AGAMOUS controls GIANT KILLER, a multifunctional chromatin modifier in reproductive organ patterning and differentiation

Abstract

The Arabidopsis homeotic protein AGAMOUS (AG), a MADS domain transcription factor, specifies reproductive organ identity during flower development. Using a binding assay and expression analysis, we identified a direct target of AG, GIANT KILLER (GIK), which fine-tunes the expression of multiple genes downstream of AG. The GIK protein contains an AT-hook DNA binding motif that is widely found in chromosomal proteins and that binds to nuclear matrix attachment regions of DNA elements. Overexpression and loss of function of GIK cause wide-ranging defects in patterning and differentiation of reproductive organs. GIK directly regulates the expression of several key transcriptional regulators, including ETTIN/AUXIN RESPONSE FACTOR 3 (ETT/ARF3) that patterns the gynoecium, by binding to the matrix attachment regions of target promoters. Overexpression of GIK causes a swift and dynamic change in repressive histone modification in the ETT promoter. We propose that GIK acts as a molecular node downstream of the homeotic protein AG, regulating patterning and differentiation of reproductive organs through chromatin organization.

Figure 1. Identification of GIK as a direct target of AG.

(A) Flow chart of the bioinformatic screening process. D = A/T/G; H = A/T/C; N = A/T/G/C, W = A/T. (B) Schematic diagram of the GIK genomic sequence and the predicted protein structure of GIK. The residues Arg (R)-Gly (G)-Arg (R)-Proline (P) are the AT-hook motif core. PPC = plant-and-prokaryote conserved domain. (C) Semi-quantitative RT-PCR analysis of GIK in wild-type tissues and ag mutant flowers. Shown at the bottom is the lipase that was amplified as a control. (D) Induction of GIK by AG in the inflorescences of ag-1 35S::AG-GR plants. Plants were mock- or DEX-treated and harvested 6 h after the treatment. Semi-quantitative RT-PCR was performed for GIK and LIPASE (LIP). (E) Induction of GIK by AG in the presence of a protein synthesis inhibitor. Inflorescences of ag-1 35S::AG-GR plants were mock-treated or treated with DEX, DEX plus cycloheximide (Cyc), or Cyc-only and harvested 2 h after the treatment. Semi-quantitative RT-PCR was performed for GIK and TUBULIN2 (TUB). Each band strength was measured using ImageJ (http://rsb.info.nih.gov/ij/), and the relative band strength was calculated from the intensity of GIK normalized to that of TUB. (F) Real-time PCR analysis of GIK induction by AG. Inflorescences from ag-1 AG-GR plants were harvested 1 (D1) and 3 (D3) d after a single DEX treatment at day 0 (D0). Expression levels were normalized to that of TUB. The expression at D0 was set as 1.0. (G) AG binds to the GIK CArG box in vivo. ChIP was performed using ag-1 35S::AG-GR inflorescences at day 7 after four DEX treatments. P1, P2, and P3 indicate primer pairs used to detect different regions of GIK genomic DNA. The asterisk shows the location of the CArG box sequences. Relative enrichment was obtained from the ratio of enrichment achieved by anti-AG to that of control IgG. Enrichment of a sequence amplified from PFK genomic DNA was used as a basal control and was set to 1.0. Standard deviation was obtained from PCR triplicates.

Figure 2. Expression pattern of GIK transcripts and GIK protein.

(A–C) Expression of GIK mRNA in serial cross-sections of wild-type inflorescence meristems shown by in situ hybridization. (D–G) Expression of GIK mRNA in longitudinal sections of the wild-type inflorescence meristem (arrow), floral primordia, and developing reproductive organs. The numbers indicate stages of the floral buds . Arrow in D indicates the inflorescence meristem. (H–J) Expression of GIK mRNA in ag-1 inflorescence meristems, floral primordial, and developing flowers. (K–Q) Staining of wild-type Arabidopsis root cell nuclei with anti-GIK (K, O), DNA dye TOPRO-3 (L), or monoclonal anti-trimethylguanosine (TMG) (P). (M) and (Q) show merged images. Arrowheads in M indicate heterochromatin-rich chromocenters (seen as blue in the merged image). Arrow in Q indicates the nucleolus (seen as green in the merged image). Despite their largely co-localized patterns, anti-GIK staining was distinguished from that of TOPRO-3 by its lack of accumulation at the heterochromatic chromocenters (Figure 2M, arrowheads) and different from anti-TMG staining by showing no detectable expression in the nucleolus (Figure 2Q, arrow). Scale bars in A (for A–G) and H (for H–J) are 100 µm. Bars in K–Q, 5.0 µm.

Figure 3. Overexpression and loss of function of GIK cause reproductive defects.

(A–C) Flowers in 35S::GIK overexpression plants show carpels with ectopic stigmatic tissue (marked by triangles in A, B), short valves (marked by asterisk in B), and excessive growth of carpelloid tissue at the lateral side of the pistil with exposed ovules (C). (D) Sepal-sepal fusion (asterisk) observed in 35S::GIK ag-1 flowers. (E–G) Scanning electron microscopic images of wild-type Arabidopsis pistil (E) and flowers from DEX-treated 35S::GIK-GR-6HA plants show carpels with excessive stigma and ovules (arrowhead in F) and bipartite carpels with outgrowth of ovules and ectopic projections (asterisks) at the upper part of the pistil (G). (H, I) Flowers of gik insertion mutant ET14389 with indehiscent anthers of stamens (H) and branched stamens (I), and defective anther differentiation showing half-petal-half-stamen morphology (inset in I). In H, sepals and petals were removed to expose inner organs. (J, K) Similar reproductive defects were observed in the flowers of GIK RNAi silencing lines showing delayed dehiscence (arrowheads) (J), petalloid anthers (inset in J), defective stamen formation (arrow in K), and branched stamens (inset in K). Scale bar for A–D and H–K, 1 mm. Scale bars for E–G, 100 µm.

Figure 4. GIK negatively regulates ETT.

(A–H) GIK (A–D) and ETT (E–H) exhibit complementary expression patterns in reproductive organs at stages 7–8 (A, E), 8 (B, F), 9 (C, G), and 12 (D, H) as shown by in situ hybridization. At stages 7, 8, and 9, GIK is expressed at the adaxial side of the developing carpels and locules of developing stamens (A–C). In contrast, ETT is expressed at the abaxial sides of the carpels and in the vasculature of the stamens (E–G). At stage 12, GIK expression was mainly observed in the funiculus (f), outer integument (oi), and chalazal megaspore (cm) of ovules (D, inset), whereas ETT expression was in inner integuments (ii) and the nucellus (n) of the ovules (H, inset). (I–L) Comparison of ETT expression in the reproductive organs of wild-type (I and K) and 35S::GIK (J and L) plants by in situ hybridization on a single slide. (M) ETT expression in an ag-1 mutant flower. Scale bars in A (for A–H) and I (for I–M) are 100 µm. (N) Time-course of ETT expression after GIK activation, as measured by real-time PCR. Inflorescences from 35S::GIK-GR-6HA plants were harvested at 0, 4, 8, 16, and 24 h after mock treatment or a single DEX treatment. ETT expression was normalized to TUB RNA levels. Relative expression in DEX-treated samples was calibrated with mock-treated samples. (O) Expression analysis of ETT in the gik mutant using real-time PCR with RNA extracted from the inflorescences of wild-type and gik mutant ET14389 plants. Expression was normalized to TUB expression. Relative expression level in the wild-type was set to 1.0. Standard deviation was obtained from three independent biological samples in N and O.

Figure 5. GIK binds to putative MARs of ETT genomic DNA to modulate its expression.

(A) GIK is localized to the nuclear matrix. Nuclear matrix was isolated from the inflorescences of 35S::GIK-GR-6HA plants treated with DEX and harvested 4 h thereafter. Total nuclear and matrix proteins were subjected to western blot analysis. The membrane was first probed with anti-HA to detect GIK and then re-probed with anti-AG. (B) Schematic representation of SMARTest-predicted MARs in the ETT upstream genomic region. Arrow indicates the transcription start site. P1, P2, P3, and P4 are primer pairs used to detect different regions of the ETT genomic DNA used in the ChIP assay. (C) In vitro MAR binding assay of GIK. Left panels, Coomassie Blue staining of a gel loaded with non-induced E. coli containing the wild-type GIK AT-hook motif construct (noninduced), with an IPTG-induced culture containing the construct for the wild-type GIK AT-hook motif (GIK-AT), and with IPTG-induced culture containing a mutated construct in the conserved residues of the GIK AT-hook motif, changing Arg-Gly-Arg-Pro to Arg-Gly-Lys-Pro (GIK-MUT). Right panels, the corresponding south-western results of the MAR binding assay probed with an ETT MAR probe. (D) GIK binds to the MARs of the ETT promoter in vivo. Inflorescences from 35S::GIK-GR-6HA plants treated with DEX were harvested 4 h after DEX induction for ChIP experiments. Anti-HA was used for immunoprecipitation. Relative enrichment was obtained from the ratio of enrichment achieved by anti-HA to that of control IgG. Enrichment of a sequence amplified from the TUB locus was used as a basal control and set to 1.0. P1, P2, P3, and P4 are primer pairs used to detect different regions of the ETT genomic DNA (as illustrated in B). (E, F) Time-course promoter analysis of the ETT gene after GIK induction. 35S::GIK-GR-6HA transgenic plants were crossed with plants transgenic for promoter constructs of wild-type pETT::GUS (E) and pETTΔMAR::GUS with a deletion of distal MARs (F). The inflorescences were treated continuously with DEX every 2 d and harvested for GUS staining at 0, 1, 2, 3, and 4 d after the initial DEX treatment. Upper panels, schematic representations of the ETT upstream genomic region fused with a GUS reporter gene. Lower panels, GUS-stained inflorescences at 0, 1, 2, 3, and 4 d after the initial GIK induction. (G) Time-course analysis of dimethylated-H3K9 level associated with the ETT genomic DNA in 35S::GIK-GR-6HA inflorescences at 0, 2, 4, and 8 h after a single GIK induction. ChIP was performed using anti-dimethylated H3K9 (Upstate). Primer pairs P1, P2, P3, and P4 are shown in Figure 5B. Relative enrichment was obtained from the ratio of bound/input achieved in the respective time points to that at 0 h. The bound/input ratio was first normalized with the bound/input ratio of a basal control, PFK, the transcription of which is not affected by GIK. The enrichment at 0 h was set as 1.0. Standard deviation was obtained from PCR triplicates in D and G.

Figure 6. GIK regulates multiple reproductive regulators.

(A–D) Time-course expression analysis of CRC (A), JAG (B), KNU (C), and LUG (D) transcripts upon GIK activation. Inflorescences from 35S::GIK-GR-6HA plants were harvested at 0, 4, 8, 16, and 24 h after a single DEX treatment for quantitative real-time PCR. Target gene expression was normalized to TUB. Relative expression in DEX-treated samples was calibrated with mock-treated samples. Standard deviation was obtained from three independent biological samples. The differences between 0 h and 8 h were statistically analyzed using paired student's t-test. *p<0.05 in (A), (B), and (C). p>0.1 in (D). (E–G) GIK binds the upstream MAR regions of CRC (E), JAG (F), and KNU (G) genomic DNA in vivo. Schematic representations of genomic regions of these genes are shown with demarcated SMARTest-predicted MAR regions. Primer sets used for quantitative PCR are shown below each graph. Arrows indicate transcription start sites. Relative enrichment was obtained from the ratio of enrichment achieved by anti-HA to that of control IgG. The enrichment value obtained from a sequence amplified from the TUB locus is shown as a control and set to 1.0. Standard deviation was obtained from PCR triplicates. The differences between the control and the primer pairs showing the highest enrichment were statistically analyzed using student's t-test. **p<0.1 in (E), ***p<0.05 in (F) and (G). (H) Expression analysis of CRC, JAG, KNU, and LUG in the gik mutant using real-time PCR as in Figure 4O.

Figure 7. Summary diagram of GIK regulation and function.

The MAR binding protein GIK is directly regulated by the floral homeotic protein AG during reproductive development. GIK modulates and refines the expression of ETT and CRC to control reproductive patterning and JAG and KNU for reproductive differentiation. GIK functions as a multifunctional determinant to coordinate gene expression during reproductive development.