Putative Arabidopsis transcriptional adaptor protein (PROPORZ1) is required to modulate histone acetylation in response to auxin

Abstract

Plant development is highly adaptable and controlled by a combination of various regulatory circuits that integrate internal and environmental cues. The phytohormone auxin mediates such growth responses, acting as a dynamic signal in the control of morphogenesis via coordinating the interplay between cell cycle progression and cell differentiation. Mutants in the chromatin-remodeling component PROPORZ1 (PRZ1; also known as AtADA2b) are impaired in auxin effects on morphogenesis, suggestive of an involvement of PRZ1-dependent control of chromatin architecture in the determination of hormone responses. Here we demonstrate that PRZ1 is required for accurate histone acetylation at auxin-controlled loci. Specifically, PRZ1 is involved in the modulation of histone modifications and corresponding adjustments in gene expression of Arabidopsis KIP RELATED PROTEIN (KRP) CDK inhibitor genes in response to auxin. Deregulated KRP expression in KRP silencer lines phenocopies prz1 hyperproliferative growth phenotypes, whereas in a KRP overexpression background some mutant phenotypes are suppressed. Collectively, our findings support a model in which translation of positional signals into developmental cues involves adjustments in chromatin modifications that orchestrate auxin effects on cell proliferation.

Fig. 1.

PRZ1 is a positive regulator of histone acetylation. Protein extracts derived from wild type and prz1-1 were normalized with anti-histone H3 and anti-histone H4 and probed with antibodies recognizing histone H3-acK9, histone H3-acK9/K14, and tetra-acetylated histone H4. Histone H3-K4me3 is another chromatin modification predominantly associated with transcribed loci. Signal quantification is shown below each panel (wild type = 1).

Fig. 2.

Expression of KRP genes. (A and B) KRP7p::KRP7:GUS in wild type (A) and prz1-1 (B) root meristem stem cell niches. (C and D) KRP7:GUS activity in adjacent root epidermis cells (arrowheads) of wild type (C) and prz1-1 (D). (E) GUS-staining of KRP7p::GUS in emerging lateral roots upon incubation on 2.5 μM NAA for 60 h. Arrowheads indicate ectopic cell proliferation of prz1-1 pericycle cells. (F) Same as (E) but after incubation on 2.5 μM NAA for 7 days. (G) Activity of KRP7p::GUS in wild type and in prz1-1 at 6 days after germination (DAG). (H) Activity of KRP7p::GUS in 6-day-old wild type and prz1-1 after treatment with 1 μM NAA for 18 h. (J) Quantitative analysis of KRP transcript levels in 6-day-old wild type and prz1-1 after treatment with 1 μM NAA for 0, 4, or 18 h. Standard deviations are indicated (n = 3). UBQ5 and TUB9 were used for normalization. (Scale bars, A, B, E−H = 100 μm; C, D = 20 μm.)

Fig. 3.

PRZ1 associates with the KRP7 locus and modulates histone acetylation. (A) Schematic drawing of the genomic KRP7 locus. Dark boxes indicate exons. Dark lines labeled with Arabic numbers indicate PCR fragments tested by ChIP performed with anti-PRZ1 and anti-myc. Gray lines labeled with Roman numbers highlight PCR fragments tested in ChIP performed with histone antibodies. (B) anti-myc ChIP performed with chromatin from Col-0 and 35S::myc:PRZ1 seedlings was tested for KRP7. (Upper) Signals obtained in the input fractions. (Lower) Signals obtained in the corresponding pull-down fractions. (C) ChIP performed with anti-PRZ1 on chromatin preparations from 10-day-old Col-0. Before IP the input fraction was put aside. The remaining sample was divided into aliquots, which were used for IP either with anti-PRZ1 (+ab.) or by omitting the antibody (w/o ab.). w/o chromatin: control IP performed in the absence of chromatin. (D) Quantification of signal intensities obtained in (B) and (C) to determine the relative enrichment (arbitrary units) of DNA fragments. Enrichment was calculated as (ChIP/Input)/(ChIP control/Input control). (E) Histone H3-acK9/K14 levels in wild type and prz1-1 grown on PNS. Amounts of acetylated histone H3 were determined after normalization to control ChIPs performed with nondiscriminating anti-histone H3 (= 100%). Standard deviations are indicated as bars (n = 3). (F) Normalized amounts of histone H3 K9/K14 in wild type and prz1-1 after treatment with 1 μM NAA for 18 h. Histone acetylation is expressed as fold change after normalization to corresponding control samples (= 1). Standard deviations are indicated as bars (n = 3).

Fig. 4.

Analysis of KRP overexpression and silencing lines. (A Left) Rosette leaves of a KRP silencer control plant (c; 22 DAG). (Center) Dedifferentiation of the shoot apical meristem in line 3.0 (30 DAG, black arrowhead). (Right) Microcallus-formation on a leaf of silencer line 2.7 (18 DAG, black arrowhead). (B) Comparison of Col-0, KRP7ox, prz1-1, and prz1-1 KRP7ox plantlets at 16 DAG. (C) Flowering prz1-1 and prz1-1 KRP7ox at 40 DAG. (D) Lugol-staining of 12-day-old wild-type, KRP7ox, prz1-1, and prz1-1 KRP7ox primary root tips, grown on PNS. Asterisks indicate position of QC. (E) Lugol-staining of 12-day-old wild type, KRP7ox, prz1-1, and prz1-1 KRP7ox primary root tips germinated on PNS for 5 days and then transferred on medium containing 2.5 μM NAA for another 7 days. (F) Primary root segments of 8-day-old prz1-1 (Upper) and prz1-1 KRP7ox (Lower) seedlings after incubation on 2.5 μM NAA for 4 days. Prz1-1 KRP7ox develops distinct lateral root primordia (white arrowheads) separated by zones with reduced pericycle proliferation (black arrowheads). (G) Root morphology of 12-day-old wild type, KRP7ox, prz1-1, and prz1-1 KRP7ox germinated on 2.5 μM NAA. Note lateral root growth in prz1-1 KRP7ox (white arrowheads), occasionally interspersed by callus formation (black arrowhead). (Scale bars, A = 2 mm; B = 10 mm; C = 20 mm; D, E = 100 μm; F = 50 μm; G = 200 μm.)