Developmental specificity of auxin response by pairs of ARF and Aux/IAA transcriptional regulators

Abstract

The plant hormone auxin elicits many specific context-dependent developmental responses. Auxin promotes degradation of Aux/IAA proteins that prevent transcription factors of the auxin response factor (ARF) family from regulating auxin-responsive target genes. Aux/IAAs and ARFs are represented by large gene families in Arabidopsis. Here we show that stabilization of BDL/IAA12 or its sister protein IAA13 prevents MP/ARF5-dependent embryonic root formation whereas stabilized SHY2/IAA3 interferes with seedling growth. Although both bdl and shy2-2 proteins inhibited MP/ARF5-dependent reporter gene activation, shy2-2 was much less efficient than bdl to interfere with embryonic root initiation when expressed from the BDL promoter. Similarly, MP was much more efficient than ARF16 in this process. When expressed from the SHY2 promoter, both shy2-2 and bdl inhibited cell elongation and auxin-induced gene expression in the seedling hypocotyl. By contrast, gravitropism and auxin-induced gene expression in the root, which were promoted by functionally redundant NPH4/ARF7 and ARF19 proteins, were inhibited by shy2-2, but not by bdl protein. Our results suggest that auxin signals are converted into specific responses by matching pairs of coexpressed ARF and Aux/IAA proteins.

Figure 1

Expression of IAA13, and analysis of an iaa13 stabilizing mutation. (A) Domain structure of Aux/IAA proteins. The four conserved domains are depicted. Below is the consensus amino-acid sequence in conserved domain II of the engineered iaa13^P80S mutation, of bdl and of shy2-2. (B) Rootless seedling homozygous for the iaa13^P80S mutation (left); inset: bdl seedling. Comparison of a wild-type (middle) and homozygous iaa13^P80S (right) globular stage embryo shows defects in hypophysis (hyp) specification; inset: abnormal division of hypophysis in bdl embryo. (C) Western blots (Myc antibody) of protein extracts from pBDL∷BDL, pBDL∷bdl, pIAA13∷IAA13 and pIAA13∷iaa13^P80S seedlings. Individual lanes represent independent transgenics. Asterisk: unspecific crossreacting band demonstrating equal loading. (D) mRNA in situ hybridization with an IAA13 antisense probe in wild-type and bdl (right) embryos. RNA signals are in red-brown. (E) GUS activity in pIAA13∷GUS embryos and seedling root tip (right).

Figure 2

BDL promoter-swap experiments. (A) Rootless pBDL∷shy2-2 and pBDL∷iaa13^P80S homozygous seedlings; inset: pBDL∷bdl seedling. (B) Flowering plants (4 weeks old) are bushy and short; inset: heterozygous bdl plant. (C) Western blots of protein extracts from pBDL∷SHY2, pBDL∷shy2-2, pBDL∷IAA13 and pBDL∷iaa13^P80S seedlings. Asterisk: unspecific crossreacting band demonstrating approximately equal loading. Percentage of rootless seedlings (%RL) is indicated for each line.

Figure 3

Repression of MP activity by SHY2 and BDL in yeast. (A) Galactosidase activity in yDR5 yeast cells expressing the empty vector (−), MP:HA (MP), MP:HA and SHY2:HA (MP+SHY2) or MP:HA and BDL:HA (MP+BDL). Values (±s.d.) are the average of 12 independent transformants. Asterisks represent statistically significant difference between MP and – (^*P<0.001, two-tailed Student's t-test), MP+SHY2 and MP (^**P<0.001, two-tailed Student's t-test), and MP+BDL and MP (^**P<0.001, two-tailed Student's t-test). (B) Western blot (HA antibody) with equal amounts of protein extracts from three independent yeast transformants for each plasmid. The regions from the same blot that represent MP:HA (105 kDa), SHY2:HA (25 kDa) and BDL:HA (29 kDa) are depicted. Note that expression levels of MP:HA protein vary between different colonies of the same genotype.

Figure 4

Inhibition of auxin responses in the shoot by stabilized Aux/IAA proteins. (A) Hypocotyl length in light-grown (white bars) or dark-grown (black bars) seedlings of wild type (COL), pSHY2∷SHY2, pSHY2∷shy2-2 (lines #27, #6 and #13), pSHY2∷BDL, pSHY2∷bdl (lines #4 and #12), pSHY2∷IAA13, pSHY2∷iaa13^P80S (lines #7 and #11) and shy2-2. Hypocotyl length (±s.d.) is represented as percentage of COL. (B) Western blot of protein extracts from light-grown seedlings in (A). Blot was probed with anti-Myc antibodies; asterisk: unspecific crossreacting band demonstrating equal loading. (C) Phenotypes of flowering plants. (D) GUS activity in hypocotyl of seedlings hemizygous for pSHY2∷GUS and pSHY2∷shy2-2, pSHY2∷bdl or wild-type controls from same cross. Seedlings were treated with IAA for 5 h and stained for GUS activity.

Figure 5

Inhibition of auxin responses in the root by shy2-2, but not by bdl. (A) Gravitropic response of pSHY2∷SHY2, pSHY2∷shy2-2, pSHY2∷BDL and pSHY2∷bdl seedlings after reorientation by 90° (data from two to five independent transgenic lines for each genotype). The percentages represent the fraction of seedlings (numbers analyzed in parentheses) with normal gravitropic response. (B) Inhibition of root growth by 2,4-D. Root length (±s.d.) was measured after 3 days of vertical growth on medium with (black bars) or without (white bars) 0.1 μM 2,4-D. Growth is represented as percentage of root length in wild type (COL) on control media. (C) Western blots of protein extracts from IAA-treated (10 μM for 5 h) or untreated roots of pSHY2∷SHY2, pSHY2∷shy2-2, pSHY2∷BDL and pSHY2∷bdl seedlings. Asterisks: unspecific crossreaction as loading control. Note that SHY2 or BDL accumulation is induced by auxin in pSHY2∷SHY2, pSHY2∷BDL and pSHY2∷bdl, but not in pSHY2∷shy2-2. (D) GUS activity in root tips of F1 seedlings from crosses between hemizygous pSHY2∷shy2-2 or pSHY2∷bdl lines and homozygous pSHY2∷GUS or pDR5(7x)∷GUS lines. Controls (WT) are wild-type segregants from the same cross. −IAA, untreated; +IAA, 10 μM IAA for 5 h. GUS staining time was 3 h for untreated and 1.5 h for IAA-treated roots.

Figure 6

NPH4 and ARF19 as targets for shy2 inhibition in the root. (A) Gravitropic response of wild-type (COL), nph4-1, arf19-4 and nph4-1 arf19-4 seedling roots (numbers analyzed in parentheses) upon reorientation by 90°. (B) Inhibition of root growth by 2,4-D. Root length (±s.d.) was measured after 3 days of vertical growth on medium with (black bars) or without (white bars) 0.1 μM 2,4-D. Growth is represented as percentage of root length in wild type (COL) on control media. (C) SHY2 mRNA expression in COL, nph4-1, arf19-4 and nph4-1 arf19-4 seedlings treated with unsupplemented medium (white bars) or with medium containing 50 μM IAA (black bars) for 3 h. Average values (±s.d.) are taken from five to six replicate real-time PCR reactions (quantitative RT–PCR); inset: semiquantitative RT–PCR experiment on dissected roots from three replicates. Expression is relative to ACT2 expression in the same cDNA samples. (D) Interaction of SHY2 or BDL with MP and ARF19 in yeast two-hybrid assays. Galactosidase activity (±s.d.) was measured in at least 12 independent colonies expressing each of the depicted plasmids (−, empty vector control).

Figure 7

Specificity of MP action during embryogenesis. Percentage of rootless seedlings in (A) 20 independent pBDL∷MP transgenic lines and (B) 11 independent pBDL∷ARF16 transgenic lines, all carrying the mp mutation. The line at 25% marks the frequency of rootless seedlings in the untransformed mp mutant.

Figure 8

Model for Aux/IAA- and ARF-mediated auxin responses. In all tissues, auxin promotes the degradation of Aux/IAA proteins. In the embryo, BDL and IAA13 likely inhibit MP. When released from inhibition, MP promotes root initiation. In hypocotyl, SHY2 inhibits yet unknown ARF(s) (ARFx,y). When released from inhibition, ARFx,y promote cell elongation and enhance the transcription of SHY2. In root tips, SHY2 inhibits NPH4 and ARF19, which, when released, promote root growth and gravitropic response. NPH4 and ARF19 activity also stimulates SHY2 transcription. When expressed from the SHY2 promoter, shy2-2 mutant protein (pSHY2∷shy2-2) constitutively inhibits ARFx,y activity in the hypocotyl and NPH4 and ARF19 activity in the root, including feedback regulation of SHY2 transcription. Mutant bdl protein, expressed from the SHY2 promoter (pSHY2∷bdl), inhibits ARFx,y activity in the hypocotyl, but not NPH4 and ARF19 activity in the root.