Ethylene Responses in Rice Roots and Coleoptiles AreDifferentially Regulated by a Carotenoid Isomerase-MediatedAbscisic Acid Pathway OPEN

Cui-Cui Yin,a,b,1 Biao Ma,a,1,2 Derek Phillip Collinge,c Barry James Pogson,c Si-Jie He,a Qing Xiong,a,b

Kai-Xuan Duan,a,b Hui Chen,a,b Chao Yang,a,b Xiang Lu,a,b Yi-Qin Wang,a Wan-Ke Zhang,a Cheng-Cai Chu,a

Xiao-Hong Sun,d Shuang Fang,d Jin-Fang Chu,d Tie-Gang Lu,e Shou-Yi Chen,a,2 and Jin-Song Zhanga,2

a State Key Lab of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101,ChinabUniversity of Chinese Academy of Sciences, Beijing 100049, Chinac Australian Research Council Centre of Excellence in Plant Energy Biology, Research School of Biology, Australian NationalUniversity, Canberra, Australian Capital Territory 0200, Australiad National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,Beijing 100101, Chinae Biotechnology Research Institute/National Key Facility for Genetic Resources and Gene Improvement, Chinese Academy ofAgricultural Sciences, Beijing 100081, China

Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA isderived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis,and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response inroots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that themutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolatedseedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, theproduction of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results inenhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5also alters the expression of ethylene-responsive genes. Genetic studies revealed that theMHZ5-mediated ABA pathway actsdownstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream butnegatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene,carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through themanipulation of these pathways in rice.

INTRODUCTION

Ethylene is synthesized from methionine by three well-definedenzymatic reactions (Lin et al., 2009). The biosynthesis pathway ofethylene is regulated via the 1-aminocyclopropane-1-carboxylatesynthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase(ACO) enzymes at either the transcriptional or posttranslationallevels (Argueso et al., 2007; Lyzenga and Stone, 2012; Lyzengaet al., 2012). Ethylene is perceived by a multimember family ofmembrane-bound receptors that possess sequence similaritywith the bacterial two-component His kinase (Hall et al., 2007).Then, the ethylene signal is transmitted via a simple signal cascade

that comprises CONSTITUTIVE TRIPLE RESPONSE1 (CTR1),ETHYLENE INSENSITIVE2 (EIN2), EIN3, and/or EIN3-LIKE1 (Anet al., 2010; Mayerhofer et al., 2012; Qiao et al., 2012; Merchanteet al., 2013). In addition to the linear pathway, several compo-nents, including REVERSION TO ETHYLENE SENSITIVITY1(RTE1) (Resnick et al., 2006; Dong et al., 2010), EIN3 Binding F-box 1 and 2 (Guo and Ecker, 2003), EIN2-TARGETING PROTEIN1(ETP1) and ETP2, also play important regulatory roles in the eth-ylene response (Qiao et al., 2009).Unlike ethylene gas, abscisic acid (ABA) has more complicated

biosynthesis and signaling pathways (Nambara and Marion-Poll,2005), comprising a core signaling pathway (Cutler et al., 2010;Finkelstein, 2013), catabolic regulation (Nambara and Marion-Poll,2005; Xu et al., 2013), and requirement for a series of transporters(Kuromori et al., 2010; Kanno et al., 2012). Because ABA is pro-duced through the cleavage of carotenoid precursors (xantho-phylls) (Cutler and Krochko, 1999; Nambara and Marion-Poll,2005), the supply of these carotenoid precursors has an importanteffect on ABA production.Plant carotenoids are tetraterpenes that are synthesized in

plastids and play different roles in plants. In photosynthetic plant

1 These authors contributed equally to this work.2 Address correspondence to mabiao@genetics.ac.cn, sychen@genetics.ac.cn, or jszhang@genetics.ac.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Jin-Song Zhang(jszhang@genetics.ac.cn).OPENArticles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.15.00080

The Plant Cell, Vol. 27: 1061–1081, April 2015, www.plantcell.org ã 2015 American Society of Plant Biologists. All rights reserved.

tissues, carotenoids contribute to light harvesting, photoprotection(Niyogi, 1999; Dall’Osto et al., 2007; Wei et al., 2010; Ballottariet al., 2014), and flower and fruit color (Hirschberg, 2001; Ruiz-Solaand Rodríguez-Concepción, 2012). Carotenoids are also presentin nonphotosynthetic plastids, such as the etioplasts of dark-grown seedlings and the leucoplasts of roots, where they providenutritional value (Howitt and Pogson, 2006).

The first committed step of the carotenoid pathway is thecondensation of two molecules of geranylgeranyldiphosphate toform phytoene, a reaction catalyzed by phytoene synthase (PSY)(Rodríguez-Villalón et al., 2009). Phytoene then undergoes fourconsecutive desaturation reactions, leading to the formation oflycopene in reactions catalyzed by phytoene desaturase andz-carotene desaturase. The production of all-trans-lycopene alsorequires Z-ISO (Chen et al., 2010) and carotenoid isomerase(CRTISO) (Isaacson et al., 2002; Park et al., 2002; Isaacson et al.,2004). Lycopene can be further converted into a-carotene and/orb-carotene, which are catalyzed by a-cyclases and b-cyclases,respectively (Cunningham et al., 1996). b-Carotene, which servesas a precursor for the plant hormone strigolactone (SL), can befurther metabolized to b,b-xanthophylls such as zeaxanthin(Nambara and Marion-Poll, 2005; Xie et al., 2010). ABA is pro-duced from violaxanthin or neoxanthin through several enzymaticreactions, including 9-cis-epoxycarotenoid dioxygenase(NCED), neoxanthin-deficient 1, alcohol dehydrogenase (ABA2)/short-chain dehydrogenase/reductase, abscisic aldehyde oxi-dase (AAO3), and sulfurated molybdenum cofactor sulfurase(ABA3) (Nambara and Marion-Poll, 2005; Finkelstein, 2013;Neuman et al., 2014).

Crosstalk between ethylene and ABA occurs at multiple levels.One of these interactions is at the level of biosynthesis. Endog-enous ABA limits ethylene production (Tal, 1979; Rakitina et al.,1994; LeNoble et al., 2004) and ethylene can inhibit ABA bio-synthesis (Hoffmann-Benning and Kende, 1992). Previous studieshave suggested that both ethylene and ABA can inhibit rootgrowth (Vandenbussche and Van Der Straeten, 2007; Arc et al.,2013). In Arabidopsis thaliana, the etr1-1 and ein2 roots are re-sistant to both ethylene and ABA, whereas the roots of the ABA-resistant mutant abi1-1 and the ABA-deficient mutant aba2 havenormal ethylene responses. This suggests that the ABA inhibitionof root growth requires a functional ethylene signaling pathwaybut that the ethylene inhibition of root growth is ABA independent(Beaudoin et al., 2000; Ghassemian et al., 2000; Cheng et al.,2009). Recent studies have indicated that ABA mediates rootgrowth by promoting ethylene biosynthesis in Arabidopsis (Luoet al., 2014). However, the interaction between ethylene and ABAin the regulation of the rice (Oryza sativa) ethylene response islargely unclear.

Rice is an extremely important cereal crop worldwide that isgrown under semiaquatic, hypoxic conditions. Rice plants haveevolved elaborate mechanisms to adapt to hypoxia stress, in-cluding coleoptile elongation, adventitious root formation, aer-enchyma development, and enhanced or repressed shootelongation (Ma et al., 2010). Ethylene plays important roles inthese adaptations (Saika et al., 2007; Steffens and Sauter, 2010;Ma et al., 2010; Steffens et al., 2012). Remarkably, in the dark,rice has a double response to ethylene (promoted coleoptileelongation and inhibited root growth) (Ma et al., 2010, 2013; Yang

et al., 2015) that is different from the Arabidopsis triple response(short hypocotyl, short root, and exaggerated apical hook)(Bleecker and Kende, 2000). Several homologous genes of Ara-bidopsis ethylene signaling components have been identified inrice, such as the receptors, RTE1-like gene, EIN2-like gene,EIN3-like gene, CTR2, and ETHYLENE RESPONSE FACTOR (ERF)(Cao et al., 2003; Jun et al., 2004; Mao et al., 2006; Rzewuski andSauter, 2008; Wuriyanghan et al., 2009; Zhang et al., 2012; Maet al., 2013; Wang et al., 2013). We previously studied the kinaseactivity of rice ETR2 and the roles of ETR2 in flowering and instarch accumulation (Wuriyanghan et al., 2009). We also isolateda set of rice ethylene response mutants (mhz) and identifiedMHZ7/EIN2 as the central component of ethylene signaling in rice(Ma et al., 2013).Here, we focused our studies on another ethylene-responsive

mutant,mhz5, which, in the presence of ethylene, exhibits reducedsensitivity of root growth but enhanced sensitivity of coleoptilegrowth. Through map-based cloning, we found that MHZ5 en-codes a carotenoid isomerase. Further physiological and geneticstudies revealed that ethylene regulates carotenoid biosynthesis inrice and that the ethylene-induced inhibition of rice root growthrequires the MHZ5/CRTISO-mediated ABA pathway. This latterfeature is different from that in Arabidopsis, in which ethyleneregulates root growth does not require ABA function. Additionally,a MHZ5/CRTISO mutation enhances ethylene production andEIN2-mediated coleoptile elongation. Our study provides impor-tant insight into the interactions of ethylene, carotenogenesis, andABA in the regulation of rice growth and development.

RESULTS

Phenotype and Ethylene Response of Dark-Grown mhz5Mutant Rice

Rice mhz5 is a previously described ethylene response mutant,and three mutant alleles of mhz5 (mhz5-1, mhz5-2, and mhz5-3)have been identified (Ma et al., 2013). Upon exposure to ethylene,root growth of wild-type etiolated rice seedlings was inhibited by;80%, but coleoptile growth was promoted (Figure 1). By con-trast, root growth of etiolated mhz5-1 seedlings was only partiallyinhibited (by ;35%) (Figures 1A, 1C, and 1D). Ethylene-inducedcoleoptile elongation was greater in mhz5-1 than that in the wildtype (Figures 1A and 1B). The two allelic mutants mhz5-2 andmhz5-3 showed a similar ethylene response (Figures 1B to 1D).These results indicate that the mhz5 mutant has hypersensitivityin ethylene-promoted coleoptile elongation but reduced sensi-tivity in ethylene-inhibited root growth. In addition, three alleles ofmhz5 display significantly (P < 0.01) shorter roots and slightly butsignificantly (P < 0.05) longer coleoptiles than those of the wildtype in the absence of ethylene (Figures 1A to 1C). The threemhz5 alleles were phenotypically indistinguishable; therefore, twoalleles, mhz5-1 and mhz5-3, were used for most of the analysesdescribed below.To further examine the ethylene response of the mhz5 mu-

tant, we analyzed the transcript level of ethylene-responsivegenes that were originally identified from a microarray assay(GSE51153). The expression of six genes, Photosystem II 10 kD

1062 The Plant Cell

Figure 1. Phenotype and Ethylene Response of mhz5.

(A) Morphology of etiolated seedlings from 3-d-old wild-type and mhz5-1 seedlings in the presence of 10 ppm ethylene or air. Bars = 10 mm.(B) Ethylene dose–response curves for the coleoptile length of 3-d-old dark-grown seedlings. The values are means 6 SD of 20 to 30 seedlings pergenotype at each dose.(C) Ethylene dose–response curves for root length. The growth condition and statistical analyses are as in (B).(D) Relative root length of the wild type and mhz5 mutants in response to ethylene (ethylene-treated versus untreated). Others are as in (B).

Ethylene, Carotenoids, and ABA in Rice 1063

polypeptide, AP2 domain-containing protein (ERF063 and ERF073),cupin domain-containing protein (Germin-like and RGLP1), andreceptor-like kinase (SHR5), was upregulated by ethylene to varyingdegrees in the wild-type shoots as detected via quantitative real-time PCR (qRT-PCR). In mhz5-1 mutant shoots, the expressionlevels of these genes were higher than those in the wild typewithout ethylene treatment and were further enhanced by ethyl-ene treatment (Figure 1E). Four other genes, including A-typeresponse regulator (RRA5), B3 DNA binding domain-containingprotein (RAP2.8), AP2 domain-containing protein (ERF002), andan auxin-responsive Aux/IAA gene family member (IAA20), werepreferentially induced by ethylene in wild-type roots but not in-duced in mhz5-1 roots (Figure 1F). Shoots instead of coleoptileswere used for gene expression analysis because rice coleoptilesand shoots have a similar ethylene response (Ku et al., 1970).These results indicate that themhz5-1mutant is hypersensitive toethylene in coleoptiles but less sensitive in roots in the expressionof the ethylene-responsive genes.

Phenotypes of Field-Grown mhz5 Mutant Rice Plants

Adult field-grown mhz5 mutant plants had excessive tillers,smaller panicles, and fewer primary and secondary branches inpanicles compared with wild-type plants (Supplemental Figure 1).The lengths of all internodes were shorter inmhz5-1 than the wildtype (Supplemental Figure 1A). At the late tillering stage, the tillernumbers of mhz5 were drastically increased compared with thewild type (Supplemental Figures 1A and 1D). After harvest, thelength and width of well-filled grains were measured, and all threeallelic mutant grains were longer and narrower than those of thewild type. Consistently, the ratio of grain length/width was alsoapparently increased in mhz5 (Supplemental Figure 1E). In addi-tion, the length of the primary roots, adventitious roots, and lateralroots of mhz5-1 seedlings were shorter than that of wild-typeseedlings. Furthermore, mhz5-1 mutants had fewer adventitiousroots but more lateral roots than the wild type (SupplementalFigure 2). These results indicate that MHZ5 disruption stronglyaffects agronomic traits.

Positional Cloning and Identification of MHZ5

We used a map-based cloning strategy to isolate the MHZ5gene. The mhz5-1 mutant was crossed with four indica cultivars(93-11, MH63, ZF802, and TN1), and F2 populations werescreened and mapped. A DNA sequence analysis of all 10 ofthe annotated genes within the mapped region revealed that theLOC_Os11g36440 had a single base pair substitution (A-T) in theeleventh exon at nucleotide 3114, and this mutation disruptedthe splicing signal, resulting in a loss of 4 bp in cDNA, generating

a premature translation termination product in mhz5-1 (Figure 2).Mutations inmhz5-2 andmhz5-3 were also identified in the samelocus by sequencing and are indicated in Figures 2A to 2C. Asingle base pair substitution (G to C) in mhz5-2 at 313 bp causeda change of Gly-105 to Arg-105 (Figures 2A and 2B). In mhz5-3,a deletion of 26 bp from nucleotides 383 to 409 disrupted thesplicing signal and resulted in aberrant splicing, causing themRNA of mhz5-3 to be 475 bp longer than that in the wild type(Figures 2A to 2C). Although this mutation does not appreciablyaffect the mRNA level (Figure 2C, left panel), it leads to a truncatedprotein of 157 amino acids. The mhz5-1 and mhz5-2 mutationswere confirmed via a derived cleaved amplified polymorphic se-quence assay using PCR (Figure 2C, right panel), and the mhz5-3mutation was confirmed via an amplified fragment length poly-morphism assay using PCR (Figure 2C, right panel). A Tos17 ret-rotransposon insertion in the seventh exon of LOC_Os11g36440(mhz5-4) (NG0489 from the rice Tos17 Insertion Mutant database,http://tos.nias.affrc.go.jp/~miyao/pub/tos17/index.html.en) com-pletely disrupted the gene and generated an altered ethylene re-sponse that was similar to that in the mhz5-1 mutant (Figures 2Aand 2B; Supplemental Figure 3).The identity of mhz5 was confirmed by genetic complemen-

tation. Plasmid pMHZ5C, harboring a 7.13-kb genomic DNAfragment consisting of the 2278-bp upstream sequence, theentire MHZ5 gene, and an 1169-bp downstream region, wasintroduced into mhz5-3. Transgenic lines harboring the entireMHZ5 genomic sequence displayed the same ethylene re-sponse and phenotypes as those of wild-type plants (Figures 2Dand 2E). These results confirm that MHZ5 is located at the locusLOC_Os11g36440, whose mutation leads to an alteration of theethylene response and agronomic traits in rice.

Disruption of the Carotenoid Biosynthesis Pathway Mimicsthe Ethylene Response Phenotypes of the mhz5 Mutant

The MHZ5 gene encodes CRTISO, which catalyzes the con-version of 7,9,99,79-tetra-cis-lycopene (prolycopene) to all-trans-lycopene in the carotenoid biosynthesis pathway in plants(Isaacson et al., 2002; Park et al., 2002; Fang et al., 2008). Wetested whether blocking the carotenoid biosynthesis pathwaywith an inhibitor of fluridone (Flu) at an early step of the conversionof phytoene to phytofluene (Hoffmann-Benning and Kende, 1992;Jamil et al., 2010) would similarly alter the ethylene response inwild-type rice seedlings. When Flu was added, the relative co-leoptile length and the relative root length of wild-type seedlingssignificantly increased in the presence of ethylene (Figure 3), sug-gesting enhanced and reduced ethylene responses in coleoptilesand in roots, respectively. The Flu-treated wild-type seedlings re-sembled the mock mhz5-1 mutant when both were subjected to

Figure 1. (continued).

(E) Relative expression level of ethylene-responsive genes in the shoots of wild-type and mhz5-1 seedlings (gene expression levels = 1 in untreatedwild-type seedlings). Three-day-old dark-grown seedlings were treated with or without 10 ppm ethylene (ET) for 8 h, and the RNA was extracted forqRT-PCR. Actin2 was used as the loading control. The values are means 6 SD of three biological replicates, and each biological replicate had two orfour technical replicates.(F) Relative expression level of ethylene-responsive genes that were preferentially induced by ethylene in the roots. Seedling growth condition, RNAextraction, and statistical analyses are as in (E). Each experiment was repeated at least three times with similar results.

1064 The Plant Cell

Figure 2. Positional Cloning of the MHZ5 Gene.

(A) Fine mapping of the MHZ5 gene. The MHZ5 locus was mapped to the long arm of chromosome 11 between markers Idl11-20.3 and Idl11-21.2.Numerals under the markers indicate the number of recombinants identified from 589 F2 mutant plants. AC136149, AC108871, AC109929, andAC137589 are BAC clones. The location of MHZ5 was then fine mapped to a 152-kb genomic DNA region between markers Idl11-20.557 and Idl11-20.709. LOC_Os11g36440 is the candidate gene for MHZ5 and mhz5-4 represents a Tos17 insertion mutant (NG0489).(B) The mutation sites of four allelic mutants of MHZ are shown superimposed on the structure of MHZ5 as predicted using SMART software (http://smart.embl-heidelberg.de). The black box represents the FAD-dependent oxidoreductase.(C) Confirmation of mutation sites in mhz5-1, mhz5-2, and mhz5-3 via PCR-based analyses. The full-length cDNA of mhz5-1 and mhz5-2 was similar tothe wild type, but that of mhz5-3 was 475 bp longer than that of the wild type (left panel). The PCR-amplified fragment from genomic DNA of mhz5-1was 25 bp longer than that of the wild type digested with PvUII, the fragment from mhz5-2 was 27 bp shorter than that of the wild type digested withHhaI, and the fragment from mhz5-3 was 26 bp shorter than that of the wild type (right panel).(D) Functional complementation of the mhz5-3 mutant. The complementation plasmid containing the entire MHZ5 (pMHZ5C) was transformed intomhz5-3 plants, rescuing the ethylene response phenotypes of mhz5-3 etiolated seedlings in transgenic lines (mhz5-3c) 6 and 14 (lower panel). Themhz5-3 mutant backgrounds in transgenic lines 6 and 14 were confirmed using PCR-based analyses with genomic DNA (upper panel). The fragment ofmhz5-3 mutant was 26 bp shorter than the wild type. Bars = 10 mm.(E) Functional complementation of the mhz5 mutant in the field. Methods are as in (D). Bar = 10 cm.

Ethylene, Carotenoids, and ABA in Rice 1065

Figure 3. Disruption of the Carotenoid Biosynthesis Pathway Mimics the Ethylene Response Phenotypes of the mhz5 Mutant.

(A) Ethylene response phenotypes of 3-d-old dark-grown wild type andmhz5-1 mutants with or without a Flu inhibitor. The Flu-treated wild-type seedlingsresembled the phenotypes of mhz5-1 in the presence of ethylene. Bars = 10 mm.(B) Relative coleoptile length (ethylene-treated versus untreated in the wild type andmhz5-1, respectively) of the wild type andmhz5-1 that were treatedwith or without Flu in the presence or absence of ethylene. Values are means 6 SD for 20 to 30 seedlings per genotype. A statistical analysis wasperformed using a one-way ANOVA (LSD t test) for ethylene-treated groups with statistical software (SPSS 18.0) (P ˂ 0.05). Values for a and b aresignificantly different at P = 0.0008; values for b and c are significantly different at P = 0.005. Different letters above each column indicate significantdifference between the compared pairs (P < 0.05).(C) Relative root length of the wild type andmhz5-1. The seedlings treatment condition and statistical analyses are as in (B). Values for b and c are significantlydifferent at P = 0.013.(D) Ethylene response of 3-d-old light-grown wild-type, mhz5-1, and ein2 seedlings in ethylene or air. Bars = 10 mm.(E) Relative root length (ethylene-treated versus untreated in the wild type and mutant, respectively) of 3-d-old light-grown rice seedlings at variousconcentrations of ethylene. Means 6 SD are shown for 20 to 30 seedlings per genotype at each dose.(F) and (G) Pigment analysis of the leaves of 4-d-old wild type and mhz5-1 mutants that were either etiolated (F) or exposed to light for 24 h (G). N, neoxanthin;V, violaxanthin; A, antheraxanthin; L, lutein; Ca, chlorophyll a; Cb, chlorophyll b; pLy, prolycopene; Ne, neurosporene; Z, zeaxanthin; tLy all-trans-lycopene;b, b-carotene. Absorbance was at 440 nm. mAU, milliabsorbance units. Each experiment was repeated at least three times with similar results.

1066 The Plant Cell

ethylene treatment (Figures 3A to 3C), demonstrating that the im-pairment of the carotenoid biosynthetic pathway affects ethyleneresponses in rice seedlings.

Light treatment can convert prolycopene to all-trans-lycopenethrough photoisomerization, partially replacing the functions ofcarotenoid isomerase (Isaacson et al., 2002; Park et al., 2002).We investigated whether light would affect the ethyleneresponse ofmhz5-1 compared with the wild type and the ethylene-insensitive mutant ein2/mhz7-1 (Ma et al., 2013). Upon expo-sure to continuous light, the roots of themhz5-1mutant had thesame ethylene response as the wild type at different concen-trations of ethylene. By contrast, the mutant ein2/mhz7-1 wasstill insensitive to ethylene in roots in the light (Figures 3D and3E). These results indicate that light can rescue the ethyleneresponse phenotype of mhz5-1 roots, indicating that car-otenogenesis mediates the regulation of ethylene responses inrice seedlings.

To elucidate the mechanisms of the different ethyleneresponses of mhz5-1 in the dark and light, we analyzed the ca-rotenoid profiles of the leaves and roots of wild-type and mhz5-1seedlings. Unlike the profile of wild-type etiolated leaves, themhz5-1 etiolated leaves accumulated prolycopene, the substrateof MHZ5/carotenoid isomerase for the conversion to all-trans-lycopene (Figure 3F). Neurosporene, a substrate for z-carotenedesaturase that is immediately upstream of the MHZ5 step, alsoaccumulated in the mhz5-1 etiolated leaves (Figure 3F). In themhz5-1 roots, only prolycopene was detected (SupplementalFigure 4). These results indicate that MHZ5 mutation leads to theaccumulation of prolycopene, the precursor of all-trans-lycopenein the leaves and roots of mhz5-1 seedlings.

Upon exposure to light, there was a rapid decrease in theprolycopene level in mhz5-1 leaves and roots (Figures 3F and3G; Supplemental Figures 4A and 4B). Furthermore, increases inthe contents of all-trans-lycopene, zeaxanthin, and antherax-anthin were apparently observed in light-treated mhz5-1 leavescompared with those in wild-type leaves (Figure 3G). Levels ofother carotenoids and the photosynthetic pigments were com-parable between the mhz5-1 and wild-type leaves, except forthe lower level of lutein in mhz5-1 compared with that of the wildtype (Figure 3G, Table 1). In the roots of light-treated mhz5-1,prolycopene has been converted to the downstream metabo-lites, and the content of neoxanthin was very similar to that inthe wild type (Supplemental Figure 4B). These results suggest

that light treatment leads to the conversion of prolycopene toall-trans-lycopene and to the further biosynthesis of down-stream metabolites, rescuing the mhz5-1 ethylene responses.In the dark, the accumulation of prolycopene leads to an

orange-yellow coloration in the mhz5-1 leaves, different from theyellow leaves of the wild-type seedlings. Additionally, the mhz5-1seedlings had a markedly delayed greening process when ex-posed to light (Supplemental Figure 5), most likely due to the lowefficiency of photoisomerization and/or the abnormal developmentof chloroplasts (Park et al., 2002).Flu inhibitor tests and light rescue experiments indicate that

the aberrant ethylene response of mhz5-1 may result from thelack of carotenoid-derived signaling molecules. Considering thatfield-grown mhz5-1 plants have more tillers than do wild-typeplants (Supplemental Figure 1), and carotenoid-derived SL in-hibits tiller development (Umehara et al., 2008), we examinedwhether SL is involved in the aberrant ethylene response of themhz5 mutant. We first analyzed 29-epi-5-deoxystrigol (epi-5DS),one compound of the SLs in the exudates of rice roots andfound that the concentration of epi-5DS in mhz5-1 was lowerthan that in the wild type (Supplemental Figure 6). We thentested the effect of the SL analog GR24 on the ethylene re-sponse and found that GR24 could not rescue the ethylene re-sponse of the mhz5-1 mutant (Supplemental Figures 6B and6C). Additionally, inhibiting the SL synthesis gene D17 encodingthe carotenoid cleavage dioxygenase (Zou et al., 2006) or the SLsignaling gene D3 encoding an F-box protein with leucine-richrepeats (Zhao et al., 2014) in transgenic rice did not alter theethylene response, although these transgenic plants had moretillers, a typical phenotype of a plant lacking SL synthesis orsignaling (Supplemental Figures 6D and 6E). These resultssuggest that the abnormal ethylene responses of mhz5-1 etio-lated seedlings do not appear to be consequences of altered SLsynthesis or signaling.

Ethylene Inhibition of Etiolated Rice Seedling Root GrowthRequires the MHZ5-Mediated ABA Biosynthesis

ABA is another important signaling molecule that is derived fromthe carotenoid biosynthesis pathway (Nambara and Marion-Poll,2005). We measured the ABA contents in wild-type and mhz5-1mutant etiolated seedlings and found that the mhz5-1 mutanthad very low levels of ABA compared with the wild type (Figure4), indicating that MHZ5/CRTISO is essential for ABA bio-synthesis in etiolated shoots and roots. Because mhz5-1 hasvery little ABA, we examined whether the addition of ABA couldcomplement the phenotypes of the mhz5-1 mutant. Withoutethylene treatment, the application of 0.04 mM ABA restored theshort roots of the mhz5 mutant to the wild-type level undernormal conditions (Figure 4B), suggesting that basal levels ofendogenous ABA are essential for the maintenance of rootgrowth. We further tested whether ABA could restore theethylene response ofmhz5-1. In the presence of 10 ppm ethylene,the application of 0.1 mM ABA could largely rescue the ethylenesensitivity ofmhz5-1 coleoptiles and roots (Figures 4C to 4E). ThisABA concentration (0.1 mM) had no effect or only a slightly in-hibitory effect on coleoptile and root growth in wild-type etiolatedseedlings (Supplemental Figure 7). These results suggest that

Table 1. Relative Pigment Content in the Leaves of Wild-Type andmhz5-1 Etiolated Seedlings after 24 h of Illumination

CompoundPeak Area Ratio formhz5-1/Wild Type

Neoxanthin 0.94 6 0.01**Violaxanthin 1.26 6 0.09**Lutein 0.18 6 0.004**Chlorophyll a 0.75 6 0.02**Chlorophyll b 0.91 6 0.01*b-Carotene 1.22 6 0.08**

Values are means 6 SD of three biological replicates. Student’s t test(**P < 0.01; *P < 0.05).

Ethylene, Carotenoids, and ABA in Rice 1067

Figure 4. Ethylene Inhibition of Etiolated Rice Seedling Root Growth Requires the MHZ5-Mediated ABA Pathway.

(A) Influence of ethylene on ABA accumulation in the shoots and roots of wild-type and mhz5-1 mutant seedlings. Three-day-old etiolated seedlingswere treated with or without ethylene (10 ppm) for 24 h. The values are the means 6 SD from three biological replicates. Asterisks represent significantdifference between ethylene-treated and untreated in wild-type seedlings.(B) The root defect of mhz5-1 is rescued by ABA. Wild-type and mhz5-1 seedlings were grown in the dark in solutions with or without 0.04 mM ABA for2.5 d. Values are means 6 SD of 30 seedlings per genotype.(C) ABA rescues the ethylene response of mhz5-1. The wild type and mhz5-1 were incubated in solutions with or without 0.1 mM ABA and treated withor without 10 ppm ethylene for 2.5 d. The coleoptiles of the wild type and mhz5-1 were sprayed once daily with 0.1 mM ABA (containing 0.001% Tween20) after germination. The mock solution contains 0.1% ethanol and 0.001% Tween 20. Bars = 10 mm.(D) Absolute coleoptile length of 2.5-d-old dark-grown wild-type andmhz5-1 seedlings that were incubated in solutions with or without 0.1 mM ABA andtreated with or without ethylene. Values are means 6 SD of 20 to 30 seedlings per genotype. Asterisks represent significant difference between mhz5with ABA, and mhz5 without ABA under ethylene-treated conditions.(E) Relative root length (ethylene-treated versus untreated in the wild type andmhz5-1, respectively). Others are as in (D). Asterisks represent significantdifference between mhz5 with ABA and mhz5 without ABA under ethylene-treated conditions.(F) MHZ5 was induced in wild-type roots by ethylene as detected using qRT-PCR. RNA was isolated from 3-d-old wild-type etiolated seedlings aftertreatment with 10 ppm ethylene, 1-MCP (an ethylene competitive inhibitor), or air for various times. The values are the means 6 SD of three biologicalreplicates.(G) MHZ5 was induced in wild-type shoots by ethylene. The seedlings growth treatment and qRT-PCR strategies are as in (F).

1068 The Plant Cell

ethylene requires ABA function to regulate the ethylene responsein etiolated rice seedlings.

We then examined the effects of ethylene on ABA concen-tration and found that ethylene inhibited ABA accumulation inwild-type shoots but promoted ABA accumulation in wild-typeroots, suggesting the tissue-specific regulation of ABA accu-mulation (Figure 4A). We further investigated the MHZ5transcript level with ethylene treatment and found that thistranscript was induced by ethylene in both the roots and shoots(Figures 4F and 4G). These results indicate that ethylene pro-motes ABA accumulation in wild-type roots, possibly, in part,through the induction of MHZ5 expression. In the wild-typeshoots, the discrepancy between ethylene-inhibited ABA accu-mulation and ethylene-induced MHZ5 expression is most likelydue to ethylene-activated ABA catabolism for homeostasis inthe shoots (Benschop et al., 2005; Saika et al., 2007).

Because ethylene induced the accumulation of ABA in wild-type roots, we further tested whether the carotenoid profile wasaltered by ethylene treatment. The contents of neoxanthin, thesubstrate of the rate-limiting enzyme NCED in the ABA bio-synthesis pathway, increased by 42% (P = 0.0024) in the wild typewith ethylene treatment (Figures 4H and 4I). By contrast, neo-xanthin was not detected in mhz5-1 roots either with or withoutethylene due to the disruption of the carotenoid biosyntheticpathway.

To further investigate the role of ethylene-triggered ABA in therice root ethylene response, we measured the effects of ABAbiosynthesis inhibitor nordihydroguaiaretic acid (NDGA) onethylene-induced ABA accumulation as well as ethylene-induciblegene expression. NDGA inhibits the enzyme NCED in the ABAbiosynthesis pathway (Creelman et al., 1992; Zhu et al., 2009). Inthe presence of NDGA, the ABA accumulation in the roots was;30% that in untreated wild type, and ethylene-triggered ABAaccumulation was completely blocked in the roots (Figure 4J).IAA20 can be induced by ethylene in the wild-type roots but not inthe mhz5-1 roots (Figure 1F). This gene can also be induced byABA in wild-type roots (Figure 4K). However, the ethylene in-duction of IAA20 was almost completely abolished in the pres-ence of NDGA (Figure 4K), suggesting that ethylene-inducedgene expression requires ABA function. In summary, the aboveresults suggest that the ethylene inhibition of rice root growthrequires MHZ5-mediated ABA biosynthesis.

mhz5 Etiolated Seedlings Produce More Ethylene, and TheirColeoptile Response to Ethylene Mainly Results fromEnhanced Ethylene Signaling

As shown in Figures 4C and 4D, the enhanced ethylene re-sponse in mhz5-1 coleoptiles was substantially inhibited byABA, suggesting that ABA deficiency is the major reason for thehypersensitivity of mhz5-1 coleoptiles to ethylene. An enhancedethylene response could result from ethylene overproductionand/or enhanced signal transduction. Thus, we examinedwhether ethylene production is altered in mhz5-1. As shown inFigure 5, mhz5-1 etiolated seedlings produced nearly 3 times asmuch ethylene than did the wild type (based on fresh weight),and ABA addition dramatically suppressed ethylene productionin the mhz5-1 mutant. These results indicate that MHZ5-mediated ABA biosynthesis inhibits ethylene production in etio-lated rice seedlings. It should be noted that ethylene production inlight-grown seedlings is very similar to that in the wild type, furtherdemonstrating that light could substitute for MHZ5 isomeraseactivity through photoisomerization as previously described(Isaacson et al., 2002; Park et al., 2002). We further studied theexpression of ethylene biosynthesis genes and found that theACS2, ACS6, and ACO5 levels were all higher in both the shootsand roots of themhz5-1 etiolated seedlings than those in the wild-type seedlings (Figure 5B). Notably, the ACO3 level was higher inthe shoots of mhz5-1 than that in the wild-type shoots. However,expression of this gene was very similar in the roots of the wildtype andmhz5-1mutant (Figure 5B). The differential expression ofACO3most likely reflects tissue-specific and/or posttranscriptionalregulation. These results suggest that enhanced ethylene emissionin mhz5-1 plants is likely due to the increased expression of eth-ylene biosynthesis-related genes.mhz5-1 had slightly but significantly (P < 0.05) longer co-

leoptiles than did the wild type in the dark in the absence ofethylene treatment (Figures 5C and 5D). L-a-(2-aminoethoxyvinyl)-glycine (AVG), the ethylene biosynthesis inhibitor, can effectivelyblock the ethylene production of the mhz5-1 mutant and wild type(Supplemental Figure 8). When AVG was included, the basal elon-gation of themhz5-1 coleoptiles was reduced to the level of the wildtype without AVG treatment (Figures 5C and 5D; SupplementalFigure 8B). These results indicate that endogenously overproducedethylene contributes to the basal coleoptile elongation of the

Figure 4. (continued).

(H) Ethylene induced neoxanthin biosynthesis in roots of etiolated rice seedlings. Pigment analysis of 3-d-old dark-grown roots in the presence of10 ppm ethylene for 24 h. Inset shows the enlargement of the HPLC trace between 10 and 14 min. Note that the retention times of this figure are differentfrom those in Figures 3F and 3G because of a different pigment extraction and analysis method used in the roots due to their low level of carotenoids.Each carotenoid profile represents the absorbance at 430 nm of pigments that were extracted from 1.2 g fresh weight of roots. N, neoxanthin; pLy,prolycopene; mAU, milliabsorbance units.(I) Relative content of neoxanthin (ethylene-treated versus untreated in wild-type roots and setting the neoxanthin content to 1 in untreated wild type).Student’s t test indicates a significant difference between ethylene-treated and untreated in wild-type roots (**P ˂ 0.01).(J) ABA contents in wild-type roots in the presence or absence of NDGA (an ABA biosynthesis inhibitor) following treatment with or without ethylene.Three-day-old etiolated seedlings that were grown in 100 mM NDGA solutions were treated with or without ethylene (10 ppm) for 24 h.(K) The ethylene induction of IAA20 requires the ABA pathway. The influence of 100 mM ABA and 10 ppm ethylene combined with or without NDGA(100 mM) on the IAA20 expression level was examined in wild-type roots using qRT-PCR. Values are means6 SD from three biological replicates. Student’st test analysis indicates a significant difference between ethylene-treated and untreated in mock wild-type roots (**P ˂ 0.01). Each experiment was repeatedat least three times with similar results.

Ethylene, Carotenoids, and ABA in Rice 1069

mhz5-1 mutant. However, in the presence of AVG, when a widerange of exogenous ethylene was applied, the coleoptile elon-gations of mhz5-1 were still greater than those of the wild type(Figures 5C and 5D). These results suggest that the endogenousethylene production of mhz5-1 does not contribute to the hy-persensitive response of mhz5-1 coleoptiles to ethylene.

We further found that the transcript level of EIN2 was higher inmhz5-1 shoots than that in the wild type in the absence ofethylene. By contrast, this transcript was not upregulated in theroots of the mhz5-1 mutant (Figure 5E). Taken together, thesedata suggest that the enhanced ethylene response of mhz5-1coleoptiles most likely results from enhanced ethylene signalingdue to higher EIN2 expression.

MHZ5 Overexpression Alters the Ethylene Response in Rice

To further elucidate the function of MHZ5, the MHZ5-codingsequence driven by the cauliflower mosaic virus 35S promoterwas introduced into wild-type plants, and four homozygous in-dependent MHZ5-overexpression lines (MHZ5-OE) were usedfor analysis (Figure 6). The four dark-grown transgenic lines alldisplayed slightly but significantly shorter coleoptiles (P < 1026)and roots (P < 1026) compared with those of the wild type in air(Figures 6B to 6D). When treated with exogenous ethylene, thecoleoptile elongation of MHZ5-OE lines was less than that in thewild type (Figures 6B and 6C), suggesting the presence of re-duced coleoptile ethylene sensitivity. However, the inhibition of

Figure 5. The mhz5 Etiolated Seedlings Produce More Ethylene Than the Wild Type, and Their Elevated Coleoptile Response to Ethylene MainlyResults from Enhanced Ethylene Signaling.

(A) Ethylene production of etiolated seedlings and green seedlings. The values are the means 6 SD from four biological replicates. Asterisks indicatea significant difference between the wild type without ABA treatment and mhz5-1 etiolated seedlings at P < 0.01 using Student’s t test.(B) Relative expression level of ethylene synthesis genes in wild-type and mhz5-1 mutant seedlings. RNA was extracted from the shoots and roots of3-d-old etiolated seedlings and used for qRT-PCR. Values are means 6 SD from three biological replicates.(C) The effect of the ethylene biosynthesis inhibitor AVG (50 mM) on the ethylene response of wild-type and mhz5-1 seedlings. Seedlings wereincubated on eight layers of cheesecloth in Petri dishes in a plastic box with or without 10 ppm ethylene for 2.5 d. Bars = 10 mm.(D) Coleoptile length of wild-type and mhz5-1 seedlings in response to ethylene following the addition of 50 mM AVG. Values are mean lengths 6 SD of20 to 30 seedlings.(E) EIN2 transcript levels in the shoots and roots of 3-d-old etiolated wild-type and mhz5-1 seedlings as detected using RT-PCR. Actin1 was used asthe loading control. Each experiment was repeated at least three times with similar results.

1070 The Plant Cell

root growth of the MHZ5-OE lines was more severe than that inthe wild type, especially under 1 ppm ethylene treatment (Fig-ures 6B, 6D, and 6E), suggesting enhanced root ethylene sen-sitivity. The roots of the MHZ5-OE lines were all shorter thanthose of the wild type under normal conditions, and thisshort-root phenotype is similar to that of the mhz5-1 mutants(Figures 1C and 6D). The short-root phenotype in these plantsmost likely resulted from altered ABA levels because a normallevel of ABA is essential for root growth in plants (Yin et al., 2009;Zhang et al., 2010; Wang et al., 2011). MHZ5 expression levelsseemed to roughly correlate with the ethylene response in thecoleoptiles and roots of the transgenic plants (Figures 6A to 6E).

To further establish the ethylene responsiveness ofMHZ5-OE,we examined the expression of ethylene-inducible genes usingqRT-PCR. Transcript levels of ethylene-inducible genes werecomparable in the wild-type and MHZ5-OE lines in the air (Fig-ures 6F and 6G). Upon exposure to ethylene, ethylene inductionof Germin-like and SHR5 was significantly lower in the MHZ5-OE shoots than those in the wild-type shoots (Figure 6F). In theroots, the induced levels of RRA5 and ERF002 were significantlyhigher in the MHZ5-OE lines than those in the wild type (Figure6G). These results indicate that the overexpression of MHZ5reduced the expression of a subset of ethylene-responsive genesin coleoptiles but promoted the expression of another subset ofethylene-responsive genes in the roots of etiolated seedlings.Furthermore, in the shoots/coleoptiles, the transcript level ofEIN2 was lower to varying degrees in the MHZ5-OE lines thanthat in the wild type (Figure 6H), suggesting that the reducedethylene responsiveness of the shoots/coleoptiles most likelyresults from the reduction of ethylene signaling. These geneexpression patterns inMHZ5-OE plants are consistent with thosein mhz5-1 mutant (Figures 1E, 1F, and 5E). Together, these re-sults indicate that MHZ5 differentially affects the ethyleneresponse of rice shoots/coleoptiles and roots at the gene ex-pression level.

Genetic Interactions of MHZ5 with Ethylene SignalingComponents in Rice

To examine the genetic interactions of MHZ5 with ethylene re-ceptor genes, double mutants were generated between mhz5-1and three ethylene receptor mutants. The three receptor singleloss-of-function rice mutants ers1, ers2, and etr2 were in thebackground of the japonica variety Dongjin (DJ), and their T-DNA insertions in the corresponding genes were identified usingPCR-based genotyping (Supplemental Figure 9). The threeethylene receptor mutants showed no significant change incoleoptile length. However, their roots were significantly shorterin the air and displayed a moderately enhanced ethylene re-sponse compared with that in the background variety DJ. Theroot ethylene responses of the three double mutants (ers1mhz5-1, ers2 mhz5-1, and etr2 mhz5-1) were very similar to thatof mhz5-1 alone (Figure 7). These results indicate that the ethyl-ene receptor single mutants require an MHZ5-mediated pathwayto display the ethylene response phenotype in the roots or thatthe MHZ5-mediated pathway acts downstream of the three eth-ylene receptors ERS1, ERS2, and ETR2 to regulate the root eth-ylene response.

A double mutant was also produced by crossing homozygousmhz5-3 with ein2. ein2/mhz7-1 was identified as an ethylene-insensitive mutant in our previous study (Ma et al., 2013). In eti-olated seedlings, ein2 completely suppressed the coleoptileelongation phenotype of mhz5-3 in a wide range of ethyleneconcentrations (Figure 8), indicating that the coleoptile ethyleneresponse ofmhz5 requires EIN2 signaling. The roots of themhz5-3ein2 double mutant displayed an absolute insensitivity to eachconcentration of exogenous ethylene (Figures 8A and 8C), sug-gesting that EIN2 and MHZ5 most likely act within the samepathway for ethylene-induced root inhibition.To further examine the genetic relationship between MHZ5

and the ethylene signaling component EIN2, we analyzed theethylene response of the mhz5-3 EIN2-OE3 plants that wereobtained by crossing homozygous mhz5-3 with EIN2-OE3(EIN2-overexpression transgenic line; Ma et al., 2013). The co-leoptiles of mhz5-3 EIN2-OE3 homozygous plants were moreelongated than the mhz5-3 and EIN2-OE3 seedlings that weretreated with or without 1 ppm ethylene (Figures 8D and 8F). Bycontrast, the root growth of mhz5-3 EIN2-OE3 homozygousplants displayed a twisted phenotype in the seminal root in theair compared with that of EIN2-OE3 seedlings (Figures 8D and8E). This phenotype was most likely due to ABA deficiency and/or ethylene overproduction. Upon exposure to ethylene, theinhibition of root growth of EIN2-OE3 seedlings was partiallyalleviated in the mhz5-3 EIN2-OE3 seedlings; however, thewaved/twisted root phenotype remained similar or was moresevere in the mhz5-3 EIN2-OE3 seedlings that were treated withethylene compared with the seedlings without ethylene treat-ment (Figures 8D, 8E, 8G, and 8H). These data suggest that theMHZ5-mediated pathway is partially required by EIN2 signalingfor the regulation of the ethylene-induced inhibition of rootgrowth.We further generated ein2 MHZ5-OE48 plants by crossing the

ein2 mutant with MHZ5-OE48 plants overexpressing the MHZ5gene (Figure 6). The coleoptiles of the double mutant seedlingswere like those of ein2 with or without ethylene (Figures 8I and8J). However, with the ethylene treatment, the relative rootlength was mildly but significantly reduced in the ein2 MHZ5-OE48 seedlings compared with that in ein2 (Figures 8I and 8K).These results indicate that MHZ5 can partially suppress rootethylene insensitivity in the ein2 mutant.

DISCUSSION

In this study, we characterized the rice ethylene response mu-tant mhz5, which displays an enhanced ethylene response incoleoptile elongation but a reduced ethylene response in rootinhibition. We determined that MHZ5 encodes a carotenoidisomerase in the carotenoid biosynthesis pathway, facilitatingmetabolic flux into the biosynthesis of ABA precursors and ABA.Ethylene induces MHZ5 expression and accumulation of theABA biosynthesis precursor neoxanthin and ABA in roots. ABAlargely rescues the ethylene response in both the coleoptilesand roots of mhz5 etiolated seedlings. Genetically, the MHZ5-mediated ABA pathway acts downstream of ethylene signalingto regulate root growth in rice. This interaction between ethyleneand ABA is different from that in Arabidopsis, where ABA

Ethylene, Carotenoids, and ABA in Rice 1071

Figure 6. MHZ5 Overexpression Alters the Ethylene Response in Rice.

(A) MHZ5 expression levels in shoots and roots of 3-d-old dark-grown wild type and four MHZ5 overexpression lines. Values are the means 6 SD ofthree biological replicates.(B) Phenotypes of the wild type and various MHZ5 overexpression lines in response to ethylene. The 2.5-d-old dark-grown seedlings of the wild typeand four independent transgenic lines were treated with or without 1 ppm ethylene. Bar = 10 mm.(C) Effect of ethylene on coleoptile length. Values are means 6 SD of 20 to 30 seedlings per genotype.(D) Effect of ethylene on root length. Values are means 6 SD of 20 to 30 seedlings per genotype.(E) Relative root length of wild-type and transgenic lines in response to ethylene (ethylene-treated versus untreated in the wild type and MHZ5-OE lines,respectively). The data are derived from (D).(F) Expression of ethylene-responsive genes in the shoots of the wild type and four transgenic lines. Three-day-old dark-grown seedlings were treatedwith or without ethylene (10 ppm) for 8 h, and total RNA was extracted for qRT-PCR. Values are means 6 SD of three biological replicates.(G) Expression levels of genes preferentially induced by ethylene in the roots. Others are as in (F).(H) EIN2 transcript levels in the shoots of 3-d-old etiolated seedlings of wild-type and MHZ5-OE lines as detected using RT-PCR. Actin1 served as theloading control. Each experiment was repeated at least three times with similar results.

requires ethylene signaling for root inhibition. By contrast, theMHZ5-mediated ABA pathway negatively regulates EIN2 sig-naling to control coleoptile growth. Our results reveal novel in-terplays among ethylene, carotenoid, and ABA in the regulationof the ethylene response in rice.

An MHZ5-Mediated ABA Pathway Acts Downstream ofEthylene Signaling for Root Growth Inhibition in EtiolatedRice Seedlings

We provide several lines of evidence to demonstrate that theMHZ5-mediated ABA pathway is required for the ethylene in-hibition of root growth in rice. First, light treatment rescues themhz5-1 root ethylene response through the photoisomerizationof prolycopene into downstream metabolites. Second, blocking

the carotenoid pathway with an inhibitor (Flu) led to aberrantethylene response phenotypes in the wild type that are similar tothe ethylene response inmhz5. Third, the exogenous applicationof ABA significantly recovers the mutant ethylene response.Fourth, ethylene induces MHZ5 expression, ABA biosynthesisprecursor neoxanthin and ABA accumulation in wild-type roots,and ethylene-induced ABA accumulation depends on MHZ5function. Fifth, ethylene-induced ABA mediates the expressionof some ethylene-responsive genes. Sixth, MHZ5 over-expression leads to an enhanced ethylene response and pro-motes ethylene-induced gene expression in the roots. Seventh,genetic analysis suggests that ethylene signaling acts upstreamof the MHZ5-mediated ABA pathway to regulate root growth(Figures 7 and 8). Additionally, other ABA-deficient mutants,such as mhz4/aba4 (Ma et al., 2014), aba1, and aba2, also

Figure 7. Genetic Interactions between mhz5-1 and Ethylene Receptor Loss-of-Function Mutants through Double Mutant Analyses.

(A) Comparison of the root ethylene response in Nipponbare (Nip), Dongjin (DJ), and the single and double mutants in the absence or presence ofethylene (1 ppm). Representative 2.5-d-old dark-grown seedlings are shown. Bars = 10 mm.(B) Ethylene dose–response curves for the root length of 2.5-d-old dark-grown seedlings of Nipponbare, Dongjin, mhz5-1, and double mutants (ers1mhz5-1, ers2 mhz5-1, and etr2 mhz5-1). The values are the means 6 SD of 20 to 30 seedlings per genotype at each dose. The experiment was repeatedat least three times with similar results.

Ethylene, Carotenoids, and ABA in Rice 1073

Figure 8. Genetic Interaction between MHZ5 and EIN2 in the Regulation of the Ethylene Response.

(A) Phenotypes of 3-d-old dark-grown seedlings in the presence or absence of ethylene (10 ppm). Bars = 10 mm.

exhibit reduced ethylene sensitivity in roots (SupplementalFigure 10). Moreover, higher concentrations of ABA inhibit rootgrowth in etiolated rice seedlings (Supplemental Figure 7). Fromthe above evidence, we propose that ethylene may exert itseffects on root inhibition at least partially through the MHZ5-mediated ABA pathway (Figure 9).

Our finding that the ethylene inhibition of root growth in rice isat least partially ABA dependent is in contrast with that obtainedin Arabidopsis, in which the ethylene inhibition of root growth isABA independent, and ABA requires ethylene biosynthesis andsignaling for root growth regulation (Beaudoin et al., 2000;Ghassemian et al., 2000; Cheng et al., 2009; Luo et al., 2014).This difference is mostly likely due to the different plant speciesthat were used. The different living conditions of their seedlings,namely, the hypoxic environment in rice versus normal aeratedsoil in Arabidopsis, may also be the reason for this result. It is notclear whether other monocotyledonous seedlings have a similarmechanism.

Themhz5mutant exhibits reduced sensitivity, but not completeinsensitivity, to ethylene in rice roots, and ethylene is still able tocause;35% reduction inmhz5 root growth (Figure 1). These datasuggest that ethylene can inhibit root growth through both anABA-dependent and ABA-independent manner. Because the re-maining ethylene response of the mhz5 roots was completelyblocked by ein2, whose loss of function makes etiolated riceseedlings completely insensitive to ethylene (Ma et al., 2013), theABA-independent ethylene response may rely on EIN2 and/or itsdownstream event. Taken together, these results demonstratethat the maximum inhibition of root growth by ethylene involvesboth ABA-dependent and ABA-independent functions and thatthe MHZ5-mediated ABA pathway may work together with theEIN2 downstream signaling pathway to coregulate the ethyleneinhibition of root growth (Figure 9A).

The Role of MHZ5 in the Ethylene Regulation of RiceColeoptile Elongation

Rice seedlings have a coleoptile for protection of emergingleaves. This feature is different from Arabidopsis seedlings.Ethylene promotes coleoptile elongation (Figure 1). ABA accu-mulation is reduced in the mhz5 mutant, whereas ethylene

production is enhanced (Figures 5 and 6). The coleoptile elon-gation of mhz5 is promoted in response to ethylene (Figure 1),indicating a hypersensitive response in etiolated rice seedlingscompared with that in the wild type. The enhanced ethyleneresponse is mostly likely due to the high expression of EIN2 inmhz5 shoots and not due to the ethylene overproductionbecause the treatment with ethylene biosynthesis inhibitor AVGdid not significantly affect the ethylene response ofmhz5 (Figure5). In addition, the hypersensitive ethylene response of mhz5 isfully dependent on EIN2 signaling through double mutant anal-ysis (Figures 8A and 8B). These findings led us to conclude thatthe MHZ5-mediated ABA pathway inhibits ethylene productionand negatively modulates ethylene signaling to control co-leoptile elongation (Figure 9B). In a feedback control manner,ethylene could decrease ABA accumulation in the shoots/co-leoptiles (Figure 4A) to release the inhibitory roles of ABA (Figure9B). ABA is also an inhibitory modulator of the ethylene-inducedmorphological changes of etiolated rice seedlings (Lee et al.,1994; Nambara and Marion-Poll, 2005).In Arabidopsis, ABA regulates root growth via ethylene sig-

naling in a synergistic regulatory manner (Beaudoin et al., 2000;Ghassemian et al., 2000; Cheng et al., 2009; Luo et al., 2014).However, we found that the MHZ5-mediated ABA pathwayantagonistically modulates ethylene signaling for coleoptile in-hibition in rice seedlings (Figure 9B). In both cases, ABA actsupstream of ethylene signaling; however, the regulatory mech-anism is different, with a synergistic regulation in Arabidopsisroots but an antagonistic regulation in rice coleoptiles. Thisdifferent regulatory mechanism may be due to the different plantspecies that are used or due to the different living conditionsthat are adopted. It should be mentioned that, considering thatother ABA-deficient mutants of aba1 and aba2 (SupplementalFigure 10) were weaker than that of mhz5 in terms of the co-leoptile ethylene response, the possibility cannot be excludedthat other carotenoid-derived molecules (e.g., SL, BYPASS,and/or uncharacterized compounds) and/or interactions amongdifferent plant growth regulators may also contribute to regula-tion of coleoptile ethylene responses in rice.In etiolated rice seedlings, crosstalk may occur at multiple

levels between ethylene and ABA, such as the biosynthesispathway, signaling pathway, or even responsive genes. Ethylene

Figure 8. (continued).

(B) Ethylene dose–response curves for the coleoptile length of 3-d-old dark-grown seedlings. The values are the means 6 SD of 20 to 30 seedlings pergenotype at each dose.(C) Ethylene dose–response curves for the root length. Others are as in (B).(D) Phenotypes of mhz5-3 EIN2-OE3 dark-grown seedlings in the presence or absence of ethylene (1 ppm) for 3 d. Bars = 10 mm.(E) Enlargement of the roots in (D). Bars = 10 mm.(F) Coleoptile length of the wild type, mhz5-3, EIN2-OE3, and mhz5-3 EIN2-OE3 in the presence or absence of ethylene (1 ppm). For each column, thevalues are the means 6 SD of 30 seedlings per genotype.(G) Root length of the wild type, mhz5-3, EIN2-OE3, and mhz5-3 EIN2-OE3. Others are as in (F).(H) Relative root length (ethylene-treated versus untreated in each genotype, respectively) derived from data in (G). Others are as in (F).(I) Phenotypes of the wild-type, ein2,MHZ5-OE48, and ein2 MHZ5-OE48 dark-grown seedlings in the presence or absence of ethylene (10 ppm) for 3 d.Bars = 10 mm.(J) Coleoptile length of the wild type, ein2, MHZ5-OE48, and ein2 MHZ5-OE48 in the presence or absence of ethylene (10 ppm). Others are as in (F).(K) Relative root length (ethylene-treated versus untreated in each genotype, respectively). Others are as in (J). Student’s t test (** P < 0.01). Eachexperiment was repeated at least three times with similar results.

Ethylene, Carotenoids, and ABA in Rice 1075

biosynthesis genes, such as ACS and ACO, were upregulated,and ethylene production increased significantly in mhz5 etio-lated seedlings, suggesting that ethylene and ABA can act an-tagonistically at the biosynthesis level. These observations areconsistent with those in the tomato mutant flacca (Tal, 1979) andthe Arabidopsis mutants aba1 and aba2 (Rakitina et al., 1994;LeNoble et al., 2004). The data mentioned above suggest thatthe ABA inhibition of ethylene biosynthesis is conserved.

Ethylene Regulates Carotenoid Biosynthesis in EtiolatedRice Seedlings

Carotenoids are essential pigments that play pivotal roles inphotoprotection (Niyogi, 1999; Dall’Osto et al., 2007; Wei et al.,2010; Ballottari et al., 2014). Carotenoid-derived compounds,such as SL, ABA, BYPASS, b-cyclocitral, and other un-characterized molecules, modulate plant developmental pro-cesses and stress responses in many organs (Xie et al., 2010;

Sieburth and Lee, 2010; Walter et al., 2010; Cazzonelli andPogson, 2010; Puig et al., 2012; Ramel et al., 2012; Avendaño-Vázquez et al., 2014; Van Norman et al., 2014; Liu et al., 2015).The regulation of carotenoid biosynthesis is interconnected withplant developmental and environmental responses, and thebiosynthesis pathway is regulated at both the transcriptional andposttranscriptional levels in plants (Ruiz-Sola and Rodríguez-Concepción, 2012). Previous studies have found that theinteraction between carotenogenesis and ethylene is mainlyassociated with tomato (Solanum lycopersicum) fruit ripening, inwhich ethylene influences multiple steps in carotenoid synthe-sis, impacting the net and relative accumulation of the com-pounds (Bramley, 2002; Alba et al., 2005). In this study, theethylene-induced expression of the carotenoid isomerase geneMHZ5 drove the metabolic flux into the formation of ABA bio-synthesis precursors, including neoxanthin, leading to ABA ac-cumulation in the roots and to the root growth inhibition ofetiolated rice seedlings (Figure 4). This conclusion is furthersupported by our recent finding that ethylene also induces theexpression of rice ABA4 (Ma et al., 2014), a gene homologous toArabidopsis ABA4, encoding a membrane protein that mightregulate the conversion of zeaxanthin to neoxanthin in the ABAbiosynthesis pathway (North et al., 2007). Additionally, ethyleneinduces the transcription of NCED in the ABA biosynthesispathway and then the accumulation of ABA to modulate fruitripening in grape berry (Vitis vinifera; Sun et al., 2010). Theseanalyses suggest that ethylene regulates the carotenoidbiosynthesis pathway at both the early steps, e.g., the con-version of prolycopene to all-trans-lycopene by carotenoidisomerase MHZ5 and the late steps in the ABA biosynthesispathway to modulate rice seedling growth and/or the fruitripening process.Root tissue is a major site of ABA biosynthesis, where the low

concentrations of carotenoid precursors may prove rate-limiting.Although only trace levels of neoxanthin and violaxanthin havebeen identified in the root tissue of plants (Parry and Horgan,1992), the trace levels of carotenoids that are induced by eth-ylene play an important role in ABA biosynthesis to synergisti-cally inhibit the root growth of etiolated rice seedlings (Figure 4).Additionally, in plant roots, the carotenoid biosynthesis rate-limiting enzyme PSY isogenes that are involved in the pro-duction of root carotenoids are induced by abiotic stress andspecifically by ABA (Welsch et al., 2008; Meier et al., 2011; Ruiz-Sola and Rodríguez-Concepción, 2012). These findings indicatethat carotenoid biosynthesis in the leucoplasts of roots iselaborately regulated by external and internal cues. It is possiblethat multiple regulation manners allow plants to be more adaptedto the complicated and changing environment at different growthand developmental stages.Shifting mhz5 seedlings from dark to light altered the carot-

enoid profile to the immediate precursors of ABA biosynthesis(Figure 3G), which is similar to those reported for light-grownseedlings of zebra2/crtiso, an allelic mutant of mhz5, when ex-posed to higher light intensities (Chai et al., 2011). However, ourstudy reveals roles for the carotenoid isomerase/MHZ5 in reg-ulation of ethylene responses. Additionally, themhz5mutant hascomplicated phenotypes in the field (Supplemental Figures 1and 2) that have not been previously reported (Chai et al., 2011).

Figure 9. A Proposed Model of the Interactions between Ethylene andthe ABA Pathway in Rice Seedlings.

(A) Ethylene signaling acts upstream of the ABA pathway to regulate rootgrowth. The inhibition of root growth in response to increased amounts ofethylene is at least partially dependent on the MHZ5/CRTISO-mediated ABApathway. The ABA pathway is required to synergize the ethylene signalingcascade and stimulate responsive genes in inhibiting root growth in riceseedlings.(B) Ethylene signaling acts downstream of the MHZ5/CRTISO-mediatedABA pathway for the regulation of coleoptile elongation. The promotionof coleoptile growth in response to increased ethylene is mediated byinhibiting endogenous ABA accumulation. ABA suppresses the ethylenesignaling cascade by suppressing EIN2 expression in etiolated riceseedlings.

1076 The Plant Cell

Field-grown mhz5 plants under environmental light conditionsdid not resemble wild-type plants, suggesting that light can onlypartially substitute for MHZ5/CRTISO activity, which is consis-tent with previous reports in Arabidopsis and tomato (Isaacsonet al., 2002; Park et al., 2002). In addition to the current roles ofthe carotenoid-derived ABA pathway in the regulation of riceseedling growth, other carotenoid-derived molecules, e.g., SL,BYPASS, and uncharacterized compounds, may be responsiblefor tiller formation (Supplemental Figure 1), root development(Supplemental Figure 2), and other phenotypic changes in field-grown mhz5 plants (Nambara and Marion-Poll, 2005; Umeharaet al., 2008; Sieburth and Lee, 2010; Kapulnik et al., 2011; Puiget al., 2012; Ramel et al., 2012; Van Norman et al., 2014).

In conclusion, we demonstrate that the carotenoid bio-synthesis of rice is regulated by ethylene. Ethylene requires theMHZ5/carotenoid isomerase-mediated ABA pathway to inhibitroot growth, and the MHZ5/carotenoid isomerase-mediatedABA pathway negatively regulates coleoptile elongation at leastin part by modulating EIN2 expression. This study demonstratesthe importance of carotenoid pathway in generating regulatorymolecules that can affect major developmental processes andfunction differentially in specific organ development. Our resultsprovide important insights into the interactions among ethylene,carotenogenesis, and ABA in rice, which are different from thosein Arabidopsis. The manipulation of the corresponding compo-nents may improve agronomic traits and adaptive growth in rice.

METHODS

Plant Materials and Growth Conditions

mhz5, ein2/mhz7-1, and EIN2-OE3 were previously identified (Ma et al.,2013). Themhz5 allelemhz5-4 was obtained from Tos17 retrotransposoninsertion lines (line number NG0489). The rice (Oryza sativa) aba1 and aba2mutants were kindly provided by Cheng-Cai Chu (Institute of Geneticsand Developmental Biology, Chinese Academy of Sciences). The T-DNAknockout mutants ers1, ers2, and etr2 are in the DJ background and wereobtained from the POSTECH Biotech Center (Yi and An, 2013). Theprimers that were used to identify homogenous ers1, ers2, and etr2 arelisted in Supplemental Table 1. The ethylene treatments were performedas previously described (Ma et al., 2013) with the following modifications:The seedlings were incubated in the dark or under continuous light(provided by fluorescentwhite-light tubes [400 to 700 nm, 250mmolm22 s21])for 2 to 4 d as indicated in each experiment. For material propagation,crossing, and investigating agronomic traits, rice plants were cultivated atthe Experimental Station of the Institute of Genetics and DevelopmentalBiology in Beijing during the natural growing seasons.

Map-Based Cloning of mhz5

To map the mhz5 locus, F2 populations were derived from the crossbetween the mutant mhz5-1 (Nipponbare and japonica) and the 93-11,MH63, ZF802, and TN1 (indica) cultivars. The genomic DNA of etiolatedseedlings from F2 progeny with a mutant phenotype was extracted usingan SDS method (Dellaporta et al., 1983). Themhz5-1 was subjected to rawand fine mapping using 589 segregated mutant individuals with insertion-deletion (Idl) and/or single sequence repeat markers that were the same asthose previously described (Ma et al., 2013). The mhz5-1 locus was pri-marily delimited to an interval of ;0.9 M between the two markers Idl11-20.3 and Idl11-21.2 on the long arm of chromosome 11. To fine-mapmhz5,additional Idl markers were generated based on the entire genomic

sequences of Nipponbare and 93-11. mhz5 was finally mapped to chro-mosome 11 between Idl11-20.557 (59-GGTCGTCGGTTGATGATAG-39and 59-TACGTCGCCACTACAAATG-39) and Idl11-20.709 (59-AGAGCA-GATTCAGCACCAGA-39 and 59-ATCAGCTGCTAACTGTCTGC-39), whichcontains 10 genes. The candidate gene was finally determined via the DNAsequencing of all of the genes in this region. The mutations of the threealleles ofmhz5were confirmed via derived cleaved amplified polymorphicsequence (mhz5-1 F, 59-TAGTTCTTCCACGTCAGGATCTAAG-39; mhz5-1R, 59-TCGGTGTGTTTTTGGTGAGCCCAGC-39; mhz5-2 F, TGCTGGA-GAAGTACGTCATCCCCGCG-39; and mhz5-2 R, CAAGATCCCCAGAA-TATACTAGCAGC-39) and amplified fragment length polymorphism (mhz5-3F, 59-TGCTGGAGAAGTACGTCATC-39, and mhz5-3 R, 59-CCCCAGAA-TATACTAGCAGC-39) assay using PCR.

Pigment Analysis and Quantification

Pigment extraction and analysis of leaf material was performed as previouslydescribed (Pogson et al., 1996; Park et al., 2002) except for the use of 300mgof fresh weight tissue, 800 mL of acetone/ethyl acetate, and 620 mL of waterduring the sample extraction process. Due to the low level of carotenoids,pigment extraction and analysis in roots were performed as previouslydescribed (Fraser et al., 2000) with the followingminor modifications: 1.2 g offresh weight tissue was used for each sample. Carotenoids were identifiedbased on their characteristic absorption spectra and typical retention timecomparedwith those of authentic standards and referring to previous reports(Fraser et al., 2000; Park et al., 2002). The relative abundance of each ca-rotenoid was obtained by showing the ratio of each peak area (the mhz5-1mutant versus the wild type after illumination or ethylene-treated versusuntreated in the wild type, respectively). Total chlorophyll was measured aspreviously described (Kong et al., 2006) with the following minor mod-ifications: Chlorophyll was extracted from fresh samples in 95%ethanol, andthe absorbance was measured at 665, 645, and 652 nm.

For carotenoid assays, etiolated wild-type andmhz5-1 seedlings weregrown in the dark for 3 to 4 d or the etiolated seedlings were treated with10 ppm ethylene or transferred to continuous light for 24 h, after which theleaves and roots were frozen in liquid nitrogen for extractions.

Vector Construction and Rice Transformation

The complementation vector was constructed as follows. First, part ofthe MHZ5 genomic DNA fragment (containing the 2278-bp upstreamsequence and a 1657-bp part of the coding region) was PCR amplifiedand ligated to a pCAMBIA2300 vector (provided by Cheng-Cai Chu) thatwas digested with XbaI and SalI to generate pMHZ5CM. The second partof the MHZ5 genomic DNA fragment (containing the 2018-bp left part ofthe coding region and the 1169-bp downstream region) was PCR am-plified and ligated to the SalI and Sse8387I sites of the pMHZ5CM vectorto form pMHZ5C. To construct the MHZ5 overexpression vector, theopen reading frame (ORF) ofMHZ5 was amplified using PCR and clonedinto the binary vector pCAMBIA2300-35S-OCS at the sites of KpnI andSalI.

To inhibit expression of the SL-relevant genes D17 and D3, D17-RNAinterference (D17-RNAi) andD3-RNA interference (D3-RNAi) vectors wereconstructed as follows. A 375-bp fragment of the D17 ORF and a 438-bpfragment of the D3 ORF were cloned in both orientations in pCam-bia2300Actin at the sites SalI and BamHI and separated by the first intronof the GA20 oxidase of potato (Solanum tuberosum) to form a hairpinstructure (Luo et al., 2005). All of the primers that were used above in thisstudy are listed in Supplemental Table 2.

The above constructs were transformed into mhz5-3 or the wild type(Nipponbare) as previously described (Wuriyanghan et al., 2009). Thetransformants were selected via PCR using kanamycin resistance (NPT II )gene-specific primers (Supplemental Table 2). Homozygous T3 or T4transgenic lines were selected via kanamycin treatment (50 mg/L).

Ethylene, Carotenoids, and ABA in Rice 1077

Measurement of ABA, Ethylene, and SL Production

For the ABA content assays, 3-d-old wild-type and mhz5-1 etiolatedseedlings were treated with or without 10 ppm ethylene for 24 h, and theshoots (containing the coleoptile and the first leaves) and roots wereharvested. For each sample, ;200 mg of fresh tissue was homogenizedunder liquid nitrogen, weighed, and extracted for 24 h with cold methanolcontaining antioxidant and 6 ng 2H6-ABA (internal standard; OlChemIm).Endogenous ABA was purified and measured as previously described (Fuet al., 2012) with some changes in detection conditions. The ultra-performance liquid chromatography-tandem mass spectrometer systemconsists of a UPLC system (ACQUITY UPLC; Waters) and a hybrid triplequadruple-linear ion trapmass spectrometer (QTRAP 5500; AB SCIEX). Thechromatographic separationwas achieved on aBEHC18 column (50mm3

2.1 mm, 1.7 mm; Waters) with the column temperature set at 25°C anda flow rate of 0.2mL/min. The linear gradient runs from95 to 85%A (solventA, 0.05% acetic acid aqueous; solvent B, acetonitrile) in 1 min, 85 to 30% Ain the next 5 min, 30 to 2% A in the following 1 min, and reequilibrated withthe initial condition for 2 min. The optimizedmass spectrometer parameterswere set as follows: curtain gas 40 p.s.i., collision gas 6 p.s.i., ion sprayvoltage 24300 V, and temperature 550°C. The declustering potential was285 V and collision energy was215 V. Multiple reaction monitoring (MRM)mode was used for quantification, and the selected MRM transitions were263.0 > 153.1 for ABA and 269.1 > 159.3 for 2H6-ABA.

For the ethylene production assays, the seedlings were grown in thedark or under continuous light in a 40-mLuncapped vial for 7 d at 28°C,after which the vials were sealed with a rubber syringe cap for 17 h, and1 mL of headspace of each vial was measured using gas chromatography(GC-2014; Shimadzu). The ethylene production of the seedlings that weretreated with AVG (50 mM) was measured in the same manner.

The SL collection, purification, and analysis were performed as pre-viously described (Jiang et al., 2013) with some changes in detectionconditions. SL was analyzed using the ultraperformance liquid chroma-tography-tandem mass spectrometer system consisting of a UPLCsystem (ACQUITY UPLC) equipped with a BEH C18 column (100 mm 3

2.1 mm, 1.7 mm; Waters) and a hybrid triple quadrupole-linear ion trapmass spectrometer (QTRAP 5500; AB SCIEX) equipped with an elec-trospray ionization source. The gradient started from 50%mobile phase A(0.05% acetic acid in water) and increased mobile phase B (0.05% aceticacid in acetonitrile) from 50 to 90% in 5 min at 25°C with a flow rate of 0.3mL/min. MS parameters were set as follows: ion spray voltage, 4500 V;desolvation temperature, 600°C; nebulizing gas, 50 p.s.i.; desolvation gas,55 p.s.i.; curtain gas, 40 p.s.i.; declustering potential, 90 V; entrance potential,7 V; collision cell exit potential, 12 V; and collision energy, 23 V. MRM modewas used for quantification, and the selected MRM transitions were 331.2 >216.1 for epi-5DS and 337.2 > 221.1 for d6-epi-5DS.

For ABA, ethylene, and SL detection, each experiment was repeatedthree times, and the averages and standard deviations are shown.

Coleoptile Elongation and Root Growth Tests

For the ABA rescue of mhz5-1 short roots, 1.5 liters of 0.04 mM ABA (mixedisomers; Sigma-Aldrich) was used. For the ABA rescue of mhz5-1 ethylenesensitivity, germinated seedswereplacedona stainless steel sieve in a 5.5-literair-tight plastic box. Then, 1.5 liters of solutionwith or without 0.1mMABAwasadded to the plastic box. ABA stock solutions were prepared in ethanol, andequivalent volumes of ethanol were added to the control. The seedlings weretreatedwith or without ethylene (10 ppm) at 28°C in the dark. The coleoptiles ofthewild type andmhz5-1were sprayedoncedailywith 0.1mMABA (containing0.001% Tween 20) after germination. For the AVG treatment, the germinatedseeds of the wild type andmhz5-1were placed on eight layers of cheesecloththat were immersed in solution in Petri dishes and incubated in a 5.5-liter air-tight plastic box. AVG was dissolved in water, and a final concentration of 50mMwas used to inhibit endogenous ethylene. The seedlings were treated with

a range of concentrations of ethylene at 28°C in the dark. After 2.5 d oftreatment, the coleoptile elongation and/or root growth were measured.

GR24 and Flu Treatment

Germinated rice seeds were placed on cheesecloth on a stainless steelsieve that was placed in a 5.5-liter air-tight plastic box and incubated at28°C in the dark. The seeds were subjected to the following treatment.The air-tight plastic box contained 700mL of water with either 0.25mMFlu(Sigma-Aldrich) or 1 mM GR24, a synthetic SL analog (Chiralix). A pre-liminary experiment showed that Flu can substantially inhibit ABA bio-synthesis in the shoots/coleoptiles and roots of etiolated rice seedlings(Supplemental Figure 11). Flu and GR24 were dissolved in acetone. Thecontrol treatments also contained 0.5% acetone. The ethylene treatmentwas performed as previously described (Ma et al., 2013).

Gene Expression Analysis Using RT-PCR

Three-day-old etiolated seedlings were treated for up to 8 h with 10 ppmethylene or air or with the application of 100 mM ABA and/or 100 mM NDGAwith or without ethylene. Equivalent volumes of ethanol were added to theABA-free or NDGA-free controls. After treatment, the shoots and roots wereharvested and immediately frozen in liquid nitrogen. The total RNA extractionand RT-PCR were performed as previously described (Ma et al., 2013). RiceActin1 orActin2was used as the internal control to quantify the relative level ofeach target gene. Thegene-specificprimers are listed inSupplemental Table 2.

Genetic Analysis

Double mutants of ers1 mhz5-1, ers2 mhz5-1, etr2 mhz5-1, mhz5-3 ein2,mhz5-3 EIN2-OE3, and ein2 MHZ5-OE48 were generated by crossingtheir respective parental lines and identified by genotyping from their F2populations, respectively, and their progeny were phenotypically and/orgenotypically analyzed in F3 or F4 populations.

Agronomic Trait Analysis

The germinated seeds were grown on a stainless steel sieve in Kimuranutrient solution in a climate chamber. Two weeks later, the maximumlength and the number of primary roots, adventitious roots, and lateralroots were measured. After harvest, agronomic traits, including the well-filled grain length/width, the number of primary and second branches perpanicle, and the tiller number of mhz5 and the wild type were analyzed.

Statistical Analysis

The relative root or coleoptile length is analyzed relative to the length ofeach genotype in untreated conditions. To analyze the gene expressionlevel, each gene expression level in untreated wild type was set to 1. All ofthe data were analyzed using a one-way ANOVA (LSD t test) for the testgroups with SPSS 18.0.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under the following accession numbers: Os11g36440 (MHZ5),Os07g06130 (EIN2), Os10g36650 (Actin2), Os08g10310 (SHR5), Os08g13440(Germin-like), Os09g11480 (ERF063), Os09g11460 (ERF073), Os07g05360(Photosystem II 10 kD polypeptide), Os08g09080 (RGLP1), Os06g08340(ERF002), Os06g07040 (IAA20), Os11g05740 (RAP2.8), Os07g26720 (RRA5),Os09g27750 (ACO3), Os05g05680 (ACO5), Os04g48850 (ACS2),Os06g03990 (ACS6), Os04g46470 (D17), Os06g06050 (D3), Os03g50885(Actin1), Os03g49500 (ERS1), Os05g06320 (ERS2), andOs04g08740 (ETR2).mhz5-4 represents a Tos17 insertion mutant (NG0489). ers1 represents a

1078 The Plant Cell

T-DNA insertionmutant (1B-08531). ers2 represents a T-DNA insertionmutant(3A-14390). etr2 represents a T-DNA insertion mutant (4A-03543).

Supplemental Data

Supplemental Figure 1. Phenotype of the mhz5 Mutant in the Field.

Supplemental Figure 2. Comparison of Root Development betweenWild-Type and mhz5-1 Mutant Plants.

Supplemental Figure 3. Phenotype and Ethylene Response ofmhz5-4.

Supplemental Figure 4. Pigment Analysis of Wild-Type and mhz5-1Roots.

Supplemental Figure 5. Greening Phenotype and Chlorophyll Accu-mulation of the Wild Type and mhz5-1 Mutant.

Supplemental Figure 6. GR24 Cannot Rescue the Ethylene Responseof mhz5-1.

Supplemental Figure 7. ABA Dose–Response Curves for Wild-TypeColeoptile and Root.

Supplemental Figure 8. Effect of AVG on Ethylene Production and theColeoptile Ethylene Response of the Wild Type and mhz5-1 Mutant.

Supplemental Figure 9. Characterization of the Ethylene ReceptorMutants of Rice.

Supplemental Figure 10. Ethylene Response of Other ABA-DeficientMutants in Rice.

Supplemental Figure 11. Quantification of the ABA Levels in Flu-Treated Wild-Type Seedlings.

Supplemental Table 1. Primers Used for Receptor Mutant Analysisvia PCR-Based Genotyping.

Supplemental Table 2. Primers Used for Gene Expression Analysisand Vector Construction.

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation ofChina (91317306), by the 973 Projects (2015CB755702, 2012CB114202,and 2013CB835205), by the CAS Project (KSCX3-EW-N-07), and by theNational Natural Science Foundation of China (91417314) and State KeyLab of Plant Genomics.

AUTHOR CONTRIBUTIONS

C.C.Y., B.M., J.S.Z., and S.Y.C. conceived and designed the experiments.C.C.Y. performed experiments. B.M. isolated the mhz5 mutant. S.J.H.carried out rice transformation. T.G.L. provided the original rice mutationpools. B.J.P., D.P.C., Y.Q.W., and C.C.C. performed the carotenoid analysis.B.J.P. and D.P.C. discussed and edited the article. J.F.C., X.H.S., and S.F.performed the ABA and SL analyses. Q.X. and others contributed to somematerial preparation. C.C.Y., J.S.Z., and B.M. wrote the article.

Received January 28, 2015; revised February 28, 2015; accepted March17, 2015; published April 3, 2015.

REFERENCES

Alba, R., Payton, P., Fei, Z., McQuinn, R., Debbie, P., Martin, G.B.,Tanksley, S.D., and Giovannoni, J.J. (2005). Transcriptome and

selected metabolite analyses reveal multiple points of ethylenecontrol during tomato fruit development. Plant Cell 17: 2954–2965.

An, F., et al. (2010). Ethylene-induced stabilization of ETHYLENEINSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal deg-radation of EIN3 binding F-box 1 and 2 that requires EIN2 in Ara-bidopsis. Plant Cell 22: 2384–2401.

Arc, E., Sechet, J., Corbineau, F., Rajjou, L., and Marion-Poll, A.(2013). ABA crosstalk with ethylene and nitric oxide in seed dor-mancy and germination. Front. Plant Sci. 4: 63.

Argueso, C.T., Hansen, M., and Kieber, J.J. (2007). Regulation ofethylene biosynthesis. J. Plant Growth Regul. 26: 92–105.

Avendaño-Vázquez, A.O., Cordoba, E., Llamas, E., San Román, C.,Nisar, N., De la Torre, S., Ramos-Vega, M., Gutiérrez-Nava, M.D.,Cazzonelli, C.I., Pogson, B.J., and León, P. (2014). An uncharacterizedapocarotenoid-derived signal generated in z-carotene desaturase mu-tants regulates leaf development and the expression of chloroplast andnuclear genes in Arabidopsis. Plant Cell 26: 2524–2537.

Ballottari, M., Alcocer, M.J., D’Andrea, C., Viola, D., Ahn, T.K.,Petrozza, A., Polli, D., Fleming, G.R., Cerullo, G., and Bassi, R.(2014). Regulation of photosystem I light harvesting by zeaxanthin.Proc. Natl. Acad. Sci. USA 111: E2431–E2438.

Beaudoin, N., Serizet, C., Gosti, F., and Giraudat, J. (2000). Inter-actions between abscisic acid and ethylene signaling cascades.Plant Cell 12: 1103–1115.

Benschop, J.J., Jackson, M.B., Gühl, K., Vreeburg, R.A., Croker,S.J., Peeters, A.J., and Voesenek, L.A. (2005). Contrasting inter-actions between ethylene and abscisic acid in Rumex species dif-fering in submergence tolerance. Plant J. 44: 756–768.

Bleecker, A.B., and Kende, H. (2000). Ethylene: a gaseous signalmolecule in plants. Annu. Rev. Cell Dev. Biol. 16: 1–18.

Bramley, P.M. (2002). Regulation of carotenoid formation during to-mato fruit ripening and development. J. Exp. Bot. 53: 2107–2113.

Cao, W.H., Dong, Y., Zhang, J.S., and Chen, S.Y. (2003). Charac-terization of an ethylene receptor homolog gene from rice. Sci.China Life Sci. 46: 370–378.

Cazzonelli, C.I., and Pogson, B.J. (2010). Source to sink: regulationof carotenoid biosynthesis in plants. Trends Plant Sci. 15: 266–274.

Chai, C., Fang, J., Liu, Y., Tong, H., Gong, Y., Wang, Y., Liu, M.,Wang, Y., Qian, Q., Cheng, Z., and Chu, C. (2011). ZEBRA2, en-coding a carotenoid isomerase, is involved in photoprotection inrice. Plant Mol. Biol. 75: 211–221.

Chen, Y., Li, F., and Wurtzel, E.T. (2010). Isolation and character-ization of the Z-ISO gene encoding a missing component of ca-rotenoid biosynthesis in plants. Plant Physiol. 153: 66–79.

Cheng, W.H., Chiang, M.H., Hwang, S.G., and Lin, P.C. (2009).Antagonism between abscisic acid and ethylene in Arabidopsis actsin parallel with the reciprocal regulation of their metabolism andsignaling pathways. Plant Mol. Biol. 71: 61–80.

Creelman, R.A., Bell, E., and Mullet, J.E. (1992). Involvement ofa lipoxygenase-like enzyme in abscisic acid biosynthesis. PlantPhysiol. 99: 1258–1260.

Cunningham, F.X., Jr., Pogson, B., Sun, Z., McDonald, K.A., DellaPenna,D., and Gantt, E. (1996). Functional analysis of the beta and epsilonlycopene cyclase enzymes of Arabidopsis reveals a mechanism forcontrol of cyclic carotenoid formation. Plant Cell 8: 1613–1626.

Cutler, A.J., and Krochko, J.E. (1999). Formation and breakdown ofABA. Trends Plant Sci. 4: 472–478.

Cutler, S.R., Rodriguez, P.L., Finkelstein, R.R., and Abrams, S.R.(2010). Abscisic acid: emergence of a core signaling network. Annu.Rev. Plant Biol. 61: 651–679.

Dall’Osto, L., Cazzaniga, S., North, H., Marion-Poll, A., and Bassi, R. (2007).The Arabidopsis aba4-1mutant reveals a specific function for neoxanthin inprotection against photooxidative stress. Plant Cell 19: 1048–1064.

Ethylene, Carotenoids, and ABA in Rice 1079

Dellaporta, S.L., Wood, J., and Hicks, J.B. (1983). A plant DNA mini-preparation version II. Plant Mol. Biol. Rep. 1: 19–21.

Dong, C.H., Jang, M., Scharein, B., Malach, A., Rivarola, M., Liesch, J.,Groth, G., Hwang, I., and Chang, C. (2010). Molecular association ofthe Arabidopsis ETR1 ethylene receptor and a regulator of ethylenesignaling, RTE1. J. Biol. Chem. 285: 40706–40713.

Fang, J., et al. (2008). Mutations of genes in synthesis of the carot-enoid precursors of ABA lead to pre-harvest sprouting and photo-oxidation in rice. Plant J. 54: 177–189.

Finkelstein, R. (2013). Abscisic acid synthesis and response. TheArabidopsis Book 11: e0166, doi/10.1199/tab.0166.

Fraser, P.D., Pinto, M.E., Holloway, D.E., and Bramley, P.M. (2000).Technical advance: application of high-performance liquid chro-matography with photodiode array detection to the metabolic pro-filing of plant isoprenoids. Plant J. 24: 551–558.

Fu, J., Chu, J., Sun, X., Wang, J., and Yan, C. (2012). Simple, rapid,and simultaneous assay of multiple carboxyl containing phyto-hormones in wounded tomatoes by UPLC-MS/MS using single SPEpurification and isotope dilution. Anal. Sci. 28: 1081–1087.

Ghassemian, M., Nambara, E., Cutler, S., Kawaide, H., Kamiya, Y., andMcCourt, P. (2000). Regulation of abscisic acid signaling by the ethyl-ene response pathway in Arabidopsis. Plant Cell 12: 1117–1126.

Guo, H., and Ecker, J.R. (2003). Plant responses to ethylene gas aremediated by SCF(EBF1/EBF2)-dependent proteolysis of EIN3transcription factor. Cell 115: 667–677.

Hall, B., Samina, N., Shakeel, S., and Schaller, G.E. (2007). Ethylenereceptors: ethylene perception and signal transduction. J. PlantGrowth Regul. 26: 118–130.

Hirschberg, J. (2001). Carotenoid biosynthesis in flowering plants.Curr. Opin. Plant Biol. 4: 210–218.

Hoffmann-Benning, S., and Kende, H. (1992). On the role of abscisicacid and gibberellin in the regulation of growth in rice. Plant Physiol.99: 1156–1161.

Howitt, C.A., and Pogson, B.J. (2006). Carotenoid accumulation andfunction in seeds and non-green tissues. Plant Cell Environ. 29: 435–445.

Isaacson, T., Ohad, I., Beyer, P., and Hirschberg, J. (2004). Analysisin vitro of the enzyme CRTISO establishes a poly-cis-carotenoidbiosynthesis pathway in plants. Plant Physiol. 136: 4246–4255.

Isaacson, T., Ronen, G., Zamir, D., and Hirschberg, J. (2002).Cloning of tangerine from tomato reveals a carotenoid isomeraseessential for the production of b-carotene and xanthophylls inplants. Plant Cell 14: 333–342.

Jamil, M., Charnikhova, T., Verstappen, F., and Bouwmeester, H.(2010). Carotenoid inhibitors reduce strigolactone production and Strigahermonthica infection in rice. Arch. Biochem. Biophys. 504: 123–131.

Jiang, L., et al. (2013). DWARF 53 acts as a repressor of strigolactonesignalling in rice. Nature 504: 401–405.

Jun, S.H., Han, M.J., Lee, S., Seo, Y.S., Kim, W.T., and An, G. (2004).OsEIN2 is a positive component in ethylene signaling in rice. PlantCell Physiol. 45: 281–289.

Kanno, Y., Hanada, A., Chiba, Y., Ichikawa, T., Nakazawa, M., Matsui,M., Koshiba, T., Kamiya, Y., and Seo, M. (2012). Identification of anabscisic acid transporter by functional screening using the receptorcomplex as a sensor. Proc. Natl. Acad. Sci. USA 109: 9653–9658.

Kapulnik, Y., et al. (2011). Strigolactones affect lateral root formationand root-hair elongation in Arabidopsis. Planta 233: 209–216.

Kong, Z., Li, M., Yang, W., Xu, W., and Xue, Y. (2006). A novel nuclear-localized CCCH-type zinc finger protein, OsDOS, is involved in delayingleaf senescence in rice. Plant Physiol. 141: 1376–1388.

Ku, H.S., Suge, H., Rappaport, L., and Pratt, H.K. (1970). Stimulationof rice coleoptile growth by ethylene. Planta 90: 333–339.

Kuromori, T., Miyaji, T., Yabuuchi, H., Shimizu, H., Sugimoto, E.,Kamiya, A., Moriyama, Y., and Shinozaki, K. (2010). ABC transporter

AtABCG25 is involved in abscisic acid transport and responses. Proc.Natl. Acad. Sci. USA 107: 2361–2366.

Lee, T.M., Lur, H.S., Shieh, Y.J., and Chu, C. (1994). Levels of ab-scisic acid in anoxia- or ethylene-treated rice (Oryza sativa L.)seedlings. Plant Sci. 95: 125–131.

LeNoble, M.E., Spollen, W.G., and Sharp, R.E. (2004). Maintenanceof shoot growth by endogenous ABA: genetic assessment of theinvolvement of ethylene suppression. J. Exp. Bot. 55: 237–245.

Lin, Z., Zhong, S., and Grierson, D. (2009). Recent advances inethylene research. J. Exp. Bot. 60: 3311–3336.

Liu, L., Shao, Z., Zhang, M., and Wang, Q. (2015). Regulation ofcarotenoid metabolism in tomato. Mol. Plant 8: 28–39.

Luo, A., Liu, L., Tang, Z., Bai, X., Cao, S., and Chu, C.C. (2005).Down-regulation of OsGRF1 gene in rice rhd1 mutant results inreduced heading date. J. Integr. Plant Biol. 47: 745–752.

Luo, X., Chen, Z., Gao, J., and Gong, Z. (2014). Abscisic acid inhibitsroot growth in Arabidopsis through ethylene biosynthesis. Plant J.79: 44–55.

Lyzenga, W.J., and Stone, S.L. (2012). Regulation of ethylene bio-synthesis through protein degradation. Plant Signal. Behav. 7:1438–1442.

Lyzenga, W.J., Booth, J.K., and Stone, S.L. (2012). The ArabidopsisRING-type E3 ligase XBAT32 mediates the proteasomal degrada-tion of the ethylene biosynthetic enzyme, 1-aminocyclopropane-1-carboxylate synthase 7. Plant J. 71: 23–34.

Ma, B., Chen, S.Y., and Zhang, J.S. (2010). Ethylene signaling in rice.Chin. Sci. Bull. 55: 2204–2210.

Ma, B., et al. (2013). Identification of rice ethylene-response mutantsand characterization of MHZ7/OsEIN2 in distinct ethylene responseand yield trait regulation. Mol. Plant 6: 1830–1848.

Ma, B., Yin, C.C., He, S.J., Lu, X., Zhang, W.K., Lu, T.G., Chen, S.Y.,and Zhang, J.S. (2014). Ethylene-induced inhibition of root growthrequires abscisic acid function in rice (Oryza sativa L.) seedlings.PLoS Genet. 10: e1004701.

Mao, C., Wang, S., Jia, Q., and Wu, P. (2006). OsEIL1, a rice homologof the Arabidopsis EIN3 regulates the ethylene response as a posi-tive component. Plant Mol. Biol. 61: 141–152.

Mayerhofer, H., Panneerselvam, S., and Mueller-Dieckmann, J.(2012). Protein kinase domain of CTR1 from Arabidopsis thalianapromotes ethylene receptor cross talk. J. Mol. Biol. 415: 768–779.

Meier, S., Tzfadia, O., Vallabhaneni, R., Gehring, C., and Wurtzel, E.T.(2011). A transcriptional analysis of carotenoid, chlorophyll and plastidialisoprenoid biosynthesis genes during development and osmotic stressresponses in Arabidopsis thaliana. BMC Syst. Biol. 5: 77.

Merchante, C., Alonso, J.M., and Stepanova, A.N. (2013). Ethylenesignaling: simple ligand, complex regulation. Curr. Opin. Plant Biol.16: 554–560.

Nambara, E., and Marion-Poll, A. (2005). Abscisic acid biosynthesisand catabolism. Annu. Rev. Plant Biol. 56: 165–185.

Neuman, H., Galpaz, N., Cunningham, F.X., Jr., Zamir, D., andHirschberg, J. (2014). The tomato mutation nxd1 reveals a gene nec-essary for neoxanthin biosynthesis and demonstrates that violaxanthin isa sufficient precursor for abscisic acid biosynthesis. Plant J. 78: 80–93.

Niyogi, K.K. (1999). Photoprotection revisited: genetic and molecularapproaches. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 333–359.

North, H.M., De Almeida, A., Boutin, J.P., Frey, A., To, A., Botran, L.,Sotta, B., and Marion-Poll, A. (2007). The Arabidopsis ABA-deficientmutant aba4 demonstrates that the major route for stress-induced ABAaccumulation is via neoxanthin isomers. Plant J. 50: 810–824.

Park, H., Kreunen, S.S., Cuttriss, A.J., DellaPenna, D., and Pogson,B.J. (2002). Identification of the carotenoid isomerase provides in-sight into carotenoid biosynthesis, prolamellar body formation, andphotomorphogenesis. Plant Cell 14: 321–332.

1080 The Plant Cell

Parry, A.D., and Horgan, R. (1992). Abscisic acid biosynthesis inroots : I. The identification of potential abscisic acid precursors, andother carotenoids. Planta 187: 185–191.

Pogson, B., McDonald, K.A., Truong, M., Britton, G., andDellaPenna, D. (1996). Arabidopsis carotenoid mutants demon-strate that lutein is not essential for photosynthesis in higher plants.Plant Cell 8: 1627–1639.

Puig, J., Pauluzzi, G., Guiderdoni, E., and Gantet, P. (2012). Regu-lation of shoot and root development through mutual signaling. Mol.Plant 5: 974–983.

Qiao, H., Chang, K.N., Yazaki, J., and Ecker, J.R. (2009). Interplay be-tween ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2triggers ethylene responses in Arabidopsis. Genes Dev. 23: 512–521.

Qiao, H., Shen, Z., Huang, S.S., Schmitz, R.J., Urich, M.A., Briggs, S.P.,and Ecker, J.R. (2012). Processing and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas. Science 338: 390–393.

Rakitina, T.Y., Vlasov, P.V., Jalilova, F.K., and Kefeli, V.I. (1994).Abscisic acid and ethylene in mutants of Arabidopsis thaliana dif-fering in their resistance to ultraviolet (UV-B) radiation stress. Russ.J. Plant Physiol. 41: 599–603.

Ramel, F., Birtic, S., Ginies, C., Soubigou-Taconnat, L., Triantaphylidès,C., and Havaux, M. (2012). Carotenoid oxidation products are stresssignals that mediate gene responses to singlet oxygen in plants. Proc.Natl. Acad. Sci. USA 109: 5535–5540.

Resnick, J.S., Wen, C.K., Shockey, J.A., and Chang, C. (2006).REVERSION-TO-ETHYLENE SENSITIVITY1, a conserved gene thatregulates ethylene receptor function in Arabidopsis. Proc. Natl.Acad. Sci. USA 103: 7917–7922.

Rodríguez-Villalón, A., Gas, E., and Rodríguez-Concepción, M.(2009). Phytoene synthase activity controls the biosynthesis ofcarotenoids and the supply of their metabolic precursors in dark-grown Arabidopsis seedlings. Plant J. 60: 424–435.

Ruiz-Sola, M.Á., and Rodríguez-Concepción, M. (2012). Carotenoidbiosynthesis in Arabidopsis: a colorful pathway. The ArabidopsisBook 10: e0158, doi/10.1199/tab.0158.

Rzewuski, G., and Sauter, M. (2008). Ethylene biosynthesis andsignaling in rice. Plant Sci. 175: 32–42.

Saika, H., et al. (2007). Ethylene promotes submergence-inducedexpression of OsABA8ox1, a gene that encodes ABA 89-hydroxylasein rice. Plant Cell Physiol. 48: 287–298.

Sieburth, L.E., and Lee, D.K. (2010). BYPASS1: how a tiny mutant tellsa big story about root-to-shoot signaling. J. Integr. Plant Biol. 52: 77–85.

Steffens, B., and Sauter, M. (2010). G proteins as regulators in ethylene-mediated hypoxia signaling. Plant Signal. Behav. 5: 375–378.

Steffens, B., Kovalev, A., Gorb, S.N., and Sauter, M. (2012).Emerging roots alter epidermal cell fate through mechanical andreactive oxygen species signaling. Plant Cell 24: 3296–3306.

Sun, L., Zhang, M., Ren, J., Qi, J., Zhang, G., and Leng, P. (2010).Reciprocity between abscisic acid and ethylene at the onset ofberry ripening and after harvest. BMC Plant Biol. 10: 257.

Tal, M. (1979). Abnormal stomatal behavior and hormonal imbalancein flacca, a wilty mutant of tomato. Plant Physiol. 63: 1044–1048.

Umehara, M., Hanada, A., Yoshida, S., Akiyama, K., Arite, T., Takeda-Kamiya, N., Magome, H., Kamiya, Y., Shirasu, K., Yoneyama, K.,Kyozuka, J., and Yamaguchi, S. (2008). Inhibition of shoot branchingby new terpenoid plant hormones. Nature 455: 195–200.

Van Norman, J.M., Zhang, J., Cazzonelli, C.I., Pogson, B.J.,Harrison, P.J., Bugg, T.D., Chan, K.X., Thompson, A.J., andBenfey, P.N. (2014). Periodic root branching in Arabidopsis requiressynthesis of an uncharacterized carotenoid derivative. Proc. Natl.Acad. Sci. USA 111: E1300–E1309.

Vandenbussche, F., and Van Der Straeten, D. (2007). One for all andall for one: crosstalk of multiple signals controlling the plant phe-notype. J. Plant Growth Regul. 26: 178–187.

Walter, M.H., Floss, D.S., and Strack, D. (2010). Apocarotenoids:hormones, mycorrhizal metabolites and aroma volatiles. Planta 232:1–17.

Wang, L., Hua, D., He, J., Duan, Y., Chen, Z., Hong, X., and Gong, Z.(2011). Auxin Response Factor2 (ARF2) and its regulated homeo-domain gene HB33 mediate abscisic acid response in Arabidopsis.PLoS Genet. 7: e1002172.

Wang, Q., Zhang, W., Yin, Z., and Wen, C.K. (2013). Rice CONSTI-TUTIVE TRIPLE-RESPONSE2 is involved in the ethylene-receptorsignalling and regulation of various aspects of rice growth anddevelopment. J. Exp. Bot. 64: 4863–4875.

Wei, J., Xu, M., Zhang, D., and Mi, H. (2010). The role of carotenoidisomerase in maintenance of photosynthetic oxygen evolution inrice plant. Acta Biochim. Biophys. Sin. (Shanghai) 42: 457–463.

Welsch, R., Wüst, F., Bär, C., Al-Babili, S., and Beyer, P. (2008). Athird phytoene synthase is devoted to abiotic stress-induced ab-scisic acid formation in rice and defines functional diversification ofphytoene synthase genes. Plant Physiol. 147: 367–380.

Wuriyanghan, H., et al. (2009). The ethylene receptor ETR2 delaysfloral transition and affects starch accumulation in rice. Plant Cell21: 1473–1494.

Xie, X., Yoneyama, K., and Yoneyama, K. (2010). The strigolactonestory. Annu. Rev. Phytopathol. 48: 93–117.

Xu, Z.Y., Kim, D.H., and Hwang, I. (2013). ABA homeostasis andsignaling involving multiple subcellular compartments and multiplereceptors. Plant Cell Rep. 32: 807–813.

Yang, C., Lu, X., Ma, B., Chen, S.Y., and Zhang, J.S. (2015). Ethylenesignaling in rice and Arabidopsis: conserved and diverged aspects.Mol. Plant doi/10.1016/j.molp.2015.01.003.

Yi, J., and An, G. (2013). Utilization of T-DNA tagging lines in rice. J.Plant Biol. 56: 85–90.

Yin, H., Zhang, X., Liu, J., Wang, Y., He, J., Yang, T., Hong, X.,Yang, Q., and Gong, Z. (2009). Epigenetic regulation, somatic ho-mologous recombination, and abscisic acid signaling are influencedby DNA polymerase epsilon mutation in Arabidopsis. Plant Cell 21:386–402.

Zhang, H., Han, W., De Smet, I., Talboys, P., Loya, R., Hassan, A.,Rong, H., Jürgens, G., Paul Knox, J., and Wang, M.H. (2010). ABApromotes quiescence of the quiescent centre and suppresses stemcell differentiation in the Arabidopsis primary root meristem. Plant J.64: 764–774.

Zhang, W., Zhou, X., and Wen, C.K. (2012). Modulation of ethyleneresponses by OsRTH1 overexpression reveals the biological sig-nificance of ethylene in rice seedling growth and development. J.Exp. Bot. 63: 4151–4164.

Zhao, J., Wang, T., Wang, M., Liu, Y., Yuan, S., Gao, Y., Yin, L., Sun,W., Peng, L., Zhang, W., Wan, J., and Li, X. (2014). DWARF3participates in an SCF complex and associates with DWARF14 tosuppress rice shoot branching. Plant Cell Physiol. 55: 1096–1109.

Zhu, G., Ye, N., and Zhang, J. (2009). Glucose-induced delay of seedgermination in rice is mediated by the suppression of ABA catab-olism rather than an enhancement of ABA biosynthesis. Plant CellPhysiol. 50: 644–651.

Zou, J., Zhang, S., Zhang, W., Li, G., Chen, Z., Zhai, W., Zhao, X.,Pan, X., Xie, Q., and Zhu, L. (2006). The rice HIGH-TILLERINGDWARF1 encoding an ortholog of Arabidopsis MAX3 is required fornegative regulation of the outgrowth of axillary buds. Plant J. 48:687–698.

Ethylene, Carotenoids, and ABA in Rice 1081

DOI 10.1105/tpc.15.00080; originally published online April 3, 2015; 2015;27;1061-1081Plant Cell

Sun, Shuang Fang, Jin-Fang Chu, Tie-Gang Lu, Shou-Yi Chen and Jin-Song ZhangDuan, Hui Chen, Chao Yang, Xiang Lu, Yi-Qin Wang, Wan-Ke Zhang, Cheng-Cai Chu, Xiao-Hong

Cui-Cui Yin, Biao Ma, Derek Phillip Collinge, Barry James Pogson, Si-Jie He, Qing Xiong, Kai-XuanIsomerase-Mediated Abscisic Acid Pathway

Ethylene Responses in Rice Roots and Coleoptiles Are Differentially Regulated by a Carotenoid

 This information is current as of March 30, 2020

 

Supplemental Data /content/suppl/2015/03/19/tpc.15.00080.DC1.html

References /content/27/4/1061.full.html#ref-list-1

This article cites 96 articles, 29 of which can be accessed free at:

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists