Ethylene (C 2 H 4 ) is a gaseous hormone with diverse actions

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1 Lorenzo, O., Piqueras, R., Sanchez-Serrano, J.J., and Solano, R. (2003). ETHYLENE RESPONSE FACTOR1 integrates signals from and jasmonate pathways in plant defense. Plant Cell 15: ; Rüžička, K., Ljung, K., Vanneste, S., Podhorská, R., Beeckman, T., Friml, J., and Benková, E. (2007). regulates root growth through effects on auxin biosynthesis and transport-dependent auxin eistribution. Plant Cell 19: Binder, B.M., O Malley, R.C., Wang, W., Moore, J.M., Parks, B.M., Spalding, E.P., and Bleecker, A.B. (2004). Arabidopsis seedling growth response and recovery to. A kinetic analysis. Plant Physiol. 136: (C 2 H 4 ) is a gaseous hormone with diverse actions Early fruit-ripening practices (control) 7 days regulates: fruit ripening organ expansion senescence gene expression stress responses in smoke has long been used to ripen fruit; this practice has included ripening pears in the smoke from incense. Gashing of unpollinated figs has also been practiced; the produced upon wounding induces ripening Cotton plants Arabidopsis Cover image from Science Vol. 241, no. 4869, 26 August 1988, reprinted with permission from AAAS; photo by Kurt Stepnitz, Michigan State University Beyer, Jr., E.M. (1976) A potent inhibitor of action in plants. Plant Physiol. 58: Image sources: British Museum; Kurt Stüber responses in Arabidopsis When germinating in the dark, impeded seedlings produce which confers a characteristic triple response The response to is very rapid -induced gene expression Inhibition of root elongation induces the triple response: reduced elongation, hypocotyl swelling, apical hook exaggeration It s thought that this response helps the seedling push past the impediment A single dark-grown Arabidopsis seedling photographed every 30 minutes over seven hours. The rapid elongation that preceded addition stopped immediately, and resumed rapidly after was removed Inhibition of leaf cell expansion Acceleration of leaf senescence By treating dark-grown seedlings with exogenous, -response mutants could be identified quickly and easily based on the triple response phenotype is an ancient hormone: Spirogyra makes & responds to it synthesis and homeostasis In 1934, Gane purified from ripening apples, demonstrating that it is an endogenous hormone The filamentous green alga Spirogyra pratensis, whose common aquatic ancestor with land plants lived more than 450 million years ago, produces and responds by cell elongation. Increasing In 1901, Dimitry Neljubow traced the source of the strange growth patterns of his dark-grown pea seedlings to the produced by gas-burning lamps Illuminating gas distilled from tar contains very high levels of In 1901, was identified as a compound that affects plant growth Reprinted from Ju. C., Van de Poel, B., Cooper, E.D., Thierer, J.H., Gibbons, T.R., Delwiche, C.F. and Chang, C. (2015) Conservation of as a plant hormone over 450 million years of evolution. Nature Plants 1: Neljubov, D.N. (1901) Uber die horizontale Nutation der Stengel von Pisum sativum und einiger anderen Pflanzen. Beih. Bot. Centralbh. 10:

2 How to measure circa 1943 The relative insensitivity of the early methods made it difficult to detect small changes in production In 1959 gas chromatography (GC) was used to measure levels This new method was a million-fold more sensitive than earlier methods. Using GC, Burg and Thimann showed that production is temperature dependent GC revealed that is a cause, not consequence, of ripening production precedes ripening and its associated CO 2 production Avocados were an early model for studying fruit ripening Pratt, H.K., Young, R.E., and Biale, J.B. (1948). The identification of as a volatile product of ripening avocados. Plant Physiol. 23: Burg, S.P., and Thimann, K.V. (1959). The physiology of formation in apples. Proc. Natl. Acad. Sci. USA 45: Burg, S.P., and Burg, E.A. (1962). Role of in fruit ripening. Plant Physiol. 37: Burg and Thimann made a key discovery about production Return to air after 4 hours oxygen deprivation Controls When an apple deprived of oxygen for four hours is returned to an aerobic environment, there is a dramatic burst of production This suggests that an precursor accumulates in oxygen-deprived cells! This precursor was called Compound X O 2 Compound X 14 C- Radiolabeled methionine was used to identify Compound X to Adams and Yang incubated apple slices in 14 C-Met to see what compound accumulated when oxygen was withheld Burg, S.P., and Thimann, K.V. (1959). The physiology of formation in apples. Proc. Natl. Acad. Sci. USA 45: Adams, D.O., and Yang, S.F. (1979). biosynthesis: Identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to. Proc. Natl. Acad. Sci. USA 76: They identified Compound X! Compound X 14 C-Met 14 C- Compound X is aminocyclopropane-carboxylic acid () synthesis to O 2 is produced from methionine (Met) via S-adenosylmethionine (AdoMet) by the action of synthase () and oxidase (ACO) to Adams, D.O., and Yang, S.F. (1979). biosynthesis: Identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to. Proc. Natl. Acad. Sci. USA 76: Reprinted from Chae, H.S., and Kieber, J.J. (2005). Eto Brute? Role of turnover in regulating biosynthesis. Trends Plant Sci.10: with permission from Elsevier. 2

3 synthesis The two key enzymes, and ACO, are rare and unstable Characterization of synthase is synthase ACO is oxidase Methionine is regenerated via the Yang cycle, elucidated by Shang Fa Yang Shang Fa Yang Reprinted from Chae, H.S., and Kieber, J.J. (2005). Eto Brute? Role of turnover in regulating biosynthesis. Trends Plant Sci.10: with permission from Elsevier.; Image sources: University of California; Crenim Isolating these proteins and the genes that encode them was a significant effort Tony Bleecker and Hans Kende made major contributions to the study of synthesis and responses Tony Bleecker Hans Kende ( ) ( ) Reprinted from Chae, H.S., and Kieber, J.J. (2005). Eto Brute? Role of turnover in regulating biosynthesis. Trends Plant Sci.10: with permission from Elsevier.; Photos courtesy of Alan Jones (University of North Carolina) and Kurt Stepnitz (Michigan State University). Proteins extracted from ripening tomatoes were used to make monoclonal antibodies Bleecker, A.B., Kenyon, W.H., Somerville, S.C., and Kende, H. (1986). Use of monoclonal antibodies in the purification and characterization of 1-aminocyclopropane-1- carboxylate synthase, an enzyme in biosynthesis. Proc. Natl. Acad. Sci. USA 83: Characterization of synthase An antibody purification scheme was used to clone an synthase cdna synthase expression levels were induced to enrich the protein extract The antibodies were screened for selectivity to synthase and then used to immunoprecipitate the enzyme synthase Antibody heavy chain Auxin cytokinin, Synthase inhibitors The other two proteins are derived from the antibody Antibody light chain Proteins were purified from ripening zucchini Uninduced protein extraction Induced protein extraction Bleecker, A.B., Kenyon, W.H., Somerville, S.C., and Kende, H. (1986). Use of monoclonal antibodies in the purification and characterization of 1-aminocyclopropane-1- carboxylate synthase, an enzyme in biosynthesis. Proc. Natl. Acad. Sci. USA 83: Sato, T., and Theologis, A. (1989). Cloning the mrna encoding 1-aminocyclopropane-1-carboxylate synthase, the key enzyme for biosynthesis in plants. Proc.Natl. Acad. Sci. USA 86: The partially purified inducedprotein extract was used to produce antiserum Y Induced protein extract The antiserum was passed over a column containing uninduced extract Y The contaminating antibodies from the antiserum were removed by absorption onto the uninduced zucchini extract, which contains very little synthase. The resulting antiserum was highly enriched for anti- synthase antibodies The anti- antibody was used to screen a cdna expression library Y Uninduced extract Induced extract Blot probed with purified antiserum A cdna expression library made from induced zucchini mrna was screened using the purified antiserum to obtain an synthase cdna Y Blot probed with crude antiserum Rabbit by Danko Sato, T., and Theologis, A. (1989). Cloning the mrna encoding 1-aminocyclopropane-1-carboxylate synthase, the key enzyme for biosynthesis in plants. Proc.Natl. Acad. Sci. USA 86: Sato, T., and Theologis, A. (1989). Cloning the mrna encoding 1-aminocyclopropane-1-carboxylate synthase, the key enzyme for biosynthesis in plants. Proc.Natl. Acad. Sci. USA 86:

4 Cloning an ACO cdna was similarly challenging. Yeast expressing the ACO cdna can make production is primarily regulated by accumulation Control ACO antisense production in ripening fruit A cdna whose kinetics matched that of accumulation was cloned introduction of an antisense construct into tomato reduced or eliminated production after wounding and during fruit ripening After these key genes were cloned, it was possible to examine how their expression was regulated is encoded by 9 genes with diverse functions and expression patterns Some proteins are strongly regulated post-translationally Reprinted by permission from Macmillan Publishers Ltd (Nature) Hamilton, A.J., Lycett, G.W., and Grierson, D. (1990). Antisense gene that inhibits synthesis of the hormone in transgenic plants. Nature 346: Copyright Type I Type II Type III is encoded by 9 genes and functions as a dimer Type I Type III Type II The gene family products can potentially form 45 homoand heterodimers of which 25 are functional S S S S S Different dimers have different catalytic properties The subset of genes are that are expressed in any cell determines the types of dimers that can form, and affects the rate of synthesis The synthase genes are differentially regulated and induced Yamagami, T., Tsuchisaka, A., Yamada, K., Haddon, W.F., Harden, L.A., and Theologis, A. (2003). Biochemical diversity among the 1-aminocyclopropane-1-carboxylate synthase isozymes encoded by the arabidopsis gene family. J. Biol. Chem. 278: Tsuchisaka, A., and Theologis, A. (2004). Unique and overlapping expression patterns among the Arabidopsis 1-amino-cyclopropane-1-carboxylate synthase gene family members. Plant Physiol. 136: genes have unique and common functions Single mutant analysis shows that each gene has a unique and specific function Higher order mutants show that there are common essential functions including effects on flowering time... Higher order mutants flower earlier: delays flowering The pentuple mutant lacks activity of 5 genes, hexuple lacks 6, etc. genes have unique and common functions Higher order mutants are more susceptible to pathogens; contributes to pathogen resistance A mutant lacking all 9 genes is not viable is necessary for plant survival AIR Genetic studies identified -overproducer (eto) mutants Wild Type ETHYLENE eto1 AIR eto mutants show a tripleresponse in air and overproduce Tsuchisaka, A., Yu, G., Jin, H., Alonso, J.M., Ecker, J.R., Zhang, X., Gao, S., and Theologis, A. (2009). A combinatorial interplay among the 1- aminocyclopropane-1-carboxylate isoforms regulates biosynthesis in Arabidopsis thaliana. Genetics 183: Tsuchisaka, A., Yu, G., Jin, H., Alonso, J.M., Ecker, J.R., Zhang, X., Gao, S., and Theologis, A. (2009). A combinatorial interplay among the 1- aminocyclopropane-1-carboxylate isoforms regulates biosynthesis in Arabidopsis thaliana. Genetics 183: Guzman, P., and Ecker, J.R. (1990). Exploiting the triple response of Arabidopsis to identify -related mutants. Plant Cell 2:

5 ETO1 is a component of a ubiquitinligase complex The eto2 and eto3 mutations affect stability of 5 and 9 proteins are normally subject to rapid proteolysis ETO1 targets proteins for ubiquitination and proteolysis by the 26S proteosome WT eto1 5 is selectively stabilized in loss-offunction eto1 5 mutants -tubulin ETO1 CUL3 26S proteasome 5 eto2 9 eto3 The mutations in eto2 and eto3 are due to changes in the C-terminal region of 5 or 9. The mutant proteins are stabilized, enhancing synthesis Translation ETO1 CUL3 Degradation by the 26S proteasome Normally is continually synthesized and continually degraded, maintaining a very low level of Reprinted by permission from Macmillan Publishers Ltd: Wang, K.L.C., Yoshida, H., Lurin, C., and Ecker, J.R. (2004). Regulation of gas biosynthesis by the Arabidopsis ETO1 protein. Nature 428: , copyright Chae, H.S., Faure, F., and Kieber, J.J. (2003). The eto1, eto2, and eto3 mutations and cytokinin treatment increase biosynthesis in Arabidopsis by increasing the stability of protein. Plant Cell 15: Liu, Y., and Zhang, S. (2004). Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces biosynthesis in Arabidopsis. Plant Cell 16: C-terminal phosphorylation stabilizes s by interfering with ETO1 action Model of regulation by ubiquitin-dependent turnover Regulation by proteolysis allows for rapid responses C-terminal serines are targets for regulated phosphorylation Type I Type II Type III Target of MAP Kinase P P P S S S S P Target of S CDP Kinase (Phosphatase) (Phosphatase) M = MAPK target sequence proteins can be stabilized by Interactions with proteins TOE = Target of ETO11 E3 Ub ligases Booker, M.A. and DeLong, A. (2015) Producing the signal: Regulation and diversification of biosynthetic enzymes. Plant Physiol. 169: 42 50; Yoon, G.M. and Kieber, J.J. (2013) regulates 1-aminocyclopropane-1-carboxylate synthase protein turnover in Arabidopsis. Plant Cell 25: Proteasome Transcription 2. RNA processing A process regulated by 3. Translation de novo transcription 4. Enzyme action has a considerable lag (steps 1 3- before beginning A process regulated by proteolysis can respond very rapidly, simply by not proteolyzing the enzyme This method however requires a constant influx of energy to maintain transport: LYSINE HISTIDINE TRANSPORTER1 transports LHT1 is an amino acid transporter, but also transports between cells to act as a mobile signal for differential synthesis. 1 µm synthesis and homeostasis - summary Biosynthesis SAM ACO proteins stabilized by wounding, other hormones response receptors and downstream signaling In the 1980s, a genetic screen was carried out by Tony Bleecker, Hans Kende and colleagues to dissect the signaling pathway at the molecular level No response insensitive LHT1 is highly expressed in the plasma membrane of leaf mesophyll cells and in lateral roots, where it can wild type lht1 take up and release to the soil The lht1 mutant is resistant to Hirner, A., Ladwig, F., Stransky, H., Okumoto, S., Keinath, M., Harms, A., Frommer, W.B., Koch, W. (2006) Arabidopsis LHT1 is a high-affinity transporter for cellular amino acid uptake in both root epidermis and leaf mesophyll. Plant Cell 18: Shin, K., Lee, S., Song, W.Y., Lee, R.A., Lee, I., Ha, K., Koo, J.C., Park, S.K., Nam, H.G., Lee, Y., Soh, M.S. (2015) Genetic identification of -RESISTANT2 reveals involvement of LYSINE HISTIDINE TRANSPORTER1 in the uptake of 1-aminocyclopropane-1-carboxylic acid in Arabidopsis thaliana. Plant Cell Physiol. 56: by permission. Simple biosynthetic pathway regulated by expression and stability of and ACO and ACO activities are tightly regulated transcriptionally and post-transcriptionally and sensitive to developmental cues, wounding and pathogen attack Normal triple response Bleecker, A.B., Estelle, M.A., Somerville, C., and Kende, H. (1988). Insensitivity to conferred by a dominant mutation in Arabidopsis thaliana. Science 241: reprinted with permission from AAAS; photo by Kurt Stepnitz, Michigan State University. 5

6 Many signaling components were identified genetically ETHYLENE RESPONSE1 () encodes an receptor The etr1-1 mutation is dominant -insensitive no triple response in -insensitive mutants etr1 etr2 ein4 ein2 ein3 ein5 ein6 was the first protein to be unambiguously identified as a phytohormone receptor (1993) binds is similar in sequence to known-receptors in animal cells is membrane localized WT WT WT WT etr1-1 etr1-1 etr1-1 Introduction of the mutant etr1-1 allele into a wild-type plant causes an insensitive phenotype Constitutive response triple response in air Constitutive-response mutants ctr1 binding GAF histidine kinase receiver air From Chang, C., Kwok, S., Bleecker, A., and Meyerowitz, E. (1993). Arabidopsis -response gene : similarity of product to two-component regulators. Science 262: ; reprinted with permission from AAAS. How can a mutant receptor have a dominant phenotype??? The receptors negatively regulate the responses No When not bound to, the receptor shuts off the response OFF When bound to, the receptor does not shut off the response ON A receptor that always shuts off signaling is dominant OFF ON OFF The dominant negative effect of etr1-1 and some other receptor mutants is because they always shut off responses, whether or not is present Arabidopsis receptors resemble hybrid histidine kinases Cytokinin receptor AHK4 CHASE domain binding GAF histidine kinase histidine kinase receiver The receptors structurally resemble the cytokinin receptors. However, unlike the cytokinin receptors, the histidine kinase domain has little role in signaling in vivo receiver Arabidopsis receptor family Subfamily I ERS1 binding 83% GAF 64% histidine kinase 64% receiver Loss-of-function mutations in receptors show constitutive responses ers1 etr1 (Loss of Subfamily 1) But different receptors have different signaling strengths Subfamily I ERS1 Subfamily II EIN4 ETR2 ERS2 etr1 etr2 ein4 EIN % 38-41% 16-29% 32% Wild-type ers1 etr1 double loss-offunction mutant Subfamily II ETR2 58% 54% 38% 52% 55% 40% 53% OFF ON ERS2 Wang, W., Hall, A.E., O'Malley, R., and Bleecker, A.B. (2003). Canonical histidine kinase activity of the transmitter domain of the receptor from Arabidopsis is not required for signal transmission. Proc. Natl. Acad. Sci. USA 100: , copyright National Academy of Sciences USA. Hall, A.E., and Bleecker, A.B. (2003). Analysis of combinatorial loss-of-function mutants in the Arabidopsis receptors reveals that the ers1 etr1 double mutant has severe developmental defects that are dependent. Plant Cell 15:

7 Resnick, J.S., Wen, C.-K., Shockey, J.A., and Chang, C. (2006). REVERSION-TO-ETHYLENE SENSITIVITY1, a conserved gene that regulates receptor function in Arabidopsis. Proc. Natl. Acad. Sci. USA 103: ; Barry, C.S. and Giovannoni, J.J. (2006) Ripening in the tomato Green-ripe mutant is inhibited by ectopic expression of a protein that disrupts signaling. Proc. Natl. Acad. Sci. USA 103: ; copyright National Academy of Sciences USA. receptor mutants have also been identified in other plants The tomato Never ripe mutant has a dominant, -insensitive phenotype, like etr1-1 Wild type Never ripe The -binding domain NH 2 binding GAF histidine kinase receiver There are three transmembrane segments in the binding domain of (four in subfamily II receptors) Mutations in the transmembrane domain abolish binding NH 2 Wild type Never ripe Abolishing binding causes a dominant insensitive phenotype From Wilkinson, J.Q., Lanahan, M.B., Yen, H.-C., Giovannoni, J.J., and Klee, H.J. (1995). An -inducible component of signal transduction encoded by Never-ripe. Science 270: , reprinted with permisison from AAAS; Lanahan, M.B., Yen, H.C., Giovannoni, J.J., and Klee, H.J. (1994). The Never ripe mutation blocks perception in tomato. Plant Cell 6: From Rodríguez, F.I., Esch, J.J., Hall, A.E., Binder, B.M., Schaller, G.E., and Bleecker, A.B. (1999). A copper cofactor for the receptor from Arabidopsis. Science 283: , Reprinted with permssion from AAAS From Rodríguez, F.I., Esch, J.J., Hall, A.E., Binder, B.M., Schaller, G.E., and Bleecker, A.B. (1999). A copper cofactor for the receptor from Arabidopsis. Science 283: , Reprinted with permssion from AAAS; Hall, A.E., Grace Chen, Q., Findell, J.L., Eric Schaller, G., and Bleecker, A.B. (1999). The relationship between binding and dominant insensitivity conferred by mutant forms of the receptor. Plant Physiol. 121: Control of receptor activity by interaction with RTE/GR Genetic epistasis studies determined the order of action of the genes The genetic pathway of signaling RTE/GR etr1-2 rte REVERSION-TO- ETHYLENE SENSITIVITY These studies suggest Loss-of-function of that RTE/GR is a RTE suppresses negative regulator of insensitive signaling etr1-2 phenotype WT Green ripe gain-offunction alleles confer a dominant, insensitive phenotype in tomato fruit etr1 + = ctr1 etr1 ctr1 The double mutant has the same phenotype as ctr1, indicating that it acts downstream from responses Receptor family C 2 H 4 ERS1 ETR2 EIN4 ERS2 (insensitive - dominant) (constitutive) EIN3 EIN5 EIN6 (insensitive - recessive) responses to is a negative regulator of signaling The ctr1 mutant has a constitutive triple response is a serine/threonine protein kinase that resembles animal Raf kinases and is predicted to act in a MAPK cascade ctr1 Wild type The receptors directly interact with and affect its activity (active) OFF In the absence of, is active and inhibits the responses (inactive) ON In the presence of, is inactive ERS The receptors directly interact with A yeast two-hybrid assay revealed a specific interaction between the C- terminal region of the receptors and the N-terminal region of Colony growth and lacz expression means the two proteins interact Reprinted from Kieber, J.J., Rothenberg, M., Roman, G., Feldmann, K.A., and Ecker, J.R. (1993)., a negative regulator of the response pathway in arabidopsis, encodes a member of the Raf family of protein kinases. Cell 72: with permission from Elsevier. Reprinted from Kendrick, M.D., and Chang, C. (2008). signaling: new levels of complexity and regulation. Curr. Opin. Plant Biol. 11: with permission from Elsevier. Clark, K.L., Larsen, P.B., Wang, X., and Chang, C. (1998). Association of the Arabidopsis Raf-like kinase with the and ERS receptors. Proc. Natl. Acad. Sci. USA 95: , copyright National Academy of Sciences USA. 7

8 Receptor complexes in the ER membrane can be heteromeric ERS1 ERS2 ETR2 EIN4 EBD GAF The basic functional unit of perception is a receptor dimer, each monomer likely bound to. However, the different receptors form high-order complexes, linked by disulfide linkages, which might amplify the response. HK Receiver S S S S Regulatory domain Kinase domain receptors have multiple potential binding sites for other proteins that remain to be identified. These might confer additional response specificity Adapted from Ju, C. and Chang, C. (2012) Advances in signalling: protein complexes at the endoplasmic reticulum membrane. AoB Plants 2012:pls031 by permission of Oxford University Press. (inactive) acts through, a positive regulator of ET signaling ON has 12 membrane spanning domains and is ER localized Genetic studies show that acts downstream of Loss-of-function mutants are insensitive has a positive role From Alonso, J., Hirayama, T., Roman, G., Nourizadeh, S., and Ecker, J. (1999)., a bifunctional transducer of and stress responses in Arabidopsis. Science 284: reprinted with permission from AAAS; Kendrick, M.D., and Chang, C. (2008). signaling: new levels of complexity and regulation. Curr. Opin. Plant Biol. 11: with permission from Elsevier. In the absence of, phosphorylates ER Without, is phosphorylated at the C-terminal domain (C-END) P P In, is inactive and is no longer phosphorylated ER Without, is phosphorylated at the C-terminal domain (C-END) P P + ER In, is inactivated, and is no longer phosphorylated lumen In, C-END is cleaved and moves to the nucleus ER Without, is phosphorylated at the C-terminal domain (C-END) P P + ER In, is inactivated, and is no longer phosphorylated, which triggers its cleavage and movement to the nucleus nucleus lumen cytoplasm C-END s C-terminal end contains a nuclear localization signal CEND NLS The C-terminal END (C-END) of the protein contains a putative nuclear localization signal (NLS) The NLS is essential for nuclear localization, because when it is mutated, the C-END fragment remains in the ER C-END-GFP C-END- NLS-GFP GFP ( C-END) DAPI (nuclear stain) Merged Reprinted by permission from Macmillan Publishers Ltd: from Wen, X., Zhang, C., Ji, Y., Zhao, Q., He, W., An, F., Jiang, L. and Guo, H. (2012) Activation of signaling is mediated by nuclear translocation of the cleaved carboxyl terminus. Cell Res. 22: Downstream of, a transcriptional cascade controls gene expression EIN3 and EIL1 are transcription factors that bind an binding site (EBS) in the promoter of ERF1. ERF1 encodes another TF that targets responsive genes? C-END In the absence of, EIN3 and EIL1 are targeted for proteolysis EBF1 and EBF2 are F-box proteins that target EIN3 and EIL1 for proteolysis Accumulation of EBF1 and EBF2 is regulated in part at the level of translation C-END EBF1/2 mrna A current model is that C-END inhibits translation of EBF1/2 mrna. EIN5, an RNA exoribonuclease, also is involved in the regulation of EBF1/2 accumulation by EBF1/2 EIN3/EIL1 EBF1/2 EIN3/EIL1 EIN3/EIL1 EBS ERF1 GCC Responsive Gene Reprinted from Chao, Q., Rothenberg, M., Solano, R., Roman, G., Terzaghi, W., and Ecker, J. (1997). Activation of the gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins Cell 89: with permission from Elsevier; Kendrick, M.D., and Chang, C. (2008). signaling: new levels of complexity and regulation. Curr. Opin. Plant Biol. 11: with permission from Elsevier. Degradation by the 26S proteasome via SCF EBF1/2 EBS ERF1 GCC Responsive Gene Reprinted from Kendrick, M.D., and Chang, C. (2008). signaling: new levels of complexity and regulation. Curr. Opin. Plant Biol. 11: with permission from Elsevier. Degradation by the 26S proteasome via SCF EBF1/2 Interfering with EBF1/2 translation results in the stabilization of EIN3/EIL1 EBS ERF1 GCC Responsive Gene Reprinted from Kendrick, M.D., and Chang, C. (2008). signaling: new levels of complexity and regulation. Curr. Opin. Plant Biol. 11: with permission from Elsevier. 8

9 No receptors activate, which phosphorylates. F-box proteins EBF1/2 degrade EIN3 and EIL1 transcription factors C-terminal domain of (C) directly or indirectly inhibits translation of EBF1 and EBF2 F-box proteins C somehow activates EIN3/EIL1 transcription factors Many forms of regulatory control Summary of synthesis and signaling SAM ACO Biosynthesis Signaling perception and signaling - summary Arabidopsis genetics, and especially the easy-to-score triple response, were instrumental in identifiying the genes encoding the signaling pathway The pathway has a novel combination of proteins acting in a mainly linear pathway Negative regulation plays an important role! Protein turnover is an important regulatory mechanism Reprinted from Salehin, M. and Estelle, M. (2015). prunes translation. Cell. 163: with permission from Elsevier. Reprinted from Ju, C. and Chang, C. (2015) Mechanistic insights in perception and signal transduction. Plant Physiol. 169: s roles in whole-plant processes Shoot and Root elongation Reproductive development Sex determination Petal and leaf senescence Fruit ripening Flooding responses Aerenchyma formation, leaf epinasty Deepwater rice Pathogen responses Interactions with other hormones restricts elongation of the shoot and root in the dark C2H 4 EBS Auxin is required for effects in the root GUS A reporter construct for -induced gene expression Auxin-signaling is required for -induced gene expression in the elongating region of the root. Stepanova, A.N., Yun, J., Likhacheva, A.V., and Alonso, J.M. (2007). Multilevel interactions between and auxin in Arabidopsis roots. Plant Cell 19: s effects are mediated by auxin in the root positively regulates apical hook formation through auxin negatively regulates gravitropism through auxin effects HOOKLESS ARF2 Differential growth wild type hookless increases auxin synthesis, transport and response on the concave side to increase growth curvature reduces gravitropic curvature by minimizing the auxin gradient across the root as shown by DR5::GFP expression on the top side The affect of on auxin in development depends on the context! Stepanova, A.N., Yun, J., Likhacheva, A.V., and Alonso, J.M. (2007). Multilevel interactions between and auxin in Arabidopsis roots. Plant Cell 19: Reprinted from Lehman, A., Black, R., and Ecker, J.R. (1996). HOOKLESS1, an response gene, is required for differential cell elongation in the Arabidopsis hypocotyl. Cell 85: and Muday, G.K., Rahman, A. and Binder, B.M. (2012) Auxin and : collaborators or competitors? Trends Plant Sci. 17: with permission from Elsevier. Reprinted from Muday, G.K., Rahman, A. and Binder, B.M. (2012) Auxin and : collaborators or competitors? Trends Plant Sci. 17: with permission from Elsevier. 9

10 Sex determination in Cucumis Female flowers arise when stamen primordia abort Genes affecting sex determination encode genes Stamen Pistil Elevated levels of production are correlated with developmental arrest of the stamen primordia Hermaphrodite Male Female Imperfect (nonhermaphroditic) flowers can lead to increased outcrossing and increased fitness. Petals Sepals Image courtesy of Abdelhafid Bendahmane, URGV - Plant Genomics Research INRA Image courtesy of Abdelhafid Bendahmane, URGV - Plant Genomics Research INRA Another sex determination gene affects receptor expression promotes petal senescence Chemical and genetic approaches can prolong petal longevity Downregulation of the receptor in stamen primordia makes these tissue more sensitive to Cell in developing pistil Wildtype STS and CACP interfere with binding to receptor Cell in developing stamen etr DAYS AFTER POLLINATION Expression of etr1-1 mutant allele represses petal responses to Klee, H.J. (2004). signal transduction. Moving beyond Arabidopsis. Plant Physiol. 135: Azad, A.K., Ishikawa, T., Ishikawa, T., Sawa, Y., and Shibata, H. (2008). Intracellular energy depletion triggers programmed cell death during petal senescence in tulip. J. Exp. Bot. 59: , by permission of Oxford University Press. Reprinted from Serek, M., Woltering, E.J., Sisler, E.C., Frello, S., and Sriskandarajah, S. (2006) Controlling responses in flowers at the receptor level. Biotech. Adv. 24: with permission from Elsevier; Wilkinson, J.Q., Lanahan, M.B., Clark, D.G., Bleecker, A.B., Chang, C., Meyerowitz, E.M., and Klee, H.J. (1997). A dominant mutant receptor from Arabidopsis confers insensitivity in heterologous plants. Nat Biotech 15: promotes age-related leaf senescence via EIN3 Increasing age Fruit ripening is induced by synthesis increases dramatically during fruit ripening The EIN3 transcription factor accumulates throughout the age of a plant. EIN3 overexpression promotes leaf senescence via regulating senescence-associated genes in response to mir164 Positive regulators EIN3 of leaf senescence Reprinted from Li, Z., Peng, J., Wen, X. and Guo, H. (2013) ETHYLENE-INSENSITIVE3 is a senescence-associated gene that accelerates age-dependent leaf senescence by directly repressing mir164 transcription in Arabidopsis. Plant Cell 25: with permission. Ripening includes: Changes in cell wall structure Pigment accumulation Flavor and aromatic volatile production Conversions of starches to sugars accumulation Giovannoni, J.J. (2004). Genetic regulation of fruit development and ripening. Plant Cell 16: S

11 induces expression of genes during ripening Fruit ripening can be controlled by controlling synthesis synthesis increases upon hypoxia caused by flooding Positive regulation steep increase in production ACO SAM Perception LE6 LE1A LE4 LE2 Developmentally regulated synthase oxidase S-adenosyl H methionine C C H H H Antisense synthase Control Normally, soil has air pockets from which plant roots can take up oxygen O2 O 2 When flooded, roots cannot take up oxygen, and become hypoxic oxygen deprived Hypoxia induces synthase and production Adapted from Barry, C.S., Llop-Tous, M.I., and Grierson, D. (2000). The regulation of 1-aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 synthesis in tomato. Plant Physiol. 123: Theologis, A., Zarembinski, T.I., Oeller, P.W., Liang, X., and Abel, S. (1992). Modification of fruit ripening by suppressing gene expression. Plant Physiol. 100: synthesis increases upon hypoxia caused by flooding moving from root to shoot induces formation and epinasty Rice is grown in regions subject to flooding O2 O 2 induces cell death or cell separation and formation of aerenchyma air channels through which oxygen can move into roots In some plants moves through the xylem into the shoot where it is converted to by oxidase Leaf epinasty, caused by differential growth of the petiole, reduces light absorption by the Cleaves 2H 4 After prolonged flooding, many strains of rice die, but submergence tolerant lines survive using either an escape or quiescence strategy Photo Author: Gordon Beakes University of Newcastle upon Tyne Image courtesy LTSN Bioscience. A darkfield micrograph of a transverse section of a stem of Hippuris spp., showing aerenchyma. Reprinted by permission from Macmillan Publishers Ltd. NATURE from Voesenek, L.A.C.J., and Bailey-Serres, J. (2009). Genetics of high-rise rice. Nature 460: copyright 2009 Rice is grown in regions subject to flooding In deepwater rice, induces internode elongation Preserved deepwater rice specimen The elongation response is encoded by two -responsive transcription factors (ERFs) Deepwater rice The escape strategy involves an response. The quiescence strategy involves a gibberellin response Gibberellin These plants can grow as much as 15m high when subjected to flooding Flooding Non-deepwater rice SNORKEL1 & 2 Transcriptional response Deepwater Flooding Non-deepwater rice does not have these genes No transcriptional response Reprinted by permission from Macmillan Publishers Ltd. NATURE from Voesenek, L.A.C.J., and Bailey-Serres, J. (2009). Genetics of high-rise rice. Nature 460: copyright 2009 Reprinted by permission from Macmillan Publishers Ltd. From Hattori, Y., et al. (2009). The response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water. Nature 460: , copyright Photo credit Moto Ashikari, Nagoya University. Reprinted by permission from Macmillan Publishers Ltd. From Hattori, Y., et al. (2009). The response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water. Nature 460: , c opyright

12 -insensitive tobacco has an impaired immune system The FLS2 gene for pathogenrecognition is regulated by is required for wound or pathogen responses Plants expressing a dominant mutant gene lack resistance to normally harmless soil-borne fungi Bacterium with flagella FLS2 Plants that do not produce or respond to fail to induce expression of proteinase inhibitor 2 (pin2) Higher order mutants are more susceptible to pathogens Plants impaired in responses are also impaired in the recognition of pathogens, and so more susceptible Plant cell Defense ACO antisense plant Hours after wounding Wounding No treatment Wounding + silver thiosulfate, an inhibitor of responses. Knoester, M., van Loon, L.C., van den Heuvel, J., Hennig, J., Bol, J.F., and Linthorst, H.J.M. (1998). -insensitive tobacco lacks nonhost resistance against soil-borne fungi. Proc. Natl. Acad. Sci. USA 95: , copyright National Academy of Sciences USA.; Tsuchisaka, A., Yu, G., Jin, H., Alonso, J.M., Ecker, J.R., Zhang, X., Gao, S., and Theologis, A. (2009). A combinatorial interplay among the 1-aminocyclopropane-1- carboxylate isoforms regulates biosynthesis in Arabidopsis thaliana. Genetics 183: Boutrot, F., Segonzac, C., Chang, K.N., Qiao, H., Ecker, J.R., Zipfel, C. and Rathjen, J.P. (2010). Direct transcriptional control of the Arabidopsis immune receptor FLS2 by the dependent transcription factors EIN3 and EIL1. Proc. Natl. Acad. Sci. USA. 107: From O'Donnell, P.J., Calvert, C., Atzorn, R., Wasternack, C., Leyser, H.M.O., and Bowles, D.J. (1996). as a signal mediating the wound response of tomato plants. Science 274: Reprinted with permission from AAAS. Some defense responsive genes respond to jasmonate and PDF1.2 is a defense gene that requires BOTH and jasmonate for induction (coi1 is a jasmonateinsensitive mutant) / JA responses are mediated by ERF1 and other TFs interacts with many other hormones Stress/ Many hormone GA development pathways converge via EIN3 as JAZ Iron and DELLA DELLA proteins, MYC2 and FIT can physically FIT interact with EIN3 works with jasmonate in defense-related gene expression. Penninckx, I.A.M.A., Thomma, B.P.H.J., Buchala, A., Metraux, J.-P., and Broekaert, W.F. (1998). Concomitant activation of jasmonate and response pathways is required for induction of a plant defensin gene in Arabidopsis. Plant Cell 10: Lorenzo, O., Piqueras, R., Sanchez-Serrano, J.J., and Solano, R. (2003). ETHYLENE RESPONSE FACTOR1 integrates signals from and jasmonate pathways in plant defense. Plant Cell 15: JA Stress/ necrotrophic pathogens JAZ EIN3 Other transcription factors? MYC2 Apical hook development Defense Leaf senescence JA Root inhibition Iron absorption Based on: Ju, C. and Chang, C. (2015) Mechanistic insights in perception and signal transduction. Plant Physiol. 169: Soil bacteria can reduce levels in plants by deactivating ETHYLENE- SUMMARY Ongoing research - 1 Stress-induced can also inhibit growth. This effect can be decreased by deaminase, produced by many soil bacteria Root cell Soil bacterium deaminase ammonia + α- ketobutyrate Growth inhibition Biosynthesis Signaling SAM ACO and others EIN3, EILs ERF1 and ERFs Cell elongation Auxin synthesis and transport Fruit ripening Senescence Pathogen defense Does itself function as a growth regulator? How can production be optimized to enhance fruit quality? What signals contribute to the posttranslational regulation of accumulation? SAM ACO What is the mechanism of production by ACO? What are the transcriptional regulators of and ACO genes? Gamalero, E. and Glick, B.R. (2015). Bacterial modulation of plant levels. Plant Physiol. 169:

13 Ongoing research - 2 What are the roles of MAP kinases in EIN3/EIL1 synthesis and signaling? How is cleaved and dephosphorylated? Many other response mutants are being characterized and integrated into the pathway what do they do? P P P S S S S What role if any is played by the histidine kinase domain in the receptors? What do the different receptor isoforms do? How can we best use this knowledge to improve access to fresh food? enhanced Robles, L.M., Wampole, J.S., Christians, M.J., and Larsen, P.B. (2007). Arabidopsis enhanced response 4 response 4 encodes an EIN3-interacting TFIID transcription factor required for proper response, including ERF1 induction. J. Exp. Bot. 58: , by permission of Oxford University Press. 13