Two homologous ATP-binding cassette transporter proteins, AtMDR1 and AtPGP1, regulate Arabidopsis photomorphogenesis and root development by mediating polar auxin transport

Abstract

Light and auxin control many aspects of plant growth and development in an overlapping manner. We report here functional characterization of two closely related ABC (ATP-binding cassette) transporter genes, AtMDR1 and AtPGP1, in light and auxin responses. We showed that loss-of-function atmdr1 and atpgp1 mutants display hypersensitivity to far-red, red, and blue-light inhibition of hypocotyl elongation, reduced chlorophyll and anthocyanin accumulation, and abnormal expression of several light-responsive genes, including CAB3, RBCS, CHS, and PORA, under both darkness and far-red light conditions. In addition, we showed that the atmdr1-100 and atmdr1-100/atpgp1-100 mutants are defective in multiple aspects of root development, including increased root-growth sensitivity to 1-naphthalene acetic acid (1-NAA), and decreased sensitivity to naphthylphthalamic acid (NPA)-mediated inhibition of root elongation. Consistent with the proposed role of AtMDR1 in basipetal auxin transport, we found that expression of the auxin responsive DR5::GUS reporter gene in the central elongation zone is significantly reduced in the atmdr1-100 mutant roots treated with 1-NAA at the root tips, compared to similarly treated wild-type plants. Moreover, atmdr1-100, atpgp1-100, and their double mutants produced fewer lateral roots, in the presence or absence of 1-NAA or NPA. The atmdr1-100 and atmdr1-100/atpgp1-100 mutants also displayed enhanced root gravitropism. Genetic-epistasis analysis revealed that mutations in phyA largely suppress the randomized-hypocotyl growth and the short-hypocotyl phenotype of the atmdr1-100 mutants under far-red light, suggesting that phyA acts downstream of AtMDR1. Together, our results suggest that AtMDR1 and AtPGP1 regulate Arabidopsis (Arabidopsis thaliana) photomorphogenesis and multiple aspects of root development by mediating polar auxin transport.

Figure 1.

Loss-of-function mutants of the AtMDR1 gene. A, The atmdr1 mutants have short hypocotyls and epinastic cotyledon under various light conditions and possess wavy hypocotyls in the dark. The seedlings were grown in continuous dark, far-red, red, and blue lights for 4 d. Bar = 1 mm. B, Structure of T-DNA insertion alleles. Black rectangles represent the exons, and lines are introns. Triangles represent T-DNA insertions. C, AtMDR1 mRNA accumulation is abolished in the T-DNA mutants analyzed by RT-PCR. Actin is shown at the bottom as a control. Lane 1, Wild type (Col); lane 2, atmdr1-100; lane 3, atmdr1-101; lane 4, atmdr1-102. D and E, The atmdr1 mutants have smaller rosettes and their rosette leaves are shorter, but wider, and wrinkled. The plants were grown in soil under long-day conditions (16 h light/8 h dark) at 22°C for 3 weeks. Bar = 1 cm.

Figure 2.

Loss-of-function mutants of the AtPGP1 gene and the atmdr1-100/atpgp1-100 double mutant. A, Schematic structure of T-DNA insertion alleles. Black rectangles represent the exons, and lines are introns. Triangles represent T-DNA insertions. B, RT-PCR analysis of the atpgp1 mutants. Actin serves as a control. Lane 1, Wild type (Col); lane 2, atpgp1-100; lane 3, atpgp1-101. C, The atpgp1 mutants possess shorter hypocotyls under different light conditions but are normal in the dark. The seedlings were grown in continuous dark, far-red, red, and blue lights for 4 d. Bar = 1 mm. D, Four-week old adult plants of wild type (Col), atmdr1-100, atpgp1-100, and the atmdr1-100/atpgp1-100 double mutant. Bar = 1 cm.

Figure 3.

Photomorphogenic defects of atmdr1-100 and atpgp1-100 mutants. A, Fluence rate responses of 4-d-old atmdr1-100, atpgp1-100, and wild-type (Col) seedlings under darkness or continuous far-red light. Bars denote sds from 20 seedlings. B, Chlorophyll content of atmdr1-100, atpgp1-100, and wild-type (Col) plants. Seedlings were grown in far-red light for 3 d, and then were transferred to white light for 2 d before chlorophyll extraction. Bars represent sds from triplicate experiments. C, Anthocyanin content of atmdr1-100, atpgp1-100, and wild-type (Col) plants. Seedlings (approximately 100) were grown in continuous far-red or blue light for 4 d before anthocyanin extraction. Bars represent sds from triplicate experiments.

Figure 4.

Abnormal expression of light-responsive genes in the atmdr1-100 and atpgp1-100 mutants. A, Reduced expression of RBCS, CAB3, CHS, and PORA. RNAs were extracted from seedlings grown in darkness (Dk) for 4 d, or seedlings grown in darkness for 4 d then exposed to far-red (FR) for 4 h. An 18S rRNA blot of the duplicating gels was shown below as a loading control. Lane 1, Col; lane 2, atmdr1-100; lane 3, atpgp1-100; lane 4, atmdr1-100/atpgp1-100. B to E, Relative expression levels of light-responsive genes quantified with a PhosphorImager, normalized against 18S rRNA. F, Light regulation of AtMDR1 expression. Wild-type (Col) or phyA-211 mutant seedlings were grown in continuous darkness (Dk), far-red (FR), red (R), or blue (B) for 4 d before total RNA extraction. An 18S rRNA blot was shown as a loading control. G, Relative AtMDR1 expression levels quantified with a PhosphorImager. The expression levels were normalized against 18S rRNA.

Figure 5.

atmdr1 and atpgp1 affect root elongation and lateral-root formation. Seedlings were grown vertically on germination plates for 3 d under white light and then transferred to new unsupplemented plates or plates supplemented with various hormones. A to C, Relative root elongation of wild type (Col), atmdr1-100, atpgp1-100, and atmdr1-100/atpgp1-100 mutants on plates supplemented with various concentrations of 2,4-D, 1-NAA, and NPA for 4 d, respectively. The percentage of relative root elongation was calculated based on control plants grown on unsupplemented media. Bars indicate the sds from 20 to 25 seedlings. The plant genotypes for A to C are indicated in A. D, Lateral root number of wild type (Col), atmdr1-100, atpgp1-100, and atmdr1-100/atpgp1-100 mutants on medium with or without 100 nm 1-NAA, 100 nm 2,4-D, and 0.1 μm NPA. Bars indicate the sds from 20 to 25 seedlings.

Figure 6.

Effects of hormone treatments on DR5::GUS expression. GUS staining of root tips of wild-type (Col) and atmdr1-100 seedlings harboring the DR5::GUS reporter gene. Seedlings were first grown vertically on germination plates for 4 d and then subjected to hormone treatments and GUS staining. A, Seedlings were treated with IAA (1.0 μm), 1-NAA (1.0 μm) or 2,4-D (1.0 μm) at the root tips for 3 h. Bar = 1 mm. CEZ, Central elongation zone; DEZ, distal elongation zone. B, Seedlings were treated with or without NPA (2.0 μm) for 24 h. Bar = 100 μm.

Figure 7.

Enhanced gravitropic responses of the atmdr1 and atmdr1/atpgp1 double mutant. A, Representative root images of wild-type (Col), atmdr1-100, atpgp1-100, and atmdr1-100/atpgp1-100 mutant seedlings 24 h after gravistimulation. Seedlings were grown vertically on unsupplemented germination plates in darkness for 2 d, and then the plates were rotated 90°. Bar = 1 cm. Arrows indicate the gravity vector. B, Time course of root gravitropic response. The angles of curvature were measured every 6 h over a 24-h period. Bars indicate sds from 35 to 40 seedlings. C to E, GUS staining of seedlings 0 (before gravistimulation), 3, and 5 h after reorientation, respectively. Seedlings of wild type (Col) and atmdr1-100 mutant harboring DR5::GUS reporter gene were grown vertically on germination plates under white light for 4 d, and then were transferred to new plates for 24 h. The plates were then rotated 90°. At the indicated times, seedlings were harvested for GUS staining. Bar = 100 μm. Arrow indicates the gravity vector.

Figure 8.

Characterization of the atmdr1-100/axr1-3 double mutant. A, Hypocotyl length of wild type, atmdr1-100, axr1-3, and atmdr1-100/axr1-3 mutants in continuous darkness or white light for 4 d. Bars represent sds from 20 seedlings. B and C, Eighteen- and 28-d-old plants grown in soil, respectively. Bar = 1 cm.

Figure 9.

phyA acts downstream of AtMDR1 in regulating hypocotyl growth. A, Representative pictures of wild type (Col, Ler), atmdr1-100, phyA-1 single mutants, and atmdr1-100/phyA-1 double mutant grown on germination plates with or without 2.0 μm NPA under far-red light for 4 d. Bar = 1 mm. B, Hypocotyl length of wild type (Col, Ler), atmdr1-100, phyA-1, and atmdr1-100/phyA-1 as shown in A. Bars represent sds from 20 seedlings. C, Hypocotyl orientation of Arabidopsis seedlings grown in the darkness or continuous far-red light for 5 d. The orientation of hypocotyls angle was measured as the sd around the vertical 0°. Values represent the pooled sd for a minimum of 100 seedlings from triplicate experiments. High sds indicate randomization of hypocotyls. D, Root elongation of wild type (Col, Ler), atmdr1-100, phyA-1, and atmdr1-100/phyA-1 mutants under far-red light. Seedlings were vertically grown in far-red for 3 d and then transferred to new plates with (10 nm or 100 nm) or without 1-NAA for further 4 d before the additional root elongation was measured. Bars denote sds from 20 seedlings. For B to D, the plant genotypes are shown in C.