- Email: [email protected]
Plant hormones: More than one way to detect ethylene
144
Dispatch
Plant hormones: More than one way to detect ethylene Athanasios Theologis
Plants have been found to use multiple forms of prokaryotic-like two-component sensors for detecting the gaseous hormone ethylene. Address: Plant Gene Expression Center, 800 Buchanan Street, Albany, California 94710, USA. Current Biology 1996, Vol 6 No 2:144–145 © Current Biology Ltd ISSN 0960-9822
Plants use the gas ethylene (C2H4) as a signaling molecule for regulating a variety of developmental processes, including fruit and flower senescence, sex determination in some monoecious species, cell elongation and pathogen responses [1]. The final reactions of the ethylene biosynthetic pathway have been elucidated [2], and information on its signaling apparatus is beginning to emerge [3,4]. Molecular genetic analysis of the ethylene signaling pathway in Arabidopsis revealed that plants sense ethylene by a protein kinase cascade [3,4]. This revelation came from the cloning of CTR1 and ETR1, two key genes in the pathway. CTR1 mutations confer a constitutive ethylene response, whereas ETR1 mutations confer ethylene insensitivity. Ecker and colleagues [5] first cloned CTR1, which encodes a putative Raf-like serine/threonine protein kinase [5]. Soon after, Meyerowitz and colleagues [6] cloned ETR1, which encodes a putative histidine protein kinase similar to prokaryotic ‘two-component’ sensors (Fig. 1). Genetic evidence indicates that ETR1 acts upstream of CTR1 and other components in the pathway [3]. In addition, ethylene-insensitive Arabidopsis plants homozygous for the etr1-1 allele do not bind ethylene effectively. Based on these properties, the ETR1 protein has been hypothesized to be the ethylene receptor [6]. Schaller and Bleecker [7] have now elegantly demonstrated that ETR1 binds ethylene and indeed qualifies as an ethylene receptor. These investigators expressed the ETR1 protein in yeast, which neither produces nor senses ethylene, and found that the transgenic yeast strain is able to bind ethylene with high affinity. The binding is saturable, and is inhibited by known competitive ethylene antagonists. By testing truncated forms of ETR1 in the yeast expression system, the receptor’s ethylene binding site has been localized to the protein’s transmembrane domain. It is of great interest that all dominant ETR1 mutations lie in this region, and when the etr1-1 mutant protein is expressed in yeast, no ethylene binding is observed [7]. For a while, it was thought that ETR1 was the only plant ethylene receptor, but Meyerowitz’s laboratory [8]
surprised us last September when they identified a second ethylene sensor expressed in Arabidopsis, termed ERS for ethylene response sensor. ERS is also similar to bacterial ‘two-component’ sensors, but more like the archetypal members of this family in that, unlike ETR1, it lacks a response regulator domain at its carboxyl terminus (Fig. 1). The introduction into the Arabidopsis ERS gene of a mutation equivalent to etr1-4 also confers dominant ethylene insensitivity, suggesting that ERS is an ethylene sensor [8]. Figure 1 (a) The classical bacterial 'two-component' signaling system Input signal
P
Output signal
P
N
CN H D Histidine protein Receiver domain kinase domain
TM Input domain
C Output domain
Response regulator
Sensor Phosphate transfer reaction
P
(b) Plant ethylene receptors C2H4 Arabidopsis ETR1
N
ERS
N
Tomato NR eTAE1
TM
H
D
C
D
C
C 72 %
62 %
70 %
61 %
94 %
75 %
C
N N
69 %
© 1996 Current Biology
(a) Bacterial ‘two-component’ signaling systems are made up of distinct modules that can be arranged in different ways in multi-domain proteins — in the archetypal systems, for example, the ‘sensor’ and ‘response regulator’ are separate proteins. Information flows from one module to another by a combination of non-covalent interactions and phosphorylation reactions involving histidine (H) and aspartate (D) residues [14]. (b) The recently discovered ethylene sensors in plants have striking structural similarities to the bacterial two-component sensors [4,7,8]. ETR1 proteins have an ethylene binding site in the transmembrane domain (TM), near the amino terminus [7], followed by putative histidine kinase domain and receiver domains fused at the carboxyl terminus [4,10]. ERS proteins are orthodox two-component sensors and lack the receiver domain [8,9]. The percentages below some domains indicate their sequence identity to the corresponding domains of the Arabidopsis ETR1 protein.
Dispatch
The excitement does not end here. Klee and colleagues [9] have found that the pleiotropic tomato ripening mutant NR is caused by a dominant mutation in the transmembrane domain of a protein, NR, which is similar to ERS (Fig. 1). More importantly, a transgenic yeast strain expressing wild-type NR protein also binds ethylene (A. Bleecker, personal communication). A tomato ETR1 homolog, eTAE1, has also been cloned [10]; eTAE1 mRNA is constitutively expressed during flower and fruit senescence, whereas NR mRNA is developmentally regulated during fruit ripening [9,10]. The emerging picture is that the ethylene sensors are encoded by multigene families whose members are differentially expressed during plant growth and development. The future looks exciting for research on plant ethylene sensors. It will be no surprise if, in the next few years, many more ethylene receptors are isolated from various monocotyledonous and dicotyledonous plant species. A first priority should be the identification of the type of metal present in the ethylene binding site of ETR1. It has been suggested [11] that Cu (I) may be the transition metal responsible for ethylene binding; biophysical and crystallographic studies of ETR1 should show if this is correct. Ethylene action requires oxygen; this may be because of a putative oxidase hypothesized to keep the metalloprotein ethylene receptor in the oxidized form [11]. A challenge for the future is to prove if such an oxidase is part of the ethylene-sensing apparatus. The identification of the components that connect the ethylene receptors to the CTR1 protein kinase — putative equivalents of bacterial response regulators — is also a task for the future. It is crucial that enzymatic verification is offered that the ethylene sensors are indeed protein kinases. Furthermore, biochemical studies are needed to test the models that have been put forward to explain the dominant nature of the ETR1, NR and ERS mutations [4]. The multiplicity of ethylene receptors in a single plant species raises the question of whether each cell expresses one or more of the ethylene sensors during development. Do the receptors bind all ethylene with the same affinity? We should remember that low levels of the gases hydrogen cyanide (HCN) and carbon monoxide (CO) initiate fruit ripening [12]. Do the ethylene receptors recognize these gases? It is possible that some of the receptors are there to sense some other gas than ethylene. For example, some of them may monitor oxygen tension under anaerobic conditions. A prominent feature of ethylene biosynthesis is that it is autocatalytic — exposure to ethylene stimulates plants to make more of the gas [13]. We need to discover which of the ethylene sensors communicates with the genes encoding the enzymes of autocatalytic ethylene production. Information about this pathway may have practical benefits too — the expression of dominant mutant ethylene receptors in agronomically important
145
crops may allow the production of flowers and vegetables that have extended shelf lives because they are unable to sense ethylene. Amidst the present flurry of excitement, we must not forget the pioneers in the ethylene receptor field, such as E. Sisler and J. Hall, who for years worked tirelessly to develop ethylene antagonists and biochemical assays to determine ethylene binding in crude plant extracts. These same assays and inhibitors were used by the molecular biologists who determined that ETR1 binds ethylene. References 1. Abeles FB, Morgan PW, Saltveit ME Jr: Ethylene in Plant Biology. New York: Academic Press; 1992. 2. Zarembinski TI, Theologis A: Ethylene biosynthesis and action: a case of conservation. Plant Mol Biol 1994, 26:1579–1597. 3. Ecker JR: The ethylene signal transduction pathway in plants. Science 1995, 268:667–674. 4. Chang C: The ethylene signal transduction pathway in Arabidopsis — an emerging paradigm? Trends Biochem Sci 1996, in press. 5. Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR: CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases. Cell 1993, 72:427–441. 6. Chang C, Kwok SF, Bleecker AB, Meyerowitz EM: Arabidopsis ethylene-response gene ETR1: similarity of product to twocomponent regulators. Science 1993, 262:539–544. 7. Schaller GE, Bleecker AB: High-affinity binding sites for ethylene are generated in yeast expressing the Arabidopsis ETR1 gene. Science 1995, 270:1809–1811. 8. Hua J, Chang C, Sun Q, Meyerowitz EM: Ethylene insensitivity conferred by Arabidopsis ERS gene. Science 1995, 269:1712–1714. 9. Wilkinson JQ, Lanahan MB, Yen HC, Giovannoni JJ, Klee HJ: Neverripe is a novel, ethylene-inducible component of plant hormone signal transduction. Science 1995, 270:1807–1809. 10. Zhou D, Mattoo AK, Tucker ML: The mRNA for an ETR1 homologue in tomato is constitutively expressed in vegetative and reproductive tissues. Plant Mol Biol 1996, in press. 11. Burg SP, Burg EA: Molecular requirements for the biological activity of ethylene. Plant Physiol 1967, 42:144–152. 12. Solomos T, Laties GG: Cellular organization and fruit ripening. Nature 1973, 245:390–392. 13. Yang SF, Hoffman NE: Ethylene biosynthesis and its regulation in higher plants. Annu Rev Plant Physiol 1984, 35:155–189. 14. Parkinson JS, Kofoid EC: Communication modules in bacterial signaling proteins. Annu Rev Genet 1992, 26:71–112.
Dispatch
Plant hormones: More than one way to detect ethylene Athanasios Theologis
Plants have been found to use multiple forms of prokaryotic-like two-component sensors for detecting the gaseous hormone ethylene. Address: Plant Gene Expression Center, 800 Buchanan Street, Albany, California 94710, USA. Current Biology 1996, Vol 6 No 2:144–145 © Current Biology Ltd ISSN 0960-9822
Plants use the gas ethylene (C2H4) as a signaling molecule for regulating a variety of developmental processes, including fruit and flower senescence, sex determination in some monoecious species, cell elongation and pathogen responses [1]. The final reactions of the ethylene biosynthetic pathway have been elucidated [2], and information on its signaling apparatus is beginning to emerge [3,4]. Molecular genetic analysis of the ethylene signaling pathway in Arabidopsis revealed that plants sense ethylene by a protein kinase cascade [3,4]. This revelation came from the cloning of CTR1 and ETR1, two key genes in the pathway. CTR1 mutations confer a constitutive ethylene response, whereas ETR1 mutations confer ethylene insensitivity. Ecker and colleagues [5] first cloned CTR1, which encodes a putative Raf-like serine/threonine protein kinase [5]. Soon after, Meyerowitz and colleagues [6] cloned ETR1, which encodes a putative histidine protein kinase similar to prokaryotic ‘two-component’ sensors (Fig. 1). Genetic evidence indicates that ETR1 acts upstream of CTR1 and other components in the pathway [3]. In addition, ethylene-insensitive Arabidopsis plants homozygous for the etr1-1 allele do not bind ethylene effectively. Based on these properties, the ETR1 protein has been hypothesized to be the ethylene receptor [6]. Schaller and Bleecker [7] have now elegantly demonstrated that ETR1 binds ethylene and indeed qualifies as an ethylene receptor. These investigators expressed the ETR1 protein in yeast, which neither produces nor senses ethylene, and found that the transgenic yeast strain is able to bind ethylene with high affinity. The binding is saturable, and is inhibited by known competitive ethylene antagonists. By testing truncated forms of ETR1 in the yeast expression system, the receptor’s ethylene binding site has been localized to the protein’s transmembrane domain. It is of great interest that all dominant ETR1 mutations lie in this region, and when the etr1-1 mutant protein is expressed in yeast, no ethylene binding is observed [7]. For a while, it was thought that ETR1 was the only plant ethylene receptor, but Meyerowitz’s laboratory [8]
surprised us last September when they identified a second ethylene sensor expressed in Arabidopsis, termed ERS for ethylene response sensor. ERS is also similar to bacterial ‘two-component’ sensors, but more like the archetypal members of this family in that, unlike ETR1, it lacks a response regulator domain at its carboxyl terminus (Fig. 1). The introduction into the Arabidopsis ERS gene of a mutation equivalent to etr1-4 also confers dominant ethylene insensitivity, suggesting that ERS is an ethylene sensor [8]. Figure 1 (a) The classical bacterial 'two-component' signaling system Input signal
P
Output signal
P
N
CN H D Histidine protein Receiver domain kinase domain
TM Input domain
C Output domain
Response regulator
Sensor Phosphate transfer reaction
P
(b) Plant ethylene receptors C2H4 Arabidopsis ETR1
N
ERS
N
Tomato NR eTAE1
TM
H
D
C
D
C
C 72 %
62 %
70 %
61 %
94 %
75 %
C
N N
69 %
© 1996 Current Biology
(a) Bacterial ‘two-component’ signaling systems are made up of distinct modules that can be arranged in different ways in multi-domain proteins — in the archetypal systems, for example, the ‘sensor’ and ‘response regulator’ are separate proteins. Information flows from one module to another by a combination of non-covalent interactions and phosphorylation reactions involving histidine (H) and aspartate (D) residues [14]. (b) The recently discovered ethylene sensors in plants have striking structural similarities to the bacterial two-component sensors [4,7,8]. ETR1 proteins have an ethylene binding site in the transmembrane domain (TM), near the amino terminus [7], followed by putative histidine kinase domain and receiver domains fused at the carboxyl terminus [4,10]. ERS proteins are orthodox two-component sensors and lack the receiver domain [8,9]. The percentages below some domains indicate their sequence identity to the corresponding domains of the Arabidopsis ETR1 protein.
Dispatch
The excitement does not end here. Klee and colleagues [9] have found that the pleiotropic tomato ripening mutant NR is caused by a dominant mutation in the transmembrane domain of a protein, NR, which is similar to ERS (Fig. 1). More importantly, a transgenic yeast strain expressing wild-type NR protein also binds ethylene (A. Bleecker, personal communication). A tomato ETR1 homolog, eTAE1, has also been cloned [10]; eTAE1 mRNA is constitutively expressed during flower and fruit senescence, whereas NR mRNA is developmentally regulated during fruit ripening [9,10]. The emerging picture is that the ethylene sensors are encoded by multigene families whose members are differentially expressed during plant growth and development. The future looks exciting for research on plant ethylene sensors. It will be no surprise if, in the next few years, many more ethylene receptors are isolated from various monocotyledonous and dicotyledonous plant species. A first priority should be the identification of the type of metal present in the ethylene binding site of ETR1. It has been suggested [11] that Cu (I) may be the transition metal responsible for ethylene binding; biophysical and crystallographic studies of ETR1 should show if this is correct. Ethylene action requires oxygen; this may be because of a putative oxidase hypothesized to keep the metalloprotein ethylene receptor in the oxidized form [11]. A challenge for the future is to prove if such an oxidase is part of the ethylene-sensing apparatus. The identification of the components that connect the ethylene receptors to the CTR1 protein kinase — putative equivalents of bacterial response regulators — is also a task for the future. It is crucial that enzymatic verification is offered that the ethylene sensors are indeed protein kinases. Furthermore, biochemical studies are needed to test the models that have been put forward to explain the dominant nature of the ETR1, NR and ERS mutations [4]. The multiplicity of ethylene receptors in a single plant species raises the question of whether each cell expresses one or more of the ethylene sensors during development. Do the receptors bind all ethylene with the same affinity? We should remember that low levels of the gases hydrogen cyanide (HCN) and carbon monoxide (CO) initiate fruit ripening [12]. Do the ethylene receptors recognize these gases? It is possible that some of the receptors are there to sense some other gas than ethylene. For example, some of them may monitor oxygen tension under anaerobic conditions. A prominent feature of ethylene biosynthesis is that it is autocatalytic — exposure to ethylene stimulates plants to make more of the gas [13]. We need to discover which of the ethylene sensors communicates with the genes encoding the enzymes of autocatalytic ethylene production. Information about this pathway may have practical benefits too — the expression of dominant mutant ethylene receptors in agronomically important
145
crops may allow the production of flowers and vegetables that have extended shelf lives because they are unable to sense ethylene. Amidst the present flurry of excitement, we must not forget the pioneers in the ethylene receptor field, such as E. Sisler and J. Hall, who for years worked tirelessly to develop ethylene antagonists and biochemical assays to determine ethylene binding in crude plant extracts. These same assays and inhibitors were used by the molecular biologists who determined that ETR1 binds ethylene. References 1. Abeles FB, Morgan PW, Saltveit ME Jr: Ethylene in Plant Biology. New York: Academic Press; 1992. 2. Zarembinski TI, Theologis A: Ethylene biosynthesis and action: a case of conservation. Plant Mol Biol 1994, 26:1579–1597. 3. Ecker JR: The ethylene signal transduction pathway in plants. Science 1995, 268:667–674. 4. Chang C: The ethylene signal transduction pathway in Arabidopsis — an emerging paradigm? Trends Biochem Sci 1996, in press. 5. Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR: CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases. Cell 1993, 72:427–441. 6. Chang C, Kwok SF, Bleecker AB, Meyerowitz EM: Arabidopsis ethylene-response gene ETR1: similarity of product to twocomponent regulators. Science 1993, 262:539–544. 7. Schaller GE, Bleecker AB: High-affinity binding sites for ethylene are generated in yeast expressing the Arabidopsis ETR1 gene. Science 1995, 270:1809–1811. 8. Hua J, Chang C, Sun Q, Meyerowitz EM: Ethylene insensitivity conferred by Arabidopsis ERS gene. Science 1995, 269:1712–1714. 9. Wilkinson JQ, Lanahan MB, Yen HC, Giovannoni JJ, Klee HJ: Neverripe is a novel, ethylene-inducible component of plant hormone signal transduction. Science 1995, 270:1807–1809. 10. Zhou D, Mattoo AK, Tucker ML: The mRNA for an ETR1 homologue in tomato is constitutively expressed in vegetative and reproductive tissues. Plant Mol Biol 1996, in press. 11. Burg SP, Burg EA: Molecular requirements for the biological activity of ethylene. Plant Physiol 1967, 42:144–152. 12. Solomos T, Laties GG: Cellular organization and fruit ripening. Nature 1973, 245:390–392. 13. Yang SF, Hoffman NE: Ethylene biosynthesis and its regulation in higher plants. Annu Rev Plant Physiol 1984, 35:155–189. 14. Parkinson JS, Kofoid EC: Communication modules in bacterial signaling proteins. Annu Rev Genet 1992, 26:71–112.