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

Abstract

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.

Fig. 1. MHZ1 positively regulates the ethylene response in rice roots.

a Ethylene response phenotype of mhz1 alleles. Etiolated seedlings were treated with various concentrations of ethylene in darkness. Representative seedlings grown in the air and in 10 ppm ethylene are shown (Left). Coleoptile (Center) and root lengths (Right) are means ± SD, n > 30. Bars indicate 10 mm. b MHZ1 genomic structure and mutation sites of different mhz1 alleles. Colored boxes indicate exons and horizontal lines indicate introns. c Schematic structure of MHZ1 and mutation sites of different mhz1 alleles. d MHZ1 gene expression in WT and mhz1-1. Actin1 was amplified as internal control. e MHZ1 overexpression lines (MHZ1-OE) have constitutive ethylene response. MHZ1 native promoter was used to drive the MHZ1 cDNA for overexpression. Etiolated seedlings were treated with various concentrations of ethylene and 10 ppm 1-MCP under darkness. Bars indicate 10 mm. Root lengths (Right) are means ± SD, n > 30. f Expression of ethylene-inducible genes OsRRA5, OsERF002 OsRAP2.8, OsERF063 and OsERF073 in mhz1-1 and MHZ1-OE lines compared with WT as revealed by qPCR. Data are means ± SD, n = 4. Source data are provided as a Source Data file.

Fig. 2. MHZ1 expression is induced by ethylene through directly binding of OsEIL1 to its promoter region.

a MHZ1 expression induced by ethylene is dependent on OsEIN2 and OsEIL1. Two-day-old etiolated seedlings of WT or Osein2 and Oseil1 were treated with 10 ppm ethylene or air. Data are means ± SD, n = 4. b Binding of OsEIL1 protein to the OsEIL1 binding site (EBS) containing region of MHZ1 promoter in vitro. GST-tagged OsEIL1 N-terminus protein was isolated and incubated with biotin-labeled probes carrying EBSs of MHZ1 promoter. An excess of unlabeled probe was added as the competitor. c Transient expression of OsEIL1 in tobacco leaves activated MHZ1 promoter activity. Relative luminescence intensity was calculated, data are means ± SD from three biological replicates. Source data are provided as a Source Data file.

Fig. 3. MHZ1-mediated phosphorelay pathway is required for ethylene-inhibited root growth.

a MHZ1 and its truncated/mutant versions used for phosphorylation analysis. H, G1, G2, and D indicate conserved residues or boxes. MBP indicates maltose-binding protein. b MHZ1 has histidine kinase activity. The radioactive band below the GST-MHZ1 indicates a degradation product. c Trans-phosphorylation between MHZ1 molecules. MBP-G1 has G588A and G590A mutations at G1 box. MBP-G2 has G618A and G620A mutations at G2 box. d MHZ1 can transfer its phosphoryl groups to OsAHP1 and OsAHP2 rather than OsPHP1 or OsPHP2. e Phosphorelay from MHZ1 to OsAHP1 and OsAHP2 was abolished when the conserved histidine was mutated in OsAHP1/2. f MHZ1 can transfer its phosphoryl groups to OsAHP1/2 and further to OsRR21. D68E mutation in OsRR21 disrupted its phosphoryl-accepting ability. g D824A mutation in MHZ1 receiver domain blocked its phosphorelay to OsAHP1/2, and OsRR26 cannot accept phosphoryl groups transferred from MHZ1 and OsAHP1/2. h Time course of the phosphorelay from MHZ1 to OsAHP1 (left panel) and further to OsRR21 (right panel). After the reaction in the left panel was finished, the OsRR21 was added and incubated for various times in the right panel. i MHZ1 but not its mutant versions rescued the ethylene-insensitive phenotype of mhz1 (mhz1-1) mutant. cDNAs of MHZ1 and its mutant versions MHZ1(G1), MHZ1(H375Q) and MHZ1(D824A), fusioned with a 3 × FLAG sequence, driven by MHZ1 native promoter, were transformed into the mhz1-1 to observe the root ethylene response. Total proteins of each line were immunoblotted for MHZ1-FLAG with anti-FLAG antibody. A non-specific band was used as a loading control. MHZ1 gene expression was examined by RT-PCR and Actin1 was amplified as control. Bar indicates 10 mm. j Ethylene response of Osahp1 Osahp2 double-mutant. Osahp1 Osahp2 double-mutant was segregated from the self-bred progenies of an Osahp1 (heterozygous)/Osahp2 (homozygous) plant. “ + ” indicates wild-type OsAHP1. k Ethylene response of OsRR21 mutants and overexpression lines (OE). For j and k, etiolated seedlings were treated with 10 ppm ethylene or air for 2.5 days. Bars indicate 10 mm. Source data are provided as a Source Data file.

Fig. 4. MHZ1 physically interacts with and inhibited by ethylene receptors.

a mhz1 suppressed the ethylene hypersensitivity of Osetr2 (Dongjin) and Osers2 (Dongjin) loss-of-function mutants. (Left) Seedlings were treated with air or 1 ppm ethylene. (Right) Data are means ± SD, n > 30. **P < 0.01; Student’s t-test. DJ, Dongjin; Nip, Nipponbare. Bars indicate 10 mm. b Ethylene response of Osers2^d/MHZ1-OE lines. Etiolated seedlings were treated with air or 10 ppm ethylene. Bars indicate 10 mm. c Root length quantification of each line in b. Total proteins of each line were immunoblotted for MHZ1-FLAG with anti-FLAG antibody. Data are means ± SD, n > 30. **P < 0.01; Student’s t-test. d Schematic structures of MHZ1 and OsERS2 and their truncated versions. e Split-ubiquitin Y2H assay for interaction of MHZ1 and OsERS2. Combination of pTSU2-APP and pNubG-Fe65 (provided in the kit) was used as a positive control. f Pull-down of MBP-MHZ1 with GST-OsERS2ΔTM in E. coli. g Co-IP assays for interaction of MHZ1 and OsERS2. The constructs were cotransformed into rice protoplasts. Total proteins were immunoprecipitated with GFP-Trap and immunoblotted with anti-GFP, anti-FLAG, and anti-UGPase antibodies. h OsERS2 facilitates the ER membrane localization of MHZ1. MHZ1-GFP and OsERS2-mCherry proteins were transiently expressed in tobacco leaf epidermal cells. Bars indicate 20 μm. i Co-IP assays for interaction domain mapping of MHZ1 and OsERS2. The constructs were cotransformed into rice protoplasts. Total proteins were immunoprecipitated with GFP-Trap and immunoblotted with anti-FLAG, anti-GFP antibodies. j OsERS2 inhibits MHZ1 kinase activity. GST was added as a control. Values at the bottom indicate relative phosphorylation levels of GST-MHZ1. k OsERS2 inhibited phosphate groups relayed to OsAHP1. Values at the bottom indicate relative phosphorylation levels of GST-AHP1. l GAF domain of OsERS2 inhibits MHZ1 kinase activity. GST was added as a control. Values at the bottom indicate relative phosphorylation levels of GST-MHZ1. m Histidine phosphorylation of MHZ1 is suppressed by OsERS2 and Osers2^d. Vectors carrying OsERS2-myc and Osers2^d-myc were transformed into protoplasts of MHZ1OE 4-4. Total protein was immunoprecipitated with anti-FLAG affinity gel and immunoblotted with anti-FLAG or anti-1-p-His (Millipore, MABS1330) antibodies. Values at the bottom indicate relative phosphorylation levels of MHZ1. Protoplasts of ProMHZ1:MHZ1(H375Q) transgenic line transformed with the control vector were used as negative control. Source data are provided as a Source Data file.

Fig. 5. Genetic interaction of MHZ1 and OsEIN2-mediated pathways.

a Ethylene response of mhz1/OsEIN2-OE in comparison with mhz1 and OsEIN2-OE. Bars indicate 10 mm. b Quantification of root length of the mutants in a. Root lengths are means ± SD, n > 30. **P < 0.01; Student’s t-test. c Enlargement of root features of the mutants treated with 10 ppm ethylene in a. d Ethylene response of Osein2/MHZ1-OE in comparison with Osein2 and MHZ1-OE. Rice seedlings were grown in dark for 2 days with air or 10 ppm ethylene. Bars indicate 10 mm. e Quantification of root length (above) and relative root length (below) of the mutants in d. Data are means ± SD, n > 30. **P < 0.01; Student’s t-test. f Comparison of MHZ1-, OsEIN2-, and OsEIL1-regulated ethylene-response genes. Etiolated seedlings of WT, mhz1, Osein2 and Oseil1 were grown for 2 days before treated with air or 10 ppm ethylene for 3 h. Roots were subjected to RNA-seq analysis with three biological replicates. Ethylene-response genes (ERGs) were identified in WT according to the gene expression levels with at least relative twofold changes (q-value < 0.05) in ethylene treatment compared to those in the air. A total of 1789 ERGs were identified. g A proposed working model for MHZ1-mediated ethylene signaling in rice. Rice has the conserved components of ethylene signaling as those in Arabidopsis, including ethylene receptors, OsCTR2, OsEIN2, and OsEIL1. Besides the conserved receptor-CTR2-OsEIN2-OsEIL1 signaling pathway, our results suggests a novel MHZ1-AHP1/2-OsRR21 phosphorelay pathway, through which ethylene receptors could regulate the root growth of rice by suppressing the kinase activity of MHZ1. In the absence of ethylene, ethylene receptors are in active conformations, which may facilitate their interaction with MHZ1 and suppress MHZ1 activity. With ethylene, ethylene receptors possibly released the repression effect on MHZ1 and the phosphorelay pathway is activated. The MHZ1-mediated phosphorelay pathway and the OsEIN2-mediated pathway may work together to regulate a subset of downstream gene to modulate root growth. MHZ1 and OsRRs can also be transcriptionally induced by ethylene through the OsEIN2-mediated pathway, which may facilitate the maintenance of MHZ1-mediated signaling after activation. The orange arrow indicates transcriptional activation. In addition, H₂O₂ may also function through MHZ1 to participate in the ethylene-regulated root growth. Source data are provided as a Source Data file.