Coordinated regulation of apical hook development by gibberellins and ethylene in etiolated Arabidopsis seedlings

Abstract

Dark-grown Arabidopsis seedlings develop an apical hook when germinating in soil, which protects the cotyledons and apical meristematic tissues when protruding through the soil. Several hormones are reported to distinctly modulate this process. Previous studies have shown that ethylene and gibberellins (GAs) coordinately regulate the hook development, although the underlying molecular mechanism is largely unknown. Here we showed that GA(3) enhanced while paclobutrazol repressed ethylene- and EIN3-overexpression (EIN3ox)-induced hook curvature, and della mutant exhibited exaggerated hook curvature, which required an intact ethylene signaling pathway. Genetic study revealed that GA-enhanced hook development was dependent on HOOKLESS 1 (HLS1), a central regulator mediating the input of the multiple signaling pathways during apical hook development. We further found that GA(3) induced (and DELLA proteins repressed) HLS1 expression in an ETHYLENE INSENSITIVE 3/EIN3-LIKE 1 (EIN3/EIL1)-dependent manner, whereby EIN3/EIL1 activated HLS1 transcription by directly binding to its promoter. Additionally, DELLA proteins were found to interact with the DNA-binding domains of EIN3/EIL1 and repress EIN3/EIL1-regulated HLS1 expression. Treatment with naphthylphthalamic acid, a polar auxin transport inhibitor, repressed the constitutively exaggerated hook curvature of EIN3ox line and della mutant, supporting that auxin functions downstream of the ethylene and GA pathways in hook development. Taken together, our results identify EIN3/EIL1 as a new class of DELLA-associated transcription factors and demonstrate that GA promotes apical hook formation in cooperation with ethylene partly by inducing the expression of HLS1 via derepression of EIN3/EIL1 functions.

Figure 1

PAC represses while GA₃ enhances the ethylene-induced hook curvature. (A) The hook phenotype of 3-day-old (top) and 6-day-old (bottom) etiolated wild-type seedlings grown on the indicated medium (1 μM PAC, 10 μM ACC, and/or 10 μM GA₃). The scale bars represent 1 mm. (B) The hook phenotype of 6-day-old etiolated seedlings of EIN3ox grown on the indicated medium (1 μM PAC and/or 10 μM GA₃). (C) The hook phenotype of 3-day-old etiolated seedlings of indicated genotypes grown on MS medium. (D) The hook phenotype of etiolated ethylene-related mutants and transgenic plants grown on MS medium supplemented with or without 1 μM PAC for 3 days.

Figure 2

The della mutant exhibits exaggerated hook curvature that can be suppressed by ein2 or ein3 eil1. (A) The exaggerated hook curvatures of della (with all the five DELLA-mutated) and penta (ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1) were not affected by AVG (ethylene biosynthesis inhibitor) treatment. Etiolated seedlings were grown on MS medium supplemented with or without 10 μM AVG for 3 days. (B) ein2 or ein3 eil1 suppressed the exaggerated hook curvature of della. Etiolated seedlings of indicated genotypes were grown on MS medium for 3 days.

Figure 3

HLS1 is required for GA- and ethylene-induced hook curvature. (A) GA₃ and/or ACC treatment did not affect the hookless phenotype of hls1. Hooks of 3-day-old etiolated seedlings grown on the indicated medium were shown. (B) hls1 prevented the hook curvature of della. Hooks of 3-day-old etiolated seedlings grown MS medium were shown.

Figure 4

The GA_3⁻ and ethylene-induced HLS1 expression is EIN3/EIL1 dependent. (A) Quantitative reverse transcription PCR (qRT-PCR) analysis of the GA₃-induced HLS1 expression in wild-type and ein3 eil1 seedlings. Seeds germinating in dark for 24 h were transferred to 0.2 μM PAC for 48 h and then treated with 100 μM GA₃ for indicated hours prior to RNA extraction for qRT-PCR. (B) qRT-PCR analysis of the ethylene-induced HLS1 expression in ein3 eil1 and EIN3ox/ein3 eil1 (overexpression of EIN3 in ein3 eil1) seedlings. 3-day-old etiolated seedlings grown on MS medium were treated with or without 100 μM ACC for 4 h. (C) qRT-PCR analysis of HLS1 expression in the indicated genotypes. 3-day-old etiolated seedlings grown on MS medium were used for RNA extraction. The HLS1 expression levels in A, B, and C were normalized with the levels of β-tubulin. Mean ± SD, n =3. All experiments were repeated at least twice with similar results.

Figure 5

HLS1 is a direct target gene of EIN3. (A) Rapid induction of HLS1 expression in iE/ein3 eil1 by estradiol. 3-day-old etiolated iE/ein3 eil1 (a transgenic plant inducibly expressing EIN3-FLAG in the ein3 eil1 background) seedlings grown on MS medium were treated with 10 μM estradiol for indicated hours before the seedlings were collected for RNA extraction. Experiments were repeated three times with similar results. (B) Oligonucleotides used in the EMSA assays. The HLS1 probe contains an EBS motif (underlined), which is a putative EBS. In the mHLS1 probe, the EBS was mutated to mEBS (underlined). Number showed the location upstream of the start codon. (C) EMSA results showing in vitro binding of GST-EIN3 to the EBS motif in the promoter of HLS1. Protein-DNA complexes were detected when GST-EIN3 was incubated with labeled HLS1 probe, and competition assays were conducted by adding 200-fold excessive unlabeled HLS1 or mHLS1 probe. GST-EIN3 was also incubated with labeled mHLS1 probe and no protein-DNA complexes were observed. (D) ChIP assays indicating in vivo binding of EIN3 to the HLS1 promoter sequence. Chromatin from ein3 eil1 and transgenic plants constitutively expressing EIN3-FLAG in ein3 eil1 was immunoprecipitated with an anti-FLAG antibody, and the quantification of the indicated DNA fragments in the precipitated chromatin was determined by quantitative real-time PCR. The amounts of DNA amplified from the EIN3-FLAG/ein3 eil1 seedlings were normalized to that from ein3 eil1 plants. The 3′-UTR fragment of HLS1 was used as a negative control. The experiment was repeated three times with similar results.

Figure 6

RGA/GAI interacts with EIN3/EIL1 and inhibits their function. (A) RGA/GAI interacted with EIN3/EIL1 in yeast cells. RGA M5 fragment (aa 209-587) and GAI M5 fragment (aa 158-533) were used in the yeast two-hybrid assays. EBF1/EIN3 interaction was used as a positive control. (B) EIN3 fragment (aa 200-500) was sufficient for the interactions with RGA and GAI in yeast cells. (C) Co-IP assays showing RGA/GAI association with EIN3 in vivo. EIN3-FLAG/ein3 eil1 (EIN3-F) was crossed with 35S:TAP-RGAΔ17 (ΔRGA) or 35S:TAP-GAIΔ17 (ΔGAI), and F1 plants were used for Co-IP. Proteins were immunoprecipitated with anti-FLAG M2 agarose beads (+Ab) and detected with either anti-MYC or anti-FLAG antibodies. (D) PAC treatment inhibited the ACC-induced 5×EBS:GUS expression. 3-day-old etiolated 5×EBS:GUS seedlings grown on the indicated medium (ACC: 10 μM, PAC: 1 μM, and GA: 10 μM) were used for GUS staining. (E) Expression of 35S:GA2ox8 in the 5×EBS:GUS background decreased the ACC-induced GUS staining. 3-day-old etiolated 5×EBS-GUS and 35S:GA2ox8 5×EBS-GUS seedlings grown on 10 μM ACC medium were used for GUS staining. (F) GA₃ treatment did not affect the protein level of EIN3 in EIN3-FLAG/ein3 eil1. 3-day-old etiolated seedlings grown on 0.2 μM PAC were treated with indicated concentrations of GA₃ for 4 h before proteins were extracted for immunoblot assay with anti-FLAG antibody.

Figure 7

Blocking of auxin transport suppresses GA- and ethylene-induced hook curvature. (A) The hook phenotype of wild-type and 35S:MYC-HLS1/hls1grown on MS medium supplemented with or without 0.1 μM PAC for 3 days. (B) The hook phenotype of 35S:MYC-HLS1/hls1 grown on MS medium supplemented with or without 10 μM ACC and/or 10 μM GA₃ for 3 days. (C) The hook phenotype of 3-day-old etiolated seedlings of indicated genotypes. Seedlings grown on MS medium and 1 μM NPA medium were shown.

Figure 8

A proposed model of GAs and ethylene co-action in promoting the hook curvature. GAs and ethylene cooperatively regulate the hook curvature partly by inducing the gene expression of HLS1, a putative N-acetyltransferase essential for hook formation. EIN3/EIL1 are the integration node linking the two hormone pathways to directly activate the HLS1 transcription, in which ethylene stabilizes EIN3/EIL1, while GAs relieve the repression of DELLA proteins on EIN3/EIL1. Meanwhile, GAs and ethylene also initiate the HLS1-independent pathways to regulate hook curvature, probably by modulating asymmetric auxin accumulation in the hook region. The solid lines indicate proved regulations, whereas the dotted lines indicate proposed regulations.