The Arabidopsis ATP-BINDING CASSETTE Transporter ABCB21 Regulates Auxin Levels in Cotyledons, the Root Pericycle, and Leaves

Abstract

The phytohormone auxin plays significant roles in regulating plant growth and development. In Arabidopsis, a subset of ATP-BINDING CASSETTE subfamily B (ABCB) transporters participate in polar movement of auxin by exclusion from and prevention of reuptake at the plasma membrane. A previous analysis identified ABCB21 as a conditional auxin uptake/efflux transporter that regulates cellular auxin levels, but clear physiological roles for ABCB21 in planta remain unknown. Here we show that ABCB21 maintains the acropetal auxin transport stream by regulating auxin levels in the pericycle. Loss of ABCB21 reduces rootward auxin transport and delays lateral root emergence. In seedling shoots, ABCB21 regulates mobilization of auxin from the photosynthetic cotyledons that is important for phototropic bending. In rosette leaves ABCB21 contributes to lateral auxin distribution. These results support a primary role for ABCB21 in regulating auxin distribution supplementary to the primary ABCB auxin transporters ABCB1 and 19.

Keywords: ABCB transporter; Arabidopsis thaliana; auxin; development; seedling.

Figure 1

Auxin transport activity and expression of ABCB21. (A) ABCB21 exhibits conditional auxin uptake/efflux in S. pombe. Data shown are means ± SD (n = 6–8 from two experiments with 3- independent transformants). (B) proABCB21:GUS expression in 4 d etiolated seedling roots. (C–G) proABCB21:GUS expression in (C) 1 d (D) 3 d, (E) 5 d, (F) 7 d, and (G) 10 d light-grown seedling roots. No GUS signal is observed in 1 d roots. Arrow indicates area of low level expression in 3 d roots. (H) Cross and (I) longitudinal sectioning confirms expression is primarily associated with the root pericycle. (J) proABCB21:GUS is absent in emerging lateral roots. (K) Treatment with cytokinins reduces proABCB21:GUS expression in the pericycle of 7 d roots. Representative images of proABCB21:GUS expression after 3 h treatment with 100 nM kinetin (kin) or trans-zeatin (zea) prior to GUS staining. (L) Quantification of proABCB21:GUS signal in (K). Images were taken between the lowest two emerged lateral roots. Data shown are means ± SD for the five most heavily stained roots. (M–O) proABCB21:GUS expression during early lateral root development. Arrows indicate lateral root primordia. Asterisks indicate statistical difference from Col-0 by Student's t-test for ** P < 0.01. Scale bars: (B–J,M–O) 50 μm; (K) 200 μm.

Figure 2

Auxin transport and levels in abcb21 roots. abcb21 mutants exhibit reduced acropetal auxin transport in roots. (A) ABCB21 gene model indicating T-DNA insertion positions for abcb21-1 (WiscDsLox1C2) and abcb21-2 (Gabi_954H06). Boxes correspond to exons with approximate positions of transmembrane domains (TMD; blue), nucleotide-binding domains (NBD; red), and linker region (yellow). (B) [³H]IAA transport from the root-shoot transition zone to the root tip in 5.5 d seedlings. Data shown are means ± SD (n = 3 pools of 10). (C) Quantification of free IAA levels in 5.5 d light grown seedlings. UR, upper root; Apex, apical 2 mm root section. Data shown are means ± SD (n = 3 pools of 10). Asterisks indicate statistical difference from Col-0 by Student's t-test for * P < 0.05 and ** P < 0.01.

Figure 3

Root phenotypes in abcb21. abcb21 mutants exhibit altered primary root elongation and lateral root development. (A) Primary root length at 5, 7, 10, and 14 d. Data shown are means ± SD (n = 40–50). (B) Emerged lateral root density in 7 and 10 d seedlings. Data shown are means ± SE (n = 40–50). (C) Distribution of stage I–IV, stage V–VI, and emerged lateral roots in 7 and 10 d seedlings. (D) DR5:GUS expression is reduced in emerging and newly emerged abcb21 lateral root tips. (E) Quantification of GUS signal in (D). Data shown are means ± SD (n ≥ 24 from two independent experiments). Asterisks indicate statistical difference from Col-0 by Student's t-test for * P < 0.05 and ** P < 0.01. Scale bar: 50 μm.

Figure 4

ABCB21 mediates cotyledon-hypocotyl auxin transport. (A) proABCB21:GUS expression at the base of petioles in 4 d etiolated seedlings. (B–D) proABCB21:GUS expression in the petioles and cotyledonary node of (B) 1 d, (C) 3 d, and (D) 5 d light-grown seedlings. (E) Cotyledon-hypocotyl [³H]IAA transport in 5.5 d seedlings. Data shown are means ± SD (n = 3 pools of 12). (F) Cotyledon areas of 3 and 5 d light-grown seedlings. Data shown are means ± SD (25 ≤ n ≤ 32). (G) Hypocotyl length in 3 d etiolated (etio.), and 3–5 d light-grown abcb21 seedlings. Data shown are means ± SD (n > 45 from 3 replicates). (H) Phototropic curvature in light-treated Col-0 and abcb21-2 seedlings. Data shown are means ± SE (n = 8 from 2 replicates). (I) Representative images of Col-0 and abcb21-2 after phototropic bending for 3 h. Asterisks indicate statistical difference from Col-0 by Student's t-test for * P < 0.05 and ** P < 0.01. Scale bars: (A–D) 500 μm.

Figure 5

Expression of ABCB21 and auxin transport activity in leaves. (A) proABCB21:GUS is expressed in rosette leaves associated with the midvein. (B) Cross section showing proABCB21:GUS expression is primarily in the bundle sheath and collenchyma on the abaxial side of the leaf. Dotted line indicates area of GUS staining. (C,D) Transport of [³H]IAA from the leaf tip to the (C) leaf midpoint or (D) petiole. (C,D) share the same scale/units. Data shown are means ± SD (n = 3 pools of 10). (E) Transport of ³H-IAA from the leaf tip to the petiole with NPA treatment. Data shown are means ± SD (n = 3 pools of 10). (F) Transport of [³H]IAA from the leaf midvein to the margin. Data shown are means ± SD (n = 3 pools of 10). (G) Free IAA levels in rosette leaves near the midvein and near the margin. (H) Free IAA levels in young leaves, mature leaves, and petioles. Data shown are means ± SD (n = 3 pools of 10). Asterisks indicate statistical difference from Col-0 by Student's t-test for * P < 0.05 and ** P < 0.01. Scale bars: (A) 2 mm; (B) 200 μm.

Figure 6

abcb21 triple mutants exhibit enhanced morphological defects in leaves. (A) Representative images of 4 weeks rosettes grown under 100 μmol m⁻² s⁻¹ white light. (B) 5th rosette leaf removed from A. Leaves were soaked in ethanol to allow curled leaves to lay flat. (C–E) Measurement of (C) length, (D) width, and (E) length/width ratio in leaves from (B). Data shown are means ± SD (n ≥ 10). Letters indicate statistical difference by ANOVA p < 0.001, Tukey's post-hoc p < 0.05. (F) Boxplot of showing 5th leaf cell size (n ≥ 219 from at least 3 leaves per line). Abaxial epidermal cells were measured at a midpoint from the leaf tip to the petiole and half way from the leaf margin. Letters indicate statistical difference by ANOVA P < 0.001, Tukey's post-hoc P < 0.05. Scale bars: 1 cm.

Figure 7

Expression of ABCB21 in abscission zones. proABCB21:GUS is expressed in the abscission zones of (A) rosette leaves, (B) cauline leaves, and (C) floral organs. (D,E) proABCB21:GUS expression domain is expanded in (D) young flowers and (E) restricted to the abscission zone in mature flowers. Plants were GUS stained for 16 h. (F) True leaf petiole angles in abcb21. Plants were grown on soil under 80 μmol m⁻² s⁻¹ white light, 16 h photoperiod. When plants reached stage 1.01 they were transferred to continuous 50 μmol m⁻² s⁻¹ red plus far-red light (50R + FR; burgundy bars), 50 μmol m⁻² s⁻¹ red light (50R; red bars), or 50 m⁻² s⁻¹ white light (50W; white bars) and allowed to grow an additional 3 d. Angle was determined by measuring the angle formed between the hypocotyl and the two first true leaf petioles minus 90°. Data shown are means ± SD (n = 60). (G) Flower petal break-strength in abcb21. Flower 1 was designated as the first flower with visible flower petals. Methods are detailed the methods section and Supplementary Figure 7. Data shown are means ± SD (n = 15). Scale bars: 1 mm.

Figure 8

proABCB21:GUS and DR5:GUS expression after wounding. (A) proABCB21:GUS expression is induced after manual wounding. Mature inflorescence stems were cut with a razor blade at the bottom internode. Attached stems were left for 2 h prior to GUS staining. (B) Initial DR5:GUS expression is not different between Col-0 and abcb21-2. Plants were treated the same as in (A) except cleared with Visikol prior to imaging to enhance visualization. Scale bars: 0.5 mm.

Figure 9

Model for ABCB21 function in seedlings and leaves. (A) ABCB21 functions primarily in restricting auxin to within the rootward auxin transport stream. Loss of ABCB21 leads to leakage of auxin from the central cylinder, resulting in reduced rootward auxin transport. E, epidermis; C, cortex; En, endodermis; P, pericycle; V, vasculature. (B) The reduction in auxin transport in the root causes pooling of auxin in the upper root and lower hypocotyl, reducing the supply needed for lateral root outgrowth. (C) ABCB21 mediates cotyledon-hypocotyl auxin transport to load the rootward auxin transport stream. (D) In leaves ABCB21 contributes to lateral auxin transport, while ABCB19 primarily mediates transport from the leaf tip to the petiole.