Current Research

Mark Estelle is interested in how the plant hormone auxin regulates diverse aspects of plant growth and development.

Like all organisms, the development of plants is under genetic control. However, because of their sessile lifestyle, plants must also be highly responsive to their environment. Changes in light and temperature, changes in water and nutrient availability, and a variety of biotic interactions result in dramatic changes in plant form. A major question in plant biology is how diverse environmental inputs are integrated with the genetic program to result in coordinated growth and development.

The plant hormone auxin is involved in every phase of the plant life cycle, from embryogenesis to senescence. It regulates cell division, cell expansion, and/or cell fate, depending on the tissue or organ. During the past decade there has been tremendous progress in our understanding of all aspects of auxin biology. The emerging picture is that of a highly integrated network in which regulatory loops connect auxin signaling, transport, and biosynthetic pathways. The complexity of this network is consistent with the diverse roles of the hormone in the life of the plant.

My lab has focused primarily on auxin signaling. Auxin rapidly regulates transcription of thousands of genes through the action of two large families of transcription factors called the ARFs (auxin response factors) and Aux/IAAs (23 and 29 members in Arabidopsis, respectively). The ARFs bind DNA and either activate or repress transcription, depending on the context. The Aux/IAA proteins interact with the ARFs and repress transcription. Auxin relieves this repression by promoting degradation of the Aux/IAAs via a ubiquitin protein ligase (E3) called SCFTIR1 (and related SCFAFBs).

Several years ago we demonstrated that auxin stimulates Aux/IAA degradation by directly binding to the F-box subunit of SCFTIR1, a protein called TIR1, and promotes an interaction with the Aux/IAA substrate. Thus auxin is perceived by a novel receptor consisting of the F-box subunit of an SCF complex. This discovery established a new paradigm for both small-molecule sensing and E3 regulation. Our work shows that a small-molecule regulator can act by promoting an interaction between two proteins. It is likely that this concept will have important implications in studies of normal cellular regulation and of various disease processes and in the agricultural chemical industry.

Despite these exciting advances, our understanding of how auxin regulates plant growth and development remains rudimentary. Many aspects of auxin receptor specificity and activity are unclear. Similarly, the activities of the Aux/IAA and ARF proteins are poorly characterized, and the transcriptional networks that mediate various auxin-regulated growth processes have not been defined. Finally, how auxin impacts the cellular machinery involved in cell growth is largely unknown. The goals of the research in my lab are to answer some of these important questions.

Auxin Perception
The discovery that the F-box protein TIR1 functions in auxin perception established an exciting new paradigm for hormone perception. One of our major projects addresses the function of this new class of hormone receptors. TIR1 consists of the F-box domain and 18 leucine-rich repeats (LRRs), a domain that is important for interaction with other proteins. Structural studies by Ning Zheng (HHMI, University of Washington), in collaboration with my group, revealed that the LRRs fold into a horseshoe-shaped structure that surrounds an open auxin-binding pocket. A sequence in the Aux/IAA protein called a degron interacts with the auxin molecule and adjacent TIR1 residues, thus covering the pocket.

Surprisingly, auxin binding does not result in significant changes in the position of any TIR1 residues. Rather, the auxin molecule provides a platform for binding to the Aux/IAA degron. We are using the insights obtained from these studies to address several fundamental issues regarding auxin perception. For example, we are using biochemical methods to determine which parts of TIR1 are important for auxin binding. Surprisingly, we find that both TIR1 and the Aux/IAA protein are required for auxin binding. The two proteins function together as coreceptors.

In other studies, we are examining the auxin-binding properties of different pairs of F-box protein and Aux/IAA protein. Since Arabidopsis has 6 TIR1/AFB proteins and 29 Aux/IAA proteins, many combinations are possible, and different coreceptor pairs may have very different binding proteins. This would dramatically increase the effective range of auxin concentrations. Such a system may contribute to the complexity of auxin response.

To further explore issues of specificity and to perform high-throughput screens, we have developed a yeast-based assay for coreceptor activity. We are using this assay to investigate the specificity of TIR1/AFB-Aux/IAA interaction, to estimate the affinity of different coreceptor pairs for IAA and other auxins, and to screen for novel mutants in both the TIR1/AFB and Aux/IAA proteins. In the latter experiments, several classes of informative mutants have been identified, including those that exhibit decreased and increased interaction between the F-box protein and the Aux/IAA protein. We also plan to use this system to perform high-throughput screens for new compounds with auxin activity, both from available chemical libraries and from plant extracts. IAA is the best-characterized natural auxin, but it is very likely that plants produce additional auxins or auxin antagonists.

Auxin Regulation of Cell Expansion
The Arabidopsis seedling stem, or hypocotyl, is a powerful tool for understanding plant growth. Hypocotyls display tremendous sensitivity to environmental cues and exhibit a relatively simple growth response. Unlike almost all other plant organs, hypocotyls grow exclusively by cell expansion, simplifying the study of auxin-regulated growth. This expansion is tightly controlled by environmental factors such as temperature and light, as well as intrinsic factors such as auxin and other hormones.

A major focus of my group is to describe the function of the auxin network in the context of hypocotyl growth. This work involves identifying the TIR1/AFB, Aux/IAA, and ARF proteins that function in the hypocotyl. Genetic studies indicate that TIR1 and AFB1–AFB3 are positive regulators of hypocotyl growth and have overlapping functions. Surprisingly, the two members of the AFB4 clade have the opposite activity. Loss of these proteins results in an increase in hypocotyl elongation, indicating that AFB4 and AFB5 function as negative regulators of growth. We are currently determining the biochemical basis for this difference. In addition, we are working to characterize the auxin-regulated transcriptional networks that mediate hypocotyl elongation. Our goal is to determine the logic of the network.

One of the most important environmental regulators of plant growth is temperature. An increase in ambient growth temperature has a dramatic effect on Arabidopsis seedling architecture. Seedlings grown at 29°C have much longer hypocotyls, roots, and petioles than those grown at 22°C. We have shown that this growth response is rapid and associated with increased auxin levels and response. One of our goals is to determine the mechanism of temperature-dependent growth regulation. We have shown that the temperature response depends on auxin biosynthesis. In addition, microarray studies indicate significant overlap between the auxin-dependent and temperature-dependent gene sets, suggesting interaction between these regulatory networks.

A grant from the National Institutes of Health provides partial support for these projects.

As of April 7, 2016

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