The Contributions of Anthony B. Bleecker to Ethylene Signaling and Beyond

How infinitesimal concentrations of a simple two-carbon gas could bring about dramatic changes in plant growth and development may have seemed to a previous generation of plant biologists like one of nature's unknowables. Now, however, ethylene signaling is understood with such clarity that it can be presented as a paradigm in textbooks. Prominent among the people responsible for this remarkable progress is Tony Bleecker, who died of cancer last year. In the sad days that followed his untimely passing, much was written and spoken about Tony's attributes and achievements. This essay is not such a eulogy but an attempt to provide context and perspective beneficial to a reader of the article authored by Tony and his colleagues presented in this issue of The Plant Cell (Wang et al., pages 3429–3442).

EARLY WORK WITH HANS KENDE AT MICHIGAN STATE

In 1982, when Tony began his PhD studies at Michigan State University under the tutelage of Hans Kende, ethylene had long been known as a regulator of cell expansion in seedlings, fruit ripening, senescence, and other processes. The enzymology of its biosynthetic pathway had been worked out and even had a name, the Yang cycle. Purification of the components, however, had yet to be accomplished. Tony's first publication on ethylene reported the purification from tomato pericarp of the enzyme that performs the rate-limiting step in ethylene biosynthesis, ACC synthase (Bleecker et al., 1986). The work also included the generation of monoclonal antibodies against the protein. Most professors probably would have judged that piece of biochemistry to be a successful PhD project, especially given how notoriously difficult ACC synthase is to work with, but much more was to come from Tony as a result of his turning to Arabidopsis, then early on its way to becoming the preeminent model plant. With colleagues at the Plant Research Laboratory at Michigan State, Tony isolated a dominant Arabidopsis mutant that was insensitive to ethylene. The seedling phenotype of etr1-1 was beautifully captured in a now iconic photograph. First published on the cover of Science (Bleecker et al., 1988), it shows a gently curving seedling with a long hypocotyl and spread cotyledons rising ridiculously high above a lawn of seedlings growing fat and slow, with closed cotyledons tucked in tight as if bracing for the worst (see figure). (This so-called triple response to ethylene is thought to aid the upward progress of seedlings caught in a jam by producing a sturdier stem that can better push through impediments.) Report of the etr1-1 mutant may have had more impact on the ethylene field than any other single publication.

Figure 1.

The Arabidopsis etr1-1 Mutant Described by Bleecker et al. (1988) and Highlighted on the Cover of Science, August 26, 1988.

Image provided by JSTOR and reproduced with permission from Science Vol. 241, No. 4869, August 26, 1988. Photo by Kurt Stepnitz. © 1988 AAAS.

ISOLATING THE ETR1 GENE AT CALTECH

Tony took the etr1-1 mutant with him to Caltech, where he endeavored to isolate the affected gene as a postdoctoral researcher in Elliott Meyerowitz's laboratory. With postdoc Caren Chang, the job was completed, and a gene as interesting as the mutant was reported in 1993, again in Science (Chang et al., 1993). The team reported that ETR1 encoded a histidine kinase–like molecule flanked on the N terminus by three α-helices predicted to span a membrane and on the other end by a response regulator domain. As such, ETR1 resembled a prokaryotic two-component sensor, and the likeness was fuel for speculation that ETR1 may in fact be an ethylene receptor. Genetic relationships between etr1-1 and other mutations that affected ethylene signaling placed ETR1 well upstream in the ethylene signaling pathway, consistent with a receptor function. However, Tony was never one to let his critical reasoning (or yours, incidentally) be seduced by a good story. Instead, he would push for the best test of the idea.

ESTABLISHING RECEPTOR STATUS FOR ETR1 AT WISCONSIN

The most pressing questions at this point were “Is ETR1 the receptor”? and, if so, “How does it bind ethylene”? These questions began to fall at the hands of G. Eric Schaller, then a new postdoc. Eric had the hallway buzzing with the first evidence that ethylene bound to membranes of yeasts expressing wild-type ETR1 but not the etr1-1 point mutant. As may be expected for an alkene ligand, the binding site(s) was found to reside in the three α-helices buried in the membrane (Schaller and Bleecker, 1995). What seemed to cinch receptor status for ETR1 was the convincing chemical explanation of how a polypeptide could so tightly bind such a gas. A Cu⁺ atom, probably coordinated by Cys residues within the membrane-spanning domains, was found to stabilize ethylene within the polypeptide. This finding, which came largely from Fernando Rodriguez's PhD work and was also published in Science (Rodriguez et al., 1999), substantiated an old and persistent theory about ethylene binding in plants being based on interaction of a transition metal with the carbon–carbon double bond. (The Ag⁺ that florists add to a vase to slow petal abscission also blocks ethylene action in seedlings, undoubtedly by displacing the natural Cu⁺ cofactor of ETR1 without replacing its function.) ETR1 now had the distinction of being the first hormone receptor identified in plants.

THE ETHYLENE SIGNALING PATHWAY

It was by no means obvious how a mutation that prevents a ligand from binding to its receptor could be dominant, but mutagenesis of the receptor gene family provided the answer (Hua and Meyerowitz, 1998). In short, the ethylene sensing system is based on a double negative, to use a grammatical analogy. ETR1 and a few homologous receptors work together to activate the CTR1 kinase (Kieber et al., 1993), which acts to turn off the ethylene pathway. The important twist, in which Tony delighted and students get lost, is that ethylene binding turns off receptor signaling, thus inactivating CTR1, which releases the pathway from inhibition. Stopping receptor signaling with ethylene, or by genetically knocking out all the receptors, releases the ethylene response pathway from inhibition (Guo and Ecker, 2004). This inverse-agonist model neatly accommodates the dominant behavior of etr1-1. When ethylene has turned off all the wild-type receptors, signaling from one copy of the nonbinding mutant protein is enough to maintain CTR1 activity, which keeps the pathway repressed, creating ethylene insensitivity. In what should have been the first half of a career, Tony Bleecker had steered a field toward an elegant exposition of a hormone signaling pathway.

Tony was adept at thinking at the level of genes and molecules, but he never lost sight of what organismal studies could add. Adopting high-resolution techniques for measuring seedling growth developed in the adjacent laboratory, Tony and postdoc Brad Binder studied the time course by which ethylene inhibited hypocotyl elongation, minute by minute. When this technique was applied to ethylene mutants with defects affecting molecules ranging from receptors to transcription factors, temporal information and mechanistic nuance was added to the genetic model of ethylene action (Binder et al., 2004a, 2004b). The approach continues to reveal interesting features of ethylene action that traditional techniques do not capture (Binder et al., 2006).

BEYOND ETHYLENE

While the ethylene signaling work may be the best known of Tony's endeavors, he and his colleagues made significant contributions in other areas. Works on apical meristem arrest, senescence, abscission, and other topics were initiated purposefully due to an ethylene connection or by chance during the early phase of his independent career (Bleecker and Patterson, 1997). Some of these lines of research continue to advance through the efforts of Sara Patterson and Vojislava Grbic, who now run their own laboratories. One project unrelated to ethylene, spawned by serendipity years ago, has a particularly bright future. During the chromosome walk to the ETR1 gene, a transmembrane receptor kinase-like gene was discovered and named TMK1 (Chang et al., 1992). Four TMK genes form a group among the hundreds of receptor-like kinases in Arabidopsis (Shiu and Bleecker, 2001). Through an elegant reverse-genetic study of these four genes, PhD student Ning Dai garnered evidence that TMKs coordinate the processes of cell production and cell expansion during development. This project particularly excited Tony. He was convinced that the tmk mutants were providing clues about how development is orchestrated at the cellular level. Betting against Tony's intuition in this case is ill advised.

Tony availed himself of all that the university had to offer in terms of relevant scientific expertise, facilities, and forums for exchanging ideas. He enjoyed a joint appointment in the Department of Genetics, but his work was all performed in a broad botany department within the liberal arts college of the University of Wisconsin. He moved easily in this environment, using his sharp intellect, wide spectrum of interests, and sparkle to engage at large in the academy. He served very ably as department chair for four years until fighting cancer became necessary. The reason for explaining this professional environment is that a reader may discern its influence in the article by Wang et al.

STRUCTURE-FUNCTION OF THE ETR1 ETHYLENE BINDING DOMAIN

In a narrow sense, the work by Wang et al. is a structure-function study of the ETR1 ethylene binding domain. In a broad sense, it is a capstone synthesis of several recent laboratory members' research, performed at multiple levels of analysis. For his PhD thesis, Jeff Esch performed a taxonomically broad survey of ethylene binding that is here meshed with a comprehensive cataloging of ethylene receptor genomic signatures by Shin-Han Shiu to make a compelling case that a cyanobacterial ethylene binding domain entered plant lineages through endosymbiosis and became fixed in the genomes of land plants. At the other end of the scale of inquiry, postdoc Wuyi Wang extended the site-directed mutation studies of PhD student Anne Hall (Hall et al., 1999) to pinpoint seven amino acid residues in the first two membrane-spanning helices that define the ETR1 ethylene binding pocket. According to the inverse-agonist model, a mutation that impairs ethylene binding by ETR1 should confer dominant ethylene insensitivity in the plant. Wang et al. tested each site-directed mutant form of the receptor by expressing it in transgenic seedlings, and the model was found to hold. More illuminating was the class of mutations that did not impair binding but which nonetheless created insensitivity in seedlings. These mutations define a discrete region of the protein near the membrane-cytoplasm boundaries of the first and third helices. This region of the protein presumably plays a special role in coupling a conformational change in the transmembrane domains triggered by ethylene binding to inactivation of signaling by the cytoplasmic domains. Now, as a result of a mutant screen performed 20 years ago, how infinitesimal quantities of a simple two-carbon gas can exert effects on plant cells does not seem like such an unknowable. That a single research article can deliver penetrating insights from the evolutionary to the molecular scale is an indication of how mature the topic of ethylene signaling has become since the discovery of etr1-1.

Knowledge of the mechanisms by which plants sense ethylene has spread from the basic research laboratory to agricultural and horticultural arenas in the forms of transgenic plants and improved practices (Wilkinson et al., 1997; Klee and Clark, 2002). That Tony will not be present to feel the pride of contributing profoundly to whatever else unfolds from ethylene research is all the evidence one should need to conclude that fairness in life is inconstant at best.