Biology Scholarship Proposal - Example 2

 

Introduction:  Plant tissues orient themselves upright in a gravitational field and have the ability to re-orient when this orientation is disrupted. This re-orientation (a process called gravitropism) is regulated by plant hormones.  Auxin is the primary plant hormone responsible for stimulating plant cellular growth and plays a central role in regulating gravitropism. When a stem is placed in a horizontal position, auxin is transported to the lower side of the stem stimulating growth and pushing the stem upright. While stem gravitropism is caused by auxin accumulated on the lower side, the gaseous hormone ethylene also increases after horizontal placement and plays a modulating role in regulating the process (Steed et al., 2004).  It is still unknown whether increased auxin on the lower side of horizontally-placed stems directly results in the increased ethylene. Ethylene usually acts as an inhibitor of shoot and root growth slowing gravitropic curvature, but has also been reported to stimulate growth under certain conditions.

 

Ethylene biosynthesis can be induced by environment stresses such as a heat-shock, flooding, change in orientation to gravity, and in response to other plant hormones, such as auxin. Ethylene is produced by the oxidation of 1‑aminocyclopropane‑1‑carboxylic acid (ACC), which is formed from S‑adenosyl‑methionine (AdoMet) in the following pathway developed by Adams and Yang (1979):

 

                   ACC synthase   ACC oxidase

Methionine  ŕ   AdoMet  ŕ     ACC    ŕ   ethylene

 

The regulation of enzyme ACC synthase (ACS) is considered to serve as the rate‑controlling step in ethylene biosynthesis. ACS enzymes are encoded by a gene family whose expression is differentially regulated in various tissues. Currently, there are eight functional ACS forms that interact as dimers (or pairs) to function in ethylene biosynthesis (Tsuchisaka and Theologis, 2004).

 

The specific project objectives are to:

·      evaluate the role of individual ACS enzymes in the regulation of  gravitropism in Arabidopsis seedlings

·      analyze expression changes for the various ACS genes during gravitropic curvature

 

Significance/uniqueness: The Arabidopsis genome project has provided “complete set of molecular genomic tools for future functional research on ethylene biosynthesis” (Tsuchisaka and Theologis, 2004). The Arabidopsis Biological Resource Center (ARBC) has a collection of mutants lacking the expression of all the ACS forms which will allow us to evaluate the role of a specific ACS enzyme in regulating gravitropic curvature. Mutants containing the regulatory region of the ACS genes attached to a reporter gene are soon to be available at the ARBC. These mutants developed by Tsuchisaka and Theologis (2004) produce either a colored or fluorescent protein product that shows tissue localization for each form of ACS enzyme.

 

Experimental Plan:

The measurement of gravitropic curvature is accomplished by image analysis. We plan to study the role of individual ACS members in stem growth and gravitropic curvature by comparing wild-type to mutants that do not express specific ACS forms. Seedlings will be grown upright in rows on square plates containing 1.2 % agar.  When the seedlings are three-days old, the plants will be rotated 90 degrees to change orientation and initiate the experiment.  We will measure the curvature at 0, 3, 5, and 7 hr time intervals using IMAGE J image analysis software. New transgenic lines that show tissue-level expression patterns for the ACS forms will be analyzed for the change in ACS expression during gravitropic curvature. These plants can be viewed by fluorescence and confocal microscopy to evaluate tissue level changes during curvature.

 

Ethylene production will be measured by gas chromatography (GC) to compare the changes in ethylene production from wild type and mutants.  For these measurements, seedlings will be planted in vials containing 1.2% agar. Vials containing three day old plants will be capped for four hours to allow ethylene to accumulate. One mL samples will be injected into the GC for ethylene analysis.

 

Specific outcomes: Preliminary research shows that mutants lacking expression of Arabidopsis-ACS4 have greatly increased ethylene as well as increased gravitropic curvature in dark-grown seedlings. Interestingly, these mutants lacking a biosynthetic enzyme produce more ethylene suggesting that removing one ACS form may cause the other ACS enzymes to form more active dimers. These preliminary results are intriguing, and we feel that complete gravitropic curvature profiles in combination of ethylene production and tissue expression changes for the ACS enzymes will provide important insight into the role of ethylene in gravitropic curvature.

 

Citations:

Adams D.O. and Yang S.F. 1979. Ethylene biosynthesis: identification of 1‑aminocyclopropane‑1‑carboxylic acid as an intermediate in the conversion of methionine to ethylene.  Proc. Natl. Acad. Sci. USA 76:170‑174.

Steed C.L., L.K. Taylor, and M.A. Harrison. 2004. Red-light regulation of ethylene biosynthesis and gravitropism in etiolated pea stems. Plant Growth Regulation:-in press.

Tsuchisaka A, Theologis A (2004) Unique and overlapping expression patterns among the Arabidopsis 1-amino-cyclopropane-1-carboxylate synthase gene family members. Plant Physiol. 136: 1-19

 

BUDGET

 

Student stipends: $1000

 

Task management: Since there are eight functional forms of ACC synthase genes, then each team member will be responsible for analyzing four functional forms. Students are expected to complete a minimum of 100 hours of research for this project.

 

We will have training sessions for each of the procedures required for experimentation.  These training sessions will include

1)    Image analysis of stem curvature using Image J software

2)    Measurement of ethylene levels using gas chromatography-flame ionization detection

3)    Analysis of gene expression using colorimetric and fluorescent reporter genes. Fluorescent images will be analyzed using the fluorescence scope (housed in the Dept. of Biological Sciences) or by confocal microscope (housed in the Chemistry Dept.)  David Neff will provide the training for the confocal microscope.

 

The students will meet with the faculty mentor weekly to discuss the data and to trouble shoot any problems.  These meetings will also include discussion of how to compile the data together to best present the results clearly in preparation for the Sigma Xi poster session.

 

Materials and supplies are available in the faculty advisor’s lab or will be obtained from the ARBC for the student use.  Therefore, no materials are requested with this project.