The embryonic leaf identity gene FUSCA3 regulates vegetative phase transitions by negatively modulating ethylene-regulated gene expression in Arabidopsis

Abstract

Background: The embryonic temporal regulator FUSCA3 (FUS3) plays major roles in the establishment of embryonic leaf identity and the regulation of developmental timing. Loss-of-function mutations of this B3 domain transcription factor result in replacement of cotyledons with leaves and precocious germination, whereas constitutive misexpression causes the conversion of leaves into cotyledon-like organs and delays vegetative and reproductive phase transitions.

Results: Herein we show that activation of FUS3 after germination dampens the expression of genes involved in the biosynthesis and response to the plant hormone ethylene, whereas a loss-of-function fus3 mutant shows many phenotypes consistent with increased ethylene signaling. This FUS3-dependent regulation of ethylene signaling also impinges on timing functions outside embryogenesis. Loss of FUS3 function results in accelerated vegetative phase change, and this is again partially dependent on functional ethylene signaling. This alteration in vegetative phase transition is dependent on both embryonic and vegetative FUS3 function, suggesting that this important transcriptional regulator controls both embryonic and vegetative developmental timing.

Conclusion: The results of this study indicate that the embryonic regulator FUS3 not only controls the embryonic-to-vegetative phase transition through hormonal (ABA/GA) regulation but also functions postembryonically to delay vegetative phase transitions by negatively modulating ethylene-regulated gene expression.

Figure 1

Microarray analysis of seedlings ectopically expressing FUS3. (A) Images showing the development of leaf 4 in 5-day-old fus3 ML1:FUS3-GR seedlings transferred to different concentrations of dexamethasone (DEX). (B) Genes that change at least twofold in expression in fus3 ML1:FUS3-GR seedlings treated with 1 μM DEX. The upper graph (2 days ± DEX) shows the ratios of fold changes in gene expression of 5-day-old seedlings grown for 2 days in the absence of DEX (black bars) or in the presence of DEX (red bars represent upregulated genes, and blue bars represent downregulated genes). The same order of genes is represented in the lower graph (4 days ± DEX), where 5-day-old seedlings were grown for 4 days in the absence of DEX (black bars) or in the presence of DEX (red and blue bars). (C) The proportions of genes associated with various molecular functions (gene ontology) are represented in the pie chart. (D) RT-PCR verification of ethylene-related genes identified by microarray analysis downregulated by FUS3 activation. Expression of four ethylene-related genes and an ACTIN7 (ACT7) control in the absence (-DEX) or presence (+DEX) of FUS3 activation.

Figure 2

Increased expression of ethylene signaling and biosynthetic genes in fus3 mutant, and ethylene-related fus3 phenotypes. (A) RT-PCR of ethylene-related genes in wild-type (WT) and fus3 seeds germinated for 12, 24 and 48 hours in minimal medium (MS). ACTIN7 (ACT7) served as a control. (B) RT-PCR of ethylene-related genes in wild-type and fus3 seeds germinated for 48 hours in MS media or MS media containing 10 μM aminoethoxyvinylglycine (AVG) or 100 μM AgNO₃ (Ag). Fold change in gene expression was normalized to ACTIN7, which served as a control. Similar trends were seen in two independent experiments. (C) GFP-EIN3 fluorescence in wild-type and fus3 roots incubated for 48 hours in minimal medium with (+) or without (-) 100 μM AgN0₃ (Ag). The inset shows the GFP-EIN3 fluorescence in wild-type roots exposed to the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Scale bar = 15.2 μm. (D) Images (right panels) and quantification (left panel) of hypocotyls length of 5-day-old fus3 and fus3 ein2-1 seedlings grown in the dark in the air. Scale bar = 0.2 cm. Averages from triplicate experiments ± SD are shown. fus3, n = 24; fus3 ein2-1, n = 21. (E) Quantification of hypocotyls length of 5-day-old wild-type, fus3 and eto1-1 seedlings grown in the dark in MS in the absence (-) or in the presence (+) of the ethylene synthesis inhibitor AVG. The eto1-1 seed, which overaccumulates ethylene gas, was used as a positive control to demonstrate rescue by AVG addition. Averages from triplicate experiments ± SD are shown. Wild type, n = 25; fus3, n = 25; eto1-1, n = 24.

Figure 3

Contributions of embryonic FUS3 to leaf identity and phase transitions. (A) Rosette leaf morphology of wild-type (top row), fus3 (middle row) and fus3 plants transformed with the FUS3:FUS3-GR construct (bottom row). fus3 FUS3:FUS3-GR parent plants were sprayed with 30 μM dexamethasone (DEX) during seed production. Leaves from ten plants were dissected, and representative profiles are shown (scale bar = 1 cm). (B) Ratios of blade-to-petiole lengths of individual rosette leaves in wild-type and fus3 and embryonically rescued fus3 FUS3:FUS3-GR plants. (C) Percentage of wild-type, fus3 and embryonically rescued fus3 FUS3:FUS3-GR (seed + DEX) rosettes that developed an abaxial trichome at each leaf position.

Figure 4

FUS3 is expressed and functions postembryonically. (A) Relative expression of the FUS3 gene at various time points after germination. Quantitative RT-PCR was performed on germinating wild-type seeds imbibed for 6 hours, 1 day, 2 days and 5 days in minimal medium (MS). Transcript levels were normalized using ACTIN7 as an internal control. Results from triplicate samples are shown with error bars (SD). Experiments were repeated twice with similar results. Data from one of these replicates are shown. (B) Histochemical staining of FUS3:GUS seedlings at 1, 2, 3 and 5 days after germination in MS. Arrows indicate emerging leaf primordia showing β-glucuronidase (GUS) staining. Scale bars = 0.2 mm (day 1) and 0.4 mm (days 2, 3 and 5). (C) Wild-type and fus3 FUS3:FUS3-GR seeds germinated in MS without dexamethasone (DEX) (-DEX) or on 10 μM DEX (+DEX) for 2 days, then transferred to soil until bolting. Of the plants analyzed, 8.3% showed a full rescue of the rosette leaf morphology, and an example is shown (full rescue). A partial rescue was obtained for 62.5% of the plants, and an example is shown (partial rescue). The morphologies of the first six to eight fus3 FUS3:FUS3-GR leaves were rescued, but those of subsequent leaves were variable. Twenty-one to twenty-four plants were analyzed, constituting a representative profile. Scale bar = 1.0 cm. (D) Percentage of abaxial trichomes in wild-type rosettes and in fus3 FUS3:FUS3-GR rosettes of plants grown with (+) or without (-) DEX.

Figure 5

Influence of ethylene signaling on fus3 vegetative phase transition. (A) Mature rosette leaf morphologies of fus3 and fus3 ein2-1 plants. Leaves from 12 plants were dissected, and a representative profile is shown. Scale bar = 1.0 cm. (B) Ratios of blade-to-petiole lengths of mature individual rosette leaves from fus3 and fus3 ein2-1 plants. (C) Percentage of rosettes displaying trichomes on the abaxial surface at each leaf position. (D) Rosette leaf morphology of fus3 germinated for 2 days in minimal medium (MS) in the presence (+) or absence (-) of 100 μM AgNO₃ (Ag) and then transferred to soil. Leaves from ten plants were dissected, and a representative profile is shown. Scale bar = 1.0 cm. (E) Ratios of blade-to-petiole lengths of individual fus3 rosette leaves of plants germinated in MS with (+) or without (-) 100 μM AgNO₃ (Ag).

Figure 6

Working model of phase change regulation by FUS3. During late embryogenesis and early germination, FUS3 negatively influences a number of factors, including ethylene signaling. As a consequence, the EIN3 protein, a key positive regulator of ethylene signaling, is reduced, thereby causing a decrease in the expression of downstream transcription factors such as ERFs and EDFs. A reduction of these ethylene-dependent transcription factors prevents the premature transition from the juvenile to the adult phase of development. In the loss-of-function fus3 mutant, ethylene signaling increases, which in turn accelerates vegetative phase transitions. The lack of full restoration of altered gene expression by ethylene inhibitors and the observation that only a subset of ethylene responsive genes are affected by FUS3 also suggest that FUS3 may have ethylene-independent effects. Consistent with this hypothesis, many ethylene-responsive genes contain FUS3-binding RY sequences in their promoter elements. It is therefore possible that FUS3 also influences these genes and some aspects of phase transitions through ethylene-independent mechanisms.