Phospho-Mutant Activity Assays Provide Evidence for the Negative Regulation of Transcriptional Regulator PRE1 by Phosphorylation

The PACLOBUTRAZOL-RESISTANCE (PRE) gene family encodes a group of atypical helix-loop-helix (HLH) proteins that act as the major hub integrating a wide range of environmental and hormonal signals to regulate plant growth and development. PRE1, as a positive regulator of cell elongation, activates HBI1 DNA binding by sequestering its inhibitor IBH1. Furthermore, PRE1 can be phosphorylated at Ser-46 and Ser-67, but how this phosphorylation regulates the functions of PRE1 remains unclear. Here, we used a phospho-mutant activity assay to reveal that the phosphorylation at Ser-67 negatively regulates the functions of PRE1 on cell elongation. Both of mutations of serine 46, either to phospho-dead alanine or phospho-mimicking glutamic acid, had no significant effects on the functions of PRE1. However, the mutation of serine 67 to glutamic acid (PRE1(S67E)-Ox), but not alanine (PRE1(S67A)-Ox), significantly reduced the promoting effects of PRE1 on cell elongation. The mutation of Ser-67 to Glu-67 impaired the interaction of PRE1 with IBH1 and resulted in PRE1 failing to inhibit the interaction between IBH1 and HBI1, losing the ability to induce the expression of the subsequent cell elongation-related genes. Furthermore, we showed that PRE1-Ox and PRE1(S67A)-Ox both suppressed but PRE1(S67E)-Ox had no strong effects on the dwarf phenotypes of IBH1-Ox. Our study demonstrated that the PRE1 activity is negatively regulated by the phosphorylation at Ser-67.