Membrane protein MHZ3 regulates the on-off switch of ethylene signaling in rice.

Ethylene regulates plant growth, development, and stress adaptation. However, the early signaling events following ethylene perception, particularly in the regulation of ethylene receptor/CTRs (CONSTITUTIVE TRIPLE RESPONSE) complex, remains less understood. Here, utilizing the rapid phospho-shift of rice OsCTR2 in response to ethylene as a sensitive readout for signal activation, we revealed that MHZ3, previously identified as a stabilizer of ETHYLENE INSENSITIVE 2 (OsEIN2), is crucial for maintaining OsCTR2 phosphorylation. Genetically, both functional MHZ3 and ethylene receptors prove essential for OsCTR2 phosphorylation. MHZ3 physically interacts with both subfamily I and II ethylene receptors, e.g., OsERS2 and OsETR2 respectively, stabilizing their association with OsCTR2 and thereby maintaining OsCTR2 activity. Ethylene treatment disrupts the interactions within the protein complex MHZ3/receptors/OsCTR2, reducing OsCTR2 phosphorylation and initiating downstream signaling. Our study unveils the dual role of MHZ3 in fine-tuning ethylene signaling activation, providing insights into the initial stages of the ethylene signaling cascade.

An RRM domain protein SOE suppresses transgene silencing in rice.

Gene expression is regulated at multiple levels, including RNA processing and DNA methylation/demethylation. How these regulations are controlled remains unclear. Here, through analysis of a suppressor for the OsEIN2 over-expressor, we identified an RNA recognition motif protein SUPPRESSOR OF EIN2 (SOE). SOE is localized in nuclear speckles and interacts with several components of the spliceosome. We find SOE associates with hundreds of targets and directly binds to a DNA glycosylase gene DNG701 pre-mRNA for efficient splicing and stabilization, allowing for subsequent DNG701-mediated DNA demethylation of the transgene promoter for proper gene expression. The V81M substitution in the suppressor mutant protein mSOE impaired its protein stability and binding activity to DNG701 pre-mRNA, leading to transgene silencing. SOE mutation enhances grain size and yield. Haplotype analysis in c. 3000 rice accessions reveals that the haplotype 1 (Hap 1) promoter is associated with high 1000-grain weight, and most of the japonica accessions, but not indica ones, have the Hap 1 elite allele. Our study discovers a novel mechanism for the regulation of gene expression and provides an elite allele for the promotion of yield potentials in rice.

A translational regulator MHZ9 modulates ethylene signaling in rice.

Ethylene plays essential roles in rice growth, development and stress adaptation. Translational control of ethylene signaling remains unclear in rice. Here, through analysis of an ethylene-response mutant mhz9, we identified a glycine-tyrosine-phenylalanine (GYF) domain protein MHZ9, which positively regulates ethylene signaling at translational level in rice. MHZ9 is localized in RNA processing bodies. The C-terminal domain of MHZ9 interacts with OsEIN2, a central regulator of rice ethylene signaling, and the N-terminal domain directly binds to the OsEBF1/2 mRNAs for translational inhibition, allowing accumulation of transcription factor OsEIL1 to activate the downstream signaling. RNA-IP seq and CLIP-seq analyses reveal that MHZ9 associates with hundreds of RNAs. Ribo-seq analysis indicates that MHZ9 is required for the regulation of ~ 90% of genes translationally affected by ethylene. Our study identifies a translational regulator MHZ9, which mediates translational regulation of genes in response to ethylene, facilitating stress adaptation and trait improvement in rice.

Histidine kinase MHZ1/OsHK1 interacts with ethylene receptors to regulate root growth in rice.

Ethylene plays essential roles during adaptive responses to water-saturating environments in rice, but knowledge of its signaling mechanism remains limited. Here, through an analysis of a rice ethylene-response mutant mhz1, we show that MHZ1 positively modulates root ethylene responses. MHZ1 encodes the rice histidine kinase OsHK1. MHZ1/OsHK1 is autophosphorylated at a conserved histidine residue and can transfer the phosphoryl signal to the response regulator OsRR21 via the phosphotransfer proteins OsAHP1/2. This phosphorelay pathway is required for root ethylene responses. Ethylene receptor OsERS2, via its GAF domain, physically interacts with MHZ1/OsHK1 and inhibits its kinase activity. Genetic analyses suggest that MHZ1/OsHK1 acts at the level of ethylene perception and works together with the OsEIN2-mediated pathway to regulate root growth. Our results suggest that MHZ1/OsHK1 mediates the ethylene response partially independently of OsEIN2, and is directly inhibited by ethylene receptors, thus revealing mechanistic details of ethylene signaling for root growth regulation.