Antagonistic relationship between AtRALF1 and brassinosteroid regulates cell expansion-related genes

Abstract

Rapid alkalinization factor (RALF) is a peptide signal that plays a role in plant cell expansion. We have recently proposed that AtRALF1 negatively regulates root cell elongation and lateral root formation by opposing the effects of brassinosteroid (BR). We reported 6 AtRALF1-inducible cell wall-related genes and 2 P450 monooxygenase -encoding genes involved in the BR biosynthetic pathway. The AtRALF1-inducible genes implicated in cell wall remodeling were not downregulated by brassinolide (BL) treatment alone; their induction was only compromised following simultaneous treatment with AtRALF1 and BL. We further examined the cell wall-remodeling gene EXPANSIN A5 (AtEXPA5), which is upregulated by BL and has been shown to positively affect root cell elongation. Herein, we report that AtEXPA5 expression is downregulated by AtRALF1 in a dose-dependent manner in the roots and hypocotyls of Arabidopsis plants. AtEXPA5 is also downregulated in plants that overexpress AtRALF1, and it is upregulated in plants in which the AtRALF1 gene is partially silenced. The AtRALF1 peptide is also able to repress AtEXPA5 induction following a pre-treatment with BL. A schematic diagram showing the gene regulatory network connecting the recently reported genes with the regulation of cell expansion by AtEXPA5 is presented.

Keywords: Arabidopsis thaliana; brassinolide; cell elongation; expansin; peptide hormone.

Figure 1.

Quantitative RT-PCR gene expression analyses of EXPANSIN A5 (AtEXPA5, At3 g29030). (A) _HisAtRALF1-treated 10-d-old wild-type plants. Total RNA was extracted from roots after 30 min of treatment with 0, 0.1, 0.5 and 1 μM of the peptide. (B) Dark-grown 4-d-old wild-type plants treated with _HisAtRALF1. Total RNA was extracted from hypocotyls after 30 min of treatment with 0, 0.1, 0.5 and 1 μM of the peptide. (C) Arabidopsis plants (10-d-old) treated with 100 nM of brassinolide (BL) for 6 h or pre-treated with 100 nM of BL for 5.5 h and treated with 1 μM of _HisAtRALF1 for 30 min. Control plants were treated with H₂O. Total RNA was extracted from the roots after each treatment. (D) AtRALF1-overexpressing (35S:AtRALF1), AtRALF1-silencing (irAtRALF1) and wild-type (WT) plants. Total RNA was extracted from roots of 10-d-old plants. Error bars indicate SD (* = p value < 0.05,** = p value < 0.01, t-test). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, At1 g13440) expression was used as a control. All experiments were repeated at least 3 times (independent biological replicates).

Figure 2.

Schematic diagram of the AtRALF1 and BL gene regulatory network for cell expansion. Arrowed solid lines represent induction, and solid lines with bars at the end represent repression. Dashed lines represent the final effects of the actions of the genes in the cell expansion process. Dashed lines ending with arrowheads represent activation, and dashed lines ending with bars represent inhibition.