A loss-of-function mutation in the nucleoporin AtNUP160 indicates that normal auxin signalling is required for a proper ethylene response in Arabidopsis

Abstract

As part of a continuing effort to elucidate mechanisms that regulate the magnitude of ethylene signalling, an Arabidopsis mutant with an enhanced ethylene response was identified. Subsequent characterization of this loss-of-function mutant revealed severe hypocotyl shortening in the presence of saturating ethylene along with increased expression in leaves of a subset of ethylene-responsive genes. It was subsequently determined by map-based cloning that the mutant (sar1-7) represents a loss-of-function mutation in the previously described nucleoporin AtNUP160 (At1g33410, SAR1). In support of previously reported results, the sar1-7 mutant partially restored auxin responsiveness to roots of an rce1 loss-of-function mutant, indicating that AtNUP160/SAR1 is required for proper expression of factors responsible for the repression of auxin signalling. Analysis of arf7-1/sar1-7 and arf19-1/sar1-7 double mutants revealed that mutations affecting either ARF7 or ARF19 function almost fully blocked manifestation of the sar1-7-dependent ethylene hypersensitivity phenotype, suggesting that ARF7- and ARF19-mediated auxin signalling is responsible for regulating the magnitude of and/or competence for the ethylene response in Arabidopsis etiolated hypocotyls. Consistent with this, addition of auxin to ethylene-treated seedlings resulted in severe hypocotyl shortening, reminiscent of that seen for other eer (enhanced ethylene response) mutants, suggesting that auxin functions in part synergistically with ethylene to control hypocotyl elongation and other ethylene-dependent phenomena.

Fig. 1.

Response of Ws wt and mutant etiolated seedlings to ethylene. (A) For hypocotyl responsiveness, seedlings were grown for 4 d in the dark with 5 μM AgNO₃ (triangles) or 5 μM 2-aminoethoxyvinyl glycine (AVG) (circles), with the latter supplemented with increasing concentrations of ethylene ranging from 0 to 100 μl l⁻¹. (B) Relative hypocotyl length (length/length at 5 μM AgNO₃), with the ethylene concentration giving 50% inhibition denoted by a dashed line. The inset shows 4-d-old dark-grown seedlings of Ws wt and mutant exposed to 100 μl l⁻¹ ethylene. (C) The ratio of mutant to Ws wt hypocotyl length for each ethylene concentration tested, with the dashed line representing the predicted ratio if the mutant was not ethylene hyper-responsive. (D, E) For root responsiveness, Ws wt and mutant seedlings were grown in the dark for 4 d in the presence of air or increasing concentrations of ethylene. Actual root length is shown in (D), whereas (E) shows relative root length (length/length in air), with the ethylene concentration giving 50% inhibition denoted by a dashed line. (F) Ethylene biosynthetic rates for 4-d-old dark-grown seedlings of Ws wt and mutant. Ethylene was measured using a gas chromatography system with production rates calculated based on tissue fresh weight. Mean ±SE values were determined from five samples of 100 seedlings each.

Fig. 2.

Ethylene-responsive gene expression in mutant leaves. (A) Four-week-old adult plants of Ws wt and the mutant were exposed to air (A) or 100 μl l⁻¹ ethylene (E) for 24 h, after which Northern blot analysis was performed to test for expression of ethylene-inducible genes. (B) As several genes are coordinately regulated by both ethylene and jasmonic acid, gene expression was also determined following exposure of leaves to air (A), 100 μM (+/–)-jasmonic acid (J) or 100 μM jasmonic acid with 100 μl l⁻¹ ethylene (JE).

Fig. 3.

A loss-of-function mutation in the nucleoporin-encoding gene AtNUP160/SAR1 causes an enhanced ethylene response. (A) Map-based cloning of the sar1-7 mutation. Solid bars represent the order of bacterial artificial chromosomes in the 11.8–12.2 Mb region of chromosome 1. The bacterial artificial chromosome containing AtNUP160/SAR1 and recombination frequencies at each CAPS marker surrounding sar1-7 are shown. The genomic structure of AtNUP160/SAR1 is illustrated either as filled boxes, which represent exons, or as intervening lines, which represent non-coding regions. The position of the sar1-7 mutation, which represents an inappropriate stop codon in exon 16, is shown. (B) Ws wt, sar1-7, and T2 progeny from sar1-7 transformed with the wt AtNUP160/SAR1 genomic construct were tested for an ethylene response after 4 d growth in the dark in the presence of 100 μl l⁻¹ ethylene. (C) The mutant sar1-6 (Col-0 background), with a T-DNA insertion in the first exon of AtNUP160/SAR1, was tested for an ethylene response following growth in the dark for 4 d in the presence of 100 μl l⁻¹ ethylene. (D) For Northern blot analysis of AtNUP160/SAR1 expression in etiolated seedlings, total RNA was isolated from dark-grown Ws wt and sar1-7 seedlings treated with 5 μM AgNO₃ or 100 μl l⁻¹ ethylene for 4 d. (E) Seedlings were grown in the light in the absence or presence of 0.1 μM 2,4-D for 7 d after which root length was measured.

Fig. 4.

Analysis of double mutants between sar1-7 and known ethylene-insensitive mutants. (A) Seedlings of Col-0 wt, Ws wt, sar1-7, ein2-5, and ein2-5/sar1-7 were grown in the dark in the presence of 5 μM AgNO₃ or 100 μl l⁻¹ ethylene for 4 d, after which hypocotyl length was determined. (B) Seedlings of Col-0 wt, Ws wt, sar1-7, ein3-1, and ein3-1/sar1-7 were grown in the dark in the presence of 5 μM AgNO₃ or 100 μl l⁻¹ ethylene for 4 d, after which hypocotyl length was assessed.

Fig. 5.

The ethylene hyper-response phenotype of sar1-7 requires functional ARF7 and ARF19. (A) An arf7-1/sar1-7 double mutant was generated and analysed for its response to ethylene by growth for 4 d in the dark in the presence of 5 μM AgNO₃ or 100 μl l⁻¹ ethylene, after which hypocotyl length was measured. (B) An arf19-1/sar1-7 double mutant was generated and analysed for ethylene responsiveness following growth for 4 d in the dark in the presence or absence of 5 μM AgNO₃ or 100 μl l⁻¹ ethylene, after which hypocotyl length was measured. (C) Photograph showing the ethylene responsiveness of the analysed mutants. (D) Leaves of arf7-1/arf19-2 have reduced ethylene-dependent expression of chiB and PDF1.2. For this analysis, 4-week-old adult plants were exposed to air (A) or 100 μl l⁻¹ ethylene (E) for 24 h, after which Northern blot analysis was performed for chiB and PDF1.2.

Fig. 6.

IAA3 is required for a normal response to ethylene. (A) Seedlings of Ler and shy2-31, which is a loss-of-function mutation affecting IAA3, were grown in the presence of 5 μM AgNO₃ or 100 μl l⁻¹ ethylene, after which hypocotyl length was measured. (B) Manifestation of the ethylene response in dark-grown seedlings of shy2-2, which represents an amino acid change that prevents auxin-dependent degradation of the repressor of auxin response IAA3, was assessed following growth for 4 d in the presence of 5 μM AgNO₃ or 100 μl l⁻¹ ethylene. (C) For the yeast two-hybrid assay, the full coding sequence of IAA3 fused to the DNA-binding domain of pLEX-NLS was tested for an interaction with the full coding sequence of ARF7 fused to the GAL4 activation domain in pACTII. The inset shows an in vitro pull-down assay in which in vitro-translated ARF7 radiolabelled with [³⁵S]methionine was tested for its ability to interact with bacterially produced MBP fused to IAA3.

Fig. 7.

Auxin signalling controls the magnitude of and competence for the ethylene response. (A–C) Wild-type seedlings were grown in the dark for 4 d with 5 μM AgNO₃ or 100 μl l⁻¹ ethylene supplemented with 0, 50 (A,), or 500 nM (B, C) IAA, after which hypocotyl length was determined for each treatment. (D) Col-0 wt seedlings were grown for 7 d hydroponically, after which all samples were treated with 10 μM (+/–)-JA for 24 h either in the absence (A) or presence (E) of 10 μM ACC. As part of this treatment, sample pairs (i.e. control and +ACC) were additionally supplemented with 1 μM IAA for the final 8 h of the ACC treatment and compared with a control pair that was treated with ethanol only. Following this, whole seedlings were collected and total RNA was isolated for Northern blot analysis for PDF1.2. (E) Wild-type seedlings were grown for 4 d in the dark with 100 μl l⁻¹ ethylene supplemented with either 0 or 50 μM PCIB, an auxin-response inhibitor. (F) Wild-type and sar1-7 etiolated seedings were grown for 4 d in the presence of 100 μl l⁻¹ ethylene supplemented with 0, 5, or 10 μM PCIB after which hypocotyl length was measured.