Ethylene Signaling Influences Light-Regulated Development in Pea

Abstract

Plant responses to light involve a complex network of interactions among multiple plant hormones. In a screen for mutants showing altered photomorphogenesis under red light, we identified a mutant with dramatically enhanced leaf expansion and delayed petal senescence. We show that this mutant exhibits reduced sensitivity to ethylene and carries a nonsense mutation in the single pea (Pisum sativum) ortholog of the ethylene signaling gene ETHYLENE INSENSITIVE2 (EIN2). Consistent with this observation, the ein2 mutation rescues the previously described effects of ethylene overproduction in mature phytochrome-deficient plants. In seedlings, ein2 confers a marked increase in leaf expansion under monochromatic red, far-red, or blue light, and interaction with phytochromeA, phytochromeB, and long1 mutants confirms that ein2 enhances both phytochrome- and cryptochrome-dependent responses in a LONG1-dependent manner. In contrast, minimal effects of ein2 on seedling development in darkness or high-irradiance white light show that ethylene is not limiting for development under these conditions. These results indicate that ethylene signaling constrains leaf expansion during deetiolation in pea and provide further evidence that down-regulation of ethylene production may be an important component mechanism in the broader control of photomorphogenic development by phytochrome and cryptochrome.

Figure 1.

Isolation of a unique mutant showing light-hypersensitive leaflet expansion and impaired petal senescence. A, Two-week-old seedlings grown under continuous red light at 15 µmol m⁻² s⁻¹ (left) or in complete darkness (right). The constitutively photomorphogenic lip1 mutant and the light hypersensitive phyA-3D mutant are shown for comparison. B, Flower senescence and pod growth. Plants were grown in the greenhouse under an 18-h extended natural photoperiod. WT, Wild type.

Figure 2.

AF145 mutant seedlings are insensitive to ethylene. Wild-type (WT) and AF145 seeds were treated with 25 µg of ethylene-releasing compound ethephon and germinated in darkness for 7 d. Values represent means ± se for n = 16 to 20.

Figure 3.

AF145 is an EIN2 mutant. A, Location of the AF145 locus and the PsEIN2 gene in pea linkage group V. B, Phylogenetic relationships of EIN2 genes in legumes. Details of all sequences and the corresponding alignment are presented in Supplemental Figure S3. C, Diagram showing the structure of PsEIN2 and MtEIN2 genes and the location of the ein2 G to A (G/A) mutation. Boxes and connecting lines represent exons and introns, respectively. Exon regions shaded gray correspond to those encoding the cleaved C-terminal fragment of AtEIN2 (Qiao et al., 2012). Cs, Cucumis sativus; FTc; Fv, Fragaria vesca.

Figure 4.

The ein2 mutation overcomes phenotypes associated with ethylene overproduction in the phyA phyB double mutant. A, Effect of the ein2 mutation on internode lengths in wild-type (WT) and phyA, phyB, and phyA phyB mutant backgrounds. Values represent means ± se for n = 8 to 12. B, Shoot apex of 4-week-old plants. C, Representative leaf from node 5 of a 4-week-old plant. Plants were grown in the greenhouse under a natural daylight photoperiod extended to 18 h.

Figure 5.

The ein2 mutation enhances leaflet expansion under multiple wavelengths of monochromatic light. Wild-type (WT) and ein2 seedlings were grown from sowing for 12 d under monochromatic far-red, red, or blue light (all 15 µmol m⁻² s⁻¹) or in complete darkness. Leaflet area was estimated as the product of length and width of the larger leaflet from the first true foliage leaf (node 3). Internode length was measured as the length between nodes 1 and 3. Values represent means ± se for n = 8 to 12.

Figure 6.

The ein2 mutation enhances the response to both phytochromes and cryptochromes. Seedlings of the wild type (WT), ein2, phyA, phyB, and their double- and triple-mutant combinations were grown from sowing for 12 d under monochromatic red or blue light (15 µmol m⁻² s⁻¹). Leaflet area was estimated as the product of length and width of the larger leaflet from the first true foliage leaf (node 3). Internode length was measured as the length between nodes 1 and 3. Values represent means ± se for n = 7 to 12.

Figure 7.

The effects of ein2 are partly mediated through the LIP1/LONG1 module. A, Interaction of ein2 and long1 in control of internode length, leaf expansion, and stem thickness. Internode length was measured as the length between nodes 1 and 3. Leaflet area was estimated as the product of length and width of the larger leaflet from the first true foliage leaf (node 3). Values represent means ± se for n = 8 to 12. B, Comparison of 12-d-old lip1 and ein2 lip1 double-mutant plants grown in darkness. C, Reversion of lip1 ein2 elongation phenotype by long1 in plants grown under natural daylight in the greenhouse. Internode length was measured as the length between nodes 1 and 6. Values represent means ± se for n = 7 to 10. WT, Wild type.