ABCG11 modulates cytokinin responses in Arabidopsis thaliana

Abstract

The Arabidopsis ABC transporter ABCG11 transports lipidic precursors of surface coating polymers at the plasma membrane of epidermal cells. Mutants in ABCG11 exhibit severe developmental defects, suggesting that ABCG11 might also participate in phytohormone-mediated development. Here, we report that ABCG11 is involved in cytokinin-mediated development. The roots of abcg11 mutant seedlings failed to respond to cytokinins and accumulated more cytokinins than wild-type roots. When grown under short-day conditions, abcg11 exhibited longer roots and shorter hypocotyls compared to wild type, similar to abcg14, a knockout mutant in a cytokinin transporter. Treatment with exogenous trans-zeatin, which inhibits primary root elongation in the wild type, enhanced abcg11 primary root elongation. It also increased the expression of cytokinin-responsive Arabidopsis response regulator (ARR) genes, and the signal of the TCS::GFP reporter in abcg11 roots compared to wild-type roots, suggesting that cytokinin signaling was enhanced in abcg11 roots. When we treated only the roots of abcg11 with trans-zeatin, their shoots showed lower ARR induction than the wild type. The abcg14 abcg11 double mutant did not have additional root phenotypes compared to abcg11. Together, these results suggest that ABCG11 is necessary for normal cytokinin-mediated root development, likely because it contributes to cytokinin transport, either directly or indirectly.

Keywords: ABC transporters; ABCG11; ABCG14; heterodimer; phytohormone; root; signaling; transport.

Figure 1

Second allele of abcg11 T-DNA insertion line cof1-3 (SALK_131624) exhibits an altered response to cytokinin, similar to abcg11. (A) cof1-3 is a knockdown mutant of ABCG11. RT-PCR analysis of AtABCG11. Actin2 (At3g18780) was amplified as an internal control. The primers are listed in Supplementary Table S3. (B–D) Photographs of the shoots of 12-day-old seedlings of Col-0, abcg11, and cof1-3. The arrows point the sites of organ fusion. Scale bar = 1.0 mm. (E) The similar responses of cof1-3 and abcg11 roots to 20 nM trans-zeatin (tZ) treatment under long-day conditions (16 h/8 h, day/night). The photographs were taken at 12 days after seed sowing. The photos are representatives of four independent experiments. Scale bars = 1 cm. (F) The average primary root length of the plants shown in (E). The error bars represent the standard error (SE; n = 40). The different letters indicate the significant differences determined by one-way ANOVA (p < 0.05).

Figure 2

Expression of phytohormone-induced genes in Col-0 and the abcg11 mutant. Relative expression levels of cytokinin- (A,B), auxin- (C,D), and abscisic acid- (E,F) induced genes in roots (A,C,E) and shoots (B,D,E). Seedlings were grown vertically on half-strength MS medium for 12 d (16 h light/8 h dark). TUBULIN8 served as an internal control; values for Col-0 were set to 1. Data are shown as means ± standard error (SE; n = 4). Asterisks represent significant differences by Student’s t-test: ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 3

The concentration of several cytokinin types is higher in abcg11 than in the wild type. Concentrations for the trans-zeatin (tZ)-type (A,B), isopentyladenine (iP)-type (C,D), and cis-zeatin (cZ)-type (E,F) cytokinins in roots (A,C,E) and shoots (B,D,F) of Col-0 and abcg11. Seedlings were grown on half-strength MS medium for 12 d under long-day conditions (16 h light/8 h dark). Data are shown as means ± SE (n = 4). Asterisks represent significant differences by Student’s t-test: ^*p < 0.05; ^**p < 0.01.

Figure 4

abcg11 behaves similarly as abcg14 in root growth on MGRL medium and in response to exogenous cytokinins. (A, left panel) The progeny of abcg11/+ plants exhibit two distinct phenotypes. The seeds were sown on MGRL medium and grown vertically for 12 days under short-day conditions (8 h light/16 h dark). Note that seedlings were rearranged according to their primary root length before taking the picture. (A, right panel) Phenotype of abcg14 under the same conditions. Scale bars, 1 cm. (B) Genotyping of the individual seedlings shown in (A), left panel. Note how the seedlings with longer roots are abcg11 homozygous mutants. The primers for genotyping are listed in Supplementary Table S2. (C) Primary root length of seedlings in (A). Data are shown as means ± SE (n = 15). Asterisks represent significant differences by Student’s t-test: ^**p < 0.01. (D) Phenotype of seedlings grown with or without 20 nM trans-zeatin (tZ) for 12 days under long-day conditions (16 h light/8 h dark) on half-strength MS medium. The photographs are representative of three independent experiments. Scale bars, 1 cm. (E) Primary root length of the seedlings in (D). Data are shown as means ± SE (n = 40). Different letters indicate significant differences, as determined by one-way ANOVA (p < 0.05).

Figure 5

Cytokinin signaling is enhanced in abcg11 seedlings upon tZ treatment. (A) Relative expression levels of ARRs in Col-0 and abcg11 roots, as determined by RT-qPCR. Seedlings were grown vertically on half-strength MS medium with or without 20 nM trans-zeatin (tZ) plate for 12 days under long-day conditions (16 h light/8 h dark). TUBULIN8 served as an internal control; values in Col-0 and abcg11 under control conditions were set to 1. Data are shown as means ± SE (n = 4). The different letters indicate the significant difference in each gene expression levels determined by one-way ANOVA (p < 0.05). (B) Fluorescence intensity of TCS::GFP in the roots of Col-0 and abcg11. Scale bars, 50 mm. (C) Fold-change in TCS::GFP fluorescence intensity induced by tZ treatment in the root tips of WT or abcg11 transgenic seedlings expressing TCS::GFP. Fluorescence intensity was determined using ImageJ. Data are shown as means ± SE from two independent experiments (total 10 biological replicates). Asterisks represent significant differences by Student’s t-test: ^*p < 0.05.

Figure 6

The abcg11 and abcg14 mutants exhibit lower root-to-shoot transfer of cytokinin signaling. (A,B) Relative expression levels of type-A ARRs in the shoots of Col-0 and abcg11 (A), and Col-4 and abcg14 (B). Only the roots of 12-d-old seedlings were soaked in half-strength MS medium or half-strength MS medium containing 1 μM trans-zeatin (tZ) for 1 h. The shoots were harvested for analysis of ARR expression levels. (C) Relative expression levels of ARRs of Col-0 and abcg11 after tZ treatment of whole seedlings. The 12-d-old seedlings were submerged in 1 μM tZ for 30 min. TUBULIN8 served as an internal control; values in Col-0 and abcg11 under control conditions were set to 1. Data are shown as means ± SE (n = 4). Different letters in (A), (B), and (C) indicate significant differences in gene expression levels, as determined by one-way ANOVA (p < 0.05).

Figure 7

The root-to-shoot transfer of [3H]tZ and T₂O. Only the roots of 12-day-old seedlings were treated with 50 nM ³H-tZ or 0.5 mCi/ml T₂O for 1 h, and then the shoots were harvested for the assay of the transferred ³H-tZ or T₂O. The error bars represent the standard deviation (SE; n = 6–8). The asterisks represent the significant differences between mutants and wild type by Student’s t-test: ^**p < 0.01, ^***p < 0.001.

Figure 8

The abcg14 abcg11 double knockout mutant exhibits a similar root length phenotype as the abcg11 single mutant. (A–C) Photographs of seedlings grown vertically on MGRL medium for 12 d under short-day conditions (8 h light/16 h dark). Scale bars, 1 cm. Note that seedlings in (A) were rearranged according to their primary root length prior to the photograph. (D) Genotyping results of abcg14 abcg11/+ and abcg14 abcg11 seedlings shown in (A). (E) Primary root length of the seedlings shown in (A–C). Data are shown as means ± SE (n = 30). Different letters represent significant differences, as determined by one-way ANOVA (p < 0.05). (F,G) Photographs of abcg14 (F) and abcg14 abcg11 (G) seedlings. The arrow points to a site of organ fusion. Scale bars, 1.0 mm.

Figure 9

The abcg14 abcg11 double knockout mutant is similar to abcg11 and abcg14 single mutants in root growth in response to cytokinins. (A) Seedlings grown with or without 20 nM trans-zeatin (tZ) treatment for 12 days under long-day conditions (16 h light/8 h dark). The photographs are representatives of four independent experiments. Scale bars, 1 cm. (B) Relative effect of tZ treatment on primary root growth. The relative effect was calculated as primary root length under tZ treatment normalized by that of the control treatment. Data are shown as means ± SE from four independent experiments. Different letters represent significant differences, as determined by one-way ANOVA (p < 0.05).