Identification of Rice Ethylene-Response Mutants and Characterization of MHZ7/OsEIN2 in Distinct Ethylene Response and Yield Trait Regulation

Molecular Plant Volume 6 Number 6 Pages 1830 1848 November 2013 RESEARCH ARTICLE Identification of Rice Ethylene-Response Mutants and Characterization of MHZ7/OsEIN2 in Distinct Ethylene Response and Yield Trait Regulation Biao Ma a, Si-Jie He a, Kai-Xuan Duan a, Cui-Cui Yin a, Hui Chen a, Chao Yang a, Qing Xiong a, Qing-Xin Song a, Xiang Lu a, Hao-Wei Chen a, Wan-Ke Zhang a, Tie-Gang Lu b, Shou-Yi Chen a,1, and Jin-Song Zhang a,1 a State Key Lab of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China b Biotechnology Research Institute/National Key Facility for Genetic Resources and Gene Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China ABSTRACT Ethylene plays essential roles in adaptive growth of rice plants in water-saturating environment; however, ethylene signaling pathway in rice is largely unclear. In this study, we report identification and characterization of ethylene-response mutants based on the specific ethylene-response phenotypes of etiolated rice seedlings, including ethylene-inhibited root growth and ethylene-promoted coleoptile elongation, which is different from the ethylene triple-response phenotype in Arabidopsis. We establish an efficient system for screening and a set of rice mutants have been identified. Genetic analysis reveals that these mutants form eight complementation groups. All the mutants show insensitivity or reduced sensitivity to ethylene in root growth but exhibit differential responses in coleoptile growth. One mutant group mhz7 has insensitivity to ethylene in both root and coleoptile growth. We identified the corresponding gene by a map-based cloning method. MHZ7 encodes a membrane protein homologous to EIN2, a central component of ethylene signaling in Arabidopsis. Upon ethylene treatment, etiolated MHZ7-overexpressing seedlings exhibit enhanced coleoptile elongation, increased mesocotyl growth and extremely twisted short roots, featuring enhanced ethyleneresponse phenotypes in rice. Grain length was promoted in MHZ7-transgenic plants and 1000-grain weight was reduced in mhz7 mutants. Leaf senescent process was also affected by MHZ7 expression. Manipulation of ethylene signaling may improve adaptive growth and yield-related traits in rice. Key words: rice; ethylene-response mutant; MHZ7; yield traits; senescence. Introduction Rice is an important monocotyledonous crop plant worldwide and lives in water-saturating environment in most of time during life cycle. As a semi-aquatic plant, rice is well adapted to hypoxia stress through multiple responses, including coleoptile elongation, adventitious root formation, aerenchyma development, and enhanced (submergence-escape) or repressed (submergence tolerance) shoot elongation (Itoh et al., 2005; Fukao and Bailey-Serres, 2008; Ma et al., 2010). Ethylene acts as a central player in these adaptations (Fukao and Bailey-Serres, 2008; Jackson, 2008; Rzewuski and Sauter, 2008; Steffens and Sauter, 2009; Ma et al., 2010; Steffens et al., 2012). In dark, ethylene promotes coleoptile growth of rice seedlings but inhibits root elongation (Ku et al., 1970; Ma et al., 2010; Kim et al., 2012). This double response is different from the triple response of dark-grown Arabidopsis seedlings, whose hypocotyls and roots are all inhibited, with an exaggerated apical hook (Bleecker and Kende, 2000). Little is known regarding the ethylene signaling pathway that regulates the adaptive responses in rice. In dicotyledonous model plant Arabidopsis, the key components in ethylene signaling pathway have been identified through analysis of ethylene-response mutants based on triple response. These include five ethylene receptors (ETR1, ETR2, EIN4, ERS1, and ERS2), a Raf-like 1 To whom correspondence should be addressed. J.-S.Z. E-mail jszhang@ genetics.ac.cn, tel. 8610-64807602, fax 8610-64806595. S.-Y.C. E-mail sychen@genetics.ac.cn, tel. 8610-64807602, fax 8610-64806595. The Author 2013. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/sst087, Advance Access publication 29 May 2013 Received 15 February 2013; accepted 21 May 2013

Ma et al. MHZ7 Regulates Ethylene Signaling in Rice 1831 ser/thr kinase CTR1, a central membrane protein EIN2, transcription factors EIN3/EIL1, and its downstream gene ERF1 (Chang et al., 1993; Kieber et al., 1993; Hua et al., 1995; Chao et al., 1997; Hua et al., 1998; Sakai et al., 1998; Solano et al., 1998; Alonso et al., 1999). Ethylene can bind to all the ethylene receptors in Arabidopsis and tomato, which negatively regulates ethylene responses (Schaller and Bleecker, 1995; Hua and Meyerowitz, 1998; Tieman et al., 2000; O Malley et al., 2005; Wang et al., 2006). Ethylene receptors from Arabidopsis, tobacco, and rice have His kinase activity and/or Ser/Thr kinase activity (Gamble et al., 1998; Xie et al., 2003; Moussatche and Klee, 2004; Zhang et al., 2004; Chen et al., 2009; Wuriyanghan et al., 2009). The kinase activity and/or phosphorylation state of these receptors plays differential roles in regulation of seedling growth, fruit ripening, and stress response (Wang et al., 2003; Binder et al., 2004; Qu and Schaller, 2004; Xie et al., 2006; Zhou et al., 2006; Chen et al., 2009; Kim et al., 2011; Hall et al., 2012; Kamiyoshihara et al., 2012). Arabidopsis ethylene receptor ETR1 signaling is regulated by RTE1 and CTR1 (Resnick et al., 2006; Zhou et al., 2007; Resnick et al., 2008; Qiu et al., 2012; Xie et al., 2012). CTR1 kinase can phosphorylate the C-terminal domain of EIN2 in the absence of ethylene and EIN2 C-terminus can be cleaved upon ethylene perception and translocated to nucleus for activation of downstream EIN3/EIL1 transcriptional cascade (Ju et al., 2012; Qiao et al., 2012; Wen et al., 2012). EIN2 and EIN3/EIL1 are regulated by proteasomal degradation (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004; Binder et al., 2007; Qiao et al., 2009; An et al., 2010). The Arabidopsis ethylene receptor ETR2 and the tomato ethylene receptor LeETR4 are also subjected to proteasomal degradation (Chen et al., 2007; Kevany et al., 2007). Ethylene receptors, EIN2, EIN3/EIL1, and ERF proteins are all involved in salt stress and other stress responses (Cao et al., 2006; Zhou et al., 2006; Cao et al., 2007; Chen et al., 2009; Lei et al., 2011; Zhang et al., 2011b; Shi et al., 2012). A MA3 domain-containing protein ECIP1 interacts with both ethylene receptors ETR2 and EIN4, and EIN2 to affect seedling growth and salt stress response (Lei et al., 2011). EIN3/EIL1 also regulates multiple other processes (Zhong et al., 2009; Zhu et al., 2011; Zhong et al., 2012). The EIN2 is a membrane protein with 12 predicted transmembrane helices and shows similarity to the Nramp family of proteins (Alonso et al., 1999). However, no metal transport activity is observed (Alonso et al., 1999). EIN2-like proteins have been identified in other plants. In petunia, transgenic plants with reduced PhEIN2 expression exhibit significant delays in flower senescence and fruit ripening, and show reduced adventitious root and seedling root hair formation (Shibuya et al., 2004). In Medicago, MtEIN2/MtSkl1 regulates early phases of the symbiotic interaction with mycorrhizal fungi, and overexpression of the C-terminal domain blocks nodulation response (Penmetsa et al., 2008). In rice, homologous genes of Arabidopsis ethylene signaling components have been identified, including ethylene receptor genes, RTE1-like gene, EIN2-like gene, and EIN3- like gene (Cao et al., 2003; Jun et al., 2004; Watanabe et al., 2004; Yau et al., 2004; Mao et al., 2006; Rzewuski and Sauter, 2008; Wuriyanghan et al., 2009; Ma et al., 2010; Zhang et al., 2012). However, only a few of these have been studied in rice. Overexpression of a subfamily II ethylene receptor gene OsETR2 reduces ethylene sensitivity and delays floral transition in transgenic rice (Wuriyanghan et al., 2009). Starch granules accumulate in internodes of the OsETR2-overexpressing plants and 1000-seed weight is enhanced in seeds of both OsETR2-RNAi plants and knockdown mutant (Wuriyanghan et al., 2009). OsRTH1, a homolog of Arabidopsis ETR1 regulator RTE1, complements loss-of-function mutation in Arabidopsis rte1-2 and inhibits ethylene-induced changes in transgenic rice seedlings (Zhang et al., 2012). Antisense expression of OsEIN2 in transgenic rice seedlings leads to inhibition of shoot elongation (Jun et al., 2004). OsEIL1 overexpression inhibits root growth in transgenic rice seedlings (Mao et al., 2006). ERF proteins including SK1/2 and Sub1A are involved in internode elongation in deepwater rice and submergence tolerance in a few rice cultivars, respectively (Fukao et al., 2006; Xu et al., 2006; Hattori et al., 2009). Considering that rice seedlings have different structure, exhibit differential ethylene response, and grow in water-saturated environment compared to Arabidopsis, it is possible that rice plants exhibit conserved and diverged mechanisms for ethylene signaling. Although roles of several ethylene signaling homologous components from rice have been studied regarding seedling growth and development, their exact functions in ethylene responses are largely unclear, and ethylene-insensitive response mutants of rice are never known. In addition, the system measuring effect of ethylene gas on water-grown rice roots is not available due to difficulty of ethylene treatment. In this study, we have established a system to screen a large population of rice T-DNA insertion lines and ethyl methanesulfonate (EMS)-mutagenized lines, and a set of ethylene-response mutants were identified. Genetic analysis reveals that these mutants form eight complementation groups representing mutations of eight genes. One group of mutant mhz7, which is insensitive to ethylene in both root and coleoptile, is further analyzed. Through map-based cloning, MHZ7 is found to encode a membrane protein homologous to EIN2, a central component of ethylene signaling in Arabidopsis. However, unlike the Arabidopsis EIN2, MHZ7 regulates distinct ethylene-response phenotype in rice. Yield-related traits including grain size, 1000-grain weight, and leaf senescence are also affected by MHZ7 expression. Our study discloses novel features of ethylene response in rice and provides valuable clues for improvement of agronomic traits in crops through manipulation of ethylene signaling.

1832 Ma et al. MHZ7 Regulates Ethylene Signaling in Rice RESULTS Screening and Genetic Analysis of Rice Ethylene- Response Mutants A T2 T-DNA insertion population of 15 000 enhancer trapping lines (Yang et al., 2004; Peng et al., 2005) and 16 000 activation tagging lines (Wan et al., 2009) were screened for rice ethylene-response mutants using an apparatus developed (Supplemental Figure 1). After ethylene treatment, wild-type (WT) seedlings showed short adventitious roots and short primary roots compared to air-treated seedlings (Figure 1A). In contrast, some segregated seedlings from a set of T-DNA insertion lines exhibited non-affected growth of adventitious roots (Figure 1B, blue circled) and these seedlings were regarded as ethylene-insensitive mutants and named as mao huzi (mhz, Chinese name with a English meaning of cat whiskers). The segregated WT seedlings had short adventitious roots (Figure 1B, orange circled). In total, 21 mutant lines were obtained and all the mutants were recessive (Table 1 and data not shown). Eight complementation groups were identified, namely mhz1, mhz2, mhz3, mhz4, mhz5, mhz6, mhz7, and mhz8 (Table 2). We further screened 23 000 lines from an EMS-mutagenized M2 population and 17 mhz mutants were obtained. Genetic allelic analysis placed 10 of these mutants into the above complementation groups, namely two in mhz1 group, one in mhz2 group, three in mhz3 group, one in mhz6 group, and three in mhz7 group (Table 2). Other mutants are still being analyzed. Comparison of Phenotypes and Ethylene Responses in Dark-Grown Ethylene-Response Mutants The eight mutant groups were further analyzed for ethylene responses. The mhz1-1 mutant had a strong ethylene-insensitive phenotype in roots (Figure 1C and 1D). However, the coleoptile of the mutant seemed to have a similar response to ethylene compared to WT seedlings, although the air-grown mhz1 seedlings had slightly longer coleoptiles (Figure 1E). The mhz3-1, mhz6-1, and mhz7-1 mutants all showed almost complete insensitivity to ethylene in roots (Figure 1C and 1D). In coleoptiles, the mhz3-1 and mhz7-1 had strong insensitivity whereas the mhz6-1 had reduced sensitivity in ethylene-promoted coleoptile elongation (Figure 1C and 1E, and Table 2). The roots of mhz4 and mhz5-1 were shorter than that of WT seedlings and showed reduced sensitivity to ethylene (Figure 1C and 1D). Coleoptiles of the two mutants were longer than that of WT seedlings in ethylene, indicating a hypersensitive response to ethylene (Figure 1E). However, the mhz5-1 had weak phenotype compared to mhz4 (Figure 1D and 1E). The mhz2-1 and mhz8 mutants were insensitive to ethylene in root growth (Figure 1C and 1D). Their coleoptiles showed ethylene responses similar to the WT seedlings (Figure 1E). It should be noted that both mutants had agravitropic roots, suggesting that the two loci are involved in both ethylene inhibition of root growth and root response to gravity. These analyses indicate that roots of the mutants have insensitivity or reduced sensitivity to ethylene inhibition whereas coleoptiles of these mutants show differential responses to ethylene. mhz1, mhz2, and mhz8 are very similar to WT in coleoptile response. mhz3, mhz6, and mhz7 are insensitive or less insensitive to ethylene in coleoptile growth. mhz4 and mhz5 are hypersensitive to ethylene in ethylenepromoted coleoptile growth. Ethylene Dose Response and Ethylene-Responsive Gene Expressions in mhz7 Mutant WT primary root growth was drastically reduced in treatments with ethylene from 0.1 to 10 ppm (µl/l) (Figure 2A and 2B). 1-Methylcyclopropene (1-MCP), a chemical structurally related to ethylene, can tightly bind to the ethylene receptors in plants and thereby block the effects of ethylene (Serek et al., 1995; Sisler and Serek, 2003). Addition of 1-MCP to the WT seedlings suppressed the ethylene-inhibited root growth, indicating that the measured root response is an ethylene response (Figure 2B). The two allelic mutants mhz7-1 and mhz7-2 were completely insensitive to ethylene in terms of ethylene inhibition of root growth. Coleoptile growth of WT seedlings was promoted after ethylene treatment and 1-MCP almost blocked the promotion (Figure 2C). At higher concentrations of ethylene (100 ppm), the 1-MCP blocking was weakly inhibited. The mhz7-1 and mhz7-2 had strong ethylene-insensitive phenotype regarding coleoptile promotion (Figure 2C). The other three mutants mhz7-3, mhz7-4, and mhz7-5 also showed insensitivity to ethylene treatment in both root and coleoptile growth (Supplemental Figure 2). These results indicate that the mhz7 mutants are insensitive to ethylene in both root inhibition and coleoptile promotion. To further examine the ethylene response of the mhz7 mutants, we studied expressions of ethylene-responsive genes originally identified from a chip analysis (GSE45751). Four genes including SHR5 (Os08g10310) encoding a receptorlike kinase, Germin-like protein gene (Os08g13440), ERF063 (Os09g11480), and ERF073 (Os09g11460) were found to be induced by ethylene in both roots and shoots of WT seedlings (Figure 2D). In shoots and roots of the mhz7-1 mutant, the expressions of the four genes remained at a very low level after ethylene treatment compared to their ethylene induction in WT, indicating unresponsiveness to ethylene. Five genes (Peptidylprolyl isomerase gene, Os01g38359; expressed protein gene, Os06g38660; DUF581 domaincontaining protein gene, Os06g03520; RGLP1 encoding a germin-like protein, Os08g09080; and ERF002, Os06g08340) were found to be induced only in roots of WT seedlings but not in roots of mhz7-1 seedlings (Figure 2E). Three genes including DUF26 kinase gene (Os07g35810), peroxidase BP1 gene (Os01g73200), and a DNA binding protein gene (Os06g12210) were induced to a higher level in WT shoots

Ma et al. MHZ7 Regulates Ethylene Signaling in Rice 1833 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.

1834 Ma et al. MHZ7 Regulates Ethylene Signaling in Rice Table 1. Genetic Segregation of Rice Ethylene-Response Mutants. Crosses F1 F2 χ 2 + + Expected ratio mhz1-1 WT 46 0 276 86 3:1 0.24 mhz2-1 WT 45 0 55 23 3:1 0.62 mhz3-1 WT 29 0 142 42 3:1 0.36 mhz4 WT 35 0 141 17 3:1 16.34 mhz5-1 WT 20 0 228 68 3:1 0.55 mhz6-1 WT 40 0 189 57 3:1 0.46 mhz7-1 WT 27 0 229 77 3:1 0 mhz8 WT 14 0 183 65 3:1 0.13 +, mutant phenotype;, WT phenotype. Please note that the ratio in mhz4 WT is not consistent with 3:1 due to abnormal germination of mutant seeds; however, later map-based cloning reveals that the mutation occurs in a single gene. than those in mhz7-1 shoots (Figure 2F). These results indicate that the mhz7-1 mutant is ethylene-insensitive in both roots and shoots for some ethylene-responsive gene expressions. However, for other genes mentioned above, MHZ7 locus may control their ethylene inductions specifically in roots or in shoots. Identification of MHZ7 Gene through Map-Based Cloning Because none of the mutant phenotype of mhz7 co-segregated with T-DNA insertion, we adopted a map-based cloning strategy to isolate the gene. The mhz7-1 mutant was crossed to four indica cultivars and 819 segregated mutant individuals Table 2. Complementation Groups of Ethylene-Response Mutants. Groups Mutants Phenotype mhz1 mhz2 mhz3 mhz1-1, mhz1-2. mhz1-3, mhz1-4 mhz2-1, mhz2-2, mhz2-3, mhz2-4, mhz2-5, mhz2-6, mhz2-7, mhz2-8 mhz3-1, mhz3-2, mhz3-3, mhz3-4, mhz3-5 Root insensitivity Root insensitivity, no root gravity response Root and coleoptile insensitivity mhz4 mhz4 Reduced sensitivity in root, coleoptile hypersensitivity mhz5 mhz6 mhz7 mhz5-1, mhz5-2, mhz5-3 mhz6-1, mhz6-2, mhz6-3, mhz6-4 mhz7-1, mhz7-2, mhz7-3, mhz7-4, mhz7-5 Reduced sensitivity in root, coleoptile hypersensitivity Root insensitivity Root and coleoptile insensitivity mhz8 mhz8 Root insensitivity, weak gravity response in root from F2 population were used for fine mapping with Indel and/ or SSR markers. Finally, mhz7-1 locus was mapped to chromosome 7 within the region between marker SNP7-2.9-DraI and Idl7-3.14 (Figure 3A). In this region, the gene Os07g06130 had 24-bp deletions in the first exon. Os07g06130 locus encoded a membrane protein similar to EIN2, which is a central component of ethylene signaling in Arabidopsis (Alonso et al., 1999). In rice, this gene has been named as OsEIN2 and analyzed through antisense approach; however, the ethyleneresponse phenotype of the antisense plants was not pursued (Jun et al., 2004). The present mhz7-1 mutation led to deletion of eight amino acid residues in MHZ7 (Figure 3B). The mutation was also confirmed by PCR through examination of cdna fragment length polymorphism (Figure 3C, left panel). The mhz7-2 harbored a mutation from C to T at the 5694-bp position, resulting in a stop codon and loss of 11 amino acid residues from the C-terminal end of MHZ7 protein (Figure 3A and 3B). This mutation was also confirmed by dcaps assay using PCR (Figure 3C, right panel). The mhz7-3 harbored a mutation from G to A at 5149 bp, and mhz7-4 had a mutation from C to T at 5547 bp. Both mutations produced stop codons (Figure 3A and 3B). The mhz7-5 harbored a mutation from G to A at 906 bp, disrupting the splicing signal and resulting in a loss of 87 bp in cdna and missing of 29 residues in protein (Figure 3A and 3B). The ethylene-response and mutant identification of mhz7-3, mhz7-4, and mhz7-5 are presented in Supplemental Figure 2. Mutation analysis in the five allelic mhz7 mutants supports that MHZ7 is Os07g06130, and reveals that all the five mutant alleles from the screen are loss-of-function mutations. This conclusion is further demonstrated by mutant complementation tests (Figure 3D, lines 22 and 36). Seedlings with longer coleoptiles were also observed in T1 segregated seedlings of some individual transgenic lines after ethylene treatment (Figure 3D, lines 10 and 32), possibly reflecting a slightly enhanced response to ethylene due to overexpression of MHZ7/Os07g06130. MHZ7 Expression Revealed by Promoter GUS Analysis The 3.3-kb promoter region of MHZ7 was cloned to drive the GUS gene and the promoter GUS construct was transformed into rice plants to reveal the gene promoter activity and gene expression by GUS staining. The MHZ7 gene was abundantly expressed in coleoptiles and roots compared to other organs (Figure 4A). It was also expressed in shoot tips (Figure 4A). In coleoptile the staining was focused on tip/top and vascular tissues (Figure 4A 4C). In roots, the staining was observed in adventitious roots (Figure 4C), vascular tissues of the seminal roots, and lateral roots (Figure 4D and 4E). The staining was relatively strong in the connecting region between vascular tissues and lateral roots; however, MHZ7 expression was not detected in root tips (Figure 4D 4F). The MHZ7 gene was also expressed in mature leaf and stem (Figure 4G and 4H). It was interesting to note that the staining was very strong in tips of adventitious roots derived from the node (Figure 4H and 4I). The GUS staining was very strong in shoot apex, young

Ma et al. MHZ7 Regulates Ethylene Signaling in Rice 1835 panicle, and anthers of a flower (Figure 4J and 4K). In pistil, the GUS staining in stigma gradually went down, and the bottom and top of the ovary were further stained (Figure 4L 4N). In developing grain, the top was heavily stained (Figure 4O). The grains were also sectioned longitudinally and a lessstained and a more-stained grain were examined. The GUS staining was mainly concentrated on top of the seed coat, the second layer from the outside fruit coat pericarp (Figure 4P and 4Q). These results indicate that MHZ7 is expressed in various organs and may play major roles in root, coleoptile and grain development. Overexpression of MHZ7 Leads to Constitutive Ethylene Response We further transformed the MHZ7 (Os07g06130) gene, with the control of 35S promoter, into WT rice plants to investigate the gene functions. Among MHZ7-overexpressing lines (Figure 5A), four (OX2, OX3, OX4, and OX44) were further analyzed. The four lines all showed short roots and longer coleoptiles in dark compared to the WT seedlings, suggesting presence of constitutive ethylene response (Figure 5B 5D). After ethylene treatment, the four transgenic lines exhibited enhanced ethylene-response phenotypes, including very short and extremely twisted roots, elongated mesocotyls, and very long coleoptiles in comparison with the WT and mhz7 mutants (Figure 5B 5D). Application of 1-MCP to etiolated seedlings of MHZ7-overexpressing lines completely eliminated the ethylene response of air-grown roots (Figure 5B 5D). However, 1-MCP only partially reduced the constitutive ethylene response in coleoptiles because the 1-MCP-treated coleoptiles of MHZ7-overexpressing plants were still longer than the WT coleoptiles (Figure 5B 5D). Examination of the ethylene-responsive gene ERF002 (Figure 2E) under normal and 1-MCP-treated conditions further proved that the MHZ7- overexpressing lines had constitutive ethylene response (Figure 5E). These results indicate that MHZ7 overexpression leads to constitutive ethylene response and confers enhanced response to ethylene treatment. Seedling Growth and Yield-Related Traits in mhz7 Mutants, MHZ7-Overexpressing Plants, and WT Plants Seedling growth from various plants was examined under light. Compared to WT seedlings with curved roots plus relatively long shoots, the two mhz7 mutants had relatively straight roots and short shoots (Figure 6A and 6B). MHZ7- overexpressing plants had extremely twisted roots compared to WT and mhz7 mutants. These results likely reflect that light-grown mhz7 seedlings have insensitive response whereas MHZ7-overexpressing plants have constitutive response especially in root growth to basal ethylene levels produced in rice seedlings during adaptive growth in water. Field-grown plants were examined and MHZ7- overexpressing plants appeared to be shorter than WT plants (Figure 6C). Panicles from mhz7 mutants and MHZ7-overexpressing plants were more erect compared to those from WT plants (Figure 6C). At maturation stage, all the plants had drooping panicles. However, mhz7 mutants seemed to have slightly more green seeds and more grains with brown regions than other plants (Figure 6D). After harvest, the yield-related and other traits were evaluated (Table 3). All the four MHZ7-overexpressing plants were shorter than WT and mhz7 mutants. The total number of grains per panicle was significantly reduced in MHZ7-overexpressing plants compared to that in WT. The 1000-grain weights of total grains in the four MHZ7-overexpressing plants were also substantially decreased (Table 3). The seed-setting rate and total grain weight per plant were all reduced in mhz7 mutants and MHZ7-overexpressing plants (Table 3), suggesting that extreme ethylene response caused by alteration of MHZ7 expression is not beneficial for these traits. All these analyses indicate that modulation of MHZ7 expression affects yieldrelated traits in rice. The grain size was further examined in mhz7 mutants and MHZ7-overexpressing plants using well-filled grains. Among the five mhz7 mutants (Table 2 and Figure 3), four mutants had reduction in grain length and five mutants showed decrease in grain width compared to WT grains (Figure 6E and 6F), indicating that MHZ7 mutation reduced grain size. In MHZ7-overexpressing plants, grain length was increased in all the four lines (MHZ7-OX2, 3, 4, and 44) compared to that in WT (Figure 6E, left panel). However, grain width was not significantly affected in three of the four MHZ7-overexpressing lines (Figure 6F, right panel). Ratio of grain length/grain width varied in mhz7 mutants (Figure 6G). In three out of four MHZ7-overexpressing lines, the ratio was increased (Figure 6G). Because the grain size was altered, we further measured the 1000-grain weight for well-filled grains. Four mhz7 mutants had significant reductions in this parameter whereas three MHZ7-overexpressing lines showed no change (Figure 6H). These results possibly indicate that MHZ7 disruption affects 1000-grain weight. Alteration of MHZ7 Expression Affects Leaf Senescence When grown in water for a longer time, the MHZ7- overexpressing plants exhibit an early senescence-like phenotype (Supplemental Figure 3). We then investigated whether the leaf senescence was affected by MHZ7. Dark treatment can mimic the senescent process. Three-week-old pot-grown seedlings were then used for dark treatment. In the dark for 5 d, the MHZ7-overexpressing plants turned yellow whereas the mhz7 mutants remained green (Figure 7A). WT plants appeared to be yellow-green. The second leaf of these plants was further examined. In light conditions, the second leaf of the four MHZ7-overexpressing lines wilted from the tip (Figure 7B, left panel). In dark, the second leaf of these overexpressing plants all became yellow and/or wilted (Figure 7B, right panel). The leaves from the two mutants were still green compared to WT leaf, which has turned yellow from the tip

1836 Ma et al. MHZ7 Regulates Ethylene Signaling in Rice 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 8 h 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).

Ma et al. MHZ7 Regulates Ethylene Signaling in Rice 1837 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 Idl7-3.14 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 (http://smart.emblheidelberg.de). 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 ethyleneinsensitive 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.

1838 Ma et al. MHZ7 Regulates Ethylene Signaling in Rice 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.

Ma et al. MHZ7 Regulates Ethylene Signaling in Rice 1839 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.

1840 Ma et al. MHZ7 Regulates Ethylene Signaling in Rice 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).

Ma et al. MHZ7 Regulates Ethylene Signaling in Rice 1841 Table 3. Yield-Related Traits in WT, mhz7 Mutants, and MHZ7-Overexpressing Rice Lines. Plant genotypes Plant height (cm) ± SD Number of panicles per plant ± SD Number of grains per plant ± SD Panicle length (cm) ± SD Number of grains per panicle ± SD Seed-setting rate (%) ± SD 1,000-grain weight (g) ± SD Grain weight per plant(g) ± SD WT 93.2 ± 2.9 7.7 ± 1.5 770.1 ± 187.7 19.1 ± 1.0 99.6 ± 10.3 82.5 ± 7.0 22.2 ± 1.2 14.1 ± 3.6 mhz7-1 95.0* ± 2.2 8.2 ± 1.6 760.0 ± 177.1 19.4 ± 0.7 93.3 ± 11.5 64.3** ± 10.5 22.1 ± 0.8 10.8** ± 3.0 mhz7-2 90.1** ± 2.4 8.8* ± 1.6 768.3 ± 152.5 19.5 ± 0.8 88.5** ± 11.8 70.9** ± 3.7 19.7** ± 1.1 10.8** ± 2.5 OX2 85.4** ± 3.1 8.6 ± 1.9 687.3 ± 148.7 18.1** ± 0.7 80.9** ± 12.9 76.2** ± 4.4 20.3** ± 0.8 10.7** ± 2.6 OX3 83.8** ± 3.1 8.3 ± 1.3 564.9* ± 127.8 16.6** ± 0.8 67.7** ± 8.5 72.9** ± 7.6 21.6 ± 1.0 8.7** ± 2.4 OX4 87.8** ± 3.6 9.3** ± 1.6 702.4 ± 128.2 19.4 ± 0.7 75.2** ± 9.0 77.3** ± 4.6 21.2** ± 0.7 11.3** ± 1.9 OX44 80.9** ± 3.2 11.1** ± 1.6 746.0 ± 118.4 18.7 ± 0.7 67.4** ± 7.1 65.3** ± 6.7 21.0** ± 1.0 10.2** ± 1.9 Each value is average of 20 plants. * and ** indicate significant difference compared to WT at P < 0.05 and P < 0.01, respectively. (Figure 7B, right panel). These results indicate that MHZ7 overexpression leads to leaf senescence whereas its disruption delays senescence of plants. Previously, we found that AtNAC2 is involved in stress responses and regulated by ethylene receptors and EIN2 of ethylene signaling (He et al., 2005). Later, the gene AtNAC2/ ORE1 was further found to be involved in leaf senescent process in Arabidopsis (Kim et al., 2009). NAC homologous genes and other senescence-related genes from rice (Lee et al., 2000) were examined in the present mhz7 mutants and MHZ7-transgenic lines. In light conditions, the MHZ7- overexpressing lines showed higher expressions of OsNAC1 (Os06g46270), OsNAC2 (Os04g38720), OsL43 (Os01g24710), OsL85 (Os07g34520), and OsL55 (Os12g41250) than WT and/ or mhz7 mutants (Figure 7C). Dark treatment enhanced the six gene expressions including Osh36 (Os05g39770) in all the plants compared. The inductions were high in the four MHZ7-overexpressing plants compared to that in WT (Figure 7D). In contrast, the inductions were relatively weak in the two mhz7 mutants compared to the WT (Figure 7D). These results suggest that MHZ7 promotes leaf senescence through up-regulation of OsNAC1/2 and other senescencerelated genes. Discussion Taking advantage of the distinct ethylene-response phenotypes of etiolated rice seedlings, namely root inhibition but coleoptile elongation, we have established an efficient system to screen rice ethylene-response mutants. All the eight complementation mutant groups exhibited insensitivity or reduced sensitivity to ethylene in roots but showed differential responses to ethylene in coleoptiles. This phenomenon may suggest that the relevant genes regulate root adaptive growth and at the same time they differentially affect adaptive growth of coleoptiles/shoots. The differential regulatory mechanism may coincide with the specific ethylene responses of etiolated rice seedlings (Figure 1A), suggesting possible presence of novel ethylene regulatory mechanism in rice. Cloning and functional analysis of the relevant genes from mutants should dissect the novel and conserved parts of the ethylene signaling pathway in rice. We have identified five mhz7 allelic mutants and all the mutants showed insensitivity to ethylene treatment in both root and coleoptile responses, suggesting that MHZ7 may control the adaptation growth in whole seedlings. MHZ7 encoded a homolog of EIN2, which is a central membrane protein of ethylene signaling in Arabidopsis (Alonso et al., 1999). This fact indicates that our system for screening ethylene-response mutants is effective. Recently, Arabidopsis EIN2-overexpressing seedling has been found to display a stunted phenotype in the presence of ACC (Ju et al., 2012). However, unlike this phenotype, MHZ7-overexpressing rice seedlings exhibit enhanced coleoptile elongation, increased mesocotyl growth, and extremely twisted short roots in the presence of ethylene (Figure 5), indicating an enhanced ethylene-response phenotype in rice, which has never been known before. These different phenotypic changes reflect plant-specific responses to ethylene, and the elongated mesocotyl and coleoptile of rice seedlings will facilitate survival in water-saturated soil environment. Without ethylene treatment, MHZ7-overexpressing lines showed constitutive ethylene-response phenotype, including short roots but long coleoptiles compared to etiolated WT seedlings (Figure 5B 5D). These phenotypes may be correlated with the high MHZ7 expression in roots and coleoptiles revealed by promoter GUS analysis (Figure 4). Ethylene-responsive gene expression analysis (Figure 5E), light-grown seedling phenotypes (Figure 6A and 6B, and Supplemental Figure 3) and leaf senescence assay (Figure 7) also support that MHZ7-overexpressing rice plants had constitutive ethylene responses. The enhanced and reduced ethylene responses in coleoptile growth have been observed in ethylene receptor OsETR2 knockdown mutants and OsETR2-overexpressing rice plants, respectively, in our previous studies (Wuriyanghan et al., 2009). These analyses suggest that MHZ7 controls a set of ethylene-response

1842 Ma et al. MHZ7 Regulates Ethylene Signaling in Rice 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. phenotype different from the triple-response phenotype in Arabidopsis. Further study may disclose the mechanisms by which the different ethylene-response phenotype was regulated in rice. It should be noted that the coleoptile and root growth in MHZ7-overexpressing lines were differentially responsive to 1-MCP treatment. Application of 1-MCP to etiolated seedlings of MHZ7-overexpressing lines completely eliminated the ethylene response of air-grown roots. However, 1-MCP only partially reduced the constitutive ethylene response in coleoptiles (Figure 5B 5D). Because 1-MCP acts on the ethylene receptors upstream of MHZ7/OsEIN2 in the signaling

Ma et al. MHZ7 Regulates Ethylene Signaling in Rice 1843 pathway, it is reasonable that 1-MCP cannot completely block the constitutive ethylene response in coleoptiles of the MHZ7- overexpressing plants. However, it is unexpected that the root length of MHZ7-overexpressing lines is completely responsive to 1-MCP. It is possible that, with 1-MCP, ethylene perception and signaling are blocked and the OsCTR1 would presumably be constitutively active, leading to inactivation of overexpressed OsEIN2, as proposed in the case of Arabidopsis ethylene signaling (Ju et al., 2012). More OsCTR1 activity but less OsEIN2 activity and/or expression in roots may then result in elimination of the constitutive ethylene response in roots of MHZ7-overexpressing lines upon 1-MCP treatment. Alternatively, other ethylene signaling pathway in rice roots may be present and characterization of mhz1 mutant, which is specifically root-insensitive to ethylene, may elucidate the mechanism. Additional MHZ7/OsEIN2 homologs may also play some subtle roles. Previously, rice OsEIN2 has been studied using a antisense transgenic approach and the seedlings with reduced OsEIN2 expression showed short shoots compared to WT (Jun et al., 2004). Our present mhz7 mutants showed similar short shoots in light at seedling stage (Figure 6A and 6B). Most importantly, our mhz7 mutants had clear and definite ethylene-insensitive response in both root growth and coleoptile growth, representing typical ethylene-insensitive phenotypes (Figure 2A 2C, and Supplemental Figure 2). This ethylene-insensitive response has not been observed in previous study (Jun et al., 2004). The ethylene insensitivity of the mhz7 mutants was further demonstrated by the absence of ethylene inductions of ethylene-responsive genes (Figure 2D). Lack of dark-induced leaf senescence in mhz7 mutant also supported the ethylene insensitivity of the mutants (Figure 7), showing that Arabidopsis EIN2 regulates the ethylene-mediated leaf senescent process (Kim et al., 2009). Light-grown mhz7 seedlings also showed phenotypes of ethylene insensitivity (Figure 6A and 6B, and Supplemental Figure 3). Considering the importance and central role of MHZ7 in ethylene signaling, the ethyleneinsensitive mhz7 mutants are a valuable tool for study of genetic interactions with other genes and/or pathways. It should be mentioned that, although both mhz7 roots and coleoptiles were insensitive to ethylene, MHZ7 regulated specific sets of genes for root and coleoptile/shoot responses, respectively (Figure 2E and 2F), in addition to a common set of genes for ethylene responses in both organs (Figure 2D). Arabidopsis EIN2 can be phosphorylated by CTR1 mainly at Ser645 and Ser924, and preventing EIN2 phosphorylation at the two residues results in constitutive ethylene response (Ju et al., 2012; Qiao et al., 2012). Ethylene triggers dephosphorylation of EIN2 and leads to cleavage and nuclear translocation of the C-terminal domain of EIN2 (Ju et al., 2012; Qiao et al., 2012; Wen et al., 2012). The present rice MHZ7 also contains the two conserved phosphorylation sites; however, whether these sites can be phosphorylated remains to be studied. In our mhz7-2 mutant, the mutation led to loss of 11 residues from the MHZ7 C-terminal end and disrupted the nuclear localization signal (Figure 3). It will be interesting to see whether the nuclear translocation of the C-terminal end of MHZ7 is affected in rice if the EIN2 signaling mechanism is conserved in plants. MHZ7 promoter GUS analysis reveals that the gene was apparently expressed in panicles and developing grains (Figure 4), suggesting that MHZ7 may regulate seed/yieldrelated traits. We found that MHZ7-overexpressing rice plants produced longer grains whereas the mhz7 mutants produced smaller grains compared to WT, indicating that MHZ7 controls grain size and may increase the sink capacity of grains. Small grains of mhz7 mutants correlated with low levels of 1000-grain weight for well-filled grains, whereas this value in the MHZ7-overexpressing plants was not enhanced along with the larger grains (Figure 6E and 6F). The inconsistency between grain size and 1000-grain weight of well-filled grains in MHZ7-transgenic plants may be due to the fact that the MHZ7-overexpressing plants produced a similar level of storage reserves to WT to fill the enlarged grains. Alternatively, changes in storage compositions, structure of starch granules, and/or other unknown factors in grains of MHZ7-overexpressing plants may also cause the inconsistency. It should be mentioned that the 1000-grain weight of total grains, including those well-filled and lessfilled grains, was even reduced in the MHZ7-overexpressing plants (Table 3). This reduction could probably result from the early leaf senescence observed in MHZ7-overexpressing plants. It is interesting to note that, while all the five mhz7 mutants showed similar ethylene-insensitive responses, they exhibited difference in yield-related traits. The mhz7-1 was different from the other four mutants in that this mutant had a slight longer grain and no decrease in 1000- grain weight for either well-filled grains or total grains compared to WT (Figure 6E 6G and Table 3). This inconsistency was probably due to the allele-specific mutation in mhz7-1, causing loss of eight amino acid residues between transmembrane regions in MHZ7, although we could not exclude other possibilities. It will be interesting to test the grain/yield change if we manipulate a seed-specific promoter-driven MHZ7 to transform the mhz7-1 mutant. In this case, the final transgenic rice plants are expected to have delayed leaf senescence and possess large grains and/ or high 1000-grain weight. Alterations of other components in ethylene signaling and ethylene biosynthesis also affect grain- and/or yield-related traits. Overexpression of ethylene receptor gene OsETR2 leads to delayed flowering and accumulation of starch granules in stems of transgenic rice plants. Reduced expression of OsETR2 causes early flowering and enlarged grains with higher 1000-grain weight compared to WT (Wuriyanghan

1844 Ma et al. MHZ7 Regulates Ethylene Signaling in Rice et al., 2009). The OsETR2 may play these roles through activation of late-flowering genes OsGI and RCN1, and inhibition of α-amylase gene Ramy3D and a monosaccharide transporter gene (Wuriyanghan et al., 2009). Transgenic canola plants expressing the bacterial ACC deaminase gene had smaller siliques with reductions in seed size and seed number, and application of ethephon restored the WT phenotype for both siliques and seeds (Walton et al., 2012). However, a biphasic effect was observed when the same level of ethephon was applied to WT plants, and this treatment diminished silique and seed development (Walton et al., 2012). Similar biphasic effects appeared to happen in a few traits of the present mhz7 mutants and MHZ7-overexpressing plants. The seedsetting rate and grain weight per plant were all reduced in the two kinds of plants with contrasting ethylene responses (Table 3). Seed size and/or shape were also altered in ethylene-response mutants of Lotus oleoptil, Medicago truncatula, and Arabidopsis (Robert et al., 2008; Cervantes et al., 2010, 2012). Overexpression of an ethylene-induced NEK6 kinase in Arabidopsis increased silique length, silique number, and seed weight per plant but reduced seed size in addition to its roles in plant growth and stress tolerance (Zhang et al., 2011a). Regarding the general roles of ethylene in rice plants, it is reasonable that, in a water-saturating environment, the accumulated ethylene will promote coleoptile/shoot elongation, adventitious root emergence, and aerenchyma development for adaptive growth and better survival (Rzewuski and Sauter, 2008; Ma et al., 2010). Ethylene inhibition of seminal roots in seedlings may also benefit plants in avoidance of risks for lacking oxygen and other resources required for root growth during hypoxia stress. However, extreme alterations of ethylene response are not beneficial for survival of rice seedlings. Ethylene insensitivity will lead to short coleoptile and/or shoots, which will be harmful for survival due to lack of oxygen under hypoxia conditions. Hypersensitivity will result in extremely abnormal root systems (Figure 5B, middle panel), which are not suitable for supporting plant structure and absorbing nutrients. At maturation stage, insensitivity tends to cause small grains but delayed leaf senescence whereas constitutive ethylene response likely increases grain length but exerts a risk of early senescence. Therefore, plants need to make a subtle and delicate balance between these extreme conditions for better survival and better grain production for later generations. Taken together, we have identified ethylene-response mutants in rice and found that MHZ7 played essential roles in regulation of distinct ethylene response of etiolated seedlings. MHZ7 overexpression resulted in enhanced coleoptile elongation, increased mesocotyl growth, and extremely twisted short roots in the presence of ethylene. Grain length was promoted and leaf senescence was affected in MHZ7- transgenic plants. Thousand-grain weight was substantially reduced for well-filled grains in mhz7 mutants. Manipulation of MHZ7 may improve adaptation and yield-related traits, and further study of other mhz mutants should disclose more novel and conserved aspects of ethylene signaling in rice. METHODS Plant Materials and Growth Conditions The rice (Oryza sativa L.) variety Nipponbare (japonica) (WT) was used in this study. For material propagation, cross, and investigation of agronomic traits, rice plants were grown in the experimental farm of the Institute of Genetics and Developmental Biology in Beijing from May to October of each year. Isolation of Ethylene-Response mhz Mutants To isolate ethylene-response mutants of rice, we screened 31 000 T-DNA insertion lines and 24 000 EMS-mutagenized lines. The T-DNA insertion populations were generated previously (Yang et al., 2004; Peng et al., 2005; Wan et al., 2009). For EMS mutagenesis, rice seeds were presoaked for 16 h at room temperature, and then treated with 0.6% EMS (Sigma, M0880) for 8 h at room temperature. The seeds were germinated at 37 C in the dark and grown in the field. T2 generation seeds of T-DNA insertion lines or M2 generation seeds of EMS-mutagenized lines were used for mutant screening. About 40 50 seeds of each individual line were placed on a stainless sieve which was placed in a 20-L air-tight plastic box (Supplemental Figure 1A and 1B). The seeds were soaked in tap water and germinated at 37 C in the dark for 2 d (the water was changed daily). For ethylene treatment, we reduced the amount of water to 1.5 1.7 cm below the seeds (Supplemental Figure 1C, this is important). Ethylene gas (10 ppm) was injected into the boxes using a syringe. Replacements of water and ethylene treatment were carried out under dim green light (Ku et al., 1970). The seedlings were cultured at 28 C in the dark for 4 d. Ethylene-response mutants were selected according to the phenotypes of roots and coleoptile of etiolated seedlings (Supplemental Figure 1D). A similar method was used in all ethylene treatments in this study. Genetic Analysis The mhz mutants were crossed with WT Nipponbare. Etiolated seedlings from F1 and F2 progenies were treated with 10 ppm of ethylene to determine the dominant/recessive characteristic and loci number of the mutations, respectively. Segregation of ethylene-response phenotype in F2 progenies were analyzed for goodness of fit to 3:1 ratio (WT:mutant) using a Chi-square test (χ 2 < χ 2 0.05,1 = 3.84). For allelic analysis, mhz mutants were crossed with each other, and complementation groups were identified based on ethylene-response phenotypes of F1 seedlings and dominant/recessive characteristics of the mutations.