AtABCG1 in the formation of the extracellular root suberin layer. Quantitative analysis of extracellular long-chain fatty acids and aliphatic alcohols in the atabcg1 mutants demonstrated altered root suberin composition, specifically a reduction in longer chain dicarboxylic acids, fatty alcohols and acids. Accordingly, the ATP-hydrolyzing activity of heterologous expressed and purified AtABCG1 was strongly stimulated by fatty alcohols (C₂₆-C₃₀) and fatty acids (C₂₄-C₃₀) in a chain length dependent manner. These results are a first indication for the function of AtABCG1 in the transport of longer chain aliphatic monomers from the cytoplasm to the apoplastic space during root suberin formation.

Figure 1

Characterization of T-DNA insertional atabcg1 mutant lines. (A) Genomic organization of the two independent T-DNA insertional atabcg1 mutant lines, atabcg1-3 and atabcg1-4. The atabcg1-3 line contains a T-DNA insertion at 791 bp and the atabcg1-4 line one at 996 bp. Arrows indicate the primers used for the mutant characterization. (B) Genetic analysis of the atabcg1 mutant lines atabcg1-3 and atabcg1-4. Genomic DNA of wildtype (WT) and mutant lines (#1) were analyzed for AtABCG1 gene specific, T-DNA specific and actin specific amplification products. N, negative control. (C) Presence of full-length AtABCG1 transcripts in T-DNA insertional atabcg1 mutant lines. Amplification of full-length AtABCG1 was performed from cDNA isolated from atabcg1-3 and atabcg1-4 lines and the depicted primer pairs. Entire gels can be found in supplemental information (Figs S5 and S6).

Figure 2

Suberin composition in Arabidopsis wildtype and atabcg1 mutant plants. (A) Suberin content in wildtype and atabcg1 mutant lines. Relative amount of released suberin after transesterification of wildtype and T-DNA insertional atabcg1 mutant lines atabcg1-3 and atabcg1-4. Data represent mean ± SD of at least 4 independent experiments. Wildtype (n = 7), atabcg1-3 (n = 4) and atabcg1-4 (n = 5) independent experiments. (B) Monomer composition of Arabidopsis wildtype and atabcg1 mutant root suberin. The relative abundance of suberin monomers in the independent T-DNA insertional mutant lines atabcg1-3 (n = 4) and atabcg1-4 (n = 4) per substance class is depicted in comparison to the wildtype (n = 4). The suberin monomers were quantified using quantifier ions and retention times from Supplemental Table S2. The red lines indicate the relative abundance of the respective monomers in the wildtype (set to 100%). Data represents mean and ±SD. Significance analysis between atabcg1 mutants and wildtype samples were performed by t test. (two-tailed): ***P ≤ 0.0001, **P ≤ 0.01, *P ≤ 0.05.

Figure 3

Calmodulin binding peptide (CBP) purification of AtABCG1. AtABCG1 was solubilized in Fos Choline-14 and purified by CBP resin. Fractions were analyzed after 10 min incubation at 65 °C via 7% SDS-PAGE (left panel) or immunoblotting (anti-His-tag antibody, right panel). AtABCG1 has a calculated molecular weight of 86 kDa. The lower 45 kDa band in the elution fractions 2–6 arises as verified by immunoblotting also from AtABCG1 and is likely a degradation product. Pre-stained molecular weight markers are shown on the left. AS, after solubilization; P, pellet; FT, flow through; W1-W2, washing fraction. The colors of the immunoblot were inverted without contrast changing.

Figure 4

Native oligomeric state of AtABCG1. CBP purified AtABCG1 was subjected to 4–16% Bis-Tris BN-PAGE. (A) Samples were analyzed in different Fos Choline-14 concentration (0× cmc, 2× cmc, 5× cmc). Native AtABCG1 partially migrates under SDS substitution mainly with 172 kDa as homodimeric (*) protein. (B) Samples were submitted with 2× cmc Fos-Choline 14 and analyzed in different SDS-concentration (1%, 2%, 3%). A molecular weight marker is shown in the left.+, samples substituted with 1% SDS; -, samples without SDS. The colors of the immunoblot were inverted without contrast changing.

Figure 5

ATPase activity of purified AtABCG1 and AtABCG1-EQ/HA mutant. The ATPase activity of CBP purified AtABCG1 (circles) and the EQ/HA mutant (squares) was measured in presence of 0 to 8 mM ATP. Data was analyzed according to Michaelis-Menten equation and represents mean and ±SD of three independent replicates.

Figure 6

Substrate stimulated AtABCG1 ATPase activity. (A) Relative ATPase activity of AtABCG1 in presence of 40 µM fatty acids and fatty alcohols (C₁₆–C₃₀). Significance analysis between basal activity and substrate stimulated ATPase activity was performed by t test. (two-tailed): ***P ≤ 0.0001, **P ≤ 0.01, *P ≤ 0.05. (B,C) Concentration dependent relative AtABCG1 activity in the presence of fatty acids (C₂₄–C₃₀) and fatty alcohols (C₂₆–C₃₀). The ATPase activity of the EQ/HA mutant was subtracted and data fitted according to Michaelis-Menten kinetics. Data represent mean and ±SD of at least three independent replicates. An ATPase activity of 100% corresponds to the basale activity of AtABCG1 in the absence of any substrate.