The ETHYLENE RESPONSE FACTORs ERF6 and ERF11 Antagonistically Regulate Mannitol-Induced Growth Inhibition in Arabidopsis

Abstract

Leaf growth is a tightly regulated and complex process, which responds in a dynamic manner to changing environmental conditions, but the mechanisms that reduce growth under adverse conditions are rather poorly understood. We previously identified a growth inhibitory pathway regulating leaf growth upon exposure to a low concentration of mannitol and characterized the ETHYLENE RESPONSE FACTOR (ERF)/APETALA2 transcription factor ERF6 as a central activator of both leaf growth inhibition and induction of stress tolerance genes. Here, we describe the role of the transcriptional repressor ERF11 in relation to the ERF6-mediated stress response in Arabidopsis (Arabidopsis thaliana). Using inducible overexpression lines, we show that ERF6 induces the expression of ERF11. ERF11 in turn molecularly counteracts the action of ERF6 and represses at least some of the ERF6-induced genes by directly competing for the target gene promoters. As a phenotypical consequence of the ERF6-ERF11 antagonism, the extreme dwarfism caused by ERF6 overexpression is suppressed by overexpression of ERF11. Together, our data demonstrate that dynamic mechanisms exist to fine-tune the stress response and that ERF11 counteracts ERF6 to maintain a balance between plant growth and stress defense.

Figure 1.

Transcriptional induction of ERF6 and ERF11 upon short-term exposure to mannitol or ACC. Expression levels of ERF6 and ERF11 in proliferating leaf tissue measured by qRT-PCR upon exposure to mild mannitol-induced stress (25 mm; A) or 5 μm ACC (B). Represented values are means of three biological repeats with their se relative to the expression value upon transfer to MS (TMS) medium as a control. *, P < 0.05 (ANOVA) for both genes. FC, Fold change.

Figure 2.

Transcriptional activation of ERF11 by ERF6 under mannitol-induced stress. A, Expression levels of ERF11 in the growing third leaf (15 DAS) of ERF6-GR plants, relative to the wild type, measured by qRT-PCR at several time points (h) upon transfer to dexamethasone-containing medium. *, P < 0.05 (ANOVA). B, Luciferase transactivation assay with cotransformation of 35S:ERF6 and pERF11:fLUC showing the activation of the ERF11 promoter by ERF6. Values are signal intensities normalized to the cotransformed normalization plasmid rLUC and relative to the negative control (35S:GUS + pERF11:fLUC). *, P < 0.05. C, Expression levels of ERF11 in wild-type (WT) plants and erf5erf6 mutants 4 h upon exposure to mannitol. D, Expression levels of ERF6 and ERF11 at different times of the day in actively growing leaves (15 DAS) of wild-type and erf5erf6 plants grown in soil. For all graphs, the represented values are the means of three biological repeats with their se. FC, Fold change.

Figure 3.

ERF11 regulates Arabidopsis leaf growth under mannitol-induced stress conditions. A, Projected rosette area at 22 DAS of the erf11 mutant and the dexamethasone-inducible overexpression lines of ERF6 (ERF6-GR) and ERF11 (ERF11-GR) and of both in ERF6-GR × ERF11-GR double homozygous plants, grown under control conditions (medium supplemented with dexamethasone for the GR lines). Represented values are the means of three biological repeats with their se. *, P < 0.05, compared with the wild type (WT). B, Size of the third true leaf over time of the GR lines described in A upon exposure to dexamethasone from 9 DAS onward. *, P < 0.05 (ANOVA), compared with the wild type. C, Rosettes of the GR lines described in A grown in soil for 22 d and watered with a dexamethasone-containing solution. D, Cellular measurements of the abaxial side of the third leaf of the wild type and ERF11-GR at 20 DAS from the plants shown in B. *, P < 0.05, compared with the wild type. E, Reduction in rosette area caused by growth for 22 d on medium supplemented with 25 mm mannitol, compared with the rosette area of the same line grown under control conditions. Error bars represent the se of three biological repeats. *, P < 0.05 (ANOVA), compared with the wild type. Bar = 2 cm.

Figure 4.

ERF6 and ERF11 antagonistically regulate common target genes. A, Expression levels of ERF6, ERF11, and the ERF6 target genes in dexamethasone-inducible overexpression lines of ERF6 (ERF6-GR), ERF11 (ERF11-GR), and both in ERF6-GR × ERF11-GR double homozygous plants. Expression values are the average of at least three biological repeats. Values are relative to the expression level in wild-type plants subjected to the same treatment. B, Protoplast activation assay with pSTZ:fLUC, pWRKY33:fLUC, and pMYB51:fLUC for binding and effect of ERF6, ERF11, and a combination of both in a 1:1 ratio. C, Protoplast activation assay on the promoter of MYB51 for the native ERF11 protein, a truncated ERF11 in which the repressive EAR domain has been removed, and a combination in a 1:1 ratio of ERF6 and the truncated ERF11. D, Titration protoplast activation assay on the promoter of MYB51 with multiple concentrations of ERF6 and ERF11. For B to D, values are signal intensities normalized to the cotransformed normalization plasmid rLUC and relative to the negative control (35S:GUS + pTARGET:fLUC, horizontal line). Values represent the average of at least two biological repeats with their se. FC, Fold change.

Figure 5.

Schematic overview of the putative roles of ERF6 and ERF11 under stress. Under basal levels of stress (plain arrows and proteins), ERF11 and ERF6 are lowly expressed. Low levels of ERF11 proteins are sufficient to repress the activation of the stress response mechanisms, as they are likely more abundant than the active, phosphorylated ERF6 proteins. Under higher stress levels (dashed arrows and proteins), ACC accumulates and MPK3/MPK6 phosphorylates and thereby activates ERF6. ERF6 now outcompetes ERF11 proteins on the promoter of the target genes, activating tolerance mechanisms and growth inhibition. However, ERF6 also transcriptionally activates ERF11, increasing ERF11 protein levels, which enables a rapid inhibition of the stress response when the stress levels decrease. P, Phosphorylation residue.