Abstract
Exposure of plants to ethylene results in drastic morphological changes. Seedlings germinated in the dark in the presence of saturating concentrations of ethylene display a characteristic phenotype known as the triple response. This phenotype is robust and easy to score. In Arabidopsis the triple response is usually evaluated at 3 days post germination in seedlings grown in the dark in rich media supplemented with 10 μM of the ethylene precursor ACC in air or in unsupplemented media in the presence of 10 ppm ethylene. The triple response in Arabidopsis consists of shortening and thickening of hypocotyls and roots and exaggeration of the curvature of apical hooks. The search for Arabidopsis mutants that fail to show this phenotype in ethylene or, vice versa, display the triple response in the absence of exogenously supplied hormone has allowed the identification of the key components of the ethylene biosynthesis and signaling pathways. Herein, we describe a simple protocol for assaying the triple response in Arabidopsis. The method can also be employed in many other dicot species, with minor modifications to account for species-specific differences in germination. We also compiled a comprehensive table of ethylene-related mutants of Arabidopsis, including many lines with auxin-related defects, as wild-type levels of auxin biosynthesis, transport, signaling, and response are necessary for the normal response of plants to ethylene.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Neljubow D (1901) Über die Horinzontale Nutation der Stengel von Pisum sativum und einiger anderer Pflanzen. Beih Bot Zentralb 10:128–139
Knight LI, Rose RC, Crocker W (1910) Effects of various gases and vapors upon etiolated seedlings of the sweet pea. Science 311(1):635–636
Ma B, He S-J, Duan K-X, Yin C-C, Chen H, Yang C et al (2013) Identification of rice ethylene-response mutants and characterization of MHZ7/OsEIN2 in distinct ethylene response and yield trait regulation. Mol Plant 6:1830–1848
Yang C, Lu X, Ma B, Chen S-Y, Zhang J-S (2015) Ethylene signaling in rice and Arabidopsis: conserved and diverged aspects. Mol Plant 8:495–505
Solano R, Ecker JR (1998) Ethylene gas: perception, signaling and response. Curr Opin Plant Biol 1:393–398
Bleecker AB, Estelle MA, Somerville C, Kende H (1988) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science 241:1086–1089
Swarup R, Perry P, Hagenbeek D, Van Der Straeten D, Beemster GTS, Sandberg G et al (2007) Ethylene upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation. Plant Cell 19:2186–2196
Alonso JM, Stepanova AN, Solano R, Wisman E, Ferrari S, Ausubel FM et al (2003) Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis. Proc Natl Acad Sci U S A 100:2992–2997
Stepanova AN, Hoyt JM, Hamilton AA, Alonso JM (2005) A link between ethylene and auxin uncovered by the characterization of two root-specific ethylene-insensitive mutants in Arabidopsis. Plant Cell 17:2230–2242
Stepanova AN, Yun J, Likhacheva AV, Alonso JM (2007) Multilevel interactions between ethylene and auxin in Arabidopsis roots. Plant Cell 19:2169–2185
Stepanova AN, Robertson-Hoyt J, Yun J, Benavente LM, Xie D-Y, Dolezal K et al (2008) TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development. Cell 133:177–191
Hobbie LJ (1998) Auxin: molecular genetic approaches in Arabidopsis. Plant Physiol Biochem 36:91–102
Collett CE, Harberd NP, Leyser O (2000) Hormonal interactions in the control of Arabidopsis hypocotyl elongation. Plant Physiol 124:553–562
Chae HS, Faure F, Kieber JJ (2003) The eto1, eto2, and eto3 mutations and cytokinin treatment increase ethylene biosynthesis in Arabidopsis by increasing the stability of ACS protein. Plant Cell 15:545–559
Hansen M, Chae HS, Kieber JJ (2009) Regulation of ACS protein stability by cytokinin and brassinosteroids. Plant J 57:606–614
Hass C, Lohrmann J, Albrecht V, Sweere U, Hummel F, Yoo S-D et al (2004) The response regulator 2 mediates ethylene signalling and hormone signal integration in Arabidopsis. EMBO J 23:3290–3302
Su W, Howell SH (1992) A single genetic locus, ckr1, defines Arabidopsis mutants in which root growth is resistant to low concentrations of cytokinin. Plant Physiol 99:1569–1574
Vogel JP, Woeste KE, Theologis A, Kieber JJ (1998) Recessive and dominant mutations in the ethylene biosynthetic gene ACS5 of Arabidopsis confer cytokinin insensitivity and ethylene overproduction, respectively. Proc Natl Acad Sci U S A 95:4766–4771
Vogel JP, Schuerman P, Woeste K, Brandstatter I, Kieber JJ (1998) Isolation and characterization of Arabidopsis mutants defective in the induction of ethylene biosynthesis by cytokinin. Genetics 149:417–427
Kushwah S, Jones AM, Laxmi A (2011) Cytokinin interplay with ethylene, auxin, and glucose signaling controls Arabidopsis seedling root directional growth. Plant Physiol 156:1851–1866
Thole JM, Beisner ER, Liu J, Venkova SV, Strader LC (2014) Abscisic acid regulates root elongation through the activities of auxin and ethylene in Arabidopsis thaliana. G3 4:1259–1274
Beaudoin N, Serizet C, Gosti F, Giraudat J (2000) Interactions between abscisic acid and ethylene signaling cascades. Plant Cell 12:1103–1115
Ghassemian M, Nambara E, Cutler S, Kawaide H, Kamiya Y, McCourt P (2000) Regulation of abscisic acid signaling by the ethylene response pathway in Arabidopsis. Plant Cell 12:1117–1126
Deslauriers SD, Larsen PB (2010) FERONIA is a key modulator of brassinosteroid and ethylene responsiveness in Arabidopsis hypocotyls. Mol Plant 3:626–640
Guzmán P, Ecker JR (1990) Exploiting the triple response of Arabidopsis to identify ethylene-related mutants. Plant Cell 2:513–523
Roman G, Lubarsky B, Kieber JJ, Rothenberg M, Ecker JR (1995) Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway. Genetics 139:1393–1409
Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR (1993) CTR1, a negative regulator of the ethylene response pathway in arabidopsis, encodes a member of the Raf family of protein kinases. Cell 72:427–441
Merchante C, Brumos J, Yun J, Hu Q, Spencer KR, Enríquez P et al (2015) Gene-specific translation regulation mediated by the hormone-signaling molecule EIN2. Cell 163:684–697
Larsen PB, Cancel JD (2003) Enhanced ethylene responsiveness in the Arabidopsis eer1 mutant results from a loss-of-function mutation in the protein phosphatase 2A A regulatory subunit, RCN1. Plant J 34:709–718
Christians MJ, Robles LM, Zeller SM, Larsen PB (2008) The eer5 mutation, which affects a novel proteasome-related subunit, indicates a prominent role for the COP9 signalosome in resetting the ethylene-signaling pathway in Arabidopsis. Plant J 55:467–477
De Paepe A, De Grauwe L, Bertrand S, Smalle J, Van Der Straeten D (2005) The Arabidopsis mutant eer2 has enhanced ethylene responses in the light. J Exp Bot 56:2409–2420
Potuschak T, Lechner E, Parmentier Y, Yanagisawa S, Grava S, Koncz C et al (2003) EIN3-dependent regulation of plant ethylene hormone signaling by two Arabidopsis F box proteins. Cell 115:679–689
Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657
Zhao Y, Wei T, Yin KQ, Chen Z, Gu H, Qu LJ et al (2012) Arabidopsis RAP2.2 plays an important role in plant resistance to Botrytis cinerea and ethylene responses. New Phytol 195:450–460
Solano R, Stepanova A, Chao Q, Ecker JR (1998) Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. Genes Dev 12:3703–3714
Rai MI, Wang X, Thibault DM, Kim HJ, Bombyk MM, Binder BM et al (2015) The ARGOS gene family functions in a negative feedback loop to desensitize plants to ethylene. BMC Plant Biol 15:157
Shi J, Habben JE, Archibald RL, Drummond BJ, Chamberlin MA, Williams RW et al (2015) Overexpression of ARGOS genes modifies plant sensitivity to ethylene, leading to improved drought tolerance in both Arabidopsis and maize. Plant Physiol 169:266–282
Wang Z, Cao H, Sun Y, Li X, Chen F, Carles A et al (2013) Arabidopsis paired amphipathic helix proteins SNL1 and SNL2 redundantly regulate primary seed dormancy via abscisic acid-ethylene antagonism mediated by histone deacetylation. Plant Cell 25:149–166
Li C, Xu J, Li J, Li Q, Yang H (2014) Involvement of Arabidopsis histone acetyltransferase HAC family genes in the ethylene signaling pathway. Plant Cell Physiol 55:426–435
Liu Z, Wu Y, Yang F, Zhang Y, Chen S, Xie Q et al (2013) BIK1 interacts with PEPRs to mediate ethylene-induced immunity. Proc Natl Acad Sci U S A 110:6205–6210
Yamaguchi Y, Huffaker A, Bryan AC, Tax FE, Ryan CA (2010) PEPR2 is a second receptor for the Pep1 and Pep2 peptides and contributes to defense responses in Arabidopsis. Plant Cell 22:508–522
Kim BC, Soh MC, Kang BJ, Furuya M, Nam HG (1996) Two dominant photomorphogenic mutations of Arabidopsis thaliana identified as suppressor mutations of hy2. Plant J 9:441–456
Reed JW (2001) Roles and activities of Aux/IAA proteins in Arabidopsis. Trends Plant Sci 6:420–425
Fukaki H, Tameda S, Masuda H, Tasaka M (2002) Lateral root formation is blocked by a gain-of-function mutation in the SOLITARY-ROOT/IAA14 gene of Arabidopsis. Plant J 29:153–168
Uehara T, Okushima Y, Mimura T, Tasaka M, Fukaki H (2008) Domain II mutations in CRANE/IAA18 suppress lateral root formation and affect shoot development in Arabidopsis thaliana. Plant Cell Physiol 49:1025–1038
Tatematsu K, Kumagai S, Muto H, Sato A, Watahiki MK, Harper RM et al (2004) MASSUGU2 encodes Aux/IAA19, an auxin-regulated protein that functions together with the transcriptional activator NPH4/ARF7 to regulate differential growth responses of hypocotyl and formation of lateral roots in Arabidopsis thaliana. Plant Cell 16:379–393
Li J, Dai X, Zhao Y (2006) A role for auxin response factor 19 in auxin and ethylene signaling in Arabidopsis. Plant Physiol 140:899–908
Tao Y, Ferrer J-L, Ljung K, Pojer F, Hong F, Long JA et al (2008) Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants. Cell 133:164–176
Won C, Shen X, Mashiguchi K, Zheng Z, Dai X, Cheng Y et al (2011) Conversion of tryptophan to indole-3-acetic acid by TRYPTOPHAN AMINOTRANSFERASES OF ARABIDOPSIS and YUCCAs in Arabidopsis. Proc Natl Acad Sci U S A 108:18518–18523
Merchante C, Vallarino JG, Osorio S, Aragüez I, Villarreal N, Ariza MT et al (2013) Ethylene is involved in strawberry fruit ripening in an organ-specific manner. J Exp Bot 64:4421–4439
Tsuchisaka A, Yu G, Jin H, Alonso JM, Ecker JR, Zhang X et al (2009) A combinatorial interplay among the 1-Aminocyclopropane-1-Carboxylate isoforms regulates ethylene biosynthesis in Arabidopsis thaliana. Genetics 183:979–1003
Liu Y, Zhang S (2004) Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis. Plant Cell 6:3386–3399
Wang KL-C, Yoshida H, Lurin C, Ecker JR (2004) Regulation of ethylene gas biosynthesis by the Arabidopsis ETO1 protein. Nature 428:945–950
Gingerich DJ, Gagne JM, Salter DW (2005) Cullin 3A and B assemble with members of the broad complex/tramtrack/bric-A-brac (BTB). J Biol Chem 280:18810–18821
Christians MJ, Gingerich DJ, Hansen M, Binder BM, Kieber JJ, Vierstra RD (2009) The BTB ubiquitin ligases ETO1, EOL1 and EOL2 act collectively to regulate ethylene biosynthesis in Arabidopsis by controlling type-2 ACC synthase levels. Plant J 57:332–345
Tan S-T, Xue H-W (2014) Casein kinase 1 regulates ethylene synthesis by phosphorylating and promoting the turnover of ACS5. Cell Rep 9:1692–1702
Yoo S-D, Cho Y-H, Tena G, Xiong Y, Sheen J (2008) Dual control of nuclear EIN3 by bifurcate MAPK cascades in C2H4 signalling. Nature 451:789–795
An F, Zhao Q, Ji Y, Li W, Jiang Z, Yu X et al (2010) Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis. Plant Cell 22:2384–2401
Xu J, Li Y, Wang Y, Liu H, Lei L, Yang H et al (2008) Activation of MAPK kinase 9 induces ethylene and camalexin biosynthesis and enhances sensitivity to salt stress in Arabidopsis. J Biol Chem 283:26996–27006
Wang H, Ngwenyama N, Liu Y, Walker JC, Zhang S (2007) Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis. Plant Cell 19:63–73
Guan Y, Lu J, Xu J, McClure B, Zhang S (2014) Two mitogen-activated protein kinases, MPK3 and MPK6, are required for funicular guidance of pollen tubes in Arabidopsis. Plant Physiol 165:528–533
Han L, Li GJ, Yang KY, Mao G, Wang R, Liu Y et al (2010) Mitogen‐activated protein kinase 3 and 6 regulate Botrytis cinerea‐induced ethylene production in Arabidopsis. Plant J 64:114–127
Zheng Z, Guo Y, Novak O, Dai X, Zhao Y, Ljung K et al (2013) Coordination of auxin and ethylene biosynthesis by the aminotransferase VAS1. Nat Chem Biol 9:244–246
Xu S-L, Rahman A, Baskin TI, Kieber JJ (2008) Two leucine-rich repeat receptor kinases mediate signaling, linking cell wall biosynthesis and ACC synthase in Arabidopsis. Plant Cell 20:3065–3079
Khanna R, Shen Y, Marion CM, Tsuchisaka A, Theologis A, Schäfer E et al (2007) The basic helix-loop-helix transcription factor PIF5 acts on ethylene biosynthesis and phytochrome signaling by distinct mechanisms. Plant Cell 19:3915–3929
Dieterle M, Thomann A, Renou J-P, Parmentier Y, Cognat V, Lemonnier G et al (2005) Molecular and functional characterization of Arabidopsis cullin 3A. Plant J 41:386–399
Thomann A, Brukhin V, Dieterle M, Gheyeselinck J, Vantard M, Grossniklaus U et al (2005) Arabidopsis CUL3A and CUL3B genes are essential for normal embryogenesis. Plant J 43:437–448
Thomann A, Lechner E, Hansen M, Dumbliauskas E, Parmentier Y, Kieber J et al (2009) Arabidopsis CULLIN3 genes regulate primary root growth and patterning by ethylene-dependent and -independent mechanisms. PLoS Genet 5:e1000328
Woodward AW, Ratzel SE, Woodward EE, Shamoo Y, Bartel B (2007) Mutation of E1-CONJUGATING ENZYME-RELATED1 decreases RELATED TO UBIQUITIN conjugation and alters auxin response and development. Plant Physiol 144:976–987
Bostick M, Lochhead SR, Honda A, Palmer S, Callis J (2004) Related to ubiquitin 1 and 2 are redundant and essential and regulate vegetative growth, auxin signaling, and ethylene production in Arabidopsis. Plant Cell 16:2418–2432
Dharmasiri S, Dharmasiri N, Hellmann H, Estelle M (2003) The RUB/Nedd8 conjugation pathway is required for early development in Arabidopsis. EMBO J 22:1762–1770
Larsen PB, Cancel JD (2004) A recessive mutation in the RUB1-conjugating enzyme, RCE1, reveals a requirement for RUB modification for control of ethylene biosynthesis and proper induction of basic chitinase and PDF1.2 in Arabidopsis. Plant J 38:626–638
Zhong R, Ripperger A, Ye ZH (2000) Ectopic deposition of lignin in the pith of stems of two Arabidopsis mutants. Plant Physiol 123:59–70
Zhong R, Kays SJ, Schroeder BP, Ye Z-H (2002) Mutation of a chitinase-like gene causes ectopic deposition of lignin, aberrant cell shapes, and overproduction of ethylene. Plant Cell 14:165–179
Chang C, Kwok SF, Bleecker AB, Meyerowitz EM (1993) Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators. Science 262:539–544
Resnick JS, Wen C-K, Shockey JA, Chang C (2006) REVERSION-TO-ETHYLENE SENSITIVITY1, a conserved gene that regulates ethylene receptor function in Arabidopsis. Proc Natl Acad Sci U S A 103:7917–7922
Hua J, Meyerowitz EM (1998) Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana. Cell 94:261–271
Cancel JD, Larsen PB (2002) Loss-of-function mutations in the ethylene receptor ETR1 cause enhanced sensitivity and exaggerated response to ethylene in Arabidopsis. Plant Physiol 129:1557–1567
Qu X, Hall BP, Gao Z, Schaller GE (2007) A strong constitutive ethylene-response phenotype conferred on Arabidopsis plants containing null mutations in the ethylene receptors ETR1 and ERS1. BMC Plant Biol 7:3
Sakai H, Hua J, Chen QG, Chang C, Bleecker AB (1998) ETR2 is an ETR1-like gene controlling ethylene signal transduction. Proc Natl Acad Sci U S A 95:5812–5817
Hua J, Sakai H, Nourizadeh S, Chen QG, Bleecker AB, Ecker JR et al (1998) EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis. Plant Cell 10:1321–1332
Hua J, Chang C, Sun Q, Meyerowitz EM (1995) Ethylene insensitivity conferred by Arabidopsis ERS gene. Science 269:1712–1714
Zhao X-C, Qu X, Mathews DE, Schaller GE (2002) Effect of ethylene pathway mutations upon expression of the ethylene receptor ETR1 from Arabidopsis. Plant Physiol 130:1983–1991
Liu Q, Xu C, Wen C-K (2010) Genetic and transformation studies reveal negative regulation of ERS1 ethylene receptor signaling in Arabidopsis. BMC Plant Biol 10:60
Liu Q, Wen C-K (2012) Arabidopsis ETR1 and ERS1 differentially repress the ethylene response in combination with other ethylene receptor genes. Plant Physiol 158:1193–1207
Hirayama T, Kieber JJ, Hirayama N, Kogan M, Guzman P, Nourizadeh S et al (1999) RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson disease–related copper transporter, is required for ethylene signaling in Arabidopsis. Cell 97:383–393
Woeste KE, Kieber JJ (2000) A strong loss-of-function mutation in RAN1 results in constitutive activation of the ethylene response pathway as well as a rosette-lethal phenotype. Plant Cell 12:443–455
Xu C, Zhou X, Wen C-K (2015) HYPER RECOMBINATION1 of the THO/TREX complex plays a role in controlling transcription of the REVERSION-TO-ETHYLENE SENSITIVITY1 gene in Arabidopsis. PLoS Genet 11:e1004956
Tao S, Zhang Y, Wang X, Xu L, Fang X, Lu ZJ et al (2016) The THO/TREX complex active in miRNA biogenesis negatively regulates root-associated acid phosphatase activity induced by phosphate starvation. Plant Physiol 171:2841–2853
Jauvion V, Elmayan T, Vaucheret H (2010) The conserved RNA trafficking proteins HPR1 and TEX1 are involved in the production of endogenous and exogenous small interfering RNA in Arabidopsis. Plant Cell 22:2697–2709
Wang H, Sun Y, Chang J, Zheng F, Pei H, Yi Y et al (2016) Regulatory function of Arabidopsis lipid transfer protein 1 (LTP1) in ethylene response and signaling. Plant Mol Biol 91:471–484
Chang J, Clay JM, Chang C (2014) Association of cytochrome b5 with ETR1 ethylene receptor signaling through RTE1 in Arabidopsis. Plant J 77:558–567
Yu J, Wen C-K (2013) Arabidopsis aux1rcr1 mutation alters AUXIN RESISTANT1 targeting and prevents expression of the auxin reporter DR5:GUS in the root apex. J Exp Bot 64:371–933
Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR (1999) EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science 284:2148–2152
Ju C, Yoon GM, Shemansky JM, Lin DY, Ying ZI, Chang J et al (2012) CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis. Proc Natl Acad Sci U S A 109:19486–19491
Chao Q, Rothenberg M, Solano R, Roman G, Terzaghi W, Ecker JR (1997) Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins. Cell 89:1133–1144
Binder BM, Walker JM, Gagne JM, Emborg TJ, Hemmann G, Bleecker AB et al (2007) The Arabidopsis EIN3 binding F-Box proteins EBF1 and EBF2 have distinct but overlapping roles in ethylene signaling. Plant Cell 19:509–523
Olmedo G, Guo H, Gregory BD, Nourizadeh SD, Aguilar-Henonin L, Li H et al (2006) ETHYLENE-INSENSITIVE5 encodes a 5´–>3´ exoribonuclease required for regulation of the EIN3-targeting F-box proteins EBF1/2. Proc Natl Acad Sci U S A 103:13286–13293
Potuschak T, Vansiri A, Binder BM, Lechner E, Vierstra RD, Genschik P (2006) The exoribonuclease XRN4 is a component of the ethylene response pathway in Arabidopsis. Plant Cell 18:3047–3057
Gazzani S, Lawrenson T, Woodward C, Headon D, Sablowski R (2004) A link between mRNA turnover and RNA interference in Arabidopsis. Science 306:1046–1048
Souret FF, Kastenmayer JP, Green PJ (2004) AtXRN4 Degrades mRNA in Arabidopsis and its substrates include selected miRNA targets. Mol Cell 15:173–183
Guo H, Ecker JR (2003) Plant responses to ethylene gas are mediated by SCFEBF1/EBF2-dependent proteolysis of EIN3 transcription factor. Cell 115:667–677
Gagne JM, Smalle J, Gingerich DJ, Walker JM, Yoo S-D, Yanagisawa S et al (2004) Arabidopsis EIN3-binding F-box 1 and 2 form ubiquitin-protein ligases that repress ethylene action and promote growth by directing EIN3 degradation. Proc Natl Acad Sci U S A 101:6803–6808
Qiao H, Chang KN, Yazaki J, Ecker JR (2009) Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis. Genes Dev 23:512–521
Arciga Reyes L, Wootton L, Kieffer M, Davies B (2006) UPF1 is required for nonsense‐mediated mRNA decay (NMD) and RNAi in Arabidopsis. Plant J 47:480–489
Li W, Ma M, Feng Y, Li H, Wang Y, Ma Y et al (2015) EIN2-directed translational regulation of ethylene signaling in Arabidopsis. Cell 163:670–683
Hori K, Watanabe Y (2005) UPF3 suppresses aberrant spliced mRNA in Arabidopsis. Plant J 43:530–540
Dufresne PJ, Ubalijoro E, Fortin MG, Laliberté J-F (2008) Arabidopsis thaliana class II poly(A)-binding proteins are required for efficient multiplication of turnip mosaic virus. J Gen Virol 89:2339–2348
Larsen PB, Chang C (2001) The Arabidopsis eer1 mutant has enhanced ethylene responses in the hypocotyl and stem. Plant Physiol 125:1061–1073
Garbers C, DeLong A, Deruére J, Bernasconi P, Söll D (1996) A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis. EMBO J 15:2115–2124
Robles LM, Wampole JS, Christians MJ, Larsen PB (2007) Arabidopsis enhanced ethylene response 4 encodes an EIN3-interacting TFIID transcription factor required for proper ethylene response, including ERF1 induction. J Exp Bot 58:2627–2639
Christians MJ, Larsen PB (2007) Mutational loss of the prohibitin AtPHB3 results in an extreme constitutive ethylene response phenotype coupled with partial loss of ethylene-inducible gene expression in Arabidopsis seedlings. J Exp Bot 58:2237–2248
Keith K, Kraml M, Dengler NG, McCourt P (1994) fusca3: a heterochronic mutation affecting late embryo development in Arabidopsis. Plant Cell 6:589–600
Luerssen H, Kirik V, Herrmann P, Miséra S (1998) FUSCA3 encodes a protein with a conserved VP1/AB13-like B3 domain which is of functional importance for the regulation of seed maturation in Arabidopsis thaliana. Plant J 15:755–764
Lumba S, Tsuchiya Y, Delmas F, Hezky J, Provart NJ, Shi Lu Q et al (2012) The embryonic leaf identity gene FUSCA3 regulates vegetative phase transitions by negatively modulating ethylene-regulated gene expression in Arabidopsis. BMC Biol 10:8
Sun J, Ma Q, Mao T (2015) Ethylene regulates the Arabidopsis microtubule-associated protein WAVE-DAMPENED2-LIKE5 in etiolated hypocotyl elongation. Plant Physiol 169:325–337
Veronese P, Nakagami H, Bluhm B, Abuqamar S, Chen X, Salmeron J et al (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens. Plant Cell 18:257–273
Laluk K, Luo H, Chai M, Dhawan R, Lai Z, Mengiste T (2011) Biochemical and genetic requirements for function of the immune response regulator BOTRYTIS-INDUCED KINASE1 in plant growth, ethylene signaling, and PAMP-triggered immunity in Arabidopsis. Plant Cell 23:2831–2849
Lehman A, Black R, Ecker JR (1996) HOOKLESS1, an ethylene response gene, is required for differential cell elongation in the Arabidopsis hypocotyl. Cell 85:183–194
Li H, Johnson P, Stepanova A, Alonso JM, Ecker JR (2004) Convergence of signaling pathways in the control of differential cell growth in Arabidopsis. Dev Cell 7:193–204
Gy I, Gasciolli V, Lauressergues D, Morel J-B, Gombert J, Proux F et al (2007) Arabidopsis FIERY1, XRN2, and XRN3 are endogenous RNA silencing suppressors. Plant Cell 19:3451–3461
Chen H, Xiong L (2010) The bifunctional abiotic stress signalling regulator and endogenous RNA silencing suppressor FIERY1 is required for lateral root formation. Plant Cell Environ 33:2180–2190
Adams E, Turner J (2010) COI1, a jasmonate receptor, is involved in ethylene-induced inhibition of Arabidopsis root growth in the light. J Exp Bot 61:4373–4386
Chen G, Bi YR, Li N (2005) EGY1 encodes a membrane-associated and ATP-independent metalloprotease that is required for chloroplast development. Plant J 41:364–375
Guo D, Gao X, Li H, Zhang T, Chen G, Huang P et al (2008) EGY1 plays a role in regulation of endodermal plastid size and number that are involved in ethylene-dependent gravitropism of light-grown Arabidopsis hypocotyls. Plant Mol Biol 66:345–360
Ding L, Pandey S, Assmann SM (2008) Arabidopsis extra-large G proteins (XLGs) regulate root morphogenesis. Plant J 53:248–263
Bueso E, Alejandro S, Carbonell P, Perez-Amador MA, Fayos J, Bellés JM et al (2007) The lithium tolerance of the Arabidopsis cat2 mutant reveals a cross-talk between oxidative stress and ethylene. Plant J 52:1052–1065
Jing H-C, Sturre MJG, Hille J, Dijkwel PP (2002) Arabidopsis onset of leaf death mutants identify a regulatory pathway controlling leaf senescence. Plant J 32:51–63
Jing H-C, Anderson L, Sturre MJG, Hille J, Dijkwel PP (2007) Arabidopsis CPR5 is a senescence-regulatory gene with pleiotropic functions as predicted by the evolutionary theory of senescence. J Exp Bot 58:3885–3894
Boch J, Verbsky ML, Robertson TL, Larkin JC, Kunkel BN (2007) Analysis of resistance gene-mediated defense responses in Arabidopsis thaliana plants carrying a mutation in CPR5. MPMI 11:1196–1206
Bouquin T, Mattsson O, Naested H, Foster R, Mundy J (2003) The Arabidopsis lue1 mutant defines a katanin p60 ortholog involved in hormonal control of microtubule orientation during cell growth. J Cell Sci 116:791–801
Ellis C, Turner JG (2001) The Arabidopsis mutant cev1 has constitutively active jasmonate and ethylene signal pathways and enhanced resistance to pathogens. Plant Cell 13:1025–1033
Ellis C, Karafyllidis I, Wasternack C, Turner JG (2002) The Arabidopsis mutant cev1 links cell wall signaling to jasmonate and ethylene responses. Plant Cell 14:1557–1566
Casson SA, Chilley PM, Topping JF, Evans IM, Souter MA, Lindsey K (2002) The POLARIS gene of Arabidopsis encodes a predicted peptide required for correct root growth and leaf vascular patterning. Plant Cell 14:1705–1721
Chilley PM, Casson SA, Tarkowski P, Hawkins N, Wang KL-C, Hussey PJ et al (2006) The POLARIS peptide of Arabidopsis regulates auxin transport and root growth via effects on ethylene signaling. Plant Cell 18:3058–3072
Clark SE, Williams RW, Meyerowitz EM (1997) The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis. Cell 89:575–585
Poulios S, Vlachonasios KE (2016) Synergistic action of histone acetyltransferase GCN5 and receptor CLAVATA1 negatively affects ethylene responses in Arabidopsis thaliana. J Exp Bot 67:905–918
Fletcher JC, Brand U, Running MP, Simon R, Meyerowitz EM (1999) Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot meristems. Science 283:1911–1914
Vlachonasios KE, Thomashow MF, Triezenberg SJ (2003) Disruption mutations of ADA2b and GCN5 transcriptional adaptor genes dramatically affect arabidopsis growth, development, and gene expression. Plant Cell 15:626–638
Li Z-G, Chen H-W, Li Q-T, Tao J-J, Bian X-H, Ma B et al (2015) Three SAUR proteins SAUR76, SAUR77 and SAUR78 promote plant growth in Arabidopsis. Sci Rep 5:12477
Hayashi S, Hirayama T (2016) ahg12 is a dominant proteasome mutant that affects multiple regulatory systems for germination of Arabidopsis. Sci Rep 6:25351
Benfey PN, Linstead PJ, Roberts K, Schiefelbein JW, Hauser MT, Aeschbacher RA (1993) Root development in Arabidopsis: four mutants with dramatically altered root morphogenesis. Development 119:57–70
Aeschbacher RA, Hauser MT, Feldmann KA, Benfey PN (1995) The SABRE gene is required for normal cell expansion in Arabidopsis. Genes Dev 9:330–340
Shin K, Lee S, Song W-Y, Lee R-A, Lee I, Ha K et al (2015) Genetic identification of ACC-RESISTANT2 reveals involvement of LYSINE HISTIDINE TRANSPORTER1 in the uptake of 1-aminocyclopropane-1-carboxylic acid in Arabidopsis thaliana. Plant Cell Physiol 56:572–582
Svennerstam H, Ganeteg U, Bellini C, Näsholm T (2007) Comprehensive screening of Arabidopsis mutants suggests the lysine histidine transporter 1 to be involved in plant uptake of amino acids. Plant Physiol 143:1853–1860
Ruegger M, Dewey E, Gray WM, Hobbie L, Turner J, Estelle M (1998) The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast Grr1p. Genes Dev 12:198–207
Tian Q, Reed JW (1999) Control of auxin-regulated root development by the Arabidopsis thaliana SHY2/IAA3 gene. Development 126:711–721
Robles LM, Deslauriers SD, Alvarez AA, Larsen PB (2012) A loss-of-function mutation in the nucleoporin AtNUP160 indicates that normal auxin signalling is required for a proper ethylene response in Arabidopsis. J Exp Bot 63:2231–2241
Rogg LE, Lasswell J, Bartel B (2001) A gain-of-function mutation in IAA28 suppresses lateral root development. Plant Cell 13:465–480
Kim T-H, Kim B-H, Yahalom A, Chamovitz DA, Arnim von AG (2004) Translational regulation via 5' mRNA leader sequences revealed by mutational analysis of the Arabidopsis translation initiation factor subunit eIF3h. Plant Cell 16:3341–3356
Hobbie L, Estelle M (1994) Genetic approaches to auxin action. Plant Cell Environ 17:525–540
Timpte C, Lincoln C, Pickett FB, Turner J, Estelle M (1995) The AXR1 and AUX1 genes of Arabidopsis function in separate auxin-response pathways. Plant J 8:561–589
Wilson AK, Pickett FB, Turner JC, Estelle M (1990) A dominant mutation in Arabidopsis confers resistance to auxin, ethylene and abscisic acid. Mol Gen Genet 222:377–383
Nagpal P, Walker LM, Young JC, Sonawala A, Timpte C, Estelle M et al (2000) AXR2 encodes a member of the Aux/IAA protein family. Plant Physiol 123:563–574
Leyser HM, Pickett FB, Dharmasiri S, Estelle M (1996) Mutations in the AXR3 gene of Arabidopsis result in altered auxin response including ectopic expression from the SAUR-AC1 promoter. Plant J 10:403–413
Rouse D, Mackay P, Stirnberg P, Estelle M, Leyser O (1998) Changes in auxin response from mutations in an AUX/IAA gene. Science 279:1371–1373
Yang X, Lee S, So J-H, Dharmasiri S, Dharmasiri N, Ge L et al (2004) The IAA1 protein is encoded by AXR5 and is a substrate of SCF(TIR1). Plant J 40:772–782
Hobbie L, McGovern M, Hurwitz LR, Pierro A, Liu NY, Bandyopadhyay A et al (2000) The axr6 mutants of Arabidopsis thaliana define a gene involved in auxin response and early development. Development 127:23–32
Hellmann H, Hobbie L, Chapman A, Dharmasiri S, Dharmasiri N, del Pozo C et al (2003) Arabidopsis AXR6 encodes CUL1 implicating SCF E3 ligases in auxin regulation of embryogenesis. EMBO J 22:3314–3325
Quint M, Ito H, Zhang W, Gray WM (2005) Characterization of a novel temperature-sensitive allele of the CUL1/AXR6 subunit of SCF ubiquitin-ligases. Plant J 43:371–383
Shen W-H, Parmentier Y, Hellmann H, Lechner E, Dong A, Masson J et al (2002) Null mutation of AtCUL1 causes arrest in early embryogenesis in Arabidopsis. Mol Biol Cell 13:1916–1928
Moon J, Zhao Y, Dai X, Zhang W, Gray WM, Huq E et al (2007) A new CULLIN 1 mutant has altered responses to hormones and light in Arabidopsis. Plant Physiol 143:684–696
Monroe-Augustus M, Zolman BK, Bartel B (2003) IBR5, a dual-specificity phosphatase-like protein modulating auxin and abscisic acid responsiveness in Arabidopsis. Plant Cell 15:2979–2991
Strader LC, Monroe-Augustus M, Bartel B (2008) The IBR5 phosphatase promotes Arabidopsis auxin responses through a novel mechanism distinct from TIR1-mediated repressor degradation. BMC Plant Biol 8:41
Fortunati A, Piconese S, Tassone P, Ferrari S, Migliaccio F (2008) A new mutant of Arabidopsis disturbed in its roots, right-handed slanting, and gravitropism defines a gene that encodes a heat-shock factor. J Exp Bot 59:1363–1374
LeClere S, Rampey RA, Bartel B (2004) IAR4, a gene required for auxin conjugate sensitivity in Arabidopsis, encodes a pyruvate dehydrogenase E1alpha homolog. Plant Physiol 135:989–999
Pickett FB, Wilson AK, Estelle M (1990) The aux1 mutation of Arabidopsis confers both auxin and ethylene resistance. Plant Physiol 94:1462–1466
Bennett MJ, Marchant A, Green HG, May ST, Ward SP, Millner PA et al (1996) Arabidopsis AUX1 gene: a permease-like regulator of root gravitropism. Science 273:948–950
Luschnig C, Gaxiola RA, Grisafi P, Fink GR (1998) EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana. Genes Dev 12:2175–2187
Dharmasiri S, Swarup R, Mockaitis K, Dharmasiri N, Singh SK, Kowalchyk M et al (2006) AXR4 is required for localization of the auxin influx facilitator AUX1. Science 312:1218–1220
Ruegger M, Dewey E, Hobbie L, Brown D, Bernasconi P, Turner J et al (1997) Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects. Plant Cell 9:745–757
Gil P, Dewey E, Friml J, Zhao Y, Snowden KC, Putterill J et al (2001) BIG: a calossin-like protein required for polar auxin transport in Arabidopsis. Genes Dev 15:1985–1997
Kanyuka K, Praekelt U, Franklin KA, Billingham OE, Hooley R, Whitelam GC et al (2003) Mutations in the huge Arabidopsis gene BIG affect a range of hormone and light responses. Plant J 35:57–70
Vandenbussche F, Smalle J, Le J, Saibo NJM, De Paepe A, Chaerle L et al (2003) The Arabidopsis mutant alh1 illustrates a cross talk between ethylene and auxin. Plant Physiol 131:1228–1238
Shin K, Lee R-A, Lee I, Lee S, Park SK, Soh M-S (2013) Genetic identification of a second site modifier of ctr1-1 that controls ethylene-responsive and gravitropic root growth in Arabidopsis thaliana. Mol Cells 36:88–96
Xu A, Zhang W, Wen C-K (2014) ENHANCING ctr1-10 ETHYLENE RESPONSE2 is a novel allele involved in CONSTITUTIVE TRIPLE-RESPONSE1-mediated ethylene receptor signaling in Arabidopsis. BMC Plant Biol 14:48
Cao XF, Linstead P, Berger F, Kieber J, Dolan L (1999) Differential ethylene sensitivity of epidermal cells is involved in the establishment of cell pattern in the Arabidopsis root. Physiol Plant 106:311–317
Ferrari S, Piconese S, Tronelli G, Migliaccio F (2000) A new Arabidopsis thaliana root gravitropism and chirality mutant. Plant Sci 158:77–85
Acknowledgments
We thank Jose Alonso, Begoña Orozco, and Delphine Pott for critical reading of the protocol and Anna Tsui for technical assistance. This work was supported by the National Science Foundation grant IOS 1444561 to A.N.S. and a Marie Curie COFUND U-Mobility postdoctoral fellowship to C.M. (cofunded by the University of Málaga and the EU 7FP GA NO. 246550).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Merchante, C., Stepanova, A.N. (2017). The Triple Response Assay and Its Use to Characterize Ethylene Mutants in Arabidopsis. In: Binder, B., Eric Schaller, G. (eds) Ethylene Signaling. Methods in Molecular Biology, vol 1573. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6854-1_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6854-1_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6852-7
Online ISBN: 978-1-4939-6854-1
eBook Packages: Springer Protocols