Ethylene regulates arabidopsis development via the modulation of DELLA protein growth repressor function

Abstract

Phytohormones regulate plant development via a poorly understood signal response network. Here, we show that the phytohormone ethylene regulates plant development at least in part via alteration of the properties of DELLA protein nuclear growth repressors, a family of proteins first identified as gibberellin (GA) signaling components. This conclusion is based on the following experimental observations. First, ethylene inhibited Arabidopsis root growth in a DELLA-dependent manner. Second, ethylene delayed the GA-induced disappearance of the DELLA protein repressor of ga1-3 from root cell nuclei via a constitutive triple response-dependent signaling pathway. Third, the ethylene-promoted "apical hook" structure of etiolated seedling hypocotyls was dependent on the relief of DELLA-mediated growth restraint. Ethylene, auxin, and GA responses now can be attributed to effects on DELLA function, suggesting that DELLA plays a key integrative role in the phytohormone signal response network.

Figure 1.

Ethylene Inhibits Root Elongation via Its Effects on GAI and RGA. (A) Representative 7-day-old primary seedling roots of wild-type (WT; Landsberg erecta), gai-t6, rga-24, and gai-t6 rga-24 plants grown in the presence (+) or absence of 10⁻⁷ M ACC. Mean lengths were determined (±se; n ≥ 20) as follows: wild type, 22.3 ± 1.4 mm; gai-t6, 21.7 ± 1.9 mm; rga-24, 22 ± 0.7 mm; gai-t6 rga-24, 22.2 ± 1.5 mm; wild type + ACC, 11.4 ± 1.6 mm; gai-t6 + ACC, 12.7 ± 1.9 mm; rga-24 + ACC, 15.6 ± 1.6 mm; and gai-t6 rga-24 + ACC, 16.6 ± 1.3 mm. (B) Dose responses of wild-type (closed circles), gai-t6 (open circles), rga-24 (closed inverted triangles), and gai-t6 rga-24 (open inverted triangles) 7-day-old plants to ACC. The data represent the mean root length (±se; n ≥ 20) expressed as a percentage of root length without ACC.

Figure 2.

Ethylene Inhibits Root Growth by Modulating GA Responses. Mean length (±se; n ≥ 20) of 7-day-old primary seedling roots of wild-type plants grown in the absence (H₂O) or presence of 0.1 μM ACC (+ACC), 0.1 μM ACC plus 1 μM GA (+ACC +GA), or 1 μM GA (+GA).

Figure 3.

Ethylene Delays the GA-Mediated Disappearance of GFP-RGA from Root Cell Nuclei. (A) GFP fluorescence of GA-treated (10 μM) primary roots of 5-day-old pRGA:GFP-RGA seedlings grown in the presence or absence of ethylene gas (10 ppm). EZ, elongation zone of the root. (B) Quantitative reverse transcriptase–mediated PCR comparison of GFP-RGA and RGA transcript levels in roots of 5-day-old seedlings grown in the presence or absence of 1 μM ACC. WT, wild type. (C) Immunoblot analysis of total proteins (20 μg) from roots of 5-day-old seedlings grown in the presence (+) or absence of ACC. GA treatment times are indicated. Mouse anti-GFP antiserum and a peroxidase-conjugated goat anti-mouse IgG were used as primary and secondary antibodies, respectively. The bottom gel shows Ponceau red staining of proteins after blotting to the membrane (loading control).

Figure 4.

Ethylene Delays the GA-Induced Disappearance of GFP-RGA via a CTR1-Dependent Pathway. (A) Cocultivation of pRGA:GFP-RGA seedlings with A. tumefaciens containing RNAi-CTR1 causes a decrease in CTR1 transcript levels, whereas cocultivation with A. tumefaciens containing the vector only does not. UBQ10 transcript was used as an RNA sample control. M, DNA size markers. (B) Silencing of CTR1 affects the GA-mediated disappearance of GFP-RGA. The GFP fluorescence of A. tumefaciens–infected pRGA:GFP-RGA primary roots after GA treatment (10 μM) is shown. EZ, elongation zone of the root. (C) GFP fluorescence of GA-treated (10 μM) ctr1-1 pRGA:GFP-RGA and pRGA:GFP-RGA primary seedling roots.

Figure 5.

Apical Hook Maintenance Is GA Dependent. (A) GA regulates apical hook maintenance via DELLA proteins. Photographs show 3-day-old seedling apices of wild-type Landsberg erecta (Ler), ga1-3, gai-t6 ga1-3, rga-24 ga1-3, and gai-t6 rga-24 ga1-3 plants grown in darkness. (B) Time-staged photographs of Ler, ga1-3, and gai-t6 rga-24 ga1-3 etiolated seedlings. Photographs were taken at 32, 48, 56, and 72 h after imbibition.

Figure 6.

DELLA Proteins Act Downstream of Ethylene and Auxin Signaling Pathways. Effects of GA, PAC, ACC, and NPA on apical hook morphology of wild-type Landsberg erecta (Ler) and Columbia (Col; control for ctr1-1), gai, ga1-3, gai-t6 rga-24 ga1-3, and ctr1-1 plants. Photographs show 3-day-old seedlings grown in darkness on 1 μM GA, 1 μM PAC, 10 μM ACC, 10 μM ACC + 1 μM GA, or 1 μM NPA.

Figure 7.

DELLA Proteins Integrate the Effects on Growth of the Ethylene, Auxin, and GA Signaling Pathways. Scheme of growth regulation via the ethylene (ETR-CTR1) and auxin signaling pathways, showing how DELLA proteins integrate information from both pathways and act as regulators of growth. The DELLA proteins restrain growth, and growth is relieved of restraint by GA-promoted DELLA protein degradation. Ethylene (perhaps via an auxin-dependent pathway) and auxin (via an ethylene-independent pathway) both alter the abundance/stability of DELLA.