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First published online February 18, 2004
doi: 10.1242/10.1242/dev.00925

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Overlapping and non-redundant functions of the Arabidopsis auxin response factors MONOPTEROS and NONPHOTOTROPIC HYPOCOTYL 4

Christian S. Hardtke^1,*, Wenzislava Ckurshumova^3,*, Danielle P. Vidaurre³, Sasha A. Singh³, George Stamatiou³, Shiv B. Tiwari², Gretchen Hagen², Tom J. Guilfoyle² and Thomas Berleth^3,

¹ McGill University, Biology Department, 1205 Docteur Penfield Avenue, Montréal, Québec H3A 1B1, Canada
² University of Missouri, Department of Biochemistry, 117 Schweitzer Hall, Columbia, MO 65211, USA
³ University of Toronto, Department of Botany, 25 Willcocks Street, Toronto, Ontario M5S 3B2, Canada

View larger version (22K): [in a new window]   Fig. 1. ARF protein interactions. (A) ARF-ARF and ARF-Aux/IAA protein interaction in yeast two hybrid assays. Relative reporter gene expression levels (ß-galactosidase units, right) in yeast cells co-expressing C-terminal regions of the indicated ARF gene-coding sequences (left) fused to DNA binding (BD) and activation (AD) domains. Line one shows ß-galactosidase expression levels in yeast cells harboring both empty vectors (pAS2-1, bait; pACT2, prey). Line two shows ß-galactosidase expression in response to the CLONTECHTM positive control. Bars represent the mean±s.e. (B) Auxin-regulated interference of Aux/IAA expression with ARF transcriptional activation in carrot suspension cell protoplasts. Quantification of GUS reporter gene activity after transfection of the reporter gene and indicated effector genes in the absence (gray) and presence (black) of 10 µM 1-NAA. Columns represent the mean±s.d. Both ARF effector genes (MP/ARF5 and NPH4/ARF7) comprise the DNA-binding domain (DBD) of yeast GAL4 (GAL4DBD) and the middle region (MR), and C-terminal domain (CTD) of the respective ARF. IAA effector genes encode full-length wild-type IAA4, IAA9, IAA19 or BDL/IAA12 proteins. Expression of effector genes was driven by the CaMV 35S promoter (Tiwari et al., 2003). The GUS reporter gene is under control of the GAL4 response element and contains a -46 minimal CaMV 35S promoter with four GAL4-binding sites fused just upstream (see Ulmasov et al., 1995; Tiwari et al., 2003).

View larger version (97K): [in a new window]   Fig. 2. Expression pattern of NPH4 and MP mRNA in wild-type plants. In situ hybridization with NPH4 (A-E) and MP (F,H J) antisense probes and with NPH4 sense probe (inset in D, G). Embryos at early heart (A,F), torpedo (B) and bent cotyledon (C,G,H) stages, vegetative leaf primordia (D,I) and flower primordia (E,J). Median longitudinal sections (A,B,F), cross-sections (C-E,G-J). Scale bars: 50 µm in A-C,F-G; 250 µm in D,E,I,J.

View larger version (59K): [in a new window]   Fig. 3. Overlapping functions of MP and NPH4 in embryos. (A) Two upper panels (dark-field optics, top; schematic, bottom) illustrate phenotype classes (from left): dicotyledonous with normal vasculature, dicotyledonous with reduced secondary vein lobes, solitary midvein, fused cotyledons, single cotyledons and club-shaped seedlings. Panel below shows the percentage of each phenotype class in single and double mutants for nph4 in various double homozygous allelic combinations with weak (mpG92; nph4-1), intermediate (mpT370; nph4-1) and strong (mpG12; nph4-1) mp alleles. Columns represent the proportion of each phenotype class in % (broken lines indicate 25, 50, 75 and 100%). Approximately 100 double mutants were evaluated for each genotype; 290 double mutants were evaluated for mpG12; nph4-1. (B) Phenotype of a `club-shaped' seedling (mpG12; nph4-1). Neither cotyledons nor any other lateral outgrowth is visible and only very few disorganized vascular cells are being produced (dark-field optics). (C) Leaf venation (xylem strands) in the first rosette leaf (left) and first cauline leaf (right) of the genotypes: Col-0 wild type, nph4-1 homozygous, 35S::MPAS in wild-type background and 35S::MPAS hemizygous in nph4-1 homozygous background. Note that the venation pattern in nph4-1 single mutants is unaffected, displaying primary (midvein), secondary (first order branches), tertiary and some quarternary veins in the area enclosed by the secondary vein arches. Tertiary and quarternary venation is strongly reduced in 35S::MPAS plants. In 35S::MPAS; nph4-1 plants, tertiary and quarternary venation is further reduced and the pattern of secondary vein lobes is disrupted (dark-field optics).

View larger version (26K): [in a new window]   Fig. 4. Non-overlapping functions of MP/ARF5 and NPH4/ARF7. (A) Quantification of cotyledon area of light-germinated seedlings of the indicated genotypes at 10 dag, grown in the presence of 0 and 1 µM 2,4-D. Sample sizes are 45 to 50 cotyledons. (B) Hypocotyl length of seedlings of the indicated genotypes germinated and grown in the dark for 5 days in the presence of 20 µM IAA. Sample sizes are 10 to 50 seedlings. On hormone-free media, hypocotyls of all genotypes elongated to 1.2 cm. (C) Transcript abundance of the Aux/IAA genes IAA2 and IAA19 in light-germinated seedlings of the indicated genotypes exposed to 0, 10 and 100 µM IAA at 7 dag for 30 minutes. Transcript abundance from three independent experiments is displayed as multiples of the wild-type level at 0 µM IAA. Columns represent the mean±s.e. (A,C) and s.d. (B).

View larger version (64K): [in a new window]   Fig. 5. Ubiquitous overexpression of MP and NPH4. (A) Hypocotyl length of seedlings of the indicated genotypes germinated and grown in the dark for 5 days in the presence of 20 µM IAA. Sample sizes are 15 to 82 seedlings. Columns represent the mean±s.d. Note that both 35S::NPH4 and 35S::MP restore auxin sensitivity. (B) Normalization of the vegetative leaf shape defects (nph4-103, left) by expression of 35S::NPH4 (center) and 35S::MP (right) in the nph4-103 background. (C) Vegetative and inflorescence phenotypes of wild-type, 35S::MP, 35S::NPH4 and 35S::NPH4; 35S::MP plants. Left column contains rosettes at 21 dag, represented by the insert in the bottom panel, because of the delayed development of 35S::NPH4; 35S::MP plants. Large rosette in this panel is at 35 dag and has bolted. Note the formation of extremely twisted leaves in 35S::NPH4; 35S::MP plants of all stages and the appearance of numerous small leaves late in vegetative development (white arrow in 35S::NPH4; 35S::MP rosette). Middle column indicates inflorescence tips at 40 dag. Inflorescences of 35S::NPH4 plants are indistinguishable from wild type, while inflorescences of 35S::MP plants produce distorted, usually sterile, flowers and may terminate in pin-shaped inflorescence tips (white arrow). Inflorescences of 35S::NPH4; 35S::MP plants do not produce any flowers. (Right column) Upper two images are scanning electron micrographs of pre-bolting inflorescence meristems. Note that there is no recognizable effect of 35S::NPH4 expression on meristem organization or size. (Lower two images) Post-bolting inflorescence meristems of 35S::NPH4; 35S::MP plants invariantly lack flower primordia, which are produced at variable density in the inflorescence meristems of 35S::MP plants (white arrows). Scale bars: 100 µm. (D) Synergistic phenotype effects associated with co-expression of NPH4 together with regions of the MP coding sequence, both under control of the CaMV 35S promoter. Synergistic effects are indistinguishable from the phenotype shown in the lower panel of C and were observed in all (yes) or none (no) of at least seven double overexpressing plants. Constructs refer to amino acid residues of the MP-coding sequence (Hardtke and Berleth, 1998): MP-289, residues 1-289; MP-785, residues 1-785; MP288+, residues 288-902.

View larger version (131K): [in a new window]   Fig. 6. Mutual suppression of 35S::MP and bdl. (A) Homozygous bdl mutant grown in soil for 5 weeks. The rosette diameter is 20 mm and a short inflorescence has been produced. (B) Homozygous bdl mutant (red arrow) and homozygous bdl mutant hemizygously carrying the 35S::MP transgene (bottom left) grown in soil for 3 weeks. (C) Inflorescence tip of a hemizygous 35S::MP plant. (D) Inflorescence tip of a homozygous bdl mutant hemizygously carrying the 35S::MP transgene. Scale bars: 1 cm.