Ethylene Signal Transduction Pathway

In fruits—tomato, avocado and banana—ethylene is known to coordinate and complete the ripening process. Tomato and, in recent years, Arabidopsis have been used as plant models to unravel mechanisms surrounding ethylene action in plants. Since ripening and senescence share many common features during cell death (Gillaspy et al., 1993), one favorite approach to unravel regulation has been the use of mutants as well as molecular genetics. In Arabidopsis, the mutations clearly support a role for ethylene in leaf senescence; however, mutations in the ethylene-signaling pathway indicate that ethylene promotes rather than initiates senescence (Bleecker and Patterson, 1997; Jing et al., 2002). Tomato ripening single-gene mutants—rin (ripening inhibitor), nor (non-ripening), Nr (never ripe) (Tigchelaar et al., 1978) and the "alcobaca" mutant (Leal and Tabim, 1974)—have contributed substantially to studies devoted to identification of ethylene receptors and the signal transduction pathway in tomato. The fruits of these mutants show an extended shelf life, absence of the ethylene-mediated climacteric rise in respiration, inability to fully soften, and inferior flavor and aroma (Giovannoni, 2001). In addition, leaves, petioles and abscission zone tissue of Nr plants exhibit greatly delayed senescence and abscission. The tomato locus Nr mapped together with an Arabidopsis ETR1 gene-RFLP probe, suggesting homology (Yen et al., 1995). The NR protein in tomato is closely related to the AtERSl ethylene receptor in Arabidopsis, indicating that pleiotropic phenotype of Nr mutation relates to ethylene insensitivity (see below; Wilkinson et al., 1995; Payton et al., 1996).

A large number of ethylene mutants, including etrl, ein2, ein3, ein4, ein5, ein6 and ein7, have been isolated from Arabidopsis, and they have significantly contributed to our current knowledge on the regulation of ethylene biosynthesis and its perception with reference to ripening, senescence and environmental stresses (see Table 8-1, and reviews by Chang and Stadler, 2001; Hall et al, 2001; Wang et al., 2002). ein5 and ein7 may be allelic, while the other loci map separately (Romano et al., 1995). The expression of rin and nor loci appears in a narrow developmental window during fruit ripening. Successful targeting of the genes via map-based cloning schemes has contributed to the understanding of the molecular nature of these lesions in nor and rin tomato mutants (Giovannoni, 2001). The rin locus has

Table 8-1. Arabidopsis Mutants Showing How Other Phytohormones Interact with the Ethylene Signaling Pathway

Mutant

Gene product

Phenotype

References

Ethylene and ABA

abi1-1 Protein phosphatase 2C

era3/ein2 Membrane bound metal sensor ctr1 Protein kinase raf family

Ethylene and sugar sensing gin1, aba2 Short chain dehydrogenase/

sis4, isi4 reductase san3, sre1

gin2 gin4, ctrl, sisl

Hexose kinase Protein kinase raf family

Ethylene and auxin auxl Auxin amino acid permease hsll surl, alfl etol ctrl pir2/ein2

N-acetyltransferase

Protein kinase raf family

Ethylene and cytokinin cin5 ACC synthase ckrl/ein2

Pleiotropic defect in ABA, response

Ethylene sensitive allelic to ein2

Reduced dormancy Constitutive triple response

Growth on 6% Glu Reduced dormancy ABA-deficient, wilty Lack triple response in dark

Growth on 6% Glu Growth on 6% Glu Reduced dormancy Constitutive triple response

Auxin resistant root growth Abolish root gravicurvature Disrupts apical hook formation Hookless

Disrupts apical hook formation

Ethylene over-producer

NPA and auxin disrupt apical hook formation

NPA and auxin disrupt apical hook formation

NPA resistant affected in root-elongation

Koorneef etal., 1984; Leung etal., 1994; Meyer et al., 1994 Alonso etal., 1999; Ghassemian et al., 2000 Kieber etal., 1993; Beaudoin et al., 2000

Zhou etal., 1998; Leon-Kloosterziel etal., 1996; Rook etal., 2001; Laby et al., 2000 Zhou etal., 1998 Zhou etal., 1998; Laby et al., 2000; Kieber etal., 1993; Beaudoin et al., 2000

Bennett et al., 1996; Romano etal., 1995; Marchant et al., 1999 Boerjan et al., 1995; Celenza etal., 1995; Lehman etal., 1996 Lehman etal., 1996

Lehman etal., 1996 Fujita and Syono, 1996

Absence of triple response in the Vogel et al., 1998 presence of kinetin

Cytokinin-resistant root growth Su and Howell, 1992

now been shown to contain two MADS-box genes—LeMADS-RIN and LeMADS-MC— that encode transcription factors (Vrebalov et al., 2002). Vrebalov and colleagues elegantly demonstrated non-hormonal regulation of ripening, upstream of ethylene in the regulatory cascade, by LeMADS-RIN. Seedlings of these mutants show normal sensitivity to ethylene (Lanahan et al., 1994). However, in the fruit, both mutants fail to synthesize climacteric ethylene or accumulate the red carotenoid lycopene (Tigchelaar et al., 1978). These mutants should aid studies for determining hormonal regulation of PCD in reproductive tissues of plants.

How do ethylene receptors regulate ethylene responses during plant growth, development and senescence? Chang et al. (1993) were the first to clone and characterize the gene, AtETR1, responsible for a dominant ethylene-insensitive mutant in Arabidopsis and discovered that it shared many similarities with two-component regulators in yeast and bacteria. Subsequently, the AtETR1 gene was expressed in yeast and the recombinant protein was found to bind ethylene in vitro with similar affinity as that estimated from the dose-response curve for ethylene inhibition of hypocotyl growth in Arabidopsis seedlings (Schaller and Bleecker, 1995). The AtETR1 ethylene receptor has three domains: a sensor, a kinase, and a receiver domain (response regulator). Ethylene binds to the N-terminal sensor domain that has three membrane-spanning helices (Schaller and Bleecker, 1995). In Arabidopsis, five genes make up a family of ethylene receptors (Bleecker, 1999). They all contain the three transmembrane domains required for ethylene binding, and a putative, GAF-like, cyclic nucleotide-binding domain (Bleecker, 1999). Interestingly, AtERS1 and AtERS2 lack a response regulator, while three of the five gene products, AtETR2, AtEIN4 and AtERS2, do not contain the target amino acids deemed necessary for the histidine kinase activity found in AtETR1 (Bleecker, 1999). Therefore, the role of the histidine kinase domain and response regulator in the ethylene signal transduction pathway remains to be elucidated. Wang et al. (2003) provide evidence that the histidine kinase domain in the ETR1 ethylene receptor is not required for ethylene signaling in Arabidopsis. Klee (unpublished data) has suggested that the proposed histidine kinase domain may in actuality be a serine-threonine kinase. Although the receptor proteins are structurally different, Hua and Meyerowitz (1998) proposed that at least four of them serve redundant functions in Arabidopsis. Analysis of the loss-of-function mutants revealed constitutive ethylene-like response, which was suggested to indicate that the ethylene response pathway is negatively regulated by the ethylene receptors in Arabidopsis. Two orthologues of the Arabidopsis ETR1 gene in tomato, eTAE1 (Zhou et al., 1996a) and TFE27 (Zhou et al., 1996b), renamed LeETR1 and LeETR2, respectively, also possess the three domains of the AtETR1 protein (sensor, histidine kinase and receiver domains), while NR (renamed LeETR3), like AtERS1, is devoid of a receiver domain. Two additional genes belonging to the tomato ethylene receptor family, LeETR4 and LeETR5 (Tieman and Klee, 1999), contain a sequence for a putative receiver domain but do not have the necessary domain for histidine kinase (Tieman and Klee, 1999). Models of how various gene products may interact to regulate ethylene action have been presented (Fig. 8-1; see reviews by Chang and Stadler, 2001; Hall et al., 2001; Wang et al., 2002) but which of these are of consequence in the various types of plant senescence is yet to be determined.

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