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.

From network to phenotype: the dynamic wiring of an Arabidopsis transcriptional network induced by osmotic stress

“…ERF59 overexpression caused a growth stimulation (increased rosette area) of 26% and 17% in two independent lines (Fig 4C and D). In addition to the already characterized ERF6‐GR dwarfed phenotype (Fig 4C; Dubois et al , 2013), the most striking growth phenotype of the GOF lines was observed in all three independent ERF9‐GR lines, which showed a size reduction of 30%, 57% and 37% (Fig 4C and D). ERF98‐GR (−12%, P  = 6.2E‐6, −11%, P  = 1.E‐4, −38%, P  < 1E‐7, Tukey's test) showed a less drastic but significant decrease in rosette area (Fig 4C and D).…”

“…Among them, ERF6 appeared to play a key role in this early stress response, enabling the inhibition of leaf growth and the simultaneous activation of stress‐inducible genes. Based on the identified mannitol‐responsive genes (Skirycz et al , 2011) and the ERF6 target genes (Dubois et al , 2013), we selected 28 genes encoding TFs with a putative role in the mannitol‐mediated growth retardation. To measure the transcriptional induction of these 28 genes by mannitol, 15‐day‐old plants grown on half‐strength Murashige and Skoog (1/2 MS) medium covered with a nylon mesh were transferred to medium containing 25 mM mannitol or control medium (Skirycz et al , 2011).…”

“…This caused abortion or decreased expression of the TF, with the exception of wrky30 , in which the T‐DNA insertion resulted in an increased expression (Appendix Figs S3–S22). Because ERF5 and ERF6 have been described to be redundant (Dubois et al , 2013), the double mutant was used for phenotypic analysis.…”

“…Previous reports (Claeys et al , 2012; Dubois et al , 2013) have shown the importance of gibberellic acid (GA) degradation in the response to mannitol treatment. To further explore this finding in our experimental setup, the expression profile of genes encoding two GA degradation ( GIBBERELLIN2‐OXIDASE4 and GIBBERELLIN2‐OXIDASE6 ) and two GA biosynthesis enzymes ( GIBBERELLIN3‐OXIDASE1 and GIBBERELLIN20‐OXIDASE1 ) was measured in expanding leaves over time (20 min, 40 min, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h after transfer to mannitol‐containing or control medium).…”

“…Upon short‐term exposure to mannitol, a gradually increasing number of genes encoding TFs is significantly upregulated, suggesting that a transcriptional cascade initiates the early response to mannitol. Few members of this transcriptional cascade have been studied previously, such as the rapidly induced ETHYLENE RESPONSE FACTOR 6 ( ERF6 ), which activates the expression of GIBBERELLIN2‐OXIDASE6 ( GA2‐OX6 ), a gene encoding a gibberellin‐inactivating enzyme (Rieu et al , 2008; Dubois et al , 2013). Because of the resulting lower levels of gibberellin, DELLA proteins are stabilized, which ensures that cells permanently exit the cell division phase and are pushed to cell differentiation (Claeys et al , 2012).…”