1. Volume 6, Issue 6, Pages 1830-1848 (November 2013)
Identification of Rice Ethylene-Response Mutants and Characterization of MHZ7/OsEIN2 in Distinct Ethylene Response and Yield Trait Regulation 
Biao Ma, Si-Jie He, Kai-Xuan Duan, Cui-Cui Yin, Hui Chen, Chao Yang, Qing Xiong, Qing-Xin Song, Xiang Lu, Hao-Wei Chen, Wan-Ke Zhang, Tie-Gang Lu, Shou-Yi Chen, Jin-Song Zhang 
Molecular Plant 
Volume 6, Issue 6, Pages (November 2013)
DOI: /mp/sst087
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 Ethylene-Response Phenotypes of mhz Mutants.
Seedlings were grown in the dark for 4 d in the absence (air) or presence of 10 ppm of ethylene.
(A) Morphological characteristics of ethylene response in WT (Nipponbare) rice seedlings.
(B) An example for mutant screening, showing segregation of mutant (mhz7-1) in T2 population in the presence of ethylene. Ethylene-insensitive adventitious roots (blue circled) were observed in etiolated mutant seedlings. Orange circles indicate WT phenotype.
(C) Ethylene-response phenotypes of various mhz mutants.
(D) Root length of WT and mhz mutants in response to ethylene. Each column is average of 20–30 seedlings and bars indicate SD.
(E) Coleoptile length of WT and mhz mutants in response to ethylene. Each column is average of 20–30 seedlings and bars indicate SD.
Molecular Plant 2013 6, DOI: ( /mp/sst087)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

3. Figure 2 Characterization of Ethylene Insensitivity in mhz7 Mutants.
(A) Ethylene-insensitive phenotypes of mhz7-1 and mhz7-2 mutants. Both coleoptiles and roots of etiolated seedlings are insensitive to ethylene.
(B) Relative root length of mhz7 mutants and WT in response to ethylene. The ethylene-binding inhibitor 1-MCP (1-methylcyclopropene, 5 ppm) was used to block ethylene perception in WT and the treatment was used as a positive control for ethylene insensitivity. Each point is average of 20–30 seedlings and bars indicate SD.
(C) Ethylene dosage-response of coleoptile elongation. Others are as in (B).
(D) Expressions of ethylene-inducible genes in both shoots and roots of WT and mhz7-1. Three-day-old etiolated seedlings were treated with or without 10 ppm of ethylene (ET) for 8h and the RNA was isolated for quantitative PCR. Data are the mean ± SD of three replicates.
(E) Expressions of genes preferentially induced in roots of WT and mhz7-1. Others are as in (D).
(F) Expression of genes preferentially induced in shoots of WT and mhz7-1. Others are as in (D).
Molecular Plant 2013 6, DOI: ( /mp/sst087)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

4. Figure 3 Map-Based Cloning of MHZ7 Gene.
(A) Fine mapping of MHZ7 gene. The locus was mapped to chromosome 7 within a 240-Kb region between SNP7-2.9-DraI and Idl markers. The numbers under the markers indicate recombinant events. Mutation sites of five allelic mutants are indicated in schematic diagram of MHZ7. Black boxes represent exons. OSJNBb0050B07 and OSJNBa0024L18 indicate BAC clone numbers.
(B) Mutation sites of five allelic mutants shown on the primary structure of MHZ7 protein predicted using the SMART software ( ). Black columns represent transmembrane domains.
(C) Confirmation of mutation sites in mhz7-1 and mhz7-2 by PCR-based analyses.
(D) Functional complementation of mhz7 mutant. MHZ7 cDNA driven by 35S promoter was transformed into mhz7-1 plants, rescuing the ethylene-insensitive phenotypes of mhz7-1 etiolated seedlings in lines 36 and 22. Lines 10 and 32 showed longer coleoptiles compared to WT, reflecting slightly enhanced ethylene response.
Molecular Plant 2013 6, DOI: ( /mp/sst087)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
MHZ7 Gene Expressions in Rice Plants Revealed by Promoter–GUS Analysis.
Transgenic plants expressing MHZ7pro::GUS were used for GUS staining.
(A) GUS staining in etiolated seedling. Bar = 10 mm.
(B) GUS staining in the coleoptile of a germinated seed.
(C) GUS staining in adventitious roots.
(D) MHZ7 expression revealed by GUS staining in vascular tissues of seminal root and lateral roots.
(E) MHZ7 expression in young lateral roots. Please note the relatively strong staining in connecting/initiation sites of lateral roots.
(F) GUS staining is not found in root tip.
(G) GUS staining in the cutting edge of leaf blade.
(H) Staining in stem and node with adventitious roots.
(I) Enlarged root tips with strong staining in the node of (H).
(J) Strong GUS staining in young panicle and the supporting stem.
(K) GUS staining is strong in anthers of a flower.
(L) Staining is apparent in style of a developing ovary.
(M) Staining is observed in style and bottom of an ovary.
(N) Staining in top and bottom of an ovary.
(O) GUS staining is strong in top of a developing grain.
(P) GUS staining distribution in longitudinal section of a less-stained grain.
(Q) GUS staining distribution in longitudinal section of a more-stained grain. Bars in (B–Q) are 1 mm.
Molecular Plant 2013 6, DOI: ( /mp/sst087)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
Overexpression of MHZ7 Gene Confers Constitutive and Enhanced Ethylene Responses in Etiolated Seedlings in the Absence and Presence of Ethylene, Respectively.
(A) MHZ7 gene expression levels in overexpressing lines detected using semiquantitative-PCR. OsActin1 was amplified as a loading control.
(B) Ethylene-response phenotypes of WT, mhz7 mutants, and MHZ7-overexpressing lines (MHZ7-OX). The etiolated seedlings were grown in the air (upper panel), 10 ppm of ethylene (middle panel), or 5 ppm of 1-MCP (lower panel) for 4 d.
(C) Root length of various plants in response to ethylene. Each column is average of 20–30 seedlings and bars indicate SD.
(D) Coleoptile length of various plants in response to ethylene. Each column is average of 20–30 seedlings and bars indicate SD.
(E) Expression of ethylene-inducible gene ERF002 in roots of WT and overexpressing lines treated with or without 10 ppm of 1-MCP. The transcript levels were detected using quantitative PCR as described in Figure 2.
Molecular Plant 2013 6, DOI: ( /mp/sst087)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
Comparison of Plant Phenotypes at Seedling, Adult, and Harvest Stages.
(A) Light-grown seedlings floating in water for 1 week.
(B) Representative individuals of WT, mhz7 mutants, and MHZ7-overexpressing lines.
(C) Phenotypic comparison of field-grown plants at flowering stage.
(D) Field-grown plants at mature stage.
(E) Comparison of grains with (left panel) or without the hull (right panel) from WT, five mhz7 allelic mutants, and four MHZ7-overexpressing lines.
(F) Grain length and width of well-filled grains. Each value is average of 20 plants and each plant has 50–100 grains. Bars indicate SD. Different letters above each column indicate significant difference between the compared pairs (P < 0.05).
(G) Comparison of the ratio of grain length/width. Others are as in (F).
(H) Comparison of 1000-grain weight from well-filled grains. Each value is average of 20 plants and each plant has 50–100 grains. Bars indicate SD. ** indicates significant difference compared to WT (P < 0.01).
Molecular Plant 2013 6, DOI: ( /mp/sst087)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

8. Figure 7
Overexpression of MHZ7 Gene Accelerates Leaf Senescence of Rice Seedlings.
(A) Phenotypes of dark-induced senescence in the seedlings of WT, mhz7 mutants, and MHZ7-overexpressing lines. The 10-day-old rice seedlings were transferred to complete dark place for 5 d to induce leaf senescence. The seedlings grown under normal photoperiod conditions were used as light control.
(B) Comparison of the second leaf blades from WT, mhz7 mutants, and MHZ7-overexpressing lines.
(C) Expressions of senescence up-regulated genes in various plants using semiquantitative-PCR.
Molecular Plant 2013 6, DOI: ( /mp/sst087)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions