The plant hormone auxin regulates many aspects of growth and development by modulating the expression of several hundred genes. Among the most rapidly and strongly auxin-induced genes are members of the Small Auxin Up-Regulated (SAUR) gene family. Although these genes appear to be present and are highly conserved as large gene families in all plants, virtually nothing is known regarding what role, if any, they play in auxin-mediated growth and development. Using a reverse genetic approach, we have found that overexpression of members of the Arabidopsis SAUR19 family containing N-terminal tags confers numerous phenotypes indicative of perturbed auxin-mediated growth. In contrast, overexpression of untagged SAUR19 results in no obvious affects on plant growth and development. Dr. Gray's findings suggest that SAUR19 is a highly unstable protein and the addition of an N-terminal tag to SAUR19 has a stabilizing effect on the protein. Studies to date lead to the hypothesis that SAUR19 plays a crucial role in auxin-mediated cell expansion, perhaps by functioning as a regulator of auxin transport. The goal of this project is to understand the function of SAUR19 and closely related SAUR family members in auxin regulated growth. Both forward and reverse genetic strategies will be employed to elucidate SAUR19 function, characterize SAUR19 interacting proteins, and investigate the auxin transport defect of SAUR19 transgenic seedlings in relationship to known components of the auxin transport machinery.
Broader Impacts: Sequence and expression analyses suggest that SAUR genes are present as large multigene families in all plants, including mosses, monocots, and dicots. Virtually nothing is known regarding the function of the SAUR genes. Since auxin plays a central role in regulating many aspects of plant growth and development, including several traits of agricultural import such as biomass, fruit set and size, and root system development, elucidation of SAUR functions is essential for understanding auxin action, and will be of broad interest. Additionally, it is expected that this work will also contribute to our understanding of the regulation of protein stability in plants and other eukaryotes. This project will provide support for one graduate student and one postdoc, both of whom will obtain broad training in genetics, molecular and cellular biological approaches. Additionally, integration of original research and undergraduate education is an integral component of this project. Dr. Paul Overvoorde and his undergraduate students at Macalester College will play vital roles in this project. Students in Dr. Overvoorde's courses will analyze the expression of SAUR genes using promoter-beta-glucuronidase reporters and will assemble this information into an online, searchable database that will be made available to the plant research community. The creation of this database will be used as a project-based learning assignment for Macalester students in computer science/bioinformatics courses taught by the co-PI and his colleague, Dr. Elizabeth Shoop. Additionally, one undergraduate student from Macalester will be selected each year to work in the Gray lab over the summer months. These students will interact closely with the PI, co-PI, and grad students and postdocs in the Gray lab where they will obtain a valuable and unique research experience in plant molecular genetics.
Like humans, plants produce hormones that regulate many aspects of their growth and development. The concept of plant hormones dates back to the pioneering studies of Charles Darwin in the 1880s, which suggested that plants produce a "signal" at their apex, which is transported down the stem of the plant causing it to bend and grow towards a light source. Since that time, scientists have discovered that Darwin’s "signal" is a small molecule called auxin and that it controls virtually every aspect of plant growth and development. The importance of this hormone in plant development was recognized immediately as indicated by its name "auxin", derived from the Greek word auxein, meaning "to grow". Some of the best-characterized examples of auxin-regulated processes include the control of plant size and shape, fruit and seed development, senescence, and responses to environmental cues such as light and gravity. One of the best-described effects of auxin is the promotion of plant cell expansion. Harnessing the ability of auxin to promote cell expansion is of great agro-economic import, as this has the potential to lead to the development of larger seeds, fruits, and vegetables for human and livestock nutrition, as well as increased biomass for biofuel production. To accomplish such tasks however, it is essential that we understand the biochemical and molecular mechanisms underlying auxin-mediated cell expansion. This project examined the possibility that Small Auxin Up-RNA (SAUR) genes mediate auxin-regulated cell expansion. SAUR genes are present as large gene families in all higher plants. SAUR gene expression is rapidly induced by auxin, but SAUR function remained elusive. To gain insight into SAUR functions, we engineered the model plant Arabidopsis thaliana to overexpress members of the SAUR19 subfamily of SAUR genes. We found that SAUR19 overexpression results in dramatic increases in plant cell expansion, leading to larger plant organs. Having demonstrated that SAUR19 family genes can indeed promote cell expansion, we sought to elucidate the mechanism. We identified a family of type 2C protein phosphatases that physically interact with SAUR19 and other SAUR proteins, and were able to demonstrate that SAUR binding inhibits the enzymatic activity of these PP2C.D phosphatases. Genetic and biochemical studies revealed that PP2C.D phosphatases function to negatively regulate the activity of plasma membrane proton ATPases (PM H+-ATPases). PM H+-ATPases have long been hypothesized to be activated by auxin and promote elongation growth by pumping protons across the plasma membrane to acidify the extracellular environment. However, this decades-old model called the "acid growth theory" has lacked molecular and biochemical support. Our findings that auxin-induced SAUR genes negatively regulate PP2C.D phosphatases to control PM H+-ATPase activity provides support for this hypothesis as well as crucial mechanistic insight. The above findings have many exciting and important implications. First, our demonstration that SAUR proteins regulate the enzymatic activity of PP2C.D family phosphatases is the first report of a biochemical/molecular function for SAUR proteins. This is a major accomplishment given that SAUR genes were first identified as primary auxin response genes over 25 years ago. Likewise, no prior functional studies of PP2C.D phosphatases had previously been reported. Secondly, we have provided a molecular framework connecting auxin signaling outputs (the induction of SAUR gene expression) to a physiological response (elongation growth). This establishes a molecular foothold on which the decades-old acid growth theory can be tested using modern molecular genetic approaches. And lastly, we have identified a novel mechanism by which plants regulate PM H+-ATPase activity. PM H+-ATPases are of extreme importance to plant life as, not only do they control cell expansion, but they also establish the electrochemical gradient that energizes secondary transport systems. Thus, it may be possible in future years to genetically manipulate SAUR or PP2C.D activity to modulate plant growth, nutrient uptake/ion transport, drought tolerance, or other PM H+-ATPase controlled processes. This project provided support for two PhD-level research associates, one high school student, and four summer exchange undergraduates from Macalester College. Resources generated during the project were also used by undergraduates in the Plant Physiology and Research-in Molecular Biology courses taught by the co-PI at Macaleter College. For example, the promoter-reporter gene lines formed the basis for inquiry-based labs in these courses. In addition, expression plasmids allowed many principles of the heterologous use of yeast as a model for molecular-genetics analysis of plant protein function to be examined.
