MHZ11 Is Localized to the ER Membrane and Positively Regulates Ethylene Signaling.

(A) MHZ11-FLAG protein rescued the ethylene-insensitive phenotype of mhz11 roots. Two-kb promoter of MHZ11 was cloned to drive the MHZ11-FLAG. Vector carrying ProMHZ11:MHZ11-FLAG was transformed into mhz11 and ethylene responses of the homozygous lines, and checked. Dark-grown seedlings were treated with 10 µL L^-1 of ethylene. Scale bar = 10 mm.

(B) Membrane association of MHZ11. Equal amounts of total protein (T), soluble protein (S), and microsomal membranes (M) were immunoblotted for MHZ11-FLAG, BiP (ER membrane marker), and UGPase (cytoplasm marker).

(C) MHZ11 is an integral membrane protein. Microsomes were prepared from roots of MHZ11-FLAG/mhz11 transgenic plants. The membrane pellet was resuspended in 0.1 M of NaCl, 0.1 of M Na₂CO₃ at pH 11, and 1% (v/v) Triton X-100 or 1% (w/v) SDS. Samples were separated into supernatant (S) and pellet (P) fractions by centrifugation at 125,000g for 1 h. Equal amounts of proteins were immunoblotted for MHZ11-FLAG.

(D) ER membrane localization of MHZ11 protein as revealed by transient expression of MHZ11-GFP in N. benthamiana leaf epidermal cells. mCherry-HDEL served as ER marker. Scale bars = 20 μm. Five randomly chosen regions of interest of infiltrated leaves were observed with similar results.

(E) Localization of different truncated versions of MHZ11 protein. TM, transmembrane motif of MHZ11. Scale bars = 20 μm. Five randomly chosen regions of interest of infiltrated leaves in each group were observed with similar results.

(F) Gene expression of MHZ11 is induced by ethylene as revealed by qPCR. Data are means ± sd, n = 3, six seedlings per replicate (^*P < 0.05, ^**P < 0.01, Student’s t test; each compared with the corresponding 0-h control). Two-d–old dark-grown seedlings were treated with 10 µL L^-1 of ethylene for various time lengths before being collected for RNA extraction.

(G) Ethylene induction of MHZ11 is abolished in Osein2 and Oseil1 mutants as revealed by qPCR. Two-d–old dark-grown seedlings were treated with 10 µL L^-1 of ethylene for 6 h. Data are means ± sd, n = 3, six seedlings per replicate (^*P < 0.05, Student’s t test; compared with the corresponding air control).

(H) Constitutive short root phenotype of MHZ11-OE lines. Vector carrying Pro35S:MHZ11-FLAG was transformed into the mhz11 mutant and three transgenic lines overexpressing MHZ11 were used for phenotype analysis. Dark-grown seedlings were treated with 10 µL L^-1 of ethylene for 2.5 d. Root lengths are means ± sd (n > 30) calculated from at least 30 seedlings (^**P < 0.01, Student’s t test; compared with the wild type [WT]).

(I) 1-MCP suppressed the short root phenotype of MHZ11-OE. Dark-grown seedlings were treated with or without 10 µL L^-1 of 1-MCP for 2 d. Scale bars = 10 mm. Root lengths are means ± sd (n > 30) calculated from at least 30 seedlings (^**P < 0.01, Student’s t test; each compared with the corresponding air control).