The gaseous phytohormone ethylene has profound effects on plant growth and development. The biosynthesis of this hormone is influenced by many other hormonal, developmental, and environmental signals, and as such acts as an important point of crosstalk in regulating plant growth and development. This project will explore the mechanisms regulating ethylene biosynthesis in the model plant Arabidopsis thaliana. Previous studies have shown that the regulation of the stability of ACC synthase, a key enzyme in the production of ethylene, is the critical factor controlling how much ethylene plants make. The research supported by this award will build on these findings by charactering the half-lives of different ACC synthase proteins from Arabidopsis to determine how rapidly each is turned-over and how pairing different isoforms together affects their turnover. How other hormones interact to regulate the stability of these different ACS proteins will also be studied. This will help understand how these various signaling pathways influence the production of ethylene. A final aim is to examine the role of proteins that were previously identified as interacting with ACC synthase, with a focus on how these proteins may further regulate the function of ACC synthase.
Broader impacts: Results from this research will lead to a deeper understanding of the regulation of ethylene biosynthesis, which may lead to the ability to manipulate the production of this hormone in agricultural settings and hence an improvement in the quality and longevity of various agricultural products. Furthermore, the regulation of protein turnover has emerged as a central mechanism underlying a variety of biological process, and this project will shed light on this fundamental process. These studies will enhance the infrastructure of research and education by providing hands-on training for undergraduate students, graduate students, and post-doctoral researchers. The PI has connections with programs aimed at broadening participation in science. In addition, graduate students are also involved in summer science camp activities for 6-8th graders, providing them hands on science experiments related to plants and ethylene.
We have analyzed the mechanisms by which the model plant Arabidopsis thaliana regulates the production of and response to the simple gaseous hormone ethylene. This hormone regulates many processes important to plant growth and development, including fruit ripening, leaf and floral abscission and senescence, as well as the response to environmental stresses such as pathogen attack and drought. The work supported by this award helped elucidate the mechanism by which plants regulate the production of ethylene. Specifically, we found that the key enzyme of ethylene biosynthesis, ACC synthase, is regulated via an interaction with 14-3-3 proteins, which are ubiquitous, but poorly understood proteins in plants that bind to targets proteins only when they are phosphorylated on serine or threonine residues. Interestingly, these 14-3-3 proteins act in two distinct ways. First, they control the stability of a protein known as an E3 ligase that tags ACS for rapid degradation via addition of a peptide known as an ubiquitin moiety. Second, 14-3-3 proteins act independently to directly stabilize ACS proteins. These results have revealed a novel, but important way that plants control the level of E3 ligase proteins, which likely underlies the control of many other proteins. One puzzling aspect of ethylene biosynthesis is the fact that the enzyme catalyzing the key step in its biosynthesis, ACC synthase, is encoded by a large gene family in almost all plant species. Our work supported by this award has shown that the multiple versions of ACS proteins have distinct properties in terms of how quickly they are degraded in the cell, and how they respond to different environmental cues. This indicates that the plant has multiple versions of this enzyme in part to allow for distinct regulatory inputs in different cells and in response to various environmental signals. For example, when the plant needs to make large amounts of ethylene, it could increase expression of a very stable ACS isoform. In contrast, in cases where a highly dynamic level of ethylene synthesis is important, it may express an ACS isoform that is very short lived. A final aspect of this project focused on how cells respond to ethylene at the molecular level. We found that a protein kinase, CTR1, directly phosphorylated a key protein in ethylene signaling called EIN2. We found that this phosphorylation of EIN2 by CTR1 blocked the ability of EIN2 to be proteolytically cleaved, which is required for its activation. When cleaved, a part of the EIN2 protein is released from a membrane in the cell and moves into the nucleus where it activates ethylene responsive genes. When phosphorylated by CTR1, EIN2 is not cleaved, and thus does not enter the nucleus and ethylene responsive genes are not turned on. These studies have substantially extended the understanding of how ethylene is perceived by plant cells. In sum, in addition to increasing our understanding of ethylene biosynthesis and signaling, these results could open up new ways to regulate ethylene function in an agricultural setting, thus increasing the quantity or quality of various crops. Finally, these studies have supported the training of undergraduate and graduate students, a postdoctoral fellow and helped expose a high school student to cutting edge plant molecular genetic studies.
