Loss of Multiple ABCB Auxin Transporters Recapitulates the Major twisted dwarf 1 Phenotypes in Arabidopsis thaliana

Abstract

FK506-BINDING PROTEIN 42/TWISTED DWARF 1 (FKBP42/TWD1) directly regulates cellular trafficking and activation of multiple ATP-BINDING CASSETTE (ABC) transporters from the ABCB and ABCC subfamilies. abcb1 abcb19 double mutants exhibit remarkable phenotypic overlap with twd1 including severe dwarfism, stamen elongation defects, and compact circinate leaves; however, twd1 mutants exhibit greater loss of polar auxin transport and additional helical twisting of roots, inflorescences, and siliques. As abcc1 abcc2 mutants do not exhibit any visible phenotypes and TWD1 does not interact with PIN or AUX1/LAX auxin transporters, loss of function of other ABCB auxin transporters is hypothesized to underly the remaining morphological phenotypes. Here, gene expression, mutant analyses, pharmacological inhibitor studies, auxin transport assays, and direct auxin quantitations were used to determine the relative contributions of loss of other reported ABCB auxin transporters (4, 6, 11, 14, 20, and 21) to twd1 phenotypes. From these analyses, the additional reduction in plant height and the twisted inflorescence, root, and silique phenotypes observed in twd1 compared to abcb1 abcb19 result from loss of ABCB6 and ABCB20 function. Additionally, abcb6 abcb20 root twisting exhibited the same sensitivity to the auxin transport inhibitor 1-napthalthalamic acid as twd1 suggesting they are the primary contributors to these auxin-dependent organ twisting phenotypes. The lack of obvious phenotypes in higher order abcb4 and abcb21 mutants suggests that the functional loss of these transporters does not contribute to twd1 root or shoot twisting. Analyses of ABCB11 and ABCB14 function revealed capacity for auxin transport; however, their activities are readily outcompeted by other substrates, suggesting alternate functions in planta, consistent with a spectrum of relative substrate affinities among ABCB transporters. Overall, the results presented here suggest that the ABCB1/19 and ABCB6/20 pairs represent the primary long-distance ABCB auxin transporters in Arabidopsis and account for all reported twd1 morphological phenotypes. Other ABCB transporters appear to participate in highly localized auxin streams or mobilize alternate transport substrates.

Keywords: ABCB transporter; Arabidopsis thaliana; FKBP42/TWD1; auxin; organ twisting.

Figure 1

twd1 exhibits additional root, shoot, and silique twisting not observed in abcb1/19. (A) Comparison of mature plant phenotypes of abcb1/19 and twd1. (B) Close-up of twisting inflorescence stems and siliques observed in twd1 but not in abcb1/19. (C) Bright field images abcb1/19 and twd1 roots. Images were converted to black and white, and brightness and contrast were adjusted to show cell files. (D) Venn diagram of the major phenotypes of abcb1/19 and twd1, and twd1 phenotypes that may be accounted for in other abcb mutants. Scale bars: (A) 1 cm; (B) 500 μm; (C) 200 μm.

Figure 2

Loss of abcb4 and abcb21 does not result in twd1-like root phenotypes. (A) Root lengths of 7-day light-grown Col-0, abcb4, abcb21, and abcb4/21 seedlings. (B) Root cell files of 7-day light-grown abcb4/21 seedlings. (C) Root lengths of 7-day light-grown Col-0, abcb1/19, abcb1/4/19, abcb1/19/21, and twd1 seedlings. (D) Root cell files of seedlings in (C). Images in (B,C) were converted to black and white, and brightness and contrast were adjusted to show cell files. Lowercase letters indicate statistical difference by pairwise Student’s t-test (p < 0.05; n = 3 replicates of 10–13). Scale bars: 200 μm.

Figure 3

Aerial tissue phenotypes observed in twd1 can be attributed to loss of ABCB1, 6, 19, and 20 function. (A) Single abcb mutant rosettes 21 days after germination. (B) twd1 and double abcb mutant rosettes 21 days after germination. (C) Mature single and double abcb mutants 35 days after germination. (D) Twisting of inflorescence stems in abcb6/20 double mutants. (E,F) Close-ups images of abcb6/20 showing (E) twisted cell files and (F) twisted siliques. (G) Occasional stem–pedicel separation defects observed in abcb6/20 mutants. (H) Cantil spur formation in abcb1/19 grown under 50 μmol m⁻² s⁻¹, 16-h photoperiod. (I) Toluidine blue O staining of hand sectioned abcb1/19 cantil spur from (H). Scale bars: (A) 1 cm; (B,I) 0.5 cm; (C) 5 cm; (D,H) 1 cm; (E–G) 1 mm.

Figure 4

Root phenotypes observed in twd1 can be attributed to loss of ABCB1/19 and 6/20 function. (A) 7-day primary root lengths (n = 3 replicates of 10–13). (B,C) Bright field images of the distal elongation and maturation zone in 7-day seedling roots. Images in (B,C) were converted to black and white, and brightness and contrast were adjusted equally within each set to show cell files. (D) Treatment with 1 μM NPA suppressed root twisting in abcb6/20 and twd1. Brightness and contrast were adjusted equally to show cell files. (E) Quantification of cell file angles in (D). Data shown are means ± SE (n = 3 replicates of 10–11). (F) Quantification of IAA and oxIAA in 5.5-day light-grown roots. Data shown are means ± SD (n = 4–5 independent pools of 25 mg tissue). Lowercase letters indicate statistical difference by all pairwise Student’s t-test (p < 0.05). Asterisks indicate statistical differences by Student’s t-test (^**p < 0.01). Scale bars: 100 μm.

Figure 5

ABCB11 is primarily expressed in the root and root tip. (A) Strong proABCB11:GUS expression is observed in the root tip. Roots were stained for 1 h. (B) Moderate proABCB11:GUS expression is observed in the epidermis, cortex, and endodermis of the mature root. (C–G) proABCB11:GUS expression is not observed in the (C) hypocotyl, shoot apex, or petioles, (D) cotyledons, (E) rosette leaves, (F) apical regions of the inflorescence stem or mature flowers, or (G) upper 3–4 cm of the inflorescence stem. Occasional staining could be observed in young unopened flowers (E, inset). Stems in (G) were GUS stained prior to hand sectioning. Scale bars: (A,F,G) 500 μm, (B,F) 1 mm, (C,E) 2 mm, (D) 100 μm.

Figure 6

ABCB11 contributes to auxin phenotypes supplementary to ABCB1/19. (A) Col-0 and abcb11 rosettes at time of bolting. (B) Col-0 and abcb11 mature plants. (C) Root cell files of 7-day light-grown abcb1/11-1/19 seedlings. Images were converted to black and white, and brightness and contrast were adjusted to show cell files. (D) 35-day rosettes and inflorescences of Col-0, abcb1/19, abcb1/11-1/19, and twd1. (E) Flower series of Col-0, abcb1/19, and abcb1/11-1/19. (F) Col-0, abcb1/19, and abcb1/11-1/19 flowers with petals removed. Scale bars: (A) 1 cm, (B,C) 5 cm, (D) 200 μm, (E,F) 1 mm.

Figure 7

abcb11-1 mutants exhibit minor defects in root auxin transport. (A,B) ³H-IAA transport in (A) upright (rootward/basipetal) and (B) inverted Col-0, abcb1, abcb11, abcb14, abcb19, and pin1 inflorescence stems segments. Assays were conducted as in Kaneda et al. (2011) with modifications as noted in the Materials and Methods section. Data shown are means ± SD (n = 3 pools independent of five segments). (C,D) ³H-IAA transport in Col-0, abcb11, abcb14, and abcb19 seedlings from the shoot apex to (C) the root–shoot transition zone (RSTZ) or (D) the root tip. Data shown are means ± SD (n = 3 independent pools of 10). (E,F) Shootward auxin transport from the root tip with ³H-IAA placed on the (E) columella or (F) quiescent center. Shootward assays were conducted as in Kubeš et al. (2012). Data shown are means ± SD (n = 3 independent pools of 10). Asterisks indicate statistical difference from Col-0 by Student’s t-test (^*p < 0.05).

Figure 8

Transport activity of ABCB11 expressed in Schizosaccharomyces pombe. (A) ³H-IAA accumulation in S. pombe expressing ABCB11 or empty vector control plasmid. Data shown are means ± SD (n = 4 independent transformations). (B) ³H-benzoic acid (BA) accumulation in S. pombe expressing ABCB11 or empty vector control plasmid. Data shown are means ± SD (n = 4 independent transformations). (C) Competition of ³H-IAA with cold benzoic acid (BA; 1:1 molar ratio) in S. pombe expressing ABCB11, ABCB19, or empty vector control plasmid. Data shown are means ± SD (n = 4 independent transformations). Asterisks indicate statistical difference by Student’s t-test (^*p < 0.05).