Arabidopsis TWISTED DWARF1 functionally interacts with auxin exporter ABCB1 on the root plasma membrane

Abstract

Plant architecture is influenced by the polar, cell-to-cell transport of auxin that is primarily provided and regulated by plasma membrane efflux catalysts of the PIN-FORMED and B family of ABC transporter (ABCB) classes. The latter were shown to require the functionality of the FK506 binding protein42 TWISTED DWARF1 (TWD1), although underlying mechanisms are unclear. By genetic manipulation of TWD1 expression, we show here that TWD1 affects shootward root auxin reflux and, thus, downstream developmental traits, such as epidermal twisting and gravitropism of the root. Using immunological assays, we demonstrate a predominant lateral, mainly outward-facing, plasma membrane location for TWD1 in the root epidermis characterized by the lateral marker ABC transporter G36/PLEIOTROPIC DRUG-RESISTANCE8/PENETRATION3. At these epidermal plasma membrane domains, TWD1 colocalizes with nonpolar ABCB1. In planta bioluminescence resonance energy transfer analysis was used to verify specific ABC transporter B1 (ABCB1)-TWD1 interaction. Our data support a model in which TWD1 promotes lateral ABCB-mediated auxin efflux via protein-protein interaction at the plasma membrane, minimizing reflux from the root apoplast into the cytoplasm.

Figure 1.

Epidermal Twisting in abcb1 abcb19 Roots Is Ecotype Dependent. (A) and (B) Primary root length (A) and LR number (B) of wild-type (Col Wt), twd1-3, HA-TWD1, and HA-TWD1-C_t lines grown under indicated light conditions and measured at growth stages 1 and R6 (Boyes et al., 2001), respectively. Mean ± sd; n ≥ 30. Significant differences (unpaired t test with Welch’s correction: P < 0.01) to wild-type are indicated by an asterisk. nd, not determined. (C) and (D) Quantification of epidermal orientation (angles to the growth direction) using agarose imprints of TWD1- and ABCB1/ABCB19 loss- and gain-of-function roots generated from Ws (Ws Wt; [C]) or Col (Col Wt; [D]) grown on solvent control, 1 µM NPA, or 100 nM IAA under 8-h light conditions. Mean ± sd; n ≥ 25. Significant differences (unpaired t test with Welch’s correction: a, P < 0.01; b, P < 0.05) to wild-type or solvent control are indicated by one or two asterisks, respectively. [See online article for color version of this figure.]

Figure 2.

TWD1 Loss and Gain of Function Alter Root Bending and Shootward Auxin Accumulation (A) and (B) Curvature (A) and curvature rates ([B]; first deviation of curvature) of TWD1 loss- (twd1-3) and gain-of-function lines with (HA-TWD1) and without the C-terminal membrane anchor (HA-TWD1-C_t). Mean ± se; n = 8. Inset, curvature at 330 min; significant differences (unpaired t test with Welch’s correction) to wild-type (Wt) control are indicated by one (P < 0.01) or two asterisks (P < 0.05), respectively. (C) to (F) Confocal images ([C] and [D]) and quantification ([E] and [F]) of columella ([C] and [E]) or lower side epidermal signals ([D] and [F]) of the auxin-responsive reporter DR5:GFP (green) in wild-type (Col Wt), twd1-3, HA-TWD1, and HA-TWD1-C_t roots with (+g) and without (−g) gravistimulation. For details of quantification, see Supplemental Figure 1 online. Note reduced expression of DR5:GFP in the columella of twd1-3 and HA-TWD1-C_t roots and enhanced basipetal accumulation in the lower side epidermis of HA-TWD1 and HA-TWD1-C_t roots. Bars = 50 μm in (C) and 100 μm in (D); means ± se; n = 12. Orientation to the gravity vector (g) is indicated by an arrow. [See online article for color version of this figure.]

Figure 3.

IAA Influx Profiles along TWD1 Loss- and Gain-of-Function Roots Measured Using an IAA-Specific Microelectrode. (A) and (B) Influx profiles (A) and heat map presentation (B) of IAA influx along wild-type (Col Wt), twd1-3, HA-TWD1, and HA-TWD1-C_t roots. Positive fluxes represent a net IAA influx. Data are means ± se (n = 12). As reference, mapping of root tissues (left) and of high-resolution IAA contents (right; Petersson et al., 2009) is given. For construction of heat maps, see Supplemental Figure 3 online; dashed lines indicate 100-μm distances from the root tip. (C) Quantification of IAA influx profiles above 0.45 mm from the root tip.

Figure 4.

TWD1 Reveals a Lateral PM Location in the Root Epidermis Where It Colocalizes with PEN3/ABCG36 and ABCB1. (A) to (H) Whole-mount immunostaining using anti-TWD1 with PM markers PEN3/ABCG36-GFP ([A] to [D]) and BRI1-GFP ([E] to [H]). Note predominant lateral PM localization ([A]; stronger at the outer polar domain) in the root epidermis, which matches outward-facing, lateral domains of polar marker PEN3/ABCG36-GFP (D) but only partially overlaps the nonpolar marker BRI1-GFP (H). Note that a weak nuclear TWD1 signal in (A) is probably caused by bleed-through of the DAPI signal because it was not found without DAPI. Bar = 15 μm. (I) to (T) TWD1 colocalizes with ABCB1-GFP ([I] to [K]) and occasionally also with ABCB19-GFP ([L] to [N]) on lateral and basal domains in the epidermis and stele, respectively. Note only partial overlap with PIN1-GFP ([O] to [Q]) and absence of overlap with PIN2-GFP ([R] to [T]). Arrowheads indicate directions of main shootward (J) and rootward (M) auxin flows in the epidermis and endodermis, correlating with main B1 (J) and B19 (M) expression, respectively. Note that B1-TWD1 and B19-TWD1 colocalization is limited to subdomains in the epidermis ([K] and [N]) and stele (N), respectively (marked by asterisks). Bars = 10 μm.

Figure 5.

TWD1 Colocalizes with PM Proteins, Including ABCB1, in BFA Compartments (A) to (F) TWD1-CFP (red) colocalizes with PM marker 29-1-GFP ([A] to [C], green) but not with ER marker ER-YFP ([D] to [F], green) in BFA-induced compartments (20 µM, 1 h). Bars = 10 μm. (G) to (R) Immunodecoration of TWD1 (red) in ABCB1-GFP ([G] to [J], green), ABCB19-GFP ([K] to [N], green), and PEN3-GFP ([O] to [R], green) lines indicate colocalization in BFA-induced compartments (50 µM, 45 min). DAPI stains are in blue. Bars = 5 μm.

Figure 6.

TWD1 Interacts with ABCB1 in Planta. (A) to (D) Expression of TWD1-YFP in the Arabidopsis root tip (A), leaf epidermis (B), root meristematic zone epidermis (C), and guard cells (D) indicating a dual location at the PM and the ER. Insets, magnification of PM and ER signals in the root meristematic zone. Bars = 20 μm in (A), 10 μm in (B), 15 μm in (C), and 5 μm in (D). (E) and (F) TWD1-YFP signals at the cell periphery colocalize with PM marker FM4-64 ([E]; see inset for close-up) and these signals are partially nonoverlapping with ER marker hexyl rhodamine B ([F]; see inset for close-up). Note, yellow (E) and green PM signals in insets of merged pictures (F) indicate PM signals in addition to ER signals for TWD1-YFP. Bar = 10 μm. (G) Phenotypes of 25 d after germination wild type (Col Wt), TWD1 loss- (twd1-3) and gain-of-function mutants, TWD1-YFP, and YFP-TWD1. DAG, days after germination. Bar = 2 cm. (H) Functional interaction between TWD1 and ABCB1 Rluc and YFP fusions using transfected tobacco leaves assayed by BRET. Note absence of significant BRET signals with unrelated PM controls, PIRK and PIP2A, and vacuolar control, TT12. Data are means ± se (n = 4).

Figure 7.

ABCBs Are Delocalized from the PM and Degraded in twd1. (A) B1-, B19-, and B4-GFP are broadly localized to the ER in twd1-3, but colocalization with PM and ER markers, FM4-64 (2.5 μΜ, 10 min staining) and hexyl rhodamine B (rhodamine B; 2 μΜ, 20 min staining), suggests that a portion resides at the PM. Wt, the wild type. Bar = 10 μm. (B) Expression of B1-, B19-, and B4-GFP is significantly downregulated in twd1-3 as revealed by confocal analysis using identical microscope settings. Bar = 50 μm.