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. 2011 Feb 1;108(5):2160-5.
doi: 10.1073/pnas.1012232108. Epub 2011 Jan 18.

Scarecrow-like 3 promotes gibberellin signaling by antagonizing master growth repressor DELLA in Arabidopsis

Zhong-Lin Zhang  1 , Mikihiro Ogawa, Christine M Fleet, Rodolfo Zentella, Jianhong Hu, Jung-Ok Heo, Jun Lim, Yuji Kamiya, Shinjiro Yamaguchi, Tai-ping Sun
Affiliations

Scarecrow-like 3 promotes gibberellin signaling by antagonizing master growth repressor DELLA in Arabidopsis

Zhong-Lin Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

The diterpenoid phytohormone gibberellin (GA) controls diverse developmental processes throughout the plant life cycle. DELLA proteins are master growth repressors that function immediately downstream of the GA receptor to inhibit GA signaling. By doing so, DELLAs also play pivotal roles as integrators of internal developmental signals from multiple hormone pathways and external cues. DELLAs are likely nuclear transcriptional regulators, which interact with other transcription factors to modulate expression of GA-responsive genes. DELLAs are also involved in maintaining GA homeostasis through feedback up-regulating expression of GA biosynthesis and receptor genes. However, the molecular mechanisms by which DELLAs restrict growth and development are largely unknown. This study reveals an important step of the mechanism. Previous microarray studies identified scarecrow-like 3 (SCL3) as a direct target gene of DELLA in Arabidopsis seedlings. SCL3 expression is induced by DELLA and repressed by GA. Unexpectedly, a scl3 null mutant displays reduced GA responses and elevated expression of GA biosynthesis genes during seed germination and seedling growth, indicating that SCL3 functions as a positive regulator of GA signaling. SCL3 seems to act as an attenuator of DELLA proteins. Transient expression, ChIP, and co-IP studies show that SCL3 autoregulates its own transcription by directly interacting with DELLA. Our data further show that SCL3 and DELLA antagonize each other in controlling both downstream GA responses and upstream GA biosynthetic genes. This work is beginning to shed light on how this complex regulatory network achieves GA homeostasis and controls GA-mediated growth and development in the plant.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Phenotypes of the scl3 mutant and SCL3 overexpression line. (A) Germination assay. Seed coat rupture after 8 d was scored as germination. (B) Hypocotyl elongation assay of etiolated seedlings in response to PAC at day 6. (C) Root elongation assay at day 10. (D) Hypocotyl elongation assay of light-grown seedlings in response to 1 μM GA4 at day 4. (E) Rosettes of ga1-3 and the ga1-3 scl3 double homozygous mutant at 45 d old on soil. (F) The major GA biosynthesis and catabolism pathways in Arabidopsis. GGDP, geranylgeranyl diphosphate. Enzyme for each step is listed above each arrow. CPS, ent-copalyl diphosphate synthase; KS, ent-kaurene synthase; KO, ent-kaurene oxidase; KAO, ent-kaurenoic acid oxidase; 20ox, GA20-oxidase; 3ox, GA3-oxidase; 2ox, GA2-oxidase. GA4 is the major active GA in Arabidopsis. (G) Relative transcript levels in 8-d-old seedlings. (Left) ga1-3 and ga1-3 scl3; (Right) Col-0 and scl3. Data represent the average of three qRT-PCR measurements ± SE. The housekeeping gene GAPC (glyceraldehyde-3-phosphate dehydrogenase C subunit), whose expression is not responsive to GA (12), was used to normalize different samples. The mRNA level of each corresponding gene in ga1-3 (for GA biosynthetic genes) or Col-0 (for GA catabolic genes) was arbitrarily set to 1. *P < 0.05; **P < 0.01.
Fig. 2.
Fig. 2.
Interactions between SCL3, RGA, and SPY. (A) rga and spy are epistatic to scl3 in the root-length assays in response to 1 μM PAC (10-d-old seedlings). (B) rga is partially epistatic to scl3 in the ga1-3 background (65-d-old plants) (Fig. S2B). (C) In vitro pull-down of RGA with recombinant GST-SCL3. Protein extracts from sly1-10 and sly1-10 rga-24 double mutant were incubated with GST or GST-SCL3 from Escherichia coli separately. − GA, in the absence of GA; + GA, in the presence of 100 μM GA4. Input and pull-down samples were analyzed by immunoblotting using affinity-purified RGA antibody. (D) co-IP of SCL3 and RGA in planta. HA-RGA was transiently expressed alone (RGA only) or coexpressed with cMyc-SCL3 (SCL3/RGA) or cMyc-GUS (GUS/RGA) in N. benthamiana. The nuclear protein extracts were immunoprecipitated with cMyc antibody-conjugated agarose beads, and the input and IP samples were analyzed by immunoblotting using antibodies for cMyc and HA, separately.
Fig. 3.
Fig. 3.
SCL3 interferes with RGA to regulate the expression of the SCL3 promoter. (A) Schematics of 35S:SCL3 in the SCL3-OE line and the endogenous SCL3 locus. The triangle symbol indicates the T-DNA insertion site in scl3. Primers P2 F+R only amplify the endogenous SCL3 transcripts, whereas primers P1 F+R amplify SCL3 transcripts produced by both the transgene and endogenous SCL3 locus. (B) Total SCL3 transcript levels in Col-0, scl3, and SCL3-OE lines. qRT-PCR analysis was performed using primers P1 F+R. (C) WT or truncated endogenous SCL3 transcripts in Col-0, scl3, and SCL3-OE lines. qRT-PCR analysis was performed using primers P2 F+R. In B and C, data represent the average of three measurements ± SE. A GA nonresponsive gene (At4g33380) (7) was used to normalize different samples. The amount of SCL3 mRNA in Col-0 was set to 1. (D) SCL3 repressed its own promoter expression. (E) SCL3 antagonized RGA-induced SCL3 promoter expression in the transient coexpression assays. In D and E, the reporter construct (PSCL3:fLUC) contains 2 kb SCL3 promoter plus the 35S minimal promoter (−45- to 1-bp region that includes the TATA box) (49) fused to fLUC. 35S:Renilla LUC (rLUC) served as an internal control for normalization of transformation efficiency. Effector constructs were 35S:RGA and/or 35S:SCL3, and the empty vector was used as a negative control. PSCL3:fLUC and 35S:rLUC constructs were cobombarded into 11-d-old triple-mutant ga1-3 rga-28 scl3 seedlings with various combinations of effector constructs using the same molar ratio. The relative fLUC activity of the empty effector control was set to 1. Data represent average value ± SE of 14 replicates from three independent experiments. Pair-wise t tests were performed. (D) **P < 0.01. (E) When two samples show different letters (a–c) above the bars, the difference between them is significant (a–c and b–c, P < 0.01; a–b, P < 0.05). Another reporter construct containing a 1-kb SCL3 promoter with its native TATA box fused to fLUC rendered similar results (Fig. S3).
Fig. 4.
Fig. 4.
Interaction of SCL3-GFP with the SCL3 promoter in vivo and a model for SCL3–DELLA interaction. (A) Schematics of the transgene PSCL3 (2.5 kb) :SCL3-GFP and the endogenous SCL3 locus. The T-DNA insertion site in scl3 is indicated by the triangle symbol. The regions tested in the ChIP-qPCR assay are indicated underneath the SCL3 genomic DNA structure. (B) SCL3 promoter scanning by ChIP-qPCR. Chromatin isolated from scl3 or scl3 PSCL3:SCL3-GFP seedlings was immunoprecipitated using anti-GFP antibody and followed by qPCR. The 18S rRNA gene was used to normalize the qPCR results in each ChIP sample. Fold enrichment of each region in the scl3 PSCL3:SCL3-GFP line was calculated by comparing with the control scl3 and then, was normalized to the copy numbers of each corresponding region (one copy for region +1,307 to +1,646 and three copies for the rest of the regions) (Fig. S4A). The normalized values of fold enrichment are the average ± SE of three qPCR reactions from one ChIP experiment. Similar results were obtained in an independent ChIP experiment. **P < 0.01 (t tests). The numbers underneath each bar indicate base pairs upstream of the ATG of the SCL3 gene. The plus symbol indicates base pairs downstream of the ATG. (C) A model for antagonistic interaction between SCL3 and DELLA in regulating upstream GA accumulation and downstream GA responses. In the root, SCL3–DELLA interaction coordinates the GA signaling activity with the developmental program controlled by the SCR/SHR pathway. Activation or inhibition could be through different modes of action. PD, protein degradation (magenta line); PPI, protein–protein interaction (orange lines); TC, transcriptional regulation (purple lines). The asterisk indicates that, in addition to PD, proteolysis-independent inactivation of DELLA by GID1 binding (PPI) has been shown to occur in the GID1 overexpression Arabidopsis line (50).
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