Ethylene Response Factor6 acts as a central regulator of leaf growth under water-limiting conditions in Arabidopsis

Abstract

Leaf growth is a complex developmental process that is continuously fine-tuned by the environment. Various abiotic stresses, including mild drought stress, have been shown to inhibit leaf growth in Arabidopsis (Arabidopsis thaliana), but the underlying mechanisms remain largely unknown. Here, we identify the redundant Arabidopsis transcription factors ETHYLENE RESPONSE FACTOR5 (ERF5) and ERF6 as master regulators that adapt leaf growth to environmental changes. ERF5 and ERF6 gene expression is induced very rapidly and specifically in actively growing leaves after sudden exposure to osmotic stress that mimics mild drought. Subsequently, enhanced ERF6 expression inhibits cell proliferation and leaf growth by a process involving gibberellin and DELLA signaling. Using an ERF6-inducible overexpression line, we demonstrate that the gibberellin-degrading enzyme GIBBERELLIN 2-OXIDASE6 is transcriptionally induced by ERF6 and that, consequently, DELLA proteins are stabilized. As a result, ERF6 gain-of-function lines are dwarfed and hypersensitive to osmotic stress, while the growth of erf5erf6 loss-of-function mutants is less affected by stress. Besides its role in plant growth under stress, ERF6 also activates the expression of a plethora of osmotic stress-responsive genes, including the well-known stress tolerance genes STZ, MYB51, and WRKY33. Interestingly, activation of the stress tolerance genes by ERF6 occurs independently from the ERF6-mediated growth inhibition. Together, these data fit into a leaf growth regulatory model in which ERF5 and ERF6 form a missing link between the previously observed stress-induced 1-aminocyclopropane-1-carboxylic acid accumulation and DELLA-mediated cell cycle exit and execute a dual role by regulating both stress tolerance and growth inhibition.

Figure 1.

The erf5erf6 double mutant is more tolerant to mild osmotic stress conditions. A, Rosette area over time of wild-type plants (WT) and erf5erf6 double mutants under standard MS medium and different stress conditions. The erf5erf6 mutant shows significant tolerance to osmotic stress (MS medium supplemented with 25 mm mannitol) only. Colored shadows indicate se. Three biological repeats were performed with at least 12 seedlings per line per treatment. B, Leaf area measurements (third leaf) of the erf5erf6 mutant and the wild type upon transfer at 9 DAS to standard or mild osmotic stress conditions. On osmotic stress, the erf5erf6 mutant is always about 50% larger than the wild type (for detailed measurements, see Supplemental Table S2). Error bars indicate se. Three biological repeats were performed with 16 leaves per repeat. H₂O₂, Hydrogen peroxide.

Figure 2.

ERF5 and ERF6 negatively regulate leaf growth. A, Growth measurements of the third leaf of inducible ERF6 overexpression plants transferred to DEX at 9 DAS to induce ERF6 overexpression. Leaf size becomes significantly smaller than that of the control at 11 DAS for ERF6^IOE-S and at 12 DAS for ERF6^IOE-W. B, Rosettes of ERF6-overexpressing plants in vitro (growth medium supplied with DEX; top panel) and in soil (plants sprayed daily with DEX; bottom panel). From left to right: GFP:IOE control line, ERF6^IOE-W, and ERF6^IOE-S. Plants are 22 d old. C, Nineteen-day-old rosettes of ERF6^IOE-W lines upon ERF6 overexpression with DEX at 9 DAS, exposure to osmotic stress (25 mm mannitol), or the combination of mannitol and DEX. **P < 0.01. For A and C, error bars indicate se of three repeats with 16 plants per repeat. [See online article for color version of this figure.]

Figure 3.

Overlap between ERF6 targets and genes rapidly induced by 25 mm mannitol. Comparison was performed of the 332 putative ERF6 targets with the previously identified list of genes specifically induced in leaf initials within hours upon exposure to osmotic stress (Skirycz et al., 2011a). Values indicated in the Venn diagram represent the number of genes induced upon 1.5- and 3-h mannitol treatment and the genes induced in leaf initials 4 h following DEX application in ERF6^IOE-S.

Figure 4.

ERF6 regulates GA levels through transcriptional control of the GA2-OX6 gene. A, Induction of GA2-OX6 following the activation of ERF6 overexpression. Two hours after transfer to DEX of ERF6^IOE-S, GA2-OX6 is significantly induced. Expression was measured in proliferating third leaves, and values are normalized to their expression in the GFP:IOE control line exposed to the same treatment. Error bars indicate se of three repeats with 64 young third leaves per repeat. B, Stabilization of the DELLA protein RGA upon the activation of ERF6 overexpression shown by western blot, targeting the GFP domain of the RGA-GFP fusion protein in pRGA:GFP-RGA × ERF6^IOE-S seedlings. DELLA stabilization first clearly appears 24 h after ERF6 activation. Three biological replicates were performed. C, Growth complementation assay. By crossing the two independent ERF6:IOE lines with a 35S-GA20-OX line (ectopic GA overproduction), the dwarfed phenotype could be partially and fully complemented in ERF6^IOE-S and ERF6^IOE-W lines, respectively. Treatment with DEX was applied at 9 DAS, and photographs were taken at 21 DAS. D, Measurements of third leaves at 21 DAS of GFP:IOE, ERF6^IOE-W, and ERF6^IOE-W × 35S-GA20-OX upon treatment with DEX at 9 DAS. Error bars indicate se of three repeats with 12 leaves per repeat. [See online article for color version of this figure.]

Figure 5.

ERF6 regulates the stress-related transcription factors STZ, MYB51, and WRKY33. A, Induction of STZ, MYB51, and WRKY33 following the activation of ERF6 overexpression. Within 2 h of the transfer of ERF6^IOE-S to DEX, STZ, WRKY33, and MYB51 are significantly induced. Expression was measured in proliferating third leaves, and values are normalized to their expression in the GFP:IOE control line exposed to the same treatment. Error bars indicate se of three repeats with 64 young third leaves per repeat. B, ERF5/ERF6-dependent activation of the promoters of MYB51, WRKY33, and STZ by the protoplast activation assay. Indicated values are luciferine detection levels normalized to the negative control. Asterisks indicate significantly different values from the control at the 1% (**) and 5% (*) significance levels. Error bars indicate se, and eight biological repeats were performed. C, Induction of STZ, MYB51, and WRKY33 expression 8 h after the activation of ERF6 overexpression in ERF6^IOE × 35S-GA20-OX plants. Although the dwarfed growth phenotype is partially and completely rescued in ERF6^IOE-S × 35S-GA20-OX and ERF6^IOE-W × 35S-GA20-OX plants, respectively (Fig. 4B), the stress-related transcription factors are still induced to the same extent as in the positive control lines (ERF6^IOE-S × GFP:IOE and ERF6^IOE-W × GFP:IOE, respectively). Expression values are normalized to their expression in the control line (GFP:IOE). Error bars indicate se of three repeats with 64 young third leaves per repeat.

Figure 6.

How ERF5 and ERF6 regulate leaf growth and stress defense under osmotic stress. A, Immediately upon exposure to osmotic stress, ACC accumulates in the actively growing leaves, where it is converted to ethylene. Ethylene further activates the signaling pathway involving MPK3 and MPK6. These kinases phosphorylate the basal amount of ERF5 and ERF6 proteins present in the cell prior to stress exposure. The activated ERF5 and ERF6 then execute their dual functions: on the one hand, the activation of the stress defense transcriptional cascade with direct transcriptional activation of WRKY33, STZ, and MYB51, and on the other hand, the activation of leaf growth inhibition. This occurs through the transcriptional activation of the gene encoding the GA-inactivating enzyme GA2-OX6, thereby decreasing the bioactive GA concentration and stabilizing the DELLA proteins. B, In accordance with the model presented in A, ERF6 transcript levels and stress-mediated activity inversely correlate with leaf growth (for discussion, see text). Bar = 1 cm. [See online article for color version of this figure.]